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Chapter 3 : Postgenomic Strategies for Genetic Analysis: Insight from Saccharomyces cerevisiae and Candida albicans
Category: Fungi and Fungal Pathogenesis
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This chapter discusses the choice of genes for study in the postgenomic era. One major limitation of using a host organism as a model for the heterologous expression of a gene of interest in another organism is differences in genetic coding between the two organisms. This problem, for example, exists when using Saccharomyces cerevisiae as a host for Candida albicans genes, where the codon CUG specifies leucine in S. cerevisiae but serine in C. albicans; thus, C. albicans proteins may be nonfunctional when translated in S. cerevisiae. The idea of considering several different screens is an extrapolation from the well-accepted idea that many different phenotypes contribute to the virulence of a pathogen. When an insertion mutant is found with a phenotype of interest, it is verified that the insertion itself is the cause of the phenotype by three approaches. First, multiple independent insertion mutant isolates are tested, all of which should have the same phenotype. Second, the insertion mutation is complemented with a clone of the complete ORF and flanking sequences, which should restore a wild-type phenotype. Third, engineered orf Δorf/orf Δ deletion strains are created with two successive transformations rather than an allelic recombination step; the deletion mutant is expected to have a phenotype similar to the initial insertion mutant. A study revealed two different kinds of phenotypic defects, thus suggesting that genetic analysis might contribute to dissection of a biofilm developmental pathway.
Key Concept Ranking
- Cell Wall Proteins
Frequencies of S. cerevisiae mutants with severe growth defects under various selection conditions. Mutant growth properties ( 17 ) are grouped by gene categories. Selection conditions and the severity of defects are as described in Table 1 . Gene categories include a representative random group (Chr7+Chr3), a group about which little is known (UKUK), the entire set of nonessential genes (All), and those with biochemically informative sequence motifs (TxnF+MbP+PrK+CWP). Abbreviations are described in Table 1 .
(A) Properties of the UAU1 cassette. UAU1 is a functional ARG4 gene flanked by nonfunctional ura3 Δ3’ and ura3 Δ5’ deletion alleles. The ura3 alleles are in direct repeat orientation and share 500 bp of homology, so they can undergo recombination. Recombination between the ura3 alleles yields a functional URA3 gene and excises ARG4. (B) Double-disruption selection. Transformation of C. albicans strain BWP17 with UAU1 results in expression of ARG4 and confers an Arg+ phenotype. If the insertion becomes homozygous through mitotic recombination or gene conversion, then a second recombination event within one cassette yields an Arg+ Ura+ segregant. These Arg+ Ura+ segregants are of two classes. One class carries a UAU1 insertion allele and a URA3 insertion allele. We refer to these segregants as homozygotes. The other class carries three copies of the gene: a UAU1 insertion allele, a URA3 insertion allele, and a wild-type allele. We refer to these segregants as allelic triplication derivatives. A trisomic genetic structure is diagrammed for simplicity, but their precise genetic structure has not been determined.
Distribution of S. cerevisiae growth-defective mutants under various selection conditions
Effect of reduced selection stringency on mutant detection