The%20Course%20of%20Development

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The Course of Development
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The Course of Development
Time
Events
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The Course of Development
Time
Events
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The Course of Development
Time
Events in time
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The Course of Development
Time
Events in time
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The Course of Development
Time
Events in time and space . . .
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The Course of Development
Time
Events in time and space . . .
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The Course of Development
Events in time and space . . .
. . . driven by patterned gene expression
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The Course of Development
Time
Events in time and space . . .
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Free-living Nostoc
Cyanobacteria
Anabaena grown on N2 / CO2
N2
CO2
O2
Matveyev and Elhai (unpublished)
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Free-living Nostoc
Cyanobacteria
Anabaena grown on N2 / CO2
NH3
N2
O2
CO2
Matveyev and Elhai (unpublished)
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Time course after N-deprivation
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Time course of differentiation
Time after after N-deprivation
Monitor what gene?

• hetR required for differentiation

• hetR sufficient for differentiation

How to monitor gene expression?
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Gene fusions to monitor expression
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Gene fusions to monitor expression
hetRRegulation
Reporter gene


NNNNNNNNNNNNNNNNNNATGNNNNNNNNNN
NNNNNN

NNNNNNNNNNNNNNNNNNTACNNN
NNNNNNNNNNNNN
5-GTA ..(8).. TACNNNNNNNNNNTANNNTNNNNNNNN
NN3-CAT ..(8).. ATGNNNNNNNNNNATNNNANNNNN
NNNNN
-N
RNA Polymerase
GTA ..(8).. TAC
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Detection of hetR gene expression through Green
Fluorescent Protein
The hydromedusa Aequoria victoria Source of
GreenFluorescent Protein
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Indicator of heterocyst differentiation
Expression of hetR visualized by GFP
hetRgfp faithfully reports heterocyst
differentiation
What about early expression?
Late expression of hetR
Late
Bill Buikema, U. Chicago
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Visualization of early hetR expression with GFP
Crude pattern ? Final pattern
How are initial cell groups chosen?
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What process can govern initiation?Hypothesis
The cell cycle
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?
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What process can govern initiation?Hypothesis
The cell cycle
Why Cell Cycle? Different cellular
statesBiochemical eventsGenes on/off Different
gene dosage Not synchronous
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What process can govern initiation?Hypothesis
The cell cycle
Why Cell Cycle? Different cellular
statesBiochemical eventsGenes on/off Different
gene dosage Not synchronous
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What process can govern initiation?Hypothesis
The cell cycle
Why Cell Cycle? Different cellular
statesBiochemical eventsGenes on/off Different
gene dosage Not synchronous
hemimethylated
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What process can govern initiation?Hypothesis
The cell cycle
Why Cell Cycle? Different cellular
statesBiochemical eventsGenes on/off Different
gene dosage Not synchronous
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What process can govern initiation?Hypothesis
The cell cycle
Why Cell Cycle? Different cellular
statesBiochemical eventsGenes on/off Different
gene dosage Not synchronous
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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
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Role of cell cycle in initial pattern?
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Role of cell cycle in initial pattern?
More than a correlation?
???
???
???
How to tell?
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Role of cell cycle in initial pattern?
Perturbing cell cycle affects heterocyst spacing?
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Regulation of initiation of DNA synthesis by
methylation
Origin of replication
origin of replication
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Regulation of initiation of DNA synthesis by
methylation
blocked
origin of replication
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Regulation of initiation of DNA synthesis by
methylation
blocked
available
origin of replication
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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

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DNA methyltransferases of Anabaena
s
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DNA methyltransferases of Anabaena
Solitary methylases
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Solitary methyltransferases in Anabaena PCC 7120
DmtA GATC Cloned DmtB GGCC Cloned DmtC
CGATCG Deduced DmtD rCCGGy Cloned DmtE
GATC Cloned
STRATEGY Knock out genesLook at DNA replication
differentiationLook at heterocyst spacing
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Formation of dmtB mutant
Phenotype
No GGCC methylation
No heterocysts
dmtB
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Complementation of dmtB mutant
ExpectCondition
Methylation Heterocysts
- plasmid - -

plasmid
ObserveCondition
Methylation Heterocysts
- plasmid - -

npt
plasmid
sll0160 dmtB trpD
dmtB
trpD
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Formation of dmtB mutant
(revisited)
sacB
Sm/Sp
Phenotype
No GGCC methylation
npt
Yes heterocysts
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Formation of dmtB mutant
(revisited)
Phenotype
npt
dmtB
trpD
trpD
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Formation of dmtB mutant
(revisited)
Phenotype
npt
dmtB
trpD
trpD
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Formation of dmtB mutant
(revisited)
Phenotype
npt
dmtB
trpD
trpD
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Formation of dmtB mutant
(revisited)
Phenotype
npt
dmtB
trpD
trpD
P
trpD
dmtB
trpD
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Deletion of dmtB-trpD region
P
trpD
dmtB
trpD
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Deletion of dmtB-trpD region
P
trpD
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Deletion of dmtB-trpD region
Het
. . . death
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What is the intergenic region?
dmtB
trpD
Blast against Anabaena genome
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What is the intergenic region?
Best Blast Hits
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What is the intergenic region?
Best Blast Hits
P
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What is the intergenic region?
Best Blast Hits
dmtB
trpD
P
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What is the intergenic region?
Best Blast Hits
Other hits (no more than 3 mismatches)
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Hypothetical regulatory RNA
Does it exist?

• Are the sequence and targets conserved?

• Is the sequence naturally transcribed?

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Is the sequence conserved?
dmtB ?
trpD2 ?
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Are the targets conserved?
P
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Are the targets conserved?
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Are the targets conserved?
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Inferred characteristics of phenomenon
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Inferred characteristics of phenomenon

• Mediated by RNA

• Secondary structure

• Inverted repeats
• Compensatory mutations

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Inferred characteristics of phenomenon

• Mediated by RNA

• Secondary structure

• Imperfect matches with putative targets

• Inverted repeats
• Compensatory mutations

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Inferred characteristics of phenomenon

• Mediated by RNA

• Secondary structure

• Imperfect matches with putative targets

• Conserved over related organisms

Is this surprising?