-j a cdc7 mutants cdc7 mutants

M EXPERIMENTAL FIGURE 9-6 Haploid yeasts carrying temperature-sensitive lethal mutations are maintained at permissive temperature and analyzed at nonpermissive temperature. (a) Genetic screen for temperature-sensitive cell-division cycle (cdc) mutants in yeast. Yeasts 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. (b) Assay of temperature-sensitive colonies for blocks at specific stages in the cell cycle. Shown here are micrographs of wild-type yeast and two different temperature-sensitive mutants after incubation at the nonpermissive temperature for 6 h. 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 (a) see L. H. Hartwell, 1967, J. Bacterial. 93:1662; part (b) from L. M. Hereford and L. H. Hartwell, 1974, J. Mol. Biol. 84:445.]

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 Saccharomyces cere-visiae 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, with each daughter cell receiving a copy of each chromosome. Exponential growth of a single yeast cell for 20-30 cell divisions forms a visible yeast colony on solid agar medium. Since mutants with a complete block in the cell cycle would not be able to form a colony, conditional mutants were required to study mutations that affect this basic cell process. To screen for such mutants, the researchers first identified mutagenized yeast cells that could grow normally at 23 °C but that could not form a colony when placed at 36 °C (Figure 9-6a).

Once temperature-sensitive mutants were isolated, further analysis revealed that they indeed were defective in cell division. In sS. cerevisiae, cell division occurs through a budding process, and the size of the bud, which is easily visualized by light microscopy, indicates a cell's position in the cell cycle. Each of the mutants that could not grow at 36 °C was examined by microscopy after several hours at the nonpermissive temperature. Examination of many different temperature-sensitive mutants revealed that about 1 percent exhibited a distinct block in the cell cycle. These mutants were therefore designated cdc (cell-division cycle) mutants. Importantly, these yeast mutants did not simply fail to grow, as they might if they carried a mutation affecting general cellular metabolism. Rather, at the nonpermissive temperature, the mutants of interest grew normally for part of the cell cycle but then arrested at a particular stage of the cell cycle, so that many cells at this stage were seen (Figure 9-6b). Most cdc mutations in yeast are recessive; that is, when haploid cdc strains are mated to wild-type haploids, the resulting heterozygous diploids are neither temperature-sensitive nor defective in cell division.

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