The procedures used to identify and isolate mutants, referred to as genetic screens, depend on whether the experimental organism is haploid or diploid and, if the latter, whether the mutation is recessive or dominant. Genes that encode proteins essential for life are among the most interesting and important ones to study. Since phenotypic expression of a mutation in an essential gene leads to death of the individual, clever genetic screens are needed to isolate and maintain organisms with a lethal mutation.

In haploid yeast cells, essential genes can be studied through the use of conditional mutations. Among the most common conditional mutations are temperature-sensitive mutations, which are useful in organisms, such as bacteria and lower eukaryotes, that can grow at a range of temperatures. For instance, a single missense mutation may cause the resulting mutant protein to have reduced thermal stability, such that the protein is fully functional at one temperature (e.g., 23 °C) but begins to denature and is thus inactive at another temperature (e.g., 36 °C), whereas the normal protein would be fully stable and functional at both temperatures. A temperature at which the mutant phenotype is observed is called nonpermissive; a permissive temperature is one at which the mutant phenotype is not observed even though the mutant allele is present. Thus mutant strains can be maintained at a permissive temperature and then subcultured at a nonpermissive temperature for analysis of the mutant phenotype.

An example of a particularly important screen for temperature-sensitive mutants in the yeast S. cerevisiae comes from the studies of L. H. Hartwell and colleagues in the late 1960s and early 1970s. They set out to identify genes important in regulation of the cell cycle (during which a cell synthesizes proteins, replicates its DNA, and then undergoes mitotic cell division). Exponential growth of a single yeast cell for 20–30 cell divisions forms a visible yeast colony on solid agar medium. Because mutants with a complete block in the cell cycle would not be able to form colonies, conditional mutants were required to study mutations that affect this basic cellular process. To screen for such mutants, the researchers first exposed yeast cells to mutagens and then identified mutant yeast cells that could grow normally at 23 °C, but could not form a colony when placed at 36 °C ( Figure 1a).

Fig1. EXPERIMENTAL FIGURE 66 Haploid yeast cells carrying temperature-sensitive lethal mutations can be maintained at per missive temperature and analyzed at nonpermissive temperature. (a) Genetic screen for temperature-sensitive cell division cycle (cdc) mutants in S. cerevisiae. Yeast cells that grow and form colonies at 23 °C (permissive temperature) but not at 36 °C (nonpermissive temperature) may carry a lethal mutation that blocks cell division. See L. H. Hartwell, 1967, J. Bacteriol. 93:1662. (b) Assay of temperature-sensitive colonies for blocks at specific stages in the cell cycle. Shown here are micro graphs of wild-type yeast and two different temperature-sensitive mutants after incubation at the nonpermissive temperature for 6 hours. Wild-type cells, which continue to grow, can be seen with all different sizes of buds, reflecting different stages of the cell cycle. In contrast, cells in the lower two micrographs exhibit a block at a specific stage in the cell cycle. The cdc28 mutants arrest at a point before emergence of a new bud and therefore appear as unbudded cells. The cdc7 mutants, which arrest just before separation of the mother cell and bud (emerging daughter cell), appear as cells with large buds. [Part (b) republished with permission of Elsevier, from Herefor, L. M, and Hartwell, L. H., “Sequential gene function in the initiation of Saccharomyces cerevisiae DNA synthesis,” Journal of Molecular Biology, 1974, 84:3, pps 445-456; permission conveyed through Copyright Clearance Center, Inc.]

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