3. The Course of Development

4. Time Events

5. The Course of Development Time Events

6. The Course of Development Time Events in time

7. The Course of Development Time Events in time

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

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

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

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

12. Free-living Nostoc heterocysts Matveyev and Elhai (unpublished) CO 2 sucrose N2N2 O2O2 Cyanobacteria Anabaena grown on N 2 / CO 2

13. Free-living Nostoc heterocysts Matveyev and Elhai (unpublished) CO 2 sucrose N2N2 NH 3 O2O2 Cyanobacteria Anabaena grown on N 2 / CO 2

14. 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

15. 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

16. 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

17. 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

18. The hydromedusa Aequoria victoria Source of Green Fluorescent Protein Detection of hetR gene expression through Green Fluorescent Protein

19. 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?

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

21. 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

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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

28. Role of cell cycle in initial pattern?

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

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

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

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

33. 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

34. 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

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

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

37. ? ? ? 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

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

39. sll0160 dmtB trpD npt dmtB trpD dmtB + + Expect Condition Methylation Heterocysts - plasmid - - + plasmid + + Observe Condition Methylation Heterocysts - plasmid - - + plasmid + Complementation of dmtB mutant -

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

41. Formation of dmtB mutant Phenotype (revisited) npt dmtB trpD npt dmtB trpD DmtB Het -- - + trpD

42. 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 - + --

43. 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

44. 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

45. P Deletion of dmtB-trpD region dmtB trpD

46. Deletion of dmtB-trpD region trpD P

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

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

49. What is the intergenic region? Best Blast Hits

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

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

52. What is the intergenic region? Best Blast Hits Other hits (no more than 3 mismatches) Cyanophycin synthetase in heterocysts

53. Hypothetical regulatory RNA Does it exist? Are the sequence and targets conserved? Is the sequence naturally transcribed?

54. dmtB  trpD2  Is the sequence conserved?

55. Are the targets conserved? P

58. Inferred characteristics of phenomenon Mediated by RNA – No reasonable orfs in area – Small intergenic region Overlap with genes unlikely – Compensatory mutations

59. Inferred characteristics of phenomenon Mediated by RNA Secondary structure – Inverted repeats – Compensatory mutations

60. Inferred characteristics of phenomenon Mediated by RNA Secondary structure Imperfect matches with putative targets – Inverted repeats – Compensatory mutations

61. Inferred characteristics of phenomenon Mediated by RNA Secondary structure Imperfect matches with putative targets Conserved over related organisms Is this surprising?