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Free-living Nostoc heterocysts Matveyev and Elhai (unpublished) CO 2 sucrose N2N2 O2O2 Cyanobacteria Anabaena grown on N 2 / CO 2
Free-living Nostoc heterocysts Matveyev and Elhai (unpublished) CO 2 sucrose N2N2 NH 3 O2O2 Cyanobacteria Anabaena grown on N 2 / CO 2
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).. TACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN 3’-CAT..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN hetR gene RNA Polymerase hetR Regulation -N GTA..(8).. TAC 5’-GTGAGTTAGCTCACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN 3’-CACTCAATCGAGTGNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN Reporter gene Gene fusions to monitor expression
GTA..(8).. TAC NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN Reporter gene 5’-GTA..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNN 3’-CAT..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNN RNA Polymerase hetR Regulation Gene fusions to monitor expression -N
The hydromedusa Aequoria victoria Source of Green Fluorescent 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 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 Cycle? Different cellular states Biochemical events Genes on/off Different gene dosage Not synchronous hemimethylated 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 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 blockedavailable 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 Restriction methylases Ancient Recent arrivals Relics Matveyev AV, Young KT, Meng A, and Elhai J (2001) Nucl Acids Res 29: DNA methyltransferases of Anabaenas
Solitary methylases DNA methyltransferases of Anabaena Matveyev AV, Young KT, Meng A, and Elhai J (2001) Nucl Acids Res 29:
? ? ? Solitary methyltransferases in Anabaena PCC 7120 STRATEGY Knock out genes Look at DNA replication & differentiation Look at heterocyst spacing DmtA GATC Cloned DmtB GGCC Cloned DmtC CGATCG Deduced DmtD rCCGGy Cloned DmtE GATC Cloned Gene Site StatusNull phenotype
npt sacB Sm/Sp Formation of dmtB mutant Phenotype No GGCC methylation dmtB npt trpD dmtB No heterocysts
sll0160 dmtB trpD npt dmtB trpD dmtB + + Expect Condition Methylation Heterocysts - plasmid plasmid + + Observe Condition Methylation Heterocysts - plasmid plasmid + Complementation of dmtB mutant -
npt sacB Sm/Sp Formation of dmtB mutant Phenotype (revisited) npt dmtB trpD No GGCC methylation Yes heterocysts
Formation of dmtB mutant Phenotype (revisited) npt dmtB trpD npt dmtB trpD DmtB Het trpD
Formation of dmtB mutant Phenotype (revisited) npt dmtB trpD npt dmtB trpD DmtB Het trpD Het + npt dmtB trpD P npt dmtB trpD TrpD Het
Formation of dmtB mutant Phenotype (revisited) npt dmtB trpD npt dmtB trpD DmtB Het trpD Het + npt dmtB trpD P npt dmtB trpD TrpD Het dmtB trpD
P Formation of dmtB mutant Phenotype (revisited) npt dmtB trpD npt dmtB trpD DmtB Het trpD Het + npt dmtB trpD npt dmtB trpD TrpD Het dmtB trpD
P Deletion of dmtB-trpD region dmtB trpD
Deletion of dmtB-trpD region trpD P
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 RNA Does it exist? Are the sequence and targets conserved? Is the sequence naturally transcribed?
dmtB trpD2 Is the sequence conserved?
Are the targets conserved? P
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?