TABLE 2.
Cell division time and cell viability of wild-type, tlc1, and tel1 tlc1 strains during vegetative subculturing in liquid mediuma
| Days of subculturingb | Genotype | Doubling time (min)c | % Viable cells
|
|
|---|---|---|---|---|
| CFUd | Dye exclusione | |||
| 1 | Wild type | 108, 84 | 100, 98 | 100, 100 |
| tlc1 | 744, 172 | 6, 29 | 97, 97 | |
| tel1 tlc1 | 180, 117 | 75, 56 | 100, 100 | |
| 2 | Wild type | 90, 90 | 100, 100 | 100, 100 |
| tlc1 | 1140, 1100 | 2, 1 | 76, 43 | |
| tel1 tlc1 | 204, 168 | 71, 22 | 97, 98 | |
| 3 | Wild type | 96, 84 | 100, 100 | 100, 100 |
| tlc1 | 96, 342 | 96, 13 | 98, 92 | |
| tel1 tlc1 | 228, 295 | 15, 9 | 97, 94 | |
A diploid strain heterozygous for the tel1 and tlc1 mutations (KRY229) was sporulated, and tetrads were dissected. Two tetratype tetrads were identified and, in two independent experiments, liquid cultures of strains of the wild-type, tlc1, and tel1 tlc1 genotypes were established. Cell growth rates and cell viability measurements were done as described below. Numbers separated by commas in each column represent data derived from two isogenic spores of different tetrads.
Spore colonies were inoculated into liquid YPD and grown overnight at 30°C. The following morning, cultures were diluted 1:100 in YPD, and growth at 30°C was continued (day 1). At the end of day 1, growth rates were calculated, and the cultures were diluted to yield a starting cell concentration of about 106 cells/ml at the start of day 2. At the end of day 2, this procedure was repeated.
At 2-h intervals for 8 h each day, the cell concentration was measured in a hemocytometer. Doubling times were determined by standard methods.
Samples from each time point were diluted and plated on solid YPD (four plates with about 200 cells/plate). After 3 days of growth at 30°C, colonies were counted. The values shown represent the number of colonies divided by the number of cells (based on hemocytometer counts), expressed as a percentage.
Cells were stained with the dye phloxine B (as described in Materials and Methods). The values represent the number of red (dye-stained) cells divided by the total number of cells examined, expressed as a percentage.