The Course of Development

The Course of Development


Time

Events


Time

Events


Time

Events in time


Time

Events in time


Time

Events in time and space . . .


Time



Events in time and space . . .. . . driven by patterned gene expression


Time


Free-living Nostoc

heterocysts

Matveyev and Elhai (unpublished)

CO2

sucroseN2

O2

CyanobacteriaAnabaena grown on N2 / CO2

Free-living Nostoc

heterocysts

Matveyev and Elhai (unpublished)

CO2

sucroseN2

NH3

NH3

O2

CyanobacteriaAnabaena grown on N2 / CO2

3 h

6 h

9 h

Earlier pattern of gene expression?

12 h

Time course after N-deprivation

0 h

Time after after N-deprivation

12 h

3 h

6 h

9 h

• hetR required for differentiation

• hetR sufficient for differentiation

Monitor what gene?

• hetR expressed early

How to monitor gene expression?

Time course of differentiation

0 h

Time after after N-deprivation

5’-GTA ..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN3’-CAT ..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN

hetR gene

RNA Polymerase

hetRRegulation

-N

GTA ..(8).. TAC

5’-GTGAGTTAGCTCACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN3’-CACTCAATCGAGTGNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN

Reporter gene

Gene fusions to monitor expression

GTA ..(8).. TAC

NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN

Reporter gene5’-GTA ..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNN3’-CAT ..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNN

RNA Polymerase

hetRRegulation

Gene fusions to monitor expression

-N

The hydromedusa Aequoria victoria

Source of GreenFluorescent Protein

Detection of hetR gene expression through Green Fluorescent Protein

Bill Buikema, U. Chicago

Expression of hetR visualized by GFP

Indicator of heterocyst differentiation

hetR::gfp faithfully reports heterocyst differentiation

Late expression of hetRLate

What about early expression?

Visualization of early hetR expression with GFP

Crude pattern Final pattern

Late expression

Early expression

How are initial cell groups chosen?

Why Cell Cycle?

Different cellular statesBiochemical eventsGenes on/off

Different gene dosage

Not synchronous

Are cells competent to differentiate chosen by their positions in the the cell cycle?

What process can govern initiation?Hypothesis: The cell cycle

Why Cell Cycle?



Not synchronous


Why Cell Cycle?



Not synchronous


Why Cell Cycle?



Not synchronous

hemimethylated


Why Cell Cycle?



Not synchronous


Why Cell Cycle?



Not synchronous


Why Cell Cycle?



Not synchronous

Regulated by:DnaA protein

Methylation


Role of cell cycle in initial pattern?

??? ??? ???

???Crude pattern of cell division Crude pattern of differentiation?

How to tell?

More than a correlation?


Perturbing cell cycle affects heterocyst spacing?

???Perturbation of cell cycle Random pattern of differentiation?


origin of replication

Regulation of initiation of DNA synthesis by methylation

Origin of replication


blocked



blocked available


C A G A T C T A G T C T A G A T

**Old DNA

C A G A T C T A G T C T A G A T *New DNA

Experimental Goal

• Gain control over DNA methylation in Anabaena

• Do perturbations affect DNA synthesis?

• Do perturbations affect heterocyst spacing?

Should be no problem• Methyltransferases easy to clone by activity

• Bacteria generally have zero or one (known) enzyme

Solitary methylases

Restrictionmethylases

Ancient

Recent arrivalsRelics

Methyltransferase Specificity Structural ClassProteins with proven activity

DmtA GATC N6mA()DmtB GGCC N4mC()DmtC CGATCG 5mCDmtD rGGCCy 5mC

DmtE/M.AvaV GATC N6mA()M.AvaI (+R.AvaI) CyCGrG N4mC()

M.AvaII (+R.AvaII) GGwCC 5mCM.AvaIII (+R.AvaIII) ATGCAT N6mA(ß)M.AvaIV (+R.AvaIV) GCTnAGC 5mC

Defective methyltransferases/restriction enzymesOrf362.31394R CTGCAG (PstI) N6mA( )

Orf275.9611 ? N4mC(ß)Orf335.3585 ? 5mC

Orf351A.9907 ? 5mCOrf376B (+.R.???) ? Type IOrf260 (+.R.???) ? Type I

Orf308A (+.R.???) ? Type IOrf308B/311A (+.R.???) ? Type I

Orf339A (+.R.???) ? Type I

Matveyev AV, Young KT, Meng A, and Elhai J (2001) Nucl Acids Res 29:1491-1506

DNA methyltransferases of Anabaenas

Solitary methylases

DNA methyltransferases of AnabaenaMethyltransferase Specificity Structural Class

Proteins with proven activityDmtA GATC N6mA()DmtB GGCC N4mC()DmtC CGATCG 5mCDmtD rGGCCy 5mC

DmtE/M.AvaV GATC N6mA()M.AvaI (+R.AvaI) CyCGrG N4mC()

M.AvaII (+R.AvaII) GGwCC 5mCM.AvaIII (+R.AvaIII) ATGCAT N6mA(ß)M.AvaIV (+R.AvaIV) GCTnAGC 5mC

Defective methyltransferases/restriction enzymesOrf362.31394R CTGCAG (PstI) N6mA( )

Orf275.9611 ? N4mC(ß)Orf335.3585 ? 5mC

Orf351A.9907 ? 5mCOrf376B (+.R.???) ? Type IOrf260 (+.R.???) ? Type I

Orf308A (+.R.???) ? Type IOrf308B/311A (+.R.???) ? Type I

Orf339A (+.R.???) ? Type I

Matveyev AV, Young KT, Meng A, and Elhai J (2001) Nucl Acids Res 29:1491-1506

? ? ?

Solitary methyltransferases in Anabaena PCC 7120

STRATEGYKnock out genes

Look at DNA replication & differentiation

Look at heterocyst spacing

DmtA GATC ClonedDmtB GGCC ClonedDmtC CGATCG DeducedDmtD rCCGGy ClonedDmtE GATC Cloned

Gene Site Status Null phenotype

npt

sacB

Sm/Sp

Formation of dmtB mutant

PhenotypeNo GGCC methylation

dmtB

npt

trpD

dmtB No heterocysts

sll0160 dmtB trpD

npt

dmtB trpD

dmtB+

+

ExpectCondition Methylation Heterocysts - plasmid - - + plasmid + +

ObserveCondition Methylation Heterocysts - plasmid - - + plasmid +

Complementation of dmtB mutant

-

npt

sacB

Sm/Sp


Phenotype

(revisited)

npt

dmtB trpD

No GGCC methylationYes heterocysts


Phenotype(revisited)

npt

dmtB trpDnpt

dmtB trpD

DmtB Het- -

DmtB Het- +

trpD



npt

dmtB trpDnpt

dmtB trpD

DmtB Het- -

DmtB Het- +

trpD

Het+

npt

dmtB trpD

trpDP

npt

dmtB trpD

TrpD Het- +

TrpD Het- -



npt

dmtB trpDnpt

dmtB trpD

DmtB Het- -

DmtB Het- +

trpD

Het+

npt

dmtB trpD

trpDP

npt

dmtB trpD

TrpD Het- +

TrpD Het- -

dmtB trpD

trpDP



npt

dmtB trpDnpt

dmtB trpD

DmtB Het- -

DmtB Het- +

trpD

Het+

npt

dmtB trpDnpt

dmtB trpD

TrpD Het- +

TrpD Het- -

dmtB trpD

trpDP

Deletion of dmtB-trpD region

dmtB trpD


trpDP


Het+++ . . . death

What is the intergenic region?

dmtB trpD

Blast against Anabaena genome

What is the intergenic region?Best Blast Hits


dmtB trpD

hetR

P


dmtB trpD

hetR

P


Other hits (no more than 3 mismatches)

Cyanophycin synthetase in heterocysts

Hypothetical regulatory RNADoes it exist?

• Are the sequence and targets conserved?

• Is the sequence naturally transcribed?

dmtB

trpD2

Is the sequence conserved?

Are the targets conserved?P

Are the targets conserved?

Are the targets conserved?

Inferred characteristics of phenomenon• Mediated by RNA

– No reasonable orfs in area– Small intergenic region Overlap with genes unlikely– Compensatory mutations


• Secondary structure– Inverted repeats– Compensatory mutations


• Secondary structure

• Imperfect matches with putative targets– Inverted repeats– Compensatory mutations


• Secondary structure

• Imperfect matches with putative targets

• Conserved over related organisms

Is this surprising?

The Course of Development

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