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Proc. Natl. Acad. Sci. USAVol. 76, No. 9, pp. 4294-4298,
September 1979Biochemistry
Structure of genes for human growth hormone and
chorionicsomatomammotropin
(in vitro packaging/filter hybridization/intervening
sequences/DNA sequences)
JOHN C. FIDDES*, PETER H. SEEBURG*t, FRANCES M. DENOTO*, ROBERT
A. HALLEWELL*,JOHN D. BAXTER*t§, AND HOWARD M. GOODMAN*ftThe Howard
Hughes Medical Institute Laboratory, Departments of *Biochemistry
and Biophysics and §Medicine, and the IMetabolic Research Unit,
Universityof California, San Francisco, California 94143
Communicated by Donald F. Steiner, June 1, 1979
ABSTRACT A 2.6-kilobase (kb) EcoRI restriction endonu-clease
fragment containing human growth hormone (hGH; so-matotropin) gene
sequences and a 2.8-kb EcoRI fragment con-taining human chorionic
somatomammotropin (hCS; cho-riomammotropin) gene sequences have
been identified by hy-bridization to cloned cDNA. Human DNA was
cleaved withEcoRI and fractionated by preparative agarose gel
electro-phoresis; DNA in the size range 2-3 kb was ligated to
XgtWES'XB DNA and viable recombinant bacteriophage wererecovered by
in vitro packaging. After infection of Escherichiacoli and
screening of phage plaques, single isolates of hGH andhCS gene
sequences were obtained. Restriction endonucleasemapping showed
that the hGH gene contains three interveningsequences interrupting
the coding sequence. Partial DNA se-quence analysis of the hGH
gene, obtained by the chain ter-mination method, confirmed the
location of the interveningsequences and the identity of the
fragment.
Human growth hormone (hGH) and human chorionic
soma-tomammotropin (hCS) are two closely related
polypeptidehormones which have more than 80% of their 191 amino
acidsin common (1, 2). They have different biological activities
andare synthesized in different tissues: hGH in the pituitary
andhCS in the placenta. The genes coding for these two
hormonesprovide a good system for studying the organization of
struc-turally related sequences and their tissue-specific
expression.A 550-base-pair Hae III fragment cDNA clone coding
for
amino acids 24-191 of the hCS sequence has been described(3).
Recently, the analogous 550-base-pair Hae III fragmentcoding for
hGH has also been cloned and its nucleotide se-quence has been
determined (unpublished data). The nucleo-tide sequences of these
two fragments are 93% homologous.
Further analysis of this system requires the isolation ofgenomic
DNA fragments that contain the entire coding se-quences and the
regulating elements that may be involved inthe differential
expression of the genes. This report describesthe construction of
genomic DNA clones for sequences of boththe hGH and hCS genes by
utilizing bacteriophage Xgt WES-XB as the vector (4) and the
bacteriophage X in vitro packagingsystem of Blattner et al.
(5).
MATERIALS AND METHODSHuman Placental DNA. High molecular weight
DNA was
extracted from human placenta obtained by caesarian section.The
frozen tissue was dispersed in a blender, treated withproteinase K,
extracted with phenol/chloroform, and then in-
cubated sequentially with RNase A and proteinase K (6).
Afterextraction with phenol/chloroform, the DNA was
precipitatedwith ethanol.
Preparative Agarose Gel Electrophoresis. EcoRI-digestedhuman
placental DNA (17.5 mg) was fractionated on a con-tinuous elution,
horizontal, 0.8% agarose gel, essentially as de-scribed by Polsky
et al. (6) except that Seakem HGT (P) agarosewas used because this
appeared to contain fewer contaminantsthat inhibit subsequent
enzymatic steps. The DNA was collectedin approximately 80
fractions, and aliquots of these were as-sayed by analytical
agarose gel electrophoresis followed by filterhybridization to a
radioactive hGH cDNA probe.
Filter Hybridization. The filter hybridization technique
ofSouthern (7) was used with the following modifications.
Filters[Schleicher & Schuell, 0.45 jim (BA85) nitrocellulose;
ap-proximately 15 X 15 cm] were preannealed, in sealed plasticbags,
at 420C in 10 ml 50% formamide/50 mM Hepes, pH7.0/5-fold
concentrated Denhardt's solution (8, 9)/0.45 MNaCl/0.045 M sodium
citrate, pH 7 (3-fold concentratedNaCl/Cit) containing 18 ,jg of
sheared and denatured Micro-coccus lysodekticus DNA and 40 jig of
yeast tRNA per ml.Heat-denatured hybridization probe (approximately
0.5-1 X106 cpm) was added in 0.5 ml of the same solution and the
filterswere hybridized at 420C for 1-2 days. The filters were
washedat 500C for 1-2 hr with two or three changes (about 200-500ml
per filter) of 0.1-strength NaCl/Cit and 0.1% sodium do-decyl
sulfate (10) and then briefly with 0.1-strength NaCl/Citat room
temperature and were exposed at -70°C to X-OmatR films and Du Pont
Cronex Lightning Plus intensifyingscreens.
Hybridization Probe. Approximately 200 ng of polyacryl-amide
gel-purified 550-base-pair hGH cDNA fragment wasincubated (room
temperature for 60-90 min) in 20 ,ul of a re-action mixture
containing 10 mM Tris.HCl at pH 7.5, 10mMMgCl2, 1 mM
dithiothreitol, 2.5 mg of calf thymus DNAoligonucleotides [prepared
by DNase I digestion (11)] per ml,dATP, dGTP, and dTTP at 10 ,jM
each, 0.5 jiM [a-32P]dCTP[2000-3000 Ci/mmol (1 Ci = 3.7 X 1010
becquerels); NEN orAmersham], and 1 unit of DNA polymerase I
(Klenow modi-fication, Boehringer Mannheim). The cDNA fragment
hadpreviously been heat denatured in the presence of the calfthymus
oligonucleotides. Routinely, specific activities in theorder of 2 X
108 cpm/,jg were obtained. The unincorporated
Abbreviations: hGH, human growth hormone (somatotropin);
hCS,human chorionic somatomammotropin (choriomammotropin);NaCl/Cit,
0.15 M NaCl/0.015 M sodium citrate, pH 7.0; kb, kilo-base(s).t
Present address: Genentech Inc., San Francisco, CA 94080.
4294
The publication costs of this article were defrayed in part by
pagecharge payment. This article must therefore be hereby marked
"ad-vertisement" in accordance with 18 U. S. C. §1734 solely to
indicatethis fact.
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Proc. Natl. Acad. Sci. USA 76 (1979) 4295
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-20.8
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5.7-53
3.2
FIG. 1. Autoradiograph of a filter hybridization between
re-striction endonuclease-digested human placental DNA
(fractionatedon a 1% agarose gel) and 32P-labeled chGH
550-base-pair DNA. Theenzymes used were: lane 1, EcoRI; lane 2, Pvu
II; lane 3, BamHI; lane4, Pst I; lane 5, Sst I; lane 6, Xba I. The
positions and sizes of thefragments produced by EcoRI digestion of
wild-type bacteriophageX DNA are shown at the right. The two
prominent EcoRI fragmentsof human DNA are 2.6 and 2.8 kilobases
(kb) long; the weakly hy-bridizing species is 9.5 kb.
3 4 5
[a-32P]dCTP was then removed by gel filtration on
SephadexG-50.
Bacteriophage X Packaging. The bacteriophage X in vitropackaging
system was used exactly as described by Blattner etal. (5). The EK2
vector Xgt WES.AB (4) was used after eitherseparation of the B
fragment from the vector arms on a 5-20%sucrose gradient (0.1 M
NaCl/10 mM Tris-HCl, pH 7.5/1 mMEDTA in an SW 40 rotor at 35,000
rpm for 4 hr) or digestionof the DNA with Sst I, for which there
are two recognition sitesin the B fragment but none in the vector
arms.
In each packaging experiment, 5.5 gg of vector DNA wasligated to
400 ng of donor DNA in a total volume of 5.5 Atl.Packaging and
subsequent cloning were performed in a P3physical containment
facility in accordance with the NationalInstitutes of Health
Guidelines for Recombinant DNA Re-search. All packaging components
were tested for packagingof endogenous DNA as well as for am+ phage
in control XgtWES.AB DNA packaging experiments. In all cases the
ratioswere
-
4296 Biochemistry: Fiddes et al.
A B
1 2 3 4 5 6 7 8 910C
U._^., _~4
_~~~~~~~~~~~O s
.
FIG. 3. Autoradiographs of filter hybridizations using the
chGH550 probe with the 2.6-kb hGH fragment (A) and the 2.8-kb
hCSfragment (B). The digests were fractionated on two separate
1%agarose gels. Lanes 1-5 were obtained with the hGH-Xgt WES
re-combinant digested with: lane 1, EcoRI; 2, EcoRI and Xba I; 3,
EcoRIand Bgl II; 4, EcoRI and Pst I; 5, EcoRI and Pvu II. Lanes
6-10 wereobtained with the hCS-Xgt WES recombinant digested with:
lane 6,EcoRI; 7, EcoRI and Pst I; 8, EcoRI and Pvu II; 9, EcoRI and
Bgl II,10, EcoRI and Xba I.
a unique Xba I site. The fact that the 2.6-kb fragment
containeda Bgl II but no Xba I site whereas the 2.8-kb fragment had
anXba I but no Bgl II site supports the identification of the
frag-ments given above.
Isolation and Identification of Recombinant DNA Con-taining hGH
and hCS Gene Sequences. In vitro packagingsystems in which
bacteriophage X DNA is ligated to exogenousDNA and used to generate
mature infective phage particleshave been described (5, 17-19). The
DNA packaged into phagecan be used to infect E. coli with a
considerably higher effi-ciency than is obtained by transfection
with the DNA itself. Inthe experiments described here, the in vitro
packaging systemof Blattner et al. (5) was used.
In several packaging experiments, a total of approximately106
recombinant phages were obtained by using bacteriophageXgt WES-XB
DNA as the vector and human DNA in the sizerange 2-3 kb. Single
isolates of the hGH 2.6-kb fragment andof the hCS 2.8-kb fragment
were obtained.To confirm the identity of the cloned human DNA
frag-
ments, their restriction endonuclease digestion pattern was
Pvu EjtI
compared with that of total human DNA (Figs. 2 and 3A andB). The
hGH recombinant contained a 2.6-kb fragment thathybridized to the
chGH 550 probe and had a restriction enzymepattern for the enzymes
EcoRI, Pvu II, Bgl II, Pst I, and XbaI (Fig. 3A) that was identical
to that observed in total humanDNA (Fig. 2). Similarly, the
digestion pattern of the 2.8-kb hCSfragment (Fig. 3B) corresponded
to that of total human DNA.This restriction enzyme data and the
specific hybridizationobserved confirms the identities of the
recombinants.
In the case of the hGH isolate, digestion of
plaque-purifiedrecombinant phage DNA with EcoRI revealed that three
EcoRIfragments of human DNA had been cloned in the same
phage,presumably as the result of multiple ligation of the
humanEcoRI DNA fragments. Two of these fragments were 2.6 kband one
was less than 2 kb. The 2.6-kb fragment that hybridizedto the chGH
550 probe was transferred to the EcoRI site of theplasmid vector
pBR322 by using X 1776 as the host strain. Thisrecombinant plasmid
has been used for most of the subsequentrestriction analysis and
DNA sequencing of the hGH gene andis designated
pBR322-ghGH(2.6).The hGH Gene Contains at Least Two Intervening
Se-
quences. A restriction enzyme cleavage map of the hGH genehas
been determined for the enzymes EcoRI, BamHI, Pst I,Pvu II, Sma I,
and Bgl II (Fig. 4). Certain features of this maplead to the
conclusion that the hGH coding sequence is inter-rupted by at least
two intervening sequences.The locations of the intervening
sequences have been con-
firmed by DNA sequence analysis. The nucleotide sequenceof the
junction between intervening sequence B and the codingregion at
approximately nucleotide 1200 was obtained by thechain terminator
method (14) using as primer a restrictionfragment corresponding to
amino acid residues 45-56 of thehGH protein. This was obtained by
Pst I and HinfI digestionof the chGH 550 recombinant. Template DNA
was preparedfrom the pBR322-ghGH(2.6) recombinant by first
digestingit with HindIII to produce a linear molecule and then
con-verting it to single-stranded DNA with E. coli exonuclease
III(15). An autoradiograph of a sequencing gel is shown in Fig.5
and the sequence of the junction between the coding andintervening
sequence is shown in Fig. 6.
In addition to locating the boundary between the codingsequence
and intervening sequence B, this confirms the identityof the 2.6-kb
fragment as hGH and not hCS because the se-quence encompasses a
region where the two hormones differin amino acid sequence.The
sequence of the junction between the coding sequence
and intervening sequence C at approximately nucleotide 1300
Elg n RyullSmaI
L A--DLAB _ A
EcoRI BamHI RsgI
L0 500
I1000
\\. T/) t fpoly A siteS.mgIttlBaramHI\\ 3' end coding region
550 H-wI cDNA
1500 2000base pairs
FIG. 4. Restriction enzyme map of the 2.6-kb EcoRI fragment
containing the hGH gene sequences. Those sites written above the
line arepresent in the cDNA; those below the line are unique to the
gene fragment. The region containing sequences homologous to the
550-base-paircDNA fragment used to probe the gene is indicated.
This extends from amino acid 24 to 48 bases into 3' untranslated
region. The 3' end of thecoding region is marked. The poly(A)
addition site is located 109 bases from the 3' end of the coding
region. Coding sequences are indicatedby shading. The intervening
sequences are labeled A, B, and C. The precise boundary at the 3'
end of region A is not clear (see text).
Ecg RI
2500
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Proc. Natl. Acad. Sci. USA 76 (1979) 4297
A G C T(+) _(+)A T G CG C _ A TT A G2 C2A2T2 A TC G TAA2 T2 C GT
A OG..G3 C3 T2A2A T C GG C T2 A2C G C GT A G2 C2G 2C A TT A \ T AC
G t IA TT A T AC2 G2 A TA T -G CG C / § IG C~A TV C G_T A T2 A2A T
C2 G2G C I T AA T OGC G2 G CT2 A2 T AG2 C2 C GT A T2 A2G3 C3 C GC G
A TG2 C T AT A A2 T2C2 G2 G CT2 A2 Ua TA
FIG. 5. Autoradiograph of an 8% polyacrylamide DNA se-quencing
gel used in the chain termination method with 2'3'-dideoxyATP,
2'3'-dideoxy CTP, 2'3'-dideoxy GTP, and 2'3'-dideoxy TTP
aschain-terminating analogues. The primer was a
29-nucleotide-longfragment obtained by digestion of the cloned
550-base-pair cDNAfragment of hGH with Pst I and Hinfl. The
template was preparedby treating HindIII-cut pBR322-ghGH(2.6) with
E. coli exonucleaseIII. The sequence read from the gel is written
adjacent to the auto-radiograph with the complementary sense strand
(+) written along-side that. The arrows indicate the two possible
splice points, and theasterisk marks the codon that differs from
the cDNA.
(Fig. 4) has been obtained by a similar strategy (data not
shown),thus confirming the existence of a second intervening
sequencein the hGH gene.
An 800-base-pair "full-length" cDNA clone of hGH (chGH800) has
been obtained recently (20). This contains, in additionto the
entire coding sequence, 29 nucleotides of the 5' untran-slated
region and all 109 nucleotides of the 3' untranslated re-gion
preceding the poly(A) sequence. When this 800base-pairfragment is
used as a hybridization probe with pBR322-ghGH(2.6), the PstI/Pvu
II fragment between positions 600and 1000 hybridizes strongly. T6is
fragment does not hybridizeto the chGH 550 probe, thus indicating
that a large portion ofthe 5' end of the hGH gene is contained in
this region (Fig. 4).A very low level of hybridization of the chGH
800 probe to the5'-terminal EcoRI/Pst I fragment has also been
detected, in-dicating the possibility of a third intervening
sequence (A) inthe hGH gene. This will require further DNA sequence
analysisfor confirmation.DNA sequence analysis has also established
that the 2.6-kb
hGH fragment contains the poly(A) addition site (Fig. 4).
DISCUSSIONEcoRI restriction endonuclease fragments 2.6 and 2.8
kb longcontaining coding sequences for the hGH gene and the
hCSgene, respectively, have been identified by hybridization to
a550-base-pair cloned Hae III fragment of hGH cDNA. In ad-dition to
these two fragments, a third fragment of about 9.5 kb,that
hybridizes weakly to the chGH 550 probe, is also detectedroutinely
(see Figs. 1 and 2). The origin of this is not clear be-cause the
other known sequence related to hGH and hCS is thepituitary hormone
prolactin which only has 16% amino acidsequence homology with hGH
and 13% homology with hCS(21). The hybridization conditions used
would not detect pro-lactin genes (assuming that the nucleotide
sequences had similarlevels of homology).
Both the 2.6-kb hGH and the 2.8-kb hCS fragments havebeen
isolated by cloning in the bacteriophage Xgt WES-XBvector with the
in vitro packaging approach. A restriction en-zyme cleavage map
(Fig. 4) derived for the hGH gene indicatesthat, in common with
other eukaryotic genes (22-26), the hGHgene contains intervening
sequences that interrupt the codingsequences.One of the two
identified intervening sequences (sequence
B) is about 220 base pairs long; the other (sequence C) is
about300 base pairs. A third intervening sequence (sequence A)
veryclose to the 5' end of the gene may also exist. The possibility
thatother very small intervening sequences are present-i.e.,
thatsome of the intervening sequences are interspersed within
shortcoding regions-has not yet been excluded.
Preliminary DNA sequence analysis of the pBR322-ghGH(2.6)
recombinant has confirmed the existence of theintervening regions
shown in Fig. 4. The sequence at the 3' endof intervening sequence
B (at approximately position 1200) isshown in Figs. 5 and 6. As in
other systems, the 3' end is markedby an A-G doublet (27).A
detailed restriction enzyme map has not yet been deter-
mined for the hCS gene but the preliminary evidence suggeststhat
the pattern of intervening sequences may be similar to thatof the
hGH gene. Total human placental DNA, when digestedwith both EcoRI
and Pvu II (Fig. 2), contains a single 980-
Intervening Sequence: C CTT GGT GGG CGG TCC TTC TCC
TAG~~~~~~~~~~~Coding Sequence: . GAG TTT GAA GAA GCC TAT ATC CTG
AAG GAG CAG AAG TAT TCA * -
Amino Acid: . GLU PHE GLU GLU ALA TYR ILE LEU LYS GLU GLN LYS
TYR SER30 9 35 40
FIG. 6. Partial sequence of an intervening region in the hGH
gene. The junction between the 3' end of the intervening sequence B
and thecoding region is shown. The codon marked with an asterisk is
CCA (proline) in the cDNA. The amino acids are numbered. The coding
sequenceto the 5' side of the intervening sequence is taken from
the cDNA. The splice point could be as shown or one nucleotide to
the right.
Biochemistry: Fiddes et al.
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Proc. Natl. Acad. Sci. USA 76 (1979)
base-pair fragment that hybridizes to the chGH 550 probe.
Thisprobe also detects hCS sequences. Because this fragment
con-tains intervening and coding sequences in the hGH gene,
theappearance of6a single band implies that the hCS gene may
alsocontain a similar structure in this region.
Several nucleotide sequence differences have been
detectedbetween the ghGH(2.6) gene and the cloned cDNA fragment.One
of these is shown in Fig. 6: a CCA proline codon at aminoacid
position 37 in the cDNA is changed to a CTG leucine codonin the
gene. The cDNA proline codon corresponds to the aminoacid sequence
established for the hGH protein (28). This codonchange is in a
region where tryptic mapping data of the hGHprotein indicates that
amino acid sequence variants occur (29).The ghGH(2.6) fragment may
therefore correspond to an allelicvariant of growth hormone.
Alternatively, the differences may-be the result of the different
sources of material used: the hGHcDNA came from a pool of pituitary
tumors and the hGH genecame from a single placenta.The amino acid
data in Fig. 6 exclude the possibility that a
growth hormone variant of molecular weight 20,000 (29),
ratherthan the normal 22,000, has been cloned. This variant,
whichexists as 5-10% of the total growth hormone, lacks the
trypticpeptide located between amino acids 38 and 41. The DNA
se-quence analysis presented in Fig. 6 shows that the coding
regioncorresponding to this peptide is present in ghGH(2.6).
Nucle-otide sequence differences between the cDNA and ghGH(2.6)have
also been detected in the 3' untranslated region (unpub-lished
data).An independently isolated 2.6-kb fragment has been
recently
cloned (unpublished data). This fragment gives rise to
anidentical hybridization pattern as the first isolate when
theenzymes Pvu II, Pst I, Bgl II, and Sma I arepused. However,
adifference between the two is noticed with the enzyme Hae III.The
possibility therefore exists that this second isolate corre-sponds
to the expressed growth hormone gene. DNA sequenceanalysis of this
isolate will be required in order to determinewhether this is the
case.
Note Added in Proof. The existence of the third intervening
sequence(A) has been confirmed by DNA sequence analysis. The splice
pointis located in the second codin of the pre-sequence of hGH.
J.C.F. is the recipient of a postdoctoral fellowship from the
AnnaFuller Fund. R.A.H. is the recipient of a postdoctoral
fellowship fromthe British Science Research Council. This work was
supported byGrants CA14026 to H.M.G. and AM19997 to H.M.G. and
J.D.B.;H.M.G. and J.D.B. are Investigators of the Howard Hughes
MedicalInstitute.
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