POSTER NO: 246

Using yeast to assign functions to human genes

¹Nianshu Zhang, ²Mike Osborn, ¹Paul Gitsham, ²Ross Miller, ¹Steve Oliver
¹The University of Manchester, Manchester, M13 9PT, UK, ²The Babraham Institute, Babraham, Cambridge CB2 4AT, UK

The recent completion of the draft genome sequence has indicated that the number of protein-encoding human genes is much smaller than many investigators had expected, at just 35,000-40,000. Nevertheless, estimates of the total number of human proteins remain high, with figures ranging from 100,000 to 500,000 being quoted in the literature. There are a number of reasons for the difference between gene number and protein number, including the trans-splicing of primary transcripts (each human gene, on average, probably encodes three mRNA species), and the post-translational processing and modification of the proteins themselves. The use of comparative genomic approaches, in which the situation in humans is compared to that in simpler eukaryotes, should be of great help in unravelling the complexity of the relationship between the human genome and proteome, and in elucidating the (probably multiple) functions played by each human gene. Such a comparative analysis should not rely exclusively on bioinformatics, since this does not cope adequately with the multidimensional nature of the proteome. Rather, it is also important to carry out functional comparisons between human gene products and those of model organisms on the experimental level. We have developed a complementation system, based on the model eukaryote Saccharomyces cerevisiae, to clone human cDNAs that can functionally complement yeast essential genes. The system employs two regulatable promoters. One promoter, tetO (determining doxycycline-repressible expression), is used to control essential S. cerevisiae genes. The other, pMET3 (which is switched off in the presence of methionine), is employed to regulate the expression of mammalian cDNAs in yeast.

We have demonstrated that this system is effective for both individual cDNA clones and for cDNA libraries, permitting the direct selection of functionally complementing clones. Three human cDNA libraries and one mouse cDNA library have been constructed and screened for clones that can complement specific essential yeast genes when their expression is switched off by the addition of doxycycline to the culture medium. The validity of each complementation was checked by showing that the yeast cells die in the presence of methionine, which represses the expression of the mammalian coding sequence. Using this system, we have screened 20 tetO replacement strains and succeeded in isolating human cDNAs complementing five essential yeast genes. In this way, we have uncovered a novel human ubiquitin-conjugating enzyme, have demonstrated that the functional segment of the human Psmd12 proteosome sub-unit contains a PINT domain, and have isolated clones for two human transcription factors that promote complementation of yeast essential genes by either direct or indirect means.