The <i>cuf2</i>Δ/<i>cuf2</i>Δ mutant is defective in FSM formation.

<p><i>A</i>, Both wild-type diploid (<i>cuf2⁺/cuf2⁺</i>) and <i>cuf2Δ/cuf2Δ</i> mutant cells expressing GFP-Psy1 were synchronously induced to undergo azygotic meiosis. At the 9 h time point, FSM formation was monitored by detecting the GFP-Psy1 signal by fluorescence microscopy. When GFP-Psy1 localized to 4 circular FSM structures, these FSMs were classified as normal (i). When GFP-Psy1 localized to either lesser or greater than 4 circular FSMs or shmoo-like structures, FSMs were classified as abnormal (ii, iii and iv). <i>B</i>, Histograms showing the percentages of each normal (i) and abnormal (ii, iii, iv) FSM structure in both wild-type (<i>cuf2⁺/cuf2⁺</i>) and <i>cuf2Δ/cuf2Δ</i> mutant cells, as well as in a diploid <i>cuf2Δ/cuf2Δ</i> disruption strain in which wild-type copies of the <i>cuf2⁺-GFP</i> fusion gene were reintegrated. <i>C</i>, Typical images of FSM structures 9 h after meiotic induction in both wild-type (<i>cuf2⁺/cuf2⁺</i>) and <i>cuf2Δ/cuf2Δ</i> mutant cells (top panels). Each strain had previously been transformed with pJK210GFP-Psy1, which encodes GFP-Psy1 that is used as an FSM-resident marker. Hoechst 33342 staining was used to visualize the chromosomal DNA (middle panels). The merged images of the GFP-Psy1 and the Hoechst 33342 dye are shown in the bottom panels. Anucleated FSM structures, or unpackaged nuclei, are indicated by the white arrows.</p

<i>cuf2</i>Δ/<i>cuf2</i>Δ mutant cells display reduced spore viability.

<p><i>A</i>, Both wild-type (<i>cuf2⁺/cuf2⁺</i>) and <i>cuf2Δ/cuf2Δ</i> mutant cells were allowed to sporulate under conditions of limiting nitrogen. Tetrad dissection analysis (>200 asci per strain; n = 210 and 214, respectively) revealed that ∼59% of the spores from <i>cuf2Δ/cuf2Δ</i> asci failed to germinate as compared to spores from wild-type (<i>cuf2+/cuf2+</i>) asci (∼98% germination). As an additional control, the mutant strain bearing the disrupted <i>cuf2Δ/cuf2Δ</i> alleles was used to reintegrate functional <i>cuf2⁺-GFP/cuf2⁺-GFP</i> alleles and was then examined for the formation of 4-spored asci. This control strain exhibited 97% of 4-spore asci (n = 220). <i>B</i>, Comparative typical results of tetrad dissection analysis in wild-type (<i>cuf2⁺/cuf2⁺</i>) and <i>cuf2Δ/cuf2Δ</i> mutant asci.</p

Effect of <i>cuf2</i>Δ/<i>cuf2</i>Δ deletion on the expression of <i>S. pombe</i> genes.

<p><i>A</i>, Venn diagram representing the numbers of genes with higher and lower expression levels in the <i>cuf2Δ/cuf2Δ</i> disruption strain as compared to the wild-type strain. The diagram also shows the overlap between middle-phase meiotic genes and all differentially expressed genes identified in <i>cuf2Δ/cuf2Δ</i> cells. <i>B</i>, Genes that exhibit higher expression levels in <i>cuf2Δ/cuf2Δ</i> cells were grouped in different functional families. <i>C</i>, <i>pat1-114</i>/<i>pat1-114 (cuf2⁺/cuf2⁺)</i> and <i>pat1-114</i>/<i>pat1-114 cuf2Δ/cuf2Δ</i> strains were pre-synchronized by nitrogen starvation and were then induced to undergo synchronous meiosis under basal conditions. At the indicated times following meiotic induction, total RNA was prepared from both the <i>cuf2⁺/cuf2⁺</i> strain and its <i>cuf2Δ/cuf2Δ</i> mutant derivative and was analyzed by RNase protection assay. The histograms shown the quantifications of three independent RNase protection experiments. Ten hours after meiotic induction, the results indicate that the levels of the <i>SPAC1B2.03c⁺</i>, <i>wtf13⁺</i>, <i>SPBC1348.01⁺</i> and <i>meu14⁺</i> transcripts in <i>cuf2Δ/cuf2Δ</i> mutant cells were derepressed above those observed in <i>cuf2⁺/cuf2⁺</i> cells under the same conditions (as indicated with arrows and brackets).</p

<i>cuf2</i>⁺ gene expression is Mei4-dependent.

<p><i>pat1-114</i>/<i>pat1-114 (mei4⁺/mei4⁺)</i> and <i>pat1-114</i>/<i>pat1-114 mei4Δ/mei4Δ</i> strains were pre-synchronized by nitrogen starvation at 25°C, and then were induced to undergo synchronous meiosis at 34°C. At the indicated time points, the <i>cuf2⁺</i> and <i>act1⁺</i> (internal control) mRNA levels were analyzed in both the control strain (<i>mei4⁺/mei4⁺</i>) and the isogenic strain lacking the <i>mei4⁺</i> alleles. To validate the absence of the <i>cuf2⁺</i> transcript during mitosis, total RNA was probed for the presence of <i>cuf2⁺</i> mRNA in vegetative cells incubated at 25°C.</p

Riboprobes used to detect steady-state levels of transcripts.

<p>Riboprobes used to detect steady-state levels of transcripts.</p

Comparison of Cuf2 with the <i>S. pombe</i> copper metalloregulatory transcription factor Cuf1.

<p><i>A</i>, Schematic representations of Cuf1 and Cuf2. The amino acid sequences of both proteins are numbered relative to their initiator codons. The locations of the domains required for Cuf1 function are indicated, including the N-terminal nuclear localization signal (NLS) (11–53) that is located within the DNA-binding module (1–174), the C-terminal Cu-sensing module (C-rich) (328–342) and the C-terminal nuclear export signal (NES) (349–358). The positions of some of the Cys (C) and His (H) residues within both Cuf1 and Cuf2 are also indicated. <i>B</i>, Amino acid alignment of the N-terminal 61 amino acid residues of Cuf1 with the N-terminal 60-residue segment of Cuf2. The black boxes indicate identical amino acids, and the gray boxes indicate amino acids that are similar between Cuf2 and Cuf1. The asterisks highlight the 7 Cys residues that are conserved between Cuf2 and Cuf1. The indicated N-terminal 1–60 amino acid region of Cuf2 exhibits 42% sequence identity and 62% sequence similarity with the N-terminal 61 amino acids of Cuf1 that are part of its DNA-binding domain.</p

Assessment of the mRNA and protein steady-state levels of Cuf2 during meiosis.

<p><i>A</i>, Representative expression profiles of the <i>cuf2</i>⁺ and <i>ctr4</i>⁺ mRNAs in <i>h</i>⁺ haploid cells that were either left untreated (−) or were treated with either 50 µM TTM or 50 µM CuSO₄ during mitosis. <i>B</i>, Cultures of <i>pat1-114/pat1-114</i> diploid cells were either maintained in vegetative growth at 25°C, or were induced to initiate and proceed through meiosis at 34°C. <i>pat1-114/pat1-114</i> diploid cells were either left untreated, or incubated in the presence of 50 µM TTM or 50 µM CuSO₄. Total RNA was isolated at the indicated time points after the induction of meiosis. Shown are representative RNase protection assays of both the <i>cuf2⁺</i> and the <i>act1⁺</i> (internal control) mRNA steady-state levels during meiosis. <i>C</i>, Cuf2-TAP protein expression during meiosis. <i>pat1-114/pat1-114 cuf2Δ/cuf2Δ</i> diploid cells expressing Cuf2-TAP were either left uninduced (25°C), or were induced (34°C) under basal conditions or in the presence of 50 µM TTM or 50 µM CuSO₄. Shown are Western blots of both Cuf2-TAP and α-tubulin (control loading) levels at different time points after meiotic induction. <i>D</i>, Meiotic progression of cells under either basal (untreated) conditions, or in the presence of TTM (50 µM) or CuSO₄ (50 µM). The values shown for each condition (TTM, basal or Cu) correspond to the percentage of cells with 1, 2, or 3–4 nuclei, and the percentage of cells with horse tails. The graphed values represent the averages of triplicate determinations +/− the standard deviations.</p

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

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

Analysis of Cuf2-GFP localization during both meiosis and sporulation.

<p>The Cuf2-GFP fluorescence signal (center left) was observed at different stages of meiosis following the azygotic meiotic induction of a <i>h⁺/h⁻ cuf2Δ/cuf2Δ cuf2⁺-GFP/cuf2⁺-GFP</i> strain. Once induced, azygotic meiotic cells were differentiated under basal conditions. Cuf2-GFP was observed at each extremity of the cell at the end of the first meiotic division (MI) that is to say at anaphase I. Cuf2-GFP followed the segregation of chromatids until late in anaphase II when the second meiotic division (MII) occurred in early anaphase II. The Cuf2-GFP fluorescence signal was detected during the FSM formation (i.e. at the end of meiosis), but disappeared during spore formation. Cells at different stages of meiosis were stained using Hoechst 33342 to visualize the DNA (centre right). The merged images are shown in the far right panels. Nomarski optics (far left) were used to monitor cell morphology.</p

<i>cuf2</i>Δ and <i>meu5</i>Δ gene disruptions have opposite effects on common meiosis-specific genes.

<p><i>A</i>, Venn diagram representing the overlap between the <i>cuf2⁺</i>- and the <i>meu5⁺</i>-dependent genes. <i>B</i>, Total RNA from both the <i>pat1-114/pat1-114</i> (<i>cuf2⁺</i>/<i>cuf2⁺</i>) and <i>pat1-114</i>/<i>pat1-114 cuf2Δ/cuf2Δ</i> disruption strains was analyzed throughout meiosis and sporulation. A representative RNase protection experiment of the effect of the absence of Cuf2 on the expression of the <i>meu5⁺</i> transcript (especially 10 to 14 h after meiotic induction) is shown. <i>C–D</i>, Cultures of <i>pat1-114/pat1-114</i>, <i>pat1-114</i>/<i>pat1-114 cuf2Δ/cuf2Δ</i>, <i>pat1-114</i>/<i>pat1-114 meu5Δ/meu5Δ</i> and <i>pat1-114</i>/<i>pat1-114 cuf2Δ/cuf2Δ meu5Δ/meu5Δ</i> diploid cells were synchronously induced into meiosis under basal conditions. Total RNA was isolated from culture aliquots taken at the indicated time points. After RNA preparation, the <i>wtf13⁺</i> and <i>SPAC1B2.03c⁺</i> steady-state mRNA levels were analyzed by RNase protection assays using actin (<i>act1</i>⁺) as an internal control. The results shown are representative of three independent experiments.</p