(Hus domesticus. Rut t y ) - UCL Discovery

T hes is f o r th e degree o f D oc to r o f P h ilo so p h y

M ito c h o n d r ia l DNA v a r ia t io n in B r i t i s h House mice (Hus domesticus. Rut t y )

C a th e rin e S Jones, B .Sc, (Hons)

U n iv e rs ity C o lle g e London, U n iv e rs ity o f London,

Gower S t re e t , London, WC1E 6BT.

J u ly 1990

ProQuest Number: 10609393

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I would l i k e t o d e d ic a te t h i s t h e s i s w i t h a l l my lo v e t o my Mother and

F a th e r , o th e rw is e known as "T u tb u ry P os tm a s te r and m is t r e s s "

THE LINNE AN SOCIETY 1780 1900 Eiectropr*ores« o* House rnouao mt DNA

- I I -

ABSTRACT!

d i iQ E b ° D d C i^ i_ J T ^ _ v ir i ation_Jjx_thB _JjJJ:j^Jp__Hou5B_m pjasB __!M u5_dpjne5ticu5i

R y tty K .

M orphom etric , k a ry o lo g ic a l and h is t o r ic a l Bvidencs in d ic a te s th a t C a ith ne ss

and OrknBy Housb micB (M us_dgmesticus. R u tty ) arB g e n e t ic a l ly d i s t in c t -from

o th s r B r i t i s h m ice, suggB sting th e y a re descended -from in t r o d u c t io n s .

M ito c h o n d r ia l DNA is a s m a ll, r a p id ly E vo lv in g , m a te rn a lly in h e r i te d m o le cu le ;

hsncB Bach mtDNA m o lecu le c a r r is s in i t s sequence thB h is to r y o f i t s lin e a g e

unco m p lica ted by rB co m b ina tio n . Thus, mtDNA RFLPs can bs ussd fo r a n a ly s in g

p o s s ib lB p a tto rn s o f c o lo n is a t io n and gene flo w in thBSB p o p u la t io n s .

H ig h ly p u r i f ie d mtDNA was is o la te d from each mouse and mapped, u s in g th e h ig h

re s o lu t io n r e s t r i c t io n method, w ith re sp e c t to the p u b lis h e d sequence o f mouse

mtDNA. T h is a llo w e d th e type s and in c id e n ce o f m u ta tio n a l change by w hich

mtDNA e vo lve s in th e House mouse to be eva lua ted . A t o t a l o f 23 mtDNA

com pos ite geno types, assayed us ing 14 r e s t r ic t io n enzymes, were re co gn ise d

among th e B r i t is h mice examined and a gene tic "b re a k " observed between

in d iv id u a ls from th e n o rth o f B r i t a in (Orkney, Ire la n d and N.E. S c o tla n d ; N.W

lin e a g e ) and those from th e south ( B r i t is h mainland, sou th o f C a ith ne ss and

S u th e rla n d ; S.E lin e a g e ) . The approxim ate lo c a tio n o f t h i s "b re a k " co rresponds

w ith th e G reat Glen f a u l t , which marks a boundary between in h o s p ita b le

m oorland, occup ied by Apodemus. Geographic o r ie n ta t io n o f mtDNA v a r i a b i l i t y

i s conco rdan t w ith da ta from o th e r sources, in c lu d in g th e p a te rn a l Y -

chromosome DNA. The House mouse i s u n l ik e ly to have s u rv iv e d th e la s t

g la c ia t io n , d a t in g th e e a r l ie s t p o s s ib le B r i t is h c o lo n is a t io n to about 10,000

B.P. An in te g ra te d approach, us ing evidence from a n th ro p o lo g ic a l,

p a la e o n to lo g ic a l, g e n e tic a l and h is t o r ic a l sources, p e rm its th e p ro g re s s io n

o f th e house mouse to be fo llo w e d th rough Europe. These da ta in d ic a te th a t

Hus domesticus p ro b a b ly reached North-W est Europe and B r i t a in in th e Iro n

- I I I -

Age. Hence, d ive rg e n ce s o f such m agnitude between th e N.W and S.E lin e a g e s a re

in c o n s is te n t w ith s u b s t i tu t io n s accum la ting in situ s in c e t h e i r a r r i v a l ;

c o n s is te n t w ith th e N.W and S.E form s o r ig in a t in g from se p a ra te in t r o d u c t io n

e ven ts from d i f f e r e n t a n c e s tra l sources.

Such a d is t in c t "b re a k " cou ld have been m a in ta ined by a number o f e i th e r

e x t r in s ic (g e o g ra p h ica l b a r r ie r s ) and /o r in t r i n s ic fa c to r s in c lu d in g ,

m ain tenance o f t e r r i t o r i e s , and s p e c i f ic mate p re fe re n c e s . A p o p u la r v ie w i s

t h a t house m ice l i v e in b e h a v io u ra lly is o la te d t r ib e s o r demes o f between 4 -

6 in d iv id u a ls , w ith v e ry l i t t l e gene f lo w between them. I t i s b e lie v e d th a t

as a consequence o f t h i s r i g id s t r u c tu re , im m ig ran ts in t o an e s ta b lis e d

p o p u la t io n a re u n l ik e ly to be re p ro d u c t iv e ly s u c c e s s fu l, and g e n e tic d r i f t

w i l l become im p o r ta n t in shap ing t h e i r p o p u la tio n s t r u c tu r e . T h is concep t may

be to o i n f l e x ib l e , as v i r t u a l l y every lo n g itu d in a l s tu d y o f f e r a l m ice has

shown some p o p u la t io n m ix in g . The I s le o f May in t r o d u c t io n e xpe rim en t

in v e s t ig a te d th e r e la t i v e im po rtance o f these i n t r i n s i c fa c to r s . House m ice

from Eday (Orkney) re le a se d in t o an e s ta b lis h e d p o p u la tio n on th e I s le o f May

( F i r t h o f F o rth ) in A p r i l 19B2, subsequen tly bred w ith th e endemic m ice . The

r e la t i v e m a terna l and p a te rn a l c o n tr ib u t io n s to th e success o f t h i s

in t r o d u c t io n were s tu d ie d us in g mtDNA and Y-chromsome m arke rs . D i f f e r e n t ia l

in t r o g r e s s io n was observed : Eday Y-chromosome a p p a re n tly spread a t a s im i la r

r a te to th e autosom al genes, w h ile Eday mtDNA in c re a se d in in c id e n c e and

d is t r ib u t io n a t o n ly o n e - th ird th e ra te . The tem poral and s p a t ia l d is t r ib u t io n

o f Eday d e r iv e d DNA showed th a t males d isperse and in t r o g r e s s more r a p id ly

than Eday fem a les , and form a s ig n i f ic a n t ly h ighe r p ro p o r t io n o f th e m a ting

p o p u la t io n than May m ales. C le a r ly , th e re seems to be no s o c ia l b a r r ie r s to

gene f lo w in t h i s f e r a l p o p u la tio n . The Is le o f May in t r o d u c t io n has a llo w e d

e v a lu a t io n o f mtDNA as a g e n e tic m arker, d e s c r ib in g p o p u la t io n s t r u c tu r e and

m a t r i l in e a l k in s h ip on a m ic rogeog raph ica l sca le .

AcknowledgementSi

I would l i k e to thank my main s u p e rv is o r , P ro fe sso r R .J B e rry f o r

in t ro d u c in g me to th e a r t o f mouse c a tc h in g from co rn r i c k s (a r a th e r

co m ica l co m b in a tio n o f g o a l-k e e p in g s k i l l s and rugby ta c k le s ! ) and f o r

re a d in g a few d r a f t s o f t h i s th e s is . A d d it io n a l ly , i f th e m a jo r i t y o f th e

f ie ld w o r k had n o t been s itu a te d in th e Orkneys, I may neve r have d is c o v e re d

th e jo y s o f a rch a e o lo g y , th e Orkneys be ing p a r t i c u la r l y s tee p ed in

p r e h is to r y , w ith th e fa s c in a t in g r u in s o f Skara B rae , L in k s o f N o lt la n d ,

Knap o f Howar, Maes Howe and many, many m ore .. . . th a n k you Sam. I would

l i k e t o ex tend my tha n ks to my jo i n t s u p e rv is o r , Dr D avid Latchm an, f o r

making a v a i la b le f a c i l i t i e s in h is la b o ra to ry e s s e n t ia l f o r th e m a jo r i t y o f

th e s e s tu d ie s , and fo r p a t ie n t ly re a d in g through a few c h a p te rs (e s p e c ia l ly

c h a p te r 3 f o r which he deserves a m e d a l! ! ) . To a l l th e p e o p le in C5

la b o ra to ry f o r h e lp and a d v is e in th e r a p id ly expand ing f i e l d o f m o le c u la r

b io lo g y , in c lu d in g Lynne Kemp, Pam and Bernado V i l l e r e a l , te c h n ic a n John,

and "sperm " A lis o n , many th a n k s . Thanks to a l l my c o lle a g u e s and fe l lo w

in h a b ita n ts o f th e "F ly House" la b o ra to ry , fo r t h e i r h e lp and f r ie n d s h ip

th ro u g h o u t my th re e years research? in p a r t ic u la r V in c e n t Bauchau, Les

Cooper, Pat Edwards, Mo F in d la y , P e te r K ing, Les N ob le , Jo P em berton, Paul

S c r iv e n , and Paul Sunnocks.

I am g r e a t ly in d e b te d to Dr Hakan Tege ls trom fo r p a t ie n t l y te a c h in g me,

d u r in g my b r ie f v i s i t to U ppsa la , th e re c e n t ly deve loped s e n s i t iv e s i l v e r

s ta in in g v is u a l is a t io n te c h n iq u e s , which became a v e ry v a lu a b le and

im p o r ta n t method th ro u g h o u t my s tu d y . For h is e n th us iasm , s u p p o r t ,

s t im u la t in g d is c u s s io n s on MtDNA tech n iq ue s and a p p l ic a t io n s , and

f r ie n d s h ip I am e te r n a l ly g r a te fu l .

I am v e ry g r a te fu l to Helen McVeigh a t the Queens U n iv e r s i t y o f B e l fa s t ,

-for her - fr ie n d s h ip and encouragement; i t was a c o n s ta n t source o f co m fo rt

and in s p ir a t io n th a t I was no t a lone in th e f r u s t r a t in g , and a t tim e s

d ep re ss in g f i e l d o f m ito c h o n d ria l g e n e tic s in B r i t a in . I would a ls o l i k e to

thank Helen and Dr Montgomery f o r t h e i r g e n e ro s ity in a r r a n g in g /c o l le c t in g

some I r is h m ice fo r use in t h is s tu d y .

Thanks to a l l th e fa rm e rs , f r ie n d s and c o lle a g u e s , to o numerous to l i s t in

d e t a i l , who e ith e r p ro v id e d th e specimens to work on o r gave t h e i r

p e rm iss io n to c o l le c t o r t ra p on t h e i r la n d , bu t e s p e c ia l ly to : Dr Graham

T r ig g s fo r w ith o u t h is con tin u ed b ia n n u a l tra p p in g seasons on th e I s le o f

May and to a l l those th a t accompanied him th ro u g h o u t th e y e a rs , c h a p te r 6

c o u ld never have been w r i t te n ; Marcus from th e W estray, O rkney, who f e r r ie d

us back and fo r th from th e I s le o f F a ray , and fo r h is e x c e lle n t

h o s p i t a l i t y ; to th e d i f f e r e n t d o c to rs a t Trenaby House, W estray over th e

y e a rs , f o r t h e i r h e lp and accommodation; to D r’ s J im and Sandy M a lle t f o r a

mouse from t h e i r son’ s n u rs e ry ;, and to Dave C la rke and Paul P ie rc e -K e lle y

f o r m ice from th e in s e c t house, London Zoo. Thanks a ls o to Dr Jean-Marc

B oyle fo r encouragement in th e e a r ly days o f my th e s is and f o r a id in some

com puting .

The acknowlegements would not be com plete w ith o u t m en tion in g th e members o f

th e "p o p u la t io n G ene tic group" in th e G ene tic departm ent a t U n iv e rs ity

C o lle g e London, fo r comments and a d v is e , and in a p a r t ic u la r case, much

needed " d i r e c t io n " , my thanks to D r’ s S teve Jones, Jim M a lle t , N ick B a rto n ,

Shahin R ouhani, Les Noble, and C h r is t ia n Raboud, and to th e pHd s tu d e n ts

Dot C u r r ie , N e il Sanderson and C h ris Beaumont.

I cannot express s u f f ic e n t ly my u n l im ite d g r a t i tu d e to my bes t f r ie n d and

c o n f id e n t, Dr Les N oble, now a t G ene tics D epartm ent, O xford U n iv e rs ity , fo r

w ith o u t a doubt I would p ro b a b ly never have f in is h e d in t h i s decade w ith o u t

h is he lp (above and beyond the c a l l o f d u ty ) , never f a i l i n g encouragement

and " fo r ju s t be ing th e re " to gu ide me th ro u g h th e peaks and tro u g h s o f th e

u lt im a te t o r t u r e in v e n te d by mankind - " th e s is w r i t i n g ! " .

F in a l ly , bu t by no means le a s t , to th e p eop le who have ke p t me sane, w ith

t h e i r co n tin u e d su p p o rt and lo v e , n o t to m ention th e c o n s ta n t nagg ings to

ensure I f in is h e d th e p a in fu l p rocess o f w r i t in g up - my p a re n ts .

- 711

TABLE OF CONTENTS

IABLE_OF_CONIENISi

T i t l e p a g e ............. ........ ............................................................................................................I

D e d ic a t io n . ...................................................................... 11

A b s t ra c t .................... . . . . . . . . . . I l l

Acknowl edgements ......................... .V

C o n te n ts ................... ........ ...................... .............................................................. ............. .. VI11

L is t o f T a b le s . . . . . ............. . . . . . . X V

L is t o f F ig u re s ............................. . .X V I I I

L is t o f P la te s ............................................................................... XX II

QH0PTER_ONEl_Introduction

1.1 General In t r o d u c t io n .....................................................................................................1

1 .2 M ito c h o n d r ia l DNA - th e d e f in i t i v e g e n e tic marker o f p o p u la t io n and

e v o lu t io n a ry b io lo g y . ......... .......... ...................................................................... ...........4

1 .2 .1 General P ro p e r t ie s and m o lecu la r c h a r a c te r is t ic s o f mtDNA.. . . 4

1 .2 .2 mtDNA as a p h y lo g e n e tic t o o l . . ...................... 11

1 .3 The house mouse (Hus domesticusf R u tty ) - th e s tu d y o rg a n is m .. . . 16

1.4 General aims o f th e s t u d y . . ............... . . . . . 2 0

QHAPIER_IWOi_Material_and_Methgds

2.1 A b b re v ia t io n s . ............. 28

2 .2 M a te r ia ls ............................................................................. 28

2 .2 .1 Sam pling p ro ce d u re s ............................................ 28

2 .2 .2 R eagents........................................................ 29

2 .2 .3 M o le cu la r w e ig h t s ta n d a rd s ............................. .30

2 .3 L a b o ra to ry m e th o d s ...................... 30

- V I I I -

TABLE OF CONTENTS

2 .3 .1 M ito c h o n d r ia l DNA is o la t io n .................................. 31

2 .3 .1 .1 R a tio n a le o f m e tho d o log ie s ..................................................... . ...............31

2 .3 .1 .2 M o d if ie d phenol e x t r a c t io n method ................. . . . . . . . . . 3 3

2 .3 .1 .3 mtDNA is o la t io n u s in g u l t r a c e n t r i f u g a t io n .................. ..................36

2 .3 .1 .3 .1 Use o f a v e r t ic a l r o t o r . ............................................................. . .3 6

2 .3 .1 .3 .2 Use o f a sw in g -o u t r o t o r ................................. .38

2 .3 .1 .4 R e s t r ic t io n endonuclease d ig e s t io n s ......................... 39

2 .3 .1 .5 Gel e le c tro p h o re s is .................... . . . . . . 4 0

2 .3 .1 .6 M ito c h o n d r ia l v is u a l is a t io n te c h n iq u e s . . ......................................40

2 .3 .1 .6 .1 S i lv e r s ta in in g ............................... . . . 4 0

2 .3 .1 .6 .2 E th id iu m brom ide s ta in in g ................................ . . . . . . 4 1

2 .3 .1 .7 Q u a n t if ic a t io n o f mtDNA...................... 41

2 .3 .2 Y-Chromosome m e th o d o lo g ie s . . . . . . . ................ 43

2 .3 .2 .1 I s o la t io n o f t o t a l genomic DNA............... 43

2 .3 .2 .2 R e s t r ic t io n enzyme d ig e s t i o n s . . . . . . .................. . . . . . 4 3

2 .3 .2 .3 Agarose ge l e le c tro p h o re s is o f genomic DNA............................... 44

2 .3 .2 .4 DNA t r a n s f e r . . ............................... 44

2 .3 .2 .5 P r e - h y b r id iz a t io n .................. .46

2 .3 .2 .6 H y b r id iz a t io n ...................................... 46

2 .3 .2 .7 R e -h y b r id is a t io n ........................................................... 47

2 .3 .2 .8 C lones ...................................................................................................... ...........48

2 .3 .2 .9 P re p a ra tio n o f com petent c e l l s ........................................................... 48

2 .3 .2 .1 0 T ra n s fo rm a tio n ...................................... 49

2 .3 .2 .1 1 M in i-p re p a ra t io n o f p lasm id DNA.. ........... . . . . . . . . . 4 9

2 .3 .2 .1 2 D ig e s tio n o f p lasm id DNA & p u r i f i c a t io n o f th e i n s e r t . . 50

2 .3 .2 .1 3 01 ig o - la b e l in g r e a c t io n ........................................................................ 30

- I X -

TABLE OF CONTENTS

2 .3 .2 .1 4 S e p a ra tio n o-f u n in c o rp o ra te d n u c le o t id e s ..................... 51

2 .3 .2 .1 5 A u to ra d io g ra p h y . .................................................................. 52

2 .4 Data A n a ly s is and in t e r p r e t a t io n ....................... . .5 2

2 .4 .1 Com posite DNA g e n o ty p e s . . . ............................... . . . . . 5 2

2 .4 .2 Fragment m o le cu la r w e ig h t e s tim a te s and s i t e m apping.................. 53

2 .4 .3 E s tim a te s o f sequence d iv e r g e n c e . . . . . ....................................... . . . . . 5 5

2 .4 .4 T ree c o n s t r u c t io n . . . ............................... 58

CHAPTER THREEs M o le cu la r e v o lu t io n o f B r i t i s h house mouse (Hus

domesticus) m ito c h o n d r ia l DNA.

3 .1 I n t r o d u c t io n . ........................................... 91

3 .2 M a te r ia ls and m ethods.................................................. ........................................... . .9 3

3 .2 .1 O rd in a t io n o f d a t a . . . . . . . ............... . . . . . 9 5

3 .3 R e s u l t s . . ................................................................................................................................ 96

3 .3 .1 Fragment d ig e s t io n p r o f i l e s . . . . . . . . . . ................................ .96

3 .3 .2 C leavage m apping.............. ........................................................ ..............................97

3 .3 .3 A n a ly s is o f s i t e g a in s .............. ................................... 98

3 .3 .4 A n a ly s is o f s i t e l o s s e s . . . . . . . . .................... 101

3 .3 .5 Summary o f s i t e o c c u r re n c e s . . ...................... 101

3 .3 .5 .1 Genomic d is t r ib u t io n s o f c leavage s i t e s ................ . . . . . 1 0 1

3 .3 .5 .2 D is t r ib u t io n o f s i t e s w ith in and between gene r e g io n s . . . . 103

3 .3 .6 Gene v a r i a b i l i t y . ........................................................................................ . . . . . 1 0 5

3 .3 .7 D e s c r ip t io n o f s i t e d i f f e r e n c e s . . . . . . . . . . .............................................. 106

3 .3 .7 .1 H e xa n u c le o tid e r e s t r i c t io n e n z y m e s . . . . . . ...................... .106

3 .3 .7 . 1.1 H ind I I I (AAGCTT).................................................................................. 106

3 .3 .7 . 1 .2 Xba I (TCTAGA).......................................... 107

TABLE OF CONTENTS

3 .3 .7 .1 .3 H inc I I (GT CPuPy] AC)................................................................ . . .1 0 8

3 .3 .7 .1 .4 Acc I (GT CA/CI CG/T3 AC)................................................................108

3 .3 .7 .2 F iv e b a s e -c u t te r r e s t r ic t io n endonuc leases.................. 109

3 .3 .7 .2 .1 Ava I I (GG CA/T3 C O ........................................................................... 109

3 .3 .7 .3 T e tra n u c le o t id e r e s t r i c t io n endonucleases.................................... 110

3 .3 .7 .3 .1 Fnud I I (CGCG).........................................................................................110

3 .3 .7 .3 .2 Hpa I I (CCGG)......................................................................................... 110

3 .3 .7 .3 .3 Taq I (TCGA)............................................................................................I l l

3 .3 .7 .3 .4 Hae I I I (GGCC).........................................................................................I l l

3 .3 .7 .3 .5 H in t I (GA CN3 T C ) . . . . . .................................................................... 113

3 .3 .7 .3 .6 Mbo I (GATC)............................................................................................115

3 .3 .7 .3 .7 Rsa I (GTAC)............................................................................................115

3 .3 .7 .3 .8 A lu I (AGCT)............................................................................................117

3 .3 .7 .3 .9 Sau 961 (GG CN3 CC).............................................................. 119

3 .4 D is c u s s io n ............................................................................................................................. 120

3 .4 .1 DNA v a r ia t io n across th e m ito c h o n d ria l g e n o m e . . . . . . . ........... . . . . 1 2 0

3 .4 .2 Base sequence v a r ia t io n in animal mtDNA................................................. 121

3 .4 .2 .1 P ro te in -c o d in g g e n e s . . . ........... . . . . . . . 1 2 1

3 .4 .2 .2 Ribosomal genes .................... . . . . 1 2 3

3. 4 .2 .3 T ra n s fe r RNA...................................................................................................... 124

3 .4 .2 .4 The d isp lace m e n t lo o p ............................... .124

3 .4 .3 Rates and ty p e s o f change in animal m tD N A ... ...................... 129

3 .4 .4 R e s t r ic t io n mapping c o m p le x it ie s and c o m p lic a t io n s ............... . . . . 1 3 6

3 .4 .4 .1 D i f f e r e n t mtDNA v is u a l is a t io n m e t h o d s . . . . . . . . ............. . . . . . . 1 3 6

3 .4 .4 .2 Cases o f m ism apping.................................................................... 138

3 .5 Summary. ............. 138

TABLE QF CONTENTS

C H A P TE R _FO yR i_P hylggeograB hic_Bgg iu la tign_5tructure_gf_B riti5h_hgu5e_(n ice

B Q Q yl§ tiQ D I_ i5sessed_by_m itgchgndria l_pN A i

4 .1 In t r o d u c t io n ............................................................................ . . . . . . . 2 3 0

4 .2 M a te r ia ls and m ethods......................................................................................................233

4 .2 .1 M ice .................................................. 233

4 .2 .2 L a b o ra to ry p ro ced u re s ................................................. .234

4 .2 .3 Data a n a ly s is .................................................................................................... . . . . 2 3 4

4 .3 Resul t s ............. 236

4 .3 .1 M ito c h o n d r ia l DNA v a r ia t io n in B r i t a in .........................................................236

4 .3 .2 M ito c h o n d r ia l DNA v a r ia t io n in Orkney and N.E. S c o t la n d . . . . . . . . 2 3 3

4 .3 .3 P h y lo g e n e tic c o n s id e ra t io n s ........................................ ................................... . .2 3 9

4 .3 .3 .1 M ic rog e og ra p h ic s t r u c tu r in g ................................ 239

4 .3 .3 .2 M acrogeograph ic s t r u c t u r in g . ............................................... . . . . . . . . 2 4 2

4 .4 Di scuss i o n ............................... 246

4 .4 .1 M ic rog e og ra p h ic s t r u c tu r in g in B r i t a in .........................................................246

4 .4 .2 M acrogeographic s t r u c tu r in g ! evidence from o th e r European mice in

th e search f o r a n c e s tra l p o p u la tio n s o f the B r i t is h house mouse............... 253

4 .4 .3 P a tte rn s o f c o lo n is a t io n suggested by mtDNA a n a ly s e s . . . . . . . . . . . 2 5 8

CHAPIER_FWE£_lntr a-sgeci_£ic_Y_chrgmg5gme_pNA_Yariat i_gn_i_n_t he_Bri_t i_sh

b 9yi§_!Dou5e_£ tfus jfpaest icusJRut t y

5 .1 In t r o d u c t io n ...................... . . . . 3 1 2

5 .2 M a te r ia ls and m ethods ......................................................................................315

5 .2 .1 Col l e c t io n s . ............... 315

5 .2 .2 D e s c r ip t io n o f th e Y -s p e c if ic probe, p Y C R 8 /B .... ............................. . .3 1 5

5 .2 .3 L a b o ra to ry p ro ce d u re s ................................. 316

TABLE OF CONTENTS

5 .2 .4 Data a n a ly s is ............................ . .3 1 7

5 .3 R e s u lts ................................................................................................................. .318

5 .3 .1 Y chromosome DNA r e s t r i c t io n fragm ent v a r i a t i o n . . . . . . ...................... 318

5 .3 .2 P h y lo g e n e tic a n a ly s e s ................................................................ 319

5 .4 Di scu ss i o n ................................................................................... 323

5 .4 .1 I n t r a - s p e c i f ic phy log e og ra p h ic c o n s id e r a t io n s . . . . .......................... . . 3 2 3

5 .4 .2 Y chromosome e v o l u t i o n . . . . . ................................................................................. 329

5 .4 .3 Y chromosome DNA v a r i a b i l i t y . ............... . . . . . . . 3 3 1

Q H A P IE R _S IX i_p iffe ren tia l_5B read_and_gene_ f lgw _gf_m itgchgndria l_D N A_and_Y

chromosome DNA in an is la n d p o p u la tio n o f th e house mouse (Hus dowesticus

R y t ty K

6 .1 I n t r o d u c t io n . ...................................................... .367

6 .1 .2 The I s le o f May s tudy a re a ........................................... 370

6 .2 M a te r ia ls and m ethods............................ 371

6 .2 .1 C o lle c t io n o f samples...............................................................................................371

6 .2 .2 M ito c h o n d r ia l DNA p re p a ra t io n s ...................................................... 372

6 .2 .3 Y chromosome p re p a ra t io n s ......................................................................................373

6 .3 R e s u lts ............... . . . . . . . . 3 7 4

6 .3 .1 M ito c h o n d r ia l DNA g e n e tic m arke rs ............................................... 374

6 .3 .2 The Y chromosome DNA g e n e tic m a rk e rs . . ................................................. . . . 3 7 6

6 .3 .3 M o n ito r in g o f the in tro d u c e d Eday genes us ing mtDNA and Y-chromosome

DNA....................................................................................................................................................... 377

6 .4 D is c u s s io n ............. . . .3 8 0

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TABLE OF CONTENTS

CHAPIER_SEyENi_PISCUSSIONi

7 .1 : D is c u s s io n . ........................... .423

7 .1 . is Phylogeography o-f th e house mouses An in te g ra te d a p p ro a c h .. . . . . 423

7 .1 .2 s F o llo w in g gene f lo w w ith sex s p e c i f ic m arkers.................... . . . . . . . . . 4 3 6

7 .1 .3 : C o lo n is a t io n and sex s p e c i f ic m arkers........................................ . . . . . . . . 4 4 0

7 .1 .4 : L im ita t io n s o f mtDNA as a p h y lo g e n e tic m a r k e r . . . . ...............................44

7 .1 .5 : F u tu re s t u d ie s . . .................. 447

7 .2 : Summary......................................................................................... 451

BIBLIOGRAPHY.....................................................................................................................................453

APPENDIX..

A. Is An im proved ra p id method fo r c reen ing m ito c h o n d ria l DNA v a r ia t io n in

c lo s e ly r e la te d P o p u la tio n s - Jones e t e l - , (1989 )............. 493

A. 2 : The th e o r e t ic a l re la t io n s h ip s between the p ro p o r t io n o f shared DNA

fra gm en ts (F) and th e number o f n u c le o t id e s u b s t i tu t io n s per s i t e ( d ) , fo r

r e s t r i c t io n enzymes re c o g n is in g 4 ,5 and 6 basepa ir s e q u e n c e s ........................500

- X I V -

TABLE OF CONTENTS

LISI_OF_JABLESi

CHAPIiR.IWQi

2 .1 : A b b re v ia t io n s . ............. 60

2 .2 : Sampling s i t e s in B r i t a in and I re la n d .......................... 62

2 .3 : L is t o-f m ajor b u f fe r s , and s o lu t io n s .................. . . . . 6 6

2 .4 : C h a ra c te r is t ic s o f r e s t r i c t io n endonucleases used............................... 69

2 .5 : M o le cu la r w e ig h t s ta n d a rd s : fragm ent s iz e s and s i t e lo c a t io n s

re cogn ised in th e p u b lis h e d mouse m ito c h o n d ria l DNA re fe re n c e

sequence .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1

2 .6 : A d d it io n a l m o le cu la r w e igh t s ta n d a rd s . . .......................................................... 79

CHAPJER_IHREEi

3 .1 : M ito c h o n d r ia l DNA r e s t r i c t io n fragm ent d ig e s t io n p r o f i l e s ..................140

3 .2 : Summary o f r e s t r i c t i o n fragm ent d ig e s t io n p r o f i l e s ..................................155

3.3A -N : V a r ia b le r e s t r i c t io n s i t e s o f mouse mtDNA d e te c te d w ith each o f th e

fo u rte e n r e s t r i c t io n endonucleases................... 157

3 .4 : Summary o f lo c a t io n s o f r e s t r ic t io n s ite s fo r 14 enzymes in Mus

domesticus mtDNA...................................................................................... ................................. 169

3 .5 : S ite g a in s f o r s p e c i f ic s u b s t i tu t io n s ............................................................... 171

3 .6 : Base s u b s t i tu t io n s rs p o n s ib le fo r s i t e ga ins in mouse mtDNA.. . . . . 176

3 .7 : Summary o f a l l s i t e g a i n s . . . . . ........................................ . .1 7 8

3 .8 : Summary o f s i t e lo s s e s d e tec ted by each o f 14 enzymes ...........179

3 .9 : R egional v a r i a b i l i t y : gene s iz e and number o f s i t e s . . . ...................... .181

3 .1 0 : Regional v a r i a b i l i t y o f mtDNA..... in Mus dowesticus............... 182

3 .1 1 : Summary o f re g io n v a r ia t io n in Mus dowesticus m tD N A .. . . .................. 184

3 .1 2 : M ito c h o n d r ia l gene v a r ia b i l i t y comparison between mouse and man.185

TABLE OF CONTENTS

QHAPIER_FOyRi

4 .1 : The mtDNA com posite genotypes (mt c lones) observed among samples o f

B r i t i s h house mice (Mus domesticus) ............... 267

4 .2 : F requenc ies o f mtDNA c lones across B r i t a in ....................................................271

4 .3 : P h y lo g e n e t ic a lly in fo rm a tiv e r e s t r i c t io n s i te s in th e B r i t i s h house

mouse us ing 14 r e s t r i c t io n e n d o n u c le a s e s . . . . . ........................................................ 273

4 .4 : M a tr ix o f sequence d ive rgence e s tim a te s between mtDNA com posite

c lo n e s from the B r i t is h house mouse u s in g 14 enzymes.................... . . .2 7 5

4 .5 : P h y lo g e n e t ic a lly in fo rm a tiv e r e s t r i c t io n s i te s o f European house mice

u s in g 11 r e s t r i c t io n endonucleases............... 277

4 .6 : P h y lo g e n e t ic a lly in fo rm a tiv e r e s t r i c t io n s i te s from w o rld -w id e samples

o f th e house mouse us ing two r e s t r i c t io n edonucleases, Mbo I S< H in f 1.279

c h a p ie r _f w e i

5 .1 : The Y chromosome DNA com posite genotypes observed among th e samples o f

Mus domesticus from B r i t a in .................................................................................................335

5 .2 : Fragment s iz e s ( in b a s e -p a irs ) c h a ra c te r is in g th e r e s t r i c t io n p r o f i le s

o f Y chromosome DNA genotypes in th e B r i t is h house mouse, us ing e ig h t

r e s t r i c t io n endonucleases..................................................................................................... 337

5 .3 : M a tr ix o f p e rcen t n u c le o tid e d ive rgence e s tim a te s and p ro p o r t io n o f

shared Y chromosome DNA r e s t r i c t io n fragm ents (Nei & L i , 1979) fo r the

B r i t i s h house mouse (Mus domesticus)............................................................................ 340

5 .4 : P h y lo g e n e t ic a lly in fo rm a tiv e r e s t r i c t io n fragm en ts o f th e B r i t is h

house mouse (Mus domesticus) Y chromosome DNA us in g 8 r e s t r i c t io n

endonucl e a s e s . ............................................................... 342

TABLE OF CONTENTS

CHAPIER_SIXi

6 .1 : F requencies o f mtDNA com posite genotypes observed among N o rth e rn

Orkney Is le s ( in c lu d in g Eday) and p re - in t r o d u c t io n I s le o f May samples o f

house mice iMus domesticus)................................................................ . ............................ 390

6 .2 : Homerange and d is p e rs a l e s tim a te s in house mouse p o p u la t io n s . . . .3 9 2

TABLE OF CONTENTS

LISI_OF_FIGL)RESi

CHAPIER_ONEi

l . l : O rg a n is a tio n o f mouse m ito c h o n d ria l DNA......................................................... .2 5

1 .2 : D is t r ib u t io n and taxonomy o f th e house mouse................. .............................. 27

CHAPIER_IWQi

2 .1 : D is t r ib u t io n o f m ajor sam pling lo c a l i t ie s in s i t e s B r i t a in and

I r e la n d ............................................. ............................. ........................ ............... 81

2 .2 : Mouse id e n t i f i c a t io n m arking s c h e m e ................................. ..................................82

2 .3 : Graph to i l l u s t r a t e th e p r o b a b i l i t y o f not d e te c t in g sequence

d iv e rg e n c e s .................................................................................................................................... 84

2 .4 : DNA q u a n t i f ic a t io n , s tandard cu rve c a l ib r a t io n : 2. Graph p l o t . . . . 85

2 .5 : S ite mapping o f s in g le d ig e s ts : High re s o lu t io n sequence com parison

method ............................................... ................................................................................. 87

CHAPIER.IHREEL

3 .1 : S im p lif ie d is o la t io n and v is u a l is a t io n s teps o f m tD N A .......................187

3 .2 : Sequence com parison s i t e mapping method b* map lo c a t io n s ....................189

3 .3 : D is t r ib u t io n s o f t r a n s i t io n s S< tra n s v e rs io n s in Mus domesticus

DNA genome. ................................................................................................. 192

3 .4A : L o c a tio n s o f c leavage s i t e s and fu n c t io n a l re g io n s in Mus

domesticus mtDNA d e te c te d w ith 11 r e s t r ic t io n e nd o n u c le a se s .. . . . . . . . . . 194

3.4B (OVERLAY): L o c a tio n s o f c leavage s i te s and fu n c t io n a l re g io n s in th e

B r i t i s h house mouse us ing th re e a d d it io n a l r e s t r i c t io n e n d o n u c le a se s ..197

3 .5 : D is t r ib u t io n o f a l l c leavage s i te s (co n s ta n t and v a r ia b le ) a c ro ss the

mtDNA genome w ith each o f 14 e n z y m e s ..................... ..................................................... 198

3 .6 : D is t r ib u t io n o f c leavage s i te s among mouse mtDNA gene r e g io n s . . . .2 0 0

3 .7 : D is t r ib u t io n o f v a r ia b le s i te s w ith in mouse mt genes .............. 203

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TABLE OF CONTENTS

3.8s O rg a n is a tio n o f th e D -loop c o n ta in in g re g io n in m ice ............................. 205

3 .9 : S u b s t i tu t io n ra te m a tr ic e s ............................................. . . . . . 2 0 7

CHAPTER FOUR:

4 .1 : L o c a tio n o f sample s i te s in Orkney and th e n e ig h b o u rin g m ain land

c o u n tie s o f C a ithness and S u th e r la n d . . . . . ...................................... 281

4 .2 : D is t r ib u t io n o f t ra p s i te s fo r th e is la n d census on F a ra y , Orkney

a rc h i p e l a g o . .................. 283

4 .3 : D is t r ib u t io n o f t ra p s i te s fo r an is la n d census, on Skskholm , on th e

Pem brokesh ire c o a s t, in autumn 1986............................................................... . . . . . . . 2 8 5

4 .4 : P h y lo g e n e tic netw orks fo r r e s t r ic t io n morphs o f each o f th e 14 enzymes

em ployed................ 287

4 .5 : Geographic d is t r ib u t io n o f r e s t r ic t io n d ig e s t io n p a t te rn morphs f o r

each v a r ia b le r e s t r i c t io n enzymes........................................................... 289

4 .6 : G eographic d is t r ib u t io n o f mtDNA com posite geno types in B r i t a i n . . 292

4 .7 : Adams consensus t r e e o f B r t is h sam ples.................................. 294

4 .8 ; Phenogram d e riv e d from a UPGMA c lu s te r a n a ly s is o f 23 mtDNA c lo n e s in

th e B r i t i s h house mouse, based on 14 enzym es..,................................... . . . . . . . . 2 9 6

4 .9 : Adams consensus t re e o f mtDNA com posite genotypes u s in g 11 enzymes

among European sam ples............................................................................................................298

4 .1 0 : Geographic d is t r ib u t io n o f mtDNA c lones in Europe................ ..................300

4 .1 1 : Parsim ony ne tw orks in te rc o n n e c tin g the com pos ite mtDNA gentypes o f

Mus domesticus u s in g a) 14 b) 11 r e s t r ic t io n enzym es.. . . . . . . . . . . . . . . . . 302

4 .1 2 : S t r i c t consensus t re e o f mtDNA com posite genotypes u s in g 2 enzymes

among w o rld -w id e sam ples................................................ .. ......................................... . . . . 3 0 5

4 .1 3 : G eographic d is t r ib u t io n o f mtDNA c lones among w o rld w id e samples u s in g

two r e s t r i c t io n enzymes............... 307

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TABLE OF CONTENTS

4 .1 4 : Map o f B r i t a in showing th e e x te n t of the ic e d u r in g maximum

g la c ia t io n and d u r in g th e la te la s t g la c ia t io n ...................................... . ..............309

4 .1 5 : D is t r ib u t io n o f mtDNA com posite genotypes from among a) Orkney & N.E.

S co tla n d b) Ire la n d & I s le o f Man, in the N-W g e n e tic assem blage in

B r i t a in ...................................................................... .310

CHAPTER_FIVEi

5 .1 : The lo c a l is a t io n and proposed o r ig in s o f the s x r re g io n o f th e mouse Y

chromosome, c o n ta in in g th e se x -d e te rm in in g g e n e s ............................................... 344

5 .2 : Southern b lo t a n a ly s is o f Y chromosome DNA RFLPs.................................... 346

5 .3 : D iagram m atic re p re s e n ta t io n o f a l l Y chromosome DNA r e s t r i c t i o n

fragm ent d ig e s t io n p r o f i le s observed in the 91 samples o f B r i t i s h house

m ice , c leaved w ith B r e s t r i c t io n endonuclease us ing th e Y - s p e c i f ic p robe ,

pYB....................................................................................................................................................... 348

5 .4 : G eographic d is t r ib u t io n o f v a r ia b le (Taq I , Hae I I I , Mbo I and H in f I )

Y chromosome genotypes in th e B r i t is h house mouse....................................... . . . . 3 5 0

5 .5 : Three e q u a lly p a rs im on ious t r e e s . . . . . . . . . . . . . . . . . ......................... . . . . . 3 5 1

5 .6 : S t r i c t consensus t re e fo r a l l tre e s of equal le n g th ............................... 353

5 .7 : A phenogram o f th e 12 Y DNA co lnes in B r i t is h house m ice by UPGMA

c lu s te r a n a ly s is o f sequence d ive rgence e s t im a te s . . ...........................................355

5 .8 : G eographic d is t r ib u t io n o f com posite DNA type s (mtDNA/ Y) across

Br i t a i n gen o typ e s ................................................................................................. 357

5 .9 : Comparison o f mtDNA and Y lin e a g e s in B r i t is h house m ice in c lu d in g A)

d iv e r s i t y o f mtDNA and Y DNA lin e a g e s estim a ted by nuc leon d iv e r s i t y B)

number o f c lo n e s per m ajor r e g io n . . . ...................... 358

CHAPIER_SIXI

6 .1 : D is t r ib u t io n o f t ra p s d u r in g an is la n d census on th e I s le o f May.394

TABLE OF CONTENTS

6 .2 : S in g le r e s t r i c t i o n enzyme parsim ony netw orks c h a ra c te r is in g p re ­

in t r o d u c t io n Eday and I s le o f May m ice........................................ 396

6 .3 : D is t r ib u t io n o f in tro d u c e d Eday nmtDNA on th e I s le o f May from

September 1985 to September 1987.................. 398

6 .4 : The tem pora l and s p a t ia l spread o f in tro d u c e d Eday gene fre q u e n c ie s

a c ro ss th e I s le o f May in Sept* 1985, 1986, and 1987: I . mtDNA I I . Y

chromosome DNA. ........................ 400

6 .5 : O v e ra ll f re q u e n c ie s o f in tro d u c e d EDay genes in September 1985, 1986 St

1987; I . mtDNA St Y chromosome DNA I I . com posite genotypes (mtDNA/Y). . .4 0 2

6 .6 : D is t r ib u t io n o f m ito c h o n d ria l DNA and Y chromosome com posites

(mtDNA/Y) a c ro ss th e I s le o f May from September 1985 to 1 9 8 7 . . . . . . . . . . 4 0 4

6 .7 : F requenc ise o f each o f the com posites (mtDNA/Y chromosome DNA) in th e

fo u r a r b i t r u a r y re g io n s o f th e is la n d (A-D) from Sept* 1985, 1986 and 1987,

d is p la y e d g r a p h ic a l ly ...................... .406

6 .8 : The d is t r ib u t io n o f mice across th e I s le o f May in September 1982 I .

and I I . O r ig in a l p re - in t ro d u c t io n mice re c a p tu re s (m ale and fem a les ,

r e s p e c t iv e ly ) , I I I . ’ H y b rid s " (as m on ito red by a llo zym e d a ta ) ....................408

CHAPIER_SEVENi

7 .1 : What i s an O r c a d ia n ? . . . . . ........................ 452

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LISI_OF_PLATESSi

CHAPJER_JWOi

2 .1 : DNA q u a n t i f ic a t io n , S tandard cu rve c a l ib r a t io n : 1. ge l photograph 89

CHAPTER THREE:

3 .1

3 .2

3 .3

3 .4

Gel pho tograph i l l u s t r a t i n g sequence com parison s i t e m apping 209

Hind I I I r e s t r i c t io n d ig e s ts o f B r i t i s h Mus domesticus mtDNA.. . . .211

Xba I " ” 214

Hpa I I " " 216

o .o : Taq I " " 218

3 .6 : Hae I I I " " " 220

3 .7 : H in f I " " 222

3 .8 : Mbo I ” " " 224

3 .9 : Rsa I " ” .226

3 .1 0 : A lu I " ” 228

c h a p ie r _f i v e i

5 .1 : Southern b lo t a n a ly s is o f a) Eco RI b) both Hae I I I & H in f I d ig e s te d

house mouse DNA from v a r io u s B r i t i s h lo c a l i t i e s u s in g th e s x r Y -s p e c if ic

p robe , p Y 8 . ............. 360

5 .2 : Southern b lo t a n a ly s is o f a) Taq I and b) Rsa I - r e s t r i c t e d house mouse

DNA us ing th e Y -s p e c if ic probe pY8................................................. .363

5 .3 : H y b r id is a t io n o f Mbo I - d ig e s te d house mouse DNAs w ith th e Y -s p e c if ic

p robe , pY8........................... 365

CHAPIER_SIXI

6 .1 : H in f I r e s t r i c t io n fragm ent d ig e s ts o f I s le o f May & Orkney house mice

(Mus domesticus) ........................... . . . . . . . . 4 1 0

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TABLE OF CONTENTS

6 .2 : A lu I r e s t r i c t i o n fragm ent d ig e s ts o f Is le o f May and Orkney Mus

domesticus mtDNA. ................................................... 412

6 .3 : Taq I and Mbo I mtDNA r e s t r ic t io n fragm ent p r o f i l e s o f May and Eday

house m ic e . ............................................................ .414

6 .4 : Rsa I and Hae I I I r e s t r i c t io n fragm ent p r o f i le s o f genomic DNA, probed

w ith a Y -s p e c if ic sequence from house mice from th e I s le s o f May and

Eday....................................................................................................................................................... 417

6 .5 : Hae I I I r e s t r i c t i o n fragm ent p a tte rn s produced by s o u th e rn b lo t

a n a ly s is and probed w ith a Y -s p e c if ic sequence from p o s t - in t r o d u c t io n I s le

o f May house mice from September 1986........................................................................ .417

6 .6 : Hae I I I r e s t r i c t i o n fragm ent p a tte rn s produced by s o u th e rn b lo t

a n a ly s is and probed w ith a Y -s p e c if ic sequence from p o s t - in t r o d u c t io n I s le

o f May house mice from September 1987.......................................... ............................. 421

- X X I I I -

CHAPIER_ONEi_INIRODUCIigNi

l i l i _ G e n e ra l_ iD tro d u c tio n i

E v o lu t io n a ry s tu d ie s ( th e s tu d y o f "descen t w ith m o d if ic a t io n " - Darw in,

1859) have t r a d i t i o n a l l y focused on two m ajor a spe c ts , th e re c o n s tru c t io n

o f th e e v o lu t io n a ry h is t o r ie s o f o rgan ism s from s tu d y o f t h e i r e x ta n t

d is t r ib u t io n s , and th e e lu c id a t io n o f th e u n d e r ly in g mechanisms o f

e v o lu t io n (N e i, 1987).

The f i r s t o b je c t iv e , was o r ig in a l l y s tu d ie d by s y s te m a t is ts and

p a la e o n to lo g is ts , who c o n s tru c te d e v o lu t io n a ry t re e s o f o rgan ism s u s in g

w e ll p rese rved f o s s i l ev idence o r , where t h i s was fra g m e n ta ry ,

m o rp h o lo g ica l c h a ra c te rs . In t h i s way th e y were a b le to id e n t i f y th e m ajor

e v o lu t io n a ry tre n d s (m a c ro e v o lu t io n ) . However, th e c o m p le x ity o f the

e v o lu t io n a ry changes d e fin e d by th e se m o rp h o lo g ica l c h a ra c te rs a re

re f le c te d in th e o fte n c o n tro v e rs a l e v o lu t io n a ry t re e s d e r iv e d from these

d a ta (Hennig, 1966).

The advent o f im m uno log ica l te c h n iq u e s , n u c le ic a c id h y b r id is a t io n , and

p ro te in sequencing, enabled s y s te m a t is ts to compare th e accum u la tio n o f

m u ta tio n s between d is t a n t ly re la te d e x ta n t groups (above th e sp e c ie s

le v e l ) , p e rm it in g c a lc u la t io n o f e v o lu t io n a ry ra te s and t im e s c a le s ( th e

"m o le c u la r c lo c k " ) (M a rg o lia sh , 1963; F itc h 8c M a rg o lia sh , 1967; W ilson et

a i . , 1977; C arlson e t al•, 1978). C o n ve rse ly , e a r ly p o p u la t io n g e n e t ic is ts

(F o rd , 1930; W rig h t, 1931; Haldane, 1932) were in te n t on fo rm u la t in g an

e m p ir ic a l b a s is f o r th e mechanism o f e v o lu t io n by n a tu ra l s e le c t io n

CHAPTER ONE

fo l lo w in g D arw in ’ s o r ig in a l th o u g h ts (D arw in , 1859), b u t th e re was l im ite d

e xpe rim en ta l v e r i f i c a t io n o f th e se th e o r ie s .

P ro te in e le c tro p h o re s is produced some common ground fo r th e se sepa ra te

approaches to e v o lu t io n a ry s tu d ie s . However, w ith th e d is c o v e ry (Lew ontin

and Hubby, 1966; H a r r is , 1966) o f s u r p r is in g ly la rg e amounts o f g e n e tic

v a r ia t io n a t th e p ro te in le v e l , w orke rs became p reoccup ied w ith th e e x te n t

and m aintenance o f t h i s v a r ia t io n in c lo s e ly re la te d groups (below th e

sp e c ie s le v e l ) . They m a in ly concerned them se lves w ith m easuring a l le le

fre q u e n c ie s o f a n c e s tra l p o p u la tio n s , e v a lu a t in g how th e se were a ffe c te d by

th e processes o f n a tu ra l s e le c t io n , g e n e tic d r i f t , gene f lo w , and changes

in p o p u la tio n s iz e (m ic ro e v o lu t io n a ry e v e n ts ) , and w ith th e un reso lved

" n e u t r a l i t y - s e le c t io n " c o n tro v e rs y (L ew on tin , 1974; Kimura & Crow, 1964;

K im ura, 1983a, 1983b). However, a lth o u g h p ro te in e le c tro p h o re s is i s q u ick

and cheap, making i t r e a d i ly a p p lic a b le to la rg e p o p u la t io n s tu d ie s , i t has

g e n e ra lly been employed in th e d e te c t io n o f n u c le a r polym orph ism s. These

genes segrega te and recom bine d u r in g sexual re p ro d u c t io n , c o m p lic a tin g and

b lu r r in g th e o v e r a l l e v o lu t io n a ry in te r p r e ta t io n . A d d i t io n a l ly , a llozym es

a re m u lt i s ta te t r a i t s (a number o f a l le le s a t each lo c u s ) and as such do

no t form g e n e t ic a l ly d is c re te da ta ( ra th e r gene f re q u e n c ie s ) , thu s

p h y lo g e n e tic o rd e r cannot be c o n f id e n t ly in fe r r e d from o bse rva b le

d if fe re n c e s in e le c tr o p h o r e t ic m o b i l i t y .

A r e c o n c i l ia t io n o f th e se d is p a ra te approaches to e v o lu t io n a ry s tu d ie s was

ach ieved o n ly when m o le cu la r b io lo g ic a l te ch n iq u e s (c lo n in g and sequencing)

became more w id e ly a v a i la b le . Indeed, th e u n i f i c a t io n o f th e two

CHAPTER ONE

d is c ip l in e s o f m o le cu la r e v o lu t io n and p o p u la t io n g e n e tic s has become known

as "m o le c u la r e v o lu t io n a ry g e n e tic s " (N e i, 1987). DNA sequences are much

more in fo rm a t iv e than t h e i r p ro te in c o u n te rp a r ts because a la rg e p ro p o r t io n

o f th e DNA sequences a re no t encoded in t o p ro te in sequences and th e g e n e tic

code i s degene ra te . These advantages a re o f f s e t by th e te c h n ic a l

d i f f i c u l t i e s in h e ra n t in c lo n in g and sequenc ing , im posing re a l l im i t a t io n s

on th e numbers o f in d iv id u a ls and p o p u la t io n s th a t can be examined. Some o f

th e s e problem s a re re s o lv e d by te ch n iq u e s w hich employ th e r e la t i v e l y

re c e n t d is c o v e ry and p u r i f i c a t io n o f r e s t r i c t io n endonucleases (Zabeau St

R o b e rts , 1979). However, a l l th e above m entioned te ch n iq u e s have

c o n tr ib u te d g r e a t ly t o th e comprehension o f th e e v o lu t io n a ry re la t io n s h ip s

between groups o f o rgan ism s, and p o p u la t io n s , y e t each te c h n iq u e i s l im ite d

in i t s u s e fu ln e s s , a p p ro p r ia te o n ly f o r a c e r ta in range o f d ive rgence

t im e s .

The e x te n t to which m ic ro e v o lu tio n a ry p rocesses among c o n s p e c if ic s can be

extended t o e x p la in m a c ro e vo lu tio n a ry e ven ts among sp ec ies has been a

re c u r r in g and c o n tro v e rs a l is su e (Dobzhansky, 1937; E ld redge Sc Gould, 1972;

G ould, 1980; Mayr, 1982). However, i t seems l i k e l y th a t by exam ining th e

h is to r y o f p a r t ic u la r genes, mechanisms common to bo th modes o f e v o lu t io n

may become apparen t (F e ls e n s te in , 1982; A v ise et al,, 1987a; A v ise , 1989).

Com parative s tu d ie s o f m ito c h o n d ria l DNA (mtDNA), a t and below th e sp e c ie s

le v e l , p e rm its fo c u s in g o f bo th m ic ro - and m a c ro e v o lu tio n a ry approaches

(A v ise et al,, 1979; A v ise and Lansman, 1983). Indeed, fo r many v e r te b ra te

ta x a , mtDNA has been s ty le d " th e d e f in i t i v e m o le cu la r m arker" (A v ise ,

1986, 1987, 1989; A v ise et aim, 1987a; W ilson et aim, 1985; H a rr is o n , 1989).

CHAPTER ONE

In t h i s s tu d y I hope to e v a lu a te some o-f th e c la im s made -for t h i s

" d e f in i t i v e m a rke r", and use i t t o in v e s t ig a te th e p o p u la tio n g e n e tic s and

e v o lu t io n a ry b io lo g y o f th e B r i t i s h house mouse (Mus dowesticus) , w ith

s p e c ia l re fe re n c e to p o p u la tio n s in Orkney and N.E. S co tla n d . The f i r s t

s e c t io n o f t h i s ch ap te r d iscu sses th e m o le cu la r fe a tu re s and p ro p e r t ie s o f

mtDNA, and th e n ex t i t s use as a to o l in in t r a s p e c i f ic p hy log e og ra p h ic

s tu d ie s , p e r t in e n t to th e R oden tia . The s tu d y sp ec ies Hus dowesticus w i l l

be d e s c r ib e d n e x t; a sp ec ies used e x te n s iv e ly in many a spec ts o f s c ie n t i f i c

re s e a rc h , p ro b a b ly one o f th e b e s t known genomes, w ith th e p o s s ib le

e x c e p tio n o f D ro s o p h ila o r Man. F in a l ly I s h a l l o u t l in e th e main o b je c t iv e s

o f t h i s s tu d y .

ljL 2 j,_ M ito ch p n d ria l DNA - th e d e f i n i t i v e g e n e tic marker of__pgpul_atign_and

e v o lu t io n a ry b io lo o y ?

In th e fo l lo w in g s e c tio n s I w i l l b r i e f l y o u t l in e th e m o le cu la r s t r u c tu r e ,

o rg a n is a t io n and fu n c t io n o f mtDNA and then go on to d is c u s s tho se

p ro p e r t ie s which make mtDNA th e id e a l p o p u la tio n g e n e tic m arker. F in a l ly I

w i l l m ention th e l im i t a t io n s o-f a p p ly in g mtDNA ana lyses as a g e n e tic m arker

in s tu d y in g house mouse p o p u la tio n s .

i i ? i ! i _G eQ era l_ fea tu re5_and_m g lecu la r_cha rac te ris tics_Q f_m tD N A L

Animal mtDNA is amongst th e bes t known and c h a ra c te r is e d re g io n s o f th e

e u k a ry o t ic genome. Here I s h a l l d is c u s s th e m o le cu la r d e ta i ls p e r t in e n t to

t h i s s tud y o f mouse mtDNA (many o f w hich a re genera l to most v e r te b ra te s ) ,

f u l l e r e x p la n a tio n s o f th e m o le cu la r b io lo g y o f th e m ito ch o n d rio n a re g ive n

in s e ve ra l re c e n t re ve iw s (W olstenholm e and C la ry , 1985; C la y to n , 1982,

CHAPTER ONE

1984; A t ta r d i , 1985; Brown, 1985; C a n ta to rre and Saccone, 1987).

Much o f th e in fo rm a tio n about th e m itochondrion comes from com p le te

sequence d a ta from human, mouse, b ov ine , Xenopus and Drosophila yakuba

(Anderson et aim, 1981; B ibb et aim, 1981; Anderson e t aim, 1982; De B r u i jn

e t aim, 1983; Roe e t aim, 1985; C la ry and W olstenholme, 1985), and la rg e

p a r ts o f r a t (Pepe e t aim, 1983) and Drosophila welanogaster (G aresse,

1988) sequences. S m a lle r segments o f the genome from a v a r ie t y o f o th e r

o rgan ism s (humans - W alberg and C la y to n , 1981; 6reenberg e t aim, 1983; r a t s

- Brown e t aim, 1981; Saccone e t aim, 1981; Brown and Simpson, 1982; K o ike

e t aim, 1982; p rim a te s -Brown e t aim, 1982) have a ls o been sequenced. In

a d d it io n , c o n s id e ra b le in fo rm a tio n has come from r e s t r i c t i o n s i t e mapping

and RFLP s tu d ie s (For re v ie w s se e - A v ise & Lansman, 1983; Brown, 1983;

W ilson e t aim, 1985; A v ise , 1986).

The house mouse m ito c h o n d ria l genome is a sm a ll, c lo s e d , c i r c u la r dup lex o f

DNA o f 16,295 b a se p a irs (B ibb e t aim, 1981) (rang ing from 16 .3 to 19.2

k ilo b a s e s in most v e r te b ra te s - Anderson e t aim, 1982; Brown, 1981. 19B3,

1985; A t ta r d i , 1985). A p p ro x im a te ly 947. o f the m ito c h o n d r ia l genome

encodes fu n c t io n a l RNA, in c lu d in g , 13 messenger RNA’ s (mRNA’ s ) , 22 t r a n s fe r

RNA’ s (tRNA’ s) and 2 ribosom al RNA’ s ( la rg e C16S3 and sm a ll C12S3). F ig u re

1.1 i l l u s t r a t e s th e g e n e tic s t r u c tu re and fu n c tio n o f mouse mtDNA. Sequence

a n a ly s is o f th e mRNA genes re ve a le d 13 reading fram es w hich code fo r known

m ito c h o n d r ia l p ro d u c ts , one o f which is coded fo r on th e l i g h t s tra n d (L -

s t ra n d ) , and th e re s t on th e heavy s tra nd (H -s tra n d ). Toge ther th e y fo rm

th e s u b u n its o f th e m ito c h o n d r ia l in n e r membrane p ro te in s , im p o rta n t in th e

e le c tro n t ra n s p o r t system , o r in p ro te in s in vo lved in th e s y n th e s is o f ATP;

CHAPTER ONE

th e y a re s u b u n its I , I I and I I I D-f cytochrom e o x idase (complex IV ) , th e

a p o p ro te in o-f cytochrom e b (complex I I I ) , 7 s u b u n its o-f th e NADH-

dehydrogenase (-fo rm erly " u n id e n t i f ie d re a d in g fram es" CURF’ sD 1 -6 , and 4L -

Chomyn et aim, 1985) and s u b u n its 6 and 8 ( fo rm e r ly URF A6L) o f ATPase

(complex V I ) .

The arrangement o f m ito c h o n d r ia l genes i s conserved a c ro ss taxonom ic groups

and i s p r a c t i c a l l y id e n t ic a l among v e r te b ra te s (Brown, 1981, 1985). The

o n ly sequences which do n o t code f o r e i th e r RNA o r p ro te in a re th e 879

n u c le o t id e s o f th e d isp lacem en t loop (D -loop - formed by th e s y n th e s is o f a

s h o r t p ie ce o f DNA, com plem entary to th e L -s tra n d , which d is p la c e s th e H-

s tra n d ) and th e o r ig in o f l i g h t s tra n d r e p l ic a t io n (0L ) . The D -loop i s

o fte n re fe r re d to as th e "c o n tro l re g io n " because i t c o n ta in s sequences

th a t a re in v o lv e d in th e i n i t i a t i o n o f r e p l ic a t io n and t r a n s c r ip t io n

(C a n ta to rre and Saccone, 19B7; A t ta r d i , 1985? C la y to n , 1984). The

r e p l ic a t io n o f anim al mtDNA i s u n id ir e c t io n a l and h ig h ly a sy m e tr ic

(C la y to n , 1982? M o ritz et aim, 1987). I t s u n u s u a lly buoyant d e n s ity , h ig h

copy number, and c o m p a rtm e n ta lis a tio n in a d is t in c t o rg a n e lle , make

m ito c h o n d r ia l DNA r e la t i v e l y easy to is o la te and p u r i f y .

In summary, th e s iz e , c o n te n t and gene o rg a n is a t io n (Brown, 1981, 1983,

1985) o f th e m ito c h o n d r ia l genome i s h ig h ly conserved . I t possesses no

in t r o n s o r r e p e t i t i v e sequences, has tRNA genes in te rs p e rs e d between rRNA

and p ro te in -c o d in g genes w ith e i th e r few o r no non -cod ing n u c le o t id e s

between cod ing sequences. D e sp ite t h i s "extrem e example o f g e n e tic economy"

( A t ta r d i , 1985), mammalian mtDNA has a r a p id ly e v o lv in g p r im a ry sequence,

CHAPTER ONE

w ith a s u b s t i t u t io n r a te 5-10 tim e s th a t o-f s in g le copy n u c le a r DNA (Brown

et aim, 1979; Brown, 1983). E ith e r h ig h m u ta tio n ra te s , o r re la x a t io n o f

fu n c t io n a l c o n s t r a in ts , have been invoked to account f o r th e ra p id ra te o f

s u b s t i tu t io n in a genome t i g h t l y packed w ith fu n c t io n a l genes (A v ise and

Lansman, 1983; Brown, 1983, 1985; Cann & W ilson , 1983; Cann et a im , 19B4).

Base s u b s t i t u t io n appears to be b iased in fa v o u r o f t r a n s i t io n s , over

tra n s v e rs io n s , in bo th cod ing and noncoding re g io n s . Yet th e g re a te r th e

d ive rge n ce between m ito c h o n d r ia l genomes th e more t h i s b ia s decreases

(Brown et a im , 1982). There are g e n e ra lly no la rg e conserved b lo c k s w ith in

th e m ito c h o n d r ia l genome, a ltho u gh c e r ta in re g io n s appear t o e vo lve fa s te r

than o th e rs (Aquadro et a im , 1984; F e r r is et a im , 1983; B ibb e t a im , 1981).

D ivergence w ith in mtDNA i s ve ry ra p id among c lo s e ly re la te d in d iv id u a ls ; a

mean ra te o f a p p ro x im a te ly two p e rce n t sequence d ive rg e n ce per m i l l io n

ye a rs (Brown et a im , 1979; H iguch i et a im , 1984; W ilson et a im , 19B5;

G y lle n s te n & W ilson , 1987b; S h ie ld s & W ilson , 1987). However, beyond 5 -10

m i l l io n ye a rs , th e base s u b s t i tu t io n r a te d e c lin e s , as th e more r a p id ly

e v o lv in g p o s i t io n s in th e genome become s a tu ra te d (Brown et a im , 1979).

M ito c h o n d r ia l DNA appears to be s t r i c t l y m a te rn a lly in h e r i te d in h ig h e r

a n im a ls (Dawid and B la c k le r , 1972; H u tch ison et a im , 1974; Hayashi et a im ,

1978; G ile s et a im , 1980). D esp ite th e fa c t th a t th e m idp iece o f th e sperm

does e n te r th e egg a t f e r t i l i s a t i o n ( S te fa n in i , e t a im , 1969), th e p a te rn a l

m ito c h o n d ria appears to be lo s t ; t h i s i s th o u g h t t o occur by e ith e r a lo s s

o f fu n c t io n o r by a c t iv e removal on th e p a r t o f th e egg (Hecht e t a im ,

1984). P a te rn a l leakage o f m ito c h o n d ria th rou g h th e sperm i s an e x tre m e ly

ra re even t, w ith no more than one p a te rn a l genome per thousand m aterna l

CHAPTER ONE

genomes (G y lle n s te n et a im , 1985; Lansman e t a im , 1983b; A v ise and

V r ije n h o e k , 1987). Thus f a r , re co m b in a tio n between mtDNA m o lecu les has no t

been re p o r te d , w ith th e m ajor e x c e p tio n o f th e D -loop in c a t t le ( O l iv io e t

a im , 1983). Hence i f s t r i c t in d iv id u a l homoplasmy and m aterna l in h e r i ta n c e

a p p ly , then in d iv d u a ls would be e f f e c t iv e ly h a p lo id and c a r ry unambiguous

in fo rm a tio n about t h e i r m aterna l lin e a g e , g iv in g r i s e to m a tr ia rc h ie s

ana logous to th e "m ale surname e v o lu t io n " seen in human s o c ie t ie s (Chapman

e t a im , 1982). Hence, mtDNA seems to be th e id e a l g e n e tic m arker.

However, i t i s u n l ik e ly th a t any one m o le cu la r m arker w i l l be th e p e r fe c t

to o l f o r p h y lo g e n e tic s tu d ie s . M ajor l im i t a t io n s may in c lu d e he te rop lasm y

(where a t le a s t two mtDNA genomes d i f f e r i n g in s iz e c o e x is t w ith in an

in d iv id u a l ) , le n g th m u ta tio n s , re v e rs a ls o r conve rgen t e v o lu t io n

(homoplasmy) and n e u t r a l i t y o f th e mtDNA (a d is c u s s io n o f th e l a t t e r two

p o in ts , d e a lt w ith in depth by A v ise and c o lle a g u e s , 1987, w i l l be d e fe rre d

to ch a p te r 7 ) .

Much in t r a s p e c i f ic mtDNA v a r ia t io n i s due to base s u b s t i tu t io n s o r to sm a ll

(o n ly a few base p a ir s ) a d d it io n s o r d e le t io n s (Cann St W ilson , 1983;

Aquadro St G reenberg, 1983). Large s c a le d if fe re n c e s in mtDNA s iz e a re known

to occur between c lo s e ly re la te d sp e c ie s ( G o r i l la and o th e r p r im a te s -

F e r r is et a im , 1981; among Drosophila sp e c ie s - Fauron St W olstenholm e,

1980). However, genome s iz e i s g e n e ra lly v e ry s ta b le w ith in a sp ec ies

(Brown, 1983, 1985) and most in d iv id u a ls a re homoplasmic w ith re s p e c t to

mtDNA, th a t i s th e y c o n ta in a s in g le mtDNA genotype (A v ise St Lansman,

1983). There a re , however, e x c e p tio n s to th e se gene ra l r u le s , both

CHAPTER ONE

he te rop lasm y and la rg e s c a le s iz e m u ta tio n s a re common among c o n s p e c if ic

in v e r te b ra te s (H a rr iso n et aim, 1985; Rand St H a rr is o n , 1986; S o lig n a c e t

a im , 1983; C la rk S< Lyckegaard, 1988) and low er v e r te b ra te s in c lu d in g

Cnemidophorus l iz a r d s (Densmore et a im , 1985), f ro g s (M onnerot e t a im ,

1984), S c a llo p s (Snyder e t a im , 1987), newts (W a ll is , 1987) and f is h

(Bermingham e t a im , 1985; Bentzen e t a l . , 1988; Buroker e t a im , 1990).

H e terop lasm y has been cons ide red an e s s e n t ia l bu t t r a n s ie n t phase in th e

e v o lu t io n o f th e m ito ch o n d ia l genome. How la rg e a problem he te rop lasm y

p re s e n ts depends upon how long th e h e te ro p la sm ic s ta te p e r s is ts and whether

mammalian ta xa are s e v e re ly a f fe c te d . The m a jo r ity o f he te rop lasm ys a re

caused by le n g th (Hale & S ingh , 1986; M o ritz e t a im , 1987) ra th e r than

p o in t m u ta tio n s , indeed th e re i s o n ly one w e ll documented case o f th e

l a t t e r in cows (H ausw irth & L a ip is , 1982), a s ta te which e x is te d fo r o n ly a

few g e n e ra tio n s , whereas hete rop lasm y fo r le n g th v a r ia t io n p e r s is ts th rough

many g e n e ra tio n s (S o lig n a c et a im , 1987; Rand & H a rr is o n , 1989). A lthough a

few is o la te d cases have been reco rded in cows (H ausw irth et a im , 1984),

humans (Greenberg et a im , 1983; Monnat & Leob, 1985), r a t (Brown &

D e s ro s ie rs , 1983) and mouse (B ourso t et a im , 1987), th e l a t t e r v a r ia n t

be ing fu n c t io n a l ly d e fe c t iv e , he te rop lasm y i s r a r e ly found in mammals and

th u s has l i t t l e im pact on ro u t in e su rveys o f anim al mtDNA (W ilson e t a im ,

1985; A v ise e t a im , 1987).

To summarise, these genera l p ro p e r t ie s (m a te rn a l, nonrecom bin ing mode o f

in h e r i ta n c e , h a p lo id y , ra p id r a te o f base sequence e v o lu t io n , e x te n s iv e

in t r a s p e c i f ic polym orphism s and ease o f is o la t io n ) make mtDNA a superb to o l

f o r s tu d ie s o f p o p u la tio n g e n e tic s as p o p u la tio n s exchange in t a c t mtDNA

CHAPTER ONE

which c a r r ie s a lin e a g e ’ s h is to r y uncom p lica ted by re c o m b in a tio n .

T h e re fo re , an in d iv id u a l ’ s mtDNA genotype can be co n s id e re d an o p e ra tio n a l

taxonom ic u n i t (OTU) and used in m a tr ia rc h ia l p h y lo g e n e tic re c o n s tru c t io n s .

There are g e n e ra lly two b a s ic approaches to th e s tu d y o f mtDNA

polym orphism s. Comparison o f e i th e r sequences from sm a ll c lo n ed re g io n s o r

o f r e s t r i c t io n enzyme c leavage s i t e s in th e e n t i r e mtDNA genome. The fo rm er

approach, a ltho u gh th e most in fo rm a tiv e method, i s tim e -consum ing and

c o s t ly , th u s o n ly a few in d iv id u a ls may be screened f o r r e la t i v e l y t in y

re g io n s , and any m u ta tio n a l changes observed may n o t be e n t i r e ly

re p re s e n ta t iv e o f th e whole genome. A l t e r n a t iv e ly , by u s in g a s e r ie s o f

ty p e I I r e s t r i c t io n endonucleases to c le a ve th e mtDNA m o le cu les th e

fragm ents can be sepa ra ted by e i th e r agarose o r p o ly a c ry la m id e gel

e le c tro p h o re s is , and v is u a lis e d by a v a r ie ty o f te c h n iq u e s ; such as e i th e r

d i r e c t s i l v e r o r e th id iu m brom ide s ta in in g o r by in d i r e c t ra d io a c t iv e end-

la b e l l in g o r so u th e rn b lo t t in g m e thodo log ies (W ilson et el., 1985;

G y lle n s te n 8c W ilson , 1987b). Using a s e t o f r e s t r i c t io n endonucleases to

assay mtDNA in t h i s manner means many in d iv id u a ls can be screened , a t a

f r a c t io n o f th e t im e o r co s t o f th e c lo n in g te c h n iq u e . D if fe re n c e s in

fragm ent s iz e s so o b ta in e d r e f le c t sequence d if fe re n c e s in re c o g n it io n

s i t e s , and these can be used to e s tim a te n u c le o t id e sequence d iv e r s i t y . In

t h i s way a mtDNA phy logeny , re p re s e n tin g th e proposed h is t o r ic a l

r e la t io n s h ip s among genotypes, can be c o n s tru c te d u s in g e i th e r th e numbers

o f m u ta tio n a l changes as c h a ra c te r s ta te s (p re se n t o r a b s e n t) , o r p e rcen t

sequence d ive rge n ce e s tim a te s ( fo r fu r th e r d e ta i ls see m a te r ia ls and

methods ch a p te r and th e re le v a n t s e c tio n s in th e r e s u l t s c h a p te rs ) .

10

CHAPTER ONE

l i 2i 2£_M tDNA_as_a_Ph^logeograBhic_togl£

Over th e la s t decade m ito c h o n d r ia l DNA has proved to be e s p e c ia l ly u s e fu l

■for e v o lu t io n a ry in fe re n c e s among c lo s e ly re la te d ta x a (W ilson et el.,

1985; A v ise , 1986; B ir le y Sc C ro f t , 1986). In p a r t i c u la r , t h i s m o lecu le has

p ro v id e d im p o rta n t in s ig h ts in t o phy logeny, h is t o r ic a l zoogeography

( in c lu d in g h y b r id is in g ta x a ) , p o p u la tio n s t r u c tu r e , m a t r i l in e a l gene f lo w

and c o lo n is a t io n (For re c e n t re v ie w s see - M o ritz e t a l« , 1987; H a rr is o n ,

1989). In th e p re v io u s s e c tio n (1 .2 .1 ) I d iscussed th e p ro p e r t ie s and

c h a r a c te r is t ic s o f anim al mtDNA which have made i t p a r t i c u la r l y u s e fu l fo r

such s tu d ie s . However, in t h i s s e c tio n I w i l l b r i e f l y re v ie w se ve ra l

e a r l ie r s tu d ie s in which mtDNA polym orphism s were used to examine

r e la t io n s h ip s among c o n s p e c if ic p o p u la tio n s .

The c h a lle n g e o f e x p la in in g a ta x o n 's p re sen t d is t r ib u t io n has long

fa s c in a te d s c ie n t is t s (Hooker, 1853). H is to r ic a l b iogeography i s

t r a d i t i o n a l l y d iv id e d in t o e c o lo g ic a l ( d is p e r s io n a l is t model; D a r lin g to n ,

1957) and h is t o r ic a l b iogeography (v ic a r ia n c e model; C ro iz a t e t a i . , 1974).

The d is p e r s io n a l is t model i s concerned w ith th e movement o f ta x a across and

around e x is t in g b a r r ie r s and i s o fte n termed " c o lo n ia l i s t i c b iogeography"

as i t im p lie s th a t ta x a a lways o r ig in a te in Dne a rea and then c o lo n is e

o th e r a reas. The v ic a r ia n c e model emphasizes th e s p l i t t i n g o r d iv is io n o f

b io ta s th rough th e developm ent o f b a r r ie r s , which does n o t deny the

e x is te n c e o f d is p e rs a l, ra th e r i t q u e s tio n s i t ' s im p o rta nce in e x p la in in g

th e p resen t d is t r ib u t io n o f b io ta s (P a tte rs o n , 1981). G e n e ra lly , th e main

aim i s no t to f in d which i s th e c o r re c t model, b u t to examine endemic ta xa

to e s ta b lis h a shared d is t r ib u t io n p a tte rn (P la tn ic k Sc N e lson, 1978; Rosen,

11

CHAPTER ONE

1978). The re c o g n it io n o f congruence between th e p h y lo g e n e tic

re c o n s tru c t io n s and g e o lo g ic a l h is to r y o f th e re g io n encompassed by th e

p a t te rn , suggests th a t th e two p a tte rn s a re c lo s e ly l in k e d and a causa l

r e la t io n s h ip can be fo rg e d . Thus, h is t o r ic a l e ven ts can p ro v id e an

e x p la n a tio n o f th e observed b io lo g ic a l p a t te rn s (P a tte rs o n , 1980). The

m a jo r i ty o f b io g e o g ra p h ica l s tu d ie s have concerned in t e r - s p e c i f ic

com parisons, however, i t has been suggested th a t c o n s p e c if ic p o p u la t io n s

may a ls o p ro v id e h is t o r ic a l in fo rm a tio n about a re g io n (Rosen, 1978).

I n t r a - s p e c i f ic mtDNA v a r i a b i l i t y can p ro v id e v a lu a b le in fo rm a tio n in two

m a jo r ways, f i r s t l y about th e m agnitude and p a t te rn o f p h y lo g e n e tic

v a r ia t io n among mtDNA genotypes and se co n d ly , on th e geog raph ic o r ie n ta t io n

o f th e mtDNA p h y lo g e n e tic assemblages. The convergence o f th e se two m ajor

p o in ts c o n s t i tu te s a new f i e l d which has been termed " mtDNA in t r a s p e c i f ic

p hy logeog raphy" (A v ise e t a i . , 1987; A v is e , 1989); t h i s d is c ip l in e a tte m p ts

t o c o n s o lid a te approaches from both p h y lo g e n e tic s y s te m a tic s and p o p u la tio n

g e n e tic s , w ith mtDNA as th e m e d ia to r. A v ise and c o lle a g u e s (1987)

fo rm u la te d se ve ra l th e o re t ic a l c a te g o r ie s o f p o s s ib le p h y lo g e n e tic outcomes

f o r any g ive n sp e c ie s . I n t r a s p e c i f ic sequence d ive rge n ces between mtDNA

genotypes may be la rg e , combined w ith s tro n g (C a tegory I ) , o r weak

(C a tegory I I ) geog ra ph ica l s t r u c tu r in g o f mtDNA c lu s te r s . A l t e r n a t iv e ly ,

sequence d ive rge n ce may be sm all between mtDNA genotypes, in c o n ju n c t io n

w ith mtDNA p h y lo g e n ie s which d is p la y m ajor geograph ic b reaks (c a te g o ry I I I )

o r u n ifo rm (ca te g o ry IV) o r nested (c a te g o ry V; in te rm e d ia te s i t u a t io n )

d is t r ib u t io n s . E m p ir ic a l examples o f a l l p o s tu la te d groups have been

documented, in c lu d in g some cases o f o v e r la p , w ith th e m ajor e x c e p tio n o f

12

CHAPTER ONE

c a te g o ry I I (mtDNA p hy log e n ie s sepa ra ted by la rg e sequence d ive rg e n ce s

w h ich co -o ccu r g e o g ra p h ic a lly ) .

The m a jo r ity o f sp e c ie s , examined th u s f a r , f a l l in t o th e f i r s t c a te g o ry ,

in which p o p u la t io n s w ith in a sp e c ie s a re c h a ra c te r is e d by p h y lo g e n e tic

d i f f e r e n t ia t io n (mean sequence d ive rge n ces o f 27. between mtDNA c la d e s )

w ith a d is t in c t geograph ic o r ie n ta t io n . P o s s ib le e x p la n a tio n s o f such

d is t r ib u t io n p a tte rn s in c lu d e long te rm h is t o r ic a l e x t r in s ic (b io g e o g ra p h ic )

b a r r ie r s t o gene exchange, such th a t c o n s p e c if ic p o p u la tio n s c lu s te r in t o

d is t in c t mtDNA assemblages on an in t r a s p e c i f ic t r e e . C o n ve rse ly ,

in te rm e d ia te genotypes in a w id e ly d is t r ib u te d sp ec ies w ith l im ite d

d is p e rs a l c a p a b i l i t ie s may become e x t in c t . A good example o f c a te g o ry I

in c lu d e s th e d e s e rt t o r t o is e iXerobates agassizi), in which th e mtDNA

genotypes were p a r t i t io n e d in to th re e m ajor p h y lo g e n e tic assem blages, each

w ith a s t r i k in g geograph ic o r ie n ta t io n (Lamb et al., 1989). Each assemblage

d i f f e r s from each o th e r by a t le a s t 17 assayed mtDNA m u ta tio n a l s te p s , w ith

p e rc e n t sequence d ive rge n ces o f between 4 .2 - 5 .6 7 . The g e o lo g ic h is to r y o f

th e C o lorado R iv e r area (which in c lu d e s e x te n s iv e m arine in c lu s io n s ) may

accoun t f o r th e marked mtDNA d ive rge n ce between e a s te rn and w estern X.

agassizi assem blages. O ther ta x a which f a l l in to t h i s ca te g o ry in c lu d e

in v e r te b ra te s iLiwulus polyphemus - Saunders e t a l 1986), ro d e n ts

(Geomys pinetis - A v ise e t a i . , 1979; Perowyscus maniculatus - Lansman e t

a l 19B3; Perowyscus leucopus - A v ise e t a l 1983), and fre s h w a te r f is h e s

iLepowis spp. - Bermingham St A v ise , 1986).

CHAPTER ONE

S eve ra l assayed sp ec ies f a l l w ith in group I I I , where th e mean sequence

d ive rg e n ce between mtDNA assemblages i s ra th e r low , much le s s than IX .

D e s p ite t h i s , th e mtDNA typ e s s t i l l rem ain g e o g ra p h ic a lly s e p a ra te as has

been dem onstra ted in th e to a d f is h e s iOpsanus tau) found a long th e A t la n t ic

c o a s t, where d is t in c t i v e mtDNA c la d e s , d is t in g u is h e d by o n ly 0 .5X sequence

d ive rg e n ce s (o n ly 1-2 assayed r e s t r i c t io n s i t e d i f fe r e n c e s ) , s t i l l tended

t o be g e o g ra p h ic a lly is o la te d in t o n o rth e rn and so u th e rn g e n e tic fo rm s

(A v is e et a l 1987b). In s ta n ce s o f such mtDNA d i f f e r e n t ia t io n have been

shown to be c o r re la te d w ith environmental im pediem nts to d is p e rs a l such as

th e long re cogn ised boundary between warm -tem perate and t r o p ic a l m arine

fa u n a . O ther n o ta b le sp e c ie s in c lu d e d in group I I I a re diamondback

te r r a p in s , and s l id e r t u r t l e s (Lamb & A v is e , in p re p ) . A v ise & c o lle a g u e s

(1987) suggested th a t most l i k e l y e x p la n a tio n fo r th e geog raph ic

o r ie n ta t io n o f such mtDNA genotypes, in th e absence o f m ajor p h y lo g e n e tic

d is c o n t in u i t ie s , in v o lv e s h is t o r i c a l l y l im i te d gene f lo w between

p o p u la tio n s which a re no t su b d iv id e d by lo n g te rm , f i r m b io g e o g ra p h ic

b a r r ie r s to d is p e rs a l.

So, i t g e n e ra lly appears th a t gene f lo w and d is p e rs a l have n o t o v e rr id d e n

h is t o r ic a l in f lu e n c e s on p o p u la tio n s u b d iv is io n s as re v e a le d by th e mtDNA

r e s t r i c t io n s i t e da ta re c o n s tru c t io n s in th e above m entioned c a te g o r ie s .

However, not o n ly a re th e sequence d ive rge n ces between genotypes ve ry sm a ll

in th e fo u r th c a te g o ry , th e re i s no, o r ve ry l i t t l e , g eog raph ic s t r u c tu r in g

o f p o p u la tio n s . M arine taxa are a ty p ic a l example ( fo r a re v ie w see A v ise ,

1987 - f i s h s to ck id e n t i f i c a t io n w orkshop), and in c lu d e th e American e e l,

Anguilla rostrata (A v ise e t al., 1986), c a t f is h e s , A r i id a e (A v is e , Reeb 8c

14

CHAPTER ONE

Saunders, 1987b), and S k ip ja c k tuna (Graves e t a l., 1984). The d iv is io n s o f

p h y lo g e n e tic groups a re somewhat a rb itu a ry , and i t m igh t be expected th a t

some sp e c ie s are in c lu d e d in s e v e ra l. For exam ple, some s p e c ie s e x h ib i t low

o v e r a l l sequence d ive rg e n ce s between mtDNA c la d e s b u t th e re i s some degree

o f g e o g ra p h ica l s t r u c tu r in g , as i s th e case in w o rld -w id e su rve ys o f man

(Brown, 1980? Cann e t al., 1984? Cann e t al., 1987) o r in b ir d s (G reat t i t s ,

Parus major - T e ge ls tro m , 1987b? Redwinged b la c k b ird s , Agelaius

phoeniceus - B a ll e t al., 1988? fo r re v ie w s see -K e s s le r 8c A v is e , 1984,

1985). Lack o f s u b s ta n t iv e d i f f e r e n t ia t io n o f mtDNA among p o p u la tio n s is

th o u g h t to r e f le c t e i th e r re c e n t p o p u la tio n and range expans ions o r

e x te n s iv e d is p e rs a l c a p a b i l i t ie s .

The geograph ic d is t r ib u t io n o f mtDNA genotypes and sequence d ive rge n ces in

deer mice from th e C a li fo rn ia n Channel Is la n d s and th e a d ja c e n t m ain land

(A sh ley & W i l ls , 1 9 8 7 ) , Columbian ground s q u i r r e ls (M acNeil 8c S trob e ck ,

1 9 8 7 ) , and Swedish house mice (G y lle n s te n 8c W ilson , 1 9 8 7 ) , a l l p ro v id e not

o n ly phy log e og ra p h ic e v idence , bu t a ls o , bo th th e t im e s and p o s s ib le

c o lo n is a t io n ro u te s in th e P le is to c e n e o r p o s t-P le is to c e n e . S o lig n a c et

al., H 9 8 6 ) u s in g mtDNA d iv e r s i t ie s i l l u s t r a t e d th a t In d o -P a c if ic i s th e

o r ig in a l home o f Drosophila simulans and i t s p re se n t w o rld w id e

d is t r ib u t io n was o n ly a tta in e d r e la t i v e ly r e c e n t ly .

CHAPTER ONE

1.3? The House mouse (Hus dowesticus. Ru t t y ) - th e s tu d y organ ism .

House mice a re th e most w id e ly used e xp e rim e n ta l a n im a ls , as a consequence

p ro b a b ly more i s known about them than any o th e r mammal w ith th e p o s s ib le

e x c e p tio n o-f man. The la b o ra to ry mouse i s p ro b a b ly so p o p u la r because i t i s

s m a ll, easy to han d le , has a good re p ro d u c t iv e perfo rm ance (average l i t t e r

s iz e s between 6 -8 ) , w ith a s h o rt g e n e ra tio n t im e ( f i r s t l i t t e r s a t th e age

o f 6 -8 weeks and fem ales breed every month th e r e a f te r ) and th e re a re a w ide

range o f r e a d i ly a v a i la b le g e n e t ic a l ly d e fin e d s t r a in s and m utants (F e s tin g

& L o v e l l , 1981). However, s tu d ie s o f w ild p o p u la t io n s o f th e house mouse

a re e q u a lly im p o r ta n t, no t o n ly fo r p ro v id in g new sou rces o f in h e r i te d

v a r ia t io n , b u t a ls o as an a id in in te r p r e t in g r e s u l t s o b ta in e d in th e

u n n a tu ra l env irom en t o f th e la b o ra to ry .

House mice a re found in an e x tre m e ly w ide range o f h a b ita ts (B e rry , 1981b»

Sage, 1981), in bo th fe r a l and commensal s i t u a t io n s . They e x h ib i t extrem e

re p ro d u c t iv e a d a p ta b i l i t y (Bronson, 1984) a llo w in g them to rep roduce in a

trem endous range o f env ironm ents . Mice a re a b le to t o le r a t e a w ide range o f

c o n d it io n s , and p o p u la tio n s in h a b it in g v e ry d i f f e r e n t h a b ita ts e x h ib i t

marked c h a r a c te r is t ic s . Mice p ro b a b ly owe t h e i r rem arkab le success to bo th

t h e i r commensal c o e x is te n c e a lo n g s id e man, and to t h e i r o ccup a tion o f

r e s t r ic t e d n ich e s which o th e r sm all mammals cannot f i l l . Most mouse

p o p u la tio n s can be lo o s e ly c la s s i f ie d as e i t h e r commensal, c h a ra c te r is e d by

h ig h p o p u la tio n d e n s it ie s w ith a s ta b le food s u p p ly , o r f e r a l , which

e x h ib i t low d e n s ity p o p u la tio n s s u b s is t in g on u n s ta b le , sparse food

re so u rce s (Bronson, 1979). In commensal h a b ita ts , m ice have become a

s ig n i f ic a n t p e s t, e s p e c ia lly in h igh d e n s ity in d o o r in fe s ta t io n s (S ou the rn ,

16

CHAPTER ONE

1954). The p e s t s ta tu s o-f t h i s sp ec ies i s aggrava ted n o t o n ly by problem s

in v o lv e d in th e c le a ra n ce o f p e rs is te n t p o p u la t io n s , b u t a ls o by th e ra p id

c o lo n is a t io n o r re - c o lo n is a t io n o f h a b ita ts ; s tu d ie s o f p es t c o n t ro l

i n i t i a t e d s c ie n t i f i c research on t h i s s p e c ie s .

However, d e s p ite th e e x te n s iv e use o f th e house mouse, i t s s y te m a tic s i s

c o n t ro v e rs ia l and p o o r ly re s o lv e d , i t s nom enc la tu re be ing f a r from

s ta n d a rd is e d ( f o r re v ie w s on house mouse taxonomy see - B e rry , 1981;

Bonhomme, 1986; Bonhomme et al., 1987; Bonhomme & Guenet, in p re s s ) .

Schwarz Sc Schwarz (1943) s im p l i f ie d th e o r ig in a l 133 d e sc rib e d fo rm s

(E lle rm a n , 1941) in to 15 subspecies o f m ice o f Old w o rld o r ig in . They

ass igned European house mice to two s u b s p e c if ic g roups , th e Hagneri group

in c lu d in g H. w. dowesticus and H. w. brevirostris, and th e Spicilegus

group in c lu d in g H. m. wusculus and ft. w. spicilegus. U n fo r tu n a te ly , t h i s

c la s s i f i c a t io n i s unw orkab le , as shown by s tu d ie s made in th e la s t decade

u s in g m orphology and a llozym e v a r ia t io n (Sage, 19B1; M a rs h a ll, 1981;

M a rsh a ll Sc Sage, 1981; T h a le r et al., 1981; Bonhomme et al., 1984),

k a ry o ty p ic v a r ia t io n (Gropp e t al., 1982; Capanna, 1982; Said et al., 1985;

W ink in g , 1986), m ajor h is to c o m p a t ib i l i t y (MHC) H-2 h a p lo typ e s and th e T / t

complex (K le in et al., 1981; Nadeau et al., 1981; K le in et al., 1986).

A d d i t io n a l ly , mtDNA been used to in f e r p h y lo g e n e tic r e la t io n s h ip s w ith in

th e genus Hus (Yonewaki et al., 1981; F e r r is et al., 1982; F e r r is et al.,

1983; B ou rso t et al., 1984; F o rt et al., 1984; B ou rso t et al., 1985; M oriw aki

et al., 1986; Yonewaki et al., 1986, 1988), as has r e p e t i t i v e DNA sequences

in c lu d in g th e Y chromosome (N is h io k a , 1988, 1989; N is k io k a S< Lamothe, 1986,

1987a, b; B o u rso t, et al., 1989).

CHAPTER ONE

Y e t, d e s p ite these numerous s tu d ie s , th e taxonom ic s ta tu s (su b spe c ie s ,

s e m i-sp e c ie s o r sp ec ies ) o f th e house mouse sp ec ies complex i s s t i l l

deba ted ; th u s th e use Df th e l a t i n b in o m ia l o r t r in o m ia l rem a ins

c o n t ro v e rs ia l (T h a le r et a l 1981 -s e m i-s p e c ie s ; M a rsh a ll & Sage, 1981;

Sage, 1981; Sage e t a l., 1986 - id e n t i f ie d a t le a s t 7 m o rp h o lo g ica l groups

term ed f u l l s p e c ie s ) . As a consequence, Bonhomme *t c o lle a g u e s (1978, 1984)

have p r o v is io n a l ly p re fe r re d n o t to make any n o m e n c la to r ia l ch o ice bu t

re p o r te d th a t th e re a re fo u r d is t in c t b io chem ica l g roups in th e Old World

(F ig u re 1 .2 a ); a u s e fu l compromise in v iew o f th e ongoing c o n tro v e rs a y .

However, r e c e n t ly these a u th o rs have dec ided th a t th e subspec ies c a te g o ry

shou ld be g iven a w ide r meaning (Bonhomme, 1986a, b ; Bonhomme it Guenet, in

p re s s ) . However, f o r conven ience I w i l l use th e b in o m ia l f o r th e European

house mouse th ro u g h o u t t h i s th e s is ; hence th e B r i t i s h fo rm becomes Hus

dowesticus (R u tty , 1772).

In g e n e ra l, th e commensal mice o f Europe a re now th o u g h t to be d iv is ib le

in t o two c lo s e ly re la te d sp ec ies w ith no s ig n i f ic a n t h y b r id is a t io n in

n a tu re . The Hus dowesticus (da rk b e l l ie d , long t a i le d - M a rsh a ll it Sage,

1981) form in c lu d e s th e common in b re d la b o ra to ry s t r a in s and w ild

p o p u la t io n s g e n e ra lly in h a b it in g Western Europe and th e M e d ite rran e an ,

fo rm e r ly c la s s i f ie d as the subspecies H. ■ . dowesticus, H. w brevirostris

and H . * . praetextus. T h is sp ec ies d i f f e r s s u b s ta n t ia l ly from Hus wusculus

(L in na e us , 1758) ( l i g h t b e l l ie d , s h o r t t a i le d mouse, w ith a b row nish y e llo w

la t e r a l l i n e ) , which in h a b its E aste rn Europe and most o f S cand inav ia

( fo rm e r ly known as th e subspecies H , » . wusculus), in m orphology, p ro te in

v a r ia t io n and behav iou r (M a rsh a ll it Sage, 1981; M a rs h a ll, 1981; Sage, 1981;

18

CHAPTER ONE

Sage et al., 1986), and mtDNA ( F e r r is e t al., 1983). F u r th e r , th e two

sp e c ie s a re la r g e ly is o la te d from each o th e r on accoun t o f t h e i r

g eo g ra ph ica l d is t r ib u t io n and male s t e r i l i t y fa c to r s ( F o re jt Sc Iv a n y i,

1974; F o r e jt , 19B1). However, a t th e h y b r id zone, d e fin e d by U rs in , (1952)

and Hunt & S e lande r, (1973) ( f o r p o s i t io n see f i g . 1 .2 ) , f e r t i l e h y b r id

fem a les a re o fte n produced, which is why th e y a re sometimes re fe re d to as

semi sp e c ie s . Y e t, th e In te rn a t io n a l Code o f Z o o lo g ic a l Nom enclature (ICZN)

g iv e s no c le a r way o f naming sem ispec ies as d is t in c t from subspec ies ,

w ith o u t caus ing c o n fu s io n between th e two in th e l i t e r a t u r e .

O ther re la te d sp ec ies a re found in sym patry w ith re p re s e n ta t iv e s o f Hus

a us cuius’, Bonhomme Sc c o lle a g u e s (1984) have shown th a t th re e o f these

a d d it io n a l sp e c ie s e x is t in Europe, nam ely, Hus spretus, Hus spicilegus,

and Hus spretoides ( th e l a t t e r two a re a l t e r n a t iv e ly named Hus hortulanus

and Hus abottif r e s p e c t iv e ly , by M a rsha ll St Sage, 1981). The geograph ic

d is t r ib u t io n o f th e f i v e taxa re fe ra b le to th e genus Hus ( f o r conven ience

term ed groups 1 -3 , 4A Sc 4B - Bonhomme et al., 1984) a re i l l u s t r a t e d in

f ig u r e 1.2B.

The e c o lo g ic a l and p o p u la tio n g e n e tic s o-f house mouse p o p u la t io n s from bo th

m ain land B r i t a in and a s s o c ia te d is la n d s have been in te n s iv e ly s tu d ie d fo r

th e la s t th re e decades (B e rry , 1964, 1968, 1978, 1983; Nash et al., 1983;

fo r re v ie w s see B e rry 1981, 1986, 1986b). B e rry (1970) concluded from

m ic rogeog raph ic s tu d ie s th a t most o f th e n o rth e rn is la n d m ice d e r iv e from

sm all fo u n d lin g p o p u la t io n s a c c id e n t ly in tro d u c e d to empty h a b ita ts by man

w ith in a p p ro x im a te ly th e la s t thousand ye a rs . On th e b a s is o f m orphom etric

19

CHAPTER ONE

e v idence , D av is (1983) has shown th e m a jo r i ty o f th e c ir c u m - B r i t is h is la n d

house mice (from th e O rkneys, S he tlands , F a roes, and H ebrides) to be q u i te

d is t in c t from th e B r i t is h m ain land ones. He concluded th a t t h i s was

p ro b a b ly due to t h e i r o r ig in o u ts id e o f B r i t a in . A d d i t io n a l ly , m orphom etric

and g e n e tic ana lyse s suggested th a t C a ithness m ice were d is c re te from those

fa r th e r s o u th , be ing more c lo s e ly re la te d to Orkney and S he tland

p o p u la t io n s , in d ic a t in g th a t th e y to o a re descendents o f in tro d u c e d

specimens from a source o th e r than th a t o f th e so u th e rn m ain land

p o p u la tio n s (Nash et aim, 1983; D av is , 1983). B rooker (1982), u s in g

k a ro ty p ic d a ta , a ls o p o s tu la te d C a ithness and th e Orkney m ice p o p u la tio n s

to have had a common a n c e s to r. I t i s these p o p u la t io n s and B r i t i s h house

mice in gene ra l th a t a re th e s u b je c t o f t h i s th e s is .

1.4s Genera l aims o f th e s tu d y*

The broad purpose o f t h i s th e s is i s to beg in a d e ta i le d c h a ra c te r is a t io n o f

m ito c h o n d r ia l DNA v a r i a b i l i t y and d i f f e r e n t ia t io n among p o p u la tio n s o f th e

house mouse (Hus dowesticus R u tty ) in th e B r i t i s h I s le s . I t has been

p o s tu la te d th a t a b e t te r u nde rs tand ing o f th e e v o lu t io n a ry p rocesses w ith in

a sp ec ies w i l l come from d e ta ile d s tu d ie s u s in g an in te g ra te d approach,

u t i l i z i n g th e most a p p ro p r ia te m ethodo log ies c u r r e n t ly and p r a c t ic a l l y

a v a i la b le . A c o n s id e ra b le body o f da ta and u n d e rs ta n d in g o f th e p o p u la tio n

g e n e tic s o f th e B r i t i s h house mouse a lre a d y e x is ts from autosomal m arkers,

th u s I hope to complement these s tu d ie s w ith those from h a p lo id , m a te rn a lly

in h e r i te d mtDNA m arkers.

The fo l lo w in g approach was adopted in th e p u r s u it o f these aims*

20

CHAPTER ONE

F i r s t l y , I hoped to e v a lu a te th e re s o lv in g power o f th e m ito c h o n d r ia l DNA

m o lecu le f o r com parisons below th e s p e c ie s le v e l . The mtDNA m o lecu le

e v o lv e s v e ry r a p id ly , ten tim e s th a t o f n u c le a r DNA and u s u a lly e x h ib i t s

e x te n s iv e in t r a s p e c i f ic r e s t r i c t io n s i t e po lym orph ism s, making i t p o s s ib le

to d is t in g u is h between c o n s p e c if ic p o p u la t io n s . However, p o p u la tio n s o f

house m ice a re a t an e a r ly s tage o f d iv e rg e n c e , th u s th e maximum sequence

d ive rg e n ce between c o n s p e c if ic s i s expected to be m in im a l. E x is t in g

m e thodo log ies a t t h i s le v e l o f re s o lu t io n n e c e s s ita te th e use o f f re q u e n t ly

c u t t in g te t r a n u c le o t id e r e s t r i c t io n endonucleases, la rg e t is s u e samples and

la b o r io u s DNA p u r i f i c a t io n p rocedures in v o lv in g expens ive in s tru m e n ta tio n

and le n g th y u l t r a c e n t r i f u g a t io n . Thus, I aim to im prove and m od ify e x is t in g

te ch n iq u e s fo r mtDNA com parisons, making them more am eniab le to p o p u la tio n

su rve ys between c lo s e ly re la te d in d iv id u a ls , u s in g r e la t i v e l y sm a ll amounts

o f t is s u e (ch a p te r tw o , s e c tio n 2 .3 .1 .2 » f o r f u l l e r d e ta i ls see Appendix 1,

Jones et al., 1988).

S econd ly , I in te n d to in v e s t ig a te th e m o le cu la r mechanisms by which mtDNA

e vo lve s in th e house mouse and to a s c e r ta in w hether i t i s t y p ic a l o f

v e r te b ra te mtDNA. One o f the p rim a ry o b je c t iv e s o f m o le cu la r g e n e tic

re se a rch i s to g iv e an a ccu ra te d e s c r ip t io n o f how th e DNA sequence changes

w ith tim e . T h is re q u ire s an assessment o f th e k in d s o f m u ta tio n a l changes

th a t occur and an a c c u ra te e s tim a te o f th e fre qu e ncy w ith which each typ e

o ccu rs . To the se ends, I in te n d to make f u l l use o f th e com plete known

mtDNA base sequence (KBS) o f th e house mouse to p e rm it th e use o f h ig h

re s o lu t io n sequence com parison r e s t r i c t i o n mapping te c h n iq u e (ch a p te r 3 ) .

R e s t r ic t io n mapping, as opposed to th e c o l le c t io n o f r e s t r i c t io n fragm ent

21

CHAPTER ONE

d a ta , n o t o n ly p ro v id e s in s ig h ts in to th e n a tu re o-f th e e v o lu t io n a ry change

th a t mtDNA undergoes, bu t a ls o g r e a t ly im proves th e accuracy w ith which

e s tim a te s o-f th e e x te n t o-f sequence d ive rge n ce can be c a lc u la te d and

g e n e a lo g ic a l t re e s b u i l t . I hope to compare th e r e s u l t s o f r e s t r i c t io n

mapping B r i t i s h house mice w ith those o b ta in e d from a b roade r b u t le s s

in te n s iv e w o rld w id e survey o f house m ice (F e r r is e t al., 1983), made u s in g

a d i f f e r e n t mtDNA v is u a l is a t io n te c h n iq u e .

T h ir d ly , w ith in th e co n ce p tio n a l fram ework as o u t l in e d by A v ise &

c o lle a g u e s (1987 ), I hope to e v a lu a te th e use o f mtDNA as a p h y lo g e o g ra p h ic

to o l f o r re c o n s tru c t in g th e e v o lu t io n a ry h is to r y o f th e B r i t i s h house mouse

(c h p a te r 4 ) . C o n s p e c if ic p o p u la tio n s o f t e r r e s t i a l and fre s h w a te r

v e r te b ra te s u s u a lly e x h ib i t c o n s id e ra b le geograph ic d i f f e r e n t ia t io n in

mtDNA. The house mouse has been documented as an e x c e p tio n to t h i s g ene ra l

r u le . However, F e r r is and c o lle a g u e s (1983) concluded from a w o rld w id e

su rve y o f mtDNA v a r ia t io n o f th e house mouse th a t a lth o u g h th e re was no

m acrogeographic s t r u c tu r in g , (p ro b a b ly th e r e s u l t o f re c e n t and e x te n s iv e

range e xp a n s io n ), th e re was s u b s ta n t ia l s t r u c tu r in g a t th e m ic rog e og ra p h ic

le v e l . M ito c h o n d r ia l DNA has been proved t o be a v a lu a b le to o l f o r t r a c in g

p a t te rn s o f c o lo n is a t io n and gene f lo w ; c lo n a l tra n s m is s io n o f mtDNA

p ro v id e s a more d e f in i t i v e m arker o f m a terna l phylogeny than does th e

s e x u a lly recom b in ing n u c le a r genome. Indeed, a llozym e da ta a re no t

e s p e c ia l ly in fo rm a tiv e w ith re g a rd to th e e v o lu t io n a ry r e la t io n s h ip s among

B r i t i s h house mouse p o p u la tio n s . However, a p a r t ic u la r aim was to use th e

d a ta a v a i la b le from n u c le a r m arkers combined w ith th a t from mtDNA to

re c o n s tru c t th e most l i k e l y e v o lu t io n a ry h is to r y f o r th e Orkney and

22

CHAPTER ONE

C a ithness m ice. An im p o rta n t is s u e th a t th e mtDNA d a ta w i l l address is

whether th e Orkney is la n d mice a re th e r e s u l t o f a s in g le i n i t i a l

c o lo n is a t io n event (m o n o p h y le tic ) o r w hether th e re have been m u lt ip le

c o lo n is a t io n even ts from se ve ra l a n c e s tra l sou rces .

M ito c h o n d ria l DNA i s m a te rn a lly in h e r i te d , hence i t i s a marker o f fem a le

d is p e rs a l o n ly . T h e re fo re , I sought to com plim ent t h i s by in v e s t ig a t in g

B r i t i s h house mouse r e la t io n s h ip s u s ing th e p a te r n a l ly in h e r i te d Y

chromosome, a com parable marker (ch a p te r 5 ) . L ik e mtDNA, th e Y chromosome

i s e f f e c t iv e ly t ra n s m it te d v ia one sex, does n o t undergo any a p p re c ia b le

re co m b ina tio n and has a r e la t i v e l y h ig h r a te o f sequence e v o lu t io n . Hence,

as th e p o te n t ia l e x is t s fo r u t i l i s i n g Y -s p e c i f ic sequences in a s im i la r

manner to mtDNA i t was my in te n t io n to screen f o r any r e s t r i c t i o n fragm ent

le n g th polym orphism s (RFLPs) w ith in B r i t i s h mouse p o p u la t io n s , u s in g a Y -

s p e c i f ic sequence as a probe w ith th e aim o f assay ing th e e x te n t and

d is t r ib u t io n o f Y chromosomal DNA v a r ia t io n , and u l t im a te ly exam ining

p a t r ia r c h ia l r e la t io n s h ip s .

Phylogeography i s o f te n d e fin e d as a fu n c t io n o f i n t r i n s i c ( l i f e h is to r y ,

d is p e rs a l and s o c ia l o rg a n is a t io n ) and e x t r in s ic (e c o lo g ic a l and

b io g e o g ra p h ic a l b a r r ie r s ) fa c to r s in f lu e n c in g th e h is t o r ic a l p a t te rn o f

gene f lo w . I n t r in s i c b a r r ie r s to gene f lo w have o fte n been a t t r ib u te d to

th e demic s t r u c tu r e and s o c ia l o rg a n is a t io n o f house mouse p o p u la tio n s , and

i t i s g e n e ra lly b e lie v e d th a t im m ig ran ts in t o an e s ta b lis h e d p o p u la tio n a re

u n l ik e ly to be re p ro d u c t iv e !y su cce ss fu l (Reimer and P e tra s , 1967). I f so

then t h i s o b s e rv a tio n may be p e r t in e n t to th e h is t o r ic a l c o lo n is a t io n o f

CHAPTER ONE

th e B r i t i s h I s le s by th e house mouse.

To t e s t t h i s h y p o th e s is a lo n g itu d in a l m ic rog e og ra p h ic s tu d y known as “ th e

I s le o-f May In t r o d u c t io n E xperim en t" was i n i t i a t e d (c h a p te r 6 ) . M ice from

Eday, in th e O rkneys, were in tro d u c e d to an endemic p o p u la t io n o f m ice on

th e I s le D f May, in th e F ir th o f F o rth . In t h i s way m ice from d i s t i n c t l y

d i f f e r e n t demes were b rough t in to c o n ta c t in a n a tu ra l h a b i ta t , where t h e i r

r e la t iv e re p ro d u c t iv e success co u ld be c lo s e ly fo l lo w e d u s in g a v a r ie ty o f

g e n e tic m arkers; bo th th e Y and mtDNA m arkers a re p a r t i c u la r l y v a lu a b le f o r

in v e s t ig a t in g th e male and fem ale c o n t r ib u t io n s . Com parisons o f p a t te rn s o f

v a r ia t io n u s in g mtDNA w ith tho se o f autosom al a n d /o r p a te r n a l ly t ra n s m it te d

genes (th e Y-chromosome) have th e p o te n t ia l to re v e a l d if fe re n c e s in fem a le

and male p o p u la tio n s t r u c tu r e , which m igh t suggest d if fe re n c e s between th e

sexes in th e spec ies* eco logy (d is p e rs a l and p o p u la t io n dynam ics) and

beh a v io u r h i t h e r t o unobserved (H a rr is o n , 1989).

F in a l ly , th e fra g m e n ta ry f o s s i l evidence makes i t d i f f i c u l t t o make any

f i r m s ta te m e n ts about c o lo n is a t io n p a tte rn s o f th e house mouse in Europe.

However, I aim to in te g r a te a g e n e tic approach to t h i s p rob lem w ith

ev idence from a n th ro p o lo g ic a l (as th e house mouse i s a commensal o f man),

p a la e o n to lo g ic a l and h is t o r ic a l sources in an a tte m p t to re c o n c i le these

d is p a ra te s tu d ie s o f th e e v o lu t io n a ry h is to r y o f th e house mouse (ch a p te r

7 ) .

24

FIGURE 1. i s O rg a n is a tio n o-f mouse m ito c h o n d r ia l DNA.

The g e n e tic o rg a n is a t io n , s t ru c tu re and fu n c t io n o f house mouse

m ito c h o n d r ia l DNA genome is p o r tra y e d . The v e r t ic a l a rrow marked 0 „ a t th e

to p in d ic a te th e o r ig in o f r e p l ic a t io n o f th e H -s tra n d s y n th e s is , s itu a te d

in th e D -loop re g io n ; th e arrow marked 0U in d ic a te s th e o r ig in o f th e L -

s tra n d s y n th e s is . L e f t and r ig h t a rrow s in d ic a te th e d ir e c t io n o f H and L

s tra n d t r a n s c r ip t io n re s p e c t iv e ly . A l l th e p ro te in cod ing genes a re H-

s tra n d encoded as i l l u s t r a t e d in th e o u te r c i r c l e (w ith c o u n te rc lo c k w is e

p o la r i t y ) , w ith th e e xce p tio n o f ND 6 , w hich i s L -s tra n d encoded as

in d ic a te d in th e in n e r c i r c le . D is ta n ces in k ilo b a s e s (kb) a re shown in s id e

th e in n e r c i r c l e , co rre sp on d ing th e th e num bering o f th e p u b lis h e d l i g h t

s tra n d sequence (B ibb e t a im , 1981) which b eg in s a t th e f i r s t 5* n u c le o t id e

o f tRNA PHe and proceeds th rough th e rRNA genes w ith in c re a s in g numbers.

The 13 p ro te in cod ing genes a re as fo l lo w s ; ND1, ND2, ND3, ND4, ND4L, ND5,

and ND6 d e p ic t s u b u n its o f NADH dehydrogenase; CO I , CO I I , and CO I I I , a re

th e s u b u n its o f I , I I and I I I o f cytochrom e o x id a se ; ATPase 6 and ATPase 8

a re th e s u b u n its 6 and 8 o f th e Fo H"* -ATPase; c y t . b i s th e cytochrom e b

gene. The genes fo r th e sm all (12S rRNA) and la rg e (16S rRNA) ribosom a l RNA

a re shown. The tRNA genes c lo ckw ise from 0M, a re fo r th e amino a c id s

p r o l in e (P ), th re o n in e (T ) , g lu ta m ic a c id (E ), le u c in e (L ) , s e r in e (S ),

h is t id in e (H ), a rg in in e (R ), g ly c in e (G ), ly s in e (K ), a s p a r t ic a c id (D ),

s e r in e (S ) , ty ro s in e (Y ), c y s te in e (C ), aspa ra g ine (N ), a la n in e (A ),

try p to p h a n (M ), m e th io n in e (M), g lu ta m in e (Q ), is o le u c in e ( I ) , le u c in e ( L ) ,

v a l in e (V ), and p h e n y la la n in e (F ) . The tRNA genes a re la b e le d on th e in n e r

o r o u te r c i r c le s ( re p re s e n tin g th e l i g h t and heavy s tra n d , r e s p e c t iv e ly ) ,

d e p ic t in g where t h e i r cod ing sequences o c c u r.

25

oH

MOUSE mtDNA ro

,c q

26

Figur*

1. 2

I*><

onom^_

of_tbf

CHAPTER TWO

CHAPIER_IWOi_MAIERIALS_AND_MEIHgDSJL

2iii_ABBREyiAII0NSi

A l l th e a b b re v ia t io n s used in t h i s th e s is t o d e s c r ib e th e ch e m ica ls ,

s u p p lie rs , s o lu t io n s , and re a g e n ts a re l i s t e d in T ab le 2 .1 .

2i2_MAIERIALSi

2i 2i li_SAMPLING_PRgCEDURESi

The m ice were c o lle c te d in se ve ra l ways depending on lo c a t io n .

A p p ro x im a te ly hal-f o f th e p o p u la tio n s were caught by 1 iv e - t r a p p in g us ing

" lo n g w o rth t ra p s " ( C h it ty & Kempson, 1949). The t ra p s were p la ced on th e

n a tu ra l runs o-f - f r e e - l iv in g fe r a l a n im a ls (eg . I s le o f May, Skokholm, and

Faray) o r in and around dom estic b u i ld in g s and fa rm s fo r commensal

p o p u la t io n s . Set near p ro ba b le mouse t r a c k s , u s u a lly e v id e n t by t r a i l s D-f

fae ce s ( fo r example B u rto n -o n -T re n t p o p u la t io n s ) th e t ra p s were b a ite d w ith

peanut b u t te r (o r any o th e r s tro n g s m e llin g fo o d ) , l in e d w ith p le n ty o-f

bedd ing s tra w and an a d d it io n a l food source (o a ts ) . The t ra p s were a lw ays

checked e a r ly in th e m orning o f th e n e x t day so as m ice were never kep t in

th e t ra p s f o r more than 24 hou rs .

As an a l te r n a t iv e to t ra p p in g , m ice were caught by hand from c o r n - r ic k s

(S outhern & L a u r ie , 1946) when th e y were d ism a n tle d fo r th re s h in g in

S p rin g (eg W estray, Eday, and Taunton p o p u la tio n s ) o r by s im p ly moving food

c o n ta in e rs a t p ig farm s (eg. N u t f ie ld ) , and c o l le c t in g th e d is tu rb e d m ice

in sacks.

28

CHAPTER TWO

T ab le 2 .2 l i s t s a l l m ice used in t h i s s tud y (c a te g o r is e d by C o u n ty /Is la n d

where the y were c a u g h t) , method and da te o-f c a p tu re , number caught and

N a tio n a l G rid re fe re n c e in m ain land B r i t a in (o r th e th re e m ile per in c h AA

map re fe re n c e fo r I r i s h sam p les). F ig u re 2.1 i l l u s t r a t e s th e m ajor

d is t r ib u t io n o f th e se samples in B r i t a in and I re la n d ( th e numbers in th e

c i r c le s re p re s e n t th e m ajor lo c a t io n s co rre sp o n d in g to th e co un ty

c a te o rg is a t io n s in ta b le 2 .2 ) . A t o t a l o f 601 mice were examined in t h i s

s tu d y from 42 B r i t i s h and I r i s h l o c a l i t i e s .

Each mouse was marked f o r la t e r id e n t i f i c a t io n u s in g a co m b in a tio n o f

e a rc lip p in g (B e rry , 1970) and to e - c l ip p in g (Tw igg, 1975),

F ig u re 2 .2 i l l u s t r a t e s th e scheme used. A l l m ice caugh t were se n t back to

th e la b o ra to ry , k i l l e d by c e rv ic a l d is lo c a t io n and s to re d a t -70 °C u n t i l

th e t is s u e s were re q u ire d .

2i 2i 2^_REAGENISi

A l l chem ical re a g e n ts used were o f s tanda rd a n a ly t ic a l g rade . Table 2 .3

summarises a l l th e re a g e n ts , chem ical s o lu t io n s and b u f fe r s used in t h i s

s tu d y .

R a d io a c t iv e ly la b e lle d d e o x y r ib o n u c le o tid e s were purchased from Amersham.

The s p e c i f ic n u c le o t id e s used were th e d e o x y r ib o n u c le o s id e o f c y to s in e and

thym ine la b e lle d w ith 32P in th e a lpha p o s i t io n .

The r e s t r i c t io n endonucleases were o b ta in e d from N o rth um bria b io lo g ic a ls

l im ite d (NBL), Bethesada rese a rch la b o ra to r ie s (BRL) and o c c a s io n a lly New

29

CHAPTER TWO

England B io la b s (NEB). Table 2 .4 l i s t s a l l th e 18 enzymes used, t h e i r

re c o g n it io n sequences, th e in c u b a tio n te m p e ra tu re s , s p e c i f ic s a l t b u f fe rs

and in d ic a te s what each enzyme was used f o r .

2i2i3i_M0LECyLAR_WEIGHI_SIANDARDSi

The a b s o lu te s iz e o f th e r e s t r ic t io n fra g m e n ts produced by ge l

e le c tro p h o re s is was e s tim a te d by com paring m o b i l i t y o f s tan d a rd s e ts o f

fra gm en ts produced by c leavage o f th e known m ito c h o n d r ia l DNA sequence o f

th e house mouse ( k . b . s . ) (B ibb e t a i . , 1981) which can be found l i s t e d in

Table 2 .5 to g e th e r w ith th e r e s t r i c t io n s i t e lo c a t io n s . Marker fragm en ts

were u s u a lly run in th e end lanes o f th e g e l , e i th e r s id e o f th e

e xp e rim e n ta l sam ples, and v is u a lis e d in th e same way as th e DNA (by e i th e r

s i l v e r o r e th id iu m brom ide s ta in in g ) . In a d d it io n m o le cu la r w e igh t m arkers

were gene ra ted by c leavage o f lambda DNA w ith H ind I I I , and lambda w ith Bgl

I o r use was made o f th e com m erc ia lly a v a i la b le 1KB la d d e r (BRL); the s iz e s

o f a l l o f th e se m arkers are l is t e d on Tab le 2 .6 .

2i 3_LAB0RAIDRY_MEIH0DSi

These can be c o n v e n ie n t ly s p l i t in to two s e c t io n s a t th e r is k o f some

o v e r la p . They in c lu d e p ro to c o ls in v o lv in g * 1. M ito c h o n d r ia l DNA is o la t io n

and v is u a l is a t io n te ch n iq u e s and 2. Y-Chromosome DNA m e thodo log ies .

CHAPTER TWO

2i 3i l_MIIOCHONDRIAL_pNA_ISOLAIIONi

2i 3i l i i_ R a tio n a le _ g f_ M e th g d g lg g ie s JL

P o p u la tio n s tu d ie s re q u ire a s im p le and in e x p e n s iv e method o-f is o la t in g

mtDNA from many in d iv id u a ls . E x is t in g methods use le n g th y u l t ra c e n t r i- fu g e

te c h n iq u e s (Lansman e t a i . , 1981) o r ra p id b u t e xpens ive bench -top u l t r a -

h igh -speed c e n t r i fu g e s (C arr St G r i f f i t h , 1987) and c o s t ly cesium c h lo r id e

g ra d ie n ts t o p u r i f y crude mtDNA f r a c t io n s and a re th u s n o t e n t i r e ly

s a t is fa c to r y . Techniques in v o lv in g r a d io la b e l l in g o f is o la te d DNA (Brown,

1980) a ls o have some d isadvan tages fo r p o p u la t io n s c re e n in g .

The phenol e x t r a c t io n p rocedu re , d esc ribe d by Pow ell Sc Zuniga (1983 ),

f u l l f i l s most o f th e c r i t e r i a f o r use in p o p u la t io n s tu d ie s , bu t i s l im i t e d

by th e low s e n s i t i v i t y o f e th id iu m brom ide s ta in in g used t o v is u a l is e th e

la rg e mtDNA fra gm en ts produced by d ig e s t io n w ith h e x a n u c le o tid e r e s t r i c t io n

endonucleases. R e s o lu tio n i s poor w ith t h i s method and a more s e n s i t iv e

d e te c t io n method i s necessary , one which p e rm its th e use o f more f re q u e n t ly

c u t t in g te t r a n u c le o t id e r e s t r i c t io n endonucleases, t o gen e ra te th e many

sm a ll fra gm en ts re q u ire d .

The advantages o f in c re a s in g th e number o f fra gm en ts in a d ig e s t i s shown

g r a p h ic a l ly in f ig u r e 2 .3 . T h is shows a p lo t o f th e p r o b a b i l i t y o f no t

d e te c t in g d if fe re n c e s between in d iv id u a ls a g a in s t th e number o f base p a ir s

which must be examined fo r d i f f e r e n t le v e ls o f pe rcen tage sequence

d ive rge n ce (d). The average d va lu e s between two in d iv id u a ls o f th e same

sp e c ie s i s u s u a lly between 0 .1 - 1.0% ie . Hus domesticus = 0.77%, Hus

CHAPTER TWO

musculus = 0.927. ( F e r r is et a l . , 1983). The d va lu e s w i l l p ro b a b ly be

c o n s id e ra b ly s m a lle r when c o n s id e r in g c o n s p e c if ic s from th e same is la n d ,

where th e sm a ll e f f e c t iv e p o p u la tio n s iz e s and s to c h a s t ic e ven ts w i l l

reduce th e number o f mtDNA typ e s p re s e n t. F ig u re 2 .3 dem onstra tes th a t a t

th e 0.057. p r o b a b i l i t y le v e l an in c re a s in g ly la rg e number o f base p a ir s

needs to be ana lysed to d e te c t low er le v e ls o f d iv e rg e n c e . T h is

n e c e s s ita te s th e use o f s e n s i t iv e s i l v e r s ta in in g p ro to c o ls (T e g e ls trom ,

1986), to d e te c t th e sm a ll fragm en ts o f mtDNA from s in g le in d iv id u a ls .

The h ig h s e n s i t i v i t y o f s i lv e r s ta in in g re q u ire s th a t th e mtDNA be

r e la t i v e l y f r e e from n u c le a r c o n ta m in a tio n . C ru c ia l m o d if ic a t io n s o f th e

o r ig in a l Pow ell and Zuniga e x tr a c t io n p rocedu re (Jones e t al», 1988J see

enclosed paper in Appendix 1) p e rm it th e r e q u is i te co m b ina tio n o f p u r i t y

and s e n s i t i v i t y w ith o u t th e need fo r u l t r a c e n t r i f u g a t io n .

L iv e r t is s u e seemed to be e s p e c ia lly s e n s i t iv e to s to ra g e c o n d it io n s

( f r e e z in g ) , g iv in g n o t o n ly poor mtDNA y ie ld s b u t samples s e v e re ly

contam ina ted w ith n u c le a r DNA. T h is was avo ided i f e i t h e r , f re s h l i v e r

t is s u e was a v a i la b le ( th e m a jo r ity o f th e samples had a lre a d y been s to re d

a t -7 0 °C ) , o r o n ly h e a r t and k idney t is s u e s were used. As a consequence o f

th e s m a lle r amount o f t is s u e u t i l i s e d mtDNA y ie ld s were reduced.

U n fo r tu n a te ly c h a r a c te r iz a t io n o f th e s tu d y p o p u la t io n mtDNA genotypes

re q u ire s la rg e amounts o f u lt r a - p u r e DNA.

32

CHAPTER TWO

The optimum s tra te g y seemed to be (-Fo llow ing Tegel s tro m , 1986) th e use o-F

d i f f e r e n t mtDNA is o la t io n te ch n iq u e s depending upon s to ra g e c o n d it io n s ,

t is s u e a v a i l a b i l i t y , and mtDNA y ie ld re q u ire d . In each case , th e DNA was

r o u t in e ly v is u a l is e d by s i l v e r s ta in in g , and o n ly in a sm a ll number o f

cases by e th id iu m brom ide s ta in in g .

Hence, d u r in g th e i n i t i a l phase o f sc re e n in g th e p o p u la t io n s w ith s u ita b le

r e s t r i c t io n enzymes to d e te c t th e g e n e tic v a r ia t io n , mtDNA was is o la te d by

u l t r a c e n t r i fu g a to n (Lansman e t 1981; M a n ia t is e t a i . , 1982) g iv in g good

y ie ld s o f h ig h ly p u r i f ie d mtDNA. Once th e mtDNA c lo n e s were c h a ra c te r iz e d

th e m o d if ie d phenol e x t r a c t io n p rocedure (Jones e t al.f 1988) was employed

to r a p id ly p rocess la rg e numbers o f in d iv id u a ls . D e ta i ls o f b o th p rocedu res

a re d e sc r ib e d below .

2i 3i l i 2_IHE_M0DIFIED_PHEN0L_EXIRACIigN^PR0CEDyRE_F0R_MIDNA_IS0LAII0N_lJones

e t aim, 1988; see appendix 1 ).

E a r l ie r e x tr a c t io n p rocedures f o r m ito c h o n d r ia l is o la t io n produced crude

f r a c t io n s con tam ina ted w ith n u c le i and o th e r c e l lu la r com ponents. The

i n i t i a l hom ogen iza tion s tep s in th e is o la t io n p rocedu re were c r i t i c a l i f

th e m ito c h o n d r ia l p e l le t s were to be r e la t i v e l y p u re . To t h i s end th e hand

Dounce hom ogenizer used by Pow ell & Zuniga (1983) was re p la c e d w ith a

m o to r-d r iv e n g la s s T e flo n hom ogenizer (Lansman et a i . , 1981). F a c to rs

c r i t i c a l f o r s u cce ss fu l hom ogen isa tion were, (a) th e c le a ra n c e between th e

p e s t le and th e tube - th e optimum appeared to be about 0.2mm ( ie . no t

t i g h t - f i t t i n g ) , a llo w in g th e p e s t le to s l id e s lo w ly and e a s i ly a long th e

33

CHAPTER TWO

tube* (b) th e number o-f s tro k e s o-f th e p e s t le - g e n e ra lly th e minimum

number o-f s tro k e s necessary to a llo w th e p e s t le to reach th e bo ttom o-f th e

tu b e (5 -8 s t ro k e s ) ; (c) th e hom ogen isa tion speed - a low speed,

a p p rx im a te ly 200 rpm were used; and (d) la rg e volumes o f hom ogenising

b u f fe r was a lw ays used where p o s s ib le .

The e x t r a c t io n p rocedu re was as fo l lo w s :

1. The t is s u e (0 .3 grams) was f in e l y chopped in c h i l le d d i s t i l l e d w a te r t o

remove any f a t t y d e p o s its o r b lo o d . The t is s u e was t ra n s fe r re d to

hom ogenizing tubes w ith a t le a s t 10ml o f p re -c h i l ie d H o m o g e n iz in g -b u ffe r.

2. The hom ogen iza tion was c a r r ie d o u t as d e s c r ib e d above.

3. The homogenate was spun a t l,0 0 0 g a t 4 °C fo r 15 m inu tes (Beckman

c e n t r i fu g e JA 2 0 .1 ). The supe rna ten t was c a r e fu l ly p ip e t te d o f f (a v o id in g

th e n u c le a r p e l le t ) and cooled on ic e .

4 . The n u c le a r p e l le t was suspended in 5 ml o f f re s h b u f fe r and respun a t

l,3 0 0 g f o r 10 m in. The supe rna ten t was p ip e t te d o f f and added to th e

o r ig in a l and spun aga in a t l,3 0 0 g f o r a n o th e r 10 m in . These s p in s were

repea ted on th e su p e rn a te n t u n t i l no n u c le a r p e l le t rem ained.

5. The f i n a l su p e rn a te n t was spun a t 15 ,000g a t 4 °C f o r 30 m inu tes to

p e l le t th e m ito c h o n d ria (Beckman c e n t r i fu g e JA 20 .1 ).

6. The s u p e rn a te n t was d isca rded and th e mt p e l le t was resuspended in 2 ml

STE b u f fe r , and warmed to 37 °C p r io r t o use.

7. A sm a ll amount o f 25% sodium dodecyl s u lp h a te (SDS) s o lu t io n was added

u n t i l th e su p e rn a te n t c le a re d , in d ic a t in g com p le te l y s i s o f th e

m ito c h o n d ria (25-50 pi fo r h e a r t /k id n e y s , 100 ^1 f o r l i v e r ) .

CHAPTER TWO

8. 30 p i o f RNase A (20 mg/ml s o lu t io n , D N ase -free , i e . , p re b o ile d fo r 10

m inu tes and coo led on ic e ) was added to th e ly s a te and in cu b a te d fo r 30

m inu tes a t 37 °C.

9. To t h i s 20 p i o f p ro te in a s e K (20 mg/ml s o lu t io n ) was added then

in cu b a te d f o r 30 m inu tes a t 37 °C.

10. The ly s a te was shaken w ith an equal volume o f T r is - e q u i l ib r a te d phenol

(M a n ia t is e t a i - , 1982) and c e n tr ifu g e d fo r 30 m in u tes a t 20 ,000g ( t h is

s tep was more e f f e c t iv e th e lo n ge r and h a rde r th e ly s a te was spun ).

11. The aqueous s u p e rn a te n t was p ip e t te d in t o a c le a n c e n t r i fu g e tub e ,

mixed w ith an equal volume o f p h e n o l-c h lo ro fo rm m ix tu re , then spun a t

12,000g fo r 20 m in u tes . T h is s tep was repea ted u n t i l th e re was no lo n g e r

any w h ite p ro te in in te rp h a s e between th e ly s a te and th e p he n o l.

12. The s u p e rn a te n t was e x tra c te d tw ic e w ith an equal volume o f c h lo ro fo rm

and tw ic e w ith an equal volume o f e th e r . The tu b e was shaken u n t i l th e

in te rp h a s e between th e e th e r and th e s u p e rn a te n t was sharp ( ie . no bubb les

o r opaque la y e r ) .

13. The e th e r was d isca rd e d and any re s id u a l e th e r a llo w e d to evapo ra te fo r

a t le a s t 15 m inu tes .

14. I f necessa ry , d i s t i l l e d w ater was added to th e s u p e rn a te n t t o b r in g th e

volume up to 2 ml ( t h is ensured th e same amount o f aqueous s o lu t io n in each

sample and low ered th e s a l t c o n c e n tra t io n ) . A b so lu te e th a n o l was added to

a p p ro x im a te ly th re e tim e s th e volume o f th e s u p e rn a te n t, th e tubes were

covered w ith p a ra f i lm and mixed by in v e rs io n , then s to re d a t -70 °C fo r a t

le a s t 2 hou rs o r o v e rn ig h t .

35

CHAPTER TWO

15. The sample was removed from -70 °C and a llo w e d t o s ta n d u n t i l i t

reached room te m p e ra tu re and mixed aga in ( i f any s a l t s had been

p r e c ip i ta te d th e y were re d is s o lv e d ) . The tubes were spun f o r 30 m inu tes a t

20 ,000g a t 4 °C to p r e c ip i ta t e th e DNA.

16. The su p e rn a te n t was d isca rd e d and th e mtDNA p e l le t vacuum d r ie d , then

d is s o lv e d in 50 ^ il o f TE b u f fe r and t ra n s fe r re d to a s t e r i l e eppendorf tube

fo r s to ra g e a t -20 °C.

The average y ie ld s o f mtDNA were 0 .5 - 1 ^ig per 0 .5 gram o f t is s u e ( f o r a

s in g le mouse).

2i 3i l i 3_intDNA_ISgLAII0N_ySING_yLIRACENIRIFyGATI0Ni

The p ro to c o ls v a r ie d s l i g h t l y depending on which r o to r was a v a i la b le f o r

u l t r a c e n t r i f u g a t io n o f th e cesium c h lo r id e g ra d ie n ts . A s w in g -o u t r o to r was

p re fe ra b le as t h i s made th e mtDNA band in th e g ra d ie n t c le a r ly v is a b le , and

hence easy to e x t r a c t . When th e v e r t ic a l r o to r had t o be used a c o n tro l

tube was spun a long s id e th e sample tu b e s . The c o n t ro l c o n ta in e d mtDNA

e x tra c te d from a fem a le la b o ra to ry mouse and her l i t t e r poo led to g e th e r .

T h is in c re ase d t is s u e w e ig h t (from 0 .5 g to 1 .5 - 2 .0 g) gave a g re a te r

y ie ld o f mtDNA and a c le a r ly marked th e mtDNA band in th e v e r t ic a l

g ra d ie n t , in d ic a t in g th e p o s i t io n o f th e f a in t o r i n v i s ib le mtDNA bands in

th e sample tub e s .

2JL3i l i 3i l_ySE_0F_A_yERIICAL_Rgi0Ri

The p ro to c o l was e s s e n t ia l ly th e same as th a t d e s c r ib e d f o r th e m o d ifie d

phenol e x tra c t io n method up to s te p 1 0 ., which produces a sem i-c rude

36

CHAPTER TWO

m ito c h o n d r ia l DNA f r a c t io n ready fo r u l t r a c e n t r i f u g a t io n a f t e r o n ly one

phenol e x t r a c t io n . F o llo w in g t h i s :

1. The volume o f th e su p e rn a te n t c o n ta in in g th e c rude mtDNA f r a c t io n was

measured. For every m i l l i l i t r e measured e x a c t ly one gram o f s o l id CsCl was

added and th e s o lu t io n mixed u n t i l a l l th e s a l t had d is s v o lv e d .

2. For every 10 ml o f CsCl s o lu t io n 0 .8 ml o f a 10 mg/ml s to c k s o lu t io n o f

e th id iu m brom ide was added. T h is gave a f i n a l d e n s ity o f 1 .55 g /m l

(p = 1 .386) and a f i n a l c o n c e n tra t io n o f e th id iu m brom ide o f 600 ^ug/ml

(M a n ia t is et a i . , 1982).

3. T h is s o lu t io n was t ra n s fe r re d to a p o ly a llo m e r q u ic k -s e a l tu b e s u ita b le

f o r c e n t r i fu g a t io n in a Beckman Type V t i 6 5 .2 r o to r .

4. The rem ainder o f th e tube was f i l l e d w ith a s tock s o lu t io n o f 1 .0 g /m l

CsCl/ 600 ^ig/ml e th id iu m brom ide.

5. A l l th e tubes were balanced to w ith in 0 .1 gram o f each o th e r and loaded

o n to th e r o to r a f te r th e top s had been sea led (Beckman s e a le r ) .

6. A l l th e tubes were spun a t 55 ,000 rpm fo r 14 hours a t 20 °C.

7. The g ra d ie n ts were v is u a lis e d under UV l i g h t . The c o v a le n t ly c losed

c i r c u la r mtDNA was seen as a v e ry f a in t band a p p ro x im a te ly 0 .5 cm below a

v a r ia b le s iz e d upper band which co n ta in e d th e n u c le a r DNA. The tub e was

p unc tu red a t th e top t o re le a s e th e p re s s u re . The lo w e r mtDNA band was

c o l le c te d (a p p ro x im a te ly 200 -7 0 0^u l) by s id e p u n c tu re im m e d ia te ly below th e

band w ith a 1 ml hypoderm ic s y r in g e and need le (th e need le was in s e r te d

w ith th e beve l uppe rm ost).

8. The sample was e x tra c te d two t o th re e tim e s w ith an equal volume o f 2 -

b u ta n o l s a tu ra te d w ith a s o lu t io n o f 5M NaCl in a 0.05M T ris -H C L b u f fe r pH

37

CHAPTER TWO

7 .5 .

9 . D is t i l l e d w ater was added to d i lu t e th e su p e rn a te n t to tw ic e th e

o r ig in a l volum e. The mtDNA was recovered by e th a n o l p r e c ip i t a t io n and

s to re d a t -2 0 °C as d e sc rib e d p re v io u s ly (p o in ts 14-16 ! s e c tio n 2 .3 . 1 .2 . ) .

2i 3i I i 3i 2_i_ySE_0F_A_SWING=0yi_R0I0Ri

A c rude mtDNA - f ra c t io n was p repared a c c o rd in g to Jones et a l 1988 up to

s te p 7. o-f th e e x tr a c t io n p rocedure ( ie . th e re was no need fo r th e RNase A,

p ro te in a s e K, o r phenol tre a tm e n ts ) . A f te r w h ich !

1. The volume o f th e crude DNA s o lu t io n was measured and 1.1 g /m l o f s o l id

CsCl was added, and s im i la r ly , e th id iu m brom ide a t a c o n c e n tra t io n o f 0 .8

ml per 10 ml o f s o lu t io n . T h is s o lu t io n was t r a n s fe r re d to a 2 .2 ml

u l t r a c e n t r i f u g e tu b e s u ita b le f o r th e TLA-55 sw in g -o u t r o to r f o r use on th e

b ench -top TLA-100 u l t r a c e n t r i f u g e ; tubes were ba lanced w ith a s to ck

s o lu t io n o f C s C l/e th id u im brom ide (1 .1 g per ml / 600 p i pe r m l) .

2 . The tubes were spun fo r 30 m inu tes a t 30 ,000 rpm, removed and th e

" p ro te in cap" (da rk p u rp le aggregate a t th e to p o f th e tu b e s ) was taken

o f f . The tu b e s were the n r e - e q u i l ib r a te d , re -b a la n c e d and spun a t 36,000

rpm fo r 42 h ou rs .

3. The low er mtDNA band v is u a lis e d by UV l i g h t was c o l le c te d by a need le

and 1 ml s y r in g e from th e top o f th e open tu b e . Again th e s o lu t io n was

b u ta n o l e x tra c te d , e th a no l p re c ip i ta te d and s to re d as p re v io u s ly d e s c r ib e d .

As an a l t e r n a t iv e t o e thano l p r e c ip i ta t io n th e mtDNA co u ld be recovered by

d ia ly s is . A lthough t h i s i s a more t im e consuming p rocedu re i t y ie ld s pure

mtDNA uncontam ina ted by s a lts which may p rove to be a problem d u r in g

33

CHAPTER TWO

r e s t r i c t i o n enzyme d ig e s t io n s (e s p e c ia l ly when u s in g th o s e enzymes

s e n s i t iv e t o s a l t c o n c e n tra t io n s eg Rsa I ) .

The mtDNA s o lu t io n was d ia ly s e d as fo l lo w s :

The s o lu t io n was p la ce d in d ia ly s is bags and d ia ly s e d a g a in s t

1 M NaCl/TE in a c o ld room o v e rn ig h t (a t le a s t 24 h o u rs ) on a m agnetic

s t i r r e r s e t on a s lo w speed. The d ia ly s is l i q u id was changed e ve ry 12

h o u rs . The d ia ly s is bags were then d ia ly s e d a g a in s t TE b u f fe r f o r o n ly 2 -6

hours (o r o v e rn ig h t w ith a change o f s o lu t io n e v e ry 12 h o u rs ) . The d ia ly s e d

mtDNA was t r a n s fe r r e d to s t e r i l e m ic ro fu g e tu b e s and s to re d a t -2 0 °C u n t i l

re q u ire d .

2i 3i l i 4_RESIRICII0N_END0NyCLEASE_DI6ESII0NSi

The enzyme d ig e s t io n s were c a r r ie d ou t in 10 ^ i l r e a c t io n m ix tu re s in 1.5m l

m ic ro fu g e tu b e s w ith th e a p p ro p r ia te b u f fe r (see ta b le 2 .4 ) a c c o rd in g to

s u p p lie rs d i r e c t io n s . When s i l v e r s ta in in g was used a p p ro x im a te ly 10-40 ng

(4 -5 / jiI o f th e 50 /J l /g o f i n i t i a l t is s u e w e ig h t; s e c t io n 2 .3 .1 .7 DNA

q u a n t i f i c a t io n ) o f mtDNA was d ig e s te d w ith 1 u n i t o f enzyme (a lth o u g h t h i s

was g r e a t ly in excess o f th e th e o r e t ic a l re q u ire m e n ts w h ich i s 1 u n i t

enzyme/ 15 m in u tes / I / jg o f DNA, p ro lon g ed d ig e s t io n seems to enhance

d ig e s t io n , th e re b y a v o id in g p a r t ia l d ig e s ts ) . When e th id iu m brom ide was

used a t le a s t 100-200 ng D f mtDNA was d ig e s te d in a 20 / j I re a c t io n volum e.

A l l d ig e s t io n s were c a r r ie d o u t a t 37 °C , w ith th e e x c e p tio n o f Taq I w hich

was used a t 65 °C. The re a c t io n was l e f t o v e rn ig h t a f t e r w hich 1-2 ^ il o f

lo a d in g b u f fe r was added and th e sample im m e d ia te ly s to re d a t -2 0 °C.

39

CHAPTER TWO

2i 3i l i 5_GEL_ELECIR0PH0RESISi

The d ig e s te d samples were sepa ra ted on 1.27. submerged h o r iz o n ta l agarose

g e ls (24 cm long x 16 cm w ide x 0 .2 cm th ic k ; see s e c t io n 2 .3 .2 .3 fo r

se tup d e ta i ls ) o r 57. v e r t ic a l p o ly a c ry la m id e g e ls (19cm lo n g x 19.5cm w ide

x 0.07cm th ic k : BRL v e r t ic a l ge l equ ipm ent, model V16) depending on th e

s iz e s o-f fragm en ts to be sepa ra ted ( fo r s iz e s 16000 - 1000 b a s e p a irs ,

u s u a lly produced by c leavage w ith s ix b a s e -c u tte r enzymes, agarose g e ls

were used; f o r 1000 -1 0 0 , b a se p a irs produced by fo u r b a s e -c u t te r enzymes,

a c ry la m id e was u sed ). The a c ry la m id e g e ls were c a s t on g la s s p la te s

p re v io u s ly s i lan e and re p e l s i la n e coated (T ege ls trom & W yoni, 1986), a

tre a tm e n t necessary f o r easy removal o f th e to p g la s s p la te and h a n d lin g o f

th e ge l d u r in g s ta in in g . B e ls were run us in g 1 x TBE b u f f e r , pH 8 .3 fo r

about 3 h ou rs , a t a p p ro x im a te ly 220 v o l t s (35 mA).

The agarose g e ls , u s u a lly run to se p a ra te la rg e fra gm en ts produced from

h e x a n u c le o tid e enzymes, were run on a TAE b u f fe r , pH 7 .4 f o r 3 -4 hours a t

80 v o l t s . S tocks o f 10 x b u f fe r c o n c e n tra t io n s were made up and s to re d a t

room tem pe ra tu re u n t i l re q u ire d , whereupon th e y were d i lu te d to normal

ru n n in g c o n c e n tra t io n s im m e d ia te ly b e fo re use.

2 i3 i l i 6_MIIOCHONDRIAL_DNA_yiSyALISAIIQN_PROCEDyRESi

2i3ili6il_SILyER_SIAININGiThe s i l v e r s ta in in g p ro to c o l (G u ille m e tte & Lew is , 1983) i s used p r im a r i ly

t o v is u a l is e th e mtDNA in p o ly a c ry la m id e g e ls y e t a v o id s th e la b o r io u s

p rocedures o f fragm ent e n d - la b e l l in g o r h y b r id is a t io n w ith s p e c i f ic p robes.

The cover p la te was removed and th e ge l im m e d ia te ly p la ced in 0.1%

40

CHAPTER TWO

c e ty l t r im e th y l ammonium brom ide (CTAB, Sigma) fo r 20 m in u te s . The ge l was

then r in s e d in d i s t i l l e d w ater -for a f u r th e r 20 m inu tes and n e x t p laced in

0.3X ammonia s o lu t io n f o r 15 m in u te s , the n 15 m inu tes in ammoniacal s i l v e r

s o lu t io n ( ta b le 2 .3 ) (0 .4g o f s i l v e r n i t r a t e d is s o lv e d in 2 ml o f d i s t i l l e d

w a te r was added to 248 ml o f d i s t i l l e d w a te r p r io r t o th e a d d it io n o f 1 ml

D f f r e s h ly p repared 1 M NaOH and 1 ml o f 255C ammonia, in t h i s o rd e r ) . The

ge l was developed fo r 10-15 m inu tes in 27. sodium ca rb on a te w ith 0 .0 27.

fo rm a ldehyde . A f te r d e v e lo p in g , th e ge l was soaked in a 27. g ly c e ro l

s o lu t io n fo r 30 m in u tes , then a llo w e d to d ry .

The d ry s i l v e r s ta in e d g e ls were r o u t in e ly pho tographed.

2i 3i l i 6i 2_ETHIDIUM_BR0MIDE_SIAININGi

E th id iu m brom ide was added to th e b o ile d agarose (from a s to c k s o lu t io n o f

10 mg/ml in d i s t i l l e d w a te r) to a f i n a l c o n c e n tra t io n o f 0 .5 ^ J l/m l. A f te r

e le c tro p h o re s is th e DNA fragm en ts can be v is u a lis e d d i r e c t l y u s in g a UV

l i g h t t r a n s i l lu m in a to r and photographed u s in g a P o la ro id ty p e 55

p o s i t iv e /n e g a t iv e f i lm . A l t e r n a t iv e ly th e g e ls cou ld be s ta in e d a f t e r th e

run by 0 .05 ^jg/m l o f e th id iu m brom ide in 1 x TBE b u f fe r f o r 15 m inu tes and

r in s e d tw ic e w ith d i s t i l l e d w a te r. The fragm en ts were v is u a l is e d w ith UV

l i g h t and photographed as b e fo re .

2jL3iliZ_0yANIIEIQAIION_gF_MIIOCHONDRIAL_DNAiThe amount o f mtDNA from each mouse was q u a n t i f ie d by ta k in g i n i t i a l

s p e c tro p h o to m e tr ic re a d in g s a t w ave leng ths o f 260nm and 280nm. A pure

p re p a ra t io n o f mtDNA shou ld have an o p t ic a l d e n s ity 0D2AO / 0D2OO o f

41

CHAPTER TWO

a p p ro x im a te ly 1 .8 . A va lu e o-f 1 f o r 260nm re a d in g co rresponds to a

c o n c e n tra t io n o f about 50ug/ml o f DNA (M a n ia t is et a i . , 1982).

However, t h i s method can o n ly be used when la rg e amounts o f h ig h ly p u r i f ie d

t is s u e a re used. More r o u t in e ly th e amount o f mtDNA per sample was

e s tim a te d from th e in t e n s i t y o f f lu o re s c e n c e e m itte d by e th id iu m b rom ide.

The amount o f f lu o re s c e n c e i s p ro p o r t io n a l to th e t o t a l mass o f DNA,

co n se q ue n tly th e q u a n t i ty o f DNA in th e sample co u ld be e s tim a te d by

com paring th e f lu o re s c e n t y ie ld s o f each uncu t mtDNA sample w ith th a t o f a

s e r ie s o f known s ta n d a rd s (uncut lambda o r lambda d ig e s te d w ith H ind 111)

u s in g th e m in ig e l method (M a n ia tis e t alr 1982).

Q u a n tify in g th e mtDNA enabled a p p ro x im a te ly th e same amount o f DNA to be

d ig e s te d and a p p lie d pe r gel t r a c k , a v o id in g c o m p lic a tio n s o f ge l

in te r p r e ta t io n caused by under o r over— lo a d in g . T y p ic a l ly 10-40ng o f mtDNA

were used per d ig e s t , th e usual y ie ld be ing a p p ro x im a te ly 400ng /0 .5 g o f

wet w e igh t t is s u e (T e g e ls trom , 1987c).

42

CHAPTER TWO

2i 2i 3_Yr CHR0M0S0ME_MEIH0D0L0GIESi

2i3i2il_ISOLAIION_OF_igiAL_GENOMIC_DNAi

1. 700 ^ i l o-f t o t a l genomic b u f fe r were added to a p p ro x im a te ly 0 .7 cm3 o f

t a i l s k in t is s u e in a 1 .5 ml m ic ro fu g e tub e and th e t is s u e m acerated w ith a

p a ir o-f s c is s o rs .

2 . To t h i s s o lu t io n 35 pi o f p ro te in a s e K (10 mg/ml s to c k s o lu t io n ) were

added, th e tu b e shaken and incuba ted a t 55 °C o v e rn ig h t .

3. Any u n d ig e s te d t is s u e was p e lle te d a t 10,000 rpm in a m ic ro fu g e fo r 5

m inu tes .

4. The su pe rn a ta n t was removed w ith a w ide-m outhed p ip e t te ( to avo id

s h e a rin g th e DNA) and t ra n s fe r re d to a new s t e r i l e tu b e c o n ta in in g 25 ^j1 o f

10 mg/ml b o i le d RNase A. F o llo w in g in c u b a tio n a t 37 °C f o r 2 h ou rs , th e

s o lu t io n was e x tra c te d w ith an equal volume o f T r is - e q u i l ib r a te d pheno l.

5. The s o lu t io n was e x tra c te d tw ic e w ith c h lo ro fo rm and th e aqueous phase

t ra n s fe r re d to a f re s h tub e and the DNA p r e c ip i ta te d by a d d it io n o f

is o p ro p a n o l.

6. The DNA was spoo led o n to a sea led g la s s c a p i l la r y tu b e , d ipped in t o 70V.

(V/V) e th a no l and the n in t o 1007. e th a n o l, d r ie d and re -d is s o lv e d in 500 pi

TE, pH 7 .4 .

2i 3i 2i 2_RESIRICII0N_ENZYME_DIGESTIQNSi

D ig e s ts o f genomic DNA were incuba ted o v e rn ig h t w ith 2 -5 f o ld excess o f

enzyme as p re v io u s ly d e s c r ib e d . D ig e s ts were stopped by th e a d d it io n o f 0 .1

volumes o f 0 .5 M EDTA o r by the a d d it io n o f 0.1 volume o f lo a d in g b u f fe r .

43

CHAPTER TWO

2i 3i 2i 3_AGAR0SE_GEL_ELECIR0PH0RESIS_0F_GEN0MIC_pNA.

Agarose (a t f in a l ge l c o n c e n tra t io n s o f 0 .75 -1 .27 . w /v) was d is s o lv e d in 1 x

TBE by b o i l in g , th e s o lu t io n a llow ed to cool to about 50 °C and e th id iu m

brom ide added to 0 .5 ^ ig /m l. The g e ls were formed in a a p la s t i c t r a y , th e

edges o f which were sea led w ith a u to c la v e tap e to p roduce a mould (M a n ia t is

e t al; 1782). The warm agarose s o lu t io n was poured in t o th e mould and th e

comb im m ed ia te ly clamped in to p o s i t io n near th e end o f th e g e l. The te e th

o f th e comb produce th e sample w e lls in t o which th e DNA, mixed w ith lo a d in g

b u f fe r , i s p ip e t te d when th e gel has s e t.

A f te r th e samples were loaded , e le c tro p h o re s is was pe rfo rm ed in 1 x TBE pH

8 .3 c o n ta in in g 0 .5 ^»g/ml e th id u im brom ide fo r 4 hours a t 80-90 v o l t s (16

v o l t s /c m . ) .

The DNA fragm en ts were v is u a lis e d under a longwave (3&5nm.) UV lamp and

photographed as d e s c r ib e d in p re v io u s s e c tio n s in o rd e r t o re c o rd the

p o s i t io n o f th e m o le cu la r w e igh t s ta n d a rd s .

2-.3i 2i 4_DNA_IRANSFERi

’’S outhern b lo t t in g " o r DNA t ra n s fe r was c a r r ie d ou t as d e s c r ib e d by

Southern (1975 ). The main o b je c t iv e was to t r a n s fe r DNA o u t o f th e agarose

ge l and b in d i t to a more conven ien t m a tr ix on which v a r io u s assays cou ld

be perfo rm ed . The DNA t r a n s fe r was perform ed as fo l lo w s :

1. The gel was in cu ba ted in d e n a tu rin g b u f fe r f o r 30 m in u tes a t room

tem pe ra tu re w ith g e n t le a g i ta t io n .

2 . The gel was then in cu b a te d in n e u t r a l iz in g b u f fe r f o r 30 m inu tes a t room

te m p e ra tu re .

CHAPTER TWO

3. W earing g lo v e s , th e GeneScreen P lu s membrane (NEN Research p ro d u c ts ) was

c u t t o th e s iz e o f th e ge l and a mark was p laced on th e B s id e . The

GeneScreen membrane has two s id e s o f which s id e B i s used f o r DNA

h y b r id is a t io n s , hence i t was im p o rta n t to use th e c o r re c t s id e . T h is was

id e n t i f ie d by th e n a tu ra l c u r l o f th e membrane when d ry <the uppermost

concave s id e was s id e B ) .

4. The cu t membrane was w e tted in d i s t i l l e d w a te r.

5 . I t was then soaked in 10 x SSC s o lu t io n fo r 15 m inu tes .

6. One l i t r e o f t r a n s fe r b u f fe r (10 x SSC) was p laced in a bak ing d is h , and

a g la s s p la te p o s it io n e d over i t to fo rm a su pp o rt s h e lf f o r th e m oistened

w ick o f Whatman 3MM pape r. The ge l was p laced on top o f t h i s w ic k , extrem e

ca re was taken to a vo id t ra p p in g any b u b b le s . The soaked membrane was

p la ce d on to p o f th e ge l so th a t s id e B was in c o n ta c t w ith th e g e l, aga in

b ubb les were c a r e fu l ly exc luded . A second la y e r o f Whatman paper was added

and th e sandwich was com ple te w ith a 2 -3 in c h s ta ck o f d ry paper to w e l l in g

(w h ich draws the b u f fe r from th e re s e rv o ir th rou g h th e w ick and g e l,

c a r ry in g th e DNA fra gm en ts in t o th e membrane where th e y become tra p p e d ) . A

sm a ll w e igh t was p la ced on top o f th e paper to w e ls and th e t r a n s fe r was

a llo w e d to proceed fo r a t le a s t 12 hou rs bu t was u s u a lly l e f t o v e rn ig h t .

The paper to w e l l in g was changed as n ecessa ry .

7. A t th e end o f th e t r a n s fe r , th e to w e ls and f i l t e r paper were c a r e fu l ly

removed, w ith o u t d is tu r b in g th e membrane.

8. The membrane was c a r e fu l ly l i f t e d away from th e ge l and immersed in an

excess o f 0 .4 M NaOH f o r 30-60 seconds to ensure com ple te d e n a tu ra t io n o f

th e im m o b ilize d DNA.

45

CHAPTER TWO

9. The membrane was then immersed in an excess o f 0 .2 M T ris -H C L , pH 7 .5 , 2

x SSC.

10. The membrane w ith th e t ra n s fe r re d DNA fa c e up was p laced Dn a p ie ce o f

f i l t e r paper and a llo w e d to d ry a t room te m p e ra tu re ( th e re was no need to

oven bake GeneScreen membrane to f i x th e DNA; see s u p p lie rs in s t r u c t io n s ) .

11. A f te r d ry in g , th e membrane was u n c u rle d a t one end and p laced in a

p la s t i c bag.

2i 3i 2i 5_PRE=HYBRIDIZAIIQNi

The membrane was p re -h y b r id iz e d by t r e a t in g i t in 10 ml o f th e fo l lo w in g

s o lu t io n : 507. form am ide (d e io n iz e d ) , 17. SDS, 1 M NaCl and 10% d ex tra n

s u lp h a te (T ab le 2 .3 s o lu t io n and b u f fe r s f o r p re p a ra t io n d e t a i ls ) .

The s o lu t io n was added t o a p la s t i c bag c o n ta in in g th e membrane. T h is was

sea led and in cu ba ted w ith co n s ta n t a g i ta t io n fo r a t le a s t 6 hours a t 42 °C.

2i 3i 2i 6_HYBRipiZATIDNi

W h ils t th e membrane was p re -h y b r id iz in g , th e probe to be used was ra d io ­

la b e l le d (s e c t io n 2 .3 .2 .1 1 fo r 01ig o - la b e l l in g and probe p r e p a ra t io n ) .

A f te r th e a d d it io n o f 300 ^1 o f Salmon sperm DNA (> 1 00 /ug /m l) to the

r a d io a c t iv e p robe , th e s o lu t io n was dena tu red by h e a tin g a t 90-100 °C fo r

10 m in u te s , then co o led on ic e f o r 5 m inu tes and f i n a l l y added to th e bag

c o n ta in in g th e p r e -h y b r id iz a t io n b u f fe r and membrane. The f in a l

c o n c e n tra t io n o f th e probe in th e bag shou ld be le s s than o r equal to

lOng/m l (10° dmp/ml) f o r optimum s ig n a l to background r a t io .

CHAPTER TWO

The bag was re se a le d and in cu ba ted w ith c o n s ta n t a g i ta t io n fo r 6-24 hou rs

a t 42 °C. The membrane was removed from th e h y b r id iz a t io n s o lu t io n and

washed as fo l lo w s :

a. 2 x 100 ml Df 2 x SSC at room temperature for 5 minutes with constant

a g ita tio n .

b. 2 x 200 ml o f a s o lu t io n c o n ta in in g 2 x SSC, 17. SDS a t 65 °C fo r 30

m inu tes w ith c o n s ta n t a g i ta t io n .

c . 2 x 100 ml o f 0 .1 x SSC a t room te m p e ra tu re f o r 30 m inu tes aga in w ith

co n s ta n t a g i ta t io n .

Tha washed membrane was p laced w ith th e DNA fa c e up on a shee t o f f i l t e r

paper and a llo w e d to d ry a t room te m p e ra tu re ( th e membrane was no t dryed

c o m p le te ly i f r e - h y b r id iz a t io n was p lanned as i r r e v e r s ib le b in d in g o f th e

probe may o c c u r; s e c t io n 2 .3 .2 .7 ) . The membrane was exposed and th e

a u to ra d io g ra p h developed as d esc rib e d in s e c tio n 2 .3 .2 .1 3 .

2i 3i 2i 7_RE-HYBRIpiZAII0N i

I f r e - h y b r id iz a t io n o f DNA was necessary th e Genescreen p lu s membrane was

in cu ba ted in 100-200 m l. o f 0 .4 M NaOH fo r 30 m in u te s , and g e n t ly a g ita te d .

I t was then in cu b a te d in 200 m l. D f 0 .1 x SSC, 0.1/C SDS, 0.2M T ris -H C l

(p .H . 7 .5 ) a t 42 °C f o r 30 m inu tes w ith g e n t le a g i ta t io n . The membrane was

then b lo t te d between paper to w e ls to remove excess s o lu t io n and

a u to ra d iog ra p he d to ensure th a t most o f th e probe had been removed (s e c t io n

2 .3 .2 .1 3 ) . The membrane can then be p re -h y b r id iz e d and h y b r id iz e d a g a in , as

p re v io u s ly d e s c r ib e d .

CHAPTER TWO

^ ^ ^ C L O N E S i .

The c lo n e pYCR/S (w hich a r r iv e d s to re d in e th a n o l) was k in d ly p ro v id e d by

C o lin B ishop from th e P asteu r I n s t i t u t e , P a r is . The DNA c lo n e was spun down

in a 1 .5 ml m ic ro fu g e tub e a t 12,000 g -for 15 m in u te s . The DNA p e l le t was

washed once in 70X e th a no l and vacuum dryed fo r one h o u r. T h is d ry p e l le t

was resuspended in 50 j i l o f d i s t i l l e d w a te r.

2i 3i 2i 9_PREPARAII0N_0F_C0MPEIENI_CELLSJL

E.coli s t r a in JM101 were s tre a k e d , from a fro z e n s to c k , o n to an L -aga r

p la te , grown a t 37 °C o v e rn ig h t . A s in g le co lo n y was used t o in o c u la te 10ml

o f L -b ro th and p laced in a shak ing in c u b a to r a t 37 °C o v e rn ig h t , to th e

m id - lo g phase. 1ml D f th e c u ltu r e was t ra n s fe r re d t o f re s h 100ml L -b ro th

(pre-warm ed to 37 °C) in a 500ml f la s k and grown t o an a pp rox im a te o p t ic a l

d e n s ity o f 0 .2 a t 650 nm. The c u ltu r e s were c h i l le d on ic e f o r te n m inu tes .

The c e l ls were p e l le te d by c e n t r i fu g a t io n a t 3500 rpm a t 4 °C f o r 10

m in u tes . The su p e rn a ta n t was d isca rd e d and th e p e l le t was resuspended in

0 .5 X volume o f c o ld 50 mM CaCl2 and l e f t on ic e f o r 20 m in u te s . The c e l ls

were respun and th e p e l le t resuspended in 0 .1 X volume o f 50 mM CaCl2 and

kep t on ic e u n t i l ready f o r use. A l t e r n a t iv e ly , 15% (V /V) g ly c e ro l was

added and 20(jul a l iq u o ts were d ispensed in t o 1.5ml e pp e nd o rfs on ic e , q u ick

fro z e n on d ry ic e and s to re d a t -7 0 °C u n t i l re q u ire d . By p re p a r in g

com petent c e l ls fo l lo w in g t h i s p ro to c o l, a p p ro x im a te ly 10* tra n s fo rm a n ts

per m icrogram o f s u p e rc o ile d p lasm id DNA were o b ta in e d .

48

CHAPTER TWO

2i 3i 2i 10_IRANSF0RMAII0Ni

The tra n s -fo rm a tio n was c a r r ie d o u t by add ing 1 j i l o-f p robe DNA to 5 0 ^ i l o f

f r e s h ly p repared com petent c e l ls (o r a 200j.il a l iq u o t thawed on ic e from th e

-7 0 °C s to c k s ) , mixed and in cu b a te d f o r 20 m inu tes on ic e , the n a t room

te m p e ra tu re fo r a f u r th e r 10 m in u te s . A p p ro x im a te ly 100 pi o f L -b ro th was

added and in cu b a te d a t 37 °C f o r 30-40 m in u tes . The c o n te n ts o f th e tu b e

were p la te d o n to aga r, spread w ith a s t e r i l e g la s s sp reade r and in cu b a te d

o v e rn ig h t a t 37 °C in a dark room.

A f te r in c u b a tio n one co lo n y was p icke d ou t w ith a s t e r i l e to o th p ic k and

mixed w ith 1ml L -b ro th and l^ il a m p ic i l l i n in a 1 .5 ml m ic ro fu g e and p laced

on a d a isy -w h ee l a t 37 °C in th e dark f o r se ve ra l h o u rs . T h is s o lu t io n p lu s

an equal volume o f g ly c e ro l was mixed and s to re d a t -7 0 °C as a s to ck o f

pYCR/8 DNA c e l ls .

2i 3i 2i l l_ M IN I=PREPARAIION_OF_PLASMID_pNAi

1. F iv e ^j1 o f th e pYCR/8 DNA c e l ls from th e -70 °C f re e z e r was added to 10

ml o f L -b ro th w ith 5 yul o f a m p ic i l l in in a s t e r i l e g la s s u n iv e r s a l, and

in cu b a te d on a shaker a t 37 °C o v e rn ig h t .

2 . The c e l l s were spun a t 3000 rpm fo r 5 -10 m inu tes a t 5 °C . The

s u p e rn a te n t poured o f f and th e p e l le t was d is s o lv e d in 200 yul o f 25mM T r is -

HCL, pH 8, 50 mM g lu c o s e , 10 mM EDTA in a 1 .5 ml m ic ro fu g e tu b e .

3. To t h i s , 400 pi o f 17. SDS, 0 .2 M NaOH s o lu t io n were added and 200 yul o f

K0AC pH 4 .8 (3M potass ium a c e ta te d is s o lv e d in h a l f th e volume o f w a ter and

g la c ia l a c e t ic a c id was added t o pH 4 .8 , to make up th e vo lum e).

49

CHAPTER TWO

4. The tu b e was vo rte xe d and spun a t 10,000 rpm in a m icro-fuge fo r 10

m in u tes . The aqueous su pe rn a te n t was c o l le c te d (a p p ro x im a te ly 800 ^ i l ) and

0 .6 x volume o-f is o p ro p a n o l added, m ixed , then l e f t a t room te m p e ra tu re f o r

15-30 m in u tes . T h is was then spun fo r 5 m in u tes to p e l le t th e DNA.

5. The p e l le t was washed w ith 70X e th a n o l, vacuum dryed and th e p e l le t

d is s o lv e d in 20 pi o f d i s t i l l e d w a te r, the n s to re d a t -2 0 °C.

2i 3i 2U2i_piGESIIQN_0F_PLASMID_DNA_AND_PyRIFICAIIDN_0F_^INSERI"i

Two ja l o f t h i s p lasm id DNA c o n ta in in g pYCR/8 were d ig e s te d w ith Eco RI

r e s t r i c t i o n enzyme as desc ribe d in p re v io u s s e c tio n s 2 .3 .2 .2 . and 3. The

fra gm en ts were sepa ra ted on a 1.27. low m e lt in g p o in t agarose ge l c o n ta in in g

5 yul o f e th id iu m brom ide (10 mg/ml s to c k s o lu t io n ) , by e le c tro p h o re s is

u s in g a TAE b u f fe r . The gel was i l lu m in a te d under UV l i g h t and th e d e s ire d

band (2 .0 kb Eco RI fragm ent in th e case o f pYCR/8) c le a n ly e xc ised and

p laced in t o a prew eighed 1.5 ml m ic ro fu g e tu b e . D is t i l l e d w a te r was added

in th e r a t i o o f 3 ml w ater per gram o f g e l. The tu b e was p laced in a

w a te rba th and b o i le d fo r 10 m inu tes to d is s o lv e th e ge l and dena tu re th e

DNA. T h is was s to re d a t -20 °C u n t i l re q u ire d . P r io r to la b e l l in g i t was

necessary t o r e b o i l th e DNA fo r a few m in u tes and in c u b a te i t a t 37 °C f o r

30 m in u tes .

2 i3 i 2i i3_OLIGO-LABELLING_REACIIONi

The DNA was la b e lle d fo r use as a probe f o r h y b r id iz a t io n . The la b e l l in g

re a c t io n was c a r r ie d ou t acco rd ing to F e in b e rg St V o g e ls te in , (1984) a t room

te m p e ra tu re by th e a d d it io n o f th e fo l lo w in g in th e s ta te d o rd e r :

50

CHAPTER TWO

1. D is t i l l e d w a te r ( to a t o t a l volume o f 50 ^ 1 ) .

2. 10 jxl o f o l ig D - la b e l l in g b u f fe r (OLE b u f fe r : ta b le 2 .3 ) .

3. 2 jlx\ o f 10 mg/ml b o v in e serum albumim.

4. A p p ro x im a te ly 20 ng DNA in agarose ie . about 10-20 yu l.

5. 3 ^1 o f a lpha 32P-dCTP (3000 c i / mmol, 10 u c i / ^ 1 ) .

6. 0 .5 ^j1 o f "K lenow " enzyme (2 u n i ts o f la rg e fragm en t o f Escherichia

coli DNA polym erase I from BRL s u p p l ie r s ) .

T h is re a c t io n was in cu b a te d o v e rn ig h t a t room te m p e ra tu re in a lead p o t.

2i 3i 2i 14_SEPARAIIQN_0F_UNI!^0RP0RAIED_NyCLE0IIDES.

The u n in c o rp o ra te d n u c le o t id e s were removed from th e la b e l le d probe by th e

spun-colum n p rocedu re d e sc rib e d by M a n ia t is e t al., 1982.

The bottom o f a 1 ml d is p o s a b le s y r in g e was p lugged w ith a sm a ll amount o f

s t e r i l e g la s s w oo l. The s y r in g e was f i l l e d w ith Sephedex G-50 s a tu ra te d

w ith 2 x SSC. T h is was p laced in a c e n t r i fu g e tub e and spun a t 1600 g f o r 4

m inu tes to pack th e sephedex. Sephedex was added and c e n t r i fu g a t io n

co n tin u e d u n t i l th e packed volume was 0 .9 m l. 200 ^ i l o f 2 x SSC was added

to th e la b e lle d DNA and t h i s s o lu t io n was p ip e t te d o n to th e sephedex column

and spun as above, c o l le c t in g o n ly th e in c o rp o ra te d n u c le o t id e s in a f re s h

c e n t r i fu g e tu b e ( th e u n in c o rp o ra te d n u c le o t id e s rem ain in th e sephedex,

which can be d is c a rd e d ) .

The probe was ready a t t h i s s tage to add to th e h y b r id iz a t io n bag as

d iscussed in s e c tio n 2 .3 .2 .6 .

51

CHAPTER TWO

ijL^iii^AyiORADIOGRAPHY^

The washed ra d io a c t iv e membranes were taped by th e edges t o f i l t e r pape r,

then covered w ith Saran wrap (o r c l in g f i lm ) and p la ce d in th e in te n s i f y in g

screen c a s s e tte s . In th e dark room Kodax X-Omat RP f i lm was c u t to s iz e and

p la ced on top o f th e membrane* th e c a s s e tte sea led and s to re d a t -70 °C (as

a l l membranes were washed in h ig h s tr in g e n c y c o n d it io n s two in te n s i f y in g

screens were r o u t in e ly used to reduce exposure t im e s ) .

The f i lm was developed a f te r th e a p p ro p r ia te exposure t im e , which v a r ie d

from 24 hours to 1 week depending on th e a c t i v i t y o f th e o f p robe .

2i 4_DAIA_ANALVSIS_AND_INIERPREIAII0Ni

Only a gene ra l o u t l in e o f th e da ta a n a ly s is perfo rm ed on b o th mtDNA and Y -

chromosome DNA in t h i s s tud y i s g iven be low . I t d id n o t seem a p p ro p r ia te to

d is c u s s each a n a ly s is s e p a ra te ly as th e re was c o n s id e ra b le o v e r la p . D e ta i ls

and any m ajor d if fe re n c e s in th e tre a tm e n t o f da ta f o r each DNA ty p e a re

g ive n in th e a p p ro p r ia te r e s u l t s c h a p te rs .

2i 4i li_CgMP0SIIE_DNA_GENQIYPESi

In i t a l l y each un ique fragm en t p a t te rn (as assessed on th e b a s is o f

c o m ig ra tio n o f fra gm en ts separa ted in th e same g e l) f o r each g iven

r e s t r i c t io n enzyme d ig e s t , was id e n t i f ie d and d e fin e d as a " r e s t r i c t i o n

morph" (Brown Sc Simpson, 1981). Each morph was g ive n a d i f f e r e n t but

a r b i t r a r y l e t t e r d e s ig n a tio n , th e l e t t e r A be ing re se rve d f o r th e fragm ent

p a t te rn appearing e i t h e r in th e known re fe re n c e sequence (B ibb et al, 1981)

o r the most common genotype found. The re s t o f th e l e t t e r s were des ign a te d

52

CHAPTER TWO

to p a t te rn s in c h ro n o lo g ic a l o rd e r as th e y were d is co ve re d (hence th e re i s

no c o r r e la t io n between a lp h a b e t ic a l p ro x im ity and degree o f s im i l a r i t y ) .

Where p o s s ib le , th e d ig e s t io n p a t te rn s were compared to th e p u b lis h e d d a ta

on Hus domesticus mtDNA (F e r r is et a i , 1983) and th e same le t t e r

d e s ig n a tio n s were used. I f t o r example each sample was d ig e s te d w ith te n

r e s t r i c t io n enzymes th e DNA co u ld be summarised as a te n l e t t e r code c a l le d

th e "com pos ite g eno type ". I f any in d iv id u a ls share th e same com pos ite f o r a

p a r t ic u la r DNA typ e assessed th e y a re assumed to be long to th e same DNA

"c lo n e " (Lansman et al, 1983).

2i 4i 2i_FRAGMENI_hgLECyLAR_WEIGHI_ESIIMAIES_AND_SIIE_MAPPINGi

The m o le cu la r w e ig h ts o f sample fra g m e n ts co u ld be e s tim a te d by com parison

w ith known s tan d a rds (s e c tio n 2 . 2 . 4 , ta b le 2 . 6 ) . T h is was ach ieved by a

co m b in a tio n o f methods depending on DNA ty p e .

S tandard c a l ib r a t io n cu rve s were gene ra ted fo r th e m o le c u la r m arkers used

in each ge l by p lo t t in g th e lo g DNA m arker fragm ent s iz e a g a in s t d is ta n c e

m ig ra te d from the o r ig in . T h is i s be i l l u s t r a t e d in f ig u r e 2 .4 . which shows

a s tan d a rd cu rve genera ted from A Bgl I fragm ent m arke rs . The d is ta n c e

m ig ra te d (cm) by th e unknown sample DNA fragm en ts ( ie I s le o f hay mouse

mtDNA c leaved w ith Dde I :s e e p la te 2 .1 ) run a lo n g s id e th e se m arkers can be

measured and read o f f th e graph to o b ta in t h e i r app rox im a te s iz e s .

The la rg e s t s iz e s (g re a te r than lOOObp) and th e t i n y fra gm en ts ( le s s than

40bp) proved to be v e ry d i f f i c u l t t o e s tim a te us ing t h i s te c h n iq u e . The use

o f a "S o n ic D ig i t iz e r " (Beckman) in c o n ju n c t io n w ith th e s o ftw a re

53

CHAPTER TWO

"M ic ro g e n ie " (IBM l t d ) a llow ed ra p id and a c c u ra te d e te rm in a t io n o f

m o le c u la r s iz e s .

The e x is te n c e o-f a com plete m ito c h o n d r ia l sequence f o r th e la b o ra to ry mouse

(B ibb et al, 1981), a llo w s t h i s t o be used as an a d d it io n a l s ta n d a rd f o r

com parison to a l l th e o th e r Mus domesticus mtDNA. C onsequen tly -for each

r e s t r i c t i o n enzyme used, an expected p a t te rn o-f fra gm en ts can be produced

from t h i s sequence ( ta b le 2 .5 ) . The fragm en t p a t te rn o b ta in e d f o r each

enzyme from each mouse mtDNA co u ld be re la te d by a sm a ll number o f p o in t

m u ta tio n s to th a t p re d ic te d from th e known base sequence ( k . b . s ) .

I t was u s u a lly p o s s ib le to s p e c ify th e e xac t lo c a t io n ( s ) in th e mtDNA

sequence a t which th e m u ta tio n (s ) must have occu rre d in o rd e r to e x p la in

how a g ive n fragm ent p a t te rn d i f f e r s fro m th a t a lre a d y known in th e k .b .s .

T h is h ig h re s o lu t io n sequence com parison method i s i l l u s t r a t e d in f ig u r e

2 .5 . T h is method has been used in p re v io u s s tu d ie s in v o lv in g humans (Cann,

1982; Cann et al.f 1984) and m ice ( F e r r is et a i , 1983).

T h is mapping te c h n iq u e i s much e a s ie r , more p re c is e and i s le s s ambiguous

than th e t r a d i t i o n a l doub le d ig e s t io n c leavage mapping methods (Nathans &

S m ith , 1975). The l a t t e r te ch n iq u e i s o n ly p r a c t ic a l when u s in g r e s t r i c t io n

enzymes, such as h e x a n u c le o tid e s , th a t produce a r e la t i v e l y sm a ll number o f

r e s t r i c t io n fra g m e n ts . Cann, e t a i» , (1982) e s tim a te th e average e r ro r w ith

which c leavage s i t e s have been mapped by c o n v e n tio n a l doub le d ig e s t io n s i s

about + 200bp, whereas th e sequence com parison method has an average e r r o r

o f a p p ro x im a te ly + 2bp.

54

CHAPTER TWO

2i 4i 3i_ESIIMAIES_0F_BEQUENCE_piyERGENCEi

A u s e fu l measure o-f s im i la r i t y between any two DNA's (w he ther o-f

m ito c h o n d r ia l, Y chromosome, o r autosom al o r ig in ) i s th e p ro p o r t io n o-f

shared r e s t r i c t i o n fra gm en ts (F ) .

F = 2 N K y / N * + N y [ 13

where N Ky i s th e t o t a l number o f fragm en ts shared by two in d iv id u a ls , and

N K St N y a re th e number o f fragm ents in in d iv id u a ls X and Y.

U pho lt (1977) was th e f i r s t to show, i f c e r ta in assum ptions a re v a l id , F

can be re la te d to P ( th e number o f base s u b s t i t u t io n s pe r n u c le o t id e ) ,

s e p a ra tin g a g ive n p a i r o f DNA ’ s by th e fo rm u la :

P = 1 - F I (F2 + 8 F )1' 2

2

1/n

[23

Where n i s th e number o f b a se p a irs re co gn ise d per c leavage s i t e (u s u a lly 4 ,

5 o r 6 b a s e -c u t te r s ) . P -va lu e s were c a lc u la te d s e p a ra te ly f o r s e ts o f

r e s t r i c t io n enzymes d i f f e r i n g in n.

Nei St L i (1979) d e r iv e t h e i r fo rm u la e in a d i f f e r e n t manner, ye t th e y y ie ld

n e a r ly id e n t ic a l d ive rg e n ce e s tim a te s <dr) when a p p lie d to most da ta s e ts

(Lansman e t a i , 1981). The re la t io n s h ip between th e f r a c t io n o f shared

fragm en ts (e q u a tio n 1» Nei St L i , 1979 -e q u a tio n 21) and th e number o f

n u c le o t id e s u b s t i tu t io n s per s i t e (d F) based on fra gm en t a n a ly s is i s a ls o

55

CHAPTER TWO

dependent upon le n g th o-f th e re c o g n it io n sequence. U sing Nei and L i ’ s

fo rm u la t io n s th e graph 2 in Appendix 2 i l l u s t r a t e s th e r e la t io n s h ip between

F and d , and e x p la in s how d -v a lu e s a re d e r iv e d from P ( p r o b a b i l i t y o f

n u c le o t id e s u b s t i tu t io n s ) in graph 1 in Appendix 2 u s in g e q u a tio n 3.

F = P* / (3-2P) C33

These fo rm u la e do no t co n s id e r back m u ta tio n s , bu t t h i s i s u n im p o rta n t

g ive n th a t th e fragm en t method can o n ly be used to e s tim a te sequence

d ive rge n ce (d ) when th e p o p u la tio n s a re c lo s e ly r e la te d (when d i s s m a ll) .

These methods o f e s t im a tin g dw from fragm en t a n a ly s is (Nei St L i , 1979?

U p h o lt, 1977) are v a l id p ro v id e d se v e ra l assum ptions a re m et, nam ely, (1)

a l l fragm ent changes must a r is e from base s u b s t i t u t io n s (2) th a t th e

d is t r ib u t io n o f c leavage s i te s i s s im i la r to th a t expected in random

sequences o f same-base c o m p o s itio n s , and (3) th a t non-hom ologous fragm en ts

a re no t scored as id e n t ic a l , assuming th a t a l l p o s s ib le fra gm en ts can be

observed . T h is f in a l assum ption may be e rroneous as th e co m p a ra tive

fragm en t method may n o t d e te c t sm a ll le n g th m u ta tio n s , Dr d e te c t s im i la r

le n g th s o f fragm en ts th a t may be produced in d i f f e r e n t p a r ts o f th e genome,

e s p e c ia l ly i f te t r a n u c le o t id e r e s t r i c t io n enzymes a re used as the se produce

many fra gm en ts . D iscu ss io n o f the se assum ptions and t h e i r v a l i d i t y in t h i s

s tu d y are p resen ted in la t e r c h a p te rs .

Use o f r e s t r i c t io n s i t e d a ta ra th e r tha n fragm ent d a ta p ro v id e s much more

in fo rm a tio n about th e exac t n a tu re o f DNA changes. A method fo r com puting

56

CHAPTER TWO

sequence d ive rg e n ce from c leavage map com parisons in v o lv e s th e

d e te rm in a t io n o f th e f r a c t io n o f shared s i t e s (S) between two in d iv id u a ls

(Nei & L i , 1979; e q u a tio n 10):

S = 2 N K y / N M + N y C41

Where NK and Ny re p re s e n t th e number o f r e s t r i c t io n s i t e s in in d iv id u a ls X

and Y and NKV is th e t o t a l number o f r e s t r i c t io n s i t e s shared between th e 2

in d iv id u a ls (X and Y ). From th e p ro p o r t io n o f shared s i t e s (S) th e

e s tim a te d number o f base s u b s t i tu t io n s per b a se p a ir (d B) by which th e

mapped mtDNA’ s o f th e two in d iv id u a ls d i f f e r can be c a lc u la te d u s in g Nei Sc

L i ’ s e q u a tio n 16:

ds = ( 2 / r ) (1 / S - 1) C53

Where r i s th e number o f b a s e p a irs in th e r e s t r i c t io n enzyme re c o g n it io n

s i t e , S i s as d e sc rib e d as above. The e q u a tio n , however, i s a ls o dependent

on a number o f assum ptions. The f i r s t assumes th a t th e m a jo r i t y o f fragm en t

d if fe re n c e s and hence r e s t r i c t i o n s i t e d if fe re n c e s , a re due to s in g le base

s u b s t i tu t io n s . Secondly th a t th e p a t te rn s o f m ethyl a t io n o f c y to s in e does

no t change, th a t i s r e s t r i c t io n s i t e d if fe re n c e s shou ld n o t be due to

m e th y la t io n p a tte rn s which rende r s p e c i f ic s i t e s w ith th e se C-G p a ir s

r e s is ta n t to d ig e s t io n . The expected G + C c o n te n t shou ld rem ain c o n s ta n t.

F in a l ly , th a t n u c le o t id e s u b s t i tu t io n s occur random ly th ro u g h o u t th e genome

and fo l lo w the po isson d is t r ib u t io n w ith a r a te o f s u b s t i t u t io n ( \ ) per

57

CHAPTER TWO

u n i t t im e ie .y e a r o r g e n e ra tio n (Nei & L i , 1979; Cann, 1982; Gotoh et a i ,

1979; Kaplan & L an g le y , 1979; Nei & T a jim a , 1981).

2i 4i 4i_IREE_C0NSIRyCII0Ni

Two methods were used -for c o n s tru c t in g t re e s in t h i s s tu d y . The advantages

and d isadvan tages o-f each te ch n iq u e a re d iscussed in th e a p p ro p r ia te

d is c u s s io n s e c tio n s .

For presence o r absence c h a ra c te r da ta (-fragm ents o r r e s t r i c t io n s i te s )

u n d ire c te d pars im ony a n a ly s is was conducted u s in g th e ” PAUP" package

developed by D.L Swof-ford (Swo-f-ford, 1985), to c o n s tru c t e v o lu t io n a ry t re e s

w ith o u t assuming a c o n s ta n t ra te o-f DNA e v o lu t io n . In g e n e ra l, those

c h a ra c te rs which a re common to a l l p o p u la t io n s o r in d iv id u a ls in q u e s tio n

a re co ns ide re d to la c k p h y lo g e n e tic in -fo rm a tio n co n ce rn in g th e

r e la t io n s h ip s between them. S im i la r ly a c h a ra c te r th a t i s un ique to a

p a r t ic u la r p o p u la tio n o r in d iv id u a l i s a ls o u n in -fo rm a tiv e . C onsequently th e

in p u t da ta c o n s is ts o f c h a ra c te rs which a re shared by a t le a s t two

p o p u la t io n s / in d iv id u a ls b u t no t by a l l . These c h a ra c te rs were d e sc rib e d as

" p h y lo g e n e t ic a l ly in fo r m a t iv e ” , and as such were removed from th e data s e t.

The s h o r te s t t re e was found fo r each d a ta s e t u s in g an o p t io n in th e PAUP

package which conducts an e xh a u s tive search fo r a l l p o s s ib le t re e s . A

consensus t r e e r e f le c t in g th e in fo rm a tio n shared by a l l t re e s was then

c o n s tru c te d when two o r more tre e s o f equal le n g th had been found . T h is was

perform ed u s in g bo th Adams (Adams, 1972) and S t r i c t (R o h lf , 1982) consensus

58

CHAPTER TWO

method - f a c i l i t i e s in PAUP. These ne tw o rks were ro o te d e i th e r a t th e

m id p o in t o-f th e path co n n e c tin g th e two most d iv e rg e n t p o p u la t io n s o r by

u s in g sequence da ta -from th e la b o ra to ry mouse as an o u tg ro u p .

Using th e "d is ta n c e in d e x " id) -from Nei & L i ’ s (1979) o r U p h o lt ’ s , (1977)

■ fo rm u la tions on e ith e r shared s i t e o r fragm ent d a ta , a m a tr ix o f a l l

p o s s ib le p a irw is e v a lu e s was c o n s tru c te d which served as th e in p u t d a ta .

These in d ic e s were then grouped by a p h e n e tic method which l in k s

p o p u la t io n s / ta x a by t h e i r s im i l a r i t y o r la c k o f s im i l a r i t y . The unw eighted

p a ir -g ro u p method (UPGMA* Sneath and S o k a l, 1973) uses th e a r ith m e t ic

averages and assumes an equal ra te o f e v o lu t io n a ry d iv e rg e n c e , th a t i s a

c o n s ta n t r a te o f n u c le o t id e s u b s t i tu t io n s . T h is method s e q u e n t ia l ly

averages th e d is ta n c e in d ic e s ac ross th e p o p u la t io n s , p ro d u c in g an

o rth o g o n a l dendrogram.

59

TABLE 2 .1 : ABBREVIATIONS.

ABBREVIATION ITEM

mtDNA M ito c h o n d r ia l DNA

BRL Bethesada Research L a b o ra to r ie sInc

NBL N o rthum bria B io lo g ic a ls L im ite d

NEB New England B io la b s

CsCl Cesium c h lo r id e

NaCl Sodium c h lo r id e

MgCl Magnesium c h lo r id e

EDTA E th y le n e d ia m in e te t r a a c e t ic a c id ,d isod ium s a l t

SDS Sodium dodecyl s u lp h a te

ug m icrogram (10 o f 1 gram)

u l m ic r o l i t e r (10 o f 1 l i t r e )

ng nanogram (10 o f 1 gram)

mCi m i l l i c u r i e

UV u l t r a v io l e t

cpm co un ts per m inu te

Kb k ilo b a s e

Z ’ -d e o x y th y m id in e - 5 - dTTP tr ip h o s p h a te

2 '-d e o xyg u a n o s in e - 5 - dGTP tr ip h o s p h a te

d e o x y r ib o n u c le ic a c idDNA

deox y r i bonuc1easeDNase

r a d io a c t iv e decays per m inu tedpm

E s c h e r ic h ia c o l iE .c o l i

nanometernm

re v o lu t io n pe r m inu terpm

NADH dehydrogenase s u b u n itsND

60

CTAB C e t ly t r im e th y l ammonium brom ide

Na2S03 Sodium s u lp h i te

APS Ammonium p e rs u lp h a te

DW d i s t i l l e d w a te r

d p e rcen tage sequence d ive rg e n ce

p d e n s ity

bp b a se p a ir

TE T r is -EDTA b u f fe r

DTT d i t h i o t h r e i t o l

g fo rc e o f g r a v i t y

mM m il l im o la r

M m olar

mA m illia m p

RNase R ibonuc lease

URF u n id e n t i f ie d re a d in g fram e

TEMED N ,N ,N ', N*- te t ra m e th y le th y le n e d i ami ne

TAE T r is a c e ta te EDTA b u f fe r

SSC S tandard s a l in e c i t r a t e

T r is 2-ami n o -2 - (h y d ro x y m e th y l) propane1 :3 d io l

w /v w e ig h t per volume

Repel s i lane 27. d im e th y l d ic h lo r o s i lan e in1 ,1 ,1 - t r ic h lo r o e th a n e

Si lan e gamma - m e th a c ry lo x y p ro p y l-t r im e th o x y s i la n e

K .B .S Known base sequence

BDH BDH ch em ica ls L td , P oo le , UK

BSA b ov ine serum a lbum in

dATP 2 ’ -deoxyadenos ine -5 - t r ip h o s p h a te

2 :‘ -d e o x y c y t id in e -5 - t r ip h o s p h a tedCTP

TABLE 2.2? SAMPLING SITES IN BRITAIN AND IRELAND.

1 S ite lo c a t io n c a te g o r is e d by C o u n ty :th e - f i r s t name i s th e is la n d o r main tow n, th e second name is the more p re c is e sample s i t e to th e n e a re s t v i l l a g e o r -farm. Numbers in c i r c le s co rrespond to main sample s i t e s d is t r ib u t io n i l l u s t r a t e d on - f ig u re 2 .1 .

2 Ordnance Survey g r id re fe re n c e .* A pprox im ate number to g iv e an in d ic a t io n o f t ra p p in g a rea whenthe p re c is e s i t e i s unknown.

The N a tio n a l G rid does no t in c lu d e I r e la n d , co n se q u e n tly th e "AA Road A t la s " ( th re e m ile s to one in c h ) o f G reat B r i t a in and I re la n d re fe re n c e i s g ive n in s te a d .

3 C ap tu re method:A M ice caugh t by hand when corn r ic k s were d is m a n tle d fo r th re s h in g .° M ice caugh t by 1iv e - t ra p p in g w ith b a ite d "Longw orth " t ra p s . c Samples co u ld a ls o be caught by hand a t p ig fa rm s s im p ly by moving th e food tro u g h s and c a tc h in g the d is tu rb e d m ice in sacks.

M id -p o in t map re fe re n c e g iven o n ly f o r j* F a ray , O rkney, S c o tla n d . F o ra ccu ra te t ra p p in g d a ta a c ro ss th e is la n d see r e s u l t s c h a p te r 4.° The I s le o f May, F i r t h o f F o r th , n o r th -e a s t S c o tla n d . P re c is e s i t e lo c a t io n s a n d e x p la n a tio n s see r e s u l t s c h a p te r 6 - " I s le o f May In t r o d u c t io n e x p e rim e n t" .6 The Is la n d o f S ko kh o lm ,o ff th e P em brokesh ire c o a s t, sou th Wales. A ccu ra te sample s i t e s a re g ive n in the a p p ro p r ia te r e s u l t s ch a p te r 4 .

7 In d iv id u a l number ass igned to each mouse.

TABLE 2 .2 : SAMPLING SITES.

SITE LOCATION 1 O.S REFERENCE N ° DATE CAPTURE INDIVIDN° 2 CAUGHT METHOD 3 N OT

1. ORKNEY ISLANDS: N o rth W estray, Noup HY 438489 11 3 /8 8 R ick caught '* 1-11

Mid W estray, Quoy HY 441469 38 3 /8 6 II 12-49

Mid W estray, Hammars HY 438441 21 3 /8 6 II 50-70

South W estray, N .G rinaby HY 483437 9 3 /8 8 II 71-9

South W estray, S ke lw ick HY 489457 8 3 /8 8 II 80-7

Papa W estray HY 488517 1 -/SO II 88

Eday, Newbiggin HY 544313 12 3 /8 0 II 89-100II 46 3 /8 6 if 101-46II 6 3 /8 8 II 147-52

Eday, Ruah HY 560360# 6 3 /8 8 II 153-58

Faray A HY 530370 * 12 9 /8 4 L iv e - tra p p e d B

II 29 9 /8 5 II159-70171-99

II 20 9 /8 6 II 200-19

S tro n s a y , Hoi 1 and HY 662222 2 4 /8 6 R ick caught 220-1

Sanday HY 722422 3 3 /8 0 II 222-22 '

2. ORKNEY MAINLAND: H a rra y , N is t house HY 313198 10 3 /8 8 II 225-34

Yaphur HY 361058 1 3 /8 8 11 235

3. CAITHNESS, SCOTLAND: John O’ G ro a ts , S ea te r ND 381725 3 9 /8 7 L iv e - tra p p e d 236-8

II 4 4 /8 6 II 239-42

Thurso, Mains o-f O lr ig ND 183667 3 9 /87 II 243-45

G reenland ND 244676 11 3 /84? R ick caught

K e iss ND 362623 12 3 /8 4 II246-56257-68

B arnaclavan ND 071647 5 9 /8 7 1iv e - tra p p e d 269-73

63

TABLE 2 . 2 : . .CONTINUED

SITE LOCATION 1

4. SUTHERLAND, SCOTLAND:A rch i emore

Armadale

5 /6 . FIRTH OF FORTH, SCOTLAND: Inchke i th

I s le o-f May ° :O r ig in a l p r e - in t r o d u c t io n Post in t r o d u c t io n

O.S REFERENCE N° 2

NC 893581

NC 791639

NT 290840*

NT 660990 *

7. DUMFRIES AND dmi_LGWAY , SCOTLAND:Curohouse u f F le e t NX 605565 *

NCAUGHT

4

1

DATE CAPTURE INDIVID METHOD 3 N OT

12356076

9 /87 » 274-77

9 /87 " 278

-/BO " 279

9 /82 L iv e - t ra p p e d 280-919 /85 " 431-659 /86 11 466-52t9 /87 " 526-601

9/88 292

8 /9 . NORTHERN IRELAND:Bel f a s tIIMoneymore

10. REPUBLIC OF IRELAND: Gal way

- D5I f

- D4

- F2

9 /878 /8 89/87

10/87

293-5296-302303

304

11. ISLE OF MAN: Linqaqne SC 220720 10/87 305

12. STAFFORDSHIRE, ENGLAND: B u r t o n - o n - t r e n t ,Hareho les Farm, T u tbu ry SK 189272 9

119/876 /88

306-314315-325

13. DERBYSHIRE, ENGLAND: H o rs ley SK 385445 7/87 326-28

14. WEST MIDLANDS, ENGLAND: Birmingham, Moseley SP 073725 6/87 329-336

15. CENTRAL LONDON, ENGLAND: London Zoo, Regents park TO 283836 9/88 337-39

64

TABLE 2 .2 : CONTINUED.

SITE LOCATION 1

K ings C ross, I s l in g to n

16. SOUTH LONDON, ENGLAND: ■fulham

Wimbledon

17. KENT, ENGLAND:East G rin s te a d , L in g f ie ld

O.S REFERENCE N N° 2

TQ 303835

TQ 240770

TQ 240720

TQ 390430

CAUGHTDATE CAPTURE INDIVID

METHOD 3 N °7

1 7 /8 8 L iv e - tra p p e d 340

3 4 /8 7

1 7 /8 7

15 6 /8 7

341-43

344

345-359

18/19. SURREY, ENGLAND: N u t- f ie ld , S a n d y h ill

West Humble, Chapel Farm

TQ 323497

TQ 160520

11 6 /8 8 Hand caught c360-70

1 6 /8 8 L iv e - tra p p e d 371

20. HAMPSHIRE, ENGLAND: W incheste r SU 490320 * 4 /8 7 372-74

21. PEMBROKESHIRE, WALES: Skokholm * SM 735050 15 9 /8 6 375-89

22. SOMERSET, ENGLAND: Taunton, Park Farm

Taunton, B rid g e w a te r, ChedzDy

Taunton, M il lv e iw I I lm in is t e r

ST 278190 9 4 /81 R ick caught 390-9827 4 /8 6 ” 399-425

ST 340380 * 2 4 /81

ST 360150 * 3 - /8 2

426-27

428-430

TOTAL NUMBER OF SAMPLING LOCALITIES =42 TOTAL NUMBER OF MAJOR AREAS/ COUNTIES =22

TOTAL NUMBER OF MICE=601

TABLE 2.3: LIST OF MAJOR BUFFERS AND SOLUTIONS.

NAME CONTENT

30% Acrylamide solution

Ammonical silver

Ampicillin

Dextran sulphate stock

EDTA, 0.5M

Ethidium bromide

Formamide, de-ionized

Homogenising buffer

L-Broth^% (w/v)

Loading buffer (lOx)

Lysis buffer

OLB buffer

0.15g bis-acrylamide, 4.85g acrylamide, lg montoed ion-exchange resin (MBI) resin in 100ml distilled water, stirred on a magnetic stirrer for an hour, filtered and stored at 4 °G

0.4g of silver nitrate dissolved in 2ml of DDW added to a final volume of 248ml DDW, prior to the addition of 1ml freshly prepared 1M NaOH and 1ml of 25% ammonia, in this order.

dissolved at a concentration of 50mg/ml in sterile water, stored at -2 0 °C.

dissolved in sterile water at 50% (w/v), stored at 4 ° C.

pH adjusted to 8.0, and autoclaved.

10 mg/ml concentration, stored at room temperature, wrapped in foil.

treated with ion-exchange resin (Bio-rad), lg per 10 ml formamide for 30 minutes, filtered through whatman n° 1 paper and stored at - 20 °C.

30mM Tris-HCL, ImM EDTA, 2.5mM CaCU, 0.25mM sucrose, autoclaved, stored at 4°C.

bactotryptone, 0.5% (w/v)bacto yeast extract, 0.5% (w/v) NaCl.

0.25% xylene cyanol, 0.25% ficoll (type 400), 0.25% bromophenol blue, in sterile water, stored at room temperature.

0.5M Na Acetate, lOmM EDTA pH 8.0, lOmM Tris-HCL pH 8.0, and 0.5% SDS. Autoclaved and stored at 4 0 C.

solution 0:1.25M Tris-HCL, 0.125M MgCl2 , pH 8.0, stored at 4 0 C. For hot T: solution A:lmlofsolutionOplus 18jil 2*mercaptoethanol, 5ul of each of dATP, dCTP, dGTP (each triphosphate previously dissolved in 0.1M TE

66

Tris -equilibrated phenol

Phenol-Chloroform

Potassium acetate stock. 3M

Pre-hybridisation buffer

Repel silane

RNase (DNase-free)

Salmon sperm DNA

ie. 3mM Tris-HCL, 0.2mM EDTA, pH 8.0), stored at -20 0 C. solution B:2M hepes pH 8.0, titrated with 4M NaOH, stored at 4 0 C. solution C:hexadeoxyribonucleotides (pl2166) suspended in T.E at 90 OD units/ml, stored at -2 0 0 C. These solutions were added together in the following quantites to make the final OLB buffer: A, 100 : B, 250 : C, 150.

removed from the freezer, allowed to warm to room temperature and melted at 65 0 C. 8, - hydroxyquino line added to a f in a l concentration of 0.1%. The melted phenol was extracted several times with an equal volume of 1M Tris-HCL, pH 8.0, then 0.1M Tris pH 8.0,

2% 2-mercaptoethanol. Stored at 4 °C, covered in foil.

redistilled phenol: chlorofrom: isoamyl alcohol (24: 24 : 1).

60% (v /v ) 5M potassium acetate, 11.5% (v /v) glacial acetic acid.

5ml formamide, 2ml sterile water, 2ml 50% dextran sulphate solution and 1ml 10% SDS, mixed by inversion and incubated at 42 ° for 10-15 minutes. 0.58g of NaCl was added to this solution, mixed and incubated again at 42 ° C for 15 minutes.

each glass plate to be treated was washed in detergent, rinsed well with DDW and left to dry. Then each plate was rinsed in repel silane, washed with running water to remove any traces of HCL which may have been formed in the treatment. Once dry these plates were stored in the dark in plastic bags.

dissolved to a concentration of lOmg/ml with sterile water and heated to 100 °C for 15 minutes and allowed to cool to room temperature, then stored at -20 °C.

5ug /u l salmon sperm DNA in lOmM Tris-HCL pH 7.5, ImMEDTA, stirred, sonicated then left stirringovernight. Storedat 4° C, boiled before use.

67

Sephedex G-50 30g sephedex G-50 added to 250ml TE pH 8.0, mixed and allowed to stand overnight at room temperature (or autoclaved for immediate use).

Si lane 0.1% solution (ie. 10ml acetone and lOulsilane),with 1ml applied to each previously washed glass plate and allowed to dry. Then rinsed in acetone alone to remove any traces of excess silane.

SodiumAcetate, 3M

Southern denaturation solution

Southern neutralising solution

SSC (20x)

STE buffer

TAE buffer

TBE buffer

TE buffer

adjusted to pH 5.2 with glacial acetic acid, autoclaved.

0.6M NaCl, 0.4M NaOH.

1.5M NaCl, 0.5M Tris-HCL, pH 7.5.

3M NaCl, 0.3M Na citrate, pH adjusted to 7.

lOOmM NaCl, lOmM Tris-HCL, pH 8.0, ImM EDTA, pH 8.0, autoclaved and stored at 4 0 C.

0.04MTris-acetate,0.001M EDTA, autoclaved, stored at room temperature.

0.089M Tris-base, 0.089M boric acid, 0.002M EDTA, pH 8.0.

lOmM Tris-HCL, 0.5mM EDTA, pH 8.0, and final solution adjusted to pH 7.5.

Total genomic buffer 50mM Tris-HCL, lOOmM EDTA, pH 8.0, lOOmM NaCl, 1% (w/v) SDS.

TABLE 2 .4 : CHARACTERISTICS OF THE RESTRICTION ENDONUCLEASES USED

B u ffe rs f o r r e s t r i c t io n endonuclease d ig e s t io n s ;

LOW; lOmM T r is Cl pH 7 .5 lOmM MgCl

ImM d i t h i o t h r e i t o l

MED; 50mM NaCllOmM T r is Cl pH 7 .5 lOmM MgCl

ImM d i t h i o t h r e i t o l

HIGH; lOOmM NaCl50mM T r is Cl pH 7 .5 lOmM MgCl

ImM d i t h i o t h r e i t o l

® A=adenine; T= thym ine; C = cy to s in e ; G=guanine; P u=purine (A o r G); P y = p y r im id in e (T o r C ); X=A o r C; Y=G o r T; Z=A o r TJ N=any base;

* Enzymes used in mtDNA s tu d ie s s c re e n in g B r i t i s h and European m ice ( th e same s e t o-f enzymes chosen by F e r r is et aim, (1983 ), a l lo w in g da ta co m p a ris o n s ).

b A d d it io n a l -four enzymes used in d is c e rn in g th e " tw ig s " ( i t any) in th e B r i t i s h Phyloqeny t r e e .

c Enzymes used in th e Y-Chromosome DNA a n a ly s is .

d M ito c h o n d r ia l DNA d ia g n o s t ic m o le cu la r m arkers used in th e " I s le o f May In t r o d u c t io n e x p e r im e n t" .

Y-Chromosome DNA d ia g n o s tic m o le cu la r m arker used in th e " I s le o f May In t r o d u c t io n e x p e r im e n t" .

69

TABLE 2 .4 1 C h a r a c te r is t ic s o-f r e s t r i c t io n endonuc leases used.

ENZYME RECOGNITION INCUBATION SALT BASE COMPSEQUENCE* TEMPERATURE ( °C) BUFFER 1 OF SITES a

G C A T

ACC I GT’ XYAC 37 MED 1.5 1. 5 1 .5 1HINC I I * - GTPu’ PyAC 37 MED 1.5 1. 5 1 .5 1HIND I I I A’ AGCTT 37 MED 1 1 2 2XBA I * p T ’ CTAGA 37 HIGH 1 1 2 2AVA I I * " G’ GZCC 37 MED 2 2 0 .5 0THA I * ’ CG’ CG 60 LOW 2 2 0 0HAE I I I — GG’ CC 37 MED 2 2 0 0TAQ I T ’ CGA 65 LOW 2 2 0 0MBO I 7 GATC 37 HIGH 1 1 1 1HINF I * ' c ' G’ ANTC 37 MED 1 1 1 1RSA I b,l,= ' GT7 AC 37 MED 1 1 1 1SAU 961 G7 GNCC 37 MED 2 2 0 0ALU I AG7CT 37 MED 1 1 1 1HPA I I * ’■ C7CGG 37 LOW 2 2 0 0DDE I C7TNAG 37 MED - - - -

SST I GAGCT7C 37 LOW - - - -

BGL I I A7 GATCT 37 LOW - - - -

ECOR I c=,‘ G’ AATTC 37 HIGH - - - -

TOTAL 20 .5 20 .5 13 137. 6 1 .2 38 .8RATIO 1 .57 : 1

BASE COMPOSITn ON LIGHT STRAND (7.)3 3 6 .7 63 .3

RATIO 1 : 1 .7

70

TABLE 2.5s M o le c u la r w e ig h t s ta n d a rd s ; -fragment s iz e s and s i t e lo c a t io n s re c o g n is e d in th e p u b lis h e d * mouse mtDNA re fe re n c e sequence.

* B ibb e t a l . 1981.

1 Number o f r e s t r i c t i o n s i te s in the p u b lis h e d * mtDNA sequence.

2 M ito c h o n d r ia l DNA fragm en t s iz e s used as th e m o le c u la r w e ig h t s ta n a rd s ,l i s t e d in descending o rd e r from th e la r g e s t to s m a lle s t fragm ent f o r eachin d iv id u a l enzyme.

3 The mapped s i t e lo c a t io n s o f each fra g m e n t2 in th e p u b lis h e d * mousere fe re n c e mtDNA sequence, in d ic a t in g th e b e g in in g and end s i t e to account f o r each fragm en t s iz e .

A Summary o f lo c a t io n s o f r e s t r i c t io n s i t e s f o r 15 endonucleases in th e mouse mtDNA o f known base sequence in ascend ing o rd e r s t a r t in g a t th e a r b itu a r y o r ig in in th e d - lo o p * .

71

TABLE 2.5s

ENZYME N° OF FRAGMENT FRAGMENT ENDS 3 SITESITES 1 SIZES 2 LOCATION

1 .ACC I 7 7358 14646 5709 57095048 9598 14646 57542304 7035 9339 6222813 6222 7035 7035468 5754 6222 9339259 9339 9598 959845 5709 5754 14646

2.HINC I I 5 8941 7718 364 3643265 1858 5123 18582266 5452 7718 51231494 364 1858 5452329 5123 5452 7718

3 . HIND I I I 3 13462 11969 9136 91361945 9136 11081 11081888 11081 11969 11969

4.XBA I 6 7576 953 8529 6125066 10907 15973 9531923 8984 10907 8529934 15973 612 8984455 3529 B9B4 10907341 612 953 15973

5.AVA I I 10 6510 6005 12515 782134 14239 78 4242046 3959 6005 20831724 12515 14239 23571659 424 2083 2871609 2371 3480 3480514 2357 2871 3959479 3480 3959 6005346 78 424 12515274 2083 2357 14239

6.THA I B 6434 4972 11406 317(FNUD I I ) 1907 14705 317 1772

1666 13039 14705 20071633 11406 13039 34101562 3410 4972 49721455 317 1772 114061403 2007 3410 13039235 1772 2007 14705

ENZYME

7. HAE I I I

8 .TAD I

N° OFSITES

35

19

FRAGMENT FRAGMENT ENDS 3 SITESIZES 2 LOCATION

2042 12666 14708 841951 4670 6621 10531362 9756 11118 1606969 84 1053 2004945 8811 9756 2215852 11526 12378 2856641 2215 2856 3335590 7592 8182 3402560 8182 B742 3871553 1053 1606 4007550 15189 15739 4129526 7015 7541 4238479 2856 3335 4670469 3402 3871 6621456 15923 84 6749448 14708 15156 7015438 4232 4670 7541398 1606 2004 7592334 11192 11526 8182266 6749 7015 8742216 12450 12666 8811211 2004 2215 9756136 3871 4007 11118128 5521 6749 11192122 15801 15923 11526122 4007 4129 12378103 4129 4232 1241774 11118 11192 1245069 8742 8811 1266667 3335 3402 1470862 15739 15801 1515651 7541 7592 1518939 1237B 12417 1573933 15156 15189 1580133 12417 12450 15923

2245 9835 12080 6341909 7668 9577 24071877 3343 5220 24291773 634 2407 33431472 12080 13552 52201403 5438 6841 52331079 15850 634 5438914 2429 3343 6841786 13562 14348 6885783 6885 7668 7668633 14620 15253 9577597 15253 15850 9835272 14348 14620 12080258 9577 9835 13552205 5233 5438 1356244 6841 6885 1434822 2407 2429 1462013 5220 5233 1525310 13552 13562 15850

ENZYME

9.MB0 I

1 0 .HINF I

N° OFSITES 1

35

30

FRAGMENT FRAGMENT ENDS 3 SITESIZES 2 LOCATION

2009 16175 1889 18891608 4276 5884 23501372 12028 13400 24381344 93B5 10729 2505845 15330 16175 3103772 8165 8937 3223705 11323 12028 3566598 2505 3103 3597581 14749 15330 4065533 13919 14452 4276529 7085 7614 5884488 7677 8165 5988468 3597 4065 6328461 1889 2350 6697439 10729 11168 6731369 6328 6697 6990343 3223 3566 7085340 5988 6328 7614301 8937 9238 7677297 14452 14749 8165259 6731 6990 8937

13592 13814 9238211 4065 4276 9385192 13400 13592 10729155 11168 11323 11168147 9238 9385 11323120 3103 3223 12028105 13814 13919 13400104 5884 5988 1359295 6990 7085 1381488 2350 2438 1391967 2438 2505 14452S3 7614 7677 1474934 6697 6731 1533031 3566 3597 16175

1993 1367 3360 2321734 9837 11571 7291329 3360 4689 13671026 13225 14251 3360946 4689 5635 4689920 7973 8893 5635379 15115 15994 6034633 729 1367 6514539 7434 7973 6882533 15994 232 7367497 232 729 7434489 12520 13009 7973485 6882 7367 8893480 6034 6514 9211

’447 14668 15115 9220417 14251 14668 9432399 5635 6034 9526368 6514 6882 9574367 11571 11938 9837318 B893 9211 11571

74

ENZYME

10.HINF

1 1 .RSA

1 2 .SAU

N° OFSITES 1

CONTINUED

37

961 25

FRAGMENT FRAGMENT ENDS 3 SITESIZES 2 LOCATION

263 9574 9837 119B8243 12277 12520 12082216 13009 13225 12277212 9220 9432 12520195 12082 12277 13009144 11938 12082 1322594 9432 9526 1425167 7367 7434 1466848 9526 9574 95749 9211 9220 9220

2925 5904 B829 59B1294 1270 2564 89512S0 4136 5416 1186994 13493 14487 1210751 10428 11179 1270720 15495 16215 2564678 16215 59B 2650658 11179 11837 2837623 14487 15110 3431594 2837 3431 3690577 9545 10122 3908495 12998 13493 4136473 5416 5889 5416455 9012 9467 5889372 11837 12209 5904308 15110 15418 8829306 10122 10428 8925297 598 895 9012291 895 1186 9467275 12209 12484 9545274 12724 12998 10122259 3431 3690 10428240 12484 12724 11179228 3908 4136 11837218 3690 3908 12209187 2650 2837 1248496 3B29 8925 12724B7 8925 9012 1299836 2564 2650 1349378 9467 9545 1448760 1210 1270 1511035 15460 15495 1541824 1186 1210 1543517 15443 15460 1544317 15418 15435 1546015 5889 5904 154958 15435 15443 16215

4572 6620 11192 782046 3959 6005 4241724 12515 14239 20831659 424 2083 22151258 11192 12450 2357615 6005 6620 2856

ENZYME

1 2 .SAU

1 3 .ALU

961

N° OFSITES 1

CONTINUED

67

FRAGMENT FRAGMENT ENDS 3 SITESIZES 2 LOCATION

583 15155 15738 2871573 15800 7B 3334499 2357 2856 3335469 14239 14708 3402463 2871 3335 3480447 14708 15155 3870390 3480 3870 3871346 78 424 3959142 2215 2357 6005132 2083 2215 662088 3871 3959 1119278 3402 3480 1245067 3335 3402 1251565 12450 12515 1423961 15739 15800 1470815 2856 2871 151551 15738 15739 157881 3870 3871 157391 3334 3335 15800

1069 13843 14912 9905 3863 4768 197709 11261 11970 474680 2463 3143 573654 5129 5733 871608 12992 13600 1033554 8490 9044 1197545 7708 8253 1321545 15759 9 1345530 1641 2171 1431451 9137 9588 1442450 14912 15362 1540450 5783 6233 1589448 9588 10036 1641381 6335 6716 2171365 10461 10326 2463352 6716 7068 3143348 3188 3536 3 IBS313 3536 3854 3536317 11970 12287 3854311 10083 10394 3863298 573 871 4768294 7331 7625 4900292 2171 2463 5075277 197 474 5129269 15490 15759 5783249 12743 12992 6233243 13600 13843 6291207 12392 12599 6335188 9 197 6716175 4900 50752 7068170 11082 11252 7115164 1033 1197 7219162 871 1033 7331144 12599 12743 7625132 4768 4900 7708

SITELOCAT

525352935409645454909044913795BB1003610083103941046110826108561097711082112521126111970122871239212599127431299213600138431491215362153881549015759

13318702519333234005725611262746388540015736

402342742874765118372508283139554989

N° OF FRAGMENT FRAGMENT ENDS 3SITES 1 SIZES 3

CONTINUED 124 1197 1321121 10856 10977116 8293 8409112 7219 7331105 12287 12392105 10977 11082104 7115 7219102 15388 1549099 474 57398 1442 154093 9044 913736 1345 143183 7625 770867 10394 1046158 6233 629154 5075 512952 1589 164149 1540 158947 10036 1008347 7068 711545 8409 845445 3143 318844 6291 633540 8253 829336 8454 849030 10826 1085626 15362 1538824 1321 134511 1431 14429 11252 122629 3854 3863

11 7336 B400 157362325 3400 57252012 6388 84001737 133 1870813 2519 3332692 15736 133649 1870 2519337 5725 6112162 6112 6274114 6274 638868 3332 3400

43 1671 337 25081124 2831 39551034 3955 4989926 5424 6350789 12164 12953780 15242 16022670 8230 8900668 11027 11695590 7120 7710520 7710 8230511 9548 10059

ENZYME

1 5 .DDE I

N° OFSITES 1

CONTINUED

FRAGMENT FRAGMENT ENDS 3 SITESIZES 3 LOCATION

496 8900 9396 5077473 6647 7120 5196472 13365 13837 5312468 14774 15242 5399451 13837 14288 5424412 12953 13365 6350384 10331 10715 6647326 511 837 7120323 2508 2831 7710312 10715 11027 8230297 6350 6647 8900272 10059 10331 9396270 14288 14558 9548241 11695 11936 10059208 16127 40 10331194 40 234 10715139 287 476 11027152 9396 9548 11695132 14642 14774 11936126 11936 12062 12062119 5077 5196 12131116 5196 5312 12164105 16022 16127 1295388 4989 5077 1336587 5312 5399 1383784 14558 14642 1428869 12062 12131 1455840 234 274 1464235 476 511 1477433 12131 12164 1524225 5399 5424 1602213 274 287 16127

TABLE 2 .6 : ADDITIONAL MOLECULAR WEIGHT STANDARDS.

A Prepared by d ig e s t in g lambda DNA to co m p le t io n w ith Hind I I I . S tored at -20°C in a s tock s o lu t i o n c o n ta in in g a p p ro x im a te ly 500ug lambda rtind I I I in lOmM Tris -H C L (pH 7 .4 ) , 5mM NaCI, 0 . lmrl EDTA. When run n in g agarose g e ls l-2^ig ofA Hind I I I i s added per lane . C o n s ide ra b ly le s s i s added when us ing a c ry la m id e q e ls to be s i l v e r s ta in e d (G u i l le m e t te and Lew is, 1983) such as 0 .2 / ig . The lambda s tan da rds were heated to 65 ° C , to d is s o c ia te the cohes ive ends, im m ed ia te ly p r i o r to use.

Prepared by d ig e s t in g lambda DNA to com p le t io n w ith B g l ' I . The sames to ra g e c o n d i t io n s and a p p l i c a t io n s as -for A Hind I I I a re a p p l ic a b le .

c A p p ro x im a te ly 5pgot 1KB ladde r i s added to 2jj1of a s o lu t io n c o n ta in in g 17. w/v SDS, 0.17. w/v bromopnenol b lu e , 100mM EDTA, 50% v /v g l y c e r o l . The s o lu t i o n i s heated a t 65°C -for 5 m inu tes and th e e n t i r e sample loaded onto the agarose g e l . When us ing ac ry la m id e g e ls t o be s i l v e r s ta in e d o n ly O.S^ig 1KB la d de r i s a p p l ie d .

D The a b b re v ia te d form o f fragm ent s iz e ( in K i lo b a se s ) u s u a l ly used asla b e ls on gel photographs.

73

TABLE 2 .6 : ADDITIONAL MOLECULAR WEIGHT STANDARDS.

PHAGEPLASMID!

ENZYME:

NAME:

FRAGMENT N(

FRAGMENTSGENERATED:

OR LAMBDA LAMBDA YEASTCIRCLE

HIND I I I BGL I -

HIND I I I A BGL I B 1KB LADDER

S 29 22

23130 (2 3 .1 )° 16173 122169416 (9 .4 ) 9649 111986557 (6 .6 ) 3009 101804361 (4 .4 ) 2481 91622322 (2 .3 ) 2256 81442027 (2 .0 ) 1650 7126

564 (0 .56 ) 1441 6108125 (0 .125) 1446 5090

1249 40721203 30541138 2036790 1635773 1018669 516621 506562 394499 344489 298447 220410 200366 154267 142210 751861261261159151

9

8 0

FIGURE_2Jj_Djstobution _of major sampling localities in Britain and

L r^ la n d .

HXMWsun

ISLE OF MAY

BELFAST

ISLC OF MAN

GAIWAY

IRELAND

IINGHAM

INCHESTER SURREY

r < ?

>IM

81

FIGURE 2.2 s Mouse identification marking schemes.

Ear-clipping, toe-dipping, -fur—clipping (Fig 2.2.1, 2, and 3 respectively),

used alone or in combination to individually mark each mouse caught.

1 EAR CLIP NUMBERING SCHEME.

LEFT EAR; TENS R IG H T EAR; DIGITS

BACK V IE W

2 TOF CLIPPING SCHEME.!°°.2ooT ^ V

BACKVIEW

300 LEFT FOOT; 1-5 HUNDRED400.500

10.00_ 8Q900 R IG H T FOOT; 6'IOHUNDRED

600 700 ^3 FUR CLIPPING SCHEME.

ARIELV IE W

1 RR—Right rump2 L R “* Left rump3 R M - R ' |h t m id 4L /V 1—Left mid 5 KF — Right fore

— Lett fore —Back

X — position of furclip

FIGURE 2.3

Probability of NOT detecting - differences between individualsagainst a number of basepairs needed to be studied for different level of differentiation of Natural populations.N.B. Broken line is level of uncertainty at 0.05% probability,i.e. chance of saying 2 individuals are identical when they are not.For P = 0.60% number of basepairs to be studied is 500.P = 0.30% number basepairs is 900. (Tegelstrom 1986)

&z>

QL

003

•o>? Percentage nucleotide divergences

s0

3

&00

8i

0.5 •

0j60

1000500100

Number of booo-pairs

EIGyRI_2i 4i_pNA_QyANIIFICAIIQNi_STANDARD_CALIBRAII0N_CyRVE_2i _GRAPH_PL0Ti

The known m o le cu la r w e ig h ts ( / Bgl I ) was p lo t te d on lo g paper a g a in s t

d is ta n c e m ig ra te d from th e o r ig in to p roduce a s ta n d a rd c u rv e from which

th e unknown v a r ia n t DNA s iz e s can be c a l ib r a te d . For exam ple in la n e 5

mouse mtDNA from th e I s le o f May ( F i r t h o f F o r th , n o r th e a s t S c o tla n d ) was

c leaved w ith Dde I (P la te 2 .1 ) th e fra gm en ts produced were o f unknown s iz e .

The f i r s t fragm ent (0) m ig ra te d 3.2cm , t h i s va lu e can be measuured o f f th e

s ta n d a rd cu rve to p roduce a m o le cu la r w e ig h t o f a p p ro x im a te ly 1670 + 20 bp.

T h is can be checked fo r any d e v ia t io n s from th e p re d ic te d re fe re n c e

fragm en t p r o f i le s : th e fra gm en t co rresponds to th e 1671bp from th e

re fe re n c e p r o f i le s . These measurements were made f o r e v e ry fragm en t in

eve ry p r o f i l e f o r eve ry enzyme s tu d ie d .

85

IOO

OO

Orx

CO

o

CO

>o

(s.d9) S1H0I3M w iro a io w

8 6

DIST

ANCE

FR

OM

ORIG

IN

(CM

)

FIGURE 2 .5 : 5ITE_MAPPING_0F_SINGLE_piGESIS_i_HIEH_RES0LlJII0N_SEDyENCE

COMPARISON.MEIHODi

R e s t r ic t io n -fragments in t h i s s tu d y se pa ra ted by g e l e le c tro p h o re s is were

mapped by th e sequence com parison method (Cann, 1982).

Type I i s a d iag ram m atic re p re s e n ta t io n o-f a mtDNA m o lecu le o f th e

re fe re n c e sequence map o f a la b o ra to ry mouse (B ibb et a i , 1981), ty p e I I

mtDNA re p re s e n ts an in d i iv id u a l f o r whom no sequence da ta e x is ts . Using a

p a r t i c u la r r e s t r i c t io n endonuclease th e mtDNA o f ty p e I in d iv id u a l can be

c le a ved a t th re e s i t e s ( la b e l le d A ,B ,C ,) p ro d u c in g th re e fra gm en ts o f known

s iz e s (For s im p l i f i c a t io n assume th e s iz e o f the mtDNA m o lecu le was

a p p ro x im a te ly 16KB), namely 8KB ( s i t e ends 0 /1 6 and 8 ) , 5Kb ( s i t e ends 3

and 8 ) , and 3KB ( s i t e p o s i t io n 0 and 3 ) .

The same enzyme c le a ve s th e unknown mtDNA ty p e I I in t o fo u r fra g m e n ts , th e

s iz e s o f w hich can be e s tim a te d from th e ge l e le c tro p h o re s is by d i r e c t

com parison to th e known m o le cu la r w e ig h ts o f typ e I ( t h is i s in d ic a te d in

th e low er p o r t io n o f th e d ia g ra m ). There i s a lo s s o f th e 8KB fragm en t

re p la c e d by a d o u b le t s ize d a p p ro x im a te ly 4KB. By d i r e c t l y m atch ing th e

fragm en t p r o f i le s in type I w ith ty p e I I we can in f e r th a t th e re has been a

m u ta tio n ca us ing a new re c o g n it io n s i t e (D ), r e s u l t in g in th e lo s s o f one

fragm en t and th e subesequent ga in o f two fra gm en ts whose s iz e s a re equal to

th e sum o f th e la rg e r lo s t fra g m e n t.

2ex.a.

ooU JOaUJ- J

2on

o

oo—j

U J

~kN

a

^ 1u j .-cZ O

U JK-to

CQ

£§§U J

IDo>U Jv/1

IO

u j

U JIDo>U Jto

Q _? ro >0 >0 I (N ff!m o<N

“ I I I I I I

If O r "

un on to >s O <n

t

2sA

pp vO "O.(N CS 'O f

m Q r«J r><

M i l I I

si «*o%L

<*)

PLAIE_2i li_DNA_QyANIIFICAIIDNi_STANDARD_CALIBRATI0N_CyRVESi_li _GEL

p h o io g r a p k

T h is f ig u r e i l l u s t r a t e s mouse mtDNA c leaved w ith r e s t r i c t i o n enzyme Dde I ,

sepa ra ted by e le c tro p h o re s is th rough a 57. p o ly a c ry la m id e ge l and s i l v e r

s ta in e d . The e xac t s iz e s o f some o f th e m o le cu la r w e ig h t m arker .ABgl I

fragm en ts a re in d ic a te d a long s id e la n e 5. The d is ta n c e m ig ra te d from th e

o r ig in (cm) f o r each m arker fragm ent was measured, and p lo t te d a g a in s t lo g

m o le cu la r w e igh t ( in bp) as in f ig u r e 2 .4 .

1ANES 1 -4 : F I , F2, Wl, E l, (Faray, W estray, and Eday, O rkney,

r e s p e c t iv e ly ) ; la n e 5: Ml ( I s le o f May, F i r t h o f F o r th , N o rth -e a s t

S c o t la n d ) .

+ in d ic a te s m ajor fragm ent d if fe re n c e s between sam ples.

83

DDE I

9 6 4 9

1650

. f

. 91

O

. o O

I

. ¥

- f

¥

CHAPTER THREE

CHAPTER THREE : MOLECULAR EVOLUTION OF BRITISH HOUSE MOUSE (HUS

DOHEST1CUS) MITOCHONDRIAL DNA.

3 .1s INTRODUCTION:

M ito c h o n d r ia l DNA i s a h ig h ly in fo r m a t iv e m o le c u le , p ro v id in g new in s ig h ts

in t o d iv e rs e to p ic s , such as m a terna l p h y lo g e n y , p o p u la t io n s t r u c tu r e ,

b io ge o g ra p h y and h is t o r ic a l c o lo n is a t io n s , a t th e le v e l o f b o th p o p u la t io n s

o r s p e c ie s , h i t h e r t o u n o b ta in a b le from n u c le a r gene system s (A v is e , 19B6;

W ilson et a l - , 19B5). However, i f mtDNA i s t o be used as an a n a ly t ic a l to o l

i t must be th o ro u g h ly unde rs tood and c h a ra c te r is e d , so th a t any r e s u l t s may

be m e a n in g fu lly in te r p r e te d . In t h i s c h a p te r , th e mechanisms by w hich

m ito c h o n d r ia l DNA has evo lved in th e B r i t i s h House mouse (Hus dowesticus)

a re d is c u s s e d .

C u r re n t ly a v a i la b le te c h n iq u e s tD s tu d y mtDNA v a r ia t io n d i f f e r g r e a t ly , in

th e p ro p o r t io n o f th e genome th a t th e y can compare, and in th e amount and

n a tu re o f in fo rm a t io n th e y p ro v id e ( F e r r is & B erg, 1987; H a rr is o n , 1989).

The most d i r e c t , p o w e rfu l method in v o lv e s DNA sequence a n a ly s is o f p a r t o r

th e w hole o f th e mt genome. However, t h i s i s h ig h ly la b o r io u s , t im e

consum ing, and c o s t ly , u s u a lly in v o lv in g c lo n in g p ro c e d u re s , and th u s

im poses se ve re l im i t a t io n s f o r p o p u la t io n s tu d ie s . In c o n t r a s t ,

h y b r id is a t io n and r e s t r i c t i o n fragm en t le n g th po lym orph ism s (RFLPs) a re

t e c h n ic a l ly s im p le r , a llo w in g la rg e r numbers o f in d iv id u a ls t o be more

r a p id ly p rocessed , y e t th e y do n o t p ro v id e d a ta on th e n a tu re o f th e

m u ta t io n a l changes c h a r a c te r is in g th e p o p u la t io n s .

CHAPTER THREE

A v a i l a b i l i t y o f a com p le te sequence o f mtDNA to r th e la b o ra to ry mouse (B ibb

et al.f 1981), ena b le s use o t th e a c c u ra te h ig h r e s o lu t io n "sequence

com parison " method o t r e s t r i c t i o n mapping (Cann, 1982; Cann, Brown, and

W ils o n , 19B2; Cann & W ils o n , 1983). T h is te c h n iq u e , p e rm its th e d e te c t io n

and ra p id mapping o t s i t e s trom s in g le d ig e s ts w ith a h ig h degree o t

p re c is io n ( to w i th in a tew b a s e -p a ir s ) , t o r a la rg e number o t sam ples,

w ith o u t re c o u rs e to th e t r a d i t i o n a l d oub le d ig e s t io n mapping m ethodology

(Danna et a i . , 1973; Nathans & S m ith , 1975). The l a t t e r te c h n iq u e has an

average mapping e r r o r o t + 200 b a s e -p a irs , and i t i s o n ly p r a c t ic a l when

u s in g r e s t r i c t i o n enzymes th a t p roduce r e la t i v e l y tew r e s t r i c t i o n

fra g m e n ts , p r im a r i ly th o se w ith s ix -b a s e p a ir r e c o g n it io n s i t e s . As

p o p u la t io n s examined in t h i s s tu d y a re c lo s e ly r e la te d , com parisons

n e c e s s ita te th e use o f f r e q u e n t ly c u t t in g te t r a n u c le o t id e r e s t r i c t i o n

endonucleases to in c re a s e th e le v e l o f r e s o lu t io n o f th e mtDNA com parisons.

The la rg e number o f fra g m e n ts th u s produced a re v i r t u a l l y im p o s s ib le to map

w ith th e c o n v e n tio n a l te c h n iq u e .

M a jo r o b je c t iv e s o f m o le c u la r e v o lu t io n a ry s tu d ie s u s u a l ly in v o lv e e i th e r

th e d e s c r ip t io n o f th e m o le c u la r b a s is o f v a r ia t io n , th e fu n c t io n and

o rg a n is a t io n o f th e g e n e tic system o r subsequent use o f t h i s v a r ia t io n as

m arkers o f g e n e t ic a l ly d i s t in c t lin e a g e s f o r p h y lo g e n e tic and e v o lu t io n a ry

a n a ly s e s . An e f f e c t iv e synerg ism o f te n o ccu rs between th e s e tw o ty p e s o f

in fo rm a t io n and in d ee d , th e employment o f sequence com parison mapping o f

house mouse mtDNA a ch ie ve s most o f th e s e a im s.

92

CHAPTER THREE

F e r r is et el,, (19B3) a p p lie d th e same mapping te c h n iq u e as used here to

W estern European, M e d ite rra n e a n , and New W orld house mouse p o p u la t io n s ,

p ro v id in g a u n ique o p p o r tu n ity f o r com parison w ith B r i t i s h sam ples examined

in t h i s s tu d y . Comparison o f t h i s da ta p ro v id e s in fo rm a t io n co n ce rn in g th e

e x te n t o f g e n e tic d iv e rg e n c e w ith in and between p o p u la t io n s in d i f f e r e n t

p a r ts o f th e w o r ld , a p r e r e q u is i te f o r a g e n e ra l u n d e rs ta n d in g o f th e

g e n e tic b a s is o f e v o lu t io n a ry change in one o f th e most in te n s iv e ly s tu d ie d

o rgan ism s a t a l l s c i e n t i f i c le v e ls .

Here I d e s c r ib e th e in c id e n c e and ty p e s o f m u ta tio n s w h ich c h a ra c te r is e th e

fra gm en t d ig e s t io n p r o f i l e s from B r i t i s h house mouse mtDNA, to e lu c id a te

th e s u s c e p t ib i l i t y o f d i f f e r e n t re g io n s o f th e mouse mt genome to base

changes. I use t h i s in fo rm a tio n in la t e r c h a p te rs f o r in v e s t ig a t io n s

ra n g in g from m a t r ia r c h ia l p h y lo g e n e tic o r ig in s o f th e B r i t i s h House mouse,

superim posed ove r m acrogeograph ica l l o c a l i t i e s in B r i t a in and w o rld w id e

(c h a p te r 4 ) , t o a m ic ro g e o g ra p h ic a l le v e l o f is la n d s tu d ie s ( in c lu d in g

B r i t a in ) in v o lv in g demography, d ip e rs a l, and p o p u la t io n dynam ics (c h a p te r

6 ) . F in a l ly , I s h a l l d is c u s s p o s s ib le v io la t io n s o f th e u n d e r ly in g

assum ptions D f p h y lo g e n e t ic models and th e a p p l ic a t io n to " r e a l1' da ta and

h ig h l ig h t any mapping c o m p le x it ie s which a r is e u s in g th e sequence

com parison te c h n iq u e s .

3 .2 : MATERIALS AND METHODS.

Four hundred and t h i r t y m ice from 42 sa m p lin g l o c a l i t i e s w ith in 17 c o u n t ie s

o f G reat B r i t a in (C hapte r 2, ta b le 2 .2 - m a jo r sam p ling s i t e s ) were used in

CHAPTER THREE

t h i s s tu d y . P u r i f ie d mtDNA was is o la te d from l i v e r , h e a r t , and k id n e ys

u s in g C s C l-e th id iu m b rom ide g ra d ie n t c e n t r i fu g a t io n (c h a p te r 2 f o r d e ta ils ?

Lansman et al.f 1983? C a rr & G r i f f i t h , 1987) o r u s in g th e m o d if ie d phenol

e x t r a c t io n p ro to c o l (Jones et al,9 1988). Each sample was d ig e s te d w ith 14

r e s t r i c t i o n endonucleases (BRL, NBL) re c o g n is in g s ix bases (H ind I I I , Xba

I , H inc I I , Acc I ) , f i v e bases (Ava I I ) and fo u r bases (Fnud I I , Hpa I I ,

Hae I I I , Taq I , Nbo I , H in f I , A lu I , Rsa I , Sau 96 I ) . T h is produced

fra g m e n ts o f mtDNA w h ich were sepa ra ted by e le c tr o p h o r e s is in e i t h e r 0 .7 -

0.87. agarose g e ls (e th id iu m brom ide s ta in e d , and v is u a l is e d under UV-

l i g h t -M a n ia t is et al,, 1982)? o r 57. p o ly a c ry la m id e g e ls ( s i l v e r s ta in e d -

T e g e ls tro m , 1986). F ig u re 3 .1 i l l u s t r a t e s th e m ajor p o in ts in th e

p re p a ra t io n , i s o la t io n , p u r i f i c a t io n , and v is u a l is a t io n o f th e mtDNA.

The s iz e o f fra gm en ts was e s tim a te d by com paring them w ith fra g m e n ts o f

known s iz e s fro m th e p u b lis h e d mouse mtDNA sequence ("known base sequence"

o r k .b . s - B ibb et a l 1981) run as a s ta n d a rd on each g e l and c u t w ith

th e a p p ro p r ia te enzyme ( ta b le 2 .5 c h a p te r 2 ) . A d d i t io n a l ly , c o m m e rc ia lly

a v a i la b le lambda DNA c u t w ith H ind I I I and th e 1 KB la d d e r (BRL) were used

as m o le c u la r w e ig h t m arke rs ( ta b le 2 .6 , c h a p te r 2 ) .

A c a p i ta l l e t t e r was ass igned to each fra gm en t d ig e s t io n " p r o f i l e "

id e n t i f ie d by a p a r t i c u la r r e s t r i c t io n enzyme, ’ A* b e in g re s e rv e d f o r th e

p a t te rn a lre a d y known in th e p u b lis h e d re fe re n c e sequence. D ig e s t io n

p a t te rn s were d e s ig n a te d c h ro n o lo g ic a l ly as th e y were d is c o v e re d , so

a lp h a b e t ic a l p ro x im ity does n o t in d ic a te g e n e tic s im i l a r i t y . Where

94

CHAPTER THREE

p o s s ib le , th e d ig e s t io n p a t te rn p r o f i l e s were compared to th e p u b lis h e d

d a ta f o r Hus dowesticus ( F e r r is e t a i * , 1983), and th e same l e t t e r

d e s ig n a t io n s used. The "sequence com parison m ethod" was used to c o n s tru c t

c le a va g e maps f o r each fragm en t p r o f i l e o f e ve ry enzyme used. The r a t io n a le

and a d e ta i le d worked example o f t h i s te c h n iq u e i s shown in f ig u r e s 2 .5

(c h a p te r 2) and p la te 3 .1 and f ig u r e 3 .2 r e s p e c t iv e ly .

3 .2 .1 O rd in a t io n o f Data?

D ive rg e nce e s tim a te s , th e (p) o f Nei and L i (1 9 7 9 ), used in th e L1PGMA

c lu s te r a n a ly s is (see c h a p te rs 4 & 5) assume a random d is t r ib u t io n o f

r e s t r i c t i o n endonuclease s i t e s around th e m ito c h o n d r ia l genome. C h i-s q u a re

s t a t i s t i c s were used to compare th e observed d is p e rs io n p a t te rn o f s i t e s

a g a in s t a p o isso n o r random d is t r ib u t io n . Where th e n u l l h y p o th e s is was

r e je c te d , d e v ia t io n from randomness was de te rm ine d by exam in ing th e

c o e f f ic e n t o f d is p e rs io n (C .D ), ta ke n as th e r a t i o o f th e v a r ia n c e to th e

mean f o r an observed fre q u e n cy d is t r i b u t io n . A t r u e ly P o isson d is t r ib u t io n

has a c o e f f ic e n t o f d is p e rs io n o f 1, clum ped p a t te rn s >1, and u n ifo rm

p a t te rn s <1. The d is t r ib u t io n o f s i t e s was te s te d f i r s t a t th e le v e l o f th e

who le genome, and then in in d iv id u a l gene re g io n s . The d is t r ib u t io n o f

enzyme c le a vag e s i t e s th ro u g h o u t th e w ho le genome were a ls o examined to

ensu re each enzyme c u ts random ly.

Where a p r e d ic t io n o f a l in e a r r e la t io n s h ip o f two v a r ia b le s from th e

re g io n a l v a r i a b i l i t y d a ta (s e c t io n 3 .3 .6 ) i s re q u ire d Model I I re g re s s io n

a n a ly s is may seem a p p ro p r ia te . Both v a r ia te s a re measured w ith e r r o r ,

v io la t in g one o f th e assum ptions o f Model I re g re s s io n a n a ly s is . However,

CHAPTER THREE

as th e in te n t io n o-f t h i s a n a ly s is i s -for pu rposes o-f p r e d ic t io n and n o t

in v e s t ig a t io n o-f t h e i r fu n c t io n a l r e la t io n s h ip , the n s im p le l in e a r

re g re s s io n te c h n iq u e s a re a p p lie d (S okal and R o h lf , 1981; p . 5 4 9 ).

5 .3 RESULTS

5 .3 .1 Fragment D ig e s t io n P r o f i le s :

U sing th e fo u r te e n r e s t r i c t i o n endonuc leases employed in t h i s s tu d y , a

t o t a l o f between 274-316 mtDNA fra g m e n ts were produced f o r each in d iv id u a l

in v e s t ig a te d . Using th e same enzymes, th e known base sequence ( k . b . s . ) o f

mouse mtDNA (p a t te rn A in each case) (B ibb e t a l . , 1981) was c le a ved in t o

29B fra g m e n ts . The s iz e s o f fra gm en ts w h ich c h a ra c te r is e th e s e mtDNA

d ig e s t io n p r o f i l e s a re l i s t e d in ta b le 3 .1 . D ig e s t io n p r o f i l e i d e n t i t i e s

were assessed on th e b a s is o f c o -m ig ra t io n o f fra g m e n ts r e la t i v e to th e

k .b . s and a d d it io n a l m o le cu la r w e ig h t m a rke rs . T y p ic a l g e ls o f e i th e r

s i l v e r s ta in e d 57. p o ly a c ry la m id e o r 0 .7 o r 0 .8 7. agarose s ta in e d w ith

e th id iu m b rom ide f o r th e same r e s t r i c t i o n enzymes used in t h i s s tu d y can be

seen in p la te s 3 .2 -3 .1 0 (m o s tly exam ples o f p r o f i l e s w ith many fra g m e n ts ,

produced w ith f r e q u e n t ly c u t t in g r e s t r i c t i o n enzymes which a re d i f f i c u l t t o

v is u a l is e fro m ju s t th e d ig e s t io n p r o f i l e d e s c r ip t io n g ive n in ta b le 3 .1 ,

a re shown). D e ta i ls o f po lym orph ism s shown in th e s e g e ls a re d iscu ssed

l a t e r .

A l l m u ta t io n a l changes observed in t h i s s tu d y were due s o le ly t o base

s u b s t i t u t io n s ; le n g th v a r ia n ts (m inor o r m a jo r) and h e te ro p la sm y , were n o t

d e te c te d in t h i s s u rv e y . Any a d d it io n a l f a i n t mtDNA bands in a r e s t r i c t i o n

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d ig e s t io n p r o f i l e was a t t r ib u te d to in c o m p le te d ig e s t io n , th e se

supernum erary bands a lw ays d isappeared when th e sam ple was re d ig e s te d .

S im i la r ly , th e t o t a l m o le c u la r w e ig h t o f a l l fra g m e n ts summed in each

r e s t r i c t i o n d ig e s t neve r exceeded 16,295 bps, th e expec ted s iz e o f th e

house mouse m ito c h o n d r ia l genome (B ibb et a J 1981).

The 128 mtDNA d ig e s t io n p r o f i le s f o r Hus dowesticus w h ich have been

re co rd e d u s in g th e ’ sequence com parison m ethod’ f o r 2 -14 r e s t r i c t io n

enzymes a re l i s t e d in ta b le 3 .1 , 58 (43.5%) o f w h ich a re found in B r i t a in .

Seventeen o f th o se observed in B r i t a in , have been p re v io u s ly d e s c r ib e d by

F e r r is et a i , , 1983) f o r p o p u la tio n s in o th e r re g io n s o f th e w o r ld , w h i ls t

th e rem a inde r (41) a re un ique to B r i t a in . F o r ty s ix p e rc e n t o f th e se un ique

d ig e s t io n p r o f i l e s were reco rded u s in g th e a d d it io n a l r e s t r i c t i o n

endonucleases (Rsa I , A lu I , fc Sau 96 I ) w h ich were n o t employed in

p re v io u s s tu d ie s ( F e r r is et a i . , 1983). T ab le 3 .2 sum m arises th e r e s u l t s

f o r th e number o f mtDNA d ig e s t io n p r o f i le s .

3 .3 .2 C leavaoe m apping:

When th e se c h a ra c te r is e d d ig e s t io n p r o f i l e s , were compared in d e ta i l w ith

th e k .b .s , i t was p o s s ib le to map an average o f 296 c le a va g e s i t e s in each

mtDNA c lo n e u s in g th e ’ sequence com parison m ethod’ (Cann, 1982; Cann &

W ilso n , 1983 f i g . 2 .5 ; p la te 3.1 and f ig u r e 3 .2 ) . Any one mouse c o n ta in s a

subse t o f th e t o t a l number o f 370 s i t e s exam ined.

The e xa c t lo c a t io n s o f th e 143 v a r ia b le s i t e s f o r each mouse mtDNA c lo n e

examined can be found in ta b le s 3 .3A-N under each r e s t r i c t i o n endonuclease

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used. There were th re e cases where i t was o b v io u s th e v a r ia b le s i t e s

re co rd e d were n o t s e p a ra te independent e ve n ts due to th e o v e r la p p in g o-f

s i t e changes. Thus th e 140 b a se p a ir p o s i t io n s a c tu a l ly accoun t -for th e 143

r e s t r i c t i o n s i t e s l i s t e d in ta b le 3 .3A -N ( f o r f u r t h e r m o le c u la r d e t a i ls o f

th e o v e r la p p in g s i t e changes, see th e ta b le legend 3 .3 A -N ). A d d i t io n a l ly in

ta b le s 3 .3A-N a n o te i s made by each v a r ia b le s i t e in th e p r o te in cod ing

re g io n s , as to w hether i t i s an amino a c id re p lacem en t o r a s i l e n t

s u b s t i t u t io n (R l-3 ; S I and 3 - see s e c t io n 3 .3 .3 ) . T a b le 3 .4 l i s t s th e 230

c o n s ta n t s i t e s found in e ve ry mouse f o r e ve ry r e s t r i c t i o n enzyme used. For

com parison purposes th e p ro te in co d ing genes a re d iv id e d in t o "C ytochrom e"

genes, in c lu d in g Cytochrom e O xidase genes I —I I I , Cytochrom e B, and ATPases

6 & 8 (p re v io u s ly r e fe r r e d to in th e l i t e r a t u r e as "known p r o te in s " ; Cann,

1982; F e r r is et al., 1983), and th e "NADH" genes in c lu d in g a l l th e NADH

dehydrogenase s u b u n its (1 -6 and 4 L ) , w h ich used t o be te rm ed u n id e n t i f ie d

re a d in g fram es (URFs 1 -6 &4L; Chomyn et al., 1985).

3 .3 .3 ? A n a ly s is o f s i t e g a in s ;

A l l s i t e s were mapped in r e la t io n to th e k .b . s , hence i f an in d iv id u a l had

an a d d it io n a l c le a vag e s i t e w ith any r e s t r i c t i o n endonuclease used when

compared w ith th e l a t t e r i t was d e s c r ib e d as a ’ s i t e g a in ’ (Cann, 1982?

Cann & W ilso n , 1983; F e r r is et a l 1983). S e v e n ty - f iv e o f th e 140 v a r ia b le

r e s t r i c t io n s i t e s a re a t t r ib u t a b le to g a in s u b s t i t u t io n s . These s i t e g a in s ,

in fe r r e d from th e re fe re n c e sequence, g iv e d e ta i le d in fo rm a t io n about th e

e xa c t n a tu re and lo c a t io n o f th e m u ta tio n s w hich cause i t . A m u ta tio n which

a f f e c t s ju s t one n u c le o t id e i s r e fe r re d to as a p o in t m u ta t io n . Thus a base

s u b s t i t u t io n , th e rep lacem en t o f one base p a i r by a n o th e r w ith o u t an

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a l t e r a t io n in th e t o t a l number o-f n u c le o t id e s i s a ls o a p o in t m u ta t io n . The

te rm ’ t r a n s i t i o n ’ d e s c r ib e s a s in g le base s u b s t i t u t io n th a t re p la c e s one

base w ith a n o th e r o t th e same ty p e ie . a p u r in e w ith a p u r in e (A-G, G -A ),

o r a p y r im id in e base w ith a no the r p y r im id in e (C -T ,T -C ); a ’ t r a n s v e r s io n ’ i s

th e re p lace m e n t o t a p u r in e base by a p y r im id in e base and v ic e v e rsa (A -

T ,A -C ,G -T ,G -C , e tc ) .

Thus, th e e x a c t lo c a t io n (base sequence number and gene in v o lv e d ) and ty p e

( t r a n s i t io n o r t ra n s v e rs io n ) o t each base change, was in te r r e d and a re

l i s t e d by r e s t r i c t io n endonuclease used to d e te c t th e base changes in ta b le

3 .5 . In th e t o t a l mt genome 53.37. o t th e g a in m u ta tio n s a re t r a n s i t i o n s ,

4 6 .7 7 t ra n s v e rs io n s ( r a t io 1.14s 1 ) . The app a ra n t r a t io s o t t r a n s i t i o n to

t ra n s v e rs io n s observed in t h i s s tu d y i s co nco rd a n t t o th o s e in s im i la r

s tu d ie s u s in g th e r e s t r i c t i o n mapping approach ( F e r r is e t a l 1983; Cann

e t al,,1984), b u t c o n s id e ra b ly lo w e r v a lu e s tha n th a t re p o r te d in

sequenc ing s tu d ie s (Brown & Simpson, 1982; Brown e t al,, 1982; G reenberg e t

al,,1983). The p ro p o r t io n o t t r a n s i t i o n s does n o t appear t o be dependent

on th e tre q u e n c y o t th e r e s t r i c t i o n s i t e ; b o th p o o r ly and w e ll re p re s e n te d

s i t e s in mouse mtDNA have low r a t io s o t t r a n s i t io n s t o t ra n s v e rs io n s (T a b le

3 .6 ) . F ig u re 3 .3 , a l in e a r map o t th e mt genome, i l l u s t r a t e s th e

d is t r ib u t io n s o t bo th to be u n ito rm ( t r a n s i t io n s - X 2 Cdt=43 = 18 * * ,

c e t t ic e n t o t D is p e rs io n CC.D3 = 0 .5 3 ; t ra n s v e rs io n s - X 2 Cdt=33 = 9 .3 8 * ,

C.D = 0 .4 ) .

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P ro te in -c o d in g genes g iv e even more d e ta i le d in fo rm a t io n in v o lv in g th e

e xa c t n a tu re o f th e base change, w hether th e y were " s i l e n t " (synonymous;

ca u s in g no a m in o -a c id rep lace m e n ts ) o r " re p la c e m e n t" (non-synonym ous)

s u b s t i t u t io n s , w h ich a m in o -ac id was in v o lv e d o r re p la c e d , and th e codon

p o s i t io n where th e y o ccu rre d ( ta b le 3 .5 ) . A p p ro x im a te ly equal numbers o f

th e 58 in f e r r a b le g a in base changes d e te c te d in th e p ro te in -c o d in g re g io n s

a re due to a m in o -a c id re p lacem en ts and s i l e n t s u b s t i t u t io n s ; 57% o f th e s e

m u ta tio n s o ccu r in th e t h i r d codon p o s i t io n . C o n s id e rin g o n ly th e p r o te in -

co d in g genes, th e app a re n t r a t i o o f t r a n s i t i o n s to t ra n s v e rs io n s in c re a s e s

s l i g h t l y to 1 .3 2 : 1, however, 757. o f th e s u b s t i t u t io n s in th e cytochrom e

p r o te in co d in g re g io n s compared t D 507. in th e NADH dehydrogenase s u b u n its

(1 -6 , 4L) p r o te in genes a re due t o t r a n s i t i o n s ( r a t io s 3 :1 ; 1:1

r e s p e c t iv e ly ) . The t r a n s i t io n s re co rd e d in a l l th e p ro te in -c o d in g genes

a re p re d o m in a n tly s i l e n t (7 5 .B7.). W h ils t th e m a jo r i t y o f t ra n s v e rs io n s

p roduce a m in o -a c id re p la ce m e n ts , o c c u r r in g more f r e q u e n t ly in NADH

dehydrogenase s u b u n its (84.27.) tha n in cytochom e p r o te in s (66 .67 .). In

re g io n s where th e re a re no open re a d in g fram es (rRNA’ s , tRNA’ s , th e D -lo o p ,

and th e o r ig in o f l i g h t s tra n d r e p l ic a t io n ) g e n e ra l ly , t ra n s v e rs io n s

(55.67.) a re more p ro m in en t than t r a n s i t i o n s (44 .47 .), in th e r a t i o 1: 0 .8 .

However, 757. were t r a n s i t io n s in th e tRNA genes as opposed t o o n ly 14.3% in

th e rRNA genes, whereas th e D -loop re g io n su p p o rte d equa l numbers o f b o th .

T ab le 3 .7 sum m arises th e da ta on g a in m u ta tio n s in each m a jo r fu n c t io n a l

re g io n .

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3 .3 .4 ? S i te lo s s e s ;

F o llo w in g th e same r a t io n a le as above a ' s i t e lo s s ' was re c o rd e d when a

m u ta tio n caused a re d u c t io n in number Df expected s i t e s r e la t i v e t o th e

k .b . s . However, i t was n o t p o s s ib le to in f e r th e e x a c t lo c a t io n and n a tu re

o f m u ta tio n s ca u s in g ' s i t e lo s s e s '. The base s u b s t i t u t io n re s p o n s ib le f o r

chang ing th e base sequence in such a way th a t i t i s no lo n g e r re c o g n is e d by

th e s p e c i f ic r e s t r i c t i o n endonuclease and hence n o t c le a ve d was unknown.

C onsequen tly o n ly th e lo c a t io n o f th e w hole r e c o g n it io n sequence can be

re co rd ed ie . 4, 5 o r 6 bases, depending on w hether a f o u r , f i v e o r s ix

b a s e -c u t te r was used, as any one o f th e se may have been th e s i t e th a t

m u ta ted .

Of th e re m a in in g v a r ia b le s i t e s , 65 a re s i t e lo s s e s w ith re s p e c t t o th e

k .b . s . The m a jo r i t y o f th e se (77.371) o ccu r in th e p ro te in - c o d in g re g io n s ,

47% o f w h ich map to th e NADH dehydrogenase s u b u n its . T ab le 3 .8 sum m arises

th e s i t e lo s s e s d e te c te d by each r e s t r i c t i o n enzyme employed in t h i s s tu d y .

3 .3 .5 : Summary o f s i t e occurences:

3 . 3 . 5 . is Genomic d is t r ib u t io n s o f c le a vao e s i t e s .

Each mouse was mapped, on average, f o r a p p ro x im a te ly 296 s i t e s (1360 b p ) ,

re p re s e n t in g 7.8% o f th e mt genome, th u s th e t o t a l mtDNA in 430 B r i t i s h

m ice su rveyed u s in g 14 enzymes was 137.3 k ilo b a s e s . The t o t a l amount o f

mtDNA a c tu a l ly compared in sc re e n in g a l l 638 (430 fro m B r i t a in - t h i s s tu d y ;

208 from W. Europe, M e d ite rra n e a n , and th e Am erica - F e r r is e t a l 1983)

M. dowesticus mtDNA u s in g th e sequence com parison mapping te c h n iq u e

em p loy ing between 2 -14 r e s t r i c t i o n endonuc leases , was about 172 k ilo b a s e s .

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W ith th e e x c e p tio n o f 5 t r a n s fe r RNA genes ( le u c in e , is o le u c in e , g ly c in e ,

t y r o s in e , and s e r in e ) , th e ATPase s u b u n it 8 (ATPase 8) and th e noncod ing

l i g h t s tra n d o r ig in o-f r e p l ic a t io n , a l l mtDNA genes possess c le a vag e s i t e s ,

f o r th e s e t o-f r e s t r i c t i o n enzymes I have used. F ig u re 3 .4A sum m arises th e

a p p ro x im a te map lo c a t io n s o-f 220 c le a va g e s i t e s (97 v a r ia b le ; 123 c o n s ta n t

s i t e s ) , in 57 com pos ite mtDNA geno types (see c h a p te r 4 -for f u r t h e r d e ta i ls )

fro m 638 in d iv id u a ls , d e te c te d w ith 2-11 enzymes. The 26 p re v io u s ly

u n re p o rte d v a r ia b le s i t e s found in B r i t i s h p o p u la t io n s , a re in d ic a te d w ith

(+) o r ( - ) t o i l l u s t r a t e th e n a tu re o f th e s i t e d i f fe r e n c e , (14 lo s s e s and

12 g a in m u ta tio n s ) r e la t i v e to th e k .b . s . F ig u re 3 .4B (o v e r la y ) show th e

lo c a t io n s o f 150 s i t e s (43 v a r ia b le C22 lo s s e s and 21 g a in s ! , 107 c o n s ta n t)

d e te c te d in B r i t i s h m ice u s in g th e th re e a d d it io n a l r e s t r i c t i o n

endonucleases (A lu I , Rsa I , & Sau 9 6 1 ), n o t p re v io u s ly used b e fo re in

sequence com parison mapping s tu d ie s o f ro d e n ts . D e s p ite th e la rg e number o f

s i t e s d e te c te d w ith th e s e e x tra enzymes o n ly 4 more co m p os ite mtDNA

geno types were id e n t i f i e d . Toge ther f ig u r e s 3 .4 a and 3 .4 b summarise th e

com p le te sc reen o f 638 tf. domesticus mtDNA’ s , 61 g en o typ e s , fro m many

g e o g ra p h ic lo c a t io n s around th e w o rld (d e te c te d u s in g 2 -1 4 enzym es),

re v e a lin g th e d is t r ib u t io n o f a l l 370 mapped r e s t r i c t i o n s i t e s (140 were

v a r ia b le ? 230 were c o n s ta n t ) .

These f ig u r e s ( f i g . 3 .4A and 3 .4 B - o v e r la y ) suggest t h a t th e s i t e s a re

w id e ly s c a tte re d th ro u g h o u t th e mt genome, indeed , th e t o t a l number o f

s i t e s (c o n s ta n t and v a r ia b le ) a re n o t s ig n i f i c a n t l y d i f f e r e n t fro m th e

p o isso n d is t r ib u t io n (X 2 C5df3 = 6 .4 5 N’ B) . However, when th e v a r ia b le

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s i t e s a lo n e (p h y lo g e n e t ic a l 1 y in fo r m a t iv e s i t e s ) a re ta ke n in t o

c o n s id e ra t io n , th e re i s a non-random d is t r ib u t io n (X 2 C2df3 = 8 .8 5 * * ) ,

th e c o e f f ic e n t o f d is p e rs io n in d ic a t in g th a t th e s i t e s a re clumped (C.D =

1 .2 6 ) . Thus, t h i s d is t r ib u t io n o n ly p a r t i a l l y f u l l f i l l s Nei and L i ’ s (1979)

assum ptions f o r c a lc u la t in g th e d iv e rg e n c e e s tim a te s , used in th e UPGMA

c lu s te r a n a ly s is in c h a p te rs 4 & 5.

In an a tte m p t to re s o lv e which r e s t r i c t i o n enzymes were c o n t r ib u t in g th e

g re a te s t t o th e o v e r a l l c lum p ing o f v a r ia b le s i t e s , th e d is t r i b u t io n o f

th e se c le a va g e s i t e s f o r each r e s t r i c t i o n enzyme used was examined f o r

d e p a r tu re fro m random d is t r ib u t io n s . F ig u re 3 .5 i l l u s t r a t e s th e

d is t r ib u t io n o f a l l c leavage s i t e s ( v e r t ic a l l in e s above and below th e

h o r iz o n ta l b a r , which re p re s e n ts th e l in e a r iz e d mt genome, a re th e v a r ia b le

and c o n s ta n t s i t e s re s p e c t iv e ly ) a c ro ss th e whole genome by each

r e s t r i c t i o n enzyme. W ith th e e x c e p tio n o f 2 enzymes, Rsa I and Sau 961,

v a r ia b le s i t e s were d is t r ib u te d random ly w ith re s p e c t to r e s t r i c t i o n

enzymes used. Both Rsa I and Sau 961 g iv e non-random d is t r ib u t io n s , th e

c o e f f ic e n t o f d is p e rs io n e s tim a te s in d ic a t in g t h e i r s i t e s were clumped (Rsa

I - X 2 C ld f 3 = 62 * * * , C.D = 1.53? Sau 961 - X 2 C Id f□ = 9 .7 8 * * , C.D =

1 .5 ) . When th e se 2 enzymes were exc lu de d fro m th e a n a ly s is b o th th e t o t a l

and v a r ia b le s i t e s showed random d is t r ib u t io n s , ( t o t a l , X 2 C5df3 = 3 .2 8

N’ s J v a r ia b le , X 2 C3df3 = 6 .52 N* s ) .

3 .3 .5 .2 i D is t r ib u t io n o f s i t e s w ith in and between gene re g io n s .

A c o m p ila t io n o f t o t a l s i t e s (3 7 0 ), in c lu d in g v a r ia b le (140) and c o n s ta n t

(230) s i t e s mapped f o r each gene re g io n ( s iz e in d ic a te d in b a s e p a irs ) ,

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d e te c te d by a l l 14 r e s t r i c t i o n enzymes, i s g iv e n in t a b le 3 .9 . E s tim a te s o f

s i t e s p e r b a s e p a ir ( t o t a l number o f c le a va g e s i t e s observed in each gene

re g io n d iv id e d by gene s iz e in b a s e p a irs ; Cann, 1982) in d ic a te s most

re g io n s were exam ined w ith an equal i n t e n s i t y (mean = 0 .0 0 2 ) , a lth o u g h

v a lu e s range fro m 0 .0 0 (ATPase 8) t o 0 .0 3 7 (ND 1 ) . G iven th e re were 370

s i t e s observed in th e genome w ith th e s e t o f enzymes used, th e expected

number o f s i t e s f o r each gene re g io n (w e ig h te d by s iz e o f gene) i s a ls o

l i s t e d in ta b le 3 .9 . O n ly th re e gene re g io n s show s ig n i f i c a n t d e v ia t io n s

from expec ted numbers o f c leavage s i t e s w i t h in them , c o n s id e r in g a random

mtDNA base sequence c o m p o s it io n . Two re g io n s (ATPase 8, and ND 6 ; X 2

C ld f3 = 4 .5 * and 4 .3 * r e s p e c t iv e ly ) , have le s s s i t e s tha n e xpe c ted ,

w hereas, o n ly one (ND 1 ; X 2 C ld f3 = 8 .9 * * ) has more. T h is i s p ro b a b ly

due to th e v a r ia t io n in base c o m p o s itio n o f s p e c i f i c gene re g io n s <7. G-C

c o n te n t ranges from 2 7 .2 - 41) and th e G-C r ic h b ia s o f th e re c o g n it io n

s i t e s o f th e s e t o f 14 endonucleases used ( ta b le 2 .4 shows th a t f o r a l l 14

enzyme re c o g n it io n sequences th e p e rce n ta g e G-C i s 6 1 .2 , compared w ith

36.77. f o r th e t o t a l mt genome. However th e re was no r e la t io n s h ip between

7G-C c o n te n t and s i t e pe r bp (R eg ress ion c o e f f ic e n t ( r ) = 0 .2 8 , fv a lu e =

1 .23 N' B) .

F u rth e rm o re , th e re i s a s ig n i f ic a n t l in e a r re g re s s io n between t o t a l ( r=

0 .6 5 , f - v a lu e = 5 8 .6 * * * ) , v a r ia b le ( r= 0 .8 7 , f - v a lu e = 5 .71 * ) , and

c o n s ta n t <r= 0 .5 4 , f - v a lu e = 4 2 .5 * * * ) c le a va g e s i t e s and th e s iz e o f gene

re g io n s (b a s e p a irs pe r re g io n ) as i l l u s t r a t e d in f ig u r e s 3 .6 A, B and C and

ta b le 3 .9 . A d d i t io n a l ly , when th e v a r ia b le s i t e s a re s u b d iv id e d in t o lo s s

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and g a in m u ta tio n s ;, b o th show s ig n i f i c a n t l in e a r re g re s s io n s w ith number o f

b a s e p a irs ( s iz e ) w i th in each m a jo r c la s s o f fu n c t io n a l re g io n (g a in

m u ta tio n s - r= 0 .9 6 , f - v a lu e = 3 6 .7 tt \ lo s s m u ta tio n s - r= 0 .9 5 , f - v a lu e

= 2 8 .4 * * ) , shown in f ig u r e 3 .6 E, and F.

F ig u re 3 .7 i l l u s t r a t e s th e a pp rox im a te p o s i t io n s o f th e v a r ia b le s i t e s

w ith in each gene re g io n d e te c te d by a l l enzymes used in th e s tu d y . The

d is t r ib u t io n o f th e s e s i t e s per gene re g io n was te s te d f o r d e v ia t io n s from

randomness u s in g e s tim a te s o f c o e f f ic e n t o f d is p e rs io n . S ix gene re g io n s

from th e 14 te s te d show s ig n i f i c a n t l y clumped d is t r ib u t io n s f o r s e v e ra l

m ajor c la s s e s o f fu n c t io n a l re g io n (Cytochrom e p r o te in s , NADH dehydrogenase

s u b u n its , 16s r ib o s o m a l RNA gene and th e n o n -co d in g D - lo o p ) . Degree o f

c lum p ing o f v a r ia b le s i t e s w ith in c e r ta in mt gene re g io n s i s n o t a fu n c t io n

o f t h e i r s iz e ( in s i t e s /b a s e p a ir » r= 0 .3 2 , f - v a lu e = 1 .4 N“ s ) .

3 .3 .6 ? Gene v a r i a b i l i t y

E xam ina tion o f th e r a te s o f base change pe r gene re g io n a re p o s s ib le once

th e d is t r ib u t io n s o f th e c leavage s i t e s a re c h a ra c te r is e d . A c o n ve n ie n t

e s tim a te o f gene v a r i a b i l i t y (Cann, Brown, and W ils o n , 1983; F e r r is e t al.r

1983) i s produced by d iv id in g th e number o f v a r ia b le s i t e s by th e t o t a l

number o f s i t e s in a gene re g io n , expressed as a p e rc e n ta g e . These

e s tim a te s a re l i s t e d in ta b le 3 .1 0 , f o r each mt gene, f o r 11 enzymes

( B r i t i s h d a ta - t h i s s tu d y , poo led w ith th e p u b lis h e d d a ta fro m tt*

douesticus - F e r r is et al 1983), 3 a d d it io n a l enzymes n o t p re v io u s ly

used b e fo re ( s i t e d a ta fro m B r i t i s h sam ples o n ly ) , and th e t o t a l 2-14

enzymes employed in c lu d in g a l l s i t e in fo rm a t io n f o r a l l m ice . T ab le 3.11

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summarises th e v a r ia t io n -for each m a jo r c la s s o f fu n c t io n a l re g io n and

ra n k s them from th e le a s t to most v a r ia b le re g io n s . Thus, th e most

conserved gene re g io n s a re th e t r a n s fe r RNA's, n ex t th e r ib o so m a l RNA’ s ,

th e n th e cytochrom e p ro te in -c o d in g genes, th e most v a r ia b le re g io n s be ing

th e NADH dehydrogenase s u b u n its (ND 1 -6 , 4L) and th e n o n -c o d in g a re a s , th e

D -lo o p . E s tim a te s o f gene v a r i a b i l i t y p e r gene re g io n in B r i t i s h m ice a re

compared w ith p re v io u s ly re co rd e d in M* douesticus p o p u la t io n s and humans,

i l l u s t r a t e d in ta b le 3 .1 2 .

3 .3 .7 ? D e s c r ip t io n s o f s i t e d i f fe re n c e s .

The fo l lo w in g d e s c r ip t io n s a re o f a l l th e s i t e d if fe re n c e s summarized in

ta b le 3 .1 (fra g m e n t d ig e s t io n p r o f i le s ) and ta b le 3 .3 A -N / t a b le 3 .4

( r e s p e c t iv e ly th e v a r ia b le and c o n s ta n t s i t e s o f e ve ry d ig e s t io n p r o f i l e ) .

These d e s c r ip t io n s a re produced by s tu d y in g pho tog raphs s im i la r to p la te s

3 .2 -3 .1 0 , f o r th e s ix , f i v e , and fo u r base c u t te r s used in t h i s s tu d y . The

sam p ling l o c a l i t i e s o f th e m ice show ing th e v a r io u s d i f f e r e n t r e s t r i c t i o n

d ig e s t io n p r o f i l e s a re m entioned in th e p la te te x t s and a re d iscu sse d

f u r t h e r in c h a p te r 4.

3 .3 .7 .1 ? H e x a n u c le o tid e R e s t r ic t io n Enzymes?

3 .3 .7 .1 .1 ? H ind I I I (AAGCTT).

T h is r e s t r i c t i o n endonuclease i s th e f i r s t o f fo u r h e x a n u c le o tid e enzymes

used to screen mtDNA fro m 430 B r i t i s h m ice . The r e c o g n it io n sequence was

o n ly found th re e t im e s in th e known base sequence (B ibb e t a l . , 1981)

(p a t te rn A) a t s i t e s 9136, 11081, and 11969 w ith th e th re e c o rre s p o n d in g

fra g m e n ts o f s iz e 13462, 1945, and 888 base p a ir s . O nly two d ig e s t io n

p r o f i l e s were seen in B r i t i s h sam ples ( ta b le 3 .1 ) , bo th o f w h ich , p a t te rn A

CHAPTER THREE

and B, have been p re v io u s ly d e s c r ib e d -for c o n t in e n ta l sam ples ( F e r r is e t

al~, 1983). P a tte rn B i s produced by a s i t e lo s s a t 9136 ( s i t e 1, ta b le

3 .3A ) w ith th e d isappearence o-f -fragm ents 13462 and 19456 w h ich jo in to

p roduce a la r g e r -fragment 15407 bp. Hence a t o t a l o-f 3 s i t e s were a ss ig n e d ,

2 c o n s ta n t , and 1 v a r ia b le , u s in g H ind I I I ( ta b le 3 .3A - v a r ia b le s i t e s ;

ta b le 3 .4 , p o in t 1 - c o n s ta n t s i t e s ) . P la te 3 .2 i l l u s t r a t e s th e s e two

d ig e s t io n p r o f i le s . T h is enzyme was im p o r ta n t f o r s ta n d a rd iz in g th e

v a r ia b le p a t te rn s d e s c r ib e d by F e r r is e t a l . (1983) w ith th o se I o b ta in e d

from B r i t i s h m ice. L a b o ra to ry s to c k s C 5 7 b l/6 J and NZB, p u b lis h e d as

p a t te rn s A and B, r e s p e c t iv e ly , p roved t o have id e n t ic a l p a t te rn s to th o se

found in th e B r i t i s h sam ples. C onsequen tly I adopted F e r r is e t a l . ’ s

n o m e n c la tu re .

3 .3 .7 .1 .2 : Xba I (TCTAGA).

U s ing t h i s r e s t r i c t i o n enzyme a l l B r i t i s h in d iv id u a ls screened p roved

t o t a l l y in v a r ia n t d is p la y in g o n ly d ig e s t io n p r o f i l e A, i l l u s t r a t i n g an

ex trem e case o f s i t e c o n s e rv a tio n w ith in Mus domesticus. T h is i s

co n co rd a n t w ith th e f in d in g s o f F e r r is et al*, 1983); th e m a jo r i t y o f Mus

domesticus mtDNA's observed in Europe and th e Am ericas have th e same

p a t te r n , w ith th e e x c e p tio n o f tw o in d iv id u a ls from S k iv e and V ib o rg ,

Denmark, w h ich have p a t te rn B. However, th e s e were d e f in e d as H* musculus

on th e b a s is o f e le c t r o p h o r e t ic and m o rphom etric e v id e n c e , b u t had M*

domesticus mtDNA th ro u g h in t r o g r e s s io n (G y lle n s te n S< W ils o n , 1987).

In t o t a l , s ix s i t e s were scored f o r t h i s enzyme, 5 c o n s ta n t and 1 v a r ia b le

( ta b le 3 .3B - v a r ia b le s i t e s ; ta b le 3 .4 , p o in t 2 - c o n s ta n t s i t e s ) . P la te

3 .3 i l l u s t r a t e s t y p ic a l Xba I d ig e s ts o f p a t te rn A from B r i t i s h m ice ,

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s e p a ra te d on s i l v e r s ta in e d 57. p o ly a c ry la m id e ;, and an e th id iu m brom ide

s ta in e d 0 .7 5 7 agarose g e l.

3 .3 .7 .1 .3 : H inc I I (GTCPuPvJAC).

The known re fe re n c e sequence i s c le a ved a t 5 s i t e s ( ta b le 2 .6 , c h a p te r 2 ) ,

th e d ig e s t io n p r o f i l e (A) i s l i s t e d in ta b le 3 .1 , a l l B r i t i s h in d iv id u a ls I

have exam ined show t h i s p a t te rn e x c lu s iv e ly . However, F e r r is and c o lle a g u e s

d e s c r ib e d 3 v a r ia n t d ig e s t io n p a t te rn s nam ely B, C, and G in M* domesticus

from Europe and th e A m ericas. Each o f th e s e a d d it io n a l v a r ia n ts d i f f e r e d

from th e k .b . s by a s in g le s i t e g a in in e v e ry case ( d e ta i ls shown in ta b le s

3.3C and 3 .4 ) . In t o t a l ft* domesticus mtDNA c le a ve d by H inc I I produced 8

s i t e s , 5 c o n s ta n t and 3 v a r ia b le .

3 .3 .7 .1 .4 : Acc I (GTCA/C3CG/T1AC).

Acc I d is c r im in a te s B r i t i s h p o p u la t io n s in t o 2 ty p e s (d ig e s t io n p r o f i l e s A

and L ) . The fo rm e r i s th e same as th e k .b . s and th e l a t t e r a u n iqu e new

p a t te rn n o t p re v io u s ly d e s c r ib e d . D ig e s t io n p a t te rn L has 8 fra g m e n ts

c h a r a c te r is in g i t (as opposed to o n ly 7 in p a t te rn A ), w h ich i s produced by

a s in g le s i t e g a in a t 4515 ( s i t e 2 , ta b le 3 .3 D ), ca us ing th e lo s s o f

fra gm en t 7358 bp, re p la c e d by fra g m e n ts 6164 and 1194 bp, th e sum o f th e

la r g e r fra gm en t lo s s ( ta b le 3.3D; ta b le 3 .4 ) . F e r r is et al*, (1983)

re c o g n ise d 6 v a r ia n t p r o f i le s (B, C, D, E, J , and K ), th e fra g m e n ts in them

a re l i s t e d in ta b le 3 .1 , and d e t a i ls o f v a r ia b le s i t e s can be found in

ta b le 3 .3D .

The t o t a l number o f r e s t r i c t i o n s i t e s asssayed f o r Acc I in ft* domesticus

i s 12 o f w h ich 6 a re c o n s ta n t and 6 a re v a r ia b le ( ta b le 3 .4 l i s t s th e t o t a l

CHAPTER THREE

as 11 .5 and th e number o-f v a r ia b le s i t e s as 5 .5 . as a s i t e g a in a t 15924

[ s i t e 6 , ta b le 3.3D3 seen in d ig e s t io n p r o f i l e s B-D, causes a co nco m ita n t

lo s s o f a Hae I I I s i t e a t 15923. For p h y lo g e n e tic p u rpo se s t h i s s i t e was

o n ly coun ted o n ce ).

3 .3 .7 .2 s F iv e b a s e -c u t te r r e s t r i c t io n endonuc leases:

3 .3 .7 .2 .1 : Ava I I (GGCA/TICC)

T h is i s th e o n ly f i v e b a s e -c u t te r used in t h i s s tu d y . A t o t a l o f 13.5 s i t e s

were assayed, 6 c o n s ta n t ( ta b le 3 .4 p o in t 5) and 7 .5 v a r ia b le ( ta b le

3 3 .3E ), 10 s i t e s com prised th e base sequence p a t te rn A ( l i s t e d ta b le 2 .6 ,

c h a p te r 2 ) . B r i t i s h p o p u la t io n s e x h ib ite d fo u r v a r ia n t d ig e s t io n p r o f i le s

J , K, L , and M, w hich have n o t been p re v io u s ly re c o rd e d , th e fragm en t s iz e s

o f each can be found in ta b le 3 .1 . P a tte rn A was n o t observed in B r i t i s h

sam ples and th e v a r ia n t d ig e s t io n p r o f i le s d i f f e r e d fro m t h i s by th e

fo l lo w in g s i t e changes. P a tte rn L has one s i t e g a in a t 12213 ( s i t e 6 , ta b le

3 .3 E ), w ith th e lo s s o f fragm en t 6510 bp and th e g a in o f two new fra gm en ts

s iz e d 6208 and 302 bp. P a t te r n 's J , M and K, a l l have t h i s l a t t e r s i t e g a in

in common as in p a t te rn L , a d d it io n a l s i t e changes fro m t h i s f o r each

p a t te rn i s d e s c r ib e d be low . P a tte rn J i s e x p la in e d by a n o th e r s i t e g a in a t

6123 ( s i t e 3 , ta b le 3 .3 E ), w h ich in co m b in a tio n w ith s i t e g a in 12213 causes

th e lo s s o f th e fra g m e n t 6510 re p la c e d by th re e fra g m e n ts 6090, 302, and

118 bp. P a tte rn M has a lo s s a t 2083 ( s i t e 1, ta b le 3 .3 E ) , in a d d it io n t o 2

s i t e g a in s a lre a d y d e s c r ib e d f o r p a t te rn J , th u s fra g m e n ts 6510, 1659 and

274 bp a re re p la c e d by 6090, 302, 118 and 1933 bp. F in a l l y p a t te rn K has a

new s i t e lo s s a t 2871 ( s i t e 2 , ta b le 3 .3 E ) , ca u s in g fra g m e n ts 609 and 514bp

to be lo s t , w ith th e g a in o f th e la rg e r fra gm en t 1123 bp in i t ' s p la c e .

CHAPTER THREE

S e ve ra l o th e r d ig e s t io n p r o f i le s n o t observed in B r i t a in have been

re p o r te d by F e r r is e t aim, (1 9 83 ), p a t te rn s B, C, D, and I , w h ich a re

summarised in ta b le s 3 .1 and 3 .3E .

3 .3 .7 .3 : T e tra n u c le o t id e r e s t r i c t i o n endonucleases:

3 .3 .7 .3 .1 ; Fnud I I (CGCB).

T h is te t r a n u c le o t id e r e s t r i c t i o n endonuclease gave th re e d ig e s t io n p a t te rn s

in B r i t i s h p o p u la t io n s . The base sequence was c le a ved a t 8 s i t e s ( ta b le

2 .6 , c h a p te r 2) p ro d u c in g 8 fra g m e n ts in th e d ig e s t io n p r o f i l e (p a tte rn A -

ta b le 3 .1 ) . The two v a r ia n t fo rm s o f d ig e s t io n p r o f i le s J and K, a re each

c h a ra c te r is e d by 7 fra g m e n ts . P a tte rn J r e s u l t s from th e lo s s o f 1 s i t e a t

14705-8 ( s i t e 5; ta b le 3 .3 F ) ca u s in g th e lo s s o f fra g m e n ts 1907 and 1666

bp, re p la c e d by th e sum o f th e se p ro d u c ts , 3573 bp. S im i la r ly , p a t te rn K

d i f f e r s from th e base sequence by 1 s i t e lo s s a t 317 ( s i t e 1; ta b le 3 .3F )

r e s u l t in g in th e g a in o f th e la r g e r fragm en t 3362 bp and th e subsequent

lo s s o f 2 s m a lle r frg a m e n ts 1907 and 1455 bp.

Three o th e r d ig e s t io n p r o f i le s have been d e s c r ib e d e lse w he re w h ich have n o t

been re co rd e d f o r B r i t i s h p o p u la t io n s , namely B, H, and I , (w h ich do n o t

resem b le any o th e r p r o f i l e s se e n ), summarised in ta b le s 3 .1 and 3 .3 F .

Com bining th e p u b lis h e d d a ta by F e r r is and c o lle a g u e s , w ith d a ta from t h i s

s tu d y , a t o t a l o f 10 s i t e s were exam ined u s in g Fnud I I in M* domesticus, 5

c o n s ta n t and 5 v a r ia b le .

3 .3 .7 .3 .2 : Hpa I I (CGG).

Two p a t te rn s , A and D, a re seen in B r i t i s h House mouse p o p u la t io n s u s in g

t h i s fo u r b a s e -c u t te r . Both o f th e se d ig e s t io n p r o f i le s have been

CHAPTER THREE

p re v io u s ly i d e n t i f i e d by F e r r is and c o lle a g u e s (c o n s e q u e n tly , th e same

l e t t e r d e s ig n a tio n s were a d o p te d ), in a d d i t io n to 2 o th e r p a t te rn s (B and

C) n o t seen in B r i t a in . In t o t a l 11 .5 s i t e s were assayed w ith t h i s enzyme,

9 c o n s ta n t ( ta b le 3 .4 ; p o in t 6) and 2 .5 v a r ia b le ( ta b le 3 .3 G ). The k .b . s .

( p a t te rn A) has 11 s i t e s and 11 c o rre s p o n d in g fra g m e n ts ( ta b le 2 .6 , c h a p te r

2 ; ta b le 3 .1 r e s p e c t iv e ly ) , p a t te rn D d i f f e r s from t h i s common p a t te rn by

th e lo s s o f 1 s i t e a t 3332 ( s i t e 2 , ta b le 3 .3 G ), w h ich causes th e

d isa p p e a ra n c e , o f fra g m e n ts 813 and 68 bp and th e appearance a new fra g m e n t

o f 881 bp. R e p re s e n ta tiv e d ig e s t io n p r o f i l e s o f th e two common p a t te rn s

observed in B r i t a in a re shown on e i t h e r agarose g e ls as in p la te 3 .2 o r in

57 p o ly a c ry la m id e in p la te 3 .4 .

3 .3 .7 .3 .3 s Tao I (TCGA).

F e r r is and c o lle a g u e s d e s c r ib e d 7 d ig e s t io n p r o f i l e s (summarised in ta b le

3 .1 ) , in M. domesticus, o f which tw o , A and B, a re commonly observed in

B r i t i s h p o p u la t io n s . The t o t a l number o f s i t e s assayed w ith Taq I was 21,

16 were c o n s ta n t ( l i s t e d ta b le 3 .4 , p o in t 9 ) , and 5 were v a r ia b le ( ta b le

3 .3 H ) . P a tte rn B d i f f e r s from th e base sequence (p a t te rn A) by a s in g le

s i t e lo s s b e g in in g a t 9577 ( s i t e 5 , ta b le 3 .3 H ) , ca us ing th e d isa pp e a ra n ce

o f 2 fra g m e n ts 1909 and 258bp, re p la c e d by 2167 bp. Hence p a t te rn B i s

c h a ra c te r is e d by 18 fra g m e n ts , as opposed t o 19 in th e base sequence. P la te

3 .5 i l l u s t r a t e s th e common B r i t i s h d ig e s t io n p a t te rn s A and B on a s i l v e r

s ta in e d 57. p o ly a c ry la m id e g e l.

3 .3 .7 .3 .4 : Hae I I I (GGCC).

F o r ty th re e s i t e s were examined w ith t h i s enzyme in M. domesticus, o f

w h ich 25 a re c o n s ta n t ( ta b le 3 .4 , p o in t 8 ) , and 18 v a r ia b le ( ta b le 3 .3 1 ) .

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CHAPTER THREE

T h is t o t a l in c lu d e s 18 d ig e s t io n p a t te rn s , 11 have been p re v io u s ly

d e s c r ib e d by F e r r is and c o lle a g u e s (A -F and M-Q; summarised in t a b le ’ s 3 .1

and 3 .3 ) . E ig h t d ig e s t io n p ro - f i le s were found in th e B r i t i s h p o p u la t io n s

in c lu d in g th e k . b . s . , namely A, R, S, U, V, X, and W. A l l d ig e s t io n

p r o f i l e s I have sco re d a re c h a ra c te r is e d by a mean o f 35 fra g m e n ts ; th e

number o f fra g m e n ts in th e k .b .s .

D if fe re n c e s r e la t i v e to th e k .b .s . in d ig e s t io n p r o f i l e s o f B r i t i s h sam ples

a re as fo l lo w s : P a tte rn S in v o lv e s 1 s i t e g a in a t 8648 ( s i t e 8 , ta b le

3 .3 1 ) , w hich causes th e lo s s o f fra gm en t 560 and th e g a in o f two s m a lle r

fra g m e n ts 465 and 95 bp. P a tte rn R in v o lv e s 3 s i t e lo s s e s and 1 s i t e g a in ,

a t p o s i t io n s 3402, 7541, 9756 p lu s 3398 ( s i t e s 3, 7 , 11 and 2 r e s p e c t iv e ly ,

ta b le 3 .3 1 ) . The lo s s a t s i t e 9756 causes th e lo s s o f fra g m e n ts 1372 and

945 bp w ith th e co n co m ita n t g a in o f th e la r g e r fra g m e n t 2307 bp. S im i la r ly ,

lo s s o f s i t e 7541 causes th e d isapp e a ra n ce o f fra g m e n ts 526 and 51 bp,

re p la c e d by fra g m e n t 577 bp. The lo s s o f 67 and 469 bp and th e appearance

o f fra g m e n ts 63 and 473 bp a re e x p la in e d by th e s im u lta n e o u s lo s s o f s i t e

3402 and g a in o f a s i t e a t 3398 (an exam ple o f t h i s p r o f i l e i s f ig u r e d in

p la te 3 .6 , la n e 2 ) .

D ig e s t io n p r o f i l e T in v o lv e s 2 s i t e lo s s e s a t B811 and 12378 ( s i t e s 9 and

10, ta b le 3 .3 1 ) , w h ich a re a d ja c e n t to each o th e r in th e mtDNA sequence,

ca u s in g th e lo s s o f fra g m e n ts 945, 560, and 69 bp, w h ich a re re p la c e d by a

fra g m e n t th e s iz e o f t h e i r sum, 1574 bp ( p la te 3 .6 , la n e 3 ) .

P a tte rn V in v o lv e s 2 s i t e lo sse s 15189 and 12378 ( s i t e s 18 and 14

r e s p e c t iv e ly , ta b le 3 .3 1 ) , w ith th e subsequent lo s s in each case o f two

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s m a lle r fra g m e n ts nam ely, 550 and 33 bp p lu s 852 and 39 bp , w ith th e g a in

o f fra g m e n ts 583 and 891 bp ( la n e 5 , p la te 3 .6 ) . P a tte rn U i s a t t r ib u t a b le

t o th e lo s s o f 1 s i t e 15189 ( s i t e 18, ta b le 3 .3 1 ) , w ith th e d isappea rance

o f fra g m e n ts 550 and 33 bp and th e g a in o f a 583 bp fra g m e n t ( la n e 4, p la te

3 .9 ) . P a tte rn X shows th e same p r o f i l e as R, d i f f e r i n g o n ly by th e presence

o f s i t e 9756 in s te a d o f a lo s s (see d e s c r ip t io n a bo ve ).

D ig e s t io n p r o f i l e W, in v o lv e s a s i t e g a in a t p o s i t io n 11210 ( s i t e 12, ta b le

3 .3 .1 ) , w ith th e lo s s o f th e la rg e r fra gm en t 334 bp and th e appearance o f 2

s m a lle r fra g m e n ts 316 and 18 bp.

3 .3 .7 .3 .5 ? H in f I (GACN3TC).

T h is te t r a n u c le o t id e r e s t r i c t i o n enzyme d e te c ts 8 d ig e s t io n p a t te rn s in

B r i t a in , p la te 3 .7 shows 5 such p r o f i le s (A, C, S, Z, and 3 r e s p e c t iv e ly ) .

F e r r is and c o lle a g u e s in t h e i r s tu d y o f European and Am erican H.

domesticus, observed 23 p a t te rn s , 4 o f w h ich (A, C, U, and Z) a re re co rd ed

in B r i t i s h sam ples. F o r ty s ix s i t e s were assayed f o r a l l d ig e s t io n p a t te rn s

d e te c te d w ith t h i s enzyme, o f w hich 19 a re c o n s ta n t ( ta b le 3 .4 , p o in t 1 1 ),

and 27 v a r ia b le ( ta b le 3 .3 K ).

B r i t i s h p o p u la t io n s fro m s e v e ra l lo c a t io n s show d ig e s t io n p r o f i l e A (p la te

3 .7 , la n e s 1 and 4 ) , th e 30 fra g m e n ts c h a r a c te r is in g th e p r o f i l e and

c o rre s p o n d in g r e s t r i c t i o n s i t e lo c a t io n s a re l i s t e d in ta b le 2 .6 (c h a p te r

2 ) . P a tte rn C d i f f e r s fro m th e known base sequence in th e lo s s o f two

a d ja c e n t s i t e s nam ely 9526 and 9574 ( s i t e s 16 and 17, ta b le 3 .3 K ),

p ro d u c in g an a d d it io n a l fragm en t o f 405 bp, w ith th e c o rre s p o n d in g lo s s o f

th re e s m a lle r fra g m e n ts , 263, 94 and 9 bp ( p la te 3 .7 , la n e 2 ) . S im i la r ly ,

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CHAPTER THREE

p a t te rn U has lo s t s i t e 9574, in a d d i t io n , th e d isa pp e a ra n ce o f fra gm en t

533 bp and th e o b s e rv a tio n o f two s m a lle r fra g m e n ts th e sum o f t h i s , o f 34B

and 185 bp, was e x p la in e d by a s i t e g a in a t p o s i t io n 16179 ( s i t e 27, ta b le

3 .3 K ) . P a tte rn Z in v o lv e d 1 s i t e lo s s a t p o s i t io n 7973 ( s i t e 12, ta b le

3 .3 k ) , th e appearance o f fragm en t 1459 bp re p la c in g fra g m e n ts o f 920 and

539 bp ( i l l u s t r a t e d in p la te 3 .7 , la n e 3 ) .

D ig e s t io n p r o f i l e s n o t p re v io u s ly re co rd e d in c lu d e p a t te rn s G, S, X, and 3,

e x p la in e d by th e fo l lo w in g base changes! G and X p r o f i l e s a re b o th no ted

f o r th e 2 a d ja c e n t s i t e lo s s e s , p re v io u s ly seen in C and U ty p e s ( d e ta i ls

see above ). However, each have a d d it io n a l s i t e changes m aking them

d is t in c t i v e . For exam ple, G has a s i t e g a in a t 11255 ( s i t e 20, ta b le

3 .3 .K ) , which p roduces 2 fra g m e n ts 1418 and 318 bp fro m th e one la rg e r 1734

bp, w h i ls t p a t te rn X su gg e s ts a co n c o m ita n t lo s s and g a in o f s i t e s 15115

and 15094 ( s i t e s 26 and 25 r e s p e c t iv e ly , ta b le 3 .3 k ) , g iv in g a d d it io n a l

fra g m e n ts o f 900 and 426 bp, in th e p la c e o f th e m is s in g 879 and 447 bps.

P a tte rn s S and 3 (see p la te 3 .7 , la n e s 5 and 6 , r e s p e c t iv e ly ) a ls o share

th e same s i t e lo s s as p r o f i l e Z, a t p o s i t io n 7973 (see d e s c r ip t io n o f

n a tu re o f fra gm en t d i f fe re n c e s above ), y e t each have a d d i t io n a l b u t

s e p a ra te s i t e lo s s e s a t p o s i t io n s 6889 and 11938 r e s p e c t iv e ly ( s i t e s 11 and

21, ta b le 3 .3 K ). The fo rm e r s i t e lo s s causes th e d isa p p e a ra n ce o f fra gm en ts

485 and 368 bp, w h i ls t th e l a t t e r in v o lv e s th e appearance o f 511 bp in th e

p la c e o f fra g m e n ts 367 and 144 bp.

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CHAPTER THREE

3 .3 .7 .3 .6 t Mbo I (BATC).

F o r ty seven c le a va g e s i t e s were seen u s in g t h i s enzyme, employed he re -for

th e purpose o-f com parison w ith p re v io u s s tu d ie s . Of th e se s i t e s 26 a re

observed in e v e ry mouse te s te d (c o n s ta n t s i t e s ) , and 21 a re v a r ia b le .

T h is enzyme d e te c ts 22 d ig e s t io n p r o - f i le s , o-f w hich 6 a re re p re s e n te d in

B r i t i s h sam ples. Two have been p re v io u s ly re p o r te d , one id e n t ic a l t o th e

k .b .s . (p a t te rn A ), th e o th e r , p a t te rn X, seen o n ly in Taunton p o p u la t io n s .

Four ty p e s a re un ique to B r i t a in ( / , 3 , 0 and W). D ig e s t io n p a t te rn X can

be de-fined by a s in g le s i t e lo s s a t 13B14, ca u s in g th e lo s s o-f -fragm ents

222 and 105 bp and th e g a in o-f fragam en t 327 bp. W h ils t p a t te rn S) i s

produced by 1 s i t e g a in w ith th e lo s s o-f th e la rg e -fragment 1344, re p la c e d

by 1179 and 165 bp. D ig e s t io n p a t te rn □ can be e x p la in e d by 2 s i t e lo s s e s

a t p o s i t io n s 2505, and 3597 and 1 s i t e g a in a t 24B9. T h is causes th e lo s s

o-f fra g m e n ts 59B + 67, 468 + 31, re p la c e d by 614 + 51, and 499

re s p e c t iv e ly ( p la te 3 .8 , la n es 2 -6 ) .

P a tte rn / has th e same s i t e changes as 0 w ith th e e x c e p tio n o f an

a d d it io n a l s i t e lo s s and g a in a t p o s i t io n s 11168 and 11305, w ith th e

subsequent g a in o f fra g m e n ts 579 and 15 bp, w ith th e lo s s o f 155 and 439

bp. P a tte rn W i s s im p ly e x p la in e d by th e o cc u rre n c e o f 1 s i t e g a in a t

10564, r e s u l t in g in th e d isappearance o f fra gm en t 1344 and th e appearance

o f 1034 and 310 bp ( p la te 3 .8 , la n e 1 ) .

3 .3 .7 .3 .7 : Rsa I (GTAC).

T h is te t r a n u c le o t id e r e s t r i c t io n endonuc lease has n o t been used in House

mouse s tu d ie s th u s f a r . In t h i s s tu d y I observed 7 d i f f e r e n t d ig e s t io n

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p r o f i le s ( p la te 3 .9 i l l u s t r a t e s fo u r d ig e s t io n p r o f i l e s , nam ely A, C, D,

and E) in B r i t i s h sam ples in c lu d in g one s im i la r t o th e k . b . s . , w h ich i s

c h a ra c te r is e d by 37 fra gm en ts ( ta b le 3 .1 , p o in t 1 3 ), th e r e s t r i c t i o n s i t e

lo c a t io n s a re l i s t e d in ta b le 2 .6 (c h a p te r 2 ) .

D ig e s t io n p r o f i l e C i s th e s im p le s t p a t te rn t o e x p la in w ith o n ly 2 s i t e

d if fe re n c e s fro m k .b . s . (p a tte rn A ), a t p o s i t io n s 9545 and 11265 ( s i t e 10

[ lo s s ! , and 11 [ g a in ] , ta b le 3 .3 N ), w ith th e lo s s o f fra g m e n ts 577 + 78 bp

and 658 bp, re p la c e d by 655 bp p lu s 572 + B6 bp r e s p e c t iv e ly . P a tte rn D

in v o lv e s 2 cases o f s im u lta n e o u s lo s s and g a in s a t 12484 ( - ) and 12492 (+ ) ,

p lu s 12998 ( - ) and 13005 (+) [ s i t e s 12, 13, 14, and 15, ta b le 3 .3N 3, w ith

th e d isappearance o f fra gm en ts 240 + 275 bp and 274 and 495 bp, and

appearance o f 283 + 232 bp and 281 and 488 bp . D ig e s t io n p r o f i l e ty p e B has

11 s i t e d i f fe re n c e s compared w ith th e k . b . s . , 5 a re g a in m u ta tio n s a t

p o s i t io n s 7B6, 2783, 6633, 11265, and 13342 ( s i t e s 3 , 4 , 8 , 11, and 16,

ta b le 3 .3 N ), and 2 lo s s e s a t 9545, and 15495 ( s i t e 10 and 21, ta b le 3 .3 N ).

T h is r e s u l t s in th e fo l lo w in g fragm en t d i f fe r e n c e s : th e lo s s o f th e la rg e r

fra gm en ts 297, 187, 2925, 658, and 495 bp w h ich a re re p la c e d by 2 s m a lle r

fra gm en ts in each s i t e g a in case ie . 188 + 109 bp; 54 + 133 bp? 2196 + 729

bp» 86 + 572 bp and 151 + 344 bp, w h i ls t on th e o th e rh an d th e 2 lo s s e s o f

s i t e s causes th e d isappearence o f 577 + 78 bp p lu s 35 + 720 bp, w ith th e

appearance o f fra g m e n ts o f t h e i r sum, 655 and 755 bp r e s p e c t iv e ly .

A d d i t io n a l ly 2 cases o f s im u lta ne o us lo s s and g a in o f s i t e s in c lo s e

p ro x im ity to each o th e r , a t s i t e s 15110 (+ ) , and 15126 ( - ) , p lu s 15418 (+ ) ,

and 1542B ( - ) [ s i t e s 17 and 18, p lu s 19 and 20, t a b le 3 .3N 3, causes th e

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lo s s o-f -fragm ents 623, 308, and 17 bp, w ith th e g a in o-f 639, 302, and 7bp

in s te a d . D ig e s t io n p r o - f i le s E, F, and G sh a re th e p re v io u s ly d e s c r ib e d s i t e

d i-f-fe re nce s in ty p e B, each however h a v in g e x tra changes as - fo llo w s :

p a t te rn E - 2 s i t e g a in s and 1 lo s s a t p o s i t io n s 3679, 3698 and 3690

r e s p e c t iv e ly ( s i t e s 5 ,7 and 6, ta b le 3 .3 N ), r e s u l t in g in th e lo s s o-f

■fragments 259 and 218 bp, w ith th e g a in o-f 248, 210 and 19 bp in t h e i r

p la c e ; p a t te rn F - 1 a d d it io n a l s i t e lo s s a t 8925 ( s i t e 9 , ta b le 3 .3 N ),

w ith th e lo s s o-f 96 and 87 bp -fragm ents p lu s th e g a in o-f th e sum o-f th e s e ,

183 bp; p a t te rn G - a s i t e g a in and lo s s a t 107 and 598 ( s i t e s 1 and 2,

ta b le 3 .3 N ), w ith th e co n co m ita n t re p lacem en t o-f -fragm ents 188 and 678 bp

w ith 184 and 682 bp.

3 .3 .7 .3 .8 ; A lu I (AGCT).

The known base sequence has 67 c leavage s i t e s ( ta b le 2 .6 , c h a p te r 2) w ith

t h i s r e s t r i c t i o n endonuclease ( th e most - fre q u e n tly c u t t in g enzyme used in

t h i s s tu d y , n o t p r e v io u s ly employed in House m ice mtDNA sequence com parison

s u rv e y s ) . 0-f th e 75 s i t e s seen in tt. domesticus d e te c te d w ith A lu I , 58

a re c o n s ta n t ( ta b le 3 .4 , p o in t 12) and 17 a re v a r ia b le ( ta b le 3 .3M ). In

B r i t a in 8 d ig e s t io n p r o f i l e ty p e s have been d is c e rn e d (A -H ), p la te 3 .1 0

i l l u s t r a t e s 3 v a r ia n t p a t te rn s B, G, and H, in a d d it io n t o th e k .b . s .

D ig e s t io n p a t te rn B c h a r a c t e r is t i c a l l y has 3 s i t e lo s s e s a t 3143, 5129,

8253 ( s i t e 3 , 7, 12, t a b le 3.3M) and 3 g a in s a t p o s i t io n s 5124, 8259, 9947

( s i t e s 4 , 6 , 13, ta b le 3 .3 M ). T h is r e s u l t s in th e fo l lo w in g fragm en t

d i f fe r e n c e s : lo s s o f 680 and 45 bp w ith th e g a in o f 725 bp; lo s s o f 54 and

654 bp, re p la c e d by 661 and 47 bp; appearance o f 551 and 34 bp in th e p la c e

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o-f 545 and 40 bp; and th e - f in a l ly th e d isa pp e a ra n ce o-f 448 bp and th e

s im u lta n e o u s g a in o-f 359 and 89 bp. Both d ig e s t io n p r o f i l e s C and D have

th e p r e v io u s ly d e s c r ib e d s ix common s i t e d i f fe re n c e s p lu s th e fo l lo w in g

a d d i t io n a l changes. P a tte rn C has 5 e x t r a s i t e d i f fe r e n c e s : th e lo s s o f th e

tw o a d ja c e n t s i t e s in th e sequence a t p o s i t io n s 6233 and 6291 ( s i t e s 10 and

11, ta b le 3.3M) r e s u l t s in th e lo s s o f 450, 58 and 44 bp, and th e

appearance o f one la r g e r fra gm en t th e sum o f th e s e , 552 bp; th e

s im u lta n e o u s lo s s and g a in o f s i t e s 1641 and 1633 ( s i t e s 1 and 2 , ta b le

3.3M) causes th e lo s s o f fra g m e n ts 52 and 530 bp, t o be re p la c e d by th o se

o f 44 and 538 bp; f i n a l l y th e g a in o f a s i t e a t 15515 ( s i t e 17, ta b le 3.3M)

w ith th e lo s s o f fra gm en t 269 bp and th e co n co m ita n t g a in o f 2 fra g m e n ts 25

and 244 bp. The l a t t e r s i t e g a in i s th e o n ly d i s t in c i t v e m u ta t io n a l change

p r o f i l e D has which s e p a ra te s i t fro m B. P a tte rn s E-H each have 2 s e p a ra te

u n iq u e s i t e d i f fe r e n c e s d e f in in g t h e i r d ig e s t io n p r o f i l e s , in th e cases o f

E, F, and G, th e se a re s im u lta n e o u s lo s s and g a in o f s i t e s , w h i ls t p a t te rn

H in v o lv e s lo s s o f 2 a d ja c e n t s i t e s . The r e s u l t o f th e s e d if fe r e n c e s i s as

f o l lo w s : p a t te rn E - a s i t e lo s s a t 5783 and g a in a t 5795 ( s i t e s 8 and 9,

t a b le 3 .3 M ), causes th e lo s s o f fra g m e n ts 645 and 450 bp, in t h e i r p la c e

a re th e 2 fra g m e n ts 662 and 438 bp; p a t te rn F - a lo s s and g a in o f s i t e s a t

3188 and 3508 ( s i t e s 3 and 4 , ta b le 3 .3 m ), w ith th e appearance o f fra g m e n ts

28 and 365 bp and th e d isa pp e a ra n ce o f 352 and 45 bp; p a t te rn G - lo c a t io n s

o f s i t e lo s s a t 10826 and g a in a t 10835 ( s i t e s 15 and 16, ta b le 3 .3 M ),

e x p la in s th e observed fra g m e n ts 374 and 21 bp in s te a d o f 365 and 30 bp;

f i n a l l y p a t te rn H w ith th e lo s s o f s i t e s 6233 and 6291, p ro d u c in g th e 552

bp fra g m e n t, in l ie u o f 450, 58 and 44 bp fra g m e n ts seen in p r o f i l e C (see

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CHAPTER THREE

d e s c r ip t io n a bove ).

5 .3 .7 .5 .9 s Sau 96 I (GGCN3CC).

P a tte rn A, th e known base sequence has 25 c le a va g e s i t e s , w h i ls t in B r i t i s h

sam ples 26 s i t e s were documented, 21 c o n s ta n t ( ta b le 3 .4 , p o in t 1 4 ), and 5

v a r ia b le ( in c lu d in g 1 s i t e g a in and 4 s i t e lo s s e s , ta b le 3 .3 L ) . T h is

r e s t r i c t i o n endonuclease has n o t been used in o th e r s tu d ie s o f th e House

mouse, hence th e p r o f i l e s (3 ; A-C) o b ta in e d a re o n ly o f B r i t i s h sam ples.

Compared w ith p a t te rn B, p a t te rn A has o n ly 1 s i t e lo s s a t p o s i t io n 1573B

( s i t e 6 , ta b le 3 .3 L ) , w ith th e subsequent lo s s o f fra g m e n ts 5B3 and 1 bp,

re p la c e d by th e s l i g h t l y la rg e r fra g m e n t 584 bp. W h ils t p a t te rn C a ls o has

t h i s s i t e lo s s in a d d it io n to a s im u lta n e o u s s i t e lo s s and g a in a t

p o s i t io n s 78 and 83 ( s i t e s 1 and 2, t a b le 3 .3 L ) , ca u s in g th e lo s s o f

fra g m e n ts 573 and 61 bp p lu s th e g a in o f fra g m e n ts 578 and 56 bp. Fragm ents

s m a lle r tha n a p p ro x im a te ly 150 bp a re v e ry d i f f i c u l t to obse rve r o u t in e ly ,

so th o s e below t h i s s iz e a re e s tim a te d fro m a know ledge o f th e k .b .s o f

mouse mt DNA.

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^.DISCUSSION:3iiiii_DNA_yariation_acrg55_the_MitDchgndrial_genome£

Double d ig e s t io n c le a vag e mapping and th e rm a l s t a b i l i t y s tu d ie s f i r s t

i l l u s t r a t e d th a t m u ta t io n a l changes were d is t r ib u t e d a l l o ve r anim al

m ito c h o n d r ia l DNA genomes. These changes were found to o ccu r a t d i f f e r e n t

fre q u e n c ie s in d i f f e r e n t re g io n s o f th e g®nome (Daw id, 1972; U p h o lt &

Dawid, 1977; Brown et a l 1979). F ig u re s 3 .4A and B view ed to g e th e r re v e a l

th e d is t r ib u t io n o f s i t e s a cross th e Hus domesticus mt genome, mapped

u s in g th e sequence com parison method, t e s t i f y i n g t o th e f a c t th a t base

s u b s t i t u t io n s a re s c a tte re d th ro u g h o u t th e mouse mt genome. Data fro m t h i s

c h a p te r i l l u s t r a t e s th a t c e r ta in gene re g io n s and n u c le o t id e p o s i t io n s seem

to be more la b i l e tha n o th e rs , p resum ab ly due to a r e la x a t io n o f fu n c t io n a l

c o n s t r a in ts . These r e s u l t s agree w ith p a t te rn s o f base s u b s t i t u t io n found

in p re v io u s s tu d ie s in v o lv in g com parisons o f c lo s e ly re la te d ta x a by d i r e c t

sequence a n a lyse s o r by in d i r e c t h ig h r e s o lu t io n sequence com parison

r e s t r i c t i o n mapping (Cann & W ils o n , 1983; Cann et a l 1984; F e r r is et al

1983), w h ich c o n f irm and extend th e e a r l i e r i n i t i a l f in d in g s , in c re a s in g

th e r e s o lu t io n o f th e m o le c u la r b a s is o f mammalian mt e v o lu t io n .

These s tu d ie s have p roved e s p e c ia l ly in fo r m a t iv e , as th e sequences compared

a re s u f f i c e n t l y c lo s e ly re la te d as to be r e la t i v e l y f r e e fro m m u lt ip le

e ve n ts o c c u r r in g a t th e same n u c le o t id e ; w h ich can obscu re p a t te rn s o f base

s u b s t i t u t io n . Thus c lo s e ly re la te d mtDNA’ s can be exam ined f o r

s u s c e p t ib i l t y o f v a r io u s p a r ts o f th e genome to base s u b s t i t u t io n w ith

c o n s id e ra b ly more c o n fid e n c e than in t e r s p e c i f i c com parisons . C om parisons o f

th e co m p le te sequences o f m ito c h o n d r ia l genomes fro m cow, mouse, and man

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(B ib b et al., 1981; Anderson et al., 1982) have re v e a le d t h i s m u lt ip le h i t

p rob lem . Brown et al., (1982) and M iya ta e t al., (1982) c a lc u la te d th e

average s i l e n t p o s i t io n has e xpe rie n ced th re e s u b s t i t u t io n s s in c e t h e i r

d iv e rg e n c e . T h is in t r a - s p e c i f i c s tu d y in Hus domesticus shows an average

o f le s s than 0 .003 pe r s i le n t base p a ir (< 0 .03 - Cann et al., 1984 - in

hum ans).

3 .4 .2 s Base sequence v a r ia t io n in anim al mtDNA.

What fo l lo w s i s a d e s c r ip t io n o f base s u b s t i t u t io n a l changes w ith in each

m a jo r fu n c t io n a l re g io n in th e mouse m ito c h o n d r ia l DNA, compared and

c o n tra s te d w ith what i s a lre a d y known fro m p r e l im in a r y s tu d ie s in t h i s

s p e c ie s and o th e r w e ll documented e v id en ce fro m a range o f s p e c ie s , u s in g

d i f f e r e n t approaches and te c h n iq u e s .

3 i4 i 2i ! i_ P r g te in - c g d in g _ r e g ig n s l

E xcept f o r th e e le v a te d ra te o f base s u b s t i t u t io n , th e g e n e ra l fe a tu re s o f

p r o te in gene e v o lu t io n appear to be s im i la r between m ito c h o n d r ia l and

n u c le a r system s (Brown, 1983, 1985; M o r itz e t al., 1987; A t t a r d i , 1985;

C a n ta to re & Saccone, 1987). Data from my s tu d y a re c o n s is te n t w ith th e

documented t re n d tow a rds th e p reponderance o f s i l e n t (synonymous)

s u b s t i t u t io n s o c c u r r in g se ve ra l t im e s more f r e q u e n t ly th a n non-synonymous

changes (am ino a c id re p la c e m e n ts ), th e l a t t e r a c c u m u la tin g a t ra te s

com parab le w ith th a t found in n u c le a r encoded genes ( r a t s - Brown &

Simpson, 1982; m ice - F e r r is et al., 1983; humans - Cann et al., 1984;

p r im a te s - Brown et al., 1982). The o rd e r o f s u b s t i t u t io n fre q u e n cy a t

d i f f e r e n t codon p o s i t io n s w ith in th e p r o te in cod ing genes i s t h i r d , f i r s t ,

the n second ( t h i r d : f i r s t : second r a t i o = 3 : 1 .3 : 1 ) . When th e p ro te in

CHAPTER THREE

co d ing genes a re s u b d iv id e d in t o c h a ra c te r is e d p ro te in s ( C O I - I I I , Cyt B,

ATPase 6 and 8) and NADH dehydrogenase s u b u n its (ND 1 -6 , 4LJ fo rm e r ly th e

u n id e n t i f ie d re a d in g fram es URF1-6, 4L - Chomyn e t al., 1985 ), t h i s r a t io

i s more pronounced in th e fo rm e r (1 4 :5 :1 as opposed to 2 . 1 : is 1 .1 ) . The

o rd e r o f fre q u e n c y i s in accordance w ith th e know ledge th a t most t h i r d

p o s i t io n s u b s t i t u t io n s a re ' s i l e n t ’ , and so do n o t cause amino a c id

re p la ce m e n ts . Changes in v e r te b ra te m ito c h o n d r ia l g e n e tic code from th e

u n iv e rs a l code enhances th e p ro p o r t io n o f s i l e n t t h i r d p o s i t io n s in th e

m ito c h o n d r ia l compared to th e n u c le a r system (B ibb et al., 19B1; Anderson

e t al., 1982; A t t a r d i , 1985; Brown, 1985). Less r a d ic a l amino a c id

re p la ce m e n ts r e s u l t fro m th e f i r s t codon s u b s t i t u t io n s ca u s in g a low ered

p r o b a b i l i t y o f im p a ire d fu n c t io n in th e p ro te in gene th a n do second codon

base changes.

E very one o f th e 13 p r o te in s in mtDNA (Cytochrom e genes and NADH

dehydrogenase s u b u n its ) e x h ib i t s i t ' s own c h a r a c te r is t ic r a te o f base

s u b s t i t u t io n . These r a te s appear to be th e same amongst v e r te b ra te s w ith

th e e x c e p tio n o f CD I I in p r im a te s , w h ich has a c o n s id e ra b ly h ig h e r s i le n t

s u b s t i t u t io n r a te th a n i s found in e i t h e r b o v in e s o r ro d e n ts (Cann et al.,

1984; F e r r is e t al., 19B3; Brown, 1985; t h i s s tu d y - w ith th e e x c e p tio n o f

tR N A 's , CO I I gene i s th e le a s t v a r ia b le re g io n in th e mouse mt genome).

A d d i t io n a l ly th e re appea rs to be a h ig h e r r a te o f change in th e n u c le a r

encoded Cytochrome C p r o te in gene o f p r im a te s (C a rlso n e t al., 1977). T h is

gene i s known to in t e r a c t d i r e c t l y w ith th e C DII gene in th e m ito c h o n d ria

(T z a g o lo f f , 1982). The observed c o r r e la t io n o f r a te a c c e le ra t io n w ith in

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b o th cytochrom e C and CO I I genes may in d ic a te c o -e v o lu t io n (Cann et al.,

1984; W ilson et al., 1985).

In t r a - s p e c i- f ic com parisons o-f th e r e la t i v e v a r i a b i l i t y e s t im a te s amongst

p r o te in genes re v e a ls th a t ND 1 and Cyt B, and CO I I I a re th e le a s t

conserved w h i ls t CO I and I I , ATPases 6 and 8 a re th e most f u n c t io n a l l y

c o n s tra in e d in m ice ( t h is s tu d y ; F e r r is et al., 1983). T h is c o n t ra s ts w ith

in t e r - s p e c i f i c com parisons g e n e ra lly show ing C O I - I I I a re th e le a s t

v a r ia b le , w h i ls t ND and ATPase fu n c t io n s a re th e most v a r ia b le (Anderson et

al., 1982; Cann et al., 1984; ta b le 3 .11 - t h i s s tu d y ) .

I t has been observed th a t in th e s i l e n t s u b s t i t u t io n s th e re was a

predom inance o f t r a n s i t io n s w ith in th e p r o te in co d in g re g io n s (Brown &

S im pson, 1982; Brown et al., 1982; Pepe et al., 1983). Indeed , Brown &

Simpson (1982) no ted a s tro n g b ia s tow a rds C-T t r a n s i t i o n s on th e l i g h t

s tra n d when exam in ing CO I I gene sequences between tw o c lo s e ly r e la te d r a t

s p e c ie s . T h is c o n t ra s ts w ith A-G t r a n s i t i o n s g r e a t ly o u tnum be ring C-T in

Hus domesticus in t h i s s tu d y (F ig u re 3 .9 ; Lanave et al., 1984, 1985).

3i4i2i2i_Ri.bg5gmai_genesiTherm al s t a b i l i t y measurements and c le a va g e map a n a ly s is i l l u s t r a t e s th a t

th e r a te o f e v o lu t io n o f th e rRNA genes i s s lo w e r tha n in th e p ro te in

co d in g genes, bu t i s a p p ro x im a te ly 10 t im e s fa s te r in mt th a n t h e i r n u c le a r

c o u n te rp a r ts (p r im a te s - F e r r is et al., 1981; m ice - F e r r is et al., 1983;

hom in iodea - Brown et al., 1979, 1982; u n g u la te s - Dawid, 1972). T h is s tu d y

i s c o n s is te n t w ith th e above, th e 16s rRNA gene i s more conse rved than th e

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CHAPTER THREE

12s rRNA gene, y e t b o th a re c o n s id e ra b ly le s s v a r ia b le tha n th e p r o te in -

co d in g genes ( ta b le 3 .1 0 and 3 .1 1 ) ; f u r t h e r th e base s u b s t i t u t io n s in b o th

r ib o s o m a l re g io n s re v e a l a b ia s tow a rds t r a n s v e rs io n s . H igh r e s o lu t io n

r e s t r i c t i o n mapping in humans (Cann et al., 1984) s u p p o rts t h i s , show ing

th e rRNA genes to be th e le a s t v a r ia b le re g io n s in th e m ito c h o n d r ia l

genome, e v o lv in g p r im a r i ly by t r a n s v e rs io n s . F o r t et al., (1984) a ls o

r e p o r ts a low t r a n s i t i o n to t ra n s v e rs io n r a t i o ( l . l : 1) u s in g sequence

a n a y ls is o f th e 16s RNA genes from v a r io u s house mouse s p e c ie s . They

p roposed th a t th e 16s rRNA 5 ’ te rm in u s i s a * h o ts p o t ’ , b e in g much more

v a r ia b le than th e r e s t o f th e gene, and suggested th e re may be some

b io lo g ic a l c o n s t r a in t m a in ta in in g th e h ig h A-T7. c o n te n t observed a t t h i s

end o f th e gene, th u s lo w e r in g th e fre q u e n c y o f t r a n s i t i o n a l e v e n ts . No

such com parab le h y p e rv a r ia b le re g io n was documented in t h i s s tu d y in Hus

domesticus* 16s rRNA gene (nD base changes in th e f i r s t 300 n u c le o t id e s ) ,

how ever, to o few s i t e s were reco rded f o r t h i s f in d in g to be m e a n in g fu l.

Y e t, F o r t et al., (1984) concluded from DNA sequence a n a ly s is , t h a t mtDNA

co d in g f o r th e f i r s t 120 n u c le o t id e s o f th e 5* end o f th e 16s rRNA gene has

v e ry d i f f e r e n t ra te s D f e v o lu t io n in d i f f e r e n t Hus l in e a g e s ; c e r t a i n t l y on

c lo s e r in s p e c t io n o f t h e i r p u b lis h e d d a ta o f Hus domesticus s p e c ie s , o n ly

1 base change was n o te d . They em phasised t h a t such c o n t ra s t in g base

s u b s t i t u t io n ra te s w ith in sm a ll re g io n s o f th e mt genome c o u ld n o t be

d e te c te d when in v e s t ig a t in g th e whole m o le cu le u s in g r e s t r i c t i o n a n a ly s is .

H ixson Sc Brown (1986) examined 12s rRNA genes by d i r e c t seqenc ing in g re a t

apes and humans, r e p o r t in g th e same g e n e ra l t re n d as F o r t and c o lle a g u e s

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CHAPTER THREE

(1984) in 16s rRNA gene re g io n . S i te d ffe re n c e s were spaced th ro u g h o u t th e

gene, bu t th e 5 ’ end was le s s co n se rve d , b e in g a p p ro x im a te ly 1 .6 x more

v a r ia b le th a n th e 3 ' end. In com p le te c o n t ra s t t o th e p re v io u s ly d e s c r ib e d

s tu d ie s , t h e i r in t e r s p e c i f i c com parisons ove r a l l d iv e rg e n c e t im e s showed a

p reponderance o-f t r a n s i t i o n s . T h is f in d in g does n o t s u p p o rt Brown and

c o l le a g u e s ' (1982) c o n c lu s io n th a t th e m agn itude o f t r a n s i t i o n a l b ia s i s

in v e r s e ly re la te d t o d iv e rg e n ce t im e in th e hom in iodea . Extrem e b ia s does

n o t , how ever, e x is t in in t e r s p e c i f i c com parisons o f more d iv e rg e n t ta x a o f

cow, mouse and man (Anderson e t 1982; B ibb e t 1981; Brown, 19B5).

W ith in Mus domesticus ( t h is s tu d y ) s i t e s a re clumped near th e 5'’ end

c o n f irm in g H ixson & Browns (1986) r e s u l t , how ever, th e re were to o few s i t e s

to be c o n f id e n t o f such a t re n d . Hence, i t appears th e re a re pa tches o f

h ig h homology in rRNA genes, in te rs p e rs e d w ith le s s conserved s t re tc h e s

p re d o m in a n tly due to p o in t m u ta t io n s . A lth o u g h sm a ll d e le t io n s (1 -2 bp)

have been d e te c te d (H ixon & Brown, 1986) in p r im a te s and man, a s s o c ia te d

w ith p o ly C t r a c t s , th e s e were n o t observed in th e house mouse.

Lower t r a n s la t io n a l c o n s t r a in ts c h a r a c t e r is t ic o f th e m ito c h o n d r ia have

been in vo ked to e x p la in th e o v e r a l l h ig h e r r a te o f e v o lu t io n and f i x a t i o n

o f m u ta tio n s in mtDNA th a n in s in g le copy n u c le a r DNA (scnDNA) (Brown,

1981, 1983, 1985). V e r te b ra te mtDNA 16s rRNA genes a re most conserved a t

th e 3 ' end and have h ig h homology to E.Coli and y e a s t mtDNA; th e n u c le o t id e

changes a re c o n s is te n t th o se w ith c o n fe r r in g re s is te n c e to c h lo ra m p h e n ic o l.

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CHAPTER THREE

li4 jL 2 i3 i_ T ra n s £ e r_ R N A i

The gene ra l fe a tu re s o f e v o lu t io n and sequence v a r ia t io n in v e r te b ra te mt

tRNA a re v i r t u a l l y th e same as in th e r ib o so m a l RNA genes.

3i5i2i.4i_The_di5Blacement_iggBiThe D -lo o p , (so c a l le d because th e nascen t H -s tra n d d is p la c e s th e p a re n ta l

H -s tra n d , ca u s in g a th re e -s tra n d e d "d is p la c e m e n t lo o p " ) th e m a jo r non­

cod ing re g io n o f th e mtDNA, spans th e re g io n from tRNA PH= t o tRNA p" ° ,

and i s v o id o f any co d in g in fo rm a t io n f o r s t r u c t u r a l genes y e t c o n ta in s a l l

th e re g u la to r y e lem en ts f o r mtDNA r e p l ic a t io n , as w e ll as p ro m o te rs f o r

t r a n s c r ip t io n o f th e heavy and l i g h t s tra n d s (C la y to n , 1982, 1984;

C a n ta to re & Saccone, 1987). Sequence s tu d ie s in d ic a te i t i s th e most

v a r ia b le re g io n o f mt genome in le n g th and base c o m p o s it io n (G reenberg et

al., 1983) and i t a ccum u la tes base changes a t r a te s c o n s id e ra b ly fa s te r

than scnDNA (Lanave et al., 1984, 1985). Sequence com parison mapping

s tu d ie s o f Hus domesticus ( t h is s tu d y ; F e r r is et al., 1983) suggest th a t

a lth o u g h th e D -loo p i s v a r ia b le i t i s n o t th e most r a p id ly chang ing re g io n

in th e mt genome. T h is i s in agreement w ith th e re c e n t f in d in g s which

suggest th a t w h i ls t in t e r s p e c i f i c com parisons show th e D -lo o p to be h ig h ly

d iv e rg e n t , in t r a s p e c i f i c com parisons re v e a l t h i s re g io n e v o lv e s a t

a p p ro x im a te ly th e same average r a te as p ro te in co d in g genes (Brown et al.,

1986; Saccone et al., 1985). T h is low ered v a r i a b i l i t y o f th e D -loo p between

c o n s p e c if ic s su g g e s ts t h i s re g io n c o n ta in s s p e c ie s - s p e c if ic fe a tu re s (Brown

et al., 1986; G reenberg et al., 1983; M o r itz et al., 1987).

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CHAPTER THREE

R ecom bina tion and r e p l ic a t io n s lip p a g e mechanisms, p ro b a b ly caused by th e

in t e r r u p t io n o f th e po lym erase enzymes a t th e secondary s t r u c tu r e , a re

proposed as mechanisms by w hich mammalian mtDNA r e g u la to r y re g io n s e v o lv e

(C a n ta to re & Saccone, 1987). U p h o lt & Dawid (1977) u s in g b o th r e s t r i c t i o n

c le a vag e mapping and h e te ro d u p le x a n a ly s is in sheep and g o a ts , were th e

• f i r s t to observe th a t bo th ends o f th e D -lo o p were h ig h ly d iv e rg e n t

r e la t i v e to th e r e s t , e s p e c ia l ly upstream o-f th e heavy s tra n d o r ig in o f

r e p l ic a t io n (0 H) . These ’’h o ts p o ts " f o r base s u b s t i t u t io n in th e c o n t ro l

re g io n re co rd ed in human mtDNA sequenc ing s tu d ie s (G reenberg et al,, 1983;

Aquadro & G reenberg, 1983), suggest th e r a te o f s u b s t i t u t io n i s n o t u n ifo rm

f o r a l l s i t e s . These a u th o rs s p e c u la te d th a t r e s t r i c t i o n mapping a n a ly s is

may n o t be s e n s i t iv e enough to re s o lv e re g io n s w ith d i f f e r i n g le v e ls o f

v a r i a b i l i t y as th e se tw o h y p e rv a r ia b le dom ains in th e D -lo o p had n o t been

p re v io u s ly d e te c te d u s in g r e s t r i c t i o n mapping in p r im a te s (Brown & Goodman,

1979; F e r r is et al,, 1981a, b) and ro d e n ts (Brown et al,, 1982; Lansman et

al,, 1983). T h is was p ro b a b ly a consequence o f th e non-random d is t r ib u t io n

D f c leavage s i t e s a lo ng th e m ito c h o n d r ia l m o lecu le (Adams & Rothman, 1982).

I t soon became e v id e n t th a t th e D -loo p can be d iv id e d in t o th re e d is t in c t

a re a s , a c e n t ra l conserved re g io n (CCR) w ith p r im a ry sequence homology f o r

up t o 80 m i l l io n ye a rs (human, mouse, r a t , and cow sequence co m parisons ;

B ibb et al,, 1981; Anderson et al,, 1982), a few conse rved sequence b lo c k s

a t 5* te rm in u s (CSB 1, 2 , 3 ) , and th e te rm in a t io n a s s o c ia te d sequences

(TAS) a t th e 3 ?end (W alberg & C la y to n , 1981; Doda et al,, 1981; a ls o see -

Brown e t al,, 1986, A t t a r d i , 1985, p lu s C a n ta to re & Saccone, 1987, f o r

re v ie w s and summary). Data from t h i s s tu d y u s in g sequence com parison

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CHAPTER THREE

r e s t r i c t i o n m apping, a re in g en e ra l agreem ent w ith t h i s ; i l l u s t r a t e d by a

c lu s te r in g o f base s u b s t i t u t io n s a t th e ends and a co n sp icu o u s la c k o f

s i t e s in th e c e n t ra l re g io n o f th e D -lo o p (see f ig u r e 3 .8 ) . D e s p ite

c o n ta in in g th e s ta b le c lo v e r le a f seconda ry s t r u c tu r e s o f th e CSB and TAS

( s t a r t and s to p s i t e s , r e s p e c t iv e ly , f o r D -lo o p s tra n d s y n th e s is ) , b o th

ends o f th e D -lo o p a re h ig h ly v a r ia b le in base sequence and le n g th , w ith

c h a r a c t e r is t i c a l l y low guan ine c o n te n t , in a l l s p e c ie s exam ined to d a te

(Saccone et al., 1985; Brown et al., 1986 ). In t h i s s tu d y , th re e v a r ia b le

s i t e s in th e mouse were mapped to th e secondary s t r u c tu r e s ( f i g 3.8s 1 and

2 -e n la rg e d d e t a i l o f th e D -loop secondary s t r u c tu r e s ) , b u t n o t t o th e

im p o r ta n t CSB1 and TAS re g io n s , w h ich a re ca p a b le o f fo rm in g " m ir r o r

sym m etry", and a re s p e c u la te d t o a c t as s p e c i f i c r e c o g n it io n s i t e s f o r

re g u la to r y p ro te in s (Saccone et al., 1987 ). In te r a c t io n between RNA

secondary s t r u c tu r e s a re known to m odu la te th e fo rm a tio n o f a p r im e r -

p re c u rs o r / te m p la te com plex, needed in th e p r im e r g e n e ra tio n by RNase H, in

th e p la sm id C oIE I system (M asukata & Tomizawa, 1984). T h is p ro ce ss may be

ana logaous t o th e system re g u la t io n o f th e D -loo p re g io n , th e proposed

model o f w h ich i s based upon a p o s s ib le in t e r a c t io n between th e c l o v e r le a f

s t r u c tu r e s a t th e 5 ? end o f th e new DNA s tra n d and an R N A /p ro te in f a c to r

(Brown et al., 1986; Saccone et al., 19B7; C a n ta to re & Saccone, 1987). In

a d d i t io n , i t has been found th a t th e h ig h ly conserved c e n t ra l domain i s

f r e e o f any complex secondary s t r u c tu r e s and c o n ta in s an open re a d in g fram e

(ORF) w ith an unknown fu n c t io n , in d ic a t iv e th a t t h i s re g io n may be under

s tro n g p r im a ry s t r u c tu r a l c o n s t r a in ts (Saccone et al., 1987).

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5iii3i_Rates_and_tyee5_gf_change_in_aQimal_mtDNAiThe m a jo r i t y o-f in t r a s p e c i- f ic mtDNA v a r ia t io n in h ig h e r a n im a ls have been

a t t r ib u t e d t o n u c le o t id e changes w ith in r e s t r i c t i o n enzyme re c o g n it io n

s i t e s (p o in t m u ta t io n s ) , o r to v e ry sm a ll (a few base p a ir s ) in s e r t io n -

d e le t io n le n g th m u ta tio n s ( f o r re v ie w s , see Brown, 1985; A t t a r d i , 1985;

C a n ta to re & Saccone, 1987). S m a ll-s c a le le n g th m u ta tio n s have been observed

in humans (Cann & W ils o n , 1983), f r u i t f l i e s (S o lig n a c et al- , 1983), r a t s

(Brown & D e s ro s ie rs , 1983), c a t t le (H a u s w irth et a l - , 1984), f ro g s

(M onnerot et a l - , 1984), c r ic k e ts (H a rr is o n e t a l - , 1985 ), and l iz a r d s

(Densmore et a l - , 1985). No le n g th m u ta tio n s were d e te c te d in th e B r i t i s h

m ice exam ined, a l l m u ta t io n a l changes re p o r te d he re a re p r im a r i ly s in g le

base s u b s t i t u t io n s . However, th e re may be m ino r le n g th v a r ia n ts among th e

B r i t i s h and European house mice sam ples, w h ich were u n d e te c ta b le w ith th e

m e th o d o lo g ie s ( r e s t r i c t i o n mapping) used. To d e te c t th e s e , a more tho ro u gh

e x a m in a tio n o f e le c t r o p h o r e t ic m o b i l i t ie s would be re q u ir e d , u s in g

d e n a tu r in g ge l c o n d it io n s (Cann, 1982; Cann & W ils o n , 1983). However,

F e r r is et a l - , (1983) re co rd ed one v e ry c le a r case o f a le n g th v a r ia n t

(s iz e d 12 bp) in th e tobacco mouse, from P osch ia vo , S w itz e r la n d ; t h i s

m o d e ra te ly s iz e d le n g th v a r ia n t s im u lta n e o u s ly a l t e r s th e d ig e s t io n

p r o f i l e s f o r a l l r e s t r i c t i o n enzymes encom passing t h i s re g io n , so i s f a r

e a s ie r to d e te c t . A ls o , F o r t & c o lle a g u e s (1 9 8 4 ), u s in g DNA sequence

a n a ly s e s , documented a few m inor le n g th m u ta tio n s w ith in Mus l in e a g e s .

M o b i l i t y v a r ia n ts in mtDNA can r e s u l t from bo th le n g th (W ris c h n ik et al. ,

1987), and c o n fo rm a tio n a l m u ta tio n s (S ingh et a l - , 1987). M o b i l i t y fragm en t

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v a r ia n ts have been documented in human (Cann St W ils o n , 1983 ), sea u rc h in

(V aw ter & Brown, 1986 ), and mouse (H o w e ll, 1985; H ow e ll e t al., 1987)

mtDNAs. S im i la r ly , anom a lous ly m ig ra t in g DNA fra g m e n ts have been w id e ly

re p o r te d in ye a s t (Snyder e t al., 1986), try p a n o s o m a tid m in i - c i r c le DNAs

( G r i f f i t h e t al., 1986 ), and S a lm o n e lla (B oss i e t al., 1984 ), in w hich

"b e n t" DNA a l t e r s th e m ig ra t io n o f r e s t r i c t i o n fra g m e n ts in p o ly a c ry la m id e

g e ls b u t n o t in agarose g e ls (S te llw a g e n , 1983). C o n fo rm a tio n a l m u ta t io n s

can e a s i ly be m is - id e n t i f ie d as in s e r t io n - d e le t io n m u ta t io n s , o r coun ted as

m u l t ip le p o ly m o rp h ic r e s t r i c t i o n s i t e s , bo th o f w h ich can le a d t o e r r o r s in

c o n s t ru c t in g r e s t r i c t i o n maps, co n se q u e n tly b ia s in g sequence d iv e rg e n c e

e s t im a te s , w h ich may d i s t o r t mtDNA p h y lo g e n ie s (Cann et al., 1987). Thus,

g re a t c a re needs t o be ta ke n when a s s ig n in g mammalian mtDNA r e s t r i c t i o n

fra g m e n ts on th e b a s is o f s in g le r e s t r i c t i o n endonuc lease d ig e s ts (H o w e ll,

1985) in f in e - s c a le r e s t r i c t i o n mapping s tu d ie s .

M ito c h o n d r ia l DNA s iz e m a c ro v a r ia t io n ( la rg e s c a le le n g th v a r ia n ts ) and

h e te ro p la s m y , ( th e p resence o f more th a n one m ito c h o n d r ia l DNA ty p e w ith in

an in d iv id u a l ) , seems t o be p re v a le n t among th e lo w e r v e r te b ra te s and

in v e r te b r a te s ( f o r a summary, see Bermingham et al., 1985, 1986 ), b u t i s

r e l a t i v e l y ra re among mammals ( c a t t le - H a u sw irth & L a ip is , 1982 and

H a u s w irth e t al., 1984; humans - G reeberg et al., 1983; r a t s - Brown &

D e s ro s ie rs , 1983). B o u rso t et al., (1987) observed h e te ro p la s m y in two Hus

musculus in d iv id u a ls ; each c o n ta in e d a m ix tu re o f norm al mtDNA and a

m ito c h o n d r ia l m utant w ith a v e ry la rg e d e le t io n in a co d in g re g io n .

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However, no in c id e n c e s o-f h e te ro p la sm y were d e te c te d in Hus domesticus

sam ples from B r i t i s h l o c a l i t i e s ( t h is s tu d y ) Dr from w o r ld -w id e c o l le c t io n s

( F e r r is et al., 1983). Any la rg e sequence h e te ro g e n e ite s w ith in a sam ple

would have been e v id e n t , by a d d it io n a l fra g m e n ts e xceed ing th e t o t a l

expected s iz e o f th e m ito c h o n d r ia l genome (16 ,295 bp in th e house mouse;

B ibb et al., 1981). However, many cases o f h e te ro p la sm y may rem ain

unobserved , e i t h e r because th e y a re m is ta k e n ly a t t r ib u t e d t o p a r t i a l enzyme

d ig e s t io n s o r re p re s e n t such a t i n y p ro p o r t io n o f th e number o f mt

m o le cu le s per sample as to be u n d e te c ta b le , even by th e most s e n s i t iv e

v is u a l is a to n te c h n iq u e s (A v ise 8c Lansman, 1983).

The o rd e r o f gene v a r i a b i l i t y ( le a s t t o most) in th e B r i t i s h house mouse i s

rRNA, tRNA, Cytochrom e p r o te in s , d is p la c e m e n t loop and NADH dehydrogenase

s u b u n its (T a b le 3 .1 1 ) . T h is i s c o n s is te n t w ith com parab le r e s t r i c t i o n

mapping a n a lyse s o f w o r ld -w id e c o l le c t io n s o f Hus domesticus ( F e r r is et

al., 1983) and sequence d a ta in th e mouse (B ibb et al., 1981). However,

Cann, Brown, 8c W ilso n , (1984) found t h a t th e D -loop was a p p ro x im a te ly 1-2

t im e s more v a r ia b le th a n bo th g roups o f p ro te in -c o d in g genes, which were

s l i g h t l y more v a r ia b le tha n th e tRNA and rRNA genes (T a b le 3 .1 2 ) .

The most s t r i k in g and w e ll documented p r o p e r t ie s o f v e r te b ra te mtDNA

in c lu d e s a ra p id r a te o f e v o lu t io n , 5 -1 0 x g re a te r tha n scnDNA (Brown et

al., 1979), and th e t r a n s i t i o n a l b ia s (Brown 8c Simpson, 1982; Brown e t al.,

1982; Greenberg et al., 1983; H ig u ch i et al., 1984, 1987).

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The e x te n t o-f p e rc e n ta g e sequence d iv e rg e n c e in v e r te b r a te mtDNA appears to

be dependent on d iv e rg e n c e t im e (Brown et al, 1979? Brown, 1983). When

c lo s e ly r e la te d ta x a a re compared th e base s u b s t i t u t io n s a re p re d o m in a n tly

t r a n s i t io n s , as seen in p r im a te s (Brown e t al., 1982; Hayaska e t al., 1988),

r a t s (Brown Sc S im pson, 1982; C as to ra e t al., 1980; Goddard e t al., 1981),

humans (Aquadro Sc G reenberg , 1983; G reenberg e t al., 1 983 ), and f r u i t - f l i e s

(W olstenholm e Sc C la ry , 1985; S a t t i e t al., 1988; D e s a lle e t al., 1987b). The

i n i t i a l h ig h r a te o f mtDNA e v o lu t io n in c lo s e ly r e la te d ta x a has been

p o s tu la te d t o be a t t r ib u t a b le t o ra p id a c c u m u la tio n o f synonymous

s u b s t i t u t io n s , fo l lo w e d by a s lo w e r a c c u m u la tio n o f non-synonym ous

s u b s t i t u t io n s (a m in o -a c id re p la c e m e n ts ), w h ich c h a r a c te r is e in c r e a s in g ly

d is t a n t ly r e la te d ta x a (Brown e t al., 1979, 1982; D e s a lle e t al., 1987b).

F u rth e rm o re , many sequenc ing s tu d ie s sugges t t h a t th e a c tu a l r a t i o o f

t r a n s i t io n s to t ra n s v e rs io n s changes as a fu n c t io n o f d iv e rg e n c e t im e

(H ig u ch i e t al., 1987; Hayaska e t al., 1988; S a t t i e t al., 1988 ). T h is i s

p ro b a b ly due t o th e tim e -d e p e n d e n t a cc u m u la tio n o f m u l t ip le s u b s t i t u t io n s

a t many o f th e n u c le o t id e p o s i t io n s and to a h ig h e r r a t e o f t r a n s i t i o n -

ca us ing m u ta tio n s , fro m two to tw e n ty - fo u r t im e s t h a t o f t ra n s v e rs io n s

depending on th e s p e c ie s , ra th e r than a h ig h e r p r o b a b i l i t y o f t h e i r

f i x a t i o n (Brown, 1981, 1983). T ra n s v e rs io n s a ccum u la te a t a p p ro x im a te ly th e

same r a te in mt and scnDNA, and th e t r a n s i t i o n t o t r a n s v e rs io n r a t i o f a l l s

w ith in c re a s in g d iv e rg e n c e . T h is drop i s expected f o r th e p ro ce ss i s b ia se d

tow a rds t r a n s i t i o n s , b u t t ra n s v e rs io n s , a lth o u g h o c c u r r in g r a r e ly , tend t o

e rase th e re c o rd o f t r a n s i t i o n s (Brown et al., 1982; D e s a lle et al., 1987b).

Thus, in te r a c t io n s between t r a n s i t io n s and t r a n v e rs io n s a re asym m etric

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(H o lm q u is t, 1976, 1983; Jukes, 1982). The h ig h r a te o f base s u b s t i t u t io n i s

p o s tu la te d t o be due to th e la c k o f m ism atch r e p a ir in m ito c h o n d r ia (W ilson

et al., 1985), th e m a jo r i t y o f m u ta tio n s a s c r ib a b le t o ta u to m e r ic s h i f t s

w h ich sh ou ld have been re p a ire d b e fo re r e p l ic a t io n as in n u c le a r DNA. As

t r a n s i t i o n s a re th e rm o d y n a m ic a lly th e most l i k e l y ty p e s o f base

s u b s t i t u t io n to occu r d u r in g r e p l ic a t io n , i t appears th a t th e r e p a ir system

i s des igned t o e xc lu d e th e se (Topal 8c F re sco , 1976; W ilson e t aim, 1985).

T r a n s i t io n a l b ia s i s n o t e v id e n t from sequence com parison r e s t r i c t i o n

mapping in e i t h e r m ice ( t h is s tu d y ; F e r r is e t al., 1983 ), o r humans (Cann,

et al., 1984; Cann, 1986; H o ria e t al., 1984), wheras d i r e c t sequencing

s tu d ie s o f hom ino idea (Brown e t al., 1982; Aquadro & G reenberg , 1983;

G reenberg e t al., 1983 ), o r r a ts (Brown Sc Sim pson, 1982; Brown e t al., 1986,

Saccone e t al., 1985; C a n ta to re Sc Saccone, 1987) do show such a b ia s . The

d if fe r e n c e s observed may be a t t r ib u t a b le t o th e r e s o lu t io n o f th e d i f f e r e n t

te c h n iq u e s ; th a t i s th e t r a n s i t io n a l b ia s may be re g io n s p e c i f i c , in w hich

case sequencing th e D -lo o p and p ro te in -c o d in g genes may e le v a te th e b ia s ,

whereas r e s t r i c t i o n mapping o f th e whole mt genome may g iv e a more

r e a l i s t i c f ig u r e . However, i t may n o t be p o s s ib le t o make in fe re n c e s about

e v o lu t io n a ry ra te s u s in g th e sequence com parison r e s t r i c t i o n mapping

methods due to th e non-random p lacem ent o f r e s t r i c t i o n endonuclease

c le a va g e s i t e s in th e m ito c h o n d r ia l genome (Adams 8c Rothman, 1982). The

fre q u e n c y o f t r a n s i t i o n s and t ra n s v e rs io n s a re th o u g h t t o be a fu n c t io n o f

th e base c o m p o s it io n o f th e n u c le o t id e sequence (Aquadro 8c G reenberg,

1983), T h is i s i l l u s t r a t e d by s e v e ra l s p e c ie s o f D ro s o p h ila w h ich have a

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h ig h A+T c o n te n t (807.), and a h ig h in c id e n c e o-f t ra n s v e rs io n s ( S a t t i , et

aim, 1988). However, th e base c o m p o s it io n o-f house mouse mtDNA sequence i s

n o t p re d o m in a n tly A+T r ic h (63 .2% ). Thus, th e q u e s tio n a r is e s , w hether

humans a n d /o r m ice co u ld d i- f - fe r e n t ia l ly r e p a ir th e s e m u ta tio n s o r i s th e re ,

p e rha p s , an e le v a te d r a te o-f e v o lu t io n in th e s e s p e c ie s .

W hether ro d e n ts ( e s p e c ia l ly m ice) e v o lv e a t c o n s is te n t ly f a s te r ra te s tha n

th o s e o f o th e r v e r te b ra te s rem a ins a c o n t r o v e r s ia l is s u e (W ilson et aim,

1987). E v idence fro m scnDNA sequences, DNA-DNA h y b r id is a t io n and

p a la e o n to g ic a l so u rces suggests th a t n o t o n ly a re th e re h ig h e r a b s o lu te

r a te s o f n u c le a r DNA s u b s t i t u t io n in ro d e n ts , sea u rc h in s , and D ro s o p h ila

compared t o p r im a te s and a r t id a c t y ls , b u t a ls o t h a t th e s e ra te s have

p ro g r e s s iv e ly slowed in th e hominoedea l in e a g e , and a re a p p ro x im a te ly equa l

in th e mouse and r a t (Wu 8c L i , 1985; B r i t t e n , 1986; Jaeger et aim, 1986; L i

8c T an im ura , 1987; L i et aim, 1987; L i 8c Wu, 1987; C a t z e f l i s et aim, 1987).

F u r th e r i t appears th a t n u c le a r and mtDNA ra te s a re s im i la r and th a t th e

t r a n s i t i o n a l b ia s does n o t h o ld f o r some s p e c ie s o f D ro s o p h ila (P ow e ll e t

aim, 1986; de B r u i jn , 1983; S o lig n a c et aim, 1986) o r s e a -u rc h in s (Vaw ter 8c

Brown, 1986 ). Vaw ter 8c Brown, (1986) conc luded t h a t th e a pp a re n t ra p id r a te

o f v e r te b ra te mtDNA e v o lu t io n is , in p a r t , an a r t i f a c t o f th e w id e ly

d iv e rg e n t r a te s o f change w ith in n u c le a r DNA. Some d o u b ts have been

expressed on th e v a l i d i t y o f th e c o n c lu s io n s drawn fro m th e se s tu d ie s due

to th e use o f in d i r e c t methods, u n c e r ta in d iv e rg e n c e t im e s , d e r iv e d from

p o o r ly re p re s e n te d f o s s i l re c o rd s , and sm a ll sam ple s iz e s (D e s a lle et aim,

1987b).

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F o s s il e v id e n ce su gg e s ts ro d e n t l in e a g e s e vo lve d r a p id ly , d iv e rg e n c e t im e

between r a t and mouse be ing o n ly 8-11 M yr. However, th e s e e s tim a te s rem ain

c o n t ro v e rs a l because o-f th e c o n f l i c t i n g e v idence fro m m o le c u la r

co m p a rison s . E ith e r th e f o s s i l re c o rd (based p r im a r i ly on cheek te e th ) has

been m is in te r p r e ta te d , o r , th e average r a te o f m o le c u la r e v o lu t io n in m ice

i s f a s te r th a n in o th e r mammals from w hich d iv e rg e n c e t im e s a re c a l ib r a te d

(W ils o n et al,, 1987). Many w o rke rs p re d ic te d DNA e v o lu t io n sh o u ld be

f a s t e r in a n im a ls w ith s h o r t g e n e ra tio n t im e s (L i et al,, 1987; L i &

Tam imura, 1987; Wu & L i , 1985; Kohne e t al,, 1972). B r i t t e n (1986)

sugges ted th a t th e ra p id d iv e rg e n c e ra te s o f n u c le a r genes a re n o t a

f u n c t io n o f g e n e ra tio n t im e as D ro s o p h ila and ro d e n ts b o th have s h o r t

tu rn o v e rs , b u t sea u rc h in s do n o t . However, th e number o f r e p l ic a t io n s per

y e a r i s la rg e f o r sea u rc h in s , c o n s e q u e n tly th e y have a la r g e r number o f

g e rm lin e r e p l ic a t io n s pe r year th a n do s p e c ie s w ith com parab le g e n e ra tio n

t im e s .

A rc h a e o lo g ic a l and g e n e tic da ta have shown th e spread o f e a r ly fa rm in g from

th e Near E ast th ro u g h th e M e d ite rra n e a n in t o n o r th e rn Europe (C la rk , 1975;

S o k a l, 1988; A u f f ra y et al,, 1988). The commensal a s s o c ia t io n o f m ice w ith

man was th o u g h t to have begun w ith th e adven t o f a g r ic u l t u r a l p r a c t ic e s ,

e s p e c ia l ly a t h ig h la t i t u d e s where m ice were dependent on human g ra in

s to r e s (S chw artz & S chw artz , 1943; Sage, 1981; G y lle n s te n & W ils o n , 1987).

F e r r is et al,, (1983) e s tim a te d th e r a te o f mtDNA d iv e rg e n c e in m ice to be

2 -4V. pe r M yr, c o n s is te n t w ith r a te s found in o th e r mammals (Brown et al,,

1979, 1982; F e r r is et al,, 1981). T h is e s t im a te i s su p p o rte d fro m f o s s i l

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e v id e n ce o-f s p r e t t i s - l i k e m ice in N o rth A f r ic a d a ted about 4 M yr. BPD

(M a rs h a ll, 1981) and from mouse f o s s i l s fro m a rc h a e lo g ic a l s i t e s about 1

M yr. BPD (B ro th w e ll , 1981; T chernov, 1983 ). To t e s t w hether ro d e n ts do

indeed have a e le v a te d r a te , i n t r a s p e c i f i c r a te s o f mtDNA d iv e rg e n c e c o u ld

be c a l ib r a te d f o r m ice , u s in g a p o p u la t io n known t o have c o lo n is e d a

s p e c i f ic g e o g ra p h ic re g io n a t a known t im e . Com parisons c o u ld th e n be made

between e s tim a te s o f d iv e rg e n c e w ith in th e g e o g ra p h ic a rea w ith e s tim a te s

fro m th e n e a re s t n e ig h b o u rs (S to n e k in g et a im , 1986; W ilson e t a im , 1987b).

Sweden i s one such p o te n t ia l exam ple, as i t was c o lo n is e d a p p ro x im a te ly

4000 ye a rs ago, v ia N o rth e rn Germany (W ils o n , P ra g e r, p e rs comm).

I f i t i s fou n d th a t mouse mtDNA does n o t e v o lv e r a p id ly , i t maybe p e r t in a n t

t o in v e s t ig a te th e e f f ic e n c y o f t h e i r DNA r e p a i r . U s ing th e same sequence

mapping te c h n iq u e , Cann and c o lle a g u e s (1984) found a lo w e r r a t i o o f

t r a n s i t i o n s t o t ra n s v e rs io n s in humans th a n e xp e c te d , b u t s t i l l s l i g h t l y

h ig h e r th a n th a t found in m ice. G e n e ra lly , i t has been suggested th a t th e

s lo w e v o lu t io n a ry r a te o f human mtDNA lin e a g e s may be a t t r ib u t a b le to an

th e e f fe c t iv e n e s s o f th e DNA r e p a ir mechanisms ( B r i t t e n , 1986).

3 i4 _ 4 £ _ R e 5 tr ic t ig n _ 5 ite _ m a B B in g _ c g m B le x it ie s _ a n d _ c g m B lic a t ig n s i

5 i5 _ ii i i_ 5 if f i ! l§ D t_ m tD N A _ y is u a lis a t ig n _ m e th g d s i

Where p o s s ib le l e t t e r d e s ig n a tio n s f o r d ig e s t io n p a t te r n s , in t h i s s tu d y ,

fo l lo w e d th o s e e s ta b lis h e d by F e r r is 8c c o lle a g u e s (1 9 8 3 ). However, a few

d ig e s t io n p a t te rn s fro m a co u p le o f enzymes were n o t d i r e c t l y r e la t a b le to

th o s e p re v io u s ly d e s c r ib e d . The in t e n t o f t h i s s tu d y has been to exam ine

th e p o p u la t io n v a r ia t io n o f mtDNA fro m as many B r i t i s h house m ice as

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p o s s ib le and n o t t o g iv e a d e ta i le d accoun t o-f th e g e n e ra l m o le c u la r

e v o lu t io n o f th e m o le c u le . Hence, o n ly one p o ly a c ry la m id e g e l c o n c e n tra t io n

was used (57.), t o s e p a ra te th e d ig e s te d DNA fra g m e n ts , r a th e r than a whole

range o f ge l c o n c e n tra t io n s . As a consequence, o n ly fra g m e n ts from th e s iz e

range 2000 to 150 b a s e p a irs co u ld be sco re d a c c u r a te ly and s u b se q u e n tly

c o n f id e n t ly s i t e mapped. Y e t, a lth o u g h n o t e v e ry fra g m e n t in th e d ig e s t io n

p r o f i le s c o u ld be d e te c te d , th e s i l v e r s ta in in g v is u a l is a t io n te c h n iq u e

(T e g e ls tro m , 19B6) does g iv e b e t te r r e s o lu t io n th a n th e e n d - la b e l l in g

method (Brown, 1980) adopted by F e r r is St c o lle a g u e s (1 9 8 3 ). They p o in te d

ou t th a t changes in Hae I I I p a t te rn s were d i f f i c u l t t o d e te c t , because th e

fragm en t lo s s e s and g a in s occur in re g io n s o f m u l t ip le superim posed D r

c lo s e ly spaced bands (hom ologous b ands), however th e s e c o u ld be re s o lv e d

u s in g s i l v e r s t a in in g . A d d i t io n a l ly , th e y examined in d iv id u a ls from Orkney

u s in g th e enzyme Mbo I , and d e s c r ib e d th e r e s u l t in g d ig e s t io n p a t te rn as

id e n t ic a l t o t h a t found o f th e k .b .s (p a t te rn A; B ibb et el,, 1981). U sing

s i l v e r s ta in in g on sam ples from th e same l o c a l i t i e s , d i s t i n c t fragm en t

d if fe re n c e s were no ted as in d ic a te d in P la te s 3 .8 & P la te 3 .1 la n e s 1 & 2,

r e s p e c t iv e ly (see ta b le 3 .3 J f o r l i s t o f v a r ia b le s ite s ? s e c t io n 3 .3 .7 .3 .6

Mbo I f o r d e s c r ip t io n s ) . A ltho u gh s im i la r t o th e k . b . s , d ig e s t io n p a t te rn s

from Orkney a re d e s ig n a te d ty p e 0 , in s te a d o f A (renamed as ty p e W by th e

w o rke rs from B e rk e le y , W ilso n , p e rs comm). However, d e s p ite d if fe re n c e s in

th e te c h n iq u e s , th e m a jo r i t y o f th e p a t te rn s d e s c r ib e d by F e r r is &

c o lle a g u e s (1983) were co ng ru e n t w ith th o s e observed in t h i s s tu d y .

137

CHAPTER THREE

3i4i4i2j._Case5_Df_mismaBEiD9i There was a p o s s ib i l t y , th a t some s i t e s may have been "m ismapped" because

o f th e d i f f i c u l t y in ch oo s in g between two o r more " s e m is i te s 11 y ie ld in g

fra g m e n ts o f a lm o s t id e n t ic a l s iz e s . T h is p rob lem was a c c e n tu a te d ,

e s p e c ia l ly w ith te t r a n u c le o t id e b a s e -c u t te rs , w ith in c re a s in g d iv e rg e n c e

between th e sam ple r e s t r i c t i o n p r o f i l e compared to th e k . b . s (B ib b et al.,

1981). However, W ilson & c o lle a g u e s (p e rs comm.,) re -e xa m in ed t h e i r

lo c a t io n o f p o s tu la te d s i t e s ( F e r r is et a l - , 1982, 1983 ), r e la t i v e to

a n o th e r p u b lis h e d sequence (Yonekawi & F is c h e r L in d a h l, 1987), and found

th e re was an e x c e l le n t agreem ent between th e sequences. They conc luded th a t

t h i s j u s t i f i e s u s in g th e r e s t r i c t i o n mapping approach to e s tim a te sequence

d iv e rg e n c e s . Any c o r r e c t io n s o f m ismapping le a d o n ly t o re f in e m e n ts ra th e r

tha n s ig n i f i c a n t r e v is io n s o f any c o n c lu s io n s drawn from th e s e com parisons.

3i 5i_Summary£

Between two to fo u r te e n r e s t r i c t i o n endonucleases were used t o screen fo r

th e presence o r absence o f c le a vag e s i t e s a t a p p ro x im a te ly 370 lo c a t io n s in

h ig h ly p u r i f ie d m ito c h o n d r ia l DNA o f 430 B r i t i s h house m ice , to g e th e r w ith

d a ta from 208 Hus domesticus from w o r ld -w id e c o l le c t io n s ( F e r r is et al.,

1983). By com paring th e DNA fra g m e n t s iz e s w ith th o se expec ted from th e

k .b .s o f mouse mtDNA (B ib b et al., 1981), u s in g th e sequence com parison

r e s t r i c t i o n mapping te c h n iq u e (Cann & W ils o n , 1983), c le a va g e maps were

c o n s tru c te d f o r a l l in d iv id u a ls . Of th e 370 s i t e s mapped, 140 were v a r ia b le

(p o ly m o rp h ic , b e in g p re s e n t in some in d iv id u a ls b u t absen t in o th e rs ) and

230 were c o n s ta n t ( ie . in v a r ia n t , found in a l l in d iv id u a ls exam ined). The

138

CHAPTER THREE

v a r ia n t s i t e s re s u lte d -from s in g le base p a i r s u b s t i t u t io n s , o c c u r r in g in

a l l - fu n c t io n a l re g io n s o f th e genome? no h e te ro p lasm y o r le n g th v a r ia n ts

were d e te c te d . In 75 cases, i t was p o s s ib le to map th e e x a c t n a tu re and

lo c a t io n o f th e m u ta tio n re s p o n s ib le f o r th e absence o f a r e s t r i c t i o n s i t e

in th e k .b .s and i t s p resence in a n o th e r mouse mtDNA. Df th e s e 75 g a in

m u ta tio n s , 58 o ccu rre d in genes c o d in g f o r p r o te in s , o f w h ich 30 were

s i le n t and 28 caused amino a c id re p la c e m e n ts . V a r i a b i l i t y o f d i f f e r e n t

fu n c t io n a l re g io n s in th e m ito c h o n d r ia l genome, from th e most conserved to

th e most v a r ia b le , in c lu d e s a l l 22 t r a n s fe r RNA genes, 12S & 16S rib o som a l

RNA genes, Cytochrom e genes (cy toch rom e o x id a s e s u b u n its I —111, cytochrom e

b , and ATPase s u b u n its 6 & 8 ) , th e d is p la c e m e n t lo o p , and l a s t l y th e NADH

dehydrogenase s u b u n it genes (1 -6 , & 4 L ) .

O nly s l i g h t l y more t r a n s i t i o n s th a n t ra n s v e rs io n s were found ( r a t io - 1.14s

1 ) , a r a t i o c o n s id e ra b ly lo w e r tha n th o s e re p o r te d fro m sequenc ing s tu d ie s

o f v a r io u s ta x a , b u t c o n s is te n t w ith com parab le s tu d ie s u s in g th e h ig h

r e s o lu t io n r e s t r i c t i o n mapping app roach . E ith e r mouse mtDNA i s e v o lv in g a

more r a p id ly r e la t i v e to o th e r ta x a o r th e re i s a d i f f e r e n t i a l DNA r e p a ir

mechanism. A l t e r n a t iv e ly , d iv e rg e n c e e s tim a te s d e r iv e d from sequence

s tu d ie s o f sm a ll gene re g io n s c o u ld be in f la t e d , o r th e non-random

p lacem ent D f r e s t r i c t i o n s i t e s a c ro s s th e genome c o u ld cause under

e s tim a te s u s in g th e r e s t r i c t i o n mapping method.

139

IABLE_3ili_MII0CH0NDRIAL_DNA_RESIRICII0N_FRAGMENI_DIGESII0N_PR0FILESi

The s iz e s ( in b a s e -p a irs ) o f -Fragments w h ich c h a r a c te r is e th e mtDNA d ig e s t io n

p r o - f i le s produced by -fo u rte e n r e s t r i c t i o n endonuc leases (Acc I , Ava I I , Fnud

I I , H inc I I , H ind I I I , H in f I , Hpa I I , Taq I , Hae I I I ,M b o I , A lu I , Rsa I , Sau

96 I , and Xba I ) i s shown. The s iz e s o-F fra g m e n ts obse rved in th e mtDNA

d ig e s t io n p r o - f i le s in e i t h e r agarose o r p o ly a c ry la m id e g e ls were e s tim a te d

fro m com parisons w ith th e known fra g m e n t s iz e s in th e p u b lis h e d re fe re n c e

sequence (B ibb e t a l , , 1981 ), p lu s a d d i t io n a l m o le c u la r w e ig h t m arke rs (1 KB

la d d e r , Lambda DNA c u t w ith H ind I I I and o r w ith Bgl I ) .

The mtDNA d ig e s t io n p a t te rn p r o f i l e s f o r each r e s t r i c t i o n endonuclease were

g iv e n a l e t t e r d e s ig n a t io n . Where p o s s ib le , th e s e p r o f i l e s were compared to

p u b lis h e d d a ta f o r Hus domesticus ( F e r r is e t a l , , 1983) and th e same l e t t e r

d e s ig n a t io n s adop ted . By c o n v e n tio n l e t t e r A i s re s e rv e d f o r th e p r o f i le s o f

th e known base seqence.

1 Fragment p r o f i l e s d e s c r ib e d by F e r r is e t a i . , (1983) b u t n o t p re v io u s ly

re c o rd e d f o r th e B r i t i s h House mouse p o p u la t io n s used in t h i s s tu d y .

2 New fragm en t p r o f i l e s n o t p r e v io u s ly d e s c r ib e d .

N .B . A l l p r o f i l e s w ith o u t a s u p e rs c r ip t deno te ty p e s n o t fo u n d in th e B r i t i s h

I s le s b u t a re d e s c r ib e d e lse w he re ( F e r r is et a l , , 1983).

Below each d ig e s t io n p r o f i l e th e number o f fra g m e n ts obse rved was re c o rd e d ,

and th e mean number o f fra g m e n ts seen when House mouse mtDNA was c le a ved w ith

t h a t p a r t i c u la r r e s t r i c t i o n endonuc lease in q u e s tio n was n o te d .

140

I£®ki_3iIl_MIIQCH0NDRIAL_DNA_RESIRICIION_FRAGMENT_DIGESIION_PROFIL.es.

ACC_I|

B C D7358 5048 6080* 6080*5048 3188* 5048 4258*2304 2892* 2304 2304

813 2304 1278* 1278*468 1278* 813 813259 813 468 790*

45 468 259 468259 45 259

45 45

7 9 8 9

MEAN=7.88

A V A _ II:

A B C D6510 4835* 8234* 10368*2134 2134 2134 20462046 2046 2046 16591724 1724 1659* 6091659 1675* 609 514609 1659 514 479514 609 479 346479 514 346 274346 479 274274 346

274

10 11 9 8

MEAN=10.22

E J K L 27358 7358 7358 6164*5048 4258* 2591* 50482563* 2304 2457* 2304

813 813 2304 1194*468 790* 813 813

45 468 468 468259 259 259

45 45 45

6 8 8 8

I J 2 K 2 L 2 M 25249* 6090* 6208* 6208* 6090*2985* 2134 2134 2134 21342134 2046 2046 2046 20462046 1724 1724 1724 1933*1659 1659 1659 1659 1724609 609 1123* 609 609514 514 479 514 514479 479 346 479 479346 346 302* 346 346274 302* 274 302* 302*

274 274 118*116*

10 12 10 11 11

141

E N y? L ii_ i lH A H i.

A x B6434 4623*1907 19071666 1811*1633 16661562 16331455 15621403 1455235 1403

235

8 9

MEAN= 7 .67

K 2 I6434 7996*3362* 19071666 16661633 16331562 14551403 1403235 235

7 7

d IN C _ IH

A 1 B C 68941 8022* 8356* 89413265 3265 3265 32652266 2266 2266 1695*1494 1494 1494 1494329 919* 585* 571*

329 329 329

5 6 6 6

MEAN= 5 .7 5

HIND_III1A 1 B 113462 15407*

1945 888888

3 2

MEAN= 2 .5

J 2 6434 3573* 1633 1562 1455 1403 235

7

142

L1993173413291242$10301946920879732$638539485480447444$417405$39936831821219567$67

9

25

143

c 1 D E F1993 1841$ 1993 19931734 1734 1734 17341329 1242$ 1026 13291026 946 920 1026946 920 915$ 946920 915$ 879 920879 879 746$ 879638 638 638 638539 539 539 539533 533 533 533497 497 497 497489 489 489 489485 485 485 485480 480 480 480447 447 447 447417 417 417 405$405$ 414$ 414$ 399399 405$ 405$ 387$368 399 399 368367 368 368 367318 367 367 318243 246$ 318 243216 243 243 216212 212 216 212195 195 212 195144 152$ 200$ 144

67 144 195 679 72 144 30$

67 67 99 9

28 30 30 29

H I J K1993 1734 1993 19931734 1632$ 1734 13291026 1326 1329 1067$946 1026 1030$ 1026920 946 1026 946915$ 920 946 920879 915$ 920 879638 638 879 638539 539 732$ 539533 533 638 533497 497 539 497489 489 485 489485 485 480 485480 480 447 480447 417 417 447417 414$ 405$ 417414$ 405$ 399 405$405$ 399 368 404$399 368 367 399368 367 318 368367 361$ 216 367318 318 212 318243 243 195 263216 216 144 243212 212 67 216195 195 9 212144 144 19567 67 144

9 9 679

29 29 26 30

H IN F_I_=CONTINUEDi

M N 0 T U 11734 1993 1734 1993 19931690* 1734 1632* 1734 17341632* 1242* 1459* 1026 13291026 946 1329 946 1026946 920 1026 920 946920 915* 946 905* 920879 879 879 879 879638 732* 638 638 638539 638 533 539 539533 539 497 533 497497 533 489 497 489489 497 485 489 485485 485 480 485 480480 480 447 480 447447 447 417 447 417417 417 399 424* 405*405* 414* 368 417 399399 405* 367 399 368368 399 361* 368 367367 368 318 367 348*318 367 263 318 318243 318 243 263 243216 212 216 243 216212 195 212 216 212195 144 195 212 195144 67 144 195 185*

67 9 94 144 1449 67 94 67

48 67 99 48

9

28 27 30 31 29

W Y Z 1 A’ (P) B ' (Q)1993 1993 1993 1993 17341418* 1734 1734 1734 1632*1026 1329 1459* 1329 1026946 1326* 1329 1284* 946920 1026 1026 1242* 920915* 946 946 1030* 915*879 920 879 946 879638 638 638 879 638539 539 533 732* 539533 533 497 638 533497 497 489 485 497489 489 485 480 489485 485 480 447 485480 480 447 417 480447 417 417 405* 447417 368 399 399 417414* 367 368 368 414*405* 327* 367 367 405*399 305* 318 318 399368 263 263 212 368318 243 243 195 367316* 216 216 175* 361*243 212 212 144 31B216 195 195 67 243212 144 144 9 216195 94* 94 212144 94 67 195

67 67 48 1449 48 9 67

9

29 29 29 25 30

V199317341242*946920915*879638539533497489485480447417414*405*39936836731824321219514467

9

28

1 4 4

HINF_I_CONIINUEDi i i i

C’ (R) S 2 X 2 3 21937* 1993 1993 19931734 1734 1734 17341026 1459* 1329 1459*946 1329 1026 1329920 1026 946 1026879 946 920 946746* 879 900* 379638 353* 638 638539 638 539 -J •-» o533 533 533 511*497 497 497 497489 489 489 489485 480 435 435480 447 480 480447 417 426* 447417 399 417 417414* 367 405 * 799405* 318 399 368399 263 368 318368 243 367 “T367 216 318 •'1?318 212 243 216243 195 216 212216 144 jl i*; 195*"> * O 1 94 195 94195 67 144 67174* •*8 481446756*

9

9 Q 9

31 23 28 28

MEAN= 28 .67

145

G 219931418*13291 026

946920379633539C77w -;049 7489485480447417405*399368367318316*24321621219514467

9

29

HAE I I I :

A 1 B 1 C D E2042 1951 2042 2042 19511951 1362 1362 1951 13621362 1229* 1230* 1362 1229*969 969 969 969 969945 945 945 945 945852 852 852 852 852641 813* 721* 641 813*590 641 641 590 641560 590 590 578* 590553 578* 560 560 560550 560 553 553 553526 553 550 550 550479 550 526 546* 526469 526 479 526 479456 479 469 469 469448 469 456 448 456438 448 448 438 448398 438 438 398 43B334 398 398 334 398266 334 334 266 334216 266 266 225* 266211 216 216 216 216136 211 211 211 211128 136 136 136 136122 128 128 128 128122 122 122 74 122103 103 122 69 122

74 74 103 62 10369 69 74 51 7467 67 69 39 6962 62 67 33 6751 51 62 33 6239 39 51 5133 33 39 3933 33 33 33

33 33

35 35 36 32 36

M N 0 P Q1951 1951 2389* 2042 20421362 1423* 1362 1362 1951969 1362 1229* 1230* 1362954* 969 969 969 969945 945 945 945 945852 852 852 852 852813* 641 813* 721* 641641 619* 641 641 590590 590 590 590 578*560 560 560 578* 560553 553 553 560 553550 550 550 553 550526 526 526 550 526479 479 479 526 479469 469 469 479 469456 456 448 469 448445* 448 398 448 438438 438 334 438 398398 398 266 398 334334 334 216 334 266275* 266 211 266 216266 216 136 216 211216 211 128 211 136211 136 122 136 128136 128 103 128 122128 122 74 122 103122 122 69 103 74122 103 67 74 69103 74 62 69 6774 69 51 67 6269 67 39 62 5167 62 33 51 3962 51 33 39 3351 39 33 3339 33 3333 3333

37 36 33 35 34

F204219511362969945852641590560553550526479469456448438398279266216211136128122122103

7469676255*51393333

36

1 4 6

HAE_IIls._CONIINyEpi i

R 2 S 2 T 2 U 2 V 2 W 2 X 22307* 2042 2042 2042 2042 2042 20422042 1951 1951 1951 1951 1951 19511951 1362 1574* 1362 1362 1362 1362969 969 1362 969 969 969 969852 945 969 945 945 945 945641 852 852 852 891* 852 852590 641 641 641 641 641 641577* 590 590 590 590 590 590560 553 553 583* 583* 560 577*553 550 550 560 560 553 560550 526 526 553 553 550 553479 479 479 526 526 526 550473* 469 469 479 479 479 479456 465* 456 469 469 469 473*448 456 448 456 456 456 456438 448 438 448 448 448 448398 438 398 438 438 438 438334 398 334 398 398 398 398266 334 266 334 334 316* 334216 266 216 266 266 266 266211 216 211 216 216 216 216136 211 136 211 211 211 211128 136 128 136 136 136 136122 128 122 128 128 128 128122 122 122 122 122 122 122103 122 103 122 122 122 122

74 103 74 103 103 103 10369 95* 67 74 74 74 7463* 74 62 69 69 69 6962 69 51 67 67 67 63*39 67 39 62 62 62 6233 62 33 51 51 51 3933 51 33 39 33 39 33

39 33 33 3333 3333 18*

33 36 33 34 34 36 34

MEAN= 3 4 .7

147

H P A _ IIi

A 1 B C7336 7336 73362325 2325 23252012 2012 20121737 1737 1737813 760* 813692 692 692649 649 649387 387 387162 162 276*114 114 68

68 6853*

11 12 10

MEAN= 10.75

IA Q _I

A 1

•

B 1 C2245 2245 22451909 2167 18771877 1877 17731773 1773 14781472 1472 14721403 1403 14031079 1079 1079914 914 786786 786 783783 783 777633 633 689597 597 633272 272 597258 205 272205 44 205

44 22 13722 13 4413 10 2210 13

10

19 IB 20

MEAN= 19

D 1 7336 2325 2012 1737 881 * 692 649 387 162 114

10

D E G2245 2245 22451877 1B77 21671773 1795 18771478 1478 17731472 1472 14721403 1403 14031079 1079 1079914 914 914786 786 786783 783 783689 689 633633 633 308597 597 289272 272 272205 205 205

44 44 4422 13 2213 10 1310 10

19 18 19

Q22451909177314721433140310799147867B3633597444272258205

44221310

20

1 4 8

MBO I :

A 1 B C D E F 6 H I J K2009 2009 2009 2009 2009 2009 2009 2009 2009 2269* 20091608 1608 1372 1819* 1608 1697* 1608 1608 1608 2009 16081372 1344 1344 1344 1372 1372 1564 1564* 1564* 1819* 1564*1344 885* 951* 885* 1344 936* 1344 1344 1344 1344 1344845 845 868* 845 860* 845 845 845 845 845 845111 772 845 772 845 772 772 772 772 772 772705 705 772 705 772 705 705 705 705 598 705598 598 705 598 705 598 598 598 598 581 598581 581 598 581 598 581 581 594* 581 533 581533 579* 581 533 581 533 533 581 529 529 529529 533 533 529 529 529 529 533 488 499* 488488 488 529 488 488 488 488 529 468 488 468468 487* 488 487* 468 468 468 488 461 461 461461 468 468 468 461 461 461 468 444* 439 444*439 461 461 461 439 439 444* 461 439 369 369369 439 439 439 369 408* 439 369 369 343 343343 369 369 369 343 369 369 343 343 340 320*340 343 343 343 340 343 343 340 320* 301 301301 340 340 340 301 340 301 301 301 297 297297 301 301 301 297 301 297 297 297 259 272*259 297 297 297 259 297 259 259 259 222 259222 259 259 259 211 259 222 222 222 155 222211 222 222 222 192 222 211 211 213* 147 213192 211 192 192 155 192 155 147 211 120 211155 192 155 155 147 155 147 120 155 105 167*147 155 147 147 120 147 120 105 147 104 155120 147 120 120 104 122* 105 104 120 95 147105 120 105 105 95 120 95 95 105 88 120104 105 104 104 88 105 88 88 95 67 10595 104 95 95 67 104 67 67 88 63 9588 88 88 88 63 95 63 63 67 34 8867 67 67 67 34 88 34 34 63 6763 63 63 63 31 67 31 31 34 6334 45* 34 34 63 31 3431 34 31 31 34 31

31 31

35 36 35 35 33 36 33 33 34 31 35

149

MBO I CONTINUED.

R S T U X 1 Y Z W 2 D 2 / 2 8 22009 2009 2009 2009 2009 2009 2009 2009 2009 2009 20091608 1608 1372 1819$ 1608 1819$ 1608 1608 1608 1608 16081564$ 1564$ 1344 1372 1372 1564$ 1372 1372 1372 1372 13721344 1344 951$ 936$ 1344 1344 1344 1034$ 1344 1344 1179$845 845 868$ 845 845 845 845 845 B45 845 845111 772 845 772 772 772 772 772 772 772 772705 705 772 705 705 705 705 705 705 705 705598 598 705 598 598 598 598 598 614$ 614$ 598581 581 594$ 581 581 581 581 581 581 581 581533 533 581 533 533 533 533 533 533 579$ 533529 529 533 529 529 529 488 529 529 533 529488 488 529 488 488 488 468 488 499$ 529 488468 468 488 468 468 468 461 468 488 499$ 468461 461 468 461 461 461 439 461 461 488 461439 369 466$ 439 439 439 369 439 439 461 439369 343 369 408$ 369 369 343 369 369 369 369343 340 343 369 343 343 340 343 343 343 343340 328$ 340 343 340 340 301 340 340 340 340301 301 301 340 327$ 301 297 310$ 301 301 301297 297 297 301 301 297 293$ 301 297 297 297259 259 259 297 297 259 259 297 259 259 259222 222 222 259 259 222 236$ 259 222 222 222211 211 211 222 211 155 222 222 211 211 211155 155 192 192 192 147 211 211 192 192 192147 147 147 155 155 120 192 192 155 147 165$120 120 120 147 147 105 155 155 147 120 155105 111$ 105 120 120 104 147 147 120 105 147104 105 104 105 104 95 120 120 105 104 12095 104 95 104 95 88 105 105 104 95 10588 95 88 95 88 67 104 104 95 88 10467 88 67 88 67 63 95 95 88 63 9563 67 63 67 63 34 88 88 63 51$ 8834 63 34 63 34 31 67 67 51$ 34 6731 34 31 34 31 63 63 34 15$ 63

31 31 34 34 31 3431 31 31

34 35 34 35 34 33 36 36 35 34 36

MEAN= 3 4 .5

1 5 0

s u l h

A 2 B 2 C 2 D 21069 1069 1069 1069905 905 905 905709 725* 725* 725*680 709 709 709654 659* 659* 659*608 608 608 608554 554 554 554545 551* 552* 551*545 545 551* 545530 530 545 530451 451 538* 451450 450 451 450450 450 450 450448 381 381 381381 365 365 365365 359* 359* 359*352 352 352 352348 348 348 348318 318 318 318317 317 317 317311 311 311 311298 298 298 298294 294 294 294292 292 292 292277 277 277 277269 269 249 269249 249 244* 249243 243 243 244*207 207 207 243188 188 188 207175 175 175 188170 170 170 175164 164 164 170162 162 162 164144 144 144 162132 132 132 144124 124 124 132121 121 121 124116 116 116 121112 112 112 116105 105 105 112105 105 105 105104 104 104 105102 102 102 10499 99 99 10298 9B 98 9993 93 93 9886 89* 89* 9383 86 86 89*67 83 83 8658 67 67 8354 58 49* 6752 52 47 5849 49* 47 52

E 2 F 2 G 2 H 21069 1069 1069 1069905 905 905 905709 709 709 709680 680 680 680662* 654 654 654608 608 608 608554 554 554 554545 545 545 552*545 545 545 545530 530 530 545451 451 451 530450 450 450 451448 450 450 450438* 448 448 448381 381 381 381365 365 374* 365352 365* 352 352348 352 348 348318 318 318 318317 317 317 317311 311 311 311298 298 298 298294 294 294 294292 292 292 292277 277 277 277269 269 269 269249 249 249 249243 243 243 243207 207 207 207188 188 188 188175 175 175 175170 170 170 170164 164 164 164162 162 162 162144 144 144 144132 132 132 132124 124 124 124121 121 121 121116 116 116 116112 112 112 112105 105 105 105105 105 105 105104 104 104 104102 102 102 10299 99 99 99^98 98 98 9893 93 93 9386 86 86 8683 83 83 8367 67 67 6758 58 58 5454 54 54 5252 52 52 4949 49 49 47

151

47 49 45 49 47 47 47 4747 47 44 49* 47 47 47 4545 47 36 47 45 45 45 4545 45 33* 47 45 44 45 4044 44 30 45 44 40 44 3640 36 26 44 40 36 40 3036 33* 25* 36 36 30 36 2630 30 24 33* 30 28* 26 2426 26 11 30 26 26 24 1124 24 9 26 24 24 21* 911 11 9 25* 11 11 11 99 9 24 9 9 99 9 11

99

9 9 9

>7 67 65 69 67 68 67 65

MEAN= 66 .88

J 2>196*1294.280994755*751729*678655*639*594572*473455372344*306302*291275274259240228218188*151*133*109*968786*866054*24171587 *

40

B.7

c 2 D 22925 29251294 12941280 1280994 994751 751720 720678 678655* 658623 623594 594572* 577495 488473 473455 455372 372308 308306 306297 297291 291275 283*274 281*259 259240 232*228 228218 218187 18796 9687 8786* 8686 7860 6035 3524 2417 1717 1715 158 8

37 37

F 2 G 22196* 2196*1294 12941280 1280994 994755* 755*751 751729* 729*678 682*655* 655*639* 639*594 594572* 572*473 473455 455372 372344* 344*306 306302* 302*291 291275 275274 274259 259240 240228 228218 218188* 184*183* 151*151* 133*133* 109*109* 9686* 8786 86*60 8654* 6024 54*17 2415 178 157* 8

7 *

39 40

E 22196*12941280994755*751729*678655*639*594572*473455372344*306302*291275274248*240228210*188*151*133*109*

968786*866054*2419*171587*

41

1 d 3

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B 245922046172416591258615584*573499469463447390346142132887867656115

11

24

24

B757650662378<

934341

5

5 .5

C 2 4592 2046 1724 1654 1258 615 584* 57B* 499 469 463 447 390 346 142 132 88 67 65 56* 15

1 1

23

154

T a b le 5 .2 : Summary o f r e s t r i c t i o n fra gm en t d i g e s t i o n pat t e rn s .

T h is ta b le summaries th e r e s t r i c t i o n fra g m e n t d ig e s t io n p r o f i l e s and th e

fra g m e n ts w h ich c h a r a c te r is e them , by each r e s t r i c t i o n endonuc lease employed

in th e s tu d y .

1 - th e t o t a l number o f d ig e s t io n p r o f i l e s re c o rd e d f o r ffus dowesticus

( F e r r is et al., 1983 - W estern European, M e d ite rra n e a n , and O ld W orld

p o p u la t io n s ; t h i s s tu d y - th e B r i t i s h I s le s ) f o r each p a r t i c u la r enzyme used.

2 - th e number o f p r o f i l e s d e s c r ib e d e lse w he re b u t n o t p r e v io u s ly re co rd ed

f o r th e B r i t i s h p o p u la t io n s exam ined in t h i s s tu d y .

3 - new B r i t i s h d ig e s t io n p r o f i l e s n o t d e s c r ib e d b e fo re .

4 - th e t o t a l number o f fra g m e n t d ig e s t io n p r o f i l e s observed in th e B r i t s h

I s le s .

5 and 6 - th e minimum and maximum numbers o f fra g m e n ts r e s p e c t iv e ly ,

c h a r a c te r is in g th e d ig e s t io n p r o f i l e s per r e s t r i c t i o n endonuc lease used.

7 - th e number o f fra g m e n ts c h a r a c te r is in g th e known base sequence d ig e s t io n

p r o f i l e s per enzyme used.

2

135

Tab l e 3 .2 : Summary o f r e s t r i c t i o n -fragment d i g e s t i o n p ro f i l e s .

Enzyme Number o f d ig e s t io n p r o f i l e s Number o f fra g m e n ts

T o ta l in in B r i t a in !Mus domesticus p r e v io u s ly u n iq u e t o t a l min max base

re co rd e d sequence

Acc I 8 1 1 2 6 9 7

Ava I I 9 - 4 4 8 12 10

Fnud 11 6 1 2 3 7 9 8

H inc I I 4 1 - 1 5 6 5

H ind I I I 2 2 - 2 2 3 3

H in f I 27 4 4 8 25 31 30

Hpa I I 4 2 - 2 10 12 11

Taq I 7 2 - 2 18 20 19

Xba I 2 1 - 1 5 6 6

Hae I I I 18 1 7 8 32 37 35

Mbo I 22 2 4 6 31 36 35

A lu I 8 - 8 65 69 67

Rsa I 7 - 7 7 37 41 37

Sau 96 I 4 - 4 4 22 25 25

T o ta ls 128 17 41 58 274 316 2

Ta b le 3.3A-N ? V a r ia b le r e s t r i c t i o n s i t e s o f mouse mtDNA d e te c te d w i th

fo u r te e n r e s t r i c t i o n endonuc leases .

U sing th e sequence com parison m ethod, c le a va g e maps f o r e v e ry mouse f o r

each o f th e fo u r te e n enzymes, were c o n s tru c te d . T a b les 3 .3 A-N l i s t s th e

hundred v a r ia b le s i t e s in fe r r e d fro m th e s e maps.

The p u b lis h e d mouse mtDNA re fe re n c e sequence (B ibb e t a l 1981) i s th e

l i g h t s tra n d (L) hence o n ly base changes o c c u r r in g on t h i s s t ra n d a re

in d ic a te d . The b a s e -p a ir num bering in t h i s sequence b e g in s a t th e f i r s t 5 '

n u c le o t id e o f th e t r a n s fe r RNApH" and p roceeds w ith in c re a s in g number

th ro u g h th e rRNA genes to th e o r ig in o f th e heavy s tra n d (H) o f

m ito c h o n d r ia l r e p l ic a t io n ( f ig u r e 1 .1 ) .

S ta r t - r e fe r s to th e f i r s t base o f th e re c o g n it io n sequence o-f th e

p a r t i c u la r r e s t r i c t i o n endonuclease used where th e v a r ia b le s i t e i s

1o ca te d .

For th e p ro te in -c o d in g re g io n s w ith g a in m u ta tio n s r e la t i v e t o th e

re fe re n c e sequence, th e e x a c t lo c a t io n and n a tu re o f th e base change can

be in fe r r e d . The s i l e n t and re p la ce m e n t s u b s t i t u t io n s (S I, S3 and R1-R3

r e s p e c t iv e ly ; th e numbers r e fe r t o th e codon p o s i t io n s o f th e base change)

a re in d ic a te d f o r th e p ro te in - c o d in g genes.

For lo s s m u ta tio n s th e e x a c t base canno t be in fe r r e d th e r e fo r e th e

lo c a t io n s o f th e w ho le re c o g n it io n sequence i s g iv e n . The fu n c t io n a l re g io n

where th e base change i s lo c a te d i s a ls o n o te d , re g a rd le s s o f w he the r i t i s

a g a in o r lo s s m u ta tio n (a b b re v ia t io n s f o r genes g iv e n in f ig u r e 3 .4 A ) .

0 o r 1 in d ic a te s a lo s s o r g a in o f a s i t e r e la t i v e t o th e re fe re n c e

sequence. The fra g m e n t p a t te rn p r o f i l e s and t h e i r a s s o c ia te d c le a v a g e maps

a re re fe r re d to by c a p i ta l l e t t e r s (see ta b le 3 .1 ) . The use o f t a b le 3 .4

(c o n s ta n t s i t e s ) in c o n ju n c t io n w ith th e v a r ia b le s i t e ta b le s a llo w s th e

c o n s t ru c t io n o f com p le te c le a va g e maps f o r each enzyme f o r each mtDNa ty p e .

1 3 ?

1 D e sc rib e d e lse w h e re ( F e r r is et aim, 1983) b u t n o t p r e v io u s ly re c o rd e d

■for th e B r i t i s h p o p u la t io n s exam ined in t h i s s tu d y .

2 New u n iqu e d ig e s t io n p r o f i l e s n o t p r e v io u s ly re c o rd e d .

A l l p r o f i l e s w ith o u t a s u p e rs c r ip t d e n o te p r o f i l e s n o t d is c o v e re d in th e

B r i t i s h I s le s and a re d e s c r ib e d e lse w h e re ( F e r r is etal•, 1 98 3 ). New la b e ls

a re g iv e n in b ra c k e ts to r e a d i ly d is t in q u is h between ty p e s f o r subsequen t

com pute r a n a ly s is where p r o f i l e s were p re v io u s ly deno ted w ith a p rim e ( ie .

A’ ) fo l lo w in g F e r r is e t al*s n o m e n c la tu re .

t in d ic a te s s i t e lo c a t io n s n o t re c o rd e d b e fo re .

* A m u ta tio n o f C to G a t p o s i t io n 15925 ( s i t e 6 ; t a b le 3 .3 D ) , lo c a te d in

th e D -loo p re g io n , p roduces an Acc I s i t e , s im u lta n e o u s ly c a u s in g th e lo s s

o f th e Hae I I I s i t e a t 15923 ( s i t e 19; ta b le 3 .3 E ). T h is s i t e change i s

o n ly coun ted once f o r p h y lo g e n e tic p u rp o se s .

b A m u ta tio n o f G t o A a t 12516 ( s i t e 7 ; ta b le 3 .3 E ) , lo c a te d in th e ND 5

re g io n , causes th e lo s s o f an Ava I I s i t e and th e c o n c o m ita n t g a in o f an

Mbo I s i t e a t 12515 ( s i t e 17? ta b le 3 .3 J ) . Afew m ice were obse rved to have

lo s t th e A v a i l s i t e w ith o u t g a in in g an Mbo I s i t e , e x p la in e d by a m u ta tio n

o f G to e i t h e r C o r T. These s i t e s were coun ted tw ic e in th e subsequent

a n a ly s e s .

c A m u ta tio n o f G t o A a t 14240 ( s i t e 8J ta b le 3 .3E ) in th e Cytochrom e B

gene, r e s u l t s in th e lo s s o f an Ava I I s i t e and s im u lta n e o u s g e n e ra tio n o f

an Mbo I s i t e a t 14239 ( s i t e 21; ta b le 3 .3 J ) . The absence o f b o th s i t e s was

a ls o n o te d , accoun ted f o r by a m u ta tio n o f G to e i t h e r T o r C a t p o s i t io n

14239. Hence t h i s s i t u a t io n was d e s c r ib e d as two s i t e s f o r th e p h y lo g e n e t ic

a n a ly s e s .

d A m u ta tio n o f G t o any o f th e o th e r bases (A, T, o r C) a t p o s t io n 3335 in

th e ND 1 re g io n , causes bo th th e lo s s e s o f th e Hpa I I s i t e ( s i t e 2 ; ta b le

3 .3G ) and th e Hae I I I s i t e ( s i t e 1; ta b le 3 .3 1 ) . However a base change a t

s i t e s 3332-4 r e s u l t s in th e lo s s o f th e Hpa I I s i t e w ith o u t th e lo s s o f th e

Hae I I I s i t e . , th u s th e se s i t e changes a re coun ted tw ic e .

* A m u ta tio n o-f T t o C a t s i t e 3788 in th e t r a n s fe r RNA010 gene e x p la in s

b o th s i t e g a in s a t 3787 (Taq I : s i t e 3 ; ta b le 3 .3H ) and 3784 (H in f I :

s i t e 7; ta b le 3 .3 K ) , w h ich was o n ly coun ted as one s i t e f o r p h y lo g e n e t ic

p u rpo se s .

* A s in g le gene m u ta tio n a t 9577 -8 causes th e s im u lta n e o u s p resence o r

absence o f b o th th e Taq I s i t e a t 9577 ( s i t e 5 ; ta b le 3 .3H ) and th e H in f I

s i t e a t 9574 ( s i t e 17; ta b le 3 .3 k ) . A ga in t h i s base change was coun ted o n ly

once.

9 A m u ta tio n o f G to A a t p o s i t io n 11168 in th e ND 4 re g io n causes th e lo s s

o f th e Mbo I s i t e ( s i t e 14; ta b le 3 .3 J ) and th e subsequent g a in o f a H in f I

s i t e ( s i t e 19; ta b le 3 .3 K ). A d d i t io n a l l y a m u ta tio n o f G t o e i t h e r T o r C

a t t h i s s i t e causes th e absence o f b o th s i t e s . However, th e d ig e s t io n

p r o f i l e s observed c o u ld a ls o be e x p la in e d by a g a in o f a h in f I s i t e

s t a r t in g a t 11168 by way o f a m u ta tio n o f C to T a t p o s i t io n 11171, p lu s a

change to A o r G a t t h i s p o s i t io n e x p la in s th e absence o f b o th s i t e s

( F e r r is e t a i . , 1983). These s i t e changes were coun ted tw ic e in th e

p h y lo g e n e t ic a n a ly s e s .

h In th e ND 3 re g io n a m u ta tio n o f G to A a t p o s i t io n 4276 causes th e lo s s

o f a Mbo I s i t e ( s i t e 5 ; ta b le 3 .3 J ) and th e c o n co m ita n t g a in o f a h in f I

s i t e ( s i t e 8 ; ta b le 3 .3 k ) . A lso a M u ta tio n o f G to e i t h e r T o r C a t t h i s

s i t e r e s u l t s in th e lo s s o f b o th s i t e s , as a consequence th e s e s i t e changes

were d e s c r ib e d as two f o r p h y lo g e n e t ic p u rp o se s .

1

TABLE 3 . 5As V a r ia b le r e s t r i c t i o n s i t e s o-f Hus dom esticus mtDNA d ig e s te dw i th Hind I I I .

L o c a tio n o-f s i t e P resence o-f s i t e

s i t e s t a r t base change ■ fun c tio na lre g io n A B

1 9136 9136-41 CO I I I 1 0

TABLE 3 .3B : V a r ia b le r e s t r i c t i o n s i t e s o f Hus domesticus mtDNA d ia e s te dw ith Xba I ,

L o c a tio n o f s i t e P resence o f s i t e

s i t e s t a r t base change fu n c t io n a l re g i on A B

1 B984 8984-9 CO I I I 1 0

t g o

TABLE 3 .3C : V a r ia b le r e s t r i c t i o n s i t e s o-f Hus dow esticus mtDNA d ig e s te dw i th H inc I I .

L o c a tio n o f s i t e P resence o f s i t e

s i t e s t a r t base change fu n c t io n a lre g io n A B C G

1 7147 7147, A-G, S3 CO I I 0 0 0 12 B637 8642, T-C, S3 CO I I I 0 1 0 03 16074 16074 C-G D-L00P 0 0 1 0

TABLE 3.3D? V a r ia b le r e s t r i c t i o n s i t e s o f Hus dowesticus mtDNA d ig e s te d w ith Acc I ,

L o c a tio n o f s i t e P resence o f s i t e

s i t e s t a r t base change fu n c t io n a lre g io n A B C D E J K L

1 2817 2822, A-C, R3 ND 1 0 1 0 0 0 0 0 0*2 4515 4515, C-G, R2 ND 2 0 0 0 0 0 0 0 13 9339 9339- 44 CO I I I 1 1 1 1 0 1 1 14 12055 12055, A-G, R2 ND 5 0 0 0 0 0 0 1 05 13856 13861, T-C, S3 ND 6 0 0 0 1 0 1 0 06* 15924 15925, C-T D-LOOP 0 1 1 1 0 0 0 0

1 5 1

TABLE 3.3Es V a r ia b le r e s t r i c t i o n s i t e s o-f Hus dow esticus mtDNA d ig e s te d w i thAva I I .

Loca t ion o f s i t e P resence o f s i t e

s i t e s t a r t base change fu n c t io n a lre g io n A B c D I J K L M

*1 2083 2083-7 16s rRNA 1 1 1 1 1 1 1 1 0*2 2871 2871-5 ND 1 1 1 1 1 1 1 0 1 1*3 6123 6124, A-G, R2 CO I 0 0 0 0 0 1 0 0 1

4 8990 8990, A-G, S3 CO I I I 0 0 0 0 1 0 0 0 05 10840 10844, A-C, S3 ND 4 0 1 0 0 0 0 0 0 0

*6 12213 12217, G-C, R2 ND 5 0 0 0 0 0 1 1 1 17b 12515 12515-9 ND 5 1 1 0 0 0 1 1 1 1Bc 14239 14239-43 CYT B 1 1 1 0 1 1 1 1 1

TABLE 3. 3F; V a r ia b le r e s t r i c t i o n s i t e s o f Hus dowesticus mtDNA d ia e s te d w ithFnu D11 -

L o c a tio n o f s i t e s Presence iD f s i t e

s i t e s t a r t base change fu n c t io n a lr eg i on A B H I J K

*1 317 317-10 12S rRNA 1 1 1 1 1 02 4972 4972-5 Trp tRNA 1 1 1 0 1 13 9505 9508, T-G, R2 ND 3 0 0 1 0 0 04 9595 9596, A—G, S3 ND 3 0 1 0 0 0 0

*5 14705 14705-8 CYT B 1 1 1 1 0 1

1 5 2

TABLE 3,5G: V a r ia b le r e s t r i c t i o n s i t e s O-f Hus dow esticus mtDNA d ig e s te dw i t h Hpa I I .

L o c a tio n o f s i t e P resence o f s i t e

s i t e s t a r t base change fu n c t io n a lre g io n A B C D

1 327? 3281, A-G, S3 ND 1 0 1 0 0* 3332 3332 -5 , ND 1 1 1 1 0

6274 6274-7 CO I 1 1 0 1

TABLE 3 . 3Hi V a r ia b le r e s t r i c t i o n s i t e s o f Mus dowesticus mtDNA d ig e s te d w ith Tag I .

L o c a tio n o f s i t e P resence o f s i t e

s i t e s t a r t base change fu n c t io n a lre g io n A B c D E G Q

41 2407 2407-10 16S rRNA 1 1 1 1 0 1 1£ 2566 2566, A-T 16S rRNA 0 0 1 0 0 0 0

37B7 3788, T-C Gin tRNA 0 0 0 0 0 0 14 8357 8358, T -C ,S3 ATP 6 0 0 1 1 1 o 05" 9577 9577-8 ND 3 1 0 0 0 0 0 16 15542 15544, A-G D-L00P 0 0 0 0 0 1 0

153

TABLE 3.51s V a r ia b le r e s t r i c t i o n s i t e s o f Hus dow esticus mtDNA d ig e s te dw i th Hae I I I .

L o c a tio n o-f s i t e P resence o-f s i t e

s i t e s t a r t base change fu n c t io n a lr eg i on ft B 1: e1 1E 1F F: s; w1 X U T■ V' n1 N C1 F► 1

*1 * 3335 3335-8 ND 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1*2 3398 3398, A -G ,S3 ND 1 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0

3 3402 3402-5 ND 1 1 1 1 1 1 1 0 1 1 0 1 1 1 1 1 1 1 14 4129 4129-32 ND 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 15 4670 4670-3 ND 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 16 5900 5900, A -G ,S3 CO I 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0

*7 7541 7541-44 CO I I 1 1 1 1 1 1 0 1 1 0 1 1 1 1 1 1 1 1*8 8647 8648, T-G R3 CO I I I 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0*9 8742 8742-5 CO I I I 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1

*10 8811 8811-4 CO I I I 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1*11 9756 9756-9 ND 3 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1*12 11210 11212, T-C,R2 ND 4 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0

13 11471 11471, A -G ,S3 ND 4 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0*14 12378 12378-81 ND 5 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1

15 13479 13482, T -C ,S I ND 5 0 1 0 0 1 0 0 0 0 0 0 0 0 1 0 1 0 016 14089 14090, A-G Glu tRNA 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 017 14433 14435, A -C ,S3 CYT’ B 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0

*18 15189 15189-92 CYT’ B 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 1 119* 15923 15923-5 D-LOOP 1 0 1 0 1 1 1 1 1 1 1 1 1 1 1 0 0 0

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TABLE 3.3Ls V a r ia b le r e s t r i c t i o n s i t e s o f Hus dow esticus mtDNA d ig e s te d w i thSau 96 I .

lo c a t io n o-f s i t e P resence o f s i t e

s i t e s t a r t base change fu n c t io n a l re g io n A B C D

*1 78 78-81 12S rRNA 1 1 0 1*2 83 83, T-G 12S rRNA 0 0 1 0*3 2357 2357-60 16S rRNA 1 1 1 0*4 2856 2856-9 ND 1 1 1 1 0*5 1573B 15738-41 D -I00P 1 0 0 1

TABLE 3.311s V a r ia b le r e s t r i c t i o n s i t e s o f ftus dowesticus rotDNA d ig e s te d w ith A lu I ,

L o c a tio n o-f s i t e P resence o-f s i t e

s i t e s t a r t base change f u n c t i ona lre g io n A B c D E F G H

*1 1633 1636, A-T 16S rRNA 0 0 1 0 0 0 0 0%2 1641 1641-4 16S rRNA 1 1 0 1 1 1 1 1*3 3143 3143-6 ND 1 1 0 0 0 1 1 1 1*4 3188 3188-91 ND 1 1 1 1 1 1 0 1 1*5 350B 3509, A—G, S3 ND 1 0 0 0 0 0 1 0 0*6 5122 5124, T-C tRNA asn 0 1 1 1 0 0 0 0*7 5129 5129-32 tRNA asn 1 0 0 0 1 1 1 1*8 5783 5783-6 CO I 1 1 1 1 0 1 1 1*9 5795 5796, T-G, R1 CO I 0 0 0 0 1 0 0 0

*10 6233 6233-6 CO I 1 1 0 1 1 1 1 0*11 6291 6291-4 CO I 1 1 0 1 1 1 1 0*12 B253 8253-6 ATP 6 1 0 0 0 1 1 1 1*13 B259 8262, C -T ,S3 ATP 6 0 1 1 1 0 0 0 0*14 9947 9948, C-G,R3 ND 4L 0 1 1 1 0 0 0 0*15 10826 10826-9 ND 4 1 1 1 1 1 1 0 1*16 10B35 10835, T-A,R3 ND 4 0 0 0 0 0 0 1 0*17 15515 15517, G-C D-L0CP 0 0 1 1 0 0 0 0

167

TABLE 3 . 3 N s V a r ia b le r e s t r i c t i o n s i t e s o-f Hus dow esticus mtDNA d ig e s te dw i th Rsa I ,

Location o-f si te Presenc o-f si te

5i te s ta rt base change functi onalr eg i on A B c D E F G

*1 104 107, A-C 12S rRNA 0 /*%V 0 0 0 0 1*2 598 598-601 12S rRNA 4

1 1 1 1 1 1 0*3 786 786, C-G 12S rRNA 0 1 0 0 41 1 1*4 2781 27B3, C-A,£3 ND 1 0 1 0 0 1 1 1*5 3679 3679, T~G,R2 ND 1 o 0 0 0 1 0 0*6 3690 3690-3 ND 1 1 1 1 1 0 1 1*7 3698 3698, A-G,S3 ND 1 0 0 0 0 1 0 0*8 6633 6633, A-G,R1 CO I 0 1 0 0 1 1 1*9 8925 8925-3 CO I I I 1 1 1 1 1 0 1*10 9545 9545-B ND 3 1 0 0 1 0 0 0*11 11265 11265, C-G,R1 ND 4 0 1 1 0 1 1 1*12 12484 12484-7 ND 5 1 1 1 0 1 1 1*13 12492 12495, G-C,R1 ND 5 0 0 0 1 0 0 0*14 12998 12998-13001 ND 5 1 1 1 0 1 1 1

*15 13005 13005, A-G,R1 ND 5 0 0 0 1 0 0 0*16 13342 13342, T-G,R2 ND 5 0 1 0 0 1 1 1

*17 15110 15110-13 CYT B 1 0 1 1 0 0 0*18 15123 15126, G-C,R1 CYT B 0 1 0 0 1 1 1

*19 154 IB 15418-21 D-LOOP 1 0 1 1 0 0 0*20 1542B 15428, T-G D-LOOP 0 1 0 0 1 1 1*21 15495 15495-3 D-LOOP 1 0 1 1 0 0 0

iss

Tab le 3 . 4: Summary o-f l o c a t i o n s o f r e s t r i c t i o n s i t e s f o r 14 enzymes in

Hus dowest icu s mtDNA.

The le -fth a n d s id e o-f th e ta b le i l l u s t r a t e s th e lo c a t io n s o-f th e 230

c o n s ta n t s i t e s -for th e 14 r e s t r i c t i o n endonuc lease em ployed in t h i s s tu d y .

The r ig h th a n d s id e sum m aries th e numbers o f a l l th e s i t e s observed fo r each

o f th e 14 endonuc leases in a l l th e Hus dowesticus mtDNA ty p e s s tu d ie d :

1 and 2 l i s t s th e t o t a l number o f o f c o n s ta n t and v a r ia b le s i t e s

r e s p e c t iv e ly , d e te c te d by each p a r t i c u la r r e s t r i c t i o n endo nu c le a se .

3 l i s t s th e t o t a l number o f s i t e s (c o n s ta n t p lu s v a r ia b le ) re c o g n is e d by

each r e s t r i c t i o n endo nu c le a se .

* shows th e t o t a l number o f s i t e s (c o n s ta n t p lu s v a r ia b le ) p e r enzyme in

th e mouse mtDNA o f known base sequence (B ibb e t a i 1981) .

co rrespond t o th e s i t e s w hich a re d e te c te d by tw o r e s t r i c t i o n

enzymes which a re co un ted as one s i t e f o r p h y lo g e n e t ic a n a ly s e s , (see t e x t

in ta b le 3 .3 f o r f u l l e r d e t a i l s ) .

1 6 9

TABLE 3 .4 * Summa ry o-f l o c a t i o n s o-f r e s t r i c t i o n s i t e s f o r 14 enzymes in tfuscfom esticus mtDNA.

ENZYME LIST OF CONSTANT SITES NUMBER OF SITES

CONSTANT1 VARIABLE2 ALL3 REF'

i i_ H lN D _ I I I i 11081, 11969, 2 1 3 3

2i _XB A _Ii 612, 953, 8529, 10907, 15973, 5 1 6 6

3 i_ H IN C _ II i 364, 1858, 5123, 5452, 7718, 5 3 8 5

4 i_ A C C _ Ii 5709, 5754, 6222, 7035, 9598, 14646, 6 5 .5 * 11.5 7

5 i_ A V A _ II i 78, 424, 2357, 3480, 3959, 6005, 6 8 14 10

^ _ F N U D _ I! i 1772, 2007, 3410, 11406, 13039, 5 5 10 8

Z i_ H P A _ II i 133, 1870, 2519, 3400, 5725, 6112, 6385, 8400, 15736, 9 3 12 11

§ i_ H A E _ II I i 84, 1053 ,1606, 2004, 2215, 2856, 5571, 4007, 4232, 6621, 6749, 7015, 7592,8182, 11118, 11192, 11526, 12417, 12450, 12666, 14708, 15156, 15739, 15801,

25 18* °a 43 35

9 i_ IA Q _ Ii 634, 2429, 3343, 5220, 5233, 5438, 6541, 6555, 7668, 9835, 1*2080, 13552, 13562, 14348, 14620, 15253, 15850,

16 5 e ,* 21 19

IQ i _M B0_Ii 1889, 2350, 2438, 3103, 3223, 3566, 4065, 5884, 6328, 6697, 6731, 6990, 7085, 7677, B165, 8937, 9238, 9385, 10729, 11323, 13592, 14452, 14749, 15330, 16175,

26 21 47 35

U iJ d IN E _ I i 729, 1367, 4689, 5635, 6034, 6514, 7367, 7434, 8893, 9220, 9432, 9837, 11571, 12082, 12277, 13009, 14251, 14668, 15994,

19 2 6 * ' * 45 30

i2 i _A LU _Ii 9 , 197, 474, 573, B71, 1033, 1197, 58 17 75 671321, 1345, 1431, 1442, 1540, 1589, 2171, 2463,3536, 3854, 3863, 4768, 4900, 5075, 6335, 6716,7068, 7115, 7219, 7331, 7625, 7708, 8293, 8409,8454, 8490, 9044, 9137, 9588, 10036, 10083,10394, 10461, 10826, 10977, 11082, 11252, 11261,11970, 12287, 12392, 12599, 12743, 12992, 13600,13843, 14912, 15362, 15388, 15490, 15759,

I 3 JLRSA_Ii 895, 1186, 1210, 1270, 2564, 2650, 2837, 28 21 49 373431, 3908, 4136, 5416, 5889, 5904, 8829, 9012,9467, 10122, 10428, 11179, 11837, 12209, 12724,13493, 14487, 15435, 15443, 15460, 16215,

14 iS A U _96_ Ii 424, 2088, 2215, 2871, 3334, 3335, 3402, 21 3480, 3870, 3871, 3959, 6005, 6020, 11192, 12450,12515, 14239, 14708, 15155, 15739, 15800,

230

5 26 25

140 370 295

1 7 0

IABLE_3i 5i_SIIE_GAINS_FgR_SPECIFIC_SyBSTIiyiI0NSi

1 R e s t r ic t io n endonuc lease used t o d e te c t th e lo c a t io n and n a tu re o-f th e

s i t e g a in obse rve d . 2 The p o s i t io n o-f th e s i t e g a in in th e la b o ra to ry

re fe re n c e sequence (B ib b et a l 19B1). 3 The gene lo c a t io n o-f th e s i t e

g a in in th e re fe re n c e sequence. * The e x a c t n a tu re o f th e b a s e -p a ir

s u b s t i t u t io n (A= A den ine ; G=Guanine; C= C y tos ine? T= th y m in e ) .

W ith in th e p ro te in - c o d in g re g io n s th e n a tu re o f th e g a in m u ta tio n change i s

in d ic a te d :

3 Replacement s u b s t i t u t io n s , fo l lo w e d by th e change in a m in o -a c id codon.

A S i le n t s u b s t i t u t io n , fo l lo w e d by in b ra c k e ts th e codon in v o lv e d .

Changes w ith in a re g io n n o t coded (no re a d in g fram e) i s i l l u s t r a t e d in th e

column marked NO * .

T57 T ra n s it io n (g a in m u ta t io n a l change i s 1. p u r in e t o p u r in e ie . A-G, G-A

o r 2 . p y r im id in e to p y r im id in e ie . T-C , C -T ).

TV° T ra n s v e rs io n (m u ta tio n a l change fro m p u r in e to p y r im id in e and v ic e

v e rs a ie . A-C, A -T , G-C, G - T . . e t c . ) . * in d ic a te s g a in m u ta t io n s n o t

p re v io u s ly d e s c r ib e d . * in d ic a te s th e same s i t e d e te c te d by two

r e s t r i c t i o n endonuc leases s im u lta n e o u s ly (see ta b le 3 .3 t e x t f o r d e t a i l s ) .

The amino a c id re p la c e m e n ts a b b re v ia t io n s in c lu d e : PHEN, p h e n ly a la n i in e ;

L E U ,le u c in e ; ILE , is o le u c in e ; MET, m e th io n in e ; VAL, v a l in e ; SER, s e r in e ;

PRO, p r o l in e ; THR, th re o n in e ; ALA, a la n in e ; TRP, try p to p h a n ? H IS ,

h is t id in e ; ASN, a s p a ra g in e ; LYS, ly s in e ; ASP, a s p a r ta te ; GLU, g lu ta m in e ;

CYS, c y s te in e ; TYR, t y r o s in e ; ARG, a r g in in e ; SER, s e r in e ; GLY, g ly c in e . For

changes in th e mouse mt g e n e t ic code fro m th e u n iv e rs a l code see B ibb et

al., (1981 ), however AGG and AGA codons, w h ich code f o r a r g in in e in th e

u n iv e rs a l code a re neve r used in re a d in g fram es in th e mtDNA, hence a re

denoted w ith a q u e s t io n mark in th e ta b le (no gene f o r a tRNA to decode

them has been i d e n t i f i e d , th u s p resum ab ly th e y become nonsense co do n s).

171

TABLE 5.3 a SITE BAINS FOR SPECIFIC SUBSTITUTIONS.

ENZYME1 SITE2 GENE3 SUB* CODON position 1 2 3

REPLACE-MENT®

SILENT® TS7 subtit

TV® M3 ▼

HINC I I 7147 CO I I A-G + * (METH) +

■ 8 6 4 2 co i n T -C + * (ASN) 4-

■ 16074 D-LOOP C-G + t

ACC I 2817 ND 1 A-C + t GLU-ASP 4-

■ 4 5 1 5 * ND 2 C-G 4- t THR-SER 4-

M 12055 ND 5 A-G 4- t TYR-CSY +

» 13861 ND 6 T -C 4- * (LEU) 4-

H 15925 * D-LOOP C -T 4- t

AVA I I 6124 * CO I A-G + * GLU-GLY 4*

m 8 9 9 0 CO I I I A-G 4- * (GLU) 4*

M 10844 ND 4 A -C 4- * (SER) 4-

H 1 2 2 1 7 * ND 5 G-C 4- * ARG-PRO 4-

FNUD I I 9 5 0 8 ND 3 T -G 4- * LEU-ARG 4-

H 9 5 9 6 ND 3 A-G + * (ALA) 4-

HPA II 3281 ND 1 A-G + * (PRO) 4-

TAQ I 2 5 6 6 16s RNA A -T + *

■ 3 7 8 8 GLN tRNA T -C 4- *

m 8 3 5 7 ATPase 6 T -C 4* t (LEU) 4-

■ 15544 D-LOOP A-G 4- t

HAE i n 3 3 9 8 * ND 1 A-G 4- t (ALA) 4-

■ 5 9 0 0 CO I A-G + * (THR) 4*

1 7 2

TABLE 3.5...C0NTINUED.

HAE in 8 6 4 8 * co m T -G + t SER—? +

M 11212* ND 4 T -C 4- * VAL-PRO +•

n 11471 ND 4 A-G » (MET) 4*

H 13482 ND 5 T -C 4- * (LEU) +

m 14090 GLU tRNA A-G +

ii 14435 CYT B A -C + * (GLY) 4*

MBO I 2 4 9 2 * 16s RNA C -T +

M 4188 ND 2 G-A 4- * GLU-ASP +

M 5019 ALA tRNA A-C +

n 7 3 7 8 CO I I I A-G + * (MET) -1-

m 9 7 9 5 ND 1 A -T + t THR-SER 4-

n 10422 * ND 4 A -C + t ASN—HIST 4-

■ 10567 * ND 4 G-C + * TRP-SER +

M 10841 ND 4 G-A + * (GLY) 4-

n 11004 ND 4 T -C + * (LEU) +

n 1 1 3 0 8 * ND 4 T -G 4- * M ET-? 4-

M 12515 * ND 5 G-A + * V A L -IL E 4-

VI 14240 CYT B G-A + * (GLY) -!-

HINF I 1523 16s RNA A-C 4-

91 2 9 9 9 ND 1 A-G + t (LEU) 4*

■ 3 3 0 8 ND 1 T -C + * (PHEN) +

H 3 5 3 7 ND 1 G -T + * ALA-SER 4-

N 4 27 6 ND 2 G-A + * (GLY) 4*

173

HINF I 5 4 3 5 CO I

01<X + t (LEU) 4-

M 5 7 2 9 CO I A-G + t (GLY) +

a B718 co in A-G + * ASN—ASP 4-

w B969 co m T -C + %(ILE) 4-

I f 10904 ND 4 T-G 4- t ILE —MET 4-

H 11168 ND 4 G-A 4- t (ARG) 4*

M 11255 ND 4 T-G 4- t (ALA) 4-

M 14639 CYT 8 C-A 4- t (GLY) 4-

H 15094 * CYT B C-G + * PRO-ARG 4-

I I 16179 D-LOOP A-G 4-

RSA I 107 * 12s rRNA A-C 4-

f f 7B6 * 12s rRNA C-G 4-

H 2 7 8 3 * ND 1 C-A + t (VAL) 4-

I f 3 6 7 9 * ND 1 T-G •»- t PHEN-CYST 4-

n 3 6 9 8 * ND 1 A-G + * (PRO) 4-

a 66 3 3 * CO I A-G + * MET-VAL 4-

■ 11265 9 ND 4 C-G + * LEU-VAL +

m 12495 * ND 5 B — C + « VAL-LEU +

m 13005 * ND 5 A-G + * MET-VAL 4-

M 13342 * ND 5 *7“I — L 3 4- t IL E —SER +

f l 15126 * CYT B G -C + t ALA-PRO 4-

H 15428 * D-LOOP T-G 4-

SAU 96 I 83

ALU I 1636

12s rRNA

16s rRNA

T-G

A -T

4- t

+ t

1 7 4

ALU I 3 5 0 9 * ND 1 A-G + t (GLLD +■

■ 5124 * ASN tRNA +

■ 5 7 9 6 * CO I T -G + * SER-ALA +

■ B 262 * ATPase 6 C -T + f (ALA) +

m 9 9 4 8 * ND 4

01U + * H IST—GLN +

n 10B35 * ND 4 T -A + t ILE —MET +

M 1 5 5 1 7 * D-LOOP G -C +

TOTAL M° COLUMNGAINS= 75 TOTALS= 14 11 3 3 2 8 3 0 4 0 3

58 1.1 si

175

TABLE 5 .6 : Base s u b s t i t u t i ons r e p o n s ib le f o r s i t e ga i n s i n mouse mtDNgL

The d is t r ib u t io n o f p o te n t ia l (e xp e c te d ) and a c tu a l (o b se rve d ) r e s t r i c t i o n

c le a va g e s i t e s f o r each r e s t r i c t i o n enzyme were te s te d f o r s ig n i f ic a n c e

fro m random and c h i-s q u a re and th e p r o b a b i l i t y v a lu e s a re g iv e n .

* Dbs r e fe r s t o th e observed number o f c le a va g e s i t e s in th e k .b . s (B ibb e t

al.f 1981) f o r each r e s t r i c t i o n e ndonuc lease .

* Exp in d ic a te s th e expec ted number o f c le a va g e s i t e s c a lc u la te d fro m th e

know ledge o f th e base c o m p o s it io n o f th e l i g h t s tra n d o f th e k . b . s C/.G+C =

3 6 .3 ) , and e q u a tio n fro m Nei and L i , (1979) where th e y c o n s id e re d a mtDNA

o f mt p a i r s (16295 b a s e -p a irs in th e house mouse) w ith a G+C c o n te n t o f g .

Assuming a random d is t r ib u t io n o f n u c le o t id e s in th e DNA sequence, th e

expec ted fre q u e n c y o f r e s t r i c t i o n c le a va g e s i t e s w ith r n u c le o t id e p a ir s

i s :

a= (g /2 ) 1-1 C (1 -g ) /23

r l i s th e numbers o f g ua n in es p lu s c y to s in e s , w h i ls t r 2 i s th e number o f

A den ines and thym in es in a r e s t r i c t i o n re c o g n it io n s i t e , a d d i t io n a l l y

r l + r2 = r . Thus th e expec ted number o f r e s t r i c t i o n s i t e s i s mt a.

W ith in th e r e c o g n it io n sequences:

A Pu = p u r in e s , e i t h e r A o r G; Py = p y r im id in e s , e i t h e r T o r C.

9 X = A o r CJ Y = G o r T.

c N = any o f th e fo u r bases.

176

TABLE 5 .6 ! Base s u b s t i t u t i o n s r e p o n s ib le fo r s i t e g a in s i n mouse mtPNA^

G ain m u ta t io n a lN ° s i t e s base changes

Enzyme r e c o g n it "sequence

obs 9 exp * X 2( p r o b /s ig n i- f )

T ra n s - T ra n s ­i t i o n v e rs io n

W ell re p re s e n te d s i t e s :2 .8 7 N’ BA lu I AGCT 67 5 4 .7 3 5

Hae I I I GGCC 35 17 .7 16 .95 * * * 6 2Xba I TCTAGA 6 5 .5 0 .0 4 N ,s 0 0H inc I I GTPuPyAC* 5 3 .1 1 .16 N*B 2 1Acc I GTCXY3AC® 7 3 .1 4 .91 * 3 2Ava I I GGCA/T3CC 10 5 .6 3 .4 6 N,B 2 2Sau 961 GGCN3CCc 25 17 .7 3 .01 N,B 0 1

s u b to ta l :

Under re p re s e n te d s i t e s

155

•■

107 2 1 .3 * * * 16 13 (1 .2 3 :1 )

Rsa I GTAC 37 5 4 .5 5 .6 2 * 3 9Hin-f I GACN3TCc 30 5 4 .5 11.01 * * * 9 6Mbo I GATC 35 5 4 .5 6 .9 8 * * 7 5Taq I TCGA 19 5 4 .5 23.1 * * * 3 1Fnud I I CGCG 8 17.7 5 .3 2 * 1 1Hpa I I CCGG 11 17 .7 2 .5 4 N*B 1 0H ind I I I AAGCTT 3 5 .5 1 .13 N‘ B 0 0

S u b to ta l: 143 259 5 1 .8 * * * 24 22 (1 .0 9 :1 )

12 .63 * * *T o ta l 298 366 40 35 (1 .1 4 :1 )

177

REGION TOTALS TRANSITIONS TRANSVERSIONS

REPLACE SILENT REPLACE SILENT

I . PROTEIN CODINGi REGIONS:A. KNOWN PROTEINS;CODON POSITION1 5 2 0 3 02 1 1 O 0 03 L4_ 0_ 11_ 1_ 2.SUBTOTAL 20 3 11 4 2

(157.) (55%) (20%) (10%)

7. MUTATION TYPE 14 (702)RATIO TS : TV

B. NADH DEHYDROGENASE SUBUNITS:CODON POSITION1 9 2 22 10 3 O3 19_ 2_ 12_TOTAL 33 5 14

(13.57.) (36.8% )

6 (30%)2 .3 : 1

574_16(42%)

7. MUTATION TYPE RATIO TS :TV

GRAND TOTAL 58

RATIO TS :TV

I I . NO READING FRAME: D-LOOP/ NON-CODING

RIBOSOMAL RNA 7

TRANSFER RNA 4

TOTAL 17

RATIO TS : TV

19 (50%)

O• o

3_3

(8%)

19 (50%)

S(13.8%)

25(43.17.)

20(34.5%)

33 (56.9%)

3(50%)1

(14.37.)3

(75%)7

(44.47.)

1 .32 : 1

(8.6%)

2 5 (43.1%)

3(50%)6(85.7%)

1(25%1>10

(55.6% )0.8

TOTAL MITOCHONDRIAL GENOME: 75 40 (53.3%) 3 5 (46.7%)

1.1 : 1

1 7 8

Ta b le 3.8? summary o t s i t e lo s s e s d e te c te d by each o t 14 r e s t r i c t i o n

iQdDnucieaseSi

A summary ta b le l i s t i n g v a r ia b le s i t e s c la s s e d as " lo s s e s " w ith re s p e c t t o

th e known base sequence, t o r each m a jo r m ito c h o n d r ia l gene re g io n , t o r each

o t th e 14 r e s t r i c t i o n endonuc lease w h ich d e te c ts them .

1 - New s i t e lo s s e s n o t p r e v io u s ly d e te c te d in th e B r i t i s h Hus domesticus

sam ples ( t h is s tu d y ) .

2 - The t o t a l number o t s i t e lo s s e s in Hus dowesticus mtDNA ( F e r r is e *

a im , 1983; t h i s s tu d y ) .

re p re s e n t th e same s i t e s w h ich a re d e te c te d by tw o enzymes

s im u lta n e o u s ly and a re hence o n ly coun ted once t o r p h y lo g e n e t ic a n a ly s e s

( t o r d e t a i ls see ta b le legend 3 .3 a -n ) .

179

T a b le 5.8s Summary o f s i t e lo s s e s d e te c te d by each o f 14 r e s t r i c t i o n endonuc leases

EnzymerRNA’ s tRNA’ s

M ito c h o n d r ia lP ro te inknown

gene re g io n co d in g genes

unknown

New1non­c o d in g

To ta ]

H ind I I I - - 1 - - - 1Xba I - - 1 - - - 1H inc I I - - - - - - -Ava I I 1 - 1 2 - 2 4Acc I - - 1 - - - 1Fnud I I 1 1 1 - - 2 3Hpa I I - - 1 1 - - 2Taq I 1 - - 0 .5 * - - 1 .5Hae I I I - - 4 6 0 .5 * 7 10.5Mbo I - - 1 8 - 2 9H in f I 1 1 3 5 .5 * - 2 0 .5Sau 96 I 2 - - 1 1 4 4A lu I 1 1 4 3 - 9 9Rsa I 1 - 2 4 2 9 9

T o ta ls 8 3 20 31 3 .5 37 6 5 .5(127.) (4.6%) (30.37.) (477.) (67.)

Table 3.9 : Reaional variability; aene size and number of sites.

Region y.G+Ccontent

Constantsites

variable total (obs) (exp)

X 2

Bp/ . s ite/ region bp

12s rRNA 35.3 16 7 23 21.1 0.08 Na

956 0.024

16s rRNA 35.6 31 8 39 35.4 0.36 Na

1582 0.025

ND 1 36.2 14 21 35 21.2 8.9 **

946 0.037

ND 2 34.4 9 6 15 23.53.1*®

1036 0.015

ND 3 33.6 3 7 10 7.8 0.62 Na

345 0.029

ND 4L 41 3 1 4 6.61.05 NO

294 0.014

ND 4 35.5 18 15 33 31.4 0.08 NO

1378 0.024

ND 5 36.7 22.5 16 38.5 41.30.2 Na

1824 0.021

ND 6 27.2 3.5 3 6.5 14.4 4.3 *

519 0.013

CO I 40.4 36 11 37 34.B 0.14 Na

1545 0.024

CO II 38.2 14 3 17 15.6 0.13 NH

684 0.025

CO III 38.3 9 12 21 17.8 0.57 NO

784 0.027

Cyt B 38.6 14 10 24 26.1 0.16 Na

1144 0.021

ATPase 6 35.4 8 4 12 15.5 0.8 NO

680 0.018

ftTP3 30 3 32.1 — — 4.54.5 *

204 -

L.S DRIGIN 36.1 — 0.2 0.01 Na

32

D-LOOP 36.4 14 9 23 20.7 0.26 Na

879 0.026

LL tRNA’s 36 25 7 32 33.6 0.08 Na

1481 0.022

Total /rtjenoirie 36.7 230 140 370 — 16295 0.023

1 3 1

Tab 1 e 3 .1 0 ; R eg iona l v a r i a b i l i t y o-f mtDNA i n Hus dowestic u s .

Data was ta k e n -from 370 r e s t r i c t i o n s i t e s d e te c te d among 57 Hus

dowesticus m ito c h o n d r ia l DNA ty p e s d ig e s te d w ith 2 -14 r e s t r i c t i o n

end onuc leases .

(a) and (d) re p re s e n t th e number o-f s i t e s ( v a r ia b le and c o n s ta n t ,

r e s p e c t iv e ly ) d e te c te d pe r Hus dowesticus m ito c h o n d r ia l DNA gene re g io n

u s in g 11 enzymes. The d a ta was p oo le d -from p a s t s tu d ie s ( F e r r is et al,f

19B3) -for m ice -from W estern E urope , th e M e d ite rra n e a n , and th e New W orld ,

w ith th o se fro m B r i t a in ( t h is s tu d y ) .

(b) and (e) i l l u s t r a t e th e number o f s i t e s ( v a r ia b le and c o n s ta n t

r e s p e c t iv e ly ) d e te c te d by 3 a d d i t io n a l r e s t r i c t i o n endonuc leases pe r mt

gene re g io n f o r th e B r i t i s h m ice o n ly .

(c ) and ( f ) show th e combined number o f s i t e s ( v a r ia b le and c o n s ta n t ,

r e s p e c t iv e ly ) d e te c te d by a l l 14 enzymes employed in th e s tu d y p e r Hus

dowesticus mt gene re g io n .

The degree o f gene v a r i a b i l i t y in each gene was c a lc u la te d by d iv id in g th e

v a r ia b le s i t e s by th e t o t a l s i t e s , th e n c o n v e rte d to p e rc e n ta g e .

1 One Taq I s i t e i s found in p a r t o f th e o v e r la p p in g re g io n s o f ND 5

and ND 6.

1 3 2

I 0 B L E L . 3 - 1 O S R e g i o n a l v a r i a t i o n o - f m t D N A i n H u s d o w e s t i c u s .

Number o f s i t e s P e rce n t v a r i a b i l i t yV a r ia b le C ons tan t 11 3 14

Region (a) (b) (c ) (d) (e) ( f ) i 101

I i Icr

1 I in 1

100 a+d 100 b+e 100 c+ f

R ibosom al RNA’ 512S 2 5 7 9 7 16 18.1 4 1 .7 3 0 .416S 5 3 8 15 16 31 2 9 .4 15.9 2 0 .5

T ra n s fe r RNA’ s 5 2 7 14 11 25 2 2 .2 18.4 2 1 .B

P r o te in cod ings I . Cytochrom e P ro te in s

CO I 6 5 11 19 7 26 2 4 .0 4 1 .7 2 9 .7CO I I 3 - 3 8 6 14 2 7 .3 - 17.7ATPase 6 2 2 4 4 4 8 3 3 .3 3 3 .3 3 3 .3ATPase 8 - - - - - - - - -

CO I I I 11 1 12 5 4 9 6 8 .8 2 0 .0 57. 1CYT B 8 2 10 10 4 14 4 4 .4 3 3 .3 4 1 .7

I I . NADH dehydrogenase s u b u n its s

ND 1 13 8 21 6 8 14 6 8 .4 5 0 .0 6 0 .0ND 2 6 - 6 5 4 9 5 4 .6 - 4 0 .0ND 3 6 1 7 1 2 3 8 5 .7 3 3 .3 7 0 .0ND 4L - 1 1 - 3 3 - 25 25ND 4 12 3 15 8 10 18 6 0 .0 23.1 4 5 .5ND 5 1 11 5 16 9 .5 13 2 2 .5 5 3 .7 2 7 .8 4 1 .6ND 6 l 3 - 3 2 .5 1 3 .5 5 4 .6 - 4 1 .2

D isp la ce m e n t 1 oop 4 5 9 7 7 14 3 6 .4 4 1 .7 39 .1L ig h t s tra n d o r ig in _

133

TABLE 3.11s Summary o-f R eg iona l va r i a t i o n 0f Hus dow esticus m it o c h o n d r ia l DNA.

Number V a r ia b le

Region (a)

o t s i t e s C o n s ta n t

(b )

V. v a r i a b i l i t y _a_ x 100 a+b

v a r i a b i l i t y Rank ( le a s t t o most v a r ia b le )A B1

T o ta l R ibosom al RNA’ s ( 12S and 16S) 15 47 2 4 .2 2 2

T ransT e r RNA’ s 7 25 2 1 .8 1 1

P ro te in co d in g re g io n s?

Cytochrom e p r o te in s 40 (CO I - I I I , CYT B, ATP 6 & 8)

71 3 6 .0 3 -

(NADH dehydrogenase s u b u n its 1 -6 , 4L) 69 73 4 8 .6 5 -

T o ta l P ro te in s 109 144 43.1 - 4

D isp la ce m e n t lo o p 9 14 39.1 4 3

T o ta l M ito c h o n d r ia lgenome 140 230 3 7 .8

1 Gene v a r i a b i l i t y ranked fro m le a s t t o most v a r ia b le t o r each gene re g io n ; A —

p r o te in co d in g genes d iv id e d in t o cy toch ro m e genes and NADH s u b u n its ; B - a l l

p ro te in - c o d in g genes re g io n s p o o le d .

184

TABLE 5 .1 2 : M i to c h o n d r ia l gene v a r i a b i l i t y com par isons between mouse and

man.

Genes and n o n -c o d in g re g io n s in th e House mouse ( t h i s s tu d y ; F e r r is et a i« ,

1983) and Human (Cann, 1982) m ito c h o n d r ia l DNA was ranked -from le a s t t o

most v a r ia b le , u s in g th e p e rce n ta g e gene v a r i a b i l i t y v a lu e s (d e fin e d as

a /b , a= number o f v a r ia b le s i t e s , b= t o t a l number o f s i t e s , th e n c o n v e rte d

t o p e rc e n ta g e ) as c a lc u la te d in ta b le 3 .1 0 .

F e r r is and c o lle a g u e s (1983) d e te c te d gene v a r i a b i l i t y in W estern European,

M e d ite r ra n e a n , and th e New W orld Hus domesticus u s in g 11 r e s t r i c t i o n

e n d o nu c le a ses , w h i ls t th e B r i t i s h m ice v a r i a b i l i t y in c lu d e d th e same 11

enzymes p lu s 3 a d d it io n a l ones ( t h is s tu d y ) . Human mtDNA gene v a r i a b i l i t y

was d e te c te d u s in g 14 e nd onuc leases , e s s e n t ia l ly th e same as th o s e in th e

B r i t i s h su rve y e xcep t Hha I and Hpa I re p la c e s Acc I and Xba I .

185

IABLE_3JLi2 j__M itgchgndria i_g0ne_variab i_ l_ itY_com £ari5D n5_bet.w een_fnD U 5e_and_m ani

GENEREGION

Rank o-f gene v a r i a b i l i t y 2 ( le a s t t o m ost) Hus dowesticus man

( t h is s tu d y ) ( F e r r is (Cann e te t a l . , 1983) a l . , 1984)

11 1 14 1 11 1 14 1

R ibosom al genes: 12s rRNA 16s rRNA

T ra n s fe r RNA genes: a l l tRNA’ s

"C ytoch rom e" p r o te in genes: CO I 4CO I I 1CO I I I 13CYT B 11ATPase 6 6NADH dehydrogenase s u b u n its ! ND 1 14ND 2 8ND 3 15ND 4 12ND 5 10ND 6 9

3414 8 6

13915 12 11 9

J113 6 8

11111581014

813 1 5 *1011

5 *4 *127915

N on -cod in g re g io n : D -lo o p 14

1 in d ic a te s number o f r e s t r i c t i o n end o nu c le a ses used in each s tu d y t o d e r iv e th e gene v a r i a b i l i t y e s t im a te upon w h ich th e ra n k in g fro m most co nse rve d t o most v a r ia b le genes i s based.

2 Gene v a r i a b i l i t y as c a lc u la te d in t a b le 3 .1 0 and 11.

* d e p ic ts m a jo r d i f fe r e n c e s in rank p o s i t io n o f gene v a r i a b i l i t y .

186

F ig u re 3.1? S im o i f ie d i s o l a t i o n and v i s u a l i s a t i o n s te p s o-f mtDNA.

M ito c h o n d r ia l DNA was is o la te d and v is u a lis e d in one o-f tw o ways dependent

on q u a l i t y and q u a n t i ty D-f v a r io u s so-ft t is s u e s . In b o th ca ses , t is s u e

(h e a r t , k id n e y , l i v e r ) was chopped up and homogenised (A ), so th a t c e l l s

were ru p tu re d to re le a s e th e in t a c t c i r c u la r o rg a n e lle s . N u c le i were

p e l le te d by low speed c e n t r i f u g a t io n , le a v in g an e n r ic h e d m ito c h o n d r ia l

su spe n s io n , w h ich can th e n be p e l le te d by h ig h speed c e n t r i f u g a t io n (B ) .

I f la rg e amounts o f t is s u e w e re re a v a i la b le , th e d i f f e r e n t a l c e n t r i fu g a t io n

s te p s a re in c re a s e d and more c a re was e x e rc is e d to a v o id as much n u c le a r

co n ta m in a tio n as p o s s ib le . The m ito c h o n d r ia l p e l le t was ly s e d t o l ib e r a te

th e c i r c u la r m ito c h o n d r ia l DNA, which was r ig o r o u s ly c le a ne d w ith many

rounds o f p h e n o l, p h e n o l-c h lo ro fo rm , c h lo ro fo rm , and e th e r e x t r a c t io n s (C

I I ) . I f t is s u e was l im i t i n g o r o f poor q u a l i t y ( l i v e r ) , th e m ito c h o n d r ia l

DNA f r a c t io n was u s u a l ly s t i l l con ta m in a te d w ith n u c le a r DNA and o th e r

c e l lu la r d e b r is , w h ich can be e f f e c t i v e ly se pa ra ted o u t by is o p y c n ic C sC l-

e th id iu m b rom ide g ra d ie n ts by U l t r a c e n t r i f u g a t io n . The lo w e r band in th e

g ra d ie n t was th e s u p e rc o ile d mtDNA (C I ) .

The p u r i f ie d mt DNA by e i t h e r is o la t io n approach was c le a ve d w ith th e

a p p ro p r ia te r e s t r i c t i o n endonucleases (D ), and th e r e s u l t in g fra gm en ts

se pa ra ted by e le c t r o p h o r e s is (E) by e i t h e r agarose o r p o ly a c ry la m id e .

Agarose was used when 5 o r 6 b a s e -c u t te rs were em ployed, g iv in g few

fra gm en ts o f la rg e s iz e , whereas p o ly a c ry la m id e was used w ith 4 base-

c u t te r s g iv in g many, s m a lle r fra g m e n ts ..1

The se pa ra ted fra g m e n ts were v is u a l is e d (F) by e i t h e r e th id iu m brom ide j1

s ta in in g (under UV l i g h t ) o r by d i r e c t s i l v e r s ta in in g . Fragm ent s iz e s were \

de te rm ined by re fe re n c e to known s iz e s ta n d a rd s .

137

tissue

(8)—-^Differencial

centrafugation

Phenol /chloroform / ether extractions

LineJr nuclear D N A

nuclear DNA m tDNA

RNA

horizontal Agarose gel

vertical Polyacrylamide gel

PurifiedmtDNA

OR

(F)Visualize

fragments

directsilverstaining

Ethidium bromide/ U V Light \

(E)Separation by gel electrophoresisi

Size standard!

mbase sa

( 0 )Endonucleaserestrictiondigestion

4KB 05KB

95KB

138

FIGLIRE_3JL2j. Sequence com parison s i t e mapping methods b).M ap lo c a t io n s .

The map lo c a t io n s o-f th e unknown v a r ia n ts o-f th e Mbo I r e s t r i c t i o n s i te s

d e p ic te d in P la te 3 .1A , by th e sequence com parison method (Cann, 1982), a re

i 1 lu s t r a te d .

A: The 1344 bp fragm en t i s gene ra ted by the Mbo I s i t e s lo c a te d a t

p o s i t io n s 9385 and 10729 o f th e re fe re n c e sequence w h ich in c lu d e s coding

sequences o f p a r t o f cytochrom e o x id ase I I I gene (CO I I I ) , th e s u b u n its o f

NADH dehydrogenase (ND3 & ND4L and p a r t o f one ND 4 ) , p lu s tw o t r a n s fe r

RNAs (tRNA g ly & tRNA a rg ) . The e r ro r a sso c ia te d w ith m easuring th e new

fragm en t bands depends upon t h e i r s iz e , a la rg e e r r o r <+ 30 bp) f o r la rg e

fra gm en ts and s im i la r l y , a s m a lle r e r ro r f o r th e s h o r te r fra g m e n ts (+ 10

b p ). The a d d it io n a l s i t e g a in e x p la in in g th e o ccu rre n ce o f th e two

fra gm en ts 1175 bp and 170 bp can be searched fo r in th e two " s e m i-s ite "

a re as , e i th e r 'A ' (between s i t e p o s it io n s 9555 and 9615 in th e ND 3 re g io n )

o r fBf (between p o s i t io n s 10499 and 10559 in th e ND4 r e g io n ) . The exact

s i t e lo c a t io n s and f u r t h e r d e t a i ls a re desc ribe d in s e c t io n 3 .3 .7 .3 .6 and

ta b le 3 .3 .J .

B: The fragm ent p a t te rn s in la n e 5), P la te 3 .1 can be mapped in two p a r ts ;

I : Fragment 468 bp is gene ra ted by Mbo I r e s t r i c t i o n s i t e s lo c a te d a t 3597

and 4065 p o s i t io n s in th e re fe re n c e sequence, s im i la r l y fragm en t 31 bp is

gene ra ted a t s i t e p o s i t io n s 3566 and 3597. Thus th e lo c a t io n o f th e s i t e

lo s s p ro d u c in g th e sum o f th e two fragm ents 499 bp can be deduced to be a t

3597 in th e ND1 re g io n , no se a rch in g f o r s e m is ite s was n e ce ssa ry .

I I : F o llo w in g th e r a t io n a le in p a r t B I, the fra g m e n ts 67 bp and 598 bp can

be found a t r e s t r i c t io n s i t e s 2438 to 2505 and 2505 and 3103, re s p e c t iv e ly ,

o f th e re fe re n c e sequence. C onsequen tly , th e s i t e lo s s can be deduced to be

a t s i t e 2505. However, th e g e n e ra tio n o f fra gm en ts o f th e a pp rox im a te s ize s

60 + 10 bp and 610 + 30 in d ic a te s a s i t e g a in w ith in th e same re g io n . Hence

189

-SITE GAINS

SEMISITES SEARCH

SITE"10729

SITE _ |o J 9 3 8 5 * = N D 3

3 g i l , ,

ND4

II75±301175 ±30

I70±30

SITE LOSS

LOSS OF SITE 3597

SITE35664 • ND I ^ SITE

cRNA ND2 i >4065tRNA tR N AGLN

468FRAGMENTS:

499

SITE LOSS & GAIN SEMISITES SEARCH'1}JE2505

SITE 2438 • N D

6I0±20FRAGMENTS:

6I0±20

t h i s a d d it io n a l Mbo I r e s t r i c t i o n s i t e can map between th e p o s i t io n s 2498

and 2538 ( s e m i- s ite re g io n A, in th e 16S rRNA r e g io n ) , o r between th e

p o s i t io n s 3003 and 3043 ( s e m i- s i te re g io n B, in th e ND1 g e n e ).

These r e s t r i c t i o n maps d e p ic te d in F ig u re 3 .2 were m o d if ie d -from S to n e k in g

e t a l - , (1 9 86 ).

191

F ig u re 5.3? D i s t r i b u t i o n o-f t r a n s i t i o n s and t r a n s v e r s io n s i n Mus

domesticus mt DNA genome.

The c i r c u la r mouse m ito c h o n d r ia l DNA genome i s p o r tra y e d in a l in e a r

■ fashion, w ith each m ajor gene re g io n id e n t i f ie d by a b b re v ia t io n s ( d e ta i ls

see f ig u r e legend 3 .4 A ).

The lo c a t io n s o f th e base changes fro m th e 75 documented s i t e g a in s a re

in d ic a te d by th e h o r iz o n ta l l in e s . The l in e s above th e mt b a r re p re s e n t 40

t r a n s i t io n s and be low , 35 t ra n s v e rs io n s .

192

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9 d i v : £ d i v

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VN d^ S3L

1

F i g u r e 3 . 4 A '• L o c a t i o n s o-f c l e a v a a e s i t e s and - f u n c t i o n a l r e g i o n s i n Hus

d o m e s t i c u s mtDNA d e t e c t e d w i t h 11 r e s t r i c t i o n e n d o n u c le a s e s .

L o c a t i o n s o-f v a r i a b l e and c o n s t a n t c l e a v a g e s i t e s i n 53 t y p e s o-f H u s

d o m e s t i c u s m i t o c h o n d r i a l DNA -from 638 i n d i v i d u a l s ( 4 3 0 - t h i s s t u d y ; 208-

F e r r i s e t a i . , 198 3 ) , d e t e c t e d w i t h 2 -1 1 ® r e s t r i c t i o n enzymes.

The h o r i z o n t a l bar r e p r e s e n t s t h e 16295 b a s e - p a i r c i r c u l a r m i t o c h o n d r i a l

genome drawn i n a l i n e a r -form, w h ich i s o r i e n t a t e d f o l l o w i n g B ib b e t a l , ,

( 1 9 8 1 ) , t h e genes a r e i n d i c a t e d as f o l l o w s : Ribosomal RNA genes (12s and

1 6 s ) ; tR N A 's and s p a c e rs den o ted by t h e shaded b l a c k a r e a s ; genes encod ing

t h e c y to c h ro m e p r o t e i n s i n c l u d i n g , c y to c h ro m e B, c y to c h ro m e o x id a s e s I I I

and I I I and ATPase s u b u n i t s 6 and 8 (Cyt B, CO I , CO I I , CO I I I , A6 and AB

r e s p e c t i v e l y ) ; NADH dehyd rogenase s u b u n i t s 1 - 6 , 4L ( 1 - 6 , and 4 L ) ; t h e

d i s p la c e m e n t lo o p p l u s a d j a c e n t n o n - c o d in g r e g i o n s d e p i c t e d by d ia g o n a l

sh ad i ng.

The l i n e s be low t h e mt genome b a r i n d i c a t e t h e mapped l o c a t i o n s o f t h e 97

v a r i a b l e s i t e s f o r H u s d o m e s t i c u s mtDNA (see t a b l e s 3 .3 A - K f o r d e t a i l s of

n a t u r e o f t h e base c h a n g e s ) . The t r i a n g l e s i n d i c a t e t h e 26 new v a r i a b l e

s i t e s d e t e c t e d i n t h e B r i t i s h p o p u l a t i o n s w i t h same p a r t i c u l a r s e t o f

enzymes, n o t p r e v i o u s l y r e c o r d e d i n a d d i t i o n t o t h o s e a l r e a d y r e p o r t e d (the

p o s i t i v e and n e g a t i v e s i g n s show w h e th e r t h e y were g a i n o r l o s s m u ta t io n s

r e l a t i v e t o t h e known base s e q u e n c e ) . L i n e s above t h e mt b a r r e p r e s e n t the

123 c o n s t a n t s i t e s i e . s i t e s p r e s e n t i n e v e r y i n d i v i d u a l examined ( t a b l e

3 . 4 , p o i n t s 1-11 f o r a co m p re h e n s iv e l i s t i n g o f t h e s e s i t e s ) .

* H i n f I , Mbo I , Hae I I I , Taq I , Hpa I I , Fnud I I , Ava I I , H in d I I I , Hinc

I I , Xba I , and Acc I .

* ND’ s 1 -6 , 4L were p r e v i o u s l y c l a s s i f i e d u n i d e n t i f i e d r e a d i n g f rames (URR

1 - 6 , 4L5 a re now c a t e g o r i s e d as 7 NADH dehyd rogenase s u b u n i t s , w h i l s t URR

A6L. i s now c la s s e d as ATPase s u b u n i t 8.

I

■ = »

196

0904

Page

iv Y-chromsome

xiv creeingxviiixix Skskholm

Brtishxx

colnesxxi nmtDNA

EDayFrequencise

2 controversy8 homoplasmy9 Scallops14 impediemnts16 adaptabitity17 Niskioka25 indicate

51 198254 Cann,57 (1/S-1)58

627987 indiividual

93 dispersal95 (p)97 1983)99 cefficent106 Bibb et aL,107 Ferris etal.,108 asssayed 125 dfferences

E. Coli130 Greeberg131

ABSTRACTY-chromosome

TABLE OF CONTENTS screening

delete "sites"SkokholmBritishdelete "genotypes"clonesmtDNAEdayFrequencies

CHAPTER 1 controversay homoplasy

scallops impediments

adaptability Nishoika

indicates

CHAPTER TWO(1982)Cann

(1/Vs-1)delete "and as such were removed from the

data set"PROPORTIONAL SPACING!! PROPORTIONAL SPACING!!

individual

CHAPTER THREE dispersalM(1983)

coefficentBibb etal.,Ferris et al.,

assayed differences E. coli Greenberg

add "in human mtDNA"

140 add "* indicates fragment differences from the k.b.s. (pattern A)n

150 turn superscripts "2" on155 Britsh British156 shift "recorded)" to second column

add superscripts "1-7" to table columns159 site., site,160-168 add superscripts "1" and "2" to tables171 phenlyalaniine phenylalanine207 transitins transitions218 [ ■1 (shaded)

CHAPTER FOUR232 Brooker, Brooker,234 details detailed

of 14 the of the 14for for 3 hours for 3 hours

238 isles of Westray. isle of Westray,249 Add "Either the observed mtDNA sequence

divergences between the two races arose251 possibilty possibility261 variabilty variability264 controversy controversay265 clone (1) clone 1267 fig. 4.4 fig. 4.5281? detials details

Snanday Sanday293 add ">"298 tree trees309 Add Campbell. J.B. (19771 The Upper

Palaeolithic of Britain. Oxford Univ. Press.

CHAPTER FIVE322 outlyer outlier326 elasped elapsed339 delete definitions358 genotuype genotype

unshdaded unshaded360 see see chapter 6365 10 g 10}ig

CHAPTER SIX368 homolouous homologous370 commomly commonly372 Eday mice Eday and Orkney mice373 specfic specific

O/ sj u.*-f 1 /o 1 .0^-/0S=0.9456 S=0.942

378 add brackets (At the timeintroduced).

379 mtDNa mtDNA387 Hence However,391 (4) (9)

BOC BOU412 add lambda symbol

0 •

4294504-^3451i+-a.4 4-a . io

501

Natufian & Aotsuka,

C.V\fOcr>£>«\ 3G tvicAsword,

1_«nrewA O-XX\svi p\

x = <f\2t

CHAPTER SEVEN Naufian (?)& Aotsuka,V\ <-o ‘■>'O Serna .\sword

<nQ

APPENDIX TWO CHECK EQ. 3

X =<72t [3]

were

J

196

F ig u re 3 .4B (o v e r la y ) s L o c a t io n s o f c le a v a g e s i t e s and - fu n c t io n a l re g io n s

in th e B r i t i s h House mouse u s in g th re e a d d i t io n a l r e s t r i c t i o n

endonuc leases .

The 16295 b a s e -p a ir c i r c u la r mouse mtDNA genome has been drawn as a l in e

s t a r t in g a t th e th e - f i r s t 5 ’ n u c le o t id e o-f th e tRNApH* gene as in th e

p u b lis h e d re fe re n c e sequence (B ib b et al., 1981), and a n n o ta te d as in

f ig u r e 3 .4A . The l in e s be low th e mt genome bar i l l u s t r a t e th e 43 v a r ia b le

(L is te d in d e ta i l in ta b le 3 .3 L -N ; + and - in d ic a te s g a in o r lo s s

m u ta t io n s ) , d e te c te d in 430 B r i t i s h House m ice u s in g th e a d d i t io n a l

r e s t r i c t i o n endonuc leases A lu I , Rsa I and Sau 96 I . The l in e s above th e

ba r re p re s e n ts th e 107 c o n s ta n t s i t e s d e te c te d (L is te d ta b le 3 .4 , p o in ts

1 2 -1 4 ).

197

EIGyR E_3^5i D is t r ib u t io n o f c le a va g e s i t e s (c o n s ta n t and v a r ia b le ) a c ro ss

th e mouse mtDNA genome f o r each o f th e fo u r te e n r e s t r i c t i o n endonuc leases .

The lo c a t io n o f 370 c le a v a g e s i t e s (140 v a r ia b le ; 230 c o n s ta n t) d e te c te d in

Hus domesticus mtDNA mapped s e p a ra te ly f o r each o f th e fo u r te e n r e s t r i c t io n

endonuc leases used. The mt genome has been l in e a r iz e d and a n n o ta te d as

i l l u s t r a t e d in f i g . 3 .4 A .

The d is t r i b u t io n o f c le a va g e s i t e s a c ro ss th e mt genome p e r r e s t r i c t io n

enzyme was te s te d t o d e te rm in e w he ther th e observed p a t te r n s d e v ia te fro m

random e x p e c ta t io n s . C h i-s q u a re , p r o b a b i l i t y and C.D v a lu e s a re g iv e n f o r each

d is t r ib u t io n by each enzyme.

1 3 8

'ND III

e>A i

INC II

CC I

VA II

NUD II

-iPA II

V - 1

% = I H ni (R)

V-1, c- 2

^ 3.1»ns ( f t )

tv-3

ir)

oo; to

KILOBASES

JAQ I

HAE llll

HINF I

MfiO I

ALU I

RSA il

SAU<96 I-

JL

■

i

.fi

i *-

m n f

t

1 1<

: Z• cc •' *-" Q

2: i__

U .

TT

iT O

i r

rf

T7!u .

i !j

- - I

■ ■ I I

+ + fi i1 JL•T.ri Mi• i

. L i - l ii T! i'l‘‘

F T! *:J l

I 1 If, 1

\ - " J lI . ' I l l

TTT

11 !! i l III ITTII II! i *! i i !

n

rp r r -wX, = 3.0lns (R)

QZ

- : i“ * *° • - i ' j y i— - . = | i*”* .*•o £ £ 6 9 Qo < < o 2 2

C- 17X --!.r (R)

9X,= 2.45ns IR)

CD= i.k

CD ='

_L 10 12 14KILOBASES

16

199

E I§yB I_3» .^ i_ D is t r ib u t io n o f c le a va g e s i t e s among th e mouse m ito c h o n d r ia l

gene re g io n s .

The number o-f c leavage s i t e s ( v a r ia b le , c o n s ta n t, t o t a l , g a in and lo s s

m u ta tio n s - g raphs A-E, r e s p e c t iv e ly ) a re shown to be h ig h ly c o r re la te d

w ith th e s iz e o f th e gene re g io n ( in b a s e -p a ir s ) .

The a b b re v ia t io n s a re as fo l lo w s f o r g ra ph s A-C:

ND 1 - 6 ,4L - NADH dehydrogenase s u b u n its ; A6 and 8 - adenos ine t r ip h o s p h a te

(ATPase’ s ) ; CO I —I I I - cy toch rom e o x id a s e s u b u n its ; CYTB - cy toch rom e B; DL

and LS - m ajor noncod ing re g io n s , th e D -lo o p and l i g h t s tra n d o r ig in o f

r e p l ic a t io n ; tRNA - a l l th e t r a n s fe r RNA’ s ; 12s and 16s -1 2 s and 16s

r ib o so m a l RNA’ s .

The s iz e o f each gene re g io n in b a s e -p a irs were c a lc u la te d from th e k .b .s

(B ibb et al.f 1981).

B .P .R . - base p a ir s pe r gene re g io n .

------------- upper and low er 95V. c o n fid e n c e l i m i t s , r e s p e c t iv e ly .

A b b re v ia t io n s f o r g raphs E & F:

NONC : n on -co d ing re g io n (D -lo o p and l i g h t s tra n d o r ig in ) .

tRNA : T ra n s fe r RNAs ( a l l 2 2 ) .

rRNA : Both 12S and 16S r ib o so m a l RNAs.

KP ; Cytochrom e p ro te in co d in g genes (CO I - I I I , Cyt B, ATPase 6 & 8 ) .

NADH ; NADH dehydrogenase s u b u n its (1 -6 & 4 L ) .

200

A. V A r \ l A b L b

NDI

ND 5

ND 4comUJ •CO/

9I6S•tRNA

CYTND 3

ND2ND4L

r=0.07, fvolue = 5.71*

400 •00 1200 IS00BPRB CONSTANT

40 H IBS

•CO/ND 5

12 SCO//uotuK- •CYTB

ND2

r=0.54> lv a lu e s 4 2 .5 * * * “

400 000 1200BPR

C TOTAL

I6S

t R N Auj

CYT

NDJND 3

ID4Lr= 0.85, f va lu e r50.4 * * *

400 800 1200BP R

5«o r s 0<95 f u i ' f Valu'*200q ---

2500 --------------------Jooo ----____3500 _<00Qflpft <500 *_

5000 - ♦ ................ •55o0 -----(5000 T “ * » .tf5oo

GA//V

HADHh o n c

t R kl A

F ia u re 5 .7 : D i s t r i b u t i o n o-f v a r i a b le s i t e s w i t h in mouse m i to c h o n d r ia l

genes.

The lo c a t io n D f 140 v a r ia b le s i t e s d e te c te d in 638 m ice w ith 2 -14

r e s t r i c t i o n e ndonuc leases , d iv id e d in t o each m ito c h o n d r ia l gene re g io n i s

shown. The le n g th o-f th e l i n e drawn re p re s e n ts th e a p p ro p r ia te gene, and i s

p r o p o r t io n a l t o th e s iz e in b a s e -p a irs o-f th a t re g io n (a p p ro x im a te s c a le ;

lcm= lOObp). Coding re g io n s a re a lig n e d to t h e i r 5 ’ ends, see - f ig u re 3 .4A

■for a b b re v ia t io n s . A l l 22 t r a n s fe r RNA’ s a re poo led and s i t e s mapped in

a p p ro x im a te p o s i t io n s o n ly .

The d is t r ib u t io n o f th e v a r ia b le s i t e s per gene re g io n was te s te d to

d e te rm in e w he ther th e observed p a t te rn d e v ia te s s ig n i f i c a n t l y fro m a random

d i s t r i b u t i o n . C h i-s q u a re v a lu e s and p r o b a b i l i t y e s tim a te s a re g iv e n f o r

each gene. The c o e f f ic e n t o f d is p e rs io n (C.D. <1 = u n ifo rm CU3; >1 -

clum ped CC3) i s shown where d is t r ib u t io n s d i f f e r s ig n i f i c a n t l y fro m random

ND5 r- JjL

16SRNA-

COI -

ND4

CYTB.

ND 2.

ND1 -

12SRN|J

• +D LOOpIL

com If. I c o n _

ATPase6.

ND6.

ND3.

ATPase 8-

1 J_L32

T TP T I 1 ill

CD=i.n(c)

CD=i 7(0

' Y r T * -* Z * 7 *x j = S.M * * C D » u (C)

OC,2= 12 46 * * * CD= 1.11(C)

= 3 7 1 * * CD= 1 76 (C)

X ,2 = i«ins ( * )

OX-f = 5 10 C D = 0 . 4 0 (U)

“X^ = 2-90nS (ft)

i 1 iy j = * . T l + * CD = 133 (C)

lllf 1= 2.93ns (R )

X jf = > » ns (R)

T 1

- ♦ ▼ 1 1

111x j = 0176ns (R )

% 1 = 1.65n s (R)

‘’X j j = 2.73 (R)

(ND)

N D 4 l . (ND)

204

FIGURE 5 .8* O rg a n is a t io n o f th e D - loo p c o n ta in in g re g io n i n m ice .

The n in e v a r ia b le s i t e s , d e te c te d by a l l r e s t r i c t io n enzymes used, a re

shown mapped ac ross th e l in e a r iz e d mt D -loo p re g io n in Mus dowesticus. The

m a jo r non -co d ing re g io n , th e D -loo p in mtDNA in th e mouse spans from the

t r a n s fe r RNA PHE gene to th e t r a n s fe r RNA PRa gene. The c e n t r a l p o r t io n

( in d ic a te d by th e heavy l in e ) i s th e most conserved re g io n c a l le d th e

" c e n t r a l conserved re g io n " (CCR). The "conserved sequence b lo c k s " (CSB)

and " te rm in a l a s s o c ia te d sequences" (TAS) a re in d ic a te d by boxes on th e

l e f t and r ig h t o f th e d - lo o p re g io n , r e s p e c t iv e ly .

1. and 2. re p re s e n t e n la rg e d d e ta i le d re g io n s o f secondary s t r u c tu r e a t

th e 5 ’ end (u n d e r lin e d sequences in d ic a te th o se lo c a te d in th e CSB1) and 3 '

end (u n d e r lin e d sequences co rrespond to th e te m p la te sequenecs im p lic a te d

in th e a r re s t o f th e D -loo p in th e TAS a rea) o f th e D -lo o p , r e s p e c t iv e ly .

On b o th 2° s t r u c tu r e s th e lo c a t io n and n a tu re o f th e v a r ia b le s i t e s noted

in Hus dowesticus a re d e p ic te d .

[D iag ram m o d if ie d from C a n ta to re & Saccone, (1 9 87 ); Brown et a l (1986 );

Saccone e t aim, (1 9 8 7 ) . ]

205

OJO0

1Q«•

ZooUJcCOz8tzozcco<z

m

COi/ll

"OcreV ^

JS * * 0 .0

u o£*o .O OJ ^c - o s > l> «j re re D > G 4o-fc o l5? yi i- -o o*" ° £ U

-re ^

uo £ Q<

c o4J £O 4J

u ulH q_

coac^ a. i o U < ^ U U H ^

I

FIGLJRE_3jl9 : S u b s t i tu t io n r a te m a tr ic e s .

A. In t r a s p e c i - f i c s i l e n t r a t e s oi s u b s t i t u t i o n w i t h in th e house mouse,

i n d i c a t i n g th e predom inance o-f A < -> G and C -> G t r a n s i t i o n s and th e slow

a c c u m u la t io n o-f C -> T t r a n s i t i o n s p lu s G -> T. C <-> A t r a n s v e r s io n s .

Dashes on th e a rro w s in d i c a t e th e a c tu a l numbers o-f each ty p e o-f base

change observed in th e p r o te in - c o d in g genes in Hus dowesticus ( t h i s

s t u d y ) . Whereas C <-> G and T < -> A a re never observed .

B. In t r a s p e c i - f i c non-synonymous (a m in o -a c id rep lace m en ts ) s u b s t i t u t i o n

m a t r ix in Hus domesticus> r e v e a l in g th e - fa s te s t r a t e s in v o lv e a b ia s

to w a rd s A -> G t r a n s i t i n s and G < -> C, p lu s T -> G t r a n s v e r s io n s . Slower

r a t e s a re seen in A <-> T, A < -> C, G -> A t r a n s v e r s io n s and C-> T

t r a n s i t i o n s . In a d d i t i o n i t appears C -> T and G -> T t r a n s i t i o n s and

t r a n s v e r s io n s r e s p e c t i v e l y a re never seen.

C. I n t e r s p e c i f i c s i l e n t r a t e s among mouse, r a t and b o v in e mtDNA’ s , showing

a p re -p o n d e ra n ce (- fas t a c c u m u la t io n ) o-f C < -> T and A < -> G t r a n s i t i o n s , in

a d d i t i o n t o a s low o cc u r re n c e A -> C o r T t r a n s v e r s io n s . Unobserved changes

(d e p ic te d by la c k o-f a rrow s in th e m a t r ix ) in c lu d e G <-> C and G < -> T

t r a n s v e r s io n s (Lanave et a l 1984, 1985; C a n ta to re & Saccone, 1987).

I n te r s p e c i - f i c and in t r a s p e c i - f i c s i l e n t (synonymous) s u b s t i t u t i o n s agree

v e ry w e l l (compare A and C m a t r ic e s ) .

207

A.

4 - T

V ....................... "*---------------------- ♦v v ✓

N x '^ X ✓

\ N /n x /

^ * Xjb /V 0✓ ' -

✓ v v/ N X✓ N X

j / V------------------------- iim m iiu i ■ — a

m --------------------

B.

\X

C.

► SLOW

» FAST

x x N N N N X

x N

T

4— -

xx N

X > NN N N NX

* ; ♦

208

PLAIE_3JLii_Seguence_com2§[:i5on_5i. te_rnaeeing_methgd5_a)_i _Gel__£icturei

Mbo I f ra g m e n ts , f rom a number o f mouse mtDNAs from d i f f e r e n t B r i t i s h

l o c a l i t i e s , were s e pa ra te d by e le c t r o p h o r e s is th ro u g h a 5V. p o ly a c ry la m id e

ge l and s i l v e r s ta in e d . The f ragm en t s iz e s o f th e known r e fe r e n c e sequence

( th e la b o r a to r y mouse; B ibb et a i 1981) c u t w i th Mbo I i s d e p ic te d

a d ja c e n t t o la n e 1.

A ( la n e 5 ) : i n d ic a t e s a s in g le base change accoun ts f o r th e d i f f e r e n c e s

between th e known re fe r e n c e p r o f i l e found in la n e s 1 & 6. The v a r ia n t

p a t te r n ( la n e 5) la c k s a 1344 bp f ragm en t wh ich i s p re s e n t in th e known

sequence ( la n e 6 ) , y e t has in i t s p la c e two new bands o f a p p ro x im a te ly 1179

bp (1175 > 30 bp) and 165 bp (170 + 30 b p ) . F ig u re 3 .2 e x p la in s how t h i s

s i t e ga in can be i n f e r r e d t o map to a p r e c is e lo c a t i o n where th e

s u b s t i t u t i o n r e s p o n s ib le f o r chang ing th e f ragm en t p a t t e r n s o c c u r re d .

B ( la n e 2 ) : T h is fragm en t p a t t e r n i s more complex t o e x p la in , i n v o lv in g

s u b s t i t u t i o n s in two a reas o f th e mtDNA genome, f i r s t l y , a s i t e lo s s i e .

th e lo s s o f f ra gm en ts 468 bp and 31 bp and th e subsequent g a in o f a

fragm en t s iz e d 499 bp, wh ich i s th e sum o f th e l a t t e r two f ra g e m ts ( f i g .

3 . 2 b l ) . The lo s s o f 598 bp and 67 bp f ra g m e n ts p lu s th e appearance o f

f ra g m en ts s iz e d 614 bp (610 + 10 bp) and 51 bp (50 + 10 bp) in v o lv e s a lo s s

o f one s i t e and a g a in o f a new s i t e (see f i g . 3 . 2 b I I , f o r a f u l l e r

e x p l a i n a t i o n ) .

Lanes 3 & 4 re p re s e n t mice w i th mtDNA w i th p r o f i l e s i d e n t i c a l t o th e

la b o r a to r y re fe re n c e sequence ( p a t te r n A ).

The mouse samples used in th e g e l in c lu d e ; la n e 1 & 6 - in b re d s t r a i n ,

C 5 7 /b l /6 ; la n e 2 - H a rra y , M a in land Orkney; Lane 3 - B u r t o n - o n - t r e n t ,

S t a f f o r d s h i r e ; lane 4 - B irm ingham ; la n e 5 - East G r in s te a d , K en t.

209

IN 6145S 598-IN 499- _SS 468 -

LO S S 67 ~ GAIN SI „ LOSS

LABORATORY MOUSE REFERENCE PROFILES

U N K N O W N N E W VARIANTS

M B O

13441179

165

Hind I I I r e s t r i c t i o n f ragment o-f B r i t i s h Hus dom esticu*

mtDNA.

Mi t o c h o n d r i a l DNA -from th e House mouse ( H u s d o m e s t i c u s ) -from v a r i o i s

B r i t i s h l o c a t i o n s , i s o l a t e d by u l t r a - c e n t r i f u g a t i o n in a sw in gou t r c t o r in

a cesium c h l o r i d e g r a d i e n t . The p u r i f i e d mtDNA was d ig e s te d w i t h

r e s t r i c t i o n endonucleases Hind I I I and HPA I I , t h e f ra g m e n ts were separated

on 0.75'/. agarose g e l s and e th id iu m bromide s t a i n e d ( M a n i a t i s e t a l . , 1982).

M o le c u la r we ig h t s tan d a rd s are i n d i c a t e d t o the r i g h t i n p i c t u r e 1 (lambda

c u t w i t h Hind I I I ; s i z e s of f ra g m e n ts in k i l o b a s e s ) and t o t h e l e f t o f the

gel p i c t u r e 2 (1 k i l o b a s e l a d d e r ; s i z e s i n b a s e - p a i r s ) .

Hind I I I : The r e fe r e n c e sequence (C57/BL/6) d ig e s te d w i t h t h i s enzyne was

run as an a d d i t i o n a l s i z e marker in bo th g e l s ( la n e 9; p i c t u r e 1 anc lane

8; p i c t u r e 2) the s i z e s o f wh ich , i n b a s e - p a i r s , a re shown a d ja c e n t t o lane

9 i n p i c t u r e 1. B r i t i s h mice show two p a t t e r n s w i t h t h i s hexanuc l eot i de

r e s t r i c t i o n enzyme, th e r e fe r e n c e sequence p a t t e r n A ( l a n e 2 ,4 ; p i c t u r e 1

and lane 5 j p i c t u r e 2) and a v a r i a n t p a t t e r n B ( la n es 1, 6 -8 ; p i c t u r e 1

and la nes 4 , 6 , 7 ; p i c t u r e 2 ) . P a t t e r n B r e s u l t s f rom a s i n g l e s i t e loss,

t h e f ra gm en ts 13462 and 1945 bp j o i n i n g t o produce th e l a r g e r f ragment

15407 bp.

HPA I I : Fragment p r o f i l e s produced by d i g e s t i o n w i t h t h i s t e t r a n u c l e o t i d e

r e s t r i c t i o n enzyme are i l l u s t r a t e d i n gel p i c t u r e 2 showing t h e two

p a t t e r n s A and D which are e x p la in e d i n more d e t a i l i n f i g u r e 3 .8 .

Lane d e s i g n a t i o n s :

2 1 . 1

212

P ic t u r e 1 - H ind I I I d ig e s t s ; la n e 1, DAN4 (D an ish mouse), la n e 2 , I .o - f.M S

( I s l e o-f May, F i r t h o-f F o r th , n o r th e a s t S c o t la n d ) , la n e 3 , B0T9 (B u r to n -o n -

T r e n t , S ta - f - fo rd s h ire ; shows a p a r t i a l d i g e s t i o n , when r e ru n p a t t e r n A was

obse rved ) , la n e 4 , NUT7 ( N u t - f ie ld , S u r re y , E n g la n d ) , la n e 5 , WH1 (West

Humble, S u r re y ; u n d e r lo ad e d sample, d e s ig n a te d p a t t e r n A when r e r u n ) , la n e

6, BARD6 (B a rn a c la v a n , C a ith n e s s , n o r th S c o t la n d ) , la n e 7, HARR10 (H a r ra y ,

M a in land O rk n e y ) , and la n e B, NG10 (N o r th G r in a b y , W es tray , O rkn ey ) .

P i c t u r e 2 - HPA I I d ig e s t s ; la n e 1, WEST4 (S k e lw ic k , W estray , O rk n e y ) , la n e

2, IRE7 (B e l- fa s t , N. I r e l a n d ) , la n e 3, C 57 /B L /6 ( l a b o r a t o r y r e fe r e n c e

sequence ); H ind I I I d ig e s t s ; la n e 4 ,7 , IRE11 and IRE7 ( B e l f a s t , N.

I r e l a n d ) , la n e 5 , B0T6 (B u r to n -o n T r e n t , S t a f f o r d s h i r e ) , and la n e 6, EDAY8

(N ew b igg in , Eday, O rkn ey ) .

213

PLATE 5 . 3? Xba I r e s t i c t io n fra g m e n ts Df B r i t i s h Hus do aes ticu s mtDNA.

The f ra g m e n ts p roduced by d ig e s t io n o f B r i t i s h Hus doaesticus mtDNA w i th

th e h e x a n u c le o t id e r e s t r i c t i o n enzyme Xba I a re shown a f t e r e le c t r o p h o r e s is

ins (1) a 57. p o ly a c ry la m id e ge l ( f i g . 3 .2 .1 ) s ta in e d w i th s i l v e r

(T e g le s tro m , 1986), and (2) a 0.77. agarose ge l ( f i g . 3 .2 .1 ) s ta in e d w i th

e th id iu m b rom ide (M a n ia t is et a i . , 1982).

A l l samples examined were i n v a r ia n t f o r t h i s enzyme, d is p la y in g o n ly th e

la b o r a t o r y re fe r e n c e sequence p a t te r n A [ C 5 7 /b l / 6 , la n e 1, f i g . 3 .2 .2 1 . In

t h i s f i g u r e p a t t e r n A i s re p re s e n te d by W15 [W es tray , Orkneys la n e s 2 in

f i g . 3 .2 .1 and 3 .2 .2 1 and T16 [T a u n to n , Somersets la n e s 3 i n bo th f i g u r e s ] .

The m o le c u la r w e ig h t m arkers a re in d ic a te d t o th e l e f t (1 K i lo b a s e la d d e r

i n b a s e -p a i r s ) and th e r i g h t hand s id e s (lambda H ind I I I i n K i lo b a s e s ) o f

th e g e ls , f i g u r e s 3 .2 .1 and 3 .2 .2 r e s p e c t i v e l y . A d d i t i o n a l l y th e e xac t

r e s t r i c t i o n fra gm en t le n g th s o f th e re fe r e n c e sequence ( p a t te r n A) a re

shown a d ja c e n t t o la n e 3 f i g . 3 .2 .1 .

T h is f i g u r e i l l u s t r a t e s i t i s p o s s ib le t o v i s u a l i z e th e d ig e s t io n p a t t e r n s

u s in g e i t h e r agarose o r p o ly a c ry la m id e g e ls . However, te n t im e s as much

mtDNA was r e q u i r e d f o r d e te c t io n in agarose g e ls (40-50 n g ) , whereas o n ly

1-5 ng mtDNA i s needed f o r s e n s i t i v e s i l v e r s t a i n i n g o f p o ly a c ry la m id e

g e ls . A lso , a l th o u g h agarose g e ls r e s o lv e f ra g m e n ts g re a te r than 5KB,

f ra gm en ts le s s than 500bp (average s iz e s o f most f ra g m e n ts produced by 4 -

base c u t t e r s ) a re more c l e a r l y d i f f e r e n t i a t e d u s in g p o ly a c ry la m id e .

?.1 a

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215

P L A T E ^ ^ f^ HPA I I r e s t r i c t i o n fra gm en t p r o f i l e s in some B r i t i s h

Hus dom esticus mtDNA.

T h is f i g u r e , o f a s i l v e r s ta in e d 57. p o ly a c ry la m id e g e l , shows th e p ro d u c ts

o f d ig e s t io n w i th th e t e t r a n u c le o t i d e r e s t r i c t i o n enzyme HPA I I . The

m o le c u la r w e ig h t marker lambda Bgl I f ra g m e n ts a re l i s t e d t o th e l e f t o f

th e gel in b a s e - p a i r s . A l l mice show th e c h a r a c t e r i s t i c p a t t e r n D which

d i f f e r s from th e r e fe r e n c e sequence ( p a t t e r n A) by a s in g le s i t e lo s s .

Hence, w i th th e lo s s o f f ra gm en ts 813 bp and 68 bp, a s in g le la r g e r

fragm en t e q u iv a le n t t o t h e i r sum, 881 bp i s ga ined (+ in d ic a t e s p o s i t i o n in

th e g e l ) .

Lane 2 - Ml ( I s l e o f May, F i r t h o f F o r t h ) ;

Lane 3 - E l (Eday, O rkney) ;

Lane 4 - W1 (W estray , O rkn e y ) ;

Lane 5 and 6 - F1,F2 (Fa ray , O rkney) ;

EA r e p r e s e n ta t i v e p r o f i l e o f HPA I I p a t t e r n A can be seen in p l a t e 3 .2 .2 ,

la n e s 2 and 3 on a 0.757. agarose ge l s ta in e d w i t h e th id iu m brom ide. 3

216

HPA II

ABGLI M

1 2 - 3 - 4 - 5 - 6 -PATTERN D

217

^aq I d ig e s ts o-f B r i t i s h ft us dcm esticus mtDNA.

E le c t r o p h o r e s is o-f Taq I mtDNA f ra g m e n ts on a s i l v e r s ta in e d 57.

p o ly a c ry la m id e g e l .

Lane 1 - Ml ( I s l e o-f May, F i r t h o-f F o r th ) shows p a t t e r n A, th e same p r o f i l e

as th e r e fe r e n c e sequence, th e s iz e s o f wh ich a re i n d ic a t e d t o th e l e f t i n

b a s e - p a i r s .

Lane 2 - E l (Eday, O rkn e y ) ; Lane 3 - W1 (W estray , O rk n e y ) ; Lane 4 and 5 -

F I , F2 (F a ra y , O rkn ey ) ; a l l show th e v a r i a n t p a t t e r n D. T h is d i f f e r s from

p a t t e r n A by a s in g le s i t e lo s s a t 9577, w i th th e r e s u l t a n t lo s s o f

f ra gm e n ts 1907 and 258 bp, and th e g a in o f a la r g e r 2167 bp f ra g m e n t .

C : i n d i c a t e s th e d i f f e r e n c e s between th e two p r e v io u s l y d e s c r ib e d

f ragm en t p a t t e r n s . 3

218

TAQ I

E, W,

22451 7 7 31403

m

1 2 3 4 5A B B B B

PLATE 5 . 6 i Hae I I I r e s t r i c t i o n p r o f i l e s o f B r i t i s h Hus dowesticus mt

DNA.

Fragments produced by th e d ig e s t io n o f mouse mtDNA w i t h th e r e s t r i c t i o n

enzyme Hae I I I on a s i l v e r s ta in e d 57. p o ly a c ry la m id e g e l . T h is ge l shows

f o u r d i f f e r e n t mtDNA p a t t e r n s from f o u r i n d i v i d u a l s each r e p r e s e n ta t i v e o f

t h e i r r e s p e c t iv e p o p u la t io n s . The re fe r e n c e sequence (C 5 7 /b l /6 ) e x h i b i t s

p a t t e r n A and was used as th e m o le c u la r w e ig h t s ta n d a rd ; i t i s shown in

la n e 1 (o n ly some o f th e fragm ent s iz e s , g ive n i n b a s e - p a i r s , a re in d ic a te d

a lo n g s id e ) . Lane 4 (EG5! East G r in s te a d , Kent) shows th e s im p le s t p a t te r n

(U ) , a t t r i b u t a b l e t o th e lo s s o f one s i t e , which la c k s f ra g m e n ts 550 bp and

33 bp bu t has ga ined a 583 bp f ra g m e n t.

EG6 (East G r in s te a d , K e n t ) , la ne 3, shows p a t t e r n T wh ich in v o lv e s two s i t e

lo s s e s . One la r g e r f ra g m e n t , 1574 bp, has been ga ined w i th th e consequent

d isa pp earence o f t h r e e s m a l le r f ra g m e n ts , 945, 5 6 0 ,and 69 bp.

BIRM5 (B irm ingham, West M id la n d s ) , la n e 5, shows p a t t e r n V, which a ls o

in v o lv e s two s i t e lo s s e s , s i t e s 15189 and 12378, w i t h th e subsequent lo s s

o f two s m a l le r f ra g m e n ts f o r each, 550 and 33 bp, 852 and 39 bp, and th e

r e s u l t a n t g a in o f two l a r g e r f ra g m e n ts , 583 bp and 891 bp r e s p e c t i v e l y .

F i n a l l y th e most complex p a t te r n (R ), la n e 2 - HARR3 (H a rray , M a in land

O rkney) , in v o lv e s th e lo s s of f ra gm en ts 1362 bp and 945 bp and th e g a in o f

a la r g e r fra gm en t 2307 bp, accounted f o r by one s i t e lo s s . A d d i t i o n a l l y ,

t h e lo s s o f s i t e 7541 r e s u l t s i n the d isa pp e a ra n ce o f f ra gm en ts 526 bp and

51 bp, re p la c e d by th e sum of th e s e , 577 bp.

2 2 0

H A E III

221

H in f I d ig e s ts o-f Hus dow esticus mtDNA.

Fragment p a t t e r n s p roduced by d ig e s t io n o-f mtDNA w i t h th e r e s t r i c t i o n

endonuc lease Hin-f I , s e p a ra te d by e le c t r o p h o r e s is on a s i l v e r s ta in e d 57.

p o ly a c ry la m id e g e l . Lane 1 shows th e re - fe re nce sequence p r o - f i l e ( p a t te r n

A ), used as th e m o le c u la r w e ig h t m arke r, th e s iz e s o-f w h ich a re in d ic a te d

t o th e r i g h t o-f th e g e l .

Lane 2 - HARR3 (H a r ra y , M a in land O rkn ey ) ; Lane 3 - B0T3 (B u r to n -o n - T r e n t ,

S ta - f - fo rd s h i re ) ; Lane 4 - DERB1 (Derby, D e r b y s h i r e ) ; Lane 5 - EG9 (East

G r in s te a d , K e n t ) ; Lane 6 - BIRM5 (B irm ingham , West M id la n d s ) .

Lanes 3 ,5 -6 (B0T3, EG9, and BIRM5 r e s p e c t i v e l y ) a l l sha re th e same s i t e

lo s s a t 11938, w h ich i s c h a r a c te r iz e d by th e d is a pp e a ra n ce o-f f ra gm en ts 539

and 920 bp and th e appearance o f a new f ra g m en t o f 1459 bp. Lane 3 (BGT3)

has o n ly t h i s s i t e lo s s and i s d e s ig n a te d p a t t e r n Z, w h i l s t EG9 ( la n e 5)

and BIRM5 ( la n e 6) b o th share t h i s lo s s p lu s an a d d i t i o n a l b u t d i f f e r e n t

s i t e lo s s each. Hence P a t te r n 3 ( la n e 6) in c o r p o r a te s th e p r e v io u s ly

d e s c r ib e d s i t e lo s s i n a d d i t i o n t o th e lo s s o f s i t e 7973 w i th th e

subsequent d isa p p e a ra n c e o f f ra g m e n ts 367 and 144 bp and th e emergence o f a

new 511 bp s iz e d f ra g m e n t . P a t te rn S ( la n e 5) i s p roduced by th e s i t e lo s s

11938 p lu s a 1055 a t s i t e 6882 which causes th e appearance o f ano the r new

f ragm en t 853 bp, th e p ro d u c t o f th e m is s in g two f ra g m e n ts 368 and 485 bp.

Lane 2 (HARR3) shows p a t t e r n C which has two e n t i r e l y d i f f e r e n t s i t e

lo s s e s , a t 9526 and 9574, r e s u l t i n g in th e lo s s o f t h r e e f ra g m e n ts 263, 946

and 48 bp and th e consequent appearance o f one l a r g e r , 405 bp f ra gm en t.

Lane 4 (DERB1) i l l u s t r a t e s th e i n v a r i a n t p a t t e r n A, i d e n t i c a l t o th e

re fe re n c e sequence t y p e ( la n e 1 ) .

2 2 2

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223

PLATE 3.B? MBO I d ig e s ts o f Mus dom esticus mtDNft.

Fragment p a t t e r n s o b ta in e d a f t e r d ig e s t io n w i t h th e t e t r a n u c le o t i d e

r e s t r i c t i o n endonuc lease Mbo I f rom s ix Mus domesticus mtDNA’ s.

Lambda DNA d ig e s te d w i t h Bgl I was used as th e f ra g m e n t s iz e marker ( lane

7 ) , th e s iz e s o f wh ich a re in d ic a te d on th e r i g h t i n b a s e - p a i r s .

Lane 1 - Ml ( I s l e o f May, F i r t h o f F o r t h ) , i l l u s t r a t e s p a t t e r n W, which

d i f f e r s from th e r e fe r e n c e sequence p a t t e r n (A) by a s i n g le s i t e ga in a t

10564. T h is r e s u l t s i n th e f i s s i o n o f th e 1344 bp f ra g m e n t i n t o two s m a l le r

f ra g m e n ts , 1034 and 310 bp.

A l l th e Orkney I s l e s have p a t t e r n 0 ( la n e s 2 -6 ; E l , W1 and W2, FI and F2

from Eday, W estray , and Faray r e s p e c t i v e l y ) . T h is d i f f e r s f ro m the

re fe r e n c e sequence p a t t e r n by th r e e s i t e d i f f e r e n c e s , two lo s s e s and one

g a in . The lo s s o f s i t e 3599 le a ve s f ra g m e n ts 468 and 31 bp jo in e d to

p roduce a 499 bp f ra g m e n t . W h i ls t th e lo s s o f s i t e 2505 accou n ts f o r th e

d isa pp e a re n ce o f f ra g m e n ts 598 and 67 bp and th e g a in o f s i t e 2489 produces

two f ra g m e n ts o f 614 and 51 bp.

224

MBO I

F 1 ABgl 1 [base pair.s]

9649

2256

1650

EL£*IE_3-.9£ Rsa I d ig e s ts o f B r i t i s h Hus dom esticus mtDNA.

E le c t r o p h o r e s is o-f Rsa I mtDNA fra g m e n ts on a s i l v e r s ta in e d 5V.

p o ly a c ry la m id e g e l , f rom f i v e m ice. Lane 1 i s th e l a b o r a to r y mouse

re fe r e n c e sequence ( p a t te r n A) f ra g m e n t s iz e s o f which a re in d ic a te d t o t h e

r i g h t ( in b a s e - p a i r s ) . Lane 4 - DERB1 (D erby, D e rb y s h ire ) a ls o shows

p a t t e r n A.

P a t te r n C seen in la n e 3, B0T3 (B u r to n -o n -T r e n t , S t a f f o r d s h i r e ) has bcth

a lo s s and g a in a t s i t e s 9545 and 11265 r e s p e c t i v e l y , c h a r a c te r iz e d by he

d isappea rance o f f ra g m e n ts 577, 658 and 78 bp and th e appearance o f

f ra gm en ts 655, 572 and 86 bp.

Lane 5 , EG9 (East G r in s te a d , K en t) shows p a t t e r n D, which has two s i t e

lo s s e s and two s i t e g a in s . Fragments 275 and 240 bp were re p la c e d by 28-

and 232 bp, s i m i l a r l y f ra g m e n ts 274 and 495 bp were re p la c e d by tho se o

281 and 488 bp.

P a t te r n E re p re s e n te d by HARR3 (H a r ra y , M a in land Orkney) has n in e g a in s and

f i v e lo s s e s o f s i t e s compared t o th e r e fe r e n c e sequence ( p a t t e r n A ).

D e t a i l s o f th e s e can be found in T a b le 3N (Rsa I v a r ia b le r e s t r i c t i o n

s i t e s ) and f i g u r e 3a - l i s t o f f ra g m e n ts .

0 : in d ic a t e s th e m ajor fra g m en t d i f f e r e n c e s .

- o r + : i n d ic a t e s a lo s s o r g a in o f a f ra g m e n t .

226

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PLATE_3i 1 0 i A lu I d ig e s ts o-f B r i t i s h Hus dom esticus mtDNA.

Fragment produced by d ig e s t i o n o-f mtDNA w i th th e t e t r a n u c le o t i d e

r e s t r i c t i o n enzyme A lu I , se p a ra te d on a s i l v e r s ta in e d 57. p o ly a c ry la m id e

g e l .

The la b o r a t o r y mouse (C 57 /BL /6) was run i n la n e 1 as th e m o le c u la r w e igh t

m a rke r, shows th e r e fe r e n c e sequence p a t t e r n A, th e s iz e s o-f some o f th e

f ra g m e n ts i l l u s t r a t e d t o th e l e f t in b a s e - p a i r s . Lanes 4, 6, DERB1

(D e r b y s h i r e ) , and BIRM5 (B irm ingham , West M id la n ds ) a ls o show th e i n v a r ia n t

p a t t e r n A.

P a t te rn G seen in la n e 5, EG9 (East G r in s te a d , Ken t) v a r ie s from p a t t e r n A

by one s i t e lo s s a t 10826 and one s i t e g a in a t 10835. T h is i s shown by th e

lo s s o f f ra g m e n ts 365 and 30 bp, re p la c e d by f ra g m e n ts 374 and 21 bp.

P a t te rn H ( la n e 3; B0T3, B u r to n -o n - T r e n t , S t a f f o r d s h i r e ) in v o lv e s two s i t e

lo s s e s a t 6233 and 6291, ca us ing th e lo s s o f t h r e e f ra g m e n ts 450, 58 and 44

bp, s u b s t i t u t e d by a l a r g e r f ra g m e n t , th e sum o f th e s e , s iz e d 552 bp.

HARR3 (H a rra y , M a in land Orkney) as seen in la n e 2 i l l u s t r a t e s p a t t e r n B,

which a r is e s f rom a s i t e lo s s and s i t e g a in a t 3143 and 9947. S ubsequen t ly

f ra g m e n ts 650 and 45 bp a re l o s t re p la c e d by 725 bp, s i m i l a r l y 448 bp

fra gm en t i s m is s in g , re p la c e d by th e two f ra g m e n ts 359 and 89 bp. In

a d d i t i o n t o th e s e , p a t t e r n B has two more s i t e lo s s e s a t 5122 and 5253,

p lu s two more s i t e g a in s a t 5129 and 8259 r e s p e c t i v e l y . Fragments 654 and

54 bp were re p la c e d by 659 and 49 bp, w h i l s t th e f ra g m e n ts 545 and 40 bp

were s u b s t i t u t e d by th o s e o f 551 and 34 bp.

+ o r - i n d i c a t e s m a jo r d i f f e r e n c e s between p r o f i l e s .

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CHAPTER FOUR

CHAPTER FOUR: P h v lo q e o q ra p h ic p o p u la t io n s t r u c t u r e o f B r i t i s h house mouse

eQ £y !§ t iD ns_ £ rg m _ an a l¥ 5 is_ g f_m itgch g nd r i.a !_D N A i

4i i i _ I o t r g d u c t i g n i

Animal m i to c h o n d r ia l DNA (mtDNA) i s s m a l l , m a te r n a l l y i n h e r i t e d ( G i le s et

aim, 1980; G y l le n s te n , e t aim, 1985), e a s i l y p u r i f i e d , la c k s re c o m b in a t io n ,

has a s im p le sequence o r g a n iz a t io n and e v o lv e s a t a h ig h e r base sequence

r a t e (1 -10 t im e s ) tha n do n u c le a r genes (Brown e t aim, 1979; Brown, 1983;

Vawter & Brown, 1986). These q u a l i t i e s make i t t h e " d e f i n i t i v e " m o le c u la r

marker f o r t r a c i n g m a te rna l g e n e a lo g ie s (W ilson e t aim, 1985, 1989; A v is e ,

1986; H a r r is o n , 1989). I t i s e s p e c ia l l y u s e fu l i n d i f f e r e n t i a t i n g among

c lo s e l y r e la t e d c o n s p e c i f i c s ; e x te n s iv e i n t r a s p e c i f i c , g e o g r a p h ic a l l y

p a r t i t i o n e d mtDNA v a r i a t i o n has been dem ons tra ted i n p o p u la t io n s o f

mammals, a m p h ib ians , f r e s h w a te r f i s h e s and c ra b s ( r e v ie w s - see A v is e et

aim, 1987a; A v is e , 1989 ). Such m a jo r " d i v i s i o n s ’' w h ich d i s t i n g u i s h some

groups o f p o p u la t io n s i l l u s t r a t e t h a t d is p e r s a l and gene f lo w have no t

o v e r r id d e n h i s t o r i c a l in f lu e n c e s on p o p u la t io n s u b d iv i s i o n s , as re v e a le d by

mtDNA p h y lo g e n e t ic r e c o n s t r u c t io n s (Saunders et aim, 1986; Bermingham &

A v is e , 1986).

The p re sen t day d i s t r i b u t i o n o f sm a ll ro d e n ts i n t h e B r i t i s h I s l e s has le d

t o innummerable d eba tes and s p e c u la t io n s about t h e i r o r i g i n s (L e v e r , 1969;

C o rb e t, 1969; Ya lden , 1982). E a r ly w o rke rs (H in to n , 1910; B a r r e t t - H a m i l t o n

& H in to n , 1910-21) suggested i s la n d p o p u la t io n s o f r o d e n ts re p re s e n t

g l a c i a l r e l i c s , some o f which were i s o la t e d when la n d - b r id g e s were b roken ,

su bse q ue n tly re p la c e d on th e m a in lands by new fo rm s m ig r a t in g from Europe

230

CHAPTER FOUR

as th e i c e r e t r e a t e d . However, t h i s c o n c lu s io n has been re p e a te d ly

c h a l le n g e d (B e i rn e , 1952: Matthews, 1952; B e r ry et a J 1967). I t i s h i g h l y

u n l i k e l y t h a t ro d e n ts -from N o rth A t l a n t i c i s la n d s c o u ld have s u r v iv e d th e

l a s t g l a c i a t i o n , as th e whole o-f S c o t la n d , t o g e th e r w i th th e m a jo r i t y o-f

I r e la n d and a s s o c ia te d i s la n d s , were co ve red by ic e d u r in g th e t im e o-f th e

P le is to c e n e g l a c i a l maximum. T h is su g g e s ts t h a t th e e a r l i e s t p o s s ib le

c o lo n i s a t i o n o f B r i t a i n would have been a p p ro x im a te ly 10,000 B.P (b e fo re

p r e s e n t ) , t h e end o f t h e W eichsch ian s ta g e o f th e P le is to c e n e . A ls o , th e

maximum e u s t a t i c lo w e r in g o f th e s e a le v e l was about 70-100 m e tre s , th u s th e

S h e t la n d s , O rkneys and th e Outer H e b r id e s were never connected t o th e

m a in land d u r in g o r s in c e th e l a s t g l a c i a l phase.

C o rbe t (1961) sugges ted an a l t e r n a t i v e h y p o th e s is , t h a t r o d e n ts (a t l e a s t

i s l a n d fo rm s ) may have been a c c id e n t a l l y in t r o d u c e d by man, a t any t im e

s in c e th e M e s o l i t h i c , i n a d v e r t a n t l y t r a n s p o r t e d i n t h e i r l i v e s t o c k fo d d e r .

T h is id e a was su p p o r te d by B e r ry (1 9 69 ) , who suggested t h a t t h e m a jo r i t y o f

is la n d f i e l d m ice (Apodeuus spp) were t r a n s p o r t e d by th e agency o f man.

Through i t s commensal a s s o c ia t io n s w i t h man, th e house mouse (Hus

domesticus Rutty) i s a h ig h l y s u c c e s s s f u l , o p p o r t u n i s t i c , c o lo n iz in g

s p e c ie s . House mice occupy h a b i t a t s v i r t u a l l y w o r ld -w id e , w i t h th e

e x c e p t io n o f p o la r r e g io n s and c e n t r a l A f r i c a (B e r ry , 1981b). T h is s p e c ie s

i s t r a d i t i o n a l l y th o u g h t t o have f i r s t in va de d Europe, f rom th e M id d le

E a s t , le s s tha n 8 ,0 0 0 ye a rs ago, as commensals o f N e o l i t h i c fa rm e rs and

fo l lo w e d th e spread o f human g r a in c u l t u r e n o r th w a rd s (Schwarz & Schwarz,

1943; B ro th w e l1, 1981; G y l le n s te n & W i ls o n , 1987).

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CHAPTER FOUR

E v idence f ro m b io c h e m ic a l , m i to c h o n d r ia l DNA and H-2 p o lym orph ism s has

d e m o n s tra te d , th u s - fa r , v e ry l i t t l e m a c ro g eo g rap h ica l s t r u c t u r i n g o-f

commensal p o p u la t io n s th ro u g h o u t t h e s p e c ie s ’ range (B e r ry 8c P e te r s , 1977;

B r i t t o n - D a v id i a n , 19B5; F e r r i s et al•, 1983; Nadeau et a l 1988 ). T h is has

been a t t r i b u t e d t o i t s re c e n t and e x te n s iv e range e x p a n s io n , and

h i s t o r i c a l l y s t r o n g gene f lo w . However, marked m ic ro g e o g ra p h ic s t r u c t u r i n g

i s e v id e n t i n most domesticus p o p u la t io n s on a more lo c a l s c a le ( K le in et

al•, 1981; F e r r i s e t aim, 1983; G y l le n s te n 8c W ils o n , 1987; N a va jas Y

N a va rro 8c B r i t t o n - D a v id i an, 1989).

The aim o f t h i s work i s t o i n v e s t i g a t e th e e x te n t and o r i g i n s o f ra c e s o f

th e B r i t i s h house mouse u s in g mtDNA a n a ly s i s . M orphom etr ic e v id e n c e (D a v is ,

1983) i n d i c a t e s t h a t C a ith n e ss and Drkney house m ice W u s domesticus

R u t ty ) a re g e n e t i c a l l y d i s t i n c t f ro m o th e r B r i t i s h m ice , s u g g e s t in g t h a t

th e s e n o r th e rn fo rm s a re descended f ro m i n t r o d u c t i o n s . T h is i s i n g e n e ra l

agreement w i t h k a r y o lo g ic a l and h i s t o r i c a l e v id e n ce (B ro o k e r , 1982; Nash

et a lm , 1983; Renfew, 1985; B e r ry , 1985b). Each mtDNA m o le c u le c a r r i e s i n

i t s sequence th e h i s t o r y o f i t s l in e a g e , unhampered by c o m p l ic a t io n s

a r i s i n g f rom re c o m b in a t io n , which have dogged a t te m p ts u s in g d i p l o i d

n u c le a r DNA m arke rs , hence, i t can be used f o r t r a c i n g p a t t e r n s o f

c o lo n i s a t i o n , i n t r o d u c t i o n and gene f l o w . T h is approach has been

s u c c e s s f u l l y a p p l ie d t o a w ide range o f s p e c ie s in c lu d in g humans (H o r ia &

Matsunaga, 1986; Cann et a im , 1987; W i lso n et a im , 1987b; S to n e k in g 8c

W ils o n , 1989; Jones, 1990; S to n e k in g et a l . , 1990; H e r tz b e rg e t a im , 1989),

sm a ll mammals (A sh le y 8c W i l l s , 1987; MacNeil 8c S tro b e c k , 1987; B o u rs o t et

a im , 1985; Yonewaki et a im , 1986, 1988 ), l a r g e mammals (Baker et a im , 1990;

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CHAPTER FOUR

S teve n s e t al,, 1989), in s e c ts (H a l l & M u ra l id h a ra n , 1989; Sm ith e t al,,

1989) and r e p t i l e s (Bowen e t al,, 1989), t o name b u t a -few.

H ig h ly p u r i f i e d mtDNA, f rom each o f 430 B r i t i s h house m ice , f ro m 7 Orkney

I s l e s and 35 l o c a l i t i e s e lsew here in th e B r i t i s h I s l e s , was i s o la t e d and

mapped, u s in g th e h ig h r e s o lu t i o n r e s t r i c t i o n method (Cann, 1982; Cann e t

al,, 1982; Cann e t al,, 1984; S to n e k in g e t al,, 1986), w i t h r e s p e c t t o th e

p u b l is h e d sequence o f mouse mtDNA (B ibb e t al,, 1981). I have used

r e s t r i c t i o n a n a ly s is o f mtDNA t o c h a r a c t e r i s e th e g e o g ra p h ic p o p u la t io n

s t r u c t u r e o f th e B r i t i s h house mouse and hence assess th e r o l e o f

h i s t o r i c a l b io g e o g ra p h ic a l f a c t o r s in shap in g i t s g e n e t ic s t r u c t u r e .

S e c o n d ly , I employed mtDNA r e s t r i c t i o n f ragm en t p o lym orph ism s as m arkers

f o r e s t im a t in g p ro b a b le p a t t e r n s o f c o lo n i s a t i o n among th e N o r th e rn I s le s

(O rkney) and a d ja c e n t m a in land p o p u la t io n s o f house m ice . In p a r t i c u l a r I

s h a l l examine th e m ic ro g e o g ra p h ic s t r u c t u r e o f th e s e mtDNA po lym orph ism s,

compared w i t h tho se o b ta in e d from n u c le a r DNA a n a ly s e s .

4i 2i _ M a te r ia ls_ a nd _ m e th g d s i

M ice^

Three hundred and t h i r t y f o u r mice were o b ta in e d by e i t h e r 1i v e - t r a p p in g

(u s in g Longworth t r a p s ) i n o r around b u i l d i n g s , in one t o two t r a p - n i g h t s ,

Dr handcaught a t t h r e s h in g f ro m 40 B r i t i s h l o c a l i t i e s , encompassing 16

c o u n t ie s . One hundred and se v e n ty f o u r o f th e se m ice were c o l l e c t e d from

s ix i s la n d s in Orkney (on W estray , Eday, and M a in land Orkney c o l l e c t i o n s

were made a t 2 -5 lo c a t i o n s per i s l a n d , each s i t e s e p a ra te d by s e v e ra l

k i lo m e t r e s - F ig u re 4 . 1 . 2 , s i t e s 1 -5 , 7 -8 & 11-12, r e s p e c t i v e l y ) , 43 mice

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CHAPTER FOUR

■from seven l o c a l i t i e s i n th e n e ig h b o u r in g m a in land c o u n t ie s o-f C a i th n e s s

and S u th e r la n d ( F i g . 4 .1 .1 ) and 117 i n d i v i d u a l s from n in e te e n sa m p lin g s i t e s

i n th e r e s t o f th e B r i t i s h I s l e s . A l i s t o f a p p ro x im a te l o c a t i o n s , sample

s iz e and c a p tu re d a te s a re o u t l i n e d i n Tab le 4.1 (more d e t a i l s sam p ling

l o c a l i t i e s a re g iv e n i n Tab le 2 .2 , Chapter 2 ) . In a d d i t i o n , m ice were

c o l l e c t e d f rom m a r k - r e le a s e - r e c a p tu r e e x p e r im e n ts f ro m tw o f e r a l , i s la n d

p o p u la t io n s o f th e house mouse; 61 m ice f ro m th e 75ha i s la n d o f Faray

(5 9 °N ) , f rom 157 t r a p s i n 14 s i t e s w i th between 5 -1 5 t r a p s p e r s i t e (F ig u re

4 . 2 ) , f rom th r e e c o n s e c u t iv e y e a rs (1984, 1985, and 1986 ); 35 mice f rom th e

100 ha Is la n d o f Skokholm (51°N ), f rom 100 t r a p s i n g roups o f f i v e a t 20

s i t e s a l l o ve r th e i s l a n d , in autumn 1986 (F ig u re 4 . 3 ) .

^iiiii-kibgratgr^procedureSiU sing d i f f e r e n t i a l c e n t r i f u g a t i o n p ro c e d u re s (Lansman et a i . , 1981) and

C s C l-e th id iu m b rom ide g r a d ie n t u l t r a - c e n t r i f u g a t i o n (C a rr & G r i f f i t h ,

1987), h ig h ly p u r i f i e d mtDNA was i s o la t e d f ro m th e 430 house m ice from

e v e ry l o c a t i o n ( i s o l a t i o n s te p s summarised i n f i g u r e 3 .1 c i , c h a p te r 3 ) .

Each i n d i v id u a l DNA sample was d ig e s te d w i t h each o f 14 th e r e s t r i c t i o n

enzymes u t i l i s e d ( l i s t e d i n Tab le 4 . 1 ) , a c c o rd in g t D m a n u fa c tu re rs

d i r e c t i o n s (BRL, Bethesda Research L a b o r a to r i e s ) . The f ra g m e n ts were

s e pa ra ted on 57. p o ly a c ry la m id e g e ls ru n f o r f o r 3 h o u rs , and v i s u a l i z e d by

s i l v e r s t a i n i n g (T e g e ls t ro m , 1986). R e s t r i c t i o n f ra g m e n ts r e s u l t i n g f rom

H ind I I I d ig e s t s o f Lambda DNA and th e c o m m e rc ia l ly bought 1 K i lo b a s e

Ladder (BRL), were used as m o le c u la r w e ig h t m a rke rs .

^ i 2 i 3i _ D a ta _ A n a ly s is i

A l l d i s t i n c t i v e mtDNA r e s t r i c t i o n f ragm en t d ig e s t io n p r o f i l e s produced by a

g iv e n r e s t r i c t i o n enzyme were d e s c r ib e d by uppe rcase l e t t e r d e s ig n a t io n s .

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CHAPTER FOUR

A lp h a b e t ic a l p r o x im ity o-f two p a t te rn s does n o t n e c e s s a r i ly r e f l e c t g e n e t ic

s i m i l a r i t y ; however ’ A* i s re s e rv e d by c o n v e n tio n f o r th e mtDNA known base

sequence (K .B .S . -B ib b e t a l . f 1981). C o n s id e r in g a l l r e s t r i c t i o n enzymes

used in t h i s s tu d y , each mouse was a ss ig ne d a co m p o s ite mtDNA geno type o f

14 l e t t e r s . Any in d iv d u a ls s h a r in g a common mtDNA c o m p o s ite geno type was

assumed t o b e lo n g t o th e same mtDNA m a t r i l i n e a l c lo n e , a lth o u g h t h i s o n ly

a p p l ie s t o th e p a r t i c u la r r e s t r i c t i o n s i t e s s u rv e y e d . The mtDNA fra gm en t

p a t te r n s on th e g e ls c o n s t i t u t e th e raw d a ta . Each r e s t r i c t i o n fra gm en t

d ig e s t io n p r o f i l e was s i t e mapped u s in g th e h ig h r e s o lu t io n sequence

com parison te c h n iq u e ( f o r d e t a i ls see F ig u re 2 .5 , C hap te r 2 & F ig u re 3 .2 ,

C hap te r 3 ; Cann e t a l 1982; Cann & W ils o n , 1983).

P a irw is e e s t im a te s o f p e rc e n t sequence d iv e rg e n c e C/.dm) between mtDNA's

were made fro m th e f r a c t io n o f shared s i t e s (S -v a lu e ) , u s in g e q u a tio n 16 o f

Nei & L i (1 9 7 9 ). S e p a ra te e s tim a te s were c a lc u la te d f o r r e s t r i c t i o n

e nd onuc leases re c o g n is in g fo u r -b a s e , f iv e - b a s e and s ix -b a s e n u c le o t id e

sequences, and th e s e v a lu e s were w e ig h te d a c c o rd in g t o th e t o t a l number o f

b a s e -p a irs re c o g n is e d by each ty p e o f r e s t r i c t i o n enzyme. Phenograms were

c o n s tru c te d fro m th e d v a lu e m a tr ic e s u s in g th e u n w e ig h te d p a i r group

method (UPGMA) u t i l i z i n g a programme w r i t t e n u s in g S t a t i s t i c a l A n a ly s is

System s (SAS I n s t i t u t e . In c , 1985) s o ftw a re .

A p re sen ce -a bse n ce d a ta m a tr ix c o n ta in in g each r e s t r i c t i o n s i t e o f e v e ry

mtDNA co m p o s ite g e n o typ e , was used in an u n d ire c te d pa rs im o ny a n a ly s is

u s in g th e PAUP program (P h y lo g e n e tic A n a ly s is U sing P ars im ony - S w o ffo rd ,

1985 ). In PAUP a n a ly s is th e s h o r te s t t r e e ( s ) was fo u n d u s in g th e 'G lo b a l '

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CHAPTER FOUR

b ra n c h -s w a p p in g o p t io n , w ith ’ M u lp a rs '. M u lp a rs o p t io n i n i t i a t e s a search

t o r m u l t ip le e q u a lly p a rs im o n io u s t r e e s v ia b ra n c h -s w a p p in g . S p e c i f ic a t io n

o f t h i s o p t io n o f te n r e s u l t s in th e d is c o v e ry o f s h o r te r t r e e s th a n co u ld

o th e rw is e be found w ith such la rg e d a ta s e ts (o p e ra t io n a l taxo n om ic u n i t s

COTU'sD > 1 2 ) . A consensus t r e e r e f le c t i n g th e in fo r m a t io n shared by a l l

t r e e s , was c o n s tru c te d , when two o r more t r e e s o f equa l le n g th were fo u n d ,

u s in g b o th th e Adams (Adams, 1972) and S t r i c t (R o h lf , 1982) consensus

m ethods in th e Paup package. N e tw orks were ro o te d a t th e m id p o in t o f th e

g r e a te s t p a t r i s t i c d is ta n c e .

A na lagous t o h e te ro z y g o s ity o r gene d iv e r s i t y a t n u c le a r lo c i (N e i, 1978 ),

th e d i v e r s i t y o f mtDNA lin e a g e s w i t h in a p o p u la t io n was e s tim a te d u s in g an

in d e x o f n u c le o n d iv e r s i t y ( h ) , fro m Nei & T a jim a ’ s (1981) e q u a tio n 9:

h = - i - " 6- -ZL\n-1 \ i = l

where X*t i s th e fre q u e n c y o f th e i t h ty p e o f mtDNA in a p o p u la t io n sam ple

o f n spec im ens and J i s th e number o f mtDNA ty p e s . N ucleon d iv e r s i t y v a lu e s

a p p ro x im a tin g z e ro in d ic a te la c k o f mtDNA co m p o s ite gen o typ e d iv e r s i t y ,

w hereas v a lu e s c lo s e to one i l l u s t r a t e h ig h d iv e r s i t y .

fL 3 i_R esu l tSjj.

5i3iIi_Mitgchgndrial_DNA_variatign_in_Bri.taini

Two r e s t r i c t i o n endonucleases (H inc I I , Xba I ) re v e a le d no v a r ia t io n in th e

B r i t i s h sam p les, and b o th enzymes showed th e same d ig e s t io n p r o f i l e as th e

in b re d la b o r a to r y s t r a in s , ’ p a t te rn A’ ( r e p r e s e n ta t iv e g e l p ic t u r e o f th e

monom orphic enzyme Xba I i s i l l u s t r a t e d in P la te 3 .3 , in c h a p te r 3 ) . The

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CHAPTER FOUR

re m a in in g 12 enzymes were p o ly m o rp h ic (- fo r exam ples see c h a p te r 3 , P la te s

3 .2 , 3 .4 - 3 .1 0 in c lu s iv e -for H ind I I I , Hpa I I , Taq I , Hae I I I , Mbo I , Rsa

I , and A lu I d ig e s ts , r e s p e c t iv e ly ) . S in g le enzyme p a rs im o ny n e tw o rk s can

be c o n s tru c te d u s in g a l l v a r ia b le s i t e s f o r each enzyme (T a b le 3 .3 A -N ,

c h a p te r 3 ) , as i l l u s t r a t e d in f ig u r e 4 .4 . A rrow s in d ic a te th e d i r e c t io n o f

r e s t r i c t i o n s i t e lo s s e s o r g a in s , and n o t th e d i r e c t io n o f e v o lu t io n . The

g e o g ra p h ic d i s t r i b u t i o n o f mtDNA g en o typ e s re v e a le d by each o f th e tw e lv e

p o ly m o rp h ic enzymes i s shown in f ig u r e 4 .5 (1 -1 2 ) . For exam ple , H ind I I I

p a t te r n ’ B* (n=291) was common t o O rkney, C a ith n e s s , S u th e r la n d , I r e la n d

and th e I s le o f Man. W h ils t H ind I I I p a t te r n 'A ' was obse rved in th e 139

in d iv id u a ls fro m th e r e s t o f th e B r i t i s h m a in la n d . The d i s t r i b u t i o n o f Taq

I g e n o typ e s was id e n t ic a l t o t h i s . O th e r enzymes g e n e r a l ly c o n firm e d th e

" n o r th - s o u th " g e n e tic d i f f e r e n t i a t i o n , a lth o u g h more th a n one p a t te r n was

obse rved f o r each N o rth /S o u th m a t r ia r c h ia l l i n e . For exam ple , geno types

fro m Mbo I show b o th ’ 0* and * /* p a t te r n s in th e n o r th e rn ty p e , w h i ls t ?A’ ,

'W ', ’ ST and ’ X* were found in th e r e s t o f B r i t a in (s o u th e rn ty p e -s e e F ig

4 .5 C83). O n ly Acc I , Hpa I I and Rsa I d e v ia te d fro m th e g e n e ra l d iv is io n .

For exam ple , p o p u la t io n s fro m th e F i r t h o f F o r th and th e M id la n d s had th e

* D* as opposed to th e ’ A* Hpa I I p a t te r n , obse rved in s o u th e rn B r i t a in

(F ig . 4 .5 C53). The s in g le enzyme p a rs im o n y n e tw o rk s (F ig . 4 .4 ) in d ic a te

t h a t o n ly a few r e s t r i c t i o n s i t e lo s s e s o r g a in s p ro du ce th e d i f fe r e n c e s

w i t h in th e " n o r th e rn '1 o r "s o u th e rn " ty p e s , whereas a la r g e number a re

obse rved between th e s e two m a jo r d iv is io n s (N and S ).

A t o t a l o f 23 d i f f e r e n t mtDNA co m p o s ite g en o typ e s o r c lo n e s (d e s c r ib e d by a

fo u r te e n l e t t e r code) were observed in B r i t a in , th e f re q u e n c ie s o f th e se

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CHAPTER FOUR

g e n o typ e s f o r each sam ple l o c a l i t y a re g iv e n ta b le 4 . 1 (T a b le 4 . 2 summaries

th e f re q u e n c ie s o f mt c lo n e s pe r l o c a l i t y ; sam ple s i t e s in each o f th e

n o r th e rn O rkney I s le s a re p o o le d ) . F ig u re 4 . 6 i l l u s t r a t e s th e g e o g ra p h ic a l

d i s t r i b u t i o n o f th e 2 3 mtDNA co m p o s ite g eno types a c ro s s B r i t a in . F iv e

g e n o typ e s (c lo n e s 1 - 5 ; n= 2 7 8 ) were obse rved in O rkney, C a ith n e s s and

S u th e r la n d , th e most common o f w h ich (n= 2 4 9 ) i s c lo n e 1 ; c lo n e s 2 - 5 are

much r a r e r (n = 2 9 ) . Three f u r t h e r g en o typ e s , c lo n e s 6 - 8 were obse rve d in

th e I r i s h sam ples <n= 1 3 ) . F if te e n g en o typ e s (c lo n e s 9 , Sc 1 1 - 2 3 ) were found

in th e re m a in d e r o f th e B r i t i s h m a in la nd sam ples (n= 1 5 2 ) .

4 jL 3 i2 £ _ M itg c h g n d ria i_ D N A _ y a ria tio n _ in _ 0 rk n e y _ a n d _ th e _ n e ig h b g u rin g _ m § iQ l§ Q d

c o u n t i§ s _ o f _C a i.thness_and_Suther£and£

F iv e mtDNA co m p o s ite g eno types were found among 2 3 5 O rkney m ice examined

f o r th e 1 4 r e s t r i c t i o n enzymes used in t h i s s tu d y ; o n ly tw o o f th e s e mtDNA

gen o typ e s (c lo n e s 1 Sc 2 ) were observed among 4 3 in d iv id u a ls fro m th e

n e ig h b o u r in g m a in land o f C a ith n e s s and S u th e r la n d . C lone 1 was observed a t

e v e ry sa m p lin g lo c a t io n , w ith th e e x c e p tio n o f one s i t e , H a rra y on M a in land

O rkney. C lone 2 was p re s e n t in low fre q u e n c ie s on th e n o r th e rn Orkney

i s le s o f W estray . Eday and F aray ( ta b le 4 . 2 ) , and o n ly fo u n d in one

in d iv id u a l in K e is s , n o r th -e a s t C a ith n e s s . C lone 3 was e x c lu s iv e ly

r e s t r i c t e d t o mid W e s tra y , s im i la r l y , c lo n e s 4 Sc 5 were o n ly found a t

H a rra y , M a in land O rkney. On Sanday, S tro n s a y and Papa W estray o n ly th e

u b iq u ito u s c lo n e 1 was seen, how ever, th e sam ple s iz e s were q u i te low (n=

2 , 3 and 1, r e p e c t iv e ly ) . The d i s t r ib u t io n o f th e s e f i v e mtDNA c lo n e s among

sam ple lo c a t io n s in O rkney and N.E S c o tla n d a re shown in f ig u r e 4 .6 , th e

f re q u e n c ie s o f each a re i l l u s t r a t e d by a p i e - c h a r t . T a b le 4 .1 i l l u s t r a t e s

th e f re q u e n c ie s o f each c lo n e in each is la n d and m a in la nd sam ple s i t e ,

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CHAPTER FOUR

sum m arised in T a b le 4 .2 ( s i t e s w ith in an is la n d were p o o le d ) . A l l mtDNA

c lo n e s obse rved d i f f e r e d -from each o th e r by 2 t o 6 r e s t r i c t i o n s i t e lo s s e s

o r g a in s .

To compare in t r a p o p u la t io n a l mtDNA co m p o s ite g en o typ e d i v e r s i t y , e s t im a te s

o-f n u c le o n d iv e r s i t y were c a lc u la te d . H e te ro g e n e ity (h) ranged fro m 0 .0 0

t o 0 .4 6 f o r O rkney is la n d sam ples, w ith a mean o f 0 .2 1 , y e t v a lu e s were

c o n s id e ra b ly lo w e r in th e n e ig h b o u r in g c o a s ta l c o u n t ie s o f C a ith n e s s and

S u th e r la n d . O n ly one s i t e (K e is s ) had more th a n one g e n o typ e ,

h e te r o g e n e it ie s ra n g in g from 0 .0 0 to 0 .0 6 , w ith a mean o f 0 .001 (T a b le

4 .2 ) . In c o n t r a s t , h e te ro g e n e ity (h) in th e re m a in in g s o u th e rn B r i t i s h

m a in land p o p u la t io n s i s com parab le t o th a t found in th e O rkneys, ra n g in g

fro m 0 .0 0 t o 0 .6 4 , w ith a mean o f 0 .2 5 .

i i 3 i 3 s ._ P h y l.g g e n e tic _ c g n 5 id e ra t ig n s i

5 i3 i 3JLi i_ M ic r g g e g g r a p h ic _ s t r u c tu r in g i

In t o t a l , 370 d i s t i n c t r e s t r i c t i o n s i t e s were mapped f o r th e 14 r e s t r i c t i o n

enzymes used in t h i s s tu d y o f 430 B r i t i s h house m ice . P r io r t o p h y lo g e n e t ic

a n a ly s is , 230 monomorphic s i t e s (p ie s io m o rp h ic - p re s e n t in a l l

p o p u la t io n s ; see T a b le 3 .4 , c h a p te r 3 f o r f u l l d e t a i l s o f c o n s ta n t s i t e s )

were removed fro m th e d a ta s e t . Of th e re m a in in g 140 v a r ia b le s i t e s (T a b le

3 .3 A -N , c h a p te r 3 ) , 78 were a u tapom orph ic ( s i t e s u n iq u e t o a p a r t i c u la r

sa m p lin g l o c a l i t y ) and were a ls o e x c lu d e d . F in a l l y , 62 p h y lo g e n e t ic a l ly

in fo r m a t iv e s i t e s (synapom orph ic - b e in g p re s e n t in o r a bsen t from a t le a s t

two p o p u la t io n s ) were coded as p re s e n t o r a b s e n t, t r e a te d as uno rde red

c h a ra c te rs and used in th e c la d i s t i c a n a ly s is . T a b le 4 .3 l i s t s th e 62

in fo r m a t iv e s i t e s by r e s t r i c t i o n enzyme, f o r a l l 23 mtDNA com p os ite c lo n e s .

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CHAPTER FOUR

The G lo b a l b ra n ch -sw a p p in g o p t io n (w ith M u lp a rs ) in PAUP was used to

c o n s t r u c t th e most p a rs im o n io u s t r e e -from th e s i t e d a ta . The t r e e was

ro o te d a t th e m id p o in t between th e most d iv e rg e n t ta x a . A minimum o-f 100

t r e e s o-f equa l le n g th (81 s te p s ) and c o n s is te n c y (c o n s is te n c y in d e x , C l =

0 .7 6 5 ) were fo u n d . F ig u re 4 .7 re p re s e n ts an Adams consensus t r e e f o r a l l

th e t r e e s o f equa l le n g th . The S t r i c t consensus t r e e was id e n t ic a l t o th e

Adams consensus t r e e . A l l t re e s gave th e same o v e r a l l to p o lo g y and o n ly

changed m in o r t o p o lo g ic a l fe a tu re s w i t h in th e m a jo r c lu s te r s o f th e n e tw o rk

shown.

Two m a jo r g e n e t ic assem blages were p roduced (d e p ic te d by th e t r ia n g le and

squa re sym bo ls in th e f ig u r e 4 .7 ) , one re p re s e n t in g c lo n e s 1 -8 & 10, th e

o th e r , c lo n e s 9 & 11 -23 . The pa rs im ony n e tw o rk was i n i t i a l l y c o n s tru c te d

w ith o u t p r io r re fe re n c e t o th e c o l le c t io n s i t e s . I t was o b v io u s , how ever,

when th e n e tw o rk was superim posed o ve r th e sam ple s i t e s , t h a t th e re was an

a p p a re n t g e o g ra p h ic o r ie n ta t io n o f c lo n e s . The mtDNA geno types 9 & 11-23

occupy a p p ro x im a te ly a d ja c e n t p la c e s in th e t r e e , r e f le c t i n g t h e i r

g e o g ra p h ic p r o x im it y and re p re s e n t in g th e "s o u th -e a s te rn assem b lage",

c o m p r is in g a l l s i t e s s o u th o f th e G rea t G len F a u lt on th e B r i t i s h m a in la n d ,

w ith th e e x c e p t io n o f th e in d iv id u a l fro m Gatehouse o f F le e t . S im i la r ly ,

c lo n e s 1 -8 ?< 10 fo rm th e "n o r th -w e s te rn " assem blage, in c lu d in g n o r th e rn

sam ples fro m O rkney (c lo n e s 1 -5 ) , C a ith n e s s (c lo n e s 1 & 2 ) , and S u th e r la n d

(c lo n e 1 ) , and w e s te rn samples (c lo n e s 6 -8 & 10) fro m n o r th & sou th

I r e la n d , I s le o f Man and Gatehouse o f F le e t , D u m frie s . The g e o g ra p h ic

d i s t r ib u t io n o f th e s e tw o m ajor g e n e tic ty p e s a re shown in f i g . 4 .7 ( in s e t )

and f ig u r e 4 .6 (shaded and unshaded c i r c l e s re p re s e n t th e "N-W" and th e "S -

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CHAPTER FOUR

E" m a t r ia r c h ia l l in e a g e s , r e s p e c t iv e ly ) . One unexpected r e s u l t o-f t h i s

i a n a ly s is i s th a t c lo n e 10 (G atehouse o-f F le e t , D u m frie s ) f a l l s in to a s u b -i

c lu s t e r w i th in th e ' n o r th e rn -w e s te rn ' g e n e t ic g ro up , y e t , 10 o u t D f th e 12

r e s t r i c t i o n enzymes w h ich produced v a r ia b le d ig e s t io n p r o f i l e s , in d ic a te d

t h a t t h i s p o p u la t io n be longed in th e ' s o u th e rn -e a s te rn ' c la d e . Enzymes Fnud

I I and Rsa I appear to be re s p o n s ib le f o r t h i s . To sum m arise, th e ’ N -S '

g e n e t ic b reak a p p a re n t fro m th e s in g le enzyme pars im ony n e tw o rks o v e r la y e d

o ve r th e g e o g ra p h ic a l l o c a l i t i e s , i s c o n co rd a n t w ith th e MN-W and S-E"!

c lu s te r s , produced fro m th e m u l t ip le enzyme n e tw o rks .

| The p ro p o r t io n o f s i t e s shared (S -v a lu e ) among in d iv id u a ls , f o r a l l

| fo u r te e n r e s t r i c t i o n enzymes used in th e s tu d y , was used t o e s tim a te th e

i mtDNA sequence d iv e rg e n c e (d B) , u s in g Nei ?< L i ' s e q u a tio n s (T a b le 4 .4 a ) ,

T a b le 4 .4 b shows th e d is ta n c e m a tr ix o f sequence d iv e rg e n c e s (d B) f o r

p a irw is e com parisons o f th e 23 d i s t i n c t mtDNA co m p os ite g en o typ e s . In th e

j UPGMA phenogram (F ig u re 4 .8 ) , c o n s tru c te d fro m th e e n t i r e m a tr ix o f

sequence d iv e rg e n c e s , tw o m a jo r g e n o ty p ic c lu s te r s , d is t in g u is h e d by

a p p ro x im a te ly 1.427. sequence d iv e rg e n c e (v a lu e s ra n g in g fro m 0 .0 7 3 to

2 .1 1 ) , were o b ta in e d , w ith n e a r ly id e n t ic a l to p o lo g y to th e Wagner

n e tw o rk s , based upon th e p resence -absence r e s t r i c t i o n s i t e d a ta (F ig u re

4 .7 ) . The mean 7.da w i t h in th e N.W and S.E c la d e s , r e s p e c t iv e ly , a re 0.277.

f and 0.487. ( ra n g in g fro m 0 .0 0 t o 0.47. f o r th e fo rm e r and 0 .0 3 8 to 1.217. f o r

th e 1a t t e r ) .

The m agnitude o f sequence d iv e rg e n c e observed between th e tw o m a jo r g e n e t ic

CHAPTER FOUR

assem blages in B r i t a in a re in c o n s is te n t w ith th e d iv e r s i t y a c c u m u l- t in g

’ i n s i t u ’ s in c e t h e i r a r r i v a l in B r i t a in , as N o rth e rn Europe was g la c ia te d

and u n in h a b ita b le by house m ice u n t i l a t th e e a r l i e s t between a p p ro x im a te ly

1 0 -8 ,0 0 0 BP (K e r r , 1983 ). Thus d a ta fro m mtDNA s u p p o r ts th e h y p o th e s is th a t

B r i t i s h m ice a re descended fro m a t le a s t tw o s e p a ra te c o lo n is a t io n e v e n ts .

4i 3 i 3 i 2 i_ M a c rg g e g g ra p h ic _ 5 tru c ty r in g i

In an a tte m p t t o f in d p o s s ib le a n c e s tra l p o p u la t io n s o f th e ’ n o r th e rn ty p e ’

in B r i t a in , d a ta fro m F e r r is e t a i - , (1983) was used. Where p o s s ib le l e t t e r

d e s ig n a t io n s f o r d ig e s t io n p a t te r n s , in t h i s s tu d y , fo l lo w e d th o s e

e s ta b l is h e d by F e r r is & c o lle a g u e s (1 9 8 3 ). They re p o r te d on mtDNA v a r ia t io n

in 190 Hus dom esticus fro m w ild caugh t (o r la b o r a to r y r a is e d desce n de n ts

o f w i ld m ic e ) , p lu s v a r io u s in b re d s t r a in s , fro m 46 l o c a l i t i e s in N.W.

Europe and th e New W o rld . However, a few d ig e s t io n p a t te r n s fro m s e v e ra l

enzymes were n o t d i r e c t l y com parab le t o th o s e d e s c r ib e d by F e r r is and

c o lle a g u e s (1 9 8 3 ). N e v e r th e le s s , d e s p ite m ino r d i f fe r e n c e s , th e m a jo r i t y o f

th e p a t te rn s d e s c r ib e d by F e r r is & c o lle a g u e s (1983) a re c o n g ru e n t w ith

th o s e obse rved in t h i s s tu d y .

T w e n ty -s ix mtDNA co m p o s ite geno types fro m 77 ft us dom esticus in d iv id u a ls

u s in g 11 r e s t r i c t i o n end o nu c le a ses , nam ely, H ind I I I , Xba I , H inc I I , Acc

I , Ava I I , FnuD I I , Hpa I I , Hae I I I , Taq I , Mbo I and H in f I , fro m s e v e ra l

w o r ld w id e l o c a l i t i e s were re c o rd e d by F e r r is e t a l (1 9 8 3 ). N in e te e n mtDNA

g e n o typ e s were observed among th e B r i t i s h m ice exam ined in t h i s s tu d y ,

u s in g th e same s e t o f enzymes, none o f w h ich had been p r e v io u s ly r e p o r te d .

A t o t a l o f 220 r e s t r i c t i o n s i t e s were mapped in th e 45 mtDNA g e n o ty p e s , 123

o f w h ich were in v a r ia n t , and th e r e fo r e e xc lu d e d fro m th e d a ta s e t .

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CHAPTER FOUR

S im i la r ly , in th e re m a in in g 97 v a r ia b le s i t e s , 48 w ere a u ta p o m o rp h ic , and

were a ls o d e le te d . The 49 p h y lo g e n e t ic a l ly in - fo rm a t iv e s i t e s ( l i s t e d in

T a b le 4 .5 ) were used in th e c la d i s t i c a n a ly s is . An Adams consensus t r e e

d e p ic t in g th e g e n e a lo g ic a l r e la t io n s h ip s fro m a l l t r e e s o f equa l le n g th (85

s te p s ) and c o n s is te n c y (0 1 = 0 .5 7 6 ), found by u s in g th e G lo b a l b ra n c h -

sw apping o p t io n , i s i l l u s t r a t e d in f ig u r e 4 .9 . The S t r i c t consensus t r e e i s

th e same as th e Adams, e xce p t t h a t c lo n e s 36, 45 and 29 ( fro m : G iza , E g yp t;

P osch ia vo , S w itz e r la n d ; and E rfo u d , M orocco, r e p e c t iv e ly ) were found

c lu s te r e d to g e th e r , as a s e p a ra te l in e a g e . G eograph ic d i s t r i b u t i o n o f th e

45 mtDNA geno types a re p o r tra y e d in f ig u r e 4 .1 0 and th e fo u r <I —IV )

a r b i t r a r i l y chosen m a jo r c lu s te r s o f th e t r e e a re d e p ic te d . I n d iv id u a ls

fro m th e "N.W" and th e "S.W" b ranches in th e B r i t i s h p h y lo g e n y , appear t o

c lu s t e r w ith sam ples fro m Y u g o s la v ia it C a l i f o r n ia (B ranch I I , F ig . 4 .9 &

4 .1 0 ) , and th e in b re d la b o r a to r y s t r a in s (B ranch I , F ig . 4 .9 & 4 .1 0 ) ,

r e s p e c t iv e ly . A hand drawn p a risom y n e tw o rk , u s in g a l l r e s t r i c t i o n s i t e

d if- fe re n c e s in c lu d in g a u tapom orph ies (u n iq u e s i t e s ) , i l l u s t r a t e s th e c lo s e

r e la t io n s h ip between mtDNA ty p e s from th e N-W ra c e w ith th o s e fro m

Y u g o s la v ia and C a l i f o r n ia (F ig u re 4.1 IB ) .

M ito c h o n d r ia l DNA fro m 113 a d d i t io n a l m ice from s e v e ra l new sam ple s i t e s ,

d ig e s te d w ith th e in fo r m a t iv e enzymes H in f I and Mbo I , re v e a le d 8 e x tr a

mtDNA geno types ( F e r r is e t a i* , 1983). A lto g e th e r , F e r r is it c o lle a g u e s

(1983) re co rd e d 31 mtDNA c lo n e s u s in g th e s e two r e s t r i c t i o n enzymes,

how ever, 3 ty p e s ( I L , KL, and IP ) gave id e n t ic a l in fo r m a t iv e s i t e s once th e

u n iqu e s i t e s had been e x c lu d e d , th u s were coun ted as o n ly one "OTU"

(o p e ra t io n a l taxo n om ic u n i t ) ; a ls o 'A T ' ty p e ( in b re d s t r a in S L /N ia ) gave

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CHAPTER FOUR

th e same s i t e s as th e common in b re d ty p e ’ AA’ a -fte r rem oval o f

au tapom orph ic s i t e s . Using o n ly Mbo I and Hin-f I enzymes, n in e

m ito c h o n d r ia l DNA c lo n e s were observed in th e B r i t i s h sam ples, y e t , f i v e

typ e s (OC, OU, AZ, AA, and XY) had been p re v io u s ly documented in th e 28

d is t in c t i v e mtDNA c lo n e s d esc rib e d by F e r r is e t e l ; (1983 ). The f in a l 32

mtDNA c lo n e s u s in g the se two enzymes gave a t o t a l o f 92 r e s t r i c t io n s i t e s ,

a f t e r removal o f in v a r ia n t and unique s i t e s , o n ly 28 in fo r m a t iv e s i t e s

( l i s t e d in T ab le 4 .6 ) were used in th e a n a ly s is (coded as p re s e n t o r absent

and tre a te d as unordered c h a ra c te rs ) . F ig u re 4 .1 2 re p re s e n ts a S t r i c t

consensus t r e e c o n s tru c te d from a l l p a rs im o n iou s t r e e s o f equal le n g th (45

s te p s ) and c o n s is te n c y (C I=0.622) found. M ito c h o n d r ia l c lo n e s from O rkney,

C a ith n e ss , S u th e rla n d and Ire la n d (N.W branch in B r i t a in ) appear to c lu s te r

w ith those from Norway (Branch I I , F ig 4 .1 2 ) , in a d d it io n to th o se from th e

sou the rn M e d ite rran e an re g io n and Am erica, as p re v io u s ly m en tioned . W h ils t ,

mtDNA c lo n e s from th e S.E lin e a g e in B r i t a in a re found grouped w ith samples

from Denmark, and Germany (Branch I , F ig . 4 .1 2 ) . F ig u re 4 .1 3 shows th e

geog raph ic d is t r ib u t io n o f these mtDNA c lo n e s .

There was concordance between th e t re e b u i l t on th e b a s is o f th e H in f I and

Mbo I mtDNA r e s t r i c t i o n p a tte rn s enzymes a lo n e (F ig . 4 .1 2 ) , and th a t based

on 11 r e s t r i c t io n endonucleases (F ig u re 4 .1 0 ) ; th e fo u r m ajor branches ( I -

IV) a re c le a r ly d is c e r n ib le , except th a t th e p o s i t io n s o f b ranches have

s h if te d lo c a t io n w ith in th e t re e s . Indeed, th e a d d it io n a l com pos ite mtDNA

genotypes d e te c te d u s in g these two enzymes m ere ly form ed e x tra ,,tw ig 5 ,,

a tta ch e d to th e m a jo r branches a lre a d y appa ren t u s in g 11 enzymes.

S im i la r ly , th e g e n e a lo g ic a l re la t io n s h ip s i l l u s t r a t e d by th e t r e e o f

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CHAPTER FOUR

B r i t i s h Hus domesticus mtDNAs on th e b a s is o-f 14 enzymes (F ig u re 4 .7 ) , a re

c o n s is te n t w ith tho se found in th e two t re e s u s in g m ice from European and

w o rld w id e l o c a l i t i e s , in th a t th e ’ N.W’ and ’ S .E ’ c lu s te r s a re conco rdan t

w ith two o-f th e m ajor branches des igna ted I I and I as d e p ic te d in F ig u re s

4 .9 & 4 .1 2 . Im p o rta n t d if- fe re n c e s in c lu d e an in d iv id u a l from Gatehouse o-f

F le e t , Dum-fries (c lo n e 10, F ig . 4 .7 ; c lo n e 6 , F ig . 4 .9 ; 'A X ' l in e a g e , F ig .

4 .1 2 ) , whose g e n e a lo g ic a l p o s it io n i s s l i g h t l y d i f f e r e n t in a l l th re e

t r e e s . I t g roups w ith th e n o rth -w e s te rn ra c e in B r i t a in , on th e b a s is o f

th e two in fo rm a t iv e enzymes Mbo I and H in f I , a ls o , w ith th e s e t o f

fo u r te e n r e s t r i c t i o n endonucleases (Branch I I , f i g . 4 .1 2 & 4 .7 ) ; whereas i t

c lu s te r s w ith th e B r i t i s h s o u th -e a s te rn ty p e s , u s in g e leven r e s t r i c t io n

enzymes (Branch I , F ig . 4 .9 ) . S im i la r ly , th e Morocco mouse ( ’ EF’ lin e a g e in

f i g . 4 .1 2 ; c lo n e 29 in F ig . 4 .10 ) s h i f t s i t s p o s i t io n from branch IV in th e

t r e e based on two enzymes to branch I I on th e b a s is o f 11 enzymes, in th e

l a t t e r t r e e i t grouped w ith mice from th e B r i t i s h N-W ra ce (Orkney & I r is h

m ic e ).

I

I

I|

II

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i jL-QiicyssioQiP re se n t p a t te rn s o-f g e n e tic v a r ia t io n are s t ro n g ly a f fe c te d by h is t o r ic a l

e v e n ts . Indeed , h is t o r ic a l c o lo n is a t io n s can o-ften be in te r r e d by exam in ing

c u r re n t p a t te rn s o f v a r ia t io n in r e la t io n to a ta x o n ’ s e x ta n t d is t r ib u t io n .

The m ajor r e s u l t s o f t h i s work a re , th e f in d in g o f m ic ro g e o g ra p h ic

s t r u c tu r in g o f mtDNA c lo n e s w ith in th e B r i t i s h house mouse and c o n f irm a tio n

o f th e g ene ra l la c k o f m acrogeographic s t r u c tu r in g over N-W Europe. A ls o ,

com b in ing mtDNA r e s t r i c t i o n fragm ent le n g th polym orph ism s d a ta w ith n u c le a r

DNA a n a ly s e s , th e most l i k e l y c o lo n is a t io n p a tte rn s among th e N o rth e rn

is le s (O rkney) and n e ig h b o u rin g m ain land a re p o s tu la te d .

4 i 3 iIi_y i£C Q Q eQ 9C iE b ic a l_ S tru c tu r in g ._ in _ B r i ta in . .

G eographic p a t te rn s o f mtDNA r e s t r i c t io n s i t e po lym orph ism s have been

documented f o r a number o f t e r r e s t r ia l (A v ise e t a l . , 1979; Lansman e t a l . ,

1983; M acNeil & S tro b e c k , 1987; T e ge ls tro m , 1987a) and f re s h w a te r sp e c ie s

(A v is e e t a l . , 1984a; Saunders e t a l . , 1986; A v is e , 1987; Bermingham &

A v is e , 1986; Ovenden e t a l . , 1988) assayed over a com parable geog raph ic

range (see A v ise e t a l . , 1987a and A v ise , 1989 f o r re v ie w s ) . However,

n o ta b le e x c e p tio n s to t h i s in c lu d e many m arine ta xa (Graves e t a l . , 1984;

A v ise e t a l . , 1986; A v ise e t a l . , 1987a; f o r a re v ie w see - A v is e , 1987),

b ir d s (K e s s le r Sc A v is e , 1984, 1985; T e ge ls tro m , 1987bj B a ll e t a l . , 1988),

and to some e x te n t man (Johnson e t a l . , 1983; Cann e t a l . , 1987; S tonek ing

e t a l . , 1990). In a d d it io n , house mice ( F e r r is e t a l . , 1983) and v o le s

(Thomas & Beckenbach, 1986; P la n te e t a l . , 1989a) have a ls o been quoted as

la c k in g any s u b s ta n t ia l geograph ic s t r u c tu r in g , suggested t o r e f l e c t re c e n t

p o p u la t io n and range expans ions, o r e x te n s iv e d is p e rs a l c a p a b i l i t ie s and

p o p u la t io n f lu c tu a t io n s . T h is i s c e r t a in ly th e case fo r w o rld w id e su rveys

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o-f Mus dom esticus , however, t h i s s tud y i l l u s t r a t e s q u i te e x te n s iv e

g eog raph ic s t r u c tu r in g on a more lo c a l, m ic rog e og ra p h ic s c a le in th e

B r i t i s h house mouse. Indeed, in B r i t a in two main fo rm s a re re co gn ise d (see

f i g . 4 .7 ) , a "n o r th e rn " and a "so u th e rn " c lo n e ; o r ra th e r "n o r th -w e s te rn "

and "s o u th -e a s te rn " c lo n e s , as, on th e b a s is o f mtDNA v a r ia t io n , I r is h m ice

a re g e n e t ic a l ly s im i la r t o Orkney m ice.

The p a t te rn o f m ito c h o n d r ia l DNA v a r ia t io n in th e B r i t i s h house mouse i s

c o n s is te n t w ith ev idence from o th e r g e n e tic m arkers. R o b e rtso n ian

t r a n s lo c a t io n s were found th roughou t C a ithness and in th re e Orkney

p o p u la t io n s , y e t , no o th e r c e n t r ic fu s io n s have been found in B r i t a in ,

sou th o f C a ith n e ss (B ro o ke r, 1982; Nash e t a l , , 1983; S c r iv e n & B rooke r,

1990). From m orphom etric d is ta n c e e s tim a te s c a lc u la te d f o r B r i t i s h m ice,

C a ith ne ss and Orkney m ice are re la te d to each D ther and to th e Hebridean

m ice, y e t a re q u i te d i s t in c t from a l l D ther B r i t i s h m a in land and is la n d

m ice (D a v is , 1983). B e rry & P e te rs (1977) examined 27 p o p u la t io n s o f house

mice from Faroe, S h e tla n d , Orkney, m ain land B r i t a in , and th re e sm all

o f fs h o re s u b - a r c t ic is la n d s , e le c t r o p h o r e t ic a l ly a t 22 a llo zym e lo c i .

N e ith e r , th e average h e te ro z y g o s it ie s nor th e d is t r ib u t io n o f a l le le

fre q u e n c ie s produced any d is c e rn ib le p a t te rn s . However, when th e B r i t i s h

p o p u la t io n s a re s u b je c t to UPGMA c lu s te r a n a ly s is o f R oger’ s (1972)

e s tim a te s Df g e n e tic d is ta n c e based on 19 a llo zym e lo c i , tw o branches

c o n s is te n t w ith th o se found by mtDNA data a re observed (Bauchau, pers

com m .,). However, th e da ta a re o f l im ite d use in e s ta b lis h in g r e la t io n s h ip s

among more lo c a l p o p u la t io n s . Thus, t h is s tu d y v e r i f i e s o th e rs in showing

th a t mtDNA is ve ry u s e fu l fo r d i f f e r e n t ia t in g among a l lo z y m a t ic a l ly s im i la r

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ta x a , in which r e la t io n s h ip s have been d i f f i c u l t to re s o lv e u s in g o th e r

m o le c lu la r te c h n iq u e s (A v ise e t a im , 1979; K e ss le r St A v is e , 1984, 1985).

The most l i k e l y e x p la n a tio n to r major g e n e tic d is c o n t in u i t ie s , and th e

geog raph ic o r ie n ta t io n th e y show, whereby c o n s p e c if ic p o p u la t io n s occupy

e a s i ly re c o n g is a b le branches on an in t r a s p e c i f ic t r e e , i s th e presence o f

lo n g te rm e x t r in s ic ( ie . zoogeograph ic) b a r r ie r s to gene f lo w . The apparent

d iv is io n o f th e tw o mtDNA groups appears to map to th e G reat G len, S co tland

(C o rb e t, 1961). Y e t, a lth o u g h t h is is not an o bv ious to p o lo g ic a l b a r r ie r ,

th e house mouse appears to be r e s t r ic te d by b io lo g ic a l ra th e r than p h y s ic a l

fa c to r s . Indeed, m ice a re though t to be poor in t e r s p e c i f ic c o m p e tito rs

( L id ic k e r , 1966; D elong, 1967; B e rry & T r ic k e r , 1969; B e rry e t aim, 1982;

Dueser and P o t te r , 1986) and areas where th e y a re n o t fo u n d , a re p ro b a b ly

occup ied by o th e r s m a ll mammals. House mice avo id open f i e l d s , w ith ve ry

l i t t l e c o v e r, c h a r a c te r is t ic o f h a b ita t around th e a rea marked by th e G reat

G len. Hence, th e poor re c o rd s o f house mice around t h i s a re a , reco rded in

th e U n ite d Kingdom Mammal Survey (C orbe t, 1971; A rn o ld , 1978), may no t be

e n t i r e ly due to la c k o f docum entation and poor sa m p lin g , b u t co u ld be

because here t h i s sp e c ie s i s g en u in e ly ra re (Y a lden , 1980). Hence, th e

h a b ita t may be u n s u ita b le f o r th e house mouse, e s p e c ia l ly as th e above

su rvey shows a preponderance o f f i e ld mice a t the se s i t e s . Thus, mice in

C a ithness and th e co as t D f S uthe rland cou ld be e f f e c t i v e ly is o la te d from

tho se in th e n e ig h b o u rin g c o u n tie s by h ig h m oorland, from w hich th e y are

p ro b a b ly exc luded by abundant Apodewus. A d d i t io n a l ly , th e re i s the

p o s s ib i l i t y th a t t i g h t s o c ia l s t ru c tu re and t e r r i t o r i a l i t y in th e house

mouse l im i t s gene f lo w between g e n e t ic a l ly d i f f e r e n t ia t e d p o p u la tio n s

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anyway (b u t see ch ap te r 6 ; B e rry , 1986; L id ic k e r & P a tto n , 1987).

M ito c h o n d r ia l DNA m u ta tio n s are though t to accum ula te s te a d i ly th rough tim e

(Brown e t aim, 1979, 1982; W ilson e t aim, 1985, 1989), th u s rough e s tim a te s

o-f t im e s s in c e mtDNA d ive rgence can be c a lc u la te d from t = d /2 A (Nei &

L i , 1979), where d i s th e es tim a ted percen t sequence d iv e rg e n c e , and A is

th e ra te o f e v o lu t io n (a p p ro x im a te ly 0 .02 - 0 .04 s u b s t i tu t io n s /base p a i r /

m i l l io n y e a rs ) . E s tim a te s suggest th a t mtDNA c lo n e s b e lo n g in g to th e N.W

and S.E groups o f th e B r i t i s h house mouse la s t shared a common a nce s to r

(mean Y.d = 1 .4 ) about 350,000 to 177,500 years ago. S im i la r ly , c lo n es

w ith in each m a jo r assemblage d ive rged about 92,500 to 46,250 yea rs ago

(mean Zd = 0 .3 7 ) . There i s a long s tan d in g debate as to whether mice

e vo lve more r a p id ly than do o th e r mammals (Jaeger e t a i 1 9 B 6 ; B r i t t e n ,

1986; L i S< Tamimura 1987; C a tz e f l is e t aim, 19B7; f o r a re v ie w see W ilson

e t aim, 1987), th u s th e low er e s tim a te s o f d ive rge n ce tim e a re p ro b a b ly th e

more r e a l i s t i c v a lu e s . However, because o f th e la rg e s tan d a rd e r ro rs

in v o lv e d in c a lc u la t in g sequence d ive rgences and th e a d d it io n a l u n c e r ta in ty

D f th e average base s u b s t i tu t io n ra te in ro d e n ts , the se da tes are p ro b a b ly

h ig h ly in a c c u ra te , and th u s should be t re a te d as a gu ide ra th e r than as

a b s o lu te e s tim a te s . Hence, th e re is no t a m ajor g e n e tic "b re a k "

d is t in g u is h in g th e two mtDNA c lones ( ie . no t g re a te r than 27. sequence

d ive rg e n ce - c a te g o ry I in A vise e t aim, 1987a; A v is e , 1989) in B r i t a in .

R a ther th e y a re s u f f ic e n t ly d is s im i la r as to be c o n s is te n t w ith A v ise k

c o lle a g u e s ’ d e f in i t io n o f p o p u la tio n s th a t have expe rienced h is t o r i c a l l y

l im i te d gene f lo w , bu t n o t subd iv ided by lo n g te rm z o o g ra p h ic a l b a r r ie r s to

d is p e r s a l.

CHAPTER FOUR

by th e a ccum u la tio n o f base s u b s t i tu t io n s in s itu - fo llo w in g t h e i r

in t r o d u c t io n , o r th e y re - f le c t v a r ia t io n p r e x is t in g in th e o r ig in a l

c o lo n is in g p o p u la t io n s , o r as th e r e s u l t o-f v e ry re c e n t m ig ra t io n .

D u rin g th e g la c ia l maximum (Devensian in B r i t a in ; s y c h ro n ise d w ith th e

W eichsel in S ca n d in a v ia , and Wurm in th e A lp s ) th e ic e sh ee ts extended as

-far sou th as th e south Wales c o a s t, a long th e eas t co a s t o f E ng land,

c o v e r in g a l l o f S c o tla n d , and most o f I re la n d (W est, 1977; see f ig u r e

4 .1 4 ) , w h i ls t th e r e s t o f England expe rienced p e r ig la c ia l c o n d it io n s . Thus,

i t seems h ig h ly im p robab le th a t any sm a ll ro d e n t co u ld have s u rv iv e d the se

c o n d it io n s , a t le a s t in N orth B r i t a in . Thus, th e e a r l ie s t p o s s ib le d a te o f

c o lo n is a t io n o f the se re g io n s would have been a p p ro x im a te ly 10,000 ye a rs

ago. However, f o s s i l ev idence has dated th e c o lo n is a t io n o f Is ra e l by Mus

douesticus t o be about 10,000 B.C. (A u ffra y e t al~, 1988), s u b s e q u e n tly ,

t h i s sp e c ie s was b e lie v e d to have been c o n fin e d to th e w este rn

M e d ite rran e an bas in d u r in g th e Bronze Age (3 ,000 B .C .) , o n ly f i n a l l y

re a c h in g th e fa r n o r th -w e s t o f Europe, in c lu d in g B r i t a in (C o rb e t, 1974) and

F rance , in th e Iro n Age (1 ,000 B .C .) (rev iew ed by A u ffra y e t a i . , 1989).

W ith th e m e tho d o log ie s a v a ila b le f o r sc re e n in g mtDNA v a r ia t io n in th e

B r i t i s h house mouse i t i s h ig h ly u n l ik e ly th a t th e few base s u b s t i t u t io n s

a ccu m u la tin g ' i n s i t u ' d u r in g th e s h o r t t im e s in c e th e e a r l ie s t p o s s ib le

p o s t - g la c ia l c o lo n is a t io n o f B r i t a in , would be d e te c ta b le . C onsequen tly , i t

would appear th a t d ive rge n ces o f th e m agnitude observed between lin e a g e s

from th e N-W and S-E suggest th e re were d if fe re n c e s between th e p o p u la t io n s

b e fo re th e y c o lo n is e d B r i t a in , th u s p ro v id in g ev idence th a t th e two fo rm s

o r ig in a te d from se pa ra te in t ro d u c t io n e ven ts from d i f f e r e n t a n c e s tra l

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sources . The p resence o f a ’’b a r r ie r " in S co tla n d p ro b a b ly p re v e n ts any

s u b s ta n t ia l in te r m ix in g D f th e two fo rm s.

E ith e r s tro n g b io g e o g ra p h ic b a r r ie r s , o r h is t o r ic a l b iogeography per se

such as p o p u la t io n s o f th e same sp ec ies w ith d i f f e r e n t e v o lu t io n a ry

h is t o r ie s co n ve rg in g on th e same landmass, sh ou ld be expected to shape th e

g e n e tic s t r u c tu r e o f o th e r in d e p e n d e n tly e v o lv in g , e c o lo g ic a l ly s im i la r

sp e c ie s ( ie . sm a ll mammals) in a conco rdan t fa s h io n , such th a t s im i la r

p a t te rn s o f g eog raph ic v a r ia t io n shou ld be appa ren t amongst them (A v ise ,

1989). There a re no s im i la r s tu d ie s o f geo g ra ph ic v a r ia t io n o f mtDNA o f

sm a ll mammals in B r i t a in , w ith which to compare and c o n tra s t th e p a tte rn s

observed in th e house mouse, a ltho u gh o th e r g e n e tic m arkers have been used.

B e rry (1975, 1985) suggested , us ing m u lt iv a r ia te m orphom etric ev idence ,

th a t th e re a re two ra ce s o f Apodemus s y lv a tic u s in B r i t a in ; a 'w e s te rn &

n o r th e rn ' fo rm and an 'e a s te rn ' fo rm , th e l a t t e r be ing c lo s e ly re la te d to

th e French Apodemus f s im i la r r e s u l t s were a ls o o b ta in e d by Del any &

W h itta k e r , (1969) f o r th e same sp e c ie s , and a ls o f o r th e common shrew

(Sorex araneus ) by S e a rle & Thorpe, (1987 ). D is t r ib u t io n s b e a rin g c lo s e

resem blance to th a t d esc rib e d above a re observed bo th in Clethrionymus

g lareo lus (C o rb e t, 1964) and Hicrotus a g re s tis (Evans, 1977), bo th

ana lyses u s in g u n iv a r ia te m orphom etries. The geog raph ic v a r ia t io n s

recoun ted above in a l l cases are o n ly s l i g h t , w eakly c o r re la te d , ra th e r

than r e f le c t in g h ig h ly d iv e rg e n t fo rm s, d is c o u n t in g th e p o s s ib i l t y o f a

m ajor p h y s ic a l b a r r ie r between p o p u la t io n s , in d ic a t in g the se p o p u la tio n s

are th e p ro d u c ts o f d i f f e r e n t c o lo n is a t io n s . M oreover, th e o r ig in s o f bo th

th e f i e l d mouse and th e bank v o le , in n o r th B r i t a in have been a t t r ib u te d to

CHAPTER FOUR

a c c id e n ta l in t r o d u c t io n s by man, and a re p ro b a b ly o f Norse descent (C o rbe t,

1961; B e rry , 1969), whereas th e so u th e rn fo rm s may o r ig in a te from the

nearby C o n tin e n t.

C o n ve rse ly , S e a rle St W ilk in s o n (1987) d e s c r ib e th re e k a ry o ty p ic races o f

Sorex araneus in B r i t a in ; a n o r th w estern ra ce (Aberdeen ra c e - a ’ C e lt ic

f r in g e ’ ) , a c e n t ra l and e a s te rn race (O xfo rd ty p e ) , and an in te rm e d ia te

ra ce (H e rm ita g e ). They conc luded , however, th a t t h i s d is t r ib u t io n s u p p o rts

th e h y p o th e s is th a t th e races spread in t o B r i t a in a t th e end o f th e la s t

g la c ia t io n in su cce ss ive waves, th e O xfo rd ra ce p a r t l y d is p la c in g the

H erm itage ra c e , which in tu rn d is p la c e d th e Aberdeen ra c e . Y e t, the y d id

acknow ledge, u s in g a llo zym e d a ta , th a t th e n o r th -w e s te rn ra c e may have had

an independent o r ig in . The r e la t i v e l y lo n g e r t im e s c a le env isaged fo r shrew

c o lo n is a t io n o f Europe (100,000 ye a rs a g a in s t B000 fo r m ice) means th e re

may have been s u f f ic e n t t im e fo r a s e r ie s o f shrew in v a s io n s (S e a r le , 1988;

S e a rle e t aim, 1990), indeed shrews were a t th e f o r e f r o n t o f p o s tg la c ia l

c o lo n is a t io n s , an im p la u s ib le s c e n a rio f o r th e house mouse c o n s id e r in g i t s

re c e n t and ra p id range expansion in Europe.

A no ther d e te rm in is t ic e x p la n a tio n o f th e geog raph ic s t r u c tu r in g o f t h i s

sp e c ie s in B r i t a in in c lu d e s th e p o s s ib i l i t y th a t n a tu ra l s e le c t io n a c tin g

on mtDNA genotypes may d i f f e r d ra m a t ic a lly from n o r th t o s o u th . M aterna l

in h e r ita n c e o f mtDNA e f f e c t iv e ly t i g h t l y l in k s a l l o f i t s genes, and

a lth o u g h i t i s assumed th a t these m arkers a re n e u tra l th e y may be c a r r ie d

to h ig h fre q u e n c ie s by h i t c h h ik in g w ith any s u f f i c e n t ly advantageous

m u ta tio n . T h is seems h ig h ly im p robab le in th e B r i t i s h p o p u la t io n s as th e re

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is no c o r r e la t io n between race typ e and la t i t u d e o r lo n g itu d e .

The a l t e r n a t iv e , s to c h a s t ic v iew , i s th a t due to random lin e a g e e x t in c t io n ,

th e in te rm e d ia te genotypes may be lo s t in a w id e ly d isp e rs e d sp e c ie s , w ith

l im i te d d is p e rs a l and gene f lo w c a p a b i l i t ie s . N e ig e l and A v ise (1986)

m odelled t h i s and concluded th a t mtDNA c lo n es would show g re a te r

s u b d iv is io n tha n n u c le a r genes; th e l a t t e r , due to sexua l re p ro d u c t io n and

re c o m b in a tio n , would n o t be expected to show any m ajor g e n e tic

d is c o n t in u i te s . Again t h i s seems im p la u s ib le f o r th e house mouse in v iew o f

i t s h ig h m o b i l i t y th ro u g h i t s commensal t ie s w ith man. I f p o p u la t io n s have

been sepa ra ted f o r lon g p e r io d s by zoogeographic b a r r ie r s (p h y s ic a l o r

b e h a v io u ra l) th e y would be expected to accum ulate co n co m ita n t d if fe re n c e s

in th e n u c le a r genome (A v ise e t a l . r 1987a). M o rphom e tric , k a ry o ty p ic and Y

chromosome DNA ev id en ce (see ch ap te r 5) fo r B r i t i s h house mouse p o p u la tio n s

are conco rdan t w ith mtDNA da ta , d iv id in g th e p o p u la t io n s in t o two d is t in c t

g e n e tic assem blages.

4i 4i 2 i_M acrog e gg ra B h ic_s tru c ty rin g i_ ev i.d e nce _ frgm _o the r_ E u rgB e an _ M ice _ in

th e _ se a rch _ f g r_ g g e u la t ig n s _ a n c e s tra l_ tg _ th e _ B r it i s h jig u s ^ m g u s e ..

There i s a p p a re n t ly l i t t l e macrogeographic s t r u c tu r in g o f mtDNA typ e s in

European and A sian c o l le c t io n s o f house mice ( t h is study? F e r r is e t a l . f

1983; M oriw aki e t aim, 1984). For example, n o r th e rn m ice ( th e N orth -W este rn

race in B r i t i s h and Norwegian samples) c lu s te re d w ith so u th e rn

M e d ite rranean p o p u la t io n s and no t w ith samples from th e in te rv e n in g

re g io n s . However, F e r r is & co lle ag u es (1983) p o s tu la te d th a t th e n o rth e rn

c lo n e s o f mtDNA may have southern ro o ts , and suggested th e fo rm e r a rose in

th e sou th in p r e g la c ia l tim es and moved no rth w a rd s in Europe as th e ic e

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sh ee ts re t re a te d . Thus, the a n c e s tra l o r ig in s o f th e N o rth e rn is la n d m ice

may be more re c e n t ly -from S cand inav ia , but u l t im a te ly from th e

M e d ite rran e an re g io n s . A l t e r n a t iv e ly , conve rgen t e v o lu t io n may account f o r

th e s im i l a r i t i e s between th e N-W race and Y u g o s la v ia , however, t h i s i s

u n l ik e ly c o n s id e r in g th e la rg e number D f r e s t r i c t io n enzymes and

r e s t r i c t i o n s i t e s in v o lv e d ( f ig u r e 4 .1 1 B ). S im i la r ly , B e rry & Rose, (1975)

u s in g da ta from e p ig e n e tic v a r ia n ts o f th e s k e le to n , showed th e Orkney v o le

p o p u la tio n s were more s im i la r to Y ugos lav ian ones than to any o th e r

c o n t in e n ta l sam ples. They concluded th a t th e v o le s p ro b a b ly o r ig in a te d from

so u th e rn o r e a s te rn M ed ite rranean re g io n s . Orkney v o le s (H icro tus a r v a lis )

are g e n e ra lly tho u gh t to have been in tro d u c e d by th e agency o f man, y e t ,

th e id e n t i t y o f th e a n c e s tra l p o p u la tio n rem a ins s p e c u la t iv e (C o rb e t,

1 9 8 6 ) .

The weak tendency f o r m acrogeographic s t r u c tu r in g o f mtDNA v a r i a b i l i t y in

Hus domesticus i s c o n s is te n t w ith i t s commensal t i e s to h ig h ly m ob ile

human p o p u la tio n s (human mtDNA a ls o e x h ib i t s l i t t l e m acrogeographic

s t r u c tu r in g - Cann Sc W ilson , 1 9 8 3 ) and is conco rdan t w ith a r e la t i v e l y

e x te n s iv e and re c e n t range expansion and th e h ig h d is p e rs a l c a p a b i l i t ie s o f

th e s p e c ie s . Indeed, Hus domesticus, as a commensal o f man, has much o f

i t s re c e n t e v o lu t io n a ry h is to r y t ie d up w ith human a c t i v i t i e s , s in c e b e fo re

th e advent o f a g r ic u l tu r a l p ra c t ic e s (B ro th w e ll, 1 9 8 1 ; Tchernov, 1 9 8 1 ) .

Thus an u n d e rs ta n d in g o f th e h is to r y and sequence o f c o lo n is a t io n o f th e

house mouse i s in co m p le te w ith o u t h is t o r ic a l and g e n e tic a l a n th ro p lo g ic a l

knowledge o f th e s e q u e n tia l demic spread o f fa rm in g in to Europe from th e

M id d le East (Sokal Sc M enozzi, 1982). T h is has been shown to be a s low and

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re g u la r p ro cess , e x te n d in g a p p ro x im a te ly from 9000 B.P to 5000 B .P , a r a te

o f advance o f about one k ilo m e tre per year (Menozzi e t aim, 1978; Bodmer 5c

C a v a l l i - S fo r z a , 1979).

There were a t le a s t f i v e main groups o-f c o lo n is ts in N orth S co tla n d and th e

Orkneys s in c e th e la s t g la c ia t io n ( M i l le r , 1976; Renfew, 1985; B e rry ,

1985b), many o f whom co u ld have b rough t th e house mouse w ith them; some are

more l i k e l y ca n d id a te s than o th e rs . Evidence suggests th a t th e f i r s t

c e r ta in s e tt le m e n t o f Orkney was begun b e fo re 3500 B.C. by N e o l i th ic

fa rm e rs . N e o l i th ic man c o lo n is e d N.W. Europe fo l lo w in g th e r e t r e a t o f th e

P le is to c e n e ic e -s h e e ts , northw a rds . I t i s p o s tu la te d th a t he was

te c h n ic a l ly i l l equipped to deal w ith th e ta sk o f fo r e s t c le a re n c e in

C e n tra l Europe and England. Thus, he advanced n o rth w a rd s from th e

M e d ite rra n e a n , by sea, g e n e ra tio n by g e n e ra tio n , fo l lo w in g c o a s t l in e s ,

in c lu d in g th e A t la n t ic , u n t i l e v e n tu a lly he reached th e f e r t i l e lo w la n ds o f

Orkney which were la r g e ly devoid o f t re e s . He e s ta b lis h e d lo c a l v e rs io n s o f

h is c u ltu r e a long th e way, le a v in g m em oria ls , such as s ta n d in g s tones and

ro ck c u t tombs (M e g a lith s ) , as ev idence o f h is p resence . House mice may

have been in a d v e r ta n t ly in tro d u ce d a long w ith dom estic l iv e s to c k , such as

goa ts and sheep, in t h e i r feed and bedd ing . Around 700 B.C. (A .D . 297) in

th e Iro n Age, th e n ex t c o lo n is ts , c a lle d th e " p ic t s " o r th e "p a in te d ones"

a r r iv e d , d e fin e d by th e numerous "B rochs" (d e r iv e d from th e word "B o rgs"

meaning d e fe n c e s ), carved symbol s tones and in s c r ip t io n s u s in g Ogam

(P ic t is h ) a lp h a b e t. E a r ly 7 th c e n tu ry b rough t th e C e lt ic C h r is ta in s from

I r e la n d , a lth o u g h t h e i r in f lu e n c e was emphemeral. Then came th e Norse

in f lu e n c e , which i n i t i a l l y took th e form o f p eace fu l s e tt le m e n ts , however,

CHAPTER FOUR

by th e b e g in in g o f th e 8 th c e n tu ry , th e re was a -flood o-f a g g re s s iv e ra id s

by th e V ik in g s , am ounting to m ass-m ig ra tion which c e r t a in ly made a g re a t

im pact on th e is la n d s (see T a y lo r , 1938 fo r a modern t r a n s la t io n o-f th e

O rkneyinga Saga, w r i t t e n 1192-1206). Orkney was used as a base fo r r a id s on

n o r th S co tla n d , th e H e b rid es , and th e I s le o f Man. The e xa c t d a te s o f th e

V ik in g e ra in th e Orkneys a re unknown, however, th e S cand inav ian in f lu e n c e

la s te d lo n g e r here than anywhere e ls e in B r i t a in . In England th e r a id on

L in d is f ra n e in 793 re p re s e n ts th e b e g in in g and th e b a t t le o f H a s tin g s in

1066 as th e end o f V ik in g in f lu e n c e . The la s t two decades o f th e 8 th

c e n tu ry is surposed t o re p re s e n t th e s t a r t o f V ik in g r a id s in I re la n d , and

th e Norman in v a s io n o f I re la n d in 1172, o r th e b a t t le o f C lo n ta r f in 1014,

th e end o f th e S cand inav ian predom inance. In th e I s le o f Man, th e re ig n o f

th e S cand inav ian k in g , Magnus, ended in 1263. The O rkneys, fo r m a lly

rem ained under th e Norse r u le u n t i l th e im p ig n o ra t io n D f th e I s le s in 1468-

9. Y e t, th e nex t phase o f c o lo n is ts , p ro b a b ly began th e p ro cess o f

’ S c o t t i f i c a t io n ’ in 1231, w ith a success ion o f S c o t t is h e a r ls in th e Orkney

Earldom .

To summarise, ju s t as C orbet (1986) b e lie v e s th e re i s no b a s is a t p re sen t

f o r id e n t i f y in g any p a r t ic u la r c o n t in e n ta l p o p u la t io n o f v o le s as be ing

a n c e s tra l t o th e Orkney v o le , so to o t h is appears to be th e case fo r th e

N-W ra ce o f th e B r i t i s h house mouse. Both g e n e tic , a n th ro p o g ic a l and

h is t o r ic a l ev idence sugges ts a Norse in f lu e n c e , conco rdan t w ith th a t

p o s tu la te d from o th e r sm a ll mammal s tu d ie s (B e rry , 1969, 1970, 1975, 1985,

1986b; Delany & W h it ta k e r , 1969; C orbe t, 1964; Evans, 1977).

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The la rg e number o-f d i f f e r e n t mtDNA c lo n e s , and h ig h d iv e r s i t y found in the

S-E ra ce in B r i t a in , i s c o n s is te n t w ith a s e r ie s o f m u lt ip le c o lo n is a t io n

e ve n ts . Hus domesticus mtDNA typ e s found in so u th e rn B r i t a in were grouped

w ith tho se from Denmark and Germany, su gg e s ting p o s s ib le c o lo n is a t io n o f

B r i t a in from th e c o n t in e n t across the E n g lish c h a n n e l, p o s s ib ly p a r a l le l in g

th e movements o f man. B e fo re t h i s the house mouse c o u ld , perhaps, have

crossed th e channel on i t s own. The e u s ta t ic lo w e r in g o f th e s e a - le v e l,

uncovered a w ide land co nn e c tion between B r i t a in and th e c o n t in e n t across

th e n o r th sea and E n g lis h channel from th e south o f Denmark to th e n o r th o f

F rance. At i t s maximum, s e a - le v e l was about 70-100 m etres below th a t o f

p re s e n t. When warmer c o n d it io n s ensued (approx. 7600 B.C) B r i t a in was s t i l l

connected to th e c o n t in e n t , a llo w in g the re p le n ish m e n t o f th e B r i t i s h fauna

by w a rm th - lo v in g s p e c ie s . I re la n d , however, d id n o t re c e iv e t h i s p o s t­

g la c ia l in v a s io n o f s p e c ie s , p ro ba b ly due to th e narrowed I r i s h sea. The

thaw ing o f th e ic e sh e e ts re s u lte d in a r is e in s e a - le v e l s u f f ic e n t to

sever B r i t a in from th e c o n t in e n t about 6600 B.C. Once B r i t a in became an

is la n d a g a in , any f u r th e r a r r iv a l o f new sp ec ies excep t f o r those th a t

co u ld f l y , would have occu rre d v ia in t ro d u c t io n s by man in boa ts o r

in d e p e n d e n tly by " r a f t in g " .

A rc h a e o lo g ic a l ev idence suggests many ro d e n ts d id manage to spread to

England b e fo re th e la n d b r id g e connec ting B r i t a in w ith th e c o n t in e n t was

severed , however, in fo rm a tio n from s u b fo s s il d a ta ar? d i f f i c u l t to

in te r p r e t because burrow s may in tru d e in to o ld e r s t r a ta . One re co rd

c o n firm s th e presence o f the house mouse in pre-Roman Iro n Age, bu t a f t e r

th e severance o f th e la n d b r id g e ; based on th e ro s tru m o f two an im a ls and

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one m and ib le , id e n t i f ie d from a s i t e a t Gussage A l l S a in ts , D o rse t. T h is

e v idence i s p ro b a b ly r e l ia b le because th e y were found in a sea led la y e r

w ith o th e r sm a ll mammals, and th e re was no s ig n o f subsequent d is tu rb a n c e

(C o rb e t, 1974; L eve r, 1985).

4i 4i 3 _ P a tte rn s _ o f _co l.gn i5a ti.gQ _5ugge5 ted_by_m itochondria l_D N A _ana lyses i

Hus domesticus in O rkney, N.E. S co tla n d (C a ith n e ss and S u th e r la n d ) ,

I re la n d and th e I s le o f Man, have mtDNA com pos ite genotypes n o t observed

among samples from th e r e s t o f th e B r i t i s h m a in land and a s s o c ia te d is la n d s

( I s le o f May, Skokholm, and In c h k e ith ) , sou th o f th e G reat Glen f a u l t (b u t

see Gatehouse o f F le e t sample, D u m frie s ). The d is t r ib u t io n o f p a r t ic u la r

r e s t r i c t io n fragm ent p a t te rn s , produced e i t h e r by s in g le r e s t r i c t io n

endnucleases o r in com b ina tion (com posite g e n o ty p e s ), can p ro v id e p o s s ib le

c lu e s about th e e v o lu t io n a ry r e la t io n s h ip s among th e races o f m ice and

suggest te n ta t iv e s c e n a rio s fo r th e sequence o f c o lo n is a t io n e ven ts .

U sing fo u rte e n r e s t r i c t io n enzymes, f i v e mtDNA c lo n e s were observed in th e

O rkneys and n e ig h b o u rin g m ain land c o u n t ie s . Based upon th e mtDNA a n a lyse s ,

th e re appears to be a c lo s e r e la t io n s h ip between a l l seven Orkney is le s

sampled and in a l l s i t e s in C a ithness and S u th e r la n d ; C lone 1 was found

p re d o m in a n tly in a l l s i t e s , even e x c lu s iv e ly in some cases. O nly one s i t e

on M ain land Orkney (H a rray) c lo n e 1 was n o t fou n d , two un ique c lo n es (4 &

5) were fou n d , however, o n ly 10km away on th e same is la n d c lo n e 1 was

observed a t th e Yaphur s i t e . Evidence from p ro te in polym orph ism d a ta , based

on 24 a llozym e lo c i , o f mice from C a ith ne ss and th re e Orkney is le s

(W estray, Eday and S tro n sa y , r e s p e c t iv e ly ) , show th e l a t t e r is la n d

p o p u la tio n s to be a subse t o f th e n e ig h b o u rin g m ain land p o p u la t io n s (Nash

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e t aim, 1983). D if fe re n c e s in a l l e le fre q u e n c ie s between is la n d p o p u la tio n s

were d e te c te d , bu t w ith one s in g le e x c e p tio n , a l l a l le le s p re s e n t in th e

is la n d p o p u la t io n s were a ls o p re s e n t in th e C a ithness m ice. The a l le le Smg-

l b (p ro te a s e lo c i , lo c a te d on chromosome 7) was observed in S tronsay and

W estray, bu t was n o t d e te c ted in th e C a ith ne ss p o p u la t io n , however, t h i s

s in g le C a ith ne ss sam pling s i t e may n o t be re p re s e n ta t iv e o f th e coun ty in

g e n e ra l.

The g e o g ra p h ic a lly a d ja ce n t n o r th e rn Orkney is la n d s o f W estray, Eday and

Faray share a no the r mtDNA com posite genotype (c lo n e 2 ) ; K e is s , N.E

C a ith n e ss , was th e o n ly o th e r s i t e where t h i s genotype was observed , over

80km away from th e N o rthe rn Orkney I s le s . I t i s h ig h ly u n l ik e ly th a t th e

complex r e s t r i c t io n fragm ent p a t te rn s c h a ra c te r is in g c lo n e 2 co u ld a r is e by

co nve rg e n t e v o lu t io n , in bo th th e n o r th e rn Is le s and th e m a in land s i t e ,

a l ik e and i s in d ic a t iv e o f a p o s s ib le c o lo n is a t io n ro u te l in k in g W estray,

Eday, F a ray , and th e N.E. co rn e r o f C a ith ne ss on th e b a s is o f c lo n e 2. T h is

mtDNA c lo n e ty p e occurs a t a much h ig h e r frequency in W estray (0 .1 2 , n=10),

and i s m a rg in a lly h ig h e r in Faray (0 .0 5 , n=3) and Eday (0 .0 4 , n = 3 ), than in

K e iss (0 .0 2 , n = l) . A s t r ik in g p a r a l le l e x is ts when t h is da ta i s compared

w ith k a ry o ty p ic in fo rm a tio n . R o b e rtso n ian t ra n s lo c a t io n s were reco rded in

th e same th re e Orkney p o p u la t io n s , and th ro u g h o u t C a ith ne ss (B ro o ke r,

1982). A l l th e o th e r Orkney is la n d s , S u th e rla n d and th e r e s t o f B r i t a in do

n o t have any c e n t r ic fu s io n s , hav ing th e normal 2n=40 k a ry o ty p e . A l l th e se

mice w ith R obertson ian fu s io n s share R b(9-12) su gg e s ting th e y have a common

o r ig in (S c riv e n & B rooke r, 1990). A d d i t io n a l ly Rb(4-10) was found on Eday

and C a ith n e s s , bu t no-where e ls e . T h is fu s io n may have a r is e n in d e p e n d e n tly

CHAPTER FOUR

in b o th p la c e s , however, th e re i s no ev idence th a t t h i s p a r t i c u la r

a s s o c ia t io n i s -favoured over any o th e r -fu s io n , o r , as seems more l i k e l y ,

th a t th e se two p o p u la tio n s share a common o r ig in .

Nash e t aim, (1983) compared Orkney p o p u la tio n s -from W estray, Eday and

S tro n sa y w ith a p o p u la tio n from C a ith n e ss , on th e b a s is o f m orphom etric ,

k a ry o ty p ic and a llozym e v a r ia t io n . They co n firm e d from t h e i r chromosomal

o b s e rv a tio n s th e c lo s e r e la t io n s h ip between Eday and C a ith n e s s , in a d d it io n

to th a t between Eday and W estray. A lso , th e y found s tro n g l in k s between

W estray and Eday, and between S tronsay and C a ith n e ss , based upon

m orphom etric ev idence . I f th e m ice a re p o s tu la te d to have a r r iv e d w ith man,

th e n th e f i r s t t ra n s lo c a t io n to have a r is e n a f te r t h e i r a r r i v a l would

p ro b a b ly have been Rb (9 -1 2 ) , hence mice would have f i r s t c o lo n is e d

W estray , and then Eday (Nash e t aim, 1983).

S p e c if ic r e s t r i c t io n fragm ent d ig e s t io n p a tte rn s suggest o th e r p o s s ib le

p a t te rn s o f c o lo n is a t io n . D is t in c t m ito c h o n d r ia l com pos ite genotypes un ique

to W estray and to H array (C lone 3 & 5, re s p e c t iv e ly ) share an A lu I

p a t te rn des igna ted "D" , which d i f f e r s from th e common * B" p a t te rn by two

s i t e d if fe re n c e s , su g g e s tin g a c lo s e r l in k than f i r s t th o u g h t. However,

t h i s p a t te rn may have a r is e n in each p o p u la tio n in d e p e n d e n tly by convergen t

e v o lu t io n , e s p e c ia l ly as th e Rsa I p a t te rn s do no t su p p o rt a c lo s e r l in k ;

c lo n e 1 shows Rsa I p a t te rn "B" , w h i ls t c lo n es 3 & 5 show p a t te rn s "G" and

"E " , r e s p e c t iv e ly , in v o lv in g s ix base d if fe re n c e s between th e l a t t e r two

p a t te rn s , to o co m p lica te d to in d ic a te a c lo s e r r e la t io n s h ip . C lones 3 & 5

appear to re p re s e n t d i f f e r e n t c o lo n is a t io n e ve n ts , from th e same a n c e s tra l

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source as th e common ty p e s found in th e O rkneys.

V a r ia b i l i t y o f mtDNA a t th e m ainland lo c a l i t i e s was c o n s id e ra b ly low er than

t h a t found in some o f th e Orkney is la n d s . Only one com pos ite mtDNA genotype

was observed among th e 7 mainland lo c a l i t i e s w ith th e e x c e p tio n o f two a t

K e is s , where th e second c lo n e was o n ly re p re se n te d by one mouse. In

com parison , th re e com pos ite genotypes were observed in two Orkney is la n d s ,

and two genotypes in a fu r th e r two is la n d s , o n ly 3 Orkney is la n d s surveyed

were monomorphic as re g a rd s c lo n a l ty p e s . Values o f h e te ro g e n e ity (h)

averaged 0.21 fo r th e is la n d s , and 0.001 fo r th e m a in land p o p u la t io n s . T h is

extrem e re d u c tio n in mtDNA v a r ia b i l i t y in C a ith n e ss , i s n o t r e f le c te d in

th e n uc lea r-co d ed a llo z y m ic v a r ia b i l i t y (B e rry 8c P e te rs , 1 9 7 7 ; Nash e t

1 9 8 3 ) ; o v e ra ll h e te ro z y g o s ity va lues per lo c u s ranges up to 1 1 .4 7 . in

C a ith ne ss p o p u la t io n s , w ith an average o f 5X among th e O rkneys. M oreover,

th e average fo r Hus domesticus p o p u la tio n s in B r i t a in i s a p p ro x im a te ly 67 .,

w h i ls t th e average f o r a l l ro d en ts i s 5 .6 7 . (S e lande r and Kaufman, 1 9 7 3 ) .

Thus, th e re appears to be no re d u c tio n o f h e te ro z y g o s ity in e i th e r m ain land

o r is la n d p o p u la t io n s . G e n e ra lly , th e main consequence o f fou n de r even ts is

e ro s io n o f g e n e tic v a r i a b i l i t y . A f i n i t e number o f c o lo n is e rs fo u n d in g a

p o p u la tio n would be expected to have le s s v a r ia t io n , w hich i s observed in

most is la n d p o p u la tio n s o f sm all ro d e n ts (Peromyscus spp - Aquadro 8c

K i lp a t r i c , 1 9 8 1 ; Hus domesticus - B e rry 8c P e te rs , 1 9 7 7 , and B e rry e t a i . ,

1 9 7 8 ) . However, e x c e p tio n s to t h is r u le have been fou n d . An in c re a s e in

a llo zym e v a r i a b i l i t y was observed in H aw aiian m ice (B e rry , e t a i» , 1 9 8 1 ) ,

a ls o Navajas Y N avarro 8c B r it to n -D a v id ia n , ( 1 9 8 9 ) showed th e re was no

re d u c t io n in a llo zym e v a r ia b i l t y in m ice from M e d ite rran e an is la n d s

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CHAPTER FOUR

compared to th e Hus domesticus p o p u la tio n s on th e n e ig h b o u rin g m a in land ,

w ith th e e x c e p tio n o-f one on a v e ry t in y is la n d (6 h a ) , in which o n ly one

t h i r d o-f th e a n c e s tra l v a r i a b i l i t y was lo s t . The s e v e r e ity o-f a founder

e f f e c t i s more dependent on th e p o s t c o lo n is a t io n r a te D f p o p u la tio n

in c re a s e , ra th e r than th e s iz e o f th e fou n de r p o p u la t io n (Nei e t a l . f

1975). The m aintenance o-f v a r ia t io n fo l lo w in g a fo u n d e r e ven t i s p ro b a b ly

due to a m u lt ip le in t r o d u c t io n p a t te rn o-f c o lo n is a t io n in th e se is la n d s ,

and ra p id p o p u la tio n g row th fo l lo w in g each c o lo n is a t io n e v e n t. Mice from

a l l o f the Orkney is la n d s have been shown to be re m a rka b ly homogenous w ith

re s p e c t to a llo zym e v a r ia t io n , which has been a t t r ib u te d to th e h igh mouse

d e n s it ie s and e x te n s iv e fa rm in g a c t i v i t e s (B e rry & P e te rs , 1977) found on

these is la n d s .

Why shou ld founde r e f f e c t s cause a g re a te r re d u c t io n in m ito c h o n d r ia l than

in n u c le a r gene v a r ia b i l i t y ? One e x p la n a tio n is th a t p o p u la t io n b o tt le n e c k s

a re much more extrem e fo r m ito c h o n d r ia l genes than f o r autosom al genes

(W ilson e t a l 1985) . T h is i s a consequence o f th e tra n s m is s io n g e n e tic s

o f m ito c h o n d r ia l DNA. A s in g le p regnant fem ale c o lo n is t w i l l c a rry a la rg e

amount o f nuclear—encoded v a r ia t io n (a u to so m a l), b u t o n ly a s in g le

m ito c h o n d r ia l geno type . M ito c h o n d r ia l DNA is m a te rn a lly in h e r i te d (F is c h e r

L in d a h l, 1985), th u s mtDNA genes can o n ly be passed th ro u g h fem ale

lin e a g e s , assuming th e re i s no p a te rn a l leakage (G y lle n s te n e t a i» , 1985)

and s t r i c t homoplasmy. H eterop lasm y (two o r more mt genotypes c o e x is t in g

w ith in an in d iv id u a l) i s e x tre m e ly unusual in anim al mtDNA (Bermingham, e t

a i . , 1986). Only one case o f he te rop lasm y has been documented in th e house

mouse, thu s f a r (B ou rso t e t a l 1987). B irk y e t a l (1983, 1989) have

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shown, assuming equal sex r a t io s , m a te rn a l, h a p lo id in h e r i ta n c e o f mtDNA,

th a t th e e f f e c t iv e p o p u la tio n s iz e f o r m ito c h o n d r ia l genes w i l l be one

q u a r te r th a t o f d ip lo id nuc lea r-e n cod e d genes. As a consequence, th e mear

t im e t o f i x a t io n o r lo s s o f new m ito c h o n d r ia l m u ta tio n s w i l l be

a p p ro x im a te ly tw ic e th a t o f n u c le a r ones. Hence p o p u la t io n s w i l l be

expec ted to be su b d iv id e d fo r m ito c h o n d r ia l genes, a t m ig ra t io n ra te s a t

w h ich th e n u c le a r genes a re p a n m ic t ic ; t h i s e f f e c t i s a cce n tu a te d i f males

d is p e rs e more than fem a les . F ie ld s tu d ie s have suggested th e re i s no

e v id e n ce o f p r e fe r e n t ia l d is p e rs a l by males in th e house mouse (B e rry ,

1968), a lth o u g h i t i s suspected (see c h a p te r 6; B u t le r , 1980). However,

equal d is p e rs a l does n o t n e c e s s a r ily equa te w ith equal gene f lo w . When

p re g n a n t fem a les d is p e rs e , t h i s causes th e f lo w o f bo th male and fem a le

genomes, w h i ls t male d is p e rs a l a llo w s th e spread o f m a le -d e r iv e d genomes

o n ly . Thus an equal system o f d is p e rs a l causes m a le -b ia sed gene f lo w , i f

th e d is p e rs in g fem a les a re in se m in a te d (D e s a lle e t a i . , 1987). P regnant

fem a les have been reco rded among house mouse e m ig ra n ts (M yers, 1974).

Sum m arising ev idence from mtDNA endonuclease d ig e s t io n p a t te rn s , to g e th e r

w ith o th e r g e n e tic d a ta , th e Orkney I s le s appear t o have have expe rie n ced

a t le a s t two se p a ra te c o lo n is a t io n s o f house m ice, from th e same a n c e s tra

so u rce . The m ajor sequence o f e ve n ts deduced from th e mtDNA d a ta appears :o

be an i n i t i a l c o lo n is a t io n even t on Mid to South W estray, fo l lo w e d by

spread to th e n e ig h b o u rin g is la n d o f Eday. The sm a ll is la n d between Eday

and W estray, Faray was p ro b a b ly c o lo n is e d by mice from bo th n e ig h b o u rin g

is la n d s , a lth o u g h mtDNA p a tte rn s have shown th a t one c lo n a l ty p e (c lo n e 2

was found p re d o m in a n tly in th e west o f th e is la n d ; y e t th e la n d in g j e t t y s

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CHAPTER FOUR

s itu a te d to th e fa r south o f th e is la n d , a p p ro x im a te ly e q u i- d is ta n t to th e

two n e ig h b o u rin g is la n d s . However, due t o i t s sm a ll s iz e (7 5 h a ), th e re is

p ro b a b ly random m ix ing o f in d iv id u a ls , over th e whole is la n d . However,

c lo n e 2 was found to p e r s is t on Faray a long th e w es te rn c o a s t, where i t was

m o n ito red in a p p ro x im a te ly th e same area in th re e c o n s e c u tiv e sam p ling

ye a rs ( f ig u r e 4 .2 ) . The is la n d has been pe rm anen tly u n in h a b ite d by man

s in c e 1940, from which t im e i t has o n ly been used as p a s tu re f o r sheep from

W estray.

M ice p ro b a b ly c o lo n is e d C a ithness o r ig in a l l y in th e N .E. c o rn e r , fo l lo w in g

a p a th from Eday v ia e i th e r e a s te rn M a in land Orkney o r S tro n s a y . On t h e i r

a r r i v a l , th e mice p ro b a b ly spread sou th and west th ro u g h C a ith n e ss to

S u th e r la n d . M ice, fo l lo w in g man, p ro b a b ly ra d ia te d o u t from th e n o rth e rn

is le s t o a l l th e o th e r is la n d s s e q u e n t ia l ly . Another independan t

c o lo n is a t io n even t may have o ccu rre d in West M ain land O rkney, founded a t

a p p ro x im a te ly th e same t im e , as un ique b u t d e r iv e d c lo n e s were observed

h e re . Yet th e y were g e n e t ic a l ly ve ry c lo s e ly re la te d to th e common c lo n e

found th ro u g h o u t th e Orkney and n e ig h b o u rin g m ain land a re a , and, c lo s e ly

r e la te d to a un ique c lo n e found on W estray. M ain land Orkney may have been

c o lo n is e d from W estray and th e documented v a r ia t io n re p re s e n ts e v o lu t io n

subsequent to t h e i r s e p a ra tio n . One l i k e l y c o lo n is a t io n s c e n a r io suggested

by t h i s s tud y i s g ive n in F ig u re 4.15A . I t i s shou ld be noted th a t t h i s i s

n o t th e o n ly pathway th a t can be env isag e d , m ere ly th e most pa rs im o n iou s .

The I r i s h fauna has long posed a prob lem fo r b iogeographers (Moore, 1987).

Indeed , th e re has been c o n tro v e rs y as to whether e x ta n t sp e c ie s e ith e r

ii

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CHAPTER FOUR

s u rv iv e d in g la c ia l r e fu g ia in p e rm a fro s t—fre e a reas o f I r e la n d , o r

d is p e rs e d to I re la n d a f t e r th e la s t g la c ia l p e r io d ove r a la n d b r id g e , o r

were in a d v e r ta n t ly in tro d u c e d by man (Y a lden , 1982). The presence o f th e

house mouse and th e bank v o le (Clethrionomyus glareolus) has been

a t t r ib u te d to th e la s t o f the se (S e a r le , 1989; F a ir le y , 1971; Smal &

F a ir le y , 1978). Indeed, th e e r r a t ic co m p o s itio n o f I r i s h fauna g e n e ra lly

sugges ts a s e r ie s o f chance in t r o d u c t io n s , ra th e r tha n an o rd e r ly sequence

o f c o lo n is a t io n s from a r t i c to tem pera te faunsa which was a b ru p t ly

te rm in a te d by th e severance o f a land c o n n e c tio n .

Three mtDNA c lo n e s were found in I re la n d , two (c lo n e s 6 & 7; n^ 11) in

N o rth e rn Ire la n d and one (c lo n e 8; n = l) in Southern I r e la n d , on th e b a s is

o f 14 r e s t r i c t io n endnucleases. The I s le o f Man and one s i t e in N o rthe rn

I re la n d shared a c lo n e ; th e l a t t e r s i t e a ls o shared a n o th e r c lo n e w ith a

s i t e fu r th e r n o r th in N .I re la n d . Too few m ice were examined to make any

f i r m s ta tem en ts about c o lo n is a t io n based upon th e p a t te rn s observed from

th e th re e c lo n a l typ e s a lo n e . However, one c lo n e (6) co u ld o n ly be

d is t in g u is h e d from c lo n e (1 ) , common in th e O rkneys, on th e b a s is o f a

s in g le r e s t r i c t io n s i t e d i f fe re n c e , re v e a lin g a c lo s e r l i n k between Orkney

and I re la n d , than between th e common Orkney c lo n e and th e o th e r 4 c lo n a l

ty p e s observed in Orkney and N.E S co tland (see f ig u r e 4 .1 1 A ).

I would t e n ta t iv e ly su gg e s t, based on mtDNA ev idence (see f ig u r e 4 .1 5 B ),

th a t th e re were p ro b a b ly a minimum o f two fo u n d in g e ven ts in I re la n d .

Assuming, th e c o lo n is a t io n s arose p r im a r i ly from th e n o r th , one event

re s u lte d in c o lo n is a t io n o f th e east coast o f I re la n d and th e I s le o f Man,

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how ever, th e d ir e c t io n o f movement between th e I r is h and Manx c o a s ts i s

d i f f i c u l t to assess. There was p ro b a b ly ano the r fo u n d in g even t on th e west

co a s t o f I r e la n d , about th e same tim e as th e fo rm er and from th e same

a n c e s tra l s o u rce . A no ther un ique c lo n e was observed on th e S c o t t is h

m a in land a d ja c e n t to I r e la n d , a t Gatehouse o f F le e t , D u m frie s ; a lth o u g h

t h i s mtDNA genotype c lu s te r s w ith th e N.W. race based upon fo u r te e n

r e s t r i c t io n enzymes, i t i s c o m p a ra tiv e ly d is t a n t ly r e la te d and p ro b a b ly

re p re s e n ts a c o m p le te ly se p a ra te in t r o d u c t io n e ven t.

In c o n c lu s io n , a lth o u g h th e re appears to be no s u b s ta n t ia l m acrogeographic

s t r u c tu r in g o f w o rld w id e house mouse p o p u la tio n s , m ic rog e og ra p h ic

s t r u c tu r in g i s apparen t in th e B r i t i s h p o p u la t io n s , d e m o n s tra tin g th a t

th e re i s a p h y lo g e o g ra p h ic component to p o p u la tio n s t r u c tu r e . T h is

in d ic a te s th a t gene f lo w i s n o t s tro n g enough to c o m p le te ly homogenise

mtDNA genotypes across th e e n t i r e sp e c ie s range. Thus, i t would appear th a t

h is t o r ic a l b iogeography i s im p o rta n t in shap ing mtDNA geog raph ic

d is t r ib u t io n s even in a h ig h ly m o b ile sp e c ie s , th a t has e xpe rienced re c e n t

and ra p id range expans ion . Indeed, mtDNA has proved e s p e c ia l ly u s e fu l f o r

re c o n s tru c t in g th e p ro b a b le c o lo n is a t io n p a tte rn s and th e e v o lu t io n a ry

h is t o r y o f th e B r i t i s h house mouse.

TABLE 4.1 ■ The mtDNA com pos ite genotypes (mt c lo n e s ) obseryed_amgnq_samples

o f B r i t i s h house m ice ( tfu s domesticus) .

1 - L e t te r s d e s c r ib in g mtDNA com posite genotypes from l e f t t o r ig h t , r e fe r

to r e s t r i c t io n fragm en t p a t te rn s fo r th e r e s t r i c t i o n endonucleases Hind

I I I , Xba I , H inc I I , Acc I , Ava I I , FnuD I I , Hpa I I , Hae I I I , Taq I , Mbo I ,

H in f I , A lu I , Rsa I and Sau 961, r e s p e c t iv e ly .

The number in b ra c k e ts r e fe r s to th e mtDNA com pos ite number (C lone number)

and th e g e o g ra p h ica l d is t r ib u t io n o f th e se a re i l l u s t r a t e d in F ig u re 4 .4 .

The number o f m ice sampled th a t show a p a r t i c u la r mtDNA com pos ite genotype

from a p a r t ic u la r c o l le c t in g s i t e a re g iv e n .

2 - Frequency o f a p a r t i c u la r mtDNA com pos ite genotype w ith in a g iven

p o p u la tio n sample per sample ye a r.

267

TABLE 4 .H

mtDNA genotype (c lo n e N ° )1 N° fre q u e n cy c a p tu re d a te?< c a p tu re lo c a t io n o f m ice o f mt c lo n e 2

(1 ) . BAAAJADXBOCBBB O rkney:

W estray, Noup W estray, Quoy W estray, Hammars W estray, G rinaby W estray, S ke lw ick Papa W estray, H o lla n d Eday, NewbigginIt II

II IIEday, Ruah FarayIIII

S tron sa y , H o lland Sanday, Newark Yaphur, M ain land Orkney

C a ith n e ss :John ’ 0 ’ G roatsIIThurso, O lr ig C a s tle to w n , G reenland Barnaclavan K e iss

S u th e rla n d :AchiemoreArmadale

(2 ) . BAAAJADXBOCCGC Orkney:

W estray, Quoy W estray, Hammars Eday. Ruah FarayIIII

C a ith n e ss :K e iss

(3 ) . BAAAJADXBOUDGC O rkney:

W estray, Quoy(4 ) . BAAAJADRBOCBEC O rkney:

H array , M ain land Orkney(5 ) . BAAAJADRBOCDEC O rkney:

H array , M ain land Orkney

11 1 .00 3 /8 829 0 .7 6 3 /8 618 0 .8 6 3 /8 69 1 .00 3 /8 88 1 .00 3 /8 81 1 .00 - /8 012 1 .00 3 /8 046 1 .00 3 /8 66 1 .00 3 /8 83 0 .5 3 /8 811 0 .91 9 /8 428 0 .9 6 9 /8 519 0 .9 5 9 /8 62 1 .00 4 /8 63 1 .00 3 /8 0I 1 .00 3 /8 8

4 1.00 4 /8 63 1 .00 9 /8 73 1.00 9 /8 7I I 1 .00 3 /8 45 1.00 9 /8 711 0 .9 2 3 /84

4 1.00 9 /8 71 1 .00 9 /87

7 0 .1 8 3 /8 63 0 .14 3 /863 0 .5 0 3 /8 81 0 .0 9 9 /841 0 .04 9 /851 0 .0 5 9 /8 6

1 0 .0 8 3 /84

3 0 .05 3 /8 6

3 0 .30 3 /8 8

7 0 .70 3 /88

(6 ) . BAAAJAAXBOCBBBN. I re la n d ,B e l f a s tII

I s le o f Man, L ingagne(7 ) . BAAAJAAXBOCBFB

N .I re la n d , B e lfa s t N. I r e la n d , Moneymore

(8 ) . BAAAMAAXB/GBFBS . I r e la n d , Galway

(9 ) . AAAALADAAWAFAA F i r t h o f F o r th :

In c h k e ith I s le o f May

(1 0 ). AAAALAAAAAXABA D u m frie s :

Gatehouse o f F le e t ,(1 1 ). AAAAKKDAAAAHBA M id la n d s :

B u r to n -o n - tre n tII

(1 2 ). AAAAKKDAAAAAAA M id la n d s :

Bur t o n - o n - t r en t D erbysh i re

(1 3 ). AAAAKKDWAAZHCA M id i and:

B u r to n -o n - tre n t(1 4 ). AAAAKKDVAASJAAA

B irm ingham , Mosley(1 5 ). AAAAKKDVAAZAAA

B irm ingham , Mosley(1 6 ), AAAALJAAAAAAAA C e n tra l London:

K ings Cross London Zoo

South London:Fulham

S u rre y :N u t f ie ld

(1 7 ). AAALLJAAAAZAAA S u rre y :

N u t f ie ld South London:

Wimbledonk e n t:

East G rin s te a d ( IB ) . AAALLJAUAAZAAA S u rre y ;

West Humble Ham pshire:

W incheste r(1 9 ). AAALLJAUA3SGDA K en t:

East G rin s te a d ,

3 1 .00 9 /8 73 0 .4 3 8 /8 81 1 .00 10/87

4 0 .57 8 /8 81 1.00 9 /87

1 1.00 10/87

1 1.00 - /8 012 1.00 - /8 0

1 1.00 9 /88

7 0 .7 8 9 /878 0 .72 6 /88

2 0 .22 9 /873 1.00 7 /87

3 0 .2 8 6 /88

2 0 .25 6 /87

6 0 .75 6 /87

1 1.00 4 /873 1.00 9 /88

3 1.00 4 /87

5 0 .55 6 /88

5 0 .45 6 /88