1. Gene Structure and Identification III BIO520 BioinformaticsJim Lund Previous reading: 1.3, 9.1-9.6 10.2, 10.4, 10.6-8

2. For real prediction we need… Solve the protein folding problem Solve the molecular docking/binding problem Develop realistic simulations of molecules in cells Simulate multicellular systems

3. Promoter/Enhancer analysis Regulatory Sequences –Known Consensus Sequences –Consensus Sequence Generation Using functional (experimental) Data HBB as an example

4. Gene Regulatory Sequences Functional sites –Consensus –Experimental tests Inferred sites –Transcriptome analysis

5. Sequence Logos http://weblogo.berkeley.edu/

7. Position Weight Matrix: PO A C G T 01 6 4 4 6 N 02 4 9 3 4 N 03 12 4 3 1 A 04 6 1 11 2 R 05 3 2 11 4 G 06 3 3 4 10 N 07 3 10 3 4 N 08 11 2 4 3 A 09 4 9 3 4 N 10 3 6 3 8 N

8. EUKARYOTES More complex signals –Basal/core promoter –Promoter –Enhancers More genes More dispersed signals –Larger promoters, distant enhancers, regulatory sites in introns. Combinatoric regulation common

9. Basal Promoter Analysis Myers and Maniatis, Genes VI, 831 TATA-box-25 to -30 TBP CCAAT-box-212 to -57 CTF/NF1 GC-box-164 to +1 SP1 K C W K Y Y Y Y+1 to +5 cap signal TATA CAATGC +1

10. Finding PolII sites (transcription start site) Promoter Scan TSSG/TSSW ( TSSP for plants) Core-Promoter FPROM BCM Search Launcher

11. Enhancer Elements OctamerOCT1, OCT2  BNF  B ATFATF AP1…AP1 ……..

12. Consensus Sequence Databases TRANSFAC TFD (transcription factor database)

13. Consensus Sequence Databases Finding sites in promoter regions: –TESS http://www.cbil.upenn.edu/cgi-bin/tess/tess –TFSEARCH http://www.cbrc.jp/research/db/TFSEARCH.html –BCM Search Launcher http://searchlauncher.bcm.tmc.edu/seq-search/gene- search.html

14. HBB promoter (TESS)

15. Sequence-based algorithms for identifying enhancer binding sites Genes from: –Microarray transcription analysis –ChIP::chip experiments –Orthologous sequences –Experimental/other Programs for finding consensus sites: –MEME analysis of clusters –AlignAce –BioProspector/CompareProspector

16. Practical Gene Finding Use ALL tools –Predictive: Stitch together a consensus ORF finders Find patterns (and WWW pattern searches) HMM: GRAIL, Genscan… –Comparative BLASTN, BLASTX Compare genomes (human:mouse) –cDNA, protein, genetic evidence

17. ORFs-aldolase gene

18. Genomic DNA-cDNA alignment DNA sequencing cDNA Align (GAP) Infer Promoter, Enhancer Test in cis P

19. Comparative Genomics Conservation of coding regions Identification of transcription signals –“words” in common Example-yeast comparisons

20. Ensembl prediction pipeline RepeatMasker Genscan Blast genscan peptides v Protein,unigene,est,vert mrna Pmatch all human Proteins and cdnas MiniGenewise MiniEst2genome Genes DNA

23. Genscan features Model both strands at once Each state may output a string of symbols (according to some probability distribution). Explicit intron/exon length modeling Advanced splice site modeling Complete intron/exon annotation for sequence Able to predict multiple genes and partial/whole genes Parameters learned from annotated genes Separate parameter training for different CpG content groups ( 57% CG content)

24. GENSCAN predictions Gn.Ex Type S.Begin...End.Len Fr Ph I/Ac Do/T CodRg P.... Tscr.. ----- ---- - ------ ------ ---- -- -- ---- ---- ----- ----- ------ 7.00 Prom + 63096 63135 40 -2.75 7.01 Init + 63183 63274 92 2 2 103 77 142 0.997 14.61 7.02 Intr + 63403 63625 223 1 1 83 96 181 0.999 15.61 7.03 Term + 64524 64652 129 2 0 101 50 83 0.373 3.00 7.04 PlyA + 64758 64763 6 1.05 8.00 Prom + 70508 70547 40 -4.75 8.01 Init + 70595 70686 92 1 2 103 77 133 0.990 13.71 8.02 Intr + 70817 71039 223 2 1 100 96 217 0.999 20.91 8.03 Term + 71890 72018 129 0 0 116 43 119 0.827 7.40 8.04 PlyA + 72126 72131 6 1.05 9.00 Prom + 74399 74438 40 -8.25 9.01 Sngl + 76602 76847 246 2 0 71 50 218 0.886 11.13 9.02 PlyA + 76928 76933 6 1.05

25. GENSCAN predicted exons

26. Annotated predicted exons

27. HBB gene HBB exons 1-3 70545..70686 70817..71039 71890..72150 GENSCAN 70595 70686 70817 71039 71890 72018