Saccharomyces cerevisiae

The first microorganism to be domesticated by humans, one of the first to be sequenced, S. cerevisiae (baker's yeast) remains a forerunner in the postgenomic era. It was used to demonstrate the feasibility of constructing archived genome-wide collections of defined mutants. The two mutagenesis methods described above, random and directed mutagenesis, were both used to construct genome-wide collection of yeast mutants. In the first approach, Ross-MacDonald et al. used a multipurpose mini-Tn3 derivative to randomly mutagenize, within Escherichia coli, a library of S. cerevisiae genomic DNA [18]. In addition to the selectable markers for yeast and bacteria, the mini-transposon also contained a promoterless lacZ reporter gene and a hemagglutinin epitope tag, allowing thus analysis of gene expression and protein localization. In a first step, E. coli strains containing mutant plasmids, i.e., harboring transposon insertions, were selected and stored individually in 96-well plates. In a second step, 92 544 plasmids were prepared from these strains, digested, and transformed in a diploid yeast strain where the interrupted fragments integrated at their corresponding genomic loci by homologous recombination. To enrich in yeast transformants containing transposon insertions within ORFs, 11 232 strains containing lacZ fusions expressed during vegetative growth were selected for further analysis and arrayed in a 96-well format. The precise location of the mini-transposon in 6358 strains was determined by sequencing the corresponding plasmid-borne insertion alleles, which indicated that insertions affected 1917 different ORFs (31% of yeast's 6200 ORFs), a large number of which were previously nonannotated. In the second approach, systematic deletion of every S. cerevisiae ORF was started via targeted mutagenesis [19], an effort underpinned by yeast's highly efficient homologous recombination. In brief, short regions of homology (45 bp) immediately upstream and downstream each of the 6200 ORFs were placed, together with unique DNA barcodes (allowing the simultaneous screening of large number of mutants similarly to signature-tagged mutagenesis), at both ends of a suitable selectable marker through a two-step PCR methodology. A consortium of yeast laboratories in Europe and North America then used the corresponding PCR products to transform the yeast in a 96-well format and to create start-to-stop codon gene deletion mutants, which were individually arrayed and centralized. In a preliminary report, deletion alleles were constructed for 2026 ORFs [19]. To date, the Saccharomyces Genome Deletion Project consortium has deleted 96% of all annotated ORFs, including nearly all ORFs larger than 100 codons (http://sequence-www.stanford.edu/group/yeast/ yeast_deletion_project/). Deletion alleles were used to generate, when possible, four different strains: haploids of both mating types, and both heterozygous and homozygous diploids, which helped demonstrate that 18.7% of yeast's genes are essential for viability since deletion alleles could not be recovered in haploid strains.

The value of these toolboxes for gene function identification, which is now undisputed, was first validated in the above original studies. The simultaneous assay of 558 homozygous deletion strains - a subset of the gene deletion mutants collection - for growth in rich and minimal media demonstrated that reliable quantitative fitness data can be simultaneously obtained under various conditions for large number of mutants [19], as was later abundantly documented. Similarly, 7680 transposon insertion haploid strains were scored, using phenotypic macroar-rays, for 20 different phenotypes, which confirmed or provided novel functional information for 407 genes [18]. In addition, protein localization was analyzed in 1340 diploid strains carrying in-frame hemagglutinin epitope tag insertions [18], illustrating the extreme versatility of this approach. Globally, these mutant collections, which can be easily obtained by any researcher, have encouraged an unparalleled blossoming of genome-wide studies that have generated a plethora of functional information, a comprehensive review of which is clearly beyond the scope of this article. However, a mere 5 years of use led to significant achievements, since more than 100 experimental conditions were assayed and more than 5000 phenotypic traits assigned to yeast genes [20]. Interestingly, as predicted, some data may actually have important therapeutic implications. For example, a study of the effects of as many as 78 commercially available compounds on pools of 3503 heterozygous deletion strains [21] provided, among other things, a clue to the cholesterol-lowering side-effect of molsidomine, a potent vasodilator used for the past 20 years to treat angina. It was found that a metabolite of molsidomine inhibits lanosterol synthase, an enzyme essential for sterol synthesis that may therefore be a safe target for the development of new cholesterol-lowering drugs. The production of a global map of gene function - an utterly fantastic goal - is therefore becoming increasingly feasible in yeast, and has been scheduled by some for the beginning of2007 [22].

Both genome-wide mutagenesis strategies have advantages and drawbacks [17]. Random mutagenesis and sequencing of the transposon insertion sites can rapidly lead to the construction of a large collection of mutants, which can immediately be used to generate a wealth of functional information. Moreover, it is relatively cost-effective and is therefore suitable for single laboratories' efforts. However, although near-saturation mutagenesis of the genome can be obtained in extremely large libraries, transposon mutagenesis generates a lot of redundancy, some genes being mutated multiple times due to their size and to characteristics inherent in the transposon itself, and exhaustiveness cannot be attained. On the other hand, systematic targeted mutagenesis of every gene is restricted to organisms with a high rate of recombination; it is laborious and expensive, and is therefore best accomplished by a consortium of laboratories. However, it can result in a collection of mutants that is actually comprehensive.

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