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The Course of Development. The Course of Development. Time. Events. The Course of Development. Time. Events. The Course of Development. Time. Events in time. The Course of Development. Time. Events in time. The Course of Development. Time. Events in time and space. - PowerPoint PPT Presentation
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The Course of Development
The Course of Development
Time
Events
The Course of Development
Time
Events
The Course of Development
Time
Events in time
The Course of Development
Time
Events in time
The Course of Development
Time
Events in time and space . . .
The Course of Development
Time
Events in time and space . . .
The Course of Development
Events in time and space . . .. . . driven by patterned gene expression
The Course of Development
Time
Events in time and space . . .
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Matveyev and Elhai (unpublished)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Matveyev and Elhai (unpublished)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?
Different cellular statesBiochemical eventsGenes on/off
Different gene dosage
Not synchronous
What process can govern initiation?Hypothesis: The cell cycle
Why Cell Cycle?
Different cellular statesBiochemical eventsGenes on/off
Different gene dosage
Not synchronous
What process can govern initiation?Hypothesis: The cell cycle
Why Cell Cycle?
Different cellular statesBiochemical eventsGenes on/off
Different gene dosage
Not synchronous
hemimethylated
What process can govern initiation?Hypothesis: The cell cycle
Why Cell Cycle?
Different cellular statesBiochemical eventsGenes on/off
Different gene dosage
Not synchronous
What process can govern initiation?Hypothesis: The cell cycle
Why Cell Cycle?
Different cellular statesBiochemical eventsGenes on/off
Different gene dosage
Not synchronous
What process can govern initiation?Hypothesis: The cell cycle
Why Cell Cycle?
Different cellular statesBiochemical eventsGenes on/off
Different gene dosage
Not synchronous
Regulated by:DnaA protein
Methylation
What process can govern initiation?Hypothesis: The cell cycle
Role of cell cycle in initial pattern?
??? ??? ???
???Crude pattern of cell division Crude pattern of differentiation?
How to tell?
More than a correlation?
Role of cell cycle in initial pattern?
Perturbing cell cycle affects heterocyst spacing?
???Perturbation of cell cycle Random pattern of differentiation?
Role of cell cycle in initial pattern?
origin of replication
Regulation of initiation of DNA synthesis by methylation
Origin of replication
origin of replication
blocked
Regulation of initiation of DNA synthesis by methylation
origin of replication
blocked available
Regulation of initiation of DNA synthesis by methylation
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
Formation of dmtB mutant
Phenotype
(revisited)
npt
dmtB trpD
No GGCC methylationYes heterocysts
Formation of dmtB mutant
Phenotype(revisited)
npt
dmtB trpDnpt
dmtB trpD
DmtB Het- -
DmtB Het- +
trpD
Formation of dmtB mutant
Phenotype(revisited)
npt
dmtB trpDnpt
dmtB trpD
DmtB Het- -
DmtB Het- +
trpD
Het+
npt
dmtB trpD
trpDP
npt
dmtB trpD
TrpD Het- +
TrpD Het- -
Formation of dmtB mutant
Phenotype(revisited)
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
Formation of dmtB mutant
Phenotype(revisited)
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
Deletion of dmtB-trpD region
trpDP
Deletion of dmtB-trpD region
Het+++ . . . death
What is the intergenic region?
dmtB trpD
Blast against Anabaena genome
What is the intergenic region?Best Blast Hits
What is the intergenic region?Best Blast Hits
dmtB trpD
hetR
P
What is the intergenic region?Best Blast Hits
dmtB trpD
hetR
P
What is the intergenic region?Best Blast Hits
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
Inferred characteristics of phenomenon• Mediated by RNA
• Secondary structure– Inverted repeats– Compensatory mutations
Inferred characteristics of phenomenon• Mediated by RNA
• Secondary structure
• Imperfect matches with putative targets– Inverted repeats– Compensatory mutations
Inferred characteristics of phenomenon• Mediated by RNA
• Secondary structure
• Imperfect matches with putative targets
• Conserved over related organisms
Is this surprising?