Abstract
Functional genomics represents a systematic approach to elucidating the function of the novel genes revealed by complete genome sequences. Such an approach should adopt a hierarchical strategy since this will both limit the number of experiments to be performed and permit a closer and closer approximation to the function of any individual gene to be achieved. Moreover, hierarchical analyses have, in their early stages, tremendous integrative power and functional genomics aims at a comprehensive and integrative view of the workings of living cells. The first draft of the human genome sequence has just been produced, and the complete genome sequences of a number of eukaryotic human pathogens (including the parasitic protozoa Plasmodium, Leishmania, and Trypanosoma) will soon be available. However, the most rapid progress in the elucidation of gene function will initially be made using model organisms. Yeast is an excellent eukaryotic model and at least 40% of single-gene determinants of human heritable diseases find homologues in yeast. We have adopted a systematic approach to the functional analysis of the Saccharomyces cerevisiae genome. A number of the approaches for the functional analysis of novel yeast genes are discussed. The different approaches are grouped into four domains: genome, transcriptome, proteome, and metabolome. The utility of genetic, biochemical, and physico-chemical methods for the analysis of these domains is discussed, and the importance of framing precise biological questions, when using these comprehensive analytical methods, is emphasized. Finally, the prospects for elucidating the function of protozoan genes by using the methods pioneered with yeast, and even exploiting Saccharomyces itself, as a surrogate, are explored.
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- Baganz F., Hayes A., Farquhar R., Butler P. R., Gardner D. C., Oliver S. G. Quantitative analysis of yeast gene function using competition experiments in continuous culture. Yeast. 1998 Nov;14(15):1417–1427. doi: 10.1002/(SICI)1097-0061(199811)14:15<1417::AID-YEA334>3.0.CO;2-N. [DOI] [PubMed] [Google Scholar]
- Baganz F., Hayes A., Marren D., Gardner D. C., Oliver S. G. Suitability of replacement markers for functional analysis studies in Saccharomyces cerevisiae. Yeast. 1997 Dec;13(16):1563–1573. doi: 10.1002/(SICI)1097-0061(199712)13:16<1563::AID-YEA240>3.0.CO;2-6. [DOI] [PubMed] [Google Scholar]
- Baudin A., Ozier-Kalogeropoulos O., Denouel A., Lacroute F., Cullin C. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 1993 Jul 11;21(14):3329–3330. doi: 10.1093/nar/21.14.3329. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bellí G., Garí E., Piedrafita L., Aldea M., Herrero E. An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast. Nucleic Acids Res. 1998 Feb 15;26(4):942–947. doi: 10.1093/nar/26.4.942. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brancia F. L., Butt A., Beynon R. J., Hubbard S. J., Gaskell S. J., Oliver S. G. A combination of chemical derivatisation and improved bioinformatic tools optimises protein identification for proteomics. Electrophoresis. 2001 Feb;22(3):552–559. doi: 10.1002/1522-2683(200102)22:3<552::AID-ELPS552>3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
- Brancia F. L., Oliver S. G., Gaskell S. J. Improved matrix-assisted laser desorption/ionization mass spectrometric analysis of tryptic hydrolysates of proteins following guanidination of lysine-containing peptides. Rapid Commun Mass Spectrom. 2000;14(21):2070–2073. doi: 10.1002/1097-0231(20001115)14:21<2070::AID-RCM133>3.0.CO;2-G. [DOI] [PubMed] [Google Scholar]
- Brazma A., Robinson A., Cameron G., Ashburner M. One-stop shop for microarray data. Nature. 2000 Feb 17;403(6771):699–700. doi: 10.1038/35001676. [DOI] [PubMed] [Google Scholar]
- Brown A. J., Planta R. J., Restuhadi F., Bailey D. A., Butler P. R., Cadahia J. L., Cerdan M. E., De Jonge M., Gardner D. C., Gent M. E. Transcript analysis of 1003 novel yeast genes using high-throughput northern hybridizations. EMBO J. 2001 Jun 15;20(12):3177–3186. doi: 10.1093/emboj/20.12.3177. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cagney G., Uetz P., Fields S. High-throughput screening for protein-protein interactions using two-hybrid assay. Methods Enzymol. 2000;328:3–14. doi: 10.1016/s0076-6879(00)28386-9. [DOI] [PubMed] [Google Scholar]
- Claverie J. M. Gene number. What if there are only 30,000 human genes? Science. 2001 Feb 16;291(5507):1255–1257. doi: 10.1126/science.1058969. [DOI] [PubMed] [Google Scholar]
- Delneri D., Gardner D. C., Oliver S. G. Analysis of the seven-member AAD gene set demonstrates that genetic redundancy in yeast may be more apparent than real. Genetics. 1999 Dec;153(4):1591–1600. doi: 10.1093/genetics/153.4.1591. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Delneri D., Tomlin G. C., Wixon J. L., Hutter A., Sefton M., Louis E. J., Oliver S. G. Exploring redundancy in the yeast genome: an improved strategy for use of the cre-loxP system. Gene. 2000 Jul 11;252(1-2):127–135. doi: 10.1016/s0378-1119(00)00217-1. [DOI] [PubMed] [Google Scholar]
- Fairhead C., Llorente B., Denis F., Soler M., Dujon B. New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using 'split-marker' recombination. Yeast. 1996 Nov;12(14):1439–1457. doi: 10.1002/(SICI)1097-0061(199611)12:14%3C1439::AID-YEA37%3E3.0.CO;2-O. [DOI] [PubMed] [Google Scholar]
- Foury F., Cazzalini O. Deletion of the yeast homologue of the human gene associated with Friedreich's ataxia elicits iron accumulation in mitochondria. FEBS Lett. 1997 Jul 14;411(2-3):373–377. doi: 10.1016/s0014-5793(97)00734-5. [DOI] [PubMed] [Google Scholar]
- Foury F. Human genetic diseases: a cross-talk between man and yeast. Gene. 1997 Aug 11;195(1):1–10. doi: 10.1016/s0378-1119(97)00140-6. [DOI] [PubMed] [Google Scholar]
- Futcher B., Latter G. I., Monardo P., McLaughlin C. S., Garrels J. I. A sampling of the yeast proteome. Mol Cell Biol. 1999 Nov;19(11):7357–7368. doi: 10.1128/mcb.19.11.7357. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Garí E., Piedrafita L., Aldea M., Herrero E. A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae. Yeast. 1997 Jul;13(9):837–848. doi: 10.1002/(SICI)1097-0061(199707)13:9<837::AID-YEA145>3.0.CO;2-T. [DOI] [PubMed] [Google Scholar]
- Giaever G., Shoemaker D. D., Jones T. W., Liang H., Winzeler E. A., Astromoff A., Davis R. W. Genomic profiling of drug sensitivities via induced haploinsufficiency. Nat Genet. 1999 Mar;21(3):278–283. doi: 10.1038/6791. [DOI] [PubMed] [Google Scholar]
- Glerum D. M., Tzagoloff A. Isolation of a human cDNA for heme A:farnesyltransferase by functional complementation of a yeast cox10 mutant. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8452–8456. doi: 10.1073/pnas.91.18.8452. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goffeau A., Barrell B. G., Bussey H., Davis R. W., Dujon B., Feldmann H., Galibert F., Hoheisel J. D., Jacq C., Johnston M. Life with 6000 genes. Science. 1996 Oct 25;274(5287):546, 563-7. doi: 10.1126/science.274.5287.546. [DOI] [PubMed] [Google Scholar]
- Gossen M., Bonin A. L., Bujard H. Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements. Trends Biochem Sci. 1993 Dec;18(12):471–475. doi: 10.1016/0968-0004(93)90009-c. [DOI] [PubMed] [Google Scholar]
- Gossen M., Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547–5551. doi: 10.1073/pnas.89.12.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gygi S. P., Rist B., Gerber S. A., Turecek F., Gelb M. H., Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol. 1999 Oct;17(10):994–999. doi: 10.1038/13690. [DOI] [PubMed] [Google Scholar]
- Gygi S. P., Rochon Y., Franza B. R., Aebersold R. Correlation between protein and mRNA abundance in yeast. Mol Cell Biol. 1999 Mar;19(3):1720–1730. doi: 10.1128/mcb.19.3.1720. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Güldener U., Heck S., Fielder T., Beinhauer J., Hegemann J. H. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 1996 Jul 1;24(13):2519–2524. doi: 10.1093/nar/24.13.2519. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kacser H., Burns J. A. The control of flux. Symp Soc Exp Biol. 1973;27:65–104. [PubMed] [Google Scholar]
- Kacser H., Burns J. A. The molecular basis of dominance. Genetics. 1981 Mar-Apr;97(3-4):639–666. doi: 10.1093/genetics/97.3-4.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koutnikova H., Campuzano V., Foury F., Dollé P., Cazzalini O., Koenig M. Studies of human, mouse and yeast homologues indicate a mitochondrial function for frataxin. Nat Genet. 1997 Aug;16(4):345–351. doi: 10.1038/ng0897-345. [DOI] [PubMed] [Google Scholar]
- Link A. J., Eng J., Schieltz D. M., Carmack E., Mize G. J., Morris D. R., Garvik B. M., Yates J. R., 3rd Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol. 1999 Jul;17(7):676–682. doi: 10.1038/10890. [DOI] [PubMed] [Google Scholar]
- Mountain H. A., Byström A. S., Larsen J. T., Korch C. Four major transcriptional responses in the methionine/threonine biosynthetic pathway of Saccharomyces cerevisiae. Yeast. 1991 Nov;7(8):781–803. doi: 10.1002/yea.320070804. [DOI] [PubMed] [Google Scholar]
- Oliver S. G. From DNA sequence to biological function: the new "Voyage of the Beagle'. Biochem Soc Trans. 1996 Feb;24(1):291–292. doi: 10.1042/bst0240291. [DOI] [PubMed] [Google Scholar]
- Oliver S. G., Winson M. K., Kell D. B., Baganz F. Systematic functional analysis of the yeast genome. Trends Biotechnol. 1998 Sep;16(9):373–378. doi: 10.1016/s0167-7799(98)01214-1. [DOI] [PubMed] [Google Scholar]
- Oliver S. G., van der Aart Q. J., Agostoni-Carbone M. L., Aigle M., Alberghina L., Alexandraki D., Antoine G., Anwar R., Ballesta J. P., Benit P. The complete DNA sequence of yeast chromosome III. Nature. 1992 May 7;357(6373):38–46. doi: 10.1038/357038a0. [DOI] [PubMed] [Google Scholar]
- Oliver S. A network approach to the systematic analysis of yeast gene function. Trends Genet. 1996 Jul;12(7):241–242. doi: 10.1016/0168-9525(96)30053-x. [DOI] [PubMed] [Google Scholar]
- Perrot M., Sagliocco F., Mini T., Monribot C., Schneider U., Shevchenko A., Mann M., Jenö P., Boucherie H. Two-dimensional gel protein database of Saccharomyces cerevisiae (update 1999). Electrophoresis. 1999 Aug;20(11):2280–2298. doi: 10.1002/(SICI)1522-2683(19990801)20:11<2280::AID-ELPS2280>3.0.CO;2-Q. [DOI] [PubMed] [Google Scholar]
- Raamsdonk L. M., Teusink B., Broadhurst D., Zhang N., Hayes A., Walsh M. C., Berden J. A., Brindle K. M., Kell D. B., Rowland J. J. A functional genomics strategy that uses metabolome data to reveal the phenotype of silent mutations. Nat Biotechnol. 2001 Jan;19(1):45–50. doi: 10.1038/83496. [DOI] [PubMed] [Google Scholar]
- Rain J. C., Selig L., De Reuse H., Battaglia V., Reverdy C., Simon S., Lenzen G., Petel F., Wojcik J., Schächter V. The protein-protein interaction map of Helicobacter pylori. Nature. 2001 Jan 11;409(6817):211–215. doi: 10.1038/35051615. [DOI] [PubMed] [Google Scholar]
- Rappsilber J., Siniossoglou S., Hurt E. C., Mann M. A generic strategy to analyze the spatial organization of multi-protein complexes by cross-linking and mass spectrometry. Anal Chem. 2000 Jan 15;72(2):267–275. doi: 10.1021/ac991081o. [DOI] [PubMed] [Google Scholar]
- Rötig A., de Lonlay P., Chretien D., Foury F., Koenig M., Sidi D., Munnich A., Rustin P. Aconitase and mitochondrial iron-sulphur protein deficiency in Friedreich ataxia. Nat Genet. 1997 Oct;17(2):215–217. doi: 10.1038/ng1097-215. [DOI] [PubMed] [Google Scholar]
- Schild D., Brake A. J., Kiefer M. C., Young D., Barr P. J. Cloning of three human multifunctional de novo purine biosynthetic genes by functional complementation of yeast mutations. Proc Natl Acad Sci U S A. 1990 Apr;87(8):2916–2920. doi: 10.1073/pnas.87.8.2916. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shoemaker D. D., Lashkari D. A., Morris D., Mittmann M., Davis R. W. Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar-coding strategy. Nat Genet. 1996 Dec;14(4):450–456. doi: 10.1038/ng1296-450. [DOI] [PubMed] [Google Scholar]
- Thon V. J., Khalil M., Cannon J. F. Isolation of human glycogen branching enzyme cDNAs by screening complementation in yeast. J Biol Chem. 1993 Apr 5;268(10):7509–7513. [PubMed] [Google Scholar]
- Uetz P., Giot L., Cagney G., Mansfield T. A., Judson R. S., Knight J. R., Lockshon D., Narayan V., Srinivasan M., Pochart P. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature. 2000 Feb 10;403(6770):623–627. doi: 10.1038/35001009. [DOI] [PubMed] [Google Scholar]
- Venter J. C., Adams M. D., Myers E. W., Li P. W., Mural R. J., Sutton G. G., Smith H. O., Yandell M., Evans C. A., Holt R. A. The sequence of the human genome. Science. 2001 Feb 16;291(5507):1304–1351. doi: 10.1126/science.1058040. [DOI] [PubMed] [Google Scholar]
- Volkman S. K., Cowman A. F., Wirth D. F. Functional complementation of the ste6 gene of Saccharomyces cerevisiae with the pfmdr1 gene of Plasmodium falciparum. Proc Natl Acad Sci U S A. 1995 Sep 12;92(19):8921–8925. doi: 10.1073/pnas.92.19.8921. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wach A., Brachat A., Pöhlmann R., Philippsen P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast. 1994 Dec;10(13):1793–1808. doi: 10.1002/yea.320101310. [DOI] [PubMed] [Google Scholar]
- Winzeler E. A., Shoemaker D. D., Astromoff A., Liang H., Anderson K., Andre B., Bangham R., Benito R., Boeke J. D., Bussey H. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science. 1999 Aug 6;285(5429):901–906. doi: 10.1126/science.285.5429.901. [DOI] [PubMed] [Google Scholar]
- Wooden J. M., Hartwell L. H., Vasquez B., Sibley C. H. Analysis in yeast of antimalaria drugs that target the dihydrofolate reductase of Plasmodium falciparum. Mol Biochem Parasitol. 1997 Mar;85(1):25–40. doi: 10.1016/s0166-6851(96)02808-3. [DOI] [PubMed] [Google Scholar]