E. coli Genome PROKARYOTES Typically, - >10 6 bp - Sequence without gaps ANIMALS Typically, >10 9 bp - Sequence with many gaps - 95+% covered The Human Genome After Sequencing and Assembly For Bioinformatics, Start with Genomics:

Who Gets Sequenced? Models Pathogens Agriculturals

Finished Genomes *** * ** * *** ** * ** * Choanoflaggelate –closest unicellular to animals

*Vertebrates Homo sapiens Pan troglodytes Mus musculus Rattus rattus Canis familiaris Bos taurus Gallus gallus Xenopus tropicalis + Fufu rubripes Tetraodon nigroviridis Orysias latipes Danio rario **Arthropods 14 Drosophila species Anopholes gambiae + Apis mellifera Ixodes - tick Who Gets Sequenced?–Animal model systems:

Genomics: READING genome sequences ASSEMBLY of the sequence ANNOTATION of the sequence carry out dideoxy sequencing connect seqs. to make whole chromosomes find the genes! For Bioinformatics, Start with:

Genomics: READING genome sequences ASSEMBLY of the sequence ANNOTATION of the sequence carry out dideoxy sequencing connect seqs. to make whole chromosomes find the genes! For Bioinformatics, Start with:

2 ways to annotate eukaryotic genomes: -ab initio gene finders: -Genes based on previous knowledge….EVIDENCE of message 2 ways to annotate eukaryotic genomes: -ab initio gene finders: Work on basic biological principles: Open reading frames Consensus splice sites Met start codons ….. -Genes based on previous knowledge….EVIDENCE of message

2 ways to annotate eukaryotic genomes: -ab initio gene finders: Work on basic biological principles: Open reading frames Consensus splice sites Met start codons ….. -Genes based on previous knowledge….EVIDENCE of message cDNA sequence of the gene’s message cDNA of a closely related gene’ message sequence Protein sequence of the known gene Same gene’s Same gene’s from another species Related gene’s protein…….

Homology based exon predictions Consensus gene structure (both strands) start and stop site predictions Splice site predictions computational exon predictions Tracking information Unique identifiers

Automatically generated annotation

A zebrafish hit shows a gene model protein encoded by a 6 exon gene. This gene structure (intron/exon) is seen in other species, as is the protein size. The proteins, if corresponding to MSP in S. gal., must be heavily glycosylated (likely). At least some have a signal peptide.

The zebrafish hit can be viewed at higher resolution, and…

The zebrafish hit can be viewed down to nucleotide resolution GO LIVE!

Genomics: READING genome sequences ASSEMBLY of the sequence ANNOTATION of the sequence carry out dideoxy sequencing connect seqs. to make whole chromosomes find the genes! But Bioinformatics is more…

TRANSCRIPTOMICS: cDNAs RNA target sample End Reads (Mates) SEQUENCE Primer cDNA Library Each cDNA provides sequence from the two ends – two ESTs & ESTs: Expressed Sequence Tags

Who Gets EST---- ed?–Animal model systems: *Vertebrates Homo sapiens Pan troglodytes Mus musculus Rattus rattus Canis familiaris Bos taurus Gallus gallus Xenopus tropicalis + Fufu rubripes Tetraodon nigroviridis Orysias latipes Danio rario **Arthropods D. melanogaster D. pseudoobscura D. simulans Anopholes gambiae + Apis mellifera Who Gets Sequenced?–Animal model systems: ***Millions **100,000s * 10,000s *** ** * * * * *

!!AA_SEQUENCE 1.0 ab peptide tenm4.pep Length: 2771 May 12, :34 Type: P Check: MDVKERKPYR SLTRRRDAER RYTSSSADSE EGKGPQKSYS SSETLKAYDQ 51 DARLAYGSRV KDMVPQEAEE FCRTGTNFTL RELGLGEMTP PHGTLYRTDI 101 GLPHCGYSMG ASSDADLEAD TVLSPEHPVR LWGRSTRSGR SSCLSSRANS 151 NLTLTDTEHE NTETDHPSSL QNHPRLRTPP PPLPHAHTPN QHHAASINSL 201 NRGNFTPRSN PSPAPTDHSL SGEPPAGSAQ EPTHAQDNWL LNSNIPLETR 251 NLGKQPFLGT LQDNLIEMDI LSASRHDGAY SDGHFLFKPG GTSPLFCTTS 301 PGYPLTSSTV YSPPPRPLPR STFSRPAFNL KKPSKYCNWK CAALSAILIS 351 ATLVILLAYF VAMHLFGLNW HLQPMEGQMQ MYEITEDTAS SWPVPTDVSL 401 YPSGGTGLET PDRKGKGAAE GKPSSLFPED SFIDSGEIDV GRRASQKIPP Protein sequence: from peptide sequencing, or from translation of sequenced nucleic acids

Structural genomics: Coordinates, rather than 1D sequence, Saved

RNA for ALL C. elegans genes

MICROARRAY ANALYSIS

Array analysis: see animation from Griffiths

Figure 4.16(1) Microarray Analysis of Those Genes Whose Expression in the Early Xenopus Embryo Is Caused by the Activin-Like Protein Nodal-Related 1 (Xnr1)

Figure 4.16(2) Microarray Analysis of Those Genes Whose Expression in the Early Xenopus Embryo Is Caused by the Activin-Like Protein Nodal-Related 1 (Xnr1)

Figure 4.15(1) Microarray Technique

Figure 4.15(2) Microarray Technique

Figure 4.23(1) Use of Antisense RNA to Examine the Roles of Genes in Development

Figure 4.23(2) Use of Antisense RNA to Examine the Roles of Genes in Development

RNAi for every C. elegans gene too! -results on the web Projects to systematically Knock-out (or pseudo-knockout) every gene, in order to establish phenotype of each gene -> function of each gene

RNAi for ALL C. elegans genes

Figure 4.24 Injection of dsRNA for E-Cadherin into the Mouse Zygote Blocks E-Cadherin Expression

Followed by INVERSE PCR to recover seqeunce adjacent to insertion. Then compare to the complete Drosophila genome sequence to know which ORF “Hit” KNOCK-OUTS OF ALL ESSENTIAL GENES – RANDOM MUTAGENESIS ATTEMPT – using transposon mobilization

About 10% of All Assumed genes “Hit” (~10/100 per interval) on Drosophila X chromosome. 1 series of random insertion experiments. ALL inset sites know, thanks to INVERSE PCR

Figure 1 The two-hybrid assay carried out by screening a protein array. a, The array of 6,000 haploid yeast transformants plated on medium lacking leucine, which allows growth of all transformants. Each transformant expresses one of the yeast ORFs expressed as a fusion to the Gal4 activation domain. b, Two-hybrid positives from a screen of the array with a Gal4 DNA-binding domain fusion of the Pcf11 protein, a component of the pre-mRNA cleavage and polyadenylation factor IA, which also consists of four other polypeptides36. Diploid colonies are shown after two weeks of growth on medium lacking tryptophan, leucine and histidine and supplemented with 3 mM 3-amino-1,2,4-triazole, thus allowing growth only of cells that express the HIS3 two-hybrid reporter gene. Three other components of factor IA, Rna14, Rna15 and Clp1, were identified as Pcf11 interactors. Positives that do not appear in Table 2 were either not reproducible or are false positives that occurred in many screens.Table 2 2-hybrid reaction between one protein and all potential interactors in Yeast Genome

Figure 2 Visualization of combined, large-scale interaction data sets in yeast. A total of 14,000 physical interactions obtained from the GRID database were represented with the Osprey network visualization system (see Each edge in the graph represents an interaction between nodes, which are coloured according to Gene Ontology (GO) functional annotation. Highly connected complexes within the data set, shown at the perimeter of the central mass, are built from nodes that share at least three interactions within other complex members. The complete graph contains 4,543 nodes of 6,000 proteins encoded by the yeast genome, 12,843 interactions and an average connectivity of 2.82 per node. The 20 highly connected complexes contain 340 genes, 1,835 connections and an average connectivity of Osprey: integrate all 2-hybrid interactions between all proteins in Yeast Genome (Proteome)