8 research outputs found
Synergistic effects of CCR4-NOT mutants on the <i>gtb1 mad2</i> double mutant.
<p>The KYPD+TBZ plate represents a permissive condition for the <i>gtb1 mad2</i> double mutant. Chromosome instability of the double mutant was enhanced on the YE plate, particularly at lower temperature. The indicated double and triple mutants were streaked on the plates and incubated at 30°C or 33°C for 3 d.</p
Colony formation from spores produced in triploid fission yeast.
<p>(A) Heterogeneously sized colonies. Colonies were incubated on YE medium at 30°C for 5 d. (B) Representative morphologies of C1- (left) and C2- (right) type microcolonies. Colonies were incubated on YE medium at 30°C for 48 h.</p
Segregation analysis of chromosome 3 disome.
(a)<p>P219 (<i>h</i><sup>−</sup><i>leu1 ade6-M210/ade6-M216</i>) was crossed with a haploid strain that was <i>h</i><sup>+</sup> with <i>ade6-M216</i> (or <i>ade6-M210</i>) and one of the indicated alleles (except <i>not2</i>, which is mapped on chromosome 3). Strain 56-1 (<i>h</i><sup>−</sup><i>leu1 ade6-M210 not2::kan/ade6-M216 not2</i><sup>+</sup>) was crossed with <i>h</i><sup>+</sup><i>ade6-M216 not2::kan</i>. Ade<sup>+</sup> segregants were selected on an EMM2 plate at 30°C.</p>(b)<p>Ade<sup>+</sup> colonies were randomly selected and tested for drug resistance. For the <i>not2</i> mutant, see (d).</p>(c)<p>All tested 12 Ade<sup>+</sup> segregants had the “unstable Ade<sup>+</sup>” phenotype, indicating a chromosome 3 disome. Note, wild-type did not produce drug-resistant segregants.</p>(d)<p>For this cross, 24 of 26 Ade<sup>+</sup> (disomic for chromosome 3) were G-418 resistant. Of these 24 segregants, 15 were homozygous for the <i>not2::kan</i> mutant, while 9 were heterozygous (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002776#s4" target="_blank">Materials and methods</a>).</p>(e)<p>G-418 resistant Ade<sup>+</sup> segregants in this mutant were generally small, and upon restreaking on YE plates only stable Ade<sup>+</sup> colonies (probably diploids) and Ade<sup>−</sup> haploid colonies were produced. Chromosome 3 disome was hardly recovered thereafter.</p
Synergistic effects of DNA repair-related mutants on the <i>gtb1 mad2</i> double mutant.
<p>See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002776#pgen-1002776-g002" target="_blank">Figure 2</a> legend for details. *Enlarged black and white image of this portion is shown on the right side.</p
Comparison of C1-type microcolony size in wild-type and <i>not3</i> mutant.
<p>Pictures are representative images of C1 type microcolonies from aneuploid spores. The difference was statistically significant for aneuploid spores (Mann-Whitney U-test; p = 4×10<sup>−7</sup>), while for the control haploid spores there was no significant difference (p = 0.41). The size estimation procedure is described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002776#s4" target="_blank">Materials and Methods</a>.</p
Characterization of chromosome 3 disomes of the CCR4-NOT mutants.
<p>(A) Temperature sensitivity of chromosome 3 disomes. 5-fold serial dilutions of the disomes with the indicated mutations were spotted on a selective EMM medium (-adenine) at 30°C for 4 d or at 36°C for 5 d. For all strains, the target cell number for the last dilution was 25. All or most of the large colonies forming after incubation at 36°C were diploid. (B) Stability of chromosome 3 disomes. The indicated strains were spotted on EMM with a low concentration of adenine. Colonies were incubated at 30°C for 4 d. Red colonies represent haploids that lost one copy of chromosome 3. Enlarged images of the third spots (*) on the right show the presence of sectored colonies in the mutants.</p
Effects of CCR4-NOT mutants on the proliferation of aneuploid cells in <i>S. cerevisiae</i>.
<p>(A) Effect of <i>not3</i> and <i>caf4</i> mutants on aneuploid spores. Randomly selected spores prepared from triploid meioses were individually plated on YPD plates and incubated at 30°C for 15.5 h. The number of cells in each microcolony was counted. A cell with an emerging bud whose diameter was smaller than approximately two-thirds that of the mother cell was counted as one. For haploid spores (wild-type: n = 226; <i>not3</i>: n = 216; <i>caf4</i>: n = 210), the number of cells was counted after 4.5 h of incubation. (B) Doubling times of <i>S. cerevisiae</i> haploid and disomes. Disomes having an extra chromosome (XV or XIII) with the indicated mutations were used. Colonies were incubated in a synthetic SD medium selective for disomy (−His+G418) at 22°C. Culture densities were measured every 2 h. Doubling time was calculated from an exponentially growing phase of each culture (OD<sub>600</sub> values, approximately from 0.15 to 1.0). Data are shown as mean ± SD (n = 3).</p
Viability of spores produced in triploid cells in <i>S. cerevisiae</i>.
<p>Viability of spores produced in triploid cells in <i>S. cerevisiae</i>.</p