2. Welcome to Introduction to Bioinformatics Friday, 12 September Introduction to Scenario 2 Finding biologically important sites in DNA How to avoid being fooled by imposters? Scenario 1: Genomic comparisons Problem Sets 1M and 1P

3. Scenario 2 Finding biologically important sites in DNA

4. You: A typical grad student

6. Your object of study: Cyanobacteria

7. How do they do it? Critical position in food web CO 2 sugar N 2 ammonia H 2 O electrons Your object of study: Cyanobacteria

8. heterocysts Matveyev and Elhai (unpublished) CO 2 sucrose N2N2 N 2 fixation in cyanobacteria O2O2

9. heterocysts Matveyev and Elhai (unpublished) CO 2 sucrose N2N2 NH 3 N 2 fixation in cyanobacteria O2O2

10. -NH 3 Differentiation in cyanobacteria Heterocysts ? ? ? ? ?

11. DNA RNA protein How do bacteria respond to the environment? From gene to protein Response to environment

12. How do bacteria respond to the environment? From gene to protein DNA RNA protein RNA Pol P

13. High N Low N How do cyanobacteria respond to NH 3 ? From gene to proteinNH 3 α-ketoglutarate glutamine DNA binding protein, NtcA RNA Pol Binding site P DNA No RNA

14. High N Low N How do cyanobacteria respond to NH 3 ? From gene to proteinNH 3 α-ketoglutarate glutamine DNA binding protein, NtcA RNA Pol Binding site P DNA No RNA

15. How do cyanobacteria respond to NH 3 ? From gene to protein DNA Low N RNA Pol NtcA Binding site P RNA protein α-ketoglutarate

16. -NH 3 Differentiation in cyanobacteria Heterocysts ? ? ? ? ?

17. -NH 3 Differentiation in cyanobacteria Heterocysts ? ? ? Activates NtcA (Nitrogen Control)

18. Differentiation in cyanobacteria What does NtcA bind to? Herrero et al (2001) J Bacteriol 183:411-425 mRNA …(20-24)…TAnnnT GTA…(8)…TAC

19. HetR Genes needed for differentiation Master regulator Differentiation in cyanobacteria Integration of signals through HetR Level of PatS Level of HetN Position in cell cycle NtcA -N ??? HetQ Strategy PCR out hetQ Random mutagenesis Look for effects on HetR expression/activity

20. cctatctccgccctatggcgatttgggcaatatatttgatgattggttag...hypothetical ttgtcagttgtcagacgtagtagcgcgtctagtctaatgtgttgttatat protein tatttgctactagaaatgaggagagggttatttttctcactgcttcccaa ttctatgagaatataaaattttccttaagtttctcatggcaataatggaa aaaaccgaccattctgatgaataagtccggttttttccaaaaaatatttt tgctttttcgctttatttatctatatttccaagttttagtacatcggtga ggggtgacaactatcttgccaatattgtcgttattgttaggttgctatcg gaaaaaatctgtaacatgagatacacaatagcatttatatttgctttagt atctctctcttgggtgggattctgcctgcaatttaaaaaccagtgttaac aattttcggctttattttccgggagttaaatcaaccaagggaaaatgtaa ctaatgtttaaatatcttcggatacacacaaagtaaaaccaatttttaca gatgtcgatgttgctcacattttttagaaatattactaaattaaaaatgt tattaaatttatgttcatagagaaccttttccaaataaaaaaataatttt cctgatgttttaagaaaattactgttgttataaattaaaggtgattcaac aaaatatagatagttctttcaataactatctacttttaccattaagtgaa cttactcatgaataatcaacaggaattaaaaataaagttcatgaatactg gttaaagattcagtaaagtttgaggaaataccggaataaatttccaccca aatatgattttttaaaagatacattggcagtacattaaaatgccgatgtt agataaatttgccttcatagctgttatctatttgctcagaactaagccaa gagtttacacaccaaacagaaattaaactatgaatccctcttcgtcgtta hetQ... Differentiation in cyanobacteria Find primers to PCR out hetQ

21. cctatctccgccctatggcgatttgggcaatatatttgatgattggttag...hypothetical ttgtcagttgtcagacgtagtagcgcgtctagtctaatgtgttgttatat protein tatttgctactagaaatgaggagagggttatttttctcactgcttcccaa ttctatgagaatataaaattttccttaagtttctcatggcaataatggaa aaaaccgaccattctgatgaataagtccggttttttccaaaaaatatttt tgctttttcgctttatttatctatatttccaagttttagtacatcggtga ggggtgacaactatcttgccaatattgtcgttattgttaggttgctatcg gaaaaaatctgtaacatgagatacacaatagcatttatatttgctttagt atctctctcttgggtgggattctgcctgcaatttaaaaaccagtgttaac aattttcggctttattttccgggagttaaatcaaccaagggaaaatgtaa ctaatgtttaaatatcttcggatacacacaaagtaaaaccaatttttaca gatgtcgatgttgctcacattttttagaaatattactaaattaaaaatgt tattaaatttatgttcatagagaaccttttccaaataaaaaaataatttt cctgatgttttaagaaaattactgttgttataaattaaaggtgattcaac aaaatatagatagttctttcaataactatctacttttaccattaagtgaa cttactcatgaataatcaacaggaattaaaaataaagttcatgaatactg gttaaagattcagtaaagtttgaggaaataccggaataaatttccaccca aatatgattttttaaaagatacattggcagtacattaaaatgccgatgtt agataaatttgccttcatagctgttatctatttgctcagaactaagccaa gagtttacacaccaaacagaaattaaactatgaatccctcttcgtcgtta hetQ...

22. ttgtcagttgtcagacgtagtagcgcgtctagtctaatgtgttgttatat tatttgctactagaaatgaggagagggttatttttctcactgcttcccaa ttctatgagaatataaaattttccttaagtttctcatggcaataatggaa aaaaccgaccattctgatgaataagtccggttttttccaaaaaatatttt tgctttttcgctttatttatctatatttccaagttttagtacatcggtga ggggtgacaactatcttgccaatattgtcgttattgttaggttgctatcg gaaaaaatcTGTAacatgagaTACAcaatagcatttatatttgctttagt atctctctcttgggtgggattctgcctgcaatttaaaaaccagtgttaac aattttcggctttattttccgggagttaaatcaaccaagggaaaatgtaa ctaatgtttaaatatcttcggatacacacaaagtaaaaccaatttttaca gatgtcgatgttgctcacattttttagaaatattactaaattaaaaatgt tattaaatttatgttcatagagaaccttttccaaataaaaaaataatttt cctgatgttttaagaaaattactgttgttataaattaaaggtgattcaac aaaatatagatagttctttcaataactatctacttttaccattaagtgaa cttactcatgaataatcaacaggaattaaaaataaagttcatgaatactg gttaaagattcagtaaagtttgaggaaataccggaataaatttccaccca aatatgattttttaaaagatacattggcagtacattaaaatgccgatgtt agataaatttgccttcatagctgttatctatttgctcagaactaagccaa gagtttacacaccaaacagaaattaaactatgaatccctcttcgtcgtta hetC... Differentiation in cyanobacteria Find primers to PCR out hetC GTA…(8)…TAC

23. ttctatgagaatataaaattttccttaagtttct aaaaccgaccattctgatgaataagtccggtttt tgctttttcgctttatttatctatatttccaagt ggggtgacaactatcttgccaatattgtcgttat gaaaaaatctGTAacatgagaTACacaatagcat ttatatttgcttTAgtaTctctctcttgggtggg Differentiation in cyanobacteria GTA…(8)…TAC NtcA binding site …(20-24)…TAnnnT Promoter

24. Level of PatS Level of HetN Position in cell cycle NtcA HetR Genes needed for differentiation -N Master regulator Differentiation in cyanobacteria Integration of signals through HetR ??? HetQ Stockholm

25. How to proceed? Choice #1 Publish Grant proposals Build a career Likely result Reviewers trash MS: too speculative

26. How to proceed? Choice #2 Forget about it Back to PCR Likely result Sometimes miss spectacular finding

27. How to proceed? Choice #3 Forget about PCR Do backbreaking NtcA binding studies Likely result Might demonstrate binding of NtcA Risky, may lose many months

28. How to proceed? Choice #4 Determine whether site is likely to be real How? N!... a! (N-a)! High school math approach

29. How to proceed? Choice #4 Determine whether site is likely to be real How? BIOINFORMATICS Simulation Exhaustive pattern search

30. End Scenario 2 Story foreach $problem (@PS1M @PS1P){ if ($problem =~ /PS1[M-1|M-5|M-9|P-3|P-5]/){ Do_problem_now($problem); } } Molecular Biology: Regulatory protein and binding sites Bioinformatics: Simulations; Nature of randomness Programming: Loops and arrays while ($time_permits);