Deletion of <i>gaf1⁺</i> causes accelerated G₁-arrest under nitrogen-starved conditions.

<p>Cells grown to mid-log phase in EMM2 for 18 h were shifted to EMM-N medium, and their DNA contents were monitored by FACS analysis at intervals. The FACS data represent typical examples of three independent experiments. (A) Cells of heterothallic (<i>h⁻</i>) WT (972) and <i>gaf1Δ</i> (KL230) strains were shifted to EMM-N medium. (B) Cells of homothallic (<i>h⁹⁰</i>) <i>gaf1Δ</i>(KL211)/pREP-Gaf1 strain were shifted to EMM-N medium with (+B₁) or without (−B₁) 20 µM thiamine. WT denotes the wild-type (<i>gaf1⁺</i>).</p

Gaf1 protein specifically binds to the GATA binding motif of <i>ste11⁺</i> promoter <i>in vitro</i>.

<p>(A) Schematic diagram of the promoter region of <i>ste11⁺</i>. The open- and closed-ovals represent binding sites for Rst2 (UASst) and Ste11 (TR1 and TR2), respectively. The major transcription start site (position number +1) is designated by a hinged arrow. The regions contained in the upstream probe P_A (−834∼−225) and the downstream probe P_B (−231∼+10) are shown as bars. The dark square represents the GATA motif spanning from −385 to −352 for which wild-type (P_W) and mutant (P_M) double-stranded oligonucleotide probes were designed. Restriction sites: Sp, <i>Sph</i>I; N, <i>Nde</i>I; RV, <i>Eco</i>RV. (B) Search for Gaf1-binding region in <i>ste11⁺</i> promoter by EMSA. GST-Gaf1 protein (1 µg) was incubated with labeled P_A probe containing the 0.6-kb upstream region of <i>ste11⁺</i> promoter in the absence (lanes 2 and 5) or presence of excess cold competitor, P_A (lane 3, 50-fold; lane 4, 100-fold) or P_B (lane 6, 50-fold; lane 7, 100-fold). (C) Analysis of Gaf1 binding to the GATA motif of <i>ste11⁺</i> promoter by EMSA. GST-Gaf1 protein (lane 3, 0.1 µg; lanes 4–6, 1 µg) was incubated with labeled P_A probe in the absence (lanes 3 and 4) or presence of 100-fold excess of cold competitor P_M (lanes 5) or P_W (lanes 6). (D) Mutational dissection of the Gaf1-binding GATA motif of <i>ste11⁺</i> promoter by EMSA. Varying amounts of GST-Gaf1 protein (lane 3, 0.1 µg; lane 4, 0.2 µg; lane 5, 0.5 µg; lanes 6, 7, 11, and 12, 1 µg) were incubated with labeled P_W oligonucleotide probe in the absence (lanes 3 and 4) or presence of 100-fold excess of cold competitor P_M (lanes 5) or P_W (lanes 6). Mock reaction mixtures without GST-Gaf1 or with GST protein were used as negative controls in (B)–(D). The closed and open arrow heads in (B)–(D) represent shifted bands and free probes, respectively.</p

Mating and sporulation frequency of <i>S. pombe</i> strains.

a<p>For analysis of homothallic strains, pre-grown cultures of <i>h⁹⁰</i> WT (JY4), <i>h⁹⁰ gaf1Δ</i> (KL211), <i>h⁹⁰ ste11Δ</i> (JZ396), and <i>h⁹⁰ gaf1Δ ste11Δ</i> (KL213) were spotted onto EMM2 (+N) and EMM-N (−N) plates, and the cells were observed by DIC microscopy to determine sporulation frequencies after 3-d incubation at 30°C. For analysis of heterothallic strains, 1∶1 mixtures of the pre-grown mating pairs, <i>h⁺</i> (ED668)×<i>h ⁻</i> (ED665), <i>h⁺ gaf1Δ</i> (KL216)×<i>h</i><b><i>⁻</i></b><i> gaf1Δ</i> (KL210), and <i>h⁺ ste11Δ</i> (KL416)×<i>h⁻ gaf1Δ</i> (KL240) were spotted onto EMM2 and EMM-N plates.</p>b<p>The values represent the average ± the standard deviation of at least three independent assays carried out in triplicate.</p

Deletion of <i>gaf1⁺</i> results in accelerated induction of <i>ste11⁺</i> expression under nitrogen-starved conditions.

<p>(A) Northern blot analysis of <i>ste11⁺</i> and <i>gaf1⁺</i> mRNA from wild-type and <i>gaf1Δ</i> cells exposed to nitrogen starvation. Cells of wild-type (972) and <i>gaf1Δ</i> (KL230) strains pre-grown in EMM2 (+N) were shifted to EMM-N (−N) and cultured with constant shaking. At indicated time points, cells were harvested and washed twice with distilled water, and total RNAs were extracted from the cells. RNA blots were hybridized with ³²P-labeled PCR-amplified <i>gaf1⁺</i> and <i>ste11⁺</i> probes. For internal control, all blots were stripped and subsequently rehybridized with ³²P-labeled actin-specific probe (<i>act1⁺</i>). (B) β-Galactosidase reporter assay for analysis of <i>ste11⁺</i> expression in wild-type and <i>gaf1Δ</i> cells subjected to nitrogen starvation. Cells of wild-type (ED665) and <i>gaf1Δ</i> (KL240) strains carrying pJLC-Ste11_(p)-LacZ were cultivated to mid-log phase in EMM2 (+N) and shifted to EMM-N (−N). At indicated time points, cells were harvested and washed twice with distilled water, and the level of <i>ste11</i>⁺ expression was estimated by measuring the activity of β-galactosidase in each sample. Values are the mean ± standard error of three independent experiments carried out in triplicate, <i>n</i> = 9. *, <i>p</i><0.01; **, <i>p</i><0.05 (two-tailed Student's t-test, <i>versus</i> wild-type). WT denotes the wild-type (<i>gaf1⁺</i>).</p

Effect of <i>gaf1Δ</i> mutation and nitrogen starvation on the global gene expression profiles of <i>S. pombe</i>.

<p>RNA samples from the nitrogen-starved (−N) and unstarved (+N) cells of wild-type (<i>gaf1⁺</i>, ED668) and <i>gaf1Δ</i> (KL216) strains were used for the transcriptome analysis with the GeneChip Yeast Genome 2.0 Array. A Venn diagram is constructed from the lists of the genes up-regulated (≥1.5-fold, p<0.05) in unstarved <i>gaf1Δ</i> cells (Group −G, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042409#pone.0042409.s001" target="_blank">Table S1</a>), nitrogen-starved wild-type cells (Group −N, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042409#pone.0042409.s002" target="_blank">Table S2</a>), and nitrogen-starved <i>gaf1Δ</i> cells (Group −N/−G, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042409#pone.0042409.s003" target="_blank">Table S3</a>). The overlapping and non-overlapping portions of the three groups (Group −G, −N, and −N/−G) are designated as Subgroup A to G. The lists of the genes included in Subgroup A–G are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042409#pone.0042409.s004" target="_blank">Tables S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042409#pone.0042409.s005" target="_blank">S5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042409#pone.0042409.s006" target="_blank">S6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042409#pone.0042409.s007" target="_blank">S7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042409#pone.0042409.s008" target="_blank">S8 </a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042409#pone.0042409.s009" target="_blank">S9</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042409#pone.0042409.s010" target="_blank">S10</a>, respectively, in the Supporting Information.</p

<i>S. pombe</i> strains used in this study.

<p><i>S. pombe</i> strains used in this study.</p

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

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 = 4×10⁻⁷), 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

Segregation analysis of chromosome 3 disome.

(a)<p>P219 (<i>h</i>⁻<i>leu1 ade6-M210/ade6-M216</i>) was crossed with a haploid strain that was <i>h</i>⁺ 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>⁻<i>leu1 ade6-M210 not2::kan/ade6-M216 not2</i>⁺) was crossed with <i>h</i>⁺<i>ade6-M216 not2::kan</i>. Ade⁺ segregants were selected on an EMM2 plate at 30°C.</p>(b)<p>Ade⁺ colonies were randomly selected and tested for drug resistance. For the <i>not2</i> mutant, see (d).</p>(c)<p>All tested 12 Ade⁺ segregants had the “unstable Ade⁺” 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⁺ (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⁺ segregants in this mutant were generally small, and upon restreaking on YE plates only stable Ade⁺ colonies (probably diploids) and Ade⁻ haploid colonies were produced. Chromosome 3 disome was hardly recovered thereafter.</p