The Genom of Homo sapiens.pdf

Harvesting the Genome’s Bounty: Integrative GenomicsP. JORGENSEN,* † B.-J. BREITKREUTZ,* K. BREITKREUTZ,* C. STARK,* G. LIU,* M. COOK,* †J. SHAROM,* † J.L. NISHIKAWA,* † T. KETELA, D. BELLOWS,* A. BREITKREUTZ, † I. RUPES,*L. BOUCHER,* † D. DEWAR,* M. VO,* M. ANGELI,* T. REGULY,* A. TONG, †B. ANDREWS, † C. BOONE, † AND M. TYERS* †*Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5; † Departmentof Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada, M4G 1A8; and Bantingand Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada M5G 1L6In this post-genomic era, we face the daunting challengeof assigning function to tens of thousands of uncharacterizedopen reading frames. Furthermore, the physical andfunctional connections between gene products must beelucidated if we are to understand how the linear DNA sequenceencodes dynamic cellular function. An unexpectedresult of the Human Genome Project is the low number ofpredicted human genes, roughly fivefold more than in single-celledyeasts and approximately double that in flies orworms (Lander et al. 2001; Venter et al. 2001). Increasedconnectivity between this relatively constant number ofgenes may underlie the massive increase in system-levelcomplexity that distinguishes yeast from humans.Genome-wide approaches to discovery of gene functionnow include systematic analysis of genetic interactions,protein interactions, genome-wide expression profiles, andmutant phenotypes. More than any single approach, eachof which is subject to caveats in interpretation and reproducibility,the intersection of orthogonal genome-scaledata sets provides a robust means to interrogate gene function.Here, we summarize advanced genome-scale methodsfor biological discovery in the budding yeast Saccharomycescerevisiae, many of which will be transportable tointerrogation of human gene function.OVERVIEW OF FUNCTIONAL GENOMICSTOOLS IN YEASTThe S. cerevisiae genome sequence, completed in 1996,was the first reported for an autonomous life form. Sincethen, the powerful molecular genetics of the budding yeastsystem has afforded the first detailed analysis of a genomeand its encoded proteins. Even though few yeast geneshave introns, reliable open reading frame (ORF) predictionhas proven non-trivial, due to both sequencing errorsand a preponderance of short ORFs. Recently, the geneannotation problem has been effectively solved by comparativegenome sequence analysis of S. cerevisiae andthree closely related yeast species, S. paradoxus, S.mikatae, and S. bayanus (Kellis et al. 2003). Through thisapproach, 5,726 conserved ORFs have been annotated asbona fide genes, including 148 new ORFs of less than 100residues. Notably, 500 of the originally predicted ORFsfail to meet the criterion of conservation and are likelyspurious. Comparative genome analysis has also pinpointedconserved sequence elements in promoter regions,which are presumed to correspond to transcription factorbindingsites (Cliften et al. 2003; Kellis et al. 2003).The yeast genome sequence has spawned a host of systematicapproaches, many of which have since been appliedto other model systems. For example, the genomesequence allowed the first genome-wide transcriptionalprofiles to be recorded by DNA microarray technology(DeRisi et al. 1997). Public databases now contain expressiondata for over 1,000 different experimental conditions(Sherlock et al. 2001). This sizable compendiumof genome-wide expression data allows many gene functionsand small-molecule targets to be assigned simply byclustering of gene expression profiles (Hughes et al.2000). Insight into transcriptional regulatory circuits hasbeen further enhanced by the development of array-basedchromatin-immunoprecipitation methods that in principleallow identification of all DNA sequence elements boundby transcription factors and other chromatin-associatedproteins (Ren et al. 2000; Iyer et al. 2001).The ease with which genes can be deleted or otherwisemanipulated by homologous recombination is a key attributeof budding yeast. This feature was exploited by aninternational consortium of laboratories to systematicallydelete ~95% of all predicted yeast genes (Giaever et al.2002). The resulting collection of ~6,000 gene deletionstrains has allowed numerous high-throughput approachesfor characterization of gene function. Phenotypicanalysis using the deletion set is augmented by animaginative design feature, namely the inclusion ofunique 20-mer oligonucleotide tags that flank each PCRdeletion cassette, aptly termed barcodes (Winzeler et al.1999). The relative abundance of all gene deletion strainsin a population pool can then be determined by PCR amplificationof all barcodes from genomic DNA usingcommon primers, followed by hybridization of labeledPCR products to a microarray of all ~12,000 barcode sequences.Competitive growth of the deletion pool underany given selection thus identifies gene deletions thatconfer sensitivity by reduction in the cognate barcode signals.This method has been used to quantify strain growthdefects and strain sensitivity to a variety of stress conditions,including osmotic stress, DNA damage, and bioac-Cold Spring Harbor Symposia on Quantitative Biology, Volume LXVIII. © 2003 Cold Spring Harbor Laboratory Press 0-87969-709-1/04. 431