1Computational Genomics and Molecular BiologyDannie DurandFall 2004Lecture 3OutlineA whirlwind review of molecular biologyAn overview of computational molecular biologyNew problems in genomicsPairwise sequence alignment (global and local)Multiple sequence alignmentlocalglobalSubstitution matricesDatabase searchingBLASTEvolutionary tree reconstructionRNA structure predictionGene FindingProtein structure predictionSequence statisticsComputational genomics…Genomes:The complete instruction setNeisseria gonorrhoeae Homo sapiensGTGCACCTGACTCCTGAG...Gene sequencesGenomic sequencesComputational GenomicsComputational implications:– Need algorithms that scale up– Genomes don’t look the way we thought they did¾ revise models– New biological questions¾ new computational problemsWhole Genome Sequencing1995 H. influenzae – 1stwhole genome sequence1997 Yeast – 1steukaryotic sequence 1998 C. elegans – 1stmulticellular organism2000 Fly, mustard weed – 1stplant 2001 Human – 1stvertebrate2002 Mouse, Ciona intestinalis2003 Mosquito, C. briggsae2004 Five more yeasts, silkworm, rat, C. merolae, white rot fungus…145 whole genome sequences: 19 eukarya, 16 archaea, 110 bacteria215 32 eukarya, 19 archaea 164 bacteriaIn progress: 522 prokaryotic genomes, 441 eukaryotic genomes www.genomesonline.org2The FantasyTGAAATAAACAACCAGGCAGCAGTTATTAACACGGGAACATGGCGGCCGCAGCCTGGGCTCCCGCGGCGGCGGCGG…Cell Function SimulatorWhole genome sequenceCell Simulator CompilerFrom Genes to OrganismsCellular pathways transcription of mating specific genesreceptor senses pheromone outside cellPheromone signaling pathwayRube Goldberg’s picture snapping machineExample: Pheromone signaling pathwaytranscription of mating specific genesreceptor senses pheromone outside cellFrom Genes to Organisms3• Predict – all genes– all gene products (protein, RNA)– regulatory motifs• Predict structure and function of individual components• Reconstruct the cellular networks– Regulatory pathways– Metabolic pathways– Signaling pathways …• Model cellular behaviorFrom Genes to OrganismsNew computational approaches– New, better algorithms– Use data in new ways • Comparative genomics– Genomic sequence– Gene content– Gene order• Combine different types of dataNew high throughput data sets– mRNA expression– Splice variants– Protein expression – Sub-cellular localization– Protein-protein interactions– Protein-DNA interactionsFrom Genes to OrganismsComputational Functional GenomicsHigh-thoughput functional assaysComputational support for • data acquisition• data analysisHigh-thoughput sequencingComputational support for • data acquisition• data analysisNew computational approaches– New, better algorithms– Use data in new ways • Comparative genomics– Genomic sequence– Gene content– Gene order• Combine different types of dataNew high throughput data sets– mRNA expression– Splice variants– Protein expression – Sub-cellular localization– Protein-protein interactions– Protein-DNA interactionsFrom Genes to OrganismsWhen are genes turned on?genesmRNAsDetermine the set of all genes being transcribed in a given cell type under particular conditionsAlternate splice forms:exon6exon1 exon2 exon3 exon5exon1 exon2 exon3 exon4DNA:mRNA:exon1 exon2 exon3 exon4exon6exon5exon1 exon2 exon3 exon4exon6exon5Determine the set of splice variants in a given cell type under particular conditions4Expressed Sequence Tags (ESTs)degradation of mRNA, synthesis of second DNA strandCATGACTCCTTGGCTAC...CCGAGTGCGGCATTTTTTGTACTGAGGAACCGATG...GGCTCACGCCGTAAAAAAdsDNACAUGACUCCUUGGCUAC...CCGAGUGCGGCAUUUUUUGTACTGAGGAACCGATG...GGCTCACGCCGTAAAAAAreverse transcriptasecDNACAUGACUCCUUGGCUAC...CCGAGUGCGGCAUUUUUUmRNAreverse primer3’ ESTforward primer5’ ESTExpressed Sequence Tags– Single-pass sequencing of “random” cDNAs– 5’ or 3’ end– Relatively low quality sequence– Tissue specific– No guarantee• that all genes are represented• that all splice forms are represented5’ ESTsmRNA3’ ESTsESTs: molecular tags for genes.ESTs– fast way to capture the coding portion of the genome. (In eukaryotes, most of the genome does not contain protein coding genes. )– provide a crude measure of transcript abundance. However, rare transcripts may be missed.– provide a crude measure of splice variants (if at the 3’ or 5’ end of the gene).When are genes turned on?DNA arrays detect mRNA transcriptsmicroarraysDNA microarraysTargets: Each well contains a cDNA oligonucelotidecorresponding to a unique subsequence of a genecgtaacgctatDNA microarrays5DNA microarraysDown regulated in tumorDNA microarraysUp regulated in tumorDNA microarraysUnchangedExpression array dataclustered array dataunsorted array dataO. Alter, P. O. Brown and D. Botstein, PNAS 97 (18), 2000New computational approaches– New, better algorithms– Use data in new ways • Comparative genomics– Genomic sequence– Gene content– Gene order• Combine different types of dataNew high throughput data sets– mRNA expression– Splice variants– Protein expression– Sub-cellular localization– Protein-protein interactions– Protein-DNA interactionsFrom Genes to OrganismsNot all mRNA transcripts are translated into proteinsmRNA:transcriptionAmino acid sequence:translationRNA polymeraseDNA:promoter gene6Protein ExpressionFind the set of all proteins being expressed in a given cell type under particular conditions• Isolate the set of proteins present– 2D gel electrophoresis• Identify the proteins in the set– Mass spectrometry– Protein chips based on antibody recognitionNew computational approaches– New, better algorithms– Use data in new ways • Comparative genomics– Genomic sequence– Gene content– Gene order• Combine different types of dataNew high throughput data sets– mRNA expression– Splice variants– Protein expression – Sub-cellular localization– Protein-protein interactions– Protein-DNA interactionsFrom Genes to OrganismsProtein Sub cellular LocalizationPlant and animal cells have compartments or “organelles”Endoplasmic Reticulum (ER)MitochondriaR. Murphy, Biological Sciences•Recognize sequence-based localization signals•Microscopy: stain protein with a “guest exon”New computational approaches– New, better algorithms– Use data in new ways • Comparative genomics– Genomic sequence– Gene content– Gene order•
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