The Course of Development The Course of Development

The Course of Development

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

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

Cyanobacteria Free-living Nostoc Anabaena grown without fixed nitrogen heterocysts N 2 NH 3 Matveyev and Elhai (unpublished)

Heterocysts only when needed Anabaena filament growing on nitrate removal of nitrate 18 hours Anabaena filament growing on N 2

Differentiation in cyanobacteria Integration of signals through Het. R Nitrogen deprivation Position in filament Position in cell cycle ? ? ? Het. R Master regulator Genes needed for differentiation

How might het. R be controlled? Presence Absence of fixed nitrogen Regulatory protein Transcription het. R gene 5’-GTANNNTACNNNNNTANNNTNNNNNNNNNNNNNNATGNNNNNNNN 3’-CATNNNATGNNNNNATNNNANNNNNNNNNNNNNNTACNNNNNNNN Promoter RNA Polymerase GTA . . (8). . TAC No Het. R protein

How might het. R be controlled? Absence of fixed nitrogen het. R gene 5’-GTANNNTACNNNNNTANNNTNNNNNNNNNNNNNNATGNNNNNNNN 3’-CATNNNATGNNNNNATNNNANNNNNNNNNNNNNNTACNNNNNNNN RNA Polymerase GTA . . (8). . TAC

How might het. R be controlled? Absence of fixed nitrogen Transcription het. R gene 5’-GTANNNTACNNNNNTANNNTNNNNNNNNNNNNNNATGNNNNNNNN 3’-CATNNNATGNNNNNATNNNANNNNNNNNNNNNNNTACNNNNNNNN RNA Polymerase GTA . . (8). . TAC Het. R protein

DNA modification and differentiation Promoter dmt. B No heterocysts! DNA methyltransferase DNA modification necessary for differentiation?

DNA modification and differentiation Promoter dmt. B trp. D 2 YES heterocysts! DNA methyltransferase DNA modification necessary for differentiation?

DNA modification and differentiation Promoter dmt. B Tryptophan biosynthesis necessary for differentiation? trp. D 2 Tryptophan biosynthesis

DNA modification and differentiation Promoter dmt. B trp. D 2 NO heterocysts! Tryptophan biosynthesis necessary for differentiation? Tryptophan biosynthesis

DNA modification and differentiation dmt. B trp. D 2 Is there a gene between dmt. B and trp. D 2 that may control heterocyst differentiation?

ORFs within intergenic region LMTVDFSVG MPLKVHKLEL dmt. B trp. D 2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC TATAAAGTTGGAGCGAAACTACTGTCAGCTAAAAAGCCATCCTATTGACGGTACGGAGAGTTTCATGTGTTTGAACTCAATACTGGTCAAGAGGATGTAG LCTLRGMAVILPKNRLSSKRG Look for pattern: (ATG | GTG | TTG). . . (TAA | TAG | TGA) start codon triplets stop codon

ORFs within intergenic region LMTVDFSVG MPLKVHKLEL dmt. B trp. D 2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC TATAAAGTTGGAGCGAAACTACTGTCAGCTAAAAAGCCATCCTATTGACGGTACGGAGAGTTTCATGTGTTTGAACTCAATACTGGTCAAGAGGATGTAG LCTLRGMAVILPKNRLSSKRG Nothing compelling What about RNA genes?

How might a noncoding gene work? dmt. B Absence of fixed nitrogen trp. D 2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC Critical gene 5’-GTANNNTACNNNNNTANNNTNNNNNNNNNNNNNNATGNNNNNNNN 3’-CATNNNATGNNNNNATNNNANNNNNNNNNNNNNNTACNNNNNNNN RNA Polymerase GTA . . (8). . TAC NO heterocysts! Find by Blast

How to find a noncoding gene? dmt. B trp. D 2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC Blast against Anabaena genome 50 64 7 25 48 61 6 19 53 66 54 67 -9 4 9 25 -15 -3 5 17 52 64 -7 5 -5 7 27 39 -5 7 53 65 51 63 55 67 -1 11 16 28 asl 0749 alr 3353 alr 0750 alr 3352 all 4762 alr 5164 alr 4509 alr 0279 alr 4028 alr 2338 alr 4029 alr 2339 alr 1301 all 1239 alr 1240 all 1663 alr 5148 all 3924 all 0048 all 0047 all 2288 alr 3885 all 0706 alr 5148 alr 5149 all 1716 all 7645 alr 7646

How to find a noncoding gene? dmt. B trp. D 2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC Blast against Anabaena genome 50 64 7 25 asl 0749 alr 3353 alr 0750 alr 3352 -9 4 9 25 alr 4028 alr 2338 alr 4029 alr 2339 5 17 all 1239 alr 1240 27 39 all 0048 all 0047 55 67 alr 5148 alr 5149 16 28 all 7645 alr 7646

How to find a noncoding gene? dmt. B trp. D 2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC Blast against Anabaena genome 50 64 7 25 asl 0749 alr 3353 -9 4 9 25 5 alr 4029 alr 2339 17 all 1239 27 39 all 0048 55 67 16 28 alr 0750 alr 1240 alr 5149 all 7645 alr 7646

How to find a noncoding gene? dmt. B trp. D 2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC Blast against Anabaena genome 50 64 7 25 asl 0749 alr 3353 -9 4 9 25 5 alr 4029 alr 2339 17 all 1239 27 39 all 0048 55 67 16 28 alr 0750 alr 1240 alr 5149 all 7645 alr 7646

How to find a noncoding gene? dmt. B trp. D 2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC Blast against Anabaena genome 50 7 -9 9 5 27 55 16 64 25 17 39 67 28 asl 0749 alr 3353 all 1239 all 0048 all 7645 alr 0750 30 S ribosomal protein hypothetical putative peptididase ~Vitamin B 12 transport heterocyst differentiation hypothetical Zam alr 4029 alr 2339 alr 1240 hypothetical alr 5149 unknown alr 7646 unknown Mob. B

How to find a noncoding gene? dmt. B trp. D 2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC Blast against Anabaena genome 7 25 9 25 alr 3353 putative peptididase alr 2339 heterocyst differentiation alr 2339 is het. R

Characterization of match between dmt. B-trp. D 2 and pre-het. R dmt. B trp. D 2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC dmt. B-trp. D 2 pre-het. R ATGACAGTCGATTTTTCGGTAGGATAACTGCCAT TTATTTTGTGATTTTTCGGTAAGATACAAGCACG transcription The match overlaps the promoter of het. R

Characterization of match between dmt. B-trp. D 2 and pre-het. R dmt. B trp. D 2 ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC dmt. B-trp. D 2 pre-het. R ATGACAGTCGATTTTTCGGTAGGATAACTGCCAT TTATTTTGTGATTTTTCGGTAAGATACAAGCACG transcription Region from Nostoc punctiforme dmt. B-trp. D 2 pre-het. R ATAGCGGTCAATTTTCCGGTAGGATAGCTCCCAT GATATCTGTAATTTTCCAGTCAGATGCAGAAACA The match is conserved in different cyanobacteria

Is there a gene between dmt. B and trp. D 2 that may control heterocyst differentiation? Coding gene? Unlikely May be a noncoding gene Striking match with het. R promoter Conserved between Anabaena and Nostoc Novel mode of bacterial regulation

Thanks to Andrey Matveyev Virginia Commonwealth University