Download A scientific tale

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 . . .
Cyanobacteria
Free-living Nostoc
Anabaena grown on N2 / CO2
heterocysts
N2
O2
Matveyev and Elhai (unpublished)
CO2
Cyanobacteria
Free-living Nostoc
Anabaena grown on N2 / CO2
NH3
heterocysts
N2
O2
NH3
Matveyev and Elhai (unpublished)
CO2
Time course after N-deprivation
Time after after N-deprivation
0h
3h
6h
9h
12 h
Earlier pattern of gene expression?
Time course of differentiation
Time after after N-deprivation
0h
3h
Monitor what gene?
• hetR required for
differentiation
6h
• hetR sufficient for
differentiation
9h
• hetR expressed early
12 h
How to monitor gene expression?
Gene fusions to monitor expression
hetR
Regulation
hetR gene
5’-GTA
3’-CAT
..(8)..
..(8)..
TACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN
ATGNNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
-N
RNA Polymerase
Reporter gene
5’-GTGAGTTAGCTCACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN
3’-CACTCAATCGAGTGNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
GTA
..(8)..
TAC
Gene fusions to monitor expression
hetR
Regulation
Reporter gene
5’-GTA
3’-CAT
..(8)..
..(8)..
TACNNNNNNNNNNTANNNTNNNNNNNNNN NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN
ATGNNNNNNNNNNATNNNANNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
-N
RNA Polymerase
GTA
..(8)..
TAC
Detection of hetR gene expression
through Green Fluorescent Protein
The hydromedusa
Aequoria victoria
Source of Green
Fluorescent Protein
Indicator of heterocyst differentiation
Expression of hetR visualized by GFP
hetR::gfp faithfully reports
heterocyst differentiation
What about
early expression?
Late expression of hetR
Bill Buikema, U. Chicago
Visualization of early hetR
expression with GFP
Early expression
Late expression
Crude pattern  Final pattern
How are initial cell groups chosen?
What process can govern initiation?
Hypothesis: The cell cycle
Why Cell Cycle?
Different cellular states
Biochemical events
Genes 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 states
Biochemical events
Genes on/off
Different gene dosage
Not synchronous
What process can govern initiation?
Hypothesis: The cell cycle
Why Cell Cycle?
Different cellular states
Biochemical events
Genes on/off
Different gene dosage
Not synchronous
What process can govern initiation?
Hypothesis: The cell cycle
Why Cell
hemimethylated
Cycle?
Different cellular states
Biochemical events
Genes on/off
Different gene dosage
Not synchronous
What process can govern initiation?
Hypothesis: The cell cycle
Why Cell Cycle?
Different cellular states
Biochemical events
Genes on/off
Different gene dosage
Not synchronous
What process can govern initiation?
Hypothesis: The cell cycle
Why Cell Cycle?
Different cellular states
Biochemical events
Genes on/off
Different gene dosage
Not synchronous
What process can govern initiation?
Hypothesis: The cell cycle
Why Cell Cycle?
Different cellular states
Biochemical events
Genes on/off
Different gene dosage
Not synchronous
Regulated by:
DnaA protein
Methylation
Role of cell cycle in initial pattern?
Role of cell cycle in initial pattern?
More than a correlation?
???
???
Crude pattern of cell division
???
???
Crude pattern of differentiation?
How to tell?
Role of cell cycle in initial pattern?
Perturbing cell cycle affects heterocyst spacing?
Perturbation of cell cycle
???
Random pattern of differentiation?
Regulation of initiation of DNA
synthesis by methylation
Origin of replication
origin of
replication
Regulation of initiation of DNA
synthesis by methylation
blocked
origin of
replication
Regulation of initiation of DNA
synthesis by methylation
blocked
available
origin of
replication
Old DNA
C A G A* T C T A
G T C T *A G A T
New DNA
C A G A T C T A
G T C T *A G A T
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
DNA methyltransferasess of Anabaena
Methyltransferase
Specificity
Proteins with proven activity
Solitary
methylases
Restriction
methylases
DmtA
DmtB
DmtC
DmtD
DmtE/M.AvaV
M.AvaI (+R.AvaI)
M.AvaII (+R.AvaII)
M.AvaIII (+R.AvaIII)
M.AvaIV (+R.AvaIV)
GATC
GGCC
CGATCG
rGGCCy
GATC
CyCGrG
GGwCC
ATGCAT
GCTnAGC
Structural Class
N6mA()
N4mC()
5mC
5mC
N6mA()
N4mC()
5mC
N6mA(ß)
5mC
Ancient
Defective methyltransferases/restriction enzymes
Relics
Orf362.31394R
Orf275.9611
Orf335.3585
Orf351A.9907
Orf376B (+.R.???)
Orf260 (+.R.???)
Orf308A (+.R.???)
Orf308B/311A (+.R.???)
Orf339A (+.R.???)
CTGCAG (PstI)
?
?
?
?
?
?
?
?
N6mA(
)
N4mC(ß)
5mC
5mC
Type I
Type I
Type I
Type I
Type I
Matveyev AV, Young KT, Meng A, and Elhai J (2001) Nucl Acids Res 29:1491-1506
Recent
arrivals
DNA methyltransferases of Anabaena
Methyltransferase
Specificity
Proteins with proven activity
Solitary
methylases
DmtA
DmtB
DmtC
DmtD
DmtE/M.AvaV
M.AvaI (+R.AvaI)
M.AvaII (+R.AvaII)
M.AvaIII (+R.AvaIII)
M.AvaIV (+R.AvaIV)
GATC
GGCC
CGATCG
rGGCCy
GATC
CyCGrG
GGwCC
ATGCAT
GCTnAGC
Structural Class
N6mA()
N4mC()
5mC
5mC
N6mA()
N4mC()
5mC
N6mA(ß)
5mC
Defective methyltransferases/restriction enzymes
Orf362.31394R
Orf275.9611
Orf335.3585
Orf351A.9907
Orf376B (+.R.???)
Orf260 (+.R.???)
Orf308A (+.R.???)
Orf308B/311A (+.R.???)
Orf339A (+.R.???)
CTGCAG (PstI)
?
?
?
?
?
?
?
?
N6mA(
)
N4mC(ß)
5mC
5mC
Type I
Type I
Type I
Type I
Type I
Matveyev AV, Young KT, Meng A, and Elhai J (2001) Nucl Acids Res 29:1491-1506
Solitary methyltransferases
in Anabaena PCC 7120
Gene
Site
Status
DmtA
GATC
Cloned
DmtB
GGCC
Cloned
DmtC
CGATCG
Deduced
DmtD
rCCGGy
Cloned
DmtE
GATC
Cloned
Null phenotype
???
STRATEGY
Knock out genes
Look at DNA replication & differentiation
Look at heterocyst spacing
Formation of dmtB mutant
sacB
Sm/Sp
No GGCC methylation
No heterocysts
npt
dmtB
npt
dmtB
Phenotype
trpD
Complementation of dmtB mutant
Expect
Condition Methylation Heterocysts
- plasmid
+ plasmid
+
+
dmtB+
+
npt
sll0160
dmtB
dmtBtrpD trpD
Observe
Condition Methylation Heterocysts
- plasmid
+ plasmid
+
-
Formation of dmtB mutant
(revisited)
sacB
Sm/Sp
Phenotype
No GGCC methylation
npt
Yes heterocysts
npt
dmtB
trpD
Formation of dmtB mutant
(revisited)
npt
trpD
dmtB trpD
Phenotype
DmtB- Het -
npt
dmtB
trpD
DmtB- Het+
Formation of dmtB mutant
(revisited)
npt
trpD
dmtB trpD
Phenotype
DmtB- Het -
npt
dmtB
trpD
P
trpD
DmtB- Het+
Het +
npt
dmtB
trpD
TrpD - Het +
npt
dmtB
trpD
TrpD - Het -
Formation of dmtB mutant
(revisited)
Phenotype
DmtB- Het -
npt
trpD
dmtB trpD
npt
dmtB
DmtB- Het+
trpD
P
trpD
npt
dmtB
trpD
Het +
dmtB
trpD
TrpD - Het +
npt
dmtB
trpD
TrpD - Het -
Formation of dmtB mutant
(revisited)
Phenotype
DmtB- Het -
npt
trpD
dmtB trpD
npt
dmtB
DmtB- Het+
trpD
P
trpD
npt
dmtB
trpD
Het +
dmtB
trpD
TrpD - Het +
npt
dmtB
trpD
TrpD - Het -
Deletion of dmtB-trpD region
P
trpD
dmtB
trpD
Deletion of dmtB-trpD region
P
trpD
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
trpD
dmtB
P
hetR
What is the intergenic region?
Best Blast Hits
trpD
dmtB
P
hetR
What is the intergenic region?
Best Blast Hits
Other hits (no more than 3 mismatches)
Cyanophycin synthetase
in heterocysts
Hypothetical regulatory RNA
Does it exist?
• Are the sequence and targets conserved?
• Is the sequence naturally transcribed?
Is the sequence conserved?
dmtB 
trpD2 
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?