by FISH across the whole genome. A subset of theresults were selected in which there was no evi-dence of the YAC having been chimeric, and theposition was resolved to a cytogenetic interval (asopposed to fractional length only). A Genbthonmarker was given for each of these YACs, whichallowed the position in centimorgans to bedetermined. Because this study contained no datafor chromosomes 19, 21, and 22, an alternativestrategy was used: Genes that are nonrecombinantwith respect to Gbnbthon markers [table 5 in C. Dibet al., Nature 380 (suppl.), iii (1996)] and havewell-known cytogenetic locations [Online Mende-lian Inheritance in Man (OMIM) at http://www.ncbi.nlm.nih.gov/] were used to establish thecross-reference.

28. N. E. Morton, Proc. Natl. Acad. Sci. U.S.A. 88, 7474(199 1).

29. Detailed instructions and examples are provided onthe Web site.

30. G. D. Schuler, J. A. Epstein, H. Ohkawa, J. A. Kans,Methods Enzymol. 266, 141 (1996).

31. Five additional assays yielded ambiguous results asa result of the presence of an interfering mouse bandor poor amplification.

32. This could be the result of either a trivial primer tubelabeling error or a sequence clustering error in whichtwo separate genes were erroneously assigned tothe same UniGene entry.

33. Typing errors in a framework marker would allow aclose EST to map with significant lod scores (loga-rithm of the odds ratio for linkage) to a correct chro-mosome location but would tend to localize themarker in a distant bin, in order to minimize "double-breaks" caused by the erroneous typings of theframework marker.

34. J. M. Craig and W. A. Bickmore, Bioessays 15, 349(1993).

35. V. Romano et al., Cytogenet. Cell Genet. 48, 148(1988).

36. S. Bahram, M. Bresnahan, D. E. Geraghty, T. Spies,Proc. Natl. Acad. Sci. U.S.A. 91, 6259 (1994).

37. G. D. Billingsley et al., Am. J. Hum. Genet. 52, 343(1993); S. S. Schneider et al., Proc. Natl. Acad. Sci.U.S.A. 92, 3147 (1995).

38. R. Sherrington et al., Nature 375, 754 (1995); D.Levitan and 1. Greenwald, ibid. 377, 351 (1995); S. A.Hahn et al., Science 271, 350 (1996).

39. S. Tugendreich et al., Hum. Mol. Genet. 3, 1509(1994); D. E. Bassett Jr., M. S. Boguski, P. Hieter,Nature 379, 589 (1996); P. Hieter, D. E. Bassett Jr.,D. Valle, Nature Genet. 13, 253 (1996).

40. The scoring systems were amino acid substitutionmatrices based on the PAM (point accepted muta-tion) model of evolutionary distance. Pam matricesmay be generated for any number of PAMs by ex-trapolation of observed mutation frequencies [M. 0.Dayhoff et aL., in Atlas of Protein Sequence andStructure, M. 0. Dayhoff, Ed. (National BiomedicalResearch Foundation, Washington, DC, 1978), vol.5, suppl. 3, pp. 345-352; S. F. Altschul, J. Mol. Evol.36, 290 (1993)]. PAM matrices were customized forscoring matches between sequences in each of the15 species pairs; for the pool of remaining proteins("Other organisms" in Table 4), the PAM120 matrixwas used because it has been shown to be good forgeneral-purpose searching [S. F. Altschul, J. Mol.Biol. 219, 555 (1991)]. The BLASTX program [W.Gish and D. J. States, Nature Genet. 3, 266 (1993)]takes a nucleotide sequence query (EST or gene),translates it into all six conceptual ORFs, and thencompares these with protein sequences in the data-base; TBLASTN performs a similar function but in-stead searches a protein query sequence againstsix-frame translations of each entry in a nucleotidesequence database. Searches were performed withE - 1 e-6 and E2 = 1 e-5 as the primary and second-ary expectation parameters.

41. M. A. Pericak-Vance et al., Am. J. Hum. Genet. 48,1034 (1991); W. J. Strittmatter et al., Proc. Natl.Acad. Sci. U.S.A. 90, 1977 (1993); R. Fishel et al.,Cell 75, 1027 (1993); N. Papadopoulos et al., Sci-ence 263, 1625 (1994); R. Shiang et al., Cell 78, 335

(1994); L. M. Mulligan et al., Nature 363, 458 (1993);P. Edery et al., ibid. 367, 378 (1994).

42. The potential effect of a comprehensive gene map iswell illustrated by the fact that 82% of genes thathave been positionally cloned to date arerepresented by one or more ESTs in GenBank (http://www.ncbi.nlm.nih.gov/dbEST/dbEST_ genes!).

43. K. H. Fasman, A. J. Cuticchia, D. T. Kingsbury, Nu-cleic Acids Res. 22, 3462 (1994).

44. M. Bonaldo et al., Genome Res. 6, 791 (1996).45. Consensus sequences from at least three overlap-

ping ESTs were generated from 294 UniGene clus-ters. Of the 230 (78%) redesigned primer pairs, 188(82%) of these yielded successful PCR assays.

46. We thank M. 0. Anderson, A. J. Collymore, D. F.Courtney, R. Devine, D. Gray, L. T. Horton Jr., V.Kouyoumjian, J. Tam, W. Ye, and 1. S. Zemstevafrom the Whitehead Institute for technical assist-ance. We thank W. Miller, E. Myers, D. J. Lipman,and A. Schaffer for essential contributions towardthe development of UniGene. Supported by NIHawards HG00098 to E.S.L., HG00206 to R.M.M.,HG00835 to J.M.S., and HGO0151 to T.C.M., andby the Whitehead Institute for Biomedical Researchand the Wellcome Trust. T.J.H. is a recipient of aClinician Scientist Award from the Medical ResearchCouncil of Canada. D.C.P. is an assistant investiga-tor of the Howard Hughes Medical Institute. TheStanford Human Genome Center and the WhiteheadInstitute-MIT Genome Center are thankful for theoligonucleotides purchased with funds donated bySandoz Pharmaceuticals. Genbthon is supported bythe Association Francaise contre les Myopathies andthe Groupement d'Etudes sur le Genome. TheSanger Centre, Genbthon, and Oxford are gratefulfor support from the European Union EVRHEST pro-gramme. The Human Genome Organization and theWellcome Trust sponsored a series of meetings fromOctober 1994 to November 1995 without which thiscollaboration would not have been possible.

Life with 6000 Genes

A. Goffeau,* B. G. Barrell, H. Bussey, R. W. Davis, B. Dujon,H. Feldmann, F. Galibert, J. D. Hoheisel, C. Jacq, M. Johnston,

E. J. Louis, H. W. Mewes, Y. Murakami, P. Philippsen,H. Tettelin, S. G. Oliver

The genome of the yeast Saccharomyces cerevisiae has been completely sequencedthrough a worldwide collaboration. The sequence of 12,068 kilobases defines 5885potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA,40 genes for small nuclear RNA molecules, and 275 transfer RNA genes. In addition, thecomplete sequence provides information about the higher order organization of yeast's16 chromosomes and allows some insight into their evolutionary history. The genome

shows a considerable amount of apparent genetic redundancy, and one of the majorproblems to be tackled during the next stage of the yeast genome project is to elucidatethe biological functions of all of these genes.

The genome of the yeast Saccharomycescerevisiae has been completely sequencedthrough an international effort involvingsome 600 scientists in Europe, North Amer-ica, and Japan. It is the largest genome to becompletely sequenced so far (a record thatwe hope will soon be bettered) and is thefirst complete genome sequence of a cu-karyote. A number of public data libraries

nucleotide and protein sequence data fromeach of the 16 yeast chromosomes (1-16)have been established (Table 1).

The position of S. cerevisiae as a modeleukaryote owes much to its intrinsic advan-tages as an experimental system. It is aunicellular organism that (unlike manymore complex eukaryotes) can be grown ondefined media, which gives the experiment-

compiling the mapping information and er complete control over its chemical and

physical environment. S. cerevisiae has a lifecycle that is ideally suited to classical ge-netic analysis, and this has permitted con-struction of a detailed genetic map thatdefines the haploid set of 16 chromosomes.Moreover, very efficient techniques havebeen developed that permit any of the 6000genes to be replaced with a mutant allele, orcompletely deleted from the genome, withabsolute accuracy (17-19). The combina-tion of a large number of chromosomes anda small genome size meant that it was pos-sible to divide sequencing responsibilitiesconveniently among the different interna-tional groups involved in the project.

Old Questions and New Answers

The genome. At the beginning of the se-quencing project, perhaps 1000 genes en-coding either RNA or protein products hadbeen defined by genetic analysis (20). Thecomplete genome sequence defines some5885 open reading frames (ORFs) that arelikely to specify protein products in theyeast cell. This means that a protein-encod-ing gene is found for every 2 kb of the yeastgenome, with almost 70% of the total se-quence consisting of ORFs (21). The yeastgenome is much more comppact than thoseof its more complex relatives in the eukary-

SCIENCCE * VOL. 274 * 25 OCTOBER 1996546

otic world. By contrast, the genome of thenematode worm contains a potential pro-tein-encoding gene every 6 kb (22), and inthe human genomne, some 30 kb or more ofsequence must be examined in order touncover such a gene. Analysis of the yeastgenome reveals the existence of 6275 ORFsthat theoretically could encode proteinslonger than 99 amino acids. However, 390ORFs are unlikely to be translated intoproteins. ThuLs, only 5885 protein-encodinggenes are believed to exist. In addition, theyeast genome contains some 140 ribosomalRNA genes in a large tandem array onchromosome XII and 40 genes encodingsmall nuclear RNAs (snRNAs) scatteredthroughout the 16 chromosomes; 275 trans-fer RNA (tRNA) genes (belonging to 43families) are also widely distributed. Table2, which provides details of the distributionof genes and other sequence elementsamong yeast's 16 chromosomes, shows thatthe genome has been completely se-quenced, with the exception of a set ofidentical genes repeated in tandem.

The compact nature of the S. cerevisiaegenome is remarkable even when comparedwith the genomes of other yeasts and fungi.CuLrrent data from the systematic sequenceanalysis of the genome of the fission yeast

A. Goffeau and H. Tettelin, Universite Catholique de Lou-vain, Unite de Biochimie Physiologique, Place Croix duSud, 2/20, 1348 Louvain-la-Neuve, Belgium.B. G. Barrell, Sanger Centre, Hinxton Hall, Hinxton, Cam-bridge CB10 1 SA, UK.H. Bussey, Department of Biology, McGill University, 1205Docteur Penfield Avenue, Montreal, H3A 1 B1, Canada.R. W. Davis, Department of Biochemistry, Stanford Uni-versity, Beckman Center, Room B400, Stanford, CA94305-5307, USA.B. Dujon, Unite de G6netique Moleculaire des Levures(URAl 149 CNRS and UPR927 Universite Pierre et MarieCurie), Institut Pasteur, 25 Rue du Docteur Roux, 75724Paris-Cedex 15, France.H. Feldmann, Universitat Munchen, Institut fOr Physiolo-gische Chemie, Schillerstrasse, 44, 80336 Munchen,Germany.F. Galibert, Laboratoire de Biochimie Moleculaire, UPR41-Faculte de M6decine, CNRS, 2 Avenue du Profes-seur Leon Bernard, 35043 Rennes Cedex, France.J. D. Hoheisel, Molecular Genetic Genome Analysis,Deutsches Krebsforschungszentrum, Im NeuenheimerFeld, 506, 69120 Heidelberg, Germany.C. Jacq, Genetique Moleculaire, Ecole Normale Su-perieure, CNRS URA 1302, 46 Rue d'Ulm, 75230 ParisCedex 05, France.M. Johnston, Department of Genetics, Box 8232, Wash-ington University Medical School, 4566 Scott Avenue, St.Louis, MO 63110, USA.E. J. Louis, Yeast Genetics, Institute of Molecular Medi-cine, John Radcliffe Hospital, Oxford OX3 9DU, UK.H. W. Mewes, Max-Planck-lnstitut fOr Biochemie, Martins-ried Institute for Protein Sciences (MIPS), Am Klopferspitz18A, 82152 Martinsried, Germany.Y. Murakami, Tsukuba Life Science Center, Division ofHuman Genome Research, RIKEN, Koyaday TsukubaScience City 3-1-1, 305 Ibaraki, Japan.P. Philippsen, Institute for Applied Microbiology, Universi-tat Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland.S. G. Oliver, Department of Biochemistry and AppliedMolecular Biology, University of Manchester Institute ofScience and Technology (UMIST), Post Office Box 88,Manchester M60 1 GD, UK.

'To whom correspondence should be addressed.

Schizosaccharomyces pombe indicate that thedensity of protein-encoding genes is ap-proximately one per 2.3 kb (23). The dif-ference between these two yeast genomescan be ascribed to the paucity of introns inS. cerevisiae. In the fission yeast, approxi-mately 40% of genes contain introns (21),whereas only 4% of protein-encoding genesin S. cerevisiae are similarly interrupted (Ta-ble 2). The Saccharomyces genes that docontain introns [notably, those encodingribosomal proteins (24)] usually have onlyone small intron close to the start of thecoding sequence (often interrupting the ini-tiator codon) (25). It has even been sug-gested (26) that many yeast genes representcDNA copies that have been generated bythe action of reverse transcriptases specifiedby retrotransposons (Ty elements).

The chromosomes. A complete genomesequence provides more information thanthe sum of all the genes (or ORFs) that itcontains. In particular, it permits an in-vestigation of the higher order organiza-tion of the S. cerevisiae genome. An ex-ample is the long-range variation in basecomposition. Many yeast chromosomesconsist of alternating large domains ofGC-rich and GC-poor DNA (21, 27),generally correlating with the variation ingene density along these chromosomes. Inthe case of chromosome III, it has beendemonstrated that the periodicity in basecomposition is paralleled by a variation inrecombination frequency along the chro-mosome arms, with the GC-rich peakscoinciding with regions of high recombi-nation in the middle of each arm of thechromosome and AT-rich troughs coin-ciding with the recombination-poor cen-tromeric and telomeric sequences. Sim-chen and co-workers (28) have demon-

strated that the relative incidence of dou-ble-strand breaks, which are thought toinitiate genetic recombination in yeast(29), correlates directly with the GC-richregions of this chromosome.

The four smallest chromosomes (I, III,VI, and IX) exhibit average recombinationfrequencies some 1 .3 to to 1.8 times greaterthan the average for the genome as a whole.Kaback (30) has suggested that high levelsof recombination have been selected for onthese very small chromosomes to ensure atleast one crossover per meiosis, and so per-mit them to segregate correctly. It is knownthat artificial chromosomes (or chromo-some fragments) of approximately 150 kb insize are mitotically unstable (31, 32), whichraises the related questions of whether thereis a minimal size for yeast chromosomes andhow the smallest chromosomes haveachieved their current size (see Table 2).The organization of chromosome I is veryunusual: The 31 kb at each of its ends arevery gene-poor, and Bussey et al. (5) havesuggested that these terminal domains mayact as "fillers" to increase the size, andhence the stability, of this smallest yeastchromosome.

Genetic redundancy is the rule at theends of yeast chromosomes. For instance,the two terminal domains of chromosomeIII show considerable nucleotide sequencehomology both to one another and to theterminal domains of other chromosomes (Vand XI). The right terminal region of chro-mosome I is duLplicated at the left end ofchromosome I (5) and at the right end ofchromosome VIII (3). The sugar fermenta-tion genes MAL, SUC, and MEL all have anumber of telomere-associated copies, notall of which are expressed (33-35). Aninteresting feature of the distribution of the

Table 1. Finding yeast genome information on the Internet. A fuller description of these and otherresources may be found in (86).

Internet addresses

FTP sites for the complete S. cerevisiae genome sequenceftp.mips.embnet.org (directory/yeast)ftp.ebi.ac.uk (directory/pub/databases/yeast)genome-ftp.stanford.edu (directory/yeast/genome-seq)

Other S. cerevisiae data librariesMIPS, Martinsried, Germany (http://www.mips.biochem.mpg.de/yeast/)Sanger Center, Hinxton, UK (http://www.sanger.ac.uk/yeast/home.html)Saccharomyces Genome Database (SGD), Stanford University, USA (http://genome-www.stanford .edu/Saccharomyces/)

SWISS-PROT, University of Geneva, Switzerland (http://expasy.hcuge.ch/sprot/sp-docu.html)Yeast Protein Database (YPD), Proteome Inc., Beverly, MA, USA

(http://www.proteome.com/YPDhome.html)GeneQuiz, European Molecular Biology Laboratory, Heidelberg, Germany

(http://www.embl-heidelberg.de/-genequiz/)XREFdb, National Center for Biological Information, Baltimore, MD, USA

(http://www.ncbi.nlm.nih.gov/XREFdb/)(Editor's note: For readers who would like a user-friendly guide to the yeast databases, please see

the special feature at the Science Web site http://www.sciencemag.org/science/feature/data/genomebase.htm)

SCIENCE * VOL. 274 * 25 OCTOBER 1996 563

MEL genes is that they are only foundassociated with one telomere of a chromo-some and not both (36). This raises theintriguing possibility that yeast chromo-somes might have an intrinsic polarity be-yond our arbitrary labeling of left and rightarms.

The left telomere of chromosome III hassome special characteristics. Like all yeasttelomeres, it contains a repeated sequenceelement called X (37, 38). It also contains apseudo-X element at an internal site about4 kb from the true X, and Voytas and Boeke(39) have suggested that the two X se-quences represent the long terminal repeats(LTRs) of a new class of yeast transposoncalled Ty5. Transposons are often found onthe healed ends of broken chromosomes inDrosophila (40, 43). The yeast genome se-quence reveals that all 19 telomere-associ-ated highly conserved repeats called Y' el-ements contain an ORF whose predictedprotein product is reminiscent of the RNAhelicases (42, 43). No Y' helicase ORFs arefound on chromosomes I, III, and XI(though there are small parts of Y's on IIIand XI near the actual telomeres) and a fewY's from tandem arrays at chromosomes XIIR and IV R have not been sequenced. Thefunctions of these ORFs are unknown; how-ever, they may have formed parts of trans-posable elements in the past (41, 44). Be-cause the synthesis of new telomeric repeatsby telomerase [see (45)] is essentially a re-verse transcription process, it may be thatcurrent mechanisms of telomere biogenesisin many eukaryotes had their origins in the

activities of retrotransposons or retroviruses.Whatever the merits of such speculla-

tion, it is evident that many of the poly-morphisms observed between homologouschromosomes in different strains of S. cer-evisiae are due to transposition events orrecombination between transposons ortheir LTRs or both (46). Fortunately forgenome analysis, and perhaps for yeast it-self, these spontaneous transposition eventsdo not appear to occur randomly along thelength of individual chromosomes. Theyeast genome that was sequenced contains52 complete Ty elements as well as 264 soloLTRs or other remnants that are the foot-prints of previous transposition events. Themajority of the Ty2 elements (11 out of 13)are found in sites that show evidence ofprevious transposition activity ("old" sites);only about half (16 out of 33) of the Tylelements are found in "new" sites (the ma-jority flanking tRNA genes). Thus, yeasttransposons appear to insert preferentiallyinto specific chromosomal regions that maybe termed transposition hot spots (46-53).

The proteome. The term "proteome" hasbeen coined to describe the complete set ofproteins that a living cell is capable ofsynthesizing (54). The completion of theyeast genome sequence means that, for thefirst time, the complete proteome of a eu-karyotic cell is accessible. Computer analy-sis of the yeast proteome allows classifica-tion of about 50% of the proteins on thebasis of their amino acid seqLmence similaritywith other proteins of known function, withthe use of simple and conservative homol-

ogy criteria. However, such assignments of-ten provide only a general description ofthe biochemical function of the predictedprotein products (such as "protein kinase"or "transcription factor") but provide noindication as to their biological role. Thus,although computational approaches pro-vide valuable guides to experimentation,they do not obviate the need to carry outreal experiments to determine protein func-tion [see (55)].

An attempt to classify yeast proteins ac-cording to their function as conservativelypredicted by such computer analyses hasbeen carried out by MIPS (56). The yeastcell devotes 11% of its proteome to metab-olism; 3% to energy production and storage;3% to DNA replication, repair, and recom-bination; 7% to transcription; and 6% totranslation. A total of 430 proteins are in-volved in intracellular trafficking or proteintargeting, and 250 proteins have structuralroles. Nearly 200 transcription factors havebeen identified (57), as well as 250 primaryand secondary transporters (58). However,these statistics refer only to yeast proteinsfor which significant homologs were found.

Another approach that is expected to begreatly facilitated by the availability of allyeast protein sequences is two-dimensionalgel electrophoretic analysis, which permitsthe resolution of more than 2000 solubleprotein species (59). Unfortunately, manyof these membrane proteins are not re-solved, and the reproducibility of the two-dimensional electrophoretograms from one

laboratory to another is still poor. Identifi-

Table 2. Distribution of genes and other sequence elements. Questionable proteins are defined in (2). Hypothetical proteins are the difference between allproteins predicted as ORFs and the questionable proteins. Introns include both experimentally verified examples and those predicted by the EXPLORAprogram. UTR, untranslated but transcribed regions.

Chromosome numberElements

11 III IV V VI VIl Vil IX X Xl XII XIII XIV XV XVI Total

Sequenced length (kb) 230 813 315 1,532 577 270 1,091 563 440 745 667 1,078 924 784 1,091 948 12,068Nonsequenced identical

repeatsName of unit ENA2 and Y' tel CUPi rDNA and Y'Length of unit (kb) 4 and 7 <1 2 9 and 7Number of units 2 and 2 1 13 ±140 and 2Length of repeats (kb) 8 and 14 <1 26 1,260 and 14 1,321

Total length (kb) 230 813 315 1,554 577 271 1,091 589 440 745 667 2,352 924 784 1,091 948 13,389ORFs (n) 110 422 172 812 291 135 572 288 231 387 334 547 487 421 569 497 6,275Questionable proteins (n) 3 30 12 65 13 5 57 12 11 29 20 41 30 23 3 36 390Hypothetical proteins (n) 107 392 160 747 278 130 515 276 220 358 314 506 457 398 566 461 5,885Introns in ORFs (n) 4 18 4 30 13 5 15 15 8 13 11 17 19 15 15 18 220Introns in UTR (n) 0 2 0 1 2 0 5 0 0 0 0 3 0 2 0 0 15Intact Ty1 (n) 1 2 0 6 1 0 4 1 0 2 0 4 4 2 2 4 33Intact Ty2 (n) 0 1 1 3 1 1 1 0 0 0 0 2 0 1 2 0 13Intact Ty3 (n) 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 2Intact Ty4 (n) 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 1 3Intact Ty5 (n) 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1tRNA genes (n) 2 13 10 27 20 10 36 1 1 10 24 16 22 21 16 20 17 275snRNAgenes (n) 1 1 2 1 2 0 3 1 1 4 1 3 8 3 7 2 40

SCIENCE * VOL. 274 * 25 OCTOBER 1996564

cation of the proteins corresponding to giv-en spots by NH2-terminal protein sequenc-ing has been slow and only about 200 as-signmnents have been made so far. Theavailability of the predicted amino acid se-quence for all yeast proteins should permit acomprehensive analysis of the proteomewith the use of rapid and accurate massspectrometric techniques, so that it shouldsoon become routine to identify all yeastproteins produced under a given set of phys-iological conditions, or those that are qual-itatively or quantitatively modified as a re-sult of the deletion of a specific gene. Anew kind of map will then emerge that ofthe direct and indirect interactions amongall of the members of the yeast proteome. Acomplete understanding of life at the mo-lecular level cannot be achieved withoutsuch knowledge.

Another consequence of defining theyeast proteome is the uncovering of geneproducts whose existence was hitherto indoubt. For instance, early views that yeastchromosomes were atypical led to the asscr-tion that yeast does not have an H 1 his-tone. In reality, yeast chromosomes containthe full repertoire of eukaryotic histones,including H1, whose gene was found onchromosome XVI (60). Another example isthe discovery of a yeast gamma tubulin geneon chromosome XII (14); this gene hadpreviously eluded yeast geneticists despiteintensive efforts by several research groups.

It has been an article of faith for sometime that a full understanding of the yeastproteome is a prerequisite for understand-ing the more complex human proteome;this has become reality with the availabilityof the complete yeast genome sequence.Nearly half of the proteins known to bedefective in human heritable diseases (61)show some amino acid sequence similarityto yeast proteins (62). Although it is evi-dent that the human genome will specifymany proteins that are not found in theyeast proteome, it is reasonable to suggestthat the majority of the yeast proteins havehuman homologs. If so, these human pro-teins could be classified on the basis of theirstructural or functional equivalence tomembers of the yeast proteome.

Genome evolution. The existence of setsof two or more genes encoding proteinswith identical or very similar sequences (re-dundancy) provides the raw material for theevolution of novel functions (63). Under-standing the true nature of redLindancy isone of the major challenges in the quest toelucidate the biological role of every genein the S. cerevisiae genome (55). An anal-ysis of the complete genome sequence sug-gests that it may have undergone duplica-tion events at some point in its evolution-ary history. The evidence for such duplica-

tions is most readily seen in the pericentricregions and in the central portions of anumber of chromosome arms. Although re-dLundancy does occur close to the ends ofchromosomes (indeed, the subtelomeric re-gions are a major repository of redundantsequences), exchanges between such re-gions are probably too frequent and toorecent to help us discern the overall historyof the yeast genome.

In S. cerevisiae, simple direct repeat clus-ters (64) take several forms, the most typi-cal being dispersed families with related butnonidentical genes scattered singly overmany chromosomes. The largest such familycomprises the 23 PAU genes, which specifythe so-called seripauperines (65), a set ofalmost identical serine-poor proteins of un-known function whose ORFs show a veryhigh codon bias and an NH2-terminal sig-nal sequence (66). The PAU genes, like thesugar fermentation genes discussed above,reside in the subtelomeric regions. Otherdispersed gene families show no obviouschromosomal positioning (such as the 15members of the PMT and KRE2 families,which encode enzymes involved in themannosylation of cell wall proteins). Clus-tered gene families are less common, but alarge family of this type occurs on chromo-some I, where six related but nonidenticalORFs (YAR023 through YAR033) specifymembrane proteins of unknown function(5). There are 16 members of this family onsix chromosomes. Some are clustered(YHLO42 through YHL046 on chromosomeVIII), others are scattered singly (YCRO07on chromosome III), and still others arelocated in subtelomeric regions (YBR302on chromosome II, YKL219 on chromo-some XI, and YHLO48 on chromosotneVIII). Functional analysis of such a complexfamily poses a challenge but one that re-mains within the capability of yeast genedisruption technology.An analysis of the numerous cluster ho-

mology regions (CHRs) revealed by theyeast genome sequence has led to a betterunderstanding of genome evolution. CHRsare large regions in which homologousgenes are arranged in the same order, withthe same relative transcriptional orienta-tions, on two or more chromosomes. Earlyreports of CHRs involved a 7.5-kb regionon chromosomes V and X (67) and a 15-kbregion from chromosomes XIV and III (68).The latter contains four ORFs that havesimilarly ordered homologs in the centro-meric regions of both chromosomes. Onehomologous pair consists of two genes, eachof which encodes citrate synthase. Howev-er, one (CIT2 on chromosome III) encodesthe peroxisomal enzyme, whereas the other(CITJ on chromosome XIV) specifies themnitochondrial enzyme. This is probably a

good example of evolution through geneduplication, but the situation is even morecomplicated as a third citrate synthase gene(CIT3) has been discovered on chromo-some XVI (68, 69). Chromosomes IV and IIshare the longest CHR, comprising a pair ofpericentric regions of 120 kb and 170 kb,respectively, that share 18 pairs of homolo-gous genes (13 ORFs and five tRNA genes).The genome has continued to evolve sincethis ancient duplication occurred: The in-sertion or deletion of genes has occurred, Tyelements and introns have been lost andgained between the two sets of sequences,and pseudogenes have been generated. Inall, at least 10 CHRs (shared with chromo-somes 11, V, VIII, XII, and XIII) can berecognized on chromosome IV. None ofthem is found in the central region of thechromosome, which, on the other hand,contains most (7 out of 9) of chromosomeIV's complement of Ty elements (Table 2);these may be the cause of the genetic plas-ticity of this region.

The example of the citrate synthasegenes suggests that much of the redundancyin the yeast genome m-ay be more apparentthan real. In this case, it was our knowledgeof the rules of protein targeting in yeast thatallowed us to discern that these genes playdifferent physiological roles. It is likely thata large number of apparently redundantyeast genes are required to deal with phys-iological challenges that are not encoun-tered in the laboratory environmnent btLtthat yeast commonly encounters in its nat-ural habitat of the rotting fig (70) or grape.Our ability to imagine these conditions andrecreate them in the laboratory is severelycompromised by our lack of knowledge ofthe ecology or natural history of S. cerevi-siae. Indeed, only very recently has it beenclearly established that S. cerevisiae is foundon the surface of the grapes used to makewine (71).

What's Next?

For yeast research. New graduate studentsare already wondering how we all managedin the "dark ages" before the seqLuence wascompleted. We MLust now tackle a MLuchlarger challenge, that of elucidating thefunction of all of the novel genes revealedby that sequence. As with the sequencingproject itself, functional analysis will re-quire a worldwide effort. In Europe, a newresearch network called EUROFAN [forEuropean Functional Analysis Network(72)] has been established to undertake thesystematic analysis of the function of novelyeast genes. Parallel activities are underwayin Germany, Canada, and Japan. In theUnited States, the National Institutes ofHealth has recently sent out a request for

SCIENCE * VOL. 274 * 25 OCTOBER 1996 565

applications for "Large-Scale FunctionalAnalysis of the Yeast Genome." Clearly, theyeast research community is mobilizing forthe next phase of the campaign to under-stand how a simple eukaryotic cell works.

In all of this, a common approach isemerging for the deletion of individualgenes by a polymerase chain reaction(PCR)-mediated gene replacement tech-nique (18, 19). This approach, which relieson the great efficiency and accuracy of mi-totic recombination in S. cerevisiae, resultsin the precise deletion of the entire geneand is economical enough to enable pro-ducton of the complete set of 6000 single-deletion mutants. This would be a majorresource for the scientific community, notonly for the functional analysis of the yeastgenome itself but also in permitting "func-tional mapping" of the genomes of higherorganisms onto that of yeast. Because of theredundancy problem, and to enable thestudy of gene interactions, it will also benecessary to construct multiply deletedstrains; easy methods to achieve this arealready in hand (73, 74). All these ap-proaches should make S. cerevisiae the eu-karyote of choice for the study of functionscommon to all eukaryotic cells, by reversingthe traditional path of genetic research toone in which the study of the gene (or DNAsequence) leads to an understanding of bio-logical function, rather than a change infunction leading to the identification of agene.

For other genomes. Before the release, on24 April 1996, of the complete yeast ge-nome sequence, two complete bacterial ge-nomes had been made public: the 1.8-Mbsequence of Haemophilus influenzae (75) andthe 0.6-Mb sequence of Mycoplasma geni-talium (76). Another prokaryotic genome,that of Methanococcus jannaschii (1.7 Mb),was subsequently released (77). The se-quences of several other bacterial genomes[Helicobacter pylori, Methanobacterium ther-moautotrophicum (1.7 Mb), Mycoplasmapneumoniae (0.8 Mb), and Synechocystis sp.(3.6 Mb)] have apparently been completedbut were not publicly available at the timethis paper went to press. The sequence of atleast two dozen other prokaryotic genomes(mostly extremophiles with genome sizesbelow 2 Mb) is underway. The shotgunseqluencing of small bacterial genomes canbe completed in less than 6 months at a costof <$0.50 per base pair (75, 77). It is noteasy to determine whether such estimatesrepresent full or marginal costs; neverthe-less, we can expect many more small ge-nomes to be completed soon. It would beunfortunate if some of these sequences,many of which will be determined throughthe use of private funds, are not made publicin a timely fashion.

For genome sizes between 2 and 6 Mb,sequencing becomes much more complexand expensive. The production of a librarycomprising a contiguous set of DNA clones(rather than the generation and assembly ofthe sequence itself) becomes the limitingfactor. In the absence of such a library,long-range PCR amplification or directPCR sequencing, or both, must be em-ployed. The sequencing of genomes largerthan 6 Mb typically requires the time-con-suming construction of clone libraries incosmids or other high-capacity vectors andthe usually tedious filling-in of the unavoid-able, sometimes numerous, and occasionallyintractable, gaps in clone coverage. Plansfor the determination of medium-sized ge-nome sequences (10 to 100 Mb) almostalways underestimate the costs of these es-sential steps. The existence of two comple-mentary, well-organized, and almost gaplesslibraries of yeast DNA in cosmid vectors(78, 79) was a major factor in the unexpect-ed speed at which the full genome sequencewas obtained. After the pilot exercise ofchromosome III (1), it took only 4 years tocomplete the remaining 11.8 Mb of theyeast genome. During 1995 alone, morethan 6 Mb of final contiguous yeast genom-ic sequence were obtained. We are confi-dent that it will soon become routine tocomplete a 10-Mb contig in a year for <$5million. Nearly complete cosmid libraries ofthe 15-Mb genome of the fission yeast S.pombe are available (80, 81), allowing itssequencing to proceed rapidly. If financialsupport is sustained, S. pombe should be oneof the next two eukaryotes to have theirgenome sequences completed [along withthe nematode C. elegans (22), probably in1998].Now that the complete sequence of a

laboratory strain of S. cerevisiae has beenobtained, the complete genome sequencesof other yeasts of industrial or medical im-portance are within our reach. Such knowl-edge should considerably accelerate the de-velopment of more productive strains andthe search for badly needed antifungaldrugs. Unfortunately, the sequence of theimportant human pathogen Candida albi-cans is proceeding slowly, with limited in-dustrial support (82, 83). Complete genomesequencing may be unnecessary when ayeast or fungal genome displays consider-able synteny (conservation of gene order)with that of S. cerevisiae. For instance, re-cent studies on Ashbya gossypii (a filamen-tous fungus that is a pathogen of cottonplants) have revealed that most of its ORFsshow homology to those of S. cerevisiae andthat at least a quarter of the clones in an A.gossypii genomic bank contain pairs orgroups of genes in the same order or relativeorientation as their S. cerevisiae counter-

parts (84). This gives considerable hope forthe rapid analysis of the genomes of a largenumber of medically and economically im-portant fungi through the use of the S.cerevisiae genome sequence as a paradigm.However, this optimism is tempered by thelack of apparent synteny between the S.cerevisiae and S. pombe genomes. This isperhaps not surprising, as the two speciesprobably diverged from a common ancestorsome 1000 million years ago (85).

The systematic sequencing of largermodel genomes, most notably those of thefruit fly Drosophila melanogaster and the di-cotyledonous plant Arabidopsis thaliana, hasnow begun. It is doubtful whether either ofthem will be completed in this century,given that <3% of these 100- to 130-Mbgenomes has been systematically sequencedso far. While we await the completion ofthe human genome sequence sometimearound the year 2005, there is danger ofdispersing sequencing power among toomany model genomes. Instead, it may bedesirable to direct sequencing capacity to-ward eukaryotic parasites (such as Plasmodi-um falciparum, Trypanosoma cruzei, Schisto-soma mansoni, and Leishmania donovani)that plague millions of people in developingcountries. These genomes are only of inter-mediate size (30 to 300 Mb) and thus areachievable objects for sequence analysis,provided that funding is increased from itspresent modest levels. On a world scale, thecost-benefit equation for such projects isoverwhelmingly positive.

Pride and Productivity

Two contrasting strategies for gathering ge-nome data have emerged, both of whichhave been applied to sequencing the yeastgenome: the "factory" and the "network"approaches. In the former, sequencing wasautomated as far as possible and was carriedout in large sequencing centers by highlyspecialized scientists and technicians whomay never have seen a yeast outside of abottle of doubly fermented beer. Their dailynotebook, put on the World Wide Web,was fully accessible to the scientific com-munity and was progressively corrected andcompleted when new information becameavailable. In the network approach, by con-trast, yeast genome sequencing was per-formed in small laboratories by scientistsand students deeply committed to the studyof particular aspects of yeast moleculalr bi-ology. These scientists had a special interestin the interpretation of the data and madepublic only verified and (they hoped) finaldata, using the same standards as for theirnormal publications.

In practice, all intermediate forms ofapproach between these two cultural ex-

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tremes have been employed in the yeastgenome sequencing project. The networksystem (to the surprise of some) workedvery well: 55% of the total genome se-quence was determined by a European Net-work in which a total of 92 laboratorieswere involved over the course of theproject. The other 45% was obtained byfive medium- to large-sized sequencing cen-ters. Over the 6 years of its life, the Euro-pean Network's performance improvedsteadily. Its 300-kb fragment of the "inter-national" chromosome XVI was sequencedand made publicly available as rapidly aswere the fragments from the three otherpartners. The sequence quality produced bythe two approaches was similar: A largefragment of chromosome XII (170 kb) wasdeliberately sequenced by both a large cen-ter and the European Network; both groupshad an extremely low error frequency (oneto two sequencing errors per 100 kb). It isestimated that an average of three errors per10 kb remain in the published version ofthe yeast genome sequence.

There are at least three reasons for thesuccess of the network. The first is the useof modern informatics technology and theInternet to coordinate the acquisition andanalysis of data, as well as to support thegeneral management of the network. Thesecond is that several small laboratories be-came extremely efficient; the most produc-tive reached 200 kb of finished sequenceper year using only two or three people andalmost no automation. The third (and mostimportant) reason is that an enthusiasticand competitive teatm spirit was built upamong the small sequencers as, month bymonth, they watched the data accumulateexponentially toward completion of the ge-nome. The general feeling amonong the net-work's participants was that their member-ship conferred considerable benefits onthemselves and on the scientific comtnuni-ty as whole.

Whether they worked in large centers orsmall laboratories, most of the 600 or soscientists involved in sequencing the yeastgenome share the feeling that the world-wide ties created by this venture are ofinestimable value to the future of yeastresearch. In Europe especially, a corporatespirit has been engendered that will permitthe sharing of data and ideas that will berequired to meet the challenge of decipher-ing the functions and interactions of thenovel genes. Nevertheless, it is doubtfulthat in the future genome sequencing willcontinue to involve many small laborato-

ries. Increasingly, large-scale sequencingwill become the province of the sequencingcenters, with the small laboratories beingenlisted to sort out problem regions wheretheir specialist knowledge of the organisminvolved may be of assistance. Enthusiasm,determination, and cooperation (forces thatare indispensable under pioneering circum-stances) drove this enterprise; we expectthese forces will continue to propel usthrough the next phase of the project.

REFERENCES AND NOTES

1 S. G. Oliver et at., Nature 357, 38 (1992).2. B. Dujon et al., ibid. 369, 371 (1994).3. M. Johnston et at., Science 265, 2077 (1994).4. H. Feldmann et at., EMBO J. 13, 5795 (1994).5. H. Bussey et al., Proc. Natl. Acad. Sci. U.S.A. 92,

3809 (1995).6. Y. Murakami et at., Nature Genet. 10, 261 (1995).7. F. Galibert et at., EMBO J. 15, 2031 (1996).8. B. Barrell et at., in preparation.9. B. Barrell et at., in preparation.

10. H. Bussey et a., in preparation.11. F. Dietrich et at., in preparation.12. B. Dujon et a., in preparation.13. C. Jacq et at., in preparation.14. M. Johnston et at., in preparation.15. P. Philippsen et at., in preparation.16. H. Tettelin et at., in preparation.17. R. J. Rothstein, Methods Enzymol. 101, 202 (1983).18. A. Baudin, 0. Ozier-Kalogeropoulos, A. Denouel, F.

Lacroute, C. Cullin, Nucleic Acids Res. 21, 3329(1993).

19. A. Wach, A. Brachat, R. Pohlmann, P. Philippsen,Yeast 10, 1793 (1994).

20. R. K. Mortimer, C. R. Contopoulou, J. S. King, ibid.8, 817 (1992).

21. B. Dujon, Trends Genet. 12, 263 (1996).22. J. Hodgkin, R. H. A. Plasterk, R. H. Waterston, Sci-

ence 270, 410 (1994).23. S. Bowman and B. Barrell, http://www.sanger.

ac.uk/yeast/pombe.html24. R. J. Planta, P. M. Goncalves, W. H. Mager, Bio-

chem. Cell Biol. 73, 825 (1996).25. B. C. Rymond and M. Rosbash, in The Molecular

Biology of the Yeast Saccharomyces: Gene Expres-sion, E. W. Jones, J. R. Pringle, J. R. Broach, Eds.(Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY, 1992), pp.143-192.

26. G. R. Fink, Cell 49, 5 (1987).27. P. M. Sharp and A. T. Lloyd, Nucleic Acids Res. 21,

179 (1993).28. D. Zenvirth et al., EMBO J. 11, 3441 (1992).29. B. De Massy, V. Rocco, A. Nicolas, ibid. 14, 4589

(1995).30. D. B. Kaback, H. Y. Steensma, P. de Jonge, Proc.

Natl. Acad. Sci. U.S.A. 86, 3694 (1989).31. P. Hieter, C. Mann, M. Snyder, R. W. Davis, Cell 40,

381 (1985).32. A. W. Murray, N. P. Schultes, J. W Szostak, ibid. 45,

529 (1986).33. H. Y. Steensma, J. C. Crowley, D. B. Kaback, Mol.

Cell. Biol. 7, 2894 (1987).34. M. J Charron, E. Read, S. R. Haut, C. A. Michels,

Genetics 122, 307 (1989).35. M. Carlson, J. L. Celenza, F. J. Eng, Mol. Cell. Biol. 5,

410 (1985).36. G. I. Naumov, E. S. Naumova, E. J. Louis, Yeast 11,

481 (1995).37. C. Chan and B.-K. Tye, Proc. Natl. Acad. Sci. U.S.A.

77, 6329 (1980).38. A. J. Link and M. V. Olson, Genetics 127, 681 (1991).

Al~~~~~~~~~~~~I

39. D. F. Voytas and J. D. Boeke, Nature 358, 717 (1992).40. 0. Danilevskaya, A. Lofsky, M.-L Pardue, Mol. Cell.

Biol. 35S, 83 (1992).41. H. Biessmann et al., ibid. 12, 3910 (1992).42. F. Foury, personal communication.43. E. J. Louis, Yeast 11, 1553 (1995).44. E. J. Louis and J. E. Haber, Genetics 131, 559

(1992).45. C. W Grieder, in The Eukaryotic Genome: Organisa-

tion and Regulation, P. M. A. Broda, S. G. Oliver, P.F. G. Sims, Eds. (Cambridge Univ. Press, Cam-bridge, 1993), pp. 31-42.

46. B. L. Wicksteed et al., Yeast 10, 39 (1992).47. A. Eigel and H. Feldmann, EMBO J. 1, 1245 (1982).48. J. Gafner, E. M. De Robertis, P. Philippsen, ibid. 2,

583 (1983).49. J. R. Warmington et al., Nucleic Acids Res. 14, 3475

(1986).50. J. R. Warmington, R. P. Green, C. S. Newlon, S. G.

Oliver, ibid. 15, 8963 (1987).51. J. Hauber, R. Stucka, R. Krieg, H. Feldmann, ibid.

16, 10623 (1988).52. H. Lochmuller, R. Stucka, H. Feldmann, Curr. Genet.

16, 247 (1989).53. H. Ji et al., Cell 73, 1007 (1993).54. M. R. Wilkins et al., BiolTechnology 14, 61 (1996).55. S. G. Oliver, Nature 379, 597 (1996).56. H. W. Mewes, personal communication.57. V. V. Svetlov and T. G. Cooper, Yeast 11, 1439

(1995).58. B. Nelissen, P. Mordant, J. L. Jonniaux, R. De

Wachter, A. Goffeau, FEBS Lett. 377, 32 (1995).59. S. Sagliocco et al., Yeast, in press.60. S. C. Ushinsky et al., ibid., in press.61. http://www3.ncbinim.nih.gov/OMIM/62. D. E. Bassett, M. S. Boguski, P. Hieter, Nature 379,

589 (1996); http://www.ncbi.nlm.gov/XREFdb/63. S. Ohno, Evolution Through Gene Duplication (Allen

and Unwin, London, 1970).64. M. V. Olson, in The Molecular Biology of the Yeast

Saccharomyces: Genome Dynamics, Protein Syn-thesis, and Energetics, J. R. Broach, J. R. Pringle, E.W. Jones, Eds. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY, 1991), pp.1-39.

65. M. Viswanathan et al., Gene 148, 149 (1994).66. B. Purnelle and A. Goffeau, Yeast, in press.67. L. Melnick and F. Sherman, J. Mol. Biol. 233, 372

(1993).68. D. Lalo, S. Stettler, S. Mariotte, P. P. Slonimski, P.

Thuriaux, C. R. Acad. Sci. Paris 316, 367 (1993).69. A.-M. Becam, Y. Jia, P. P. Slonimski, C. J. Herbert,

Yeast 11, S300 (1995).70. R. K. Mortimer and J. R. Johnston, Genetics 113, 35

(1989).71. R. K. Mortimer, T. Torok, P. Romano, G. Suzzi, M.

Polsinelli, Yeast 11, S574 (1995).72. S. G. Oliver, Trends Genet. 12, 241 (1996).73. F. Langle-Rouault and E. Jacobs, NucleicAcids Res.

23, 3079 (1995).74. U. Guldener, S. Heck, T. Fiedler, J. Beinhauer, J. H.

Hegemann, ibid. 24, 2519 (1996).75. R. D. Fleischmann et al., Science 269, 496 (1995).76. C. M. Frazer et al., ibid. 270, 397 (1995).77. C. Bult et al., ibid. 273, 1058 (1996).78. L. Riles et al., Genetics 134, 81 (1993).79. A. Thierry, L. Gaillon, F. Galibert, B. Dujon Yeast 11,

121 (1995).80. J. D. Hoheisel et al., Cell 73, 109 (1993).81. T. Mizukami et al., ibid., p. 121.82. W. S. Chu, B. B. Magee, P. T. Magee, J. Bacteriol.

175, 6637 (1993).83. http://alces.med.umn.edu/Candida.html84. R. Altman-Johl and P. Philippsen, Molec. Gen. Gen-

et. 250, 69 (1996).85. P. Russell and P. Nurse, Cell 45, 781 (1986).86. S. Walsh and B. Barrell, Trends Genet. 12, 276

(1996).

SCIENCE * VOL. 274 * 25 OCTOBER 1996 567