Normal Yeast Genes Can Be Replaced with Mutant Alleles by Homologous Recombination

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Modifying the genome of the yeast S. cerevisiae is particularly easy for two reasons: yeast cells readily take up exogenous DNA under certain conditions, and the introduced DNA is efficiently exchanged for the homologous chromosomal site in the recipient cell. This specific, targeted recombination of identical stretches of DNA allows any gene in yeast chromosomes to be replaced with a mutant allele. (As we saw in Section 6.1, recombination between homologous chromosomes also occurs naturally during meiosis.)

In one popular method for disrupting yeast genes in this fashion, PCR is used to generate a disruption construct containing a selectable marker, which is subsequently transfected into yeast cells. As shown in Figure 6-36a, the two primers for PCR amplification of the selectable marker are each designed to include about 20 nucleotides identical to sequences flanking the yeast gene to be replaced. The resulting amplified construct comprises the selectable marker (e.g., the kanMX gene, which, like neor, confers resistance to G-418) flanked by about 20 bp at each end that match the ends of the target yeast gene. Transformed diploid yeast cells in which one of the two copies of the target endogenous gene has been replaced by the disruption construct can be identified by their resistance to G-418 or other selectable phenotype. These heterozygous diploid yeast cells generally grow normally regardless of the function of the target gene, but half the haploid spores derived from these cells will carry only the disrupted allele (Figure 6-36b). If a gene is essential for viability, then spores carrying a disrupted allele will not survive.

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EXPERIMENTAL FIGURE 6-36 Homologous recombination with transfected disruption constructs can inactivate specific target genes in yeast. (a) A suitable construct for disrupting a target gene can be prepared using PCR. The two primers designed for this purpose each contain a sequence of about 20 nucleotides (nt) that is homologous to one end of the target yeast gene as well as sequences needed to amplify a segment of DNA carrying a selectable marker gene such as kanMX, which confers resistance to G-418. (b) When recipient diploid Saccharomyces cells are transformed with the disruption construct, homologous recombination between the ends of the construct and the corresponding chromosomal sequences integrates the marker gene into the chromosome, replacing the target-gene sequence. The recombinant diploid cells will grow on a medium containing G-418, whereas untransformed cells will not. If the target gene is essential for viability, half the haploid spores that form after sporulation of recombinant diploid cells will be nonviable.

Disruption of genes by this method is proving particularly useful in assessing the roles of proteins identified by analysis of the entire genomic sequence of S. cerevisiae (see Chapter 8). Each of the approximately 6000 genes has been disrupted with the kanMX construct in diploids, and gene disruptions in haploid spores have also been produced. These analyses have shown that about 4500 of the 6000 yeast gene disruptions can reside in viable haploid spores, revealing an unexpectedly large number of apparently nonessential genes. In some cases, disruption of a particular gene may give rise to subtle defects that do not compromise the viability of yeast cells growing under laboratory conditions. Alternatively, cells carrying a disrupted gene may be viable because of the operation of backup or compensatory pathways. To investigate this possibility, yeast geneticists are currently testing all possible double-mutant combinations for synthetic lethal effects that might reveal nonessential genes with redundant functions (see Figure 6-9c).