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The Course of Development

The Course of Development. The Course of Development. Complicated. Time. Events in time and space. The Course of Development. Really. Complicated. Time. Events in time and space. heterocysts. NH 3. Cyanobacteria. Free-living Nostoc. Anabaena grown without fixed nitrogen. N 2.

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The Course of Development

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  1. The Course of Development

  2. The Course of Development Complicated Time Events in time and space . . .

  3. The Course of Development Really Complicated Time Events in time and space . . .

  4. heterocysts NH3 Cyanobacteria Free-living Nostoc Anabaena grown without fixed nitrogen N2 Matveyev and Elhai (unpublished)

  5. removal of nitrate 18hours Anabaena filament growing on N2 Heterocysts only when needed Anabaena filament growing on nitrate

  6. Differentiation in cyanobacteriaIntegration of signals through HetR Nitrogen deprivation ?????? Genes needed for differentiation HetR Position in filament Master regulator Position in cell cycle

  7. Regulatory protein RNA Polymerase How might hetR be controlled? Presence of fixed nitrogen Absence of fixed nitrogen Transcription hetR gene NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN 5’-GTANNNTACNNNNNNNNNNTANNNTNNNNNNNNNN3’-CATNNNATGNNNNNNNNNNATNNNANNNNNNNNNN Promoter No HetR protein GTA ..(8).. TAC

  8. How might hetR be controlled? Absence of fixed nitrogen hetR gene NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN 5’-GTANNNTACNNNNNNNNNNTANNNTNNNNNNNNNN3’-CATNNNATGNNNNNNNNNNATNNNANNNNNNNNNN RNA Polymerase GTA ..(8).. TAC

  9. How might hetR be controlled? Absence of fixed nitrogen Transcription hetR gene NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN 5’-GTANNNTACNNNNNNNNNNTANNNTNNNNNNNNNN3’-CATNNNATGNNNNNNNNNNATNNNANNNNNNNNNN HetR protein RNA Polymerase GTA ..(8).. TAC

  10. dmtB DNA methyltransferase DNA modification and differentiation Promoter No heterocysts! DNA modification necessary for differentiation?

  11. dmtB DNA methyltransferase DNA modification and differentiation Promoter trpD2 YES heterocysts! DNA modification necessary for differentiation?

  12. Tryptophan biosynthesis DNA modification and differentiation Promoter dmtB trpD2 Tryptophan biosynthesis necessary for differentiation?

  13. Tryptophan biosynthesis DNA modification and differentiation Promoter dmtB trpD2 NO heterocysts! Tryptophan biosynthesis necessary for differentiation?

  14. DNA modification and differentiation dmtB trpD2 Is there a gene between dmtB and trpD2 that may control heterocyst differentiation?

  15. LMTVDFSVG MPLKVHKLEL TATAAAGTTGGAGCGAAACTACTGTCAGCTAAAAAGCCATCCTATTGACGGTACGGAGAGTTTCATGTGTTTGAACTCAATACTGGTCAAGAGGATGTAG LCTLRGMAVILPKNRLSSKRG Look for pattern: (ATG | GTG | TTG)... ... (TAA | TAG | TGA) start codon triplets stop codon ORFs within intergenic region dmtB trpD2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC

  16. LMTVDFSVG MPLKVHKLEL TATAAAGTTGGAGCGAAACTACTGTCAGCTAAAAAGCCATCCTATTGACGGTACGGAGAGTTTCATGTGTTTGAACTCAATACTGGTCAAGAGGATGTAG LCTLRGMAVILPKNRLSSKRG ORFs within intergenic region dmtB trpD2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC Nothing compelling What about RNA genes?

  17. dmtB trpD2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC How might a noncoding gene work? Absence of fixed nitrogen Critical gene NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN 5’-GTANNNTACNNNNNNNNNNTANNNTNNNNNNNNNN3’-CATNNNATGNNNNNNNNNNATNNNANNNNNNNNNN NO heterocysts! Find by Blast RNA Polymerase GTA ..(8).. TAC

  18. dmtB trpD2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC 50 64 asl0749 alr0750 7 25 alr3353 alr3352 48 61 all4762 6 19 alr5164 53 66 alr4509 54 67 alr0279 -9 4 alr4028 alr4029 9 25 alr2338 alr2339 -15 -3 alr1301 5 17 all1239 alr1240 52 64 all1663 -7 5 alr5148 -5 7 all3924 27 39 all0048 all0047 -5 7 all2288 53 65 alr3885 51 63 all0706 55 67 alr5148 alr5149 -1 11 all1716 16 28 all7645 alr7646 How to find a noncoding gene? Blast against Anabaena genome

  19. dmtB trpD2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC How to find a noncoding gene? Blast against Anabaena genome 50 64 asl0749 alr0750 7 25 alr3353 alr3352 -9 4 alr4028 alr4029 9 25 alr2338 alr2339 5 17 all1239 alr1240 27 39 all0048 all0047 55 67 alr5148 alr5149 16 28 all7645 alr7646

  20. dmtB trpD2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC How to find a noncoding gene? Blast against Anabaena genome 50 64 asl0749 alr0750 7 25 alr3353 -9 4 alr4029 9 25 alr2339 5 17 all1239 alr1240 27 39 all0048 55 67 alr5149 16 28 all7645 alr7646

  21. dmtB trpD2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC How to find a noncoding gene? Blast against Anabaena genome 50 64 asl0749 alr0750 7 25 alr3353 -9 4 alr4029 9 25 alr2339 5 17 all1239 alr1240 27 39 all0048 55 67 alr5149 16 28 all7645 alr7646

  22. dmtB trpD2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC How to find a noncoding gene? Blast against Anabaena genome 50 64 asl0749 alr0750 30S ribosomal protein hypothetical 7 25 alr3353 putative peptididase -9 4 alr4029 ~Vitamin B12 transport 9 25 alr2339 heterocyst differentiation 5 17 all1239 alr1240 hypothetical Zam 27 39 all0048 hypothetical 55 67 alr5149 unknown 16 28 all7645 alr7646 unknown MobB

  23. dmtB trpD2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC How to find a noncoding gene? Blast against Anabaena genome 7 25 alr3353 putative peptididase 9 25 alr2339 heterocyst differentiation alr2339 is hetR

  24. dmtB trpD2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC dmtB-trpD2ATGACAGTCGATTTTTCGGTAGGATAACTGCCATpre-hetR TTATTTTGTGATTTTTCGGTAAGATACAAGCACG transcription Characterization of matchbetween dmtB-trpD2 and pre-hetR The match overlaps the promoter of hetR

  25. dmtB trpD2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC dmtB-trpD2ATGACAGTCGATTTTTCGGTAGGATAACTGCCATpre-hetR TTATTTTGTGATTTTTCGGTAAGATACAAGCACG transcription dmtB-trpD2ATAGCGGTCAATTTTCCGGTAGGATAGCTCCCAT pre-hetR GATATCTGTAATTTTCCAGTCAGATGCAGAAACA Characterization of matchbetween dmtB-trpD2 and pre-hetR Region from Nostoc punctiforme The match is conserved in different cyanobacteria

  26. Is there a gene between dmtB and trpD2 that may control heterocyst differentiation? Coding gene? Unlikely May be a noncoding gene Striking match with hetR promoter Conserved between Anabaena and Nostoc Novel mode of bacterial regulation

  27. Thanks to Andrey Matveyev Virginia Commonwealth University

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