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The Course of Development

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.

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The Course of Development

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  1. The Course of Development

  2. The Course of Development Time Events

  3. The Course of Development Time Events

  4. The Course of Development Time Events in time

  5. The Course of Development Time Events in time

  6. The Course of Development Time Events in time and space . . .

  7. The Course of Development Time Events in time and space . . .

  8. The Course of Development Events in time and space . . . . . . driven by patterned gene expression

  9. The Course of Development Time Events in time and space . . .

  10. heterocysts sucrose Cyanobacteria Free-living Nostoc Anabaena grown on N2 / CO2 N2 CO2 O2 Matveyev and Elhai (unpublished)

  11. heterocysts sucrose NH3 Cyanobacteria Free-living Nostoc Anabaena grown on N2 / CO2 NH3 N2 O2 CO2 Matveyev and Elhai (unpublished)

  12. Time after after N-deprivation 0 h 3 h 6 h 9 h 12 h Earlier pattern of gene expression? Time course after N-deprivation

  13. 0 h 3 h 6 h 9 h • hetR expressed early 12 h 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?

  14. hetRRegulation hetR gene 5’-GTA ..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN3’-CAT ..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN -N RNA Polymerase Reporter gene 5’-GTGAGTTAGCTCACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN3’-CACTCAATCGAGTGNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN GTA ..(8).. TAC Gene fusions to monitor expression

  15. Gene fusions to monitor expression hetRRegulation Reporter gene NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN 5’-GTA ..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNN3’-CAT ..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNN -N RNA Polymerase GTA ..(8).. TAC

  16. Detection of hetR gene expression through Green Fluorescent Protein The hydromedusaAequoria victoria Source of GreenFluorescent Protein

  17. Indicator of heterocyst differentiation Expression of hetR visualized by GFP hetR::gfp faithfully reports heterocyst differentiation What about early expression? Late expression of hetR Late Bill Buikema, U. Chicago

  18. Early expression Late expression Visualization of early hetR expression with GFP Crude pattern  Final pattern How are initial cell groups chosen?

  19. 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?

  20. What process can govern initiation?Hypothesis: The cell cycle Why Cell Cycle? Different cellular statesBiochemical eventsGenes on/off Different gene dosage Not synchronous

  21. What process can govern initiation?Hypothesis: The cell cycle Why Cell Cycle? Different cellular statesBiochemical eventsGenes on/off Different gene dosage Not synchronous

  22. What process can govern initiation?Hypothesis: The cell cycle Why Cell Cycle? Different cellular statesBiochemical eventsGenes on/off Different gene dosage Not synchronous hemimethylated

  23. What process can govern initiation?Hypothesis: The cell cycle Why Cell Cycle? Different cellular statesBiochemical eventsGenes on/off Different gene dosage Not synchronous

  24. What process can govern initiation?Hypothesis: The cell cycle Why Cell Cycle? Different cellular statesBiochemical eventsGenes on/off Different gene dosage Not synchronous

  25. DnaA protein Methylation 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:

  26. Role of cell cycle in initial pattern?

  27. Role of cell cycle in initial pattern? ??? Crude pattern of cell division Crude pattern of differentiation? More than a correlation? ??? ??? ??? How to tell?

  28. Role of cell cycle in initial pattern? ??? Perturbation of cell cycle Random pattern of differentiation? Perturbing cell cycle affects heterocyst spacing?

  29. Regulation of initiation of DNA synthesis by methylation Origin of replication origin of replication

  30. Regulation of initiation of DNA synthesis by methylation blocked origin of replication

  31. * C A G A T C T A Old DNA * G T C T A G A T C A G A T C T A New DNA * G T C T A G A T Regulation of initiation of DNA synthesis by methylation blocked available origin of replication

  32. 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

  33. Solitary methylases Ancient Restrictionmethylases Recent arrivals Relics Matveyev AV, Young KT, Meng A, and Elhai J (2001) Nucl Acids Res 29:1491-1506 DNA methyltransferases of Anabaena s

  34. Matveyev AV, Young KT, Meng A, and Elhai J (2001) Nucl Acids Res 29:1491-1506 DNA methyltransferases of Anabaena Solitary methylases

  35. Null phenotype Gene Site Status ? ? ? 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

  36. sacB Sm/Sp npt npt dmtB trpD Formation of dmtB mutant Phenotype No GGCC methylation No heterocysts dmtB

  37. dmtB+ + - Complementation of dmtB mutant ExpectConditionMethylationHeterocysts - plasmid - - + plasmid + + ObserveConditionMethylationHeterocysts - plasmid - - npt + plasmid + sll0160 dmtB trpD dmtB trpD

  38. npt dmtB trpD Formation of dmtB mutant (revisited) sacB Sm/Sp Phenotype No GGCC methylation npt Yes heterocysts

  39. - - DmtBHet npt - + DmtBHet dmtB trpD Formation of dmtB mutant (revisited) Phenotype npt dmtB trpD trpD

  40. - - - - TrpDHet DmtBHet npt + Het - - + + DmtBHet TrpDHet P dmtB trpD trpD npt trpD dmtB npt trpD dmtB Formation of dmtB mutant (revisited) Phenotype npt dmtB trpD trpD

  41. - - - - TrpDHet DmtBHet npt + dmtB trpD Het - - + + TrpDHet DmtBHet P dmtB trpD trpD npt trpD dmtB npt trpD dmtB Formation of dmtB mutant (revisited) Phenotype npt dmtB trpD trpD

  42. - - - - TrpDHet DmtBHet npt + Het - - + + TrpDHet DmtBHet dmtB trpD npt trpD dmtB npt trpD dmtB Formation of dmtB mutant (revisited) Phenotype npt dmtB trpD trpD P trpD dmtB trpD

  43. Deletion of dmtB-trpD region P trpD dmtB trpD

  44. Deletion of dmtB-trpD region P trpD

  45. Deletion of dmtB-trpD region Het+++ . . . death

  46. What is the intergenic region? dmtB trpD Blast against Anabaena genome

  47. What is the intergenic region? Best Blast Hits

  48. dmtB trpD hetR What is the intergenic region? Best Blast Hits P

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