Number of Yeast Genes

Until recently, the total number of functional genes in an organism was estimated by saturation mutagenesis. What this means is that any gene that is associated with a function can be identified by the loss of function after mutagenesis. Therefore, if an organism is mutagenized and enough mutants are screened, the total number of mutants can give a rough estimate of the total number of functional genes. The yeast genome sequence revealed about 6200 genes, two times more than that predicted from saturation mutagenesis. A second approach to analyze gene function is by targeted disruption of genes, possible only after the genes have been identified and their sequences determined. R.W. Davis and his colleagues disrupted 5916 genes (96.5% of total) and analyzed the behavior of mutants under a variety of nutritional and environmental conditions (Glaever et al., 2002). In their experiments, each gene was precisely deleted from the start to the stop codon and replaced with a "deletion cassette" containing a selectable marker kanamycin. The kanamycin gene in each deletion cassette was flanked on either side by two unique 20-nucleotide sequences. These unique sequence tags can be viewed as a barcode, a permanent identifier of each deletion mutant. This clever trick of tagging every deleted gene permitted the researchers to carry out growth experiments with many deletion mutants in parallel. In a typical experiment the relative contribution of genes for growth on galactose was examined with the objective of assigning functions to as of yet uncharacterized genes (Figure 6.13).

Twelve deletion mutants were grown in a medium with galactose as the sole carbon source with the relative proportion of each mutant cell in the mixed culture determined by quantitating the relative amount of each tag present in the culture by microarray technology (described in the next section). Analysis of growth characteristics of each mutant in galactose media revealed the impact of individual genes on the utilization of galactose. By this method, the functions of two novel genes YML090W and YML077W in

Figure 6.13 Measuring fitness of deletion strains of S. cerevisiae in galactose medium. Strains numbered from 1 to 12 were grown together in the same tube. The growth of each strain was quantitated by quantifying the barcodes associated with each mutant using an oligonucleotide array (microarray) as described in the text. (From Glaever et al. (2002).)

Figure 6.13 Measuring fitness of deletion strains of S. cerevisiae in galactose medium. Strains numbered from 1 to 12 were grown together in the same tube. The growth of each strain was quantitated by quantifying the barcodes associated with each mutant using an oligonucleotide array (microarray) as described in the text. (From Glaever et al. (2002).)

the utilization of galactose were revealed. The collection of deletion mutants is being used as a functional genomic strategy for the analysis of complex cellular processes and metabolic pathways.

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