Telomere binding of the Rap1 protein is required for meiosis in fission yeast.

Telomeres are essential for chromosome integrity, protecting the ends of eukaryotic linear chromosomes during cell proliferation. Telomeres also function in meiosis; a characteristic clustering of telomeres beneath the nuclear membrane is observed during meiotic prophase in many organisms from yeasts to plants and humans, and the role of the telomeres in meiotic pairing and the recombination of homologous chromosomes has been demonstrated in the fission yeast Schizosaccharomyces pombe and in the budding yeast Saccharomyces cerevisiae. Here we report that S. pombe Rap1 is a telomeric protein essential for meiosis. While Rap1 is conserved in budding yeast and humans, schemes for telomere binding vary among species: human RAP1 binds to the telomere through interaction with the telomere binding protein TRF2; S. cerevisiae Rap1, however, binds telomeric DNA directly, and no orthologs of TRF proteins have been identified in this organism. In S. pombe, unlike in S. cerevisiae, an ortholog of human TRF has been identified. This ortholog, Taz1, binds directly to telomere repeats [18] and is necessary for telomere clustering in meiotic prophase. Our results demonstrate that S. pombe Rap1 binds to telomeres through interaction with Taz1, similar to human Rap1-TRF2, and that Taz1-mediated telomere localization of Rap1 is necessary for telomere clustering and for the successful completion of meiosis. Moreover, in taz1-disrupted cells, molecular fusion of Rap1 with the Taz1 DNA binding domain recovers telomere clustering and largely complements defects in meiosis, indicating that telomere localization of Rap1 is a key requirement for meiosis.

Characterization of fission yeast meiotic mutants based on live observation of meiotic prophase nuclear movement.

We characterized four meiotic mutants of the fission yeast Schizosaccharomyces pombe by live observation of nuclear movement. Nuclei were stained with either the DNA-specific fluorescent dye Hoechst 33342 or jellyfish green fluorescent protein (GFP) fused with the N-terminal portion of DNA polymerase alpha. We first followed nuclear dynamics in wild-type cells to determine the temporal sequence of meiotic events: nuclear fusion in the conjugated zygote is immediately followed by oscillatory nuclear movements that continue for 146 min; then, after coming to rest, the nucleus remains in the center of the cell for 26 min before the first meiotic division. Next we examined nuclear dynamics in four meiotic mutants: mei1 (also called mat2), mei4, dhc1, and taz1. Mei1 and mei4 both arrest during meiotic prophase; our observations, however, show that the timing of mei1 arrest is quite different from that of mei4: the mei1 mutant arrests after nuclear fusion but before starting the oscillatory nuclear movements, while the mei4 mutant arrests after the nucleus has completed the oscillatory movements but before the first meiotic division. We also show examples of the dynamic phenotypes of dhc1 and taz1, both of which complete meiosis but exhibit impaired nuclear movement and reduced frequencies of homologous recombination: the dhc1 mutant exhibits no nuclear movement after nuclear fusion, while the taz1 mutant exhibits severely impaired nuclear movement after nuclear fusion.

Large-scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP-fusion genomic DNA library.

BACKGROUND: Intracellular localization is an important part of the characterization of a gene product. In an attempt to search for genes based on the intracellular localization of their products, we constructed a green fluorescent protein (GFP)-fusion genomic DNA library of S. pombe. RESULTS: We constructed the S. pombe GFP-fusion genomic DNA library by fusing, in all three reading frames, random fragments of genomic DNA to the 5' end of the GFP gene in such a way that expression of potential GFP-fusion proteins would be under the control of the own promoters contained in the genomic DNA fragments. Fission yeast cells were transformed with this plasmid library, and microscopic screening of 49 845 transformants yielded 6954 transformants which exhibited GFP fluorescence, of which 728 transformants showed fluorescence localized to distinct intracellular structures such as the nucleus, the nuclear membrane, and cytoskeletal structures. Plasmids were isolated from 516 of these transformants, and a determination of their DNA sequences identified 250 independent genes. The intracellular localizations of the 250 GFP-fusion constructs was categorized as an image database; using this database, DNA sequences can be searched for based on the localizations of their products. CONCLUSIONS: A number of new intracellular structural components were found in this library. The library of GFP-fusion constructs also provides useful fluorescent markers for various intracellular structures and cellular activities, which can be readily used for microscopic observation in living cells.

Dynamics of centromeres during metaphase-anaphase transition in fission yeast: Dis1 is implicated in force balance in metaphase bipolar spindle.

In higher eukaryotic cells, the spindle forms along with chromosome condensation in mitotic prophase. In metaphase, chromosomes are aligned on the spindle with sister kinetochores facing toward the opposite poles. In anaphase A, sister chromatids separate from each other without spindle extension, whereas spindle elongation takes place during anaphase B. We have critically examined whether such mitotic stages also occur in a lower eukaryote, Schizosaccharomyces pombe. Using the green fluorescent protein tagging technique, early mitotic to late anaphase events were observed in living fission yeast cells. S. pombe has three phases in spindle dynamics, spindle formation (phase 1), constant spindle length (phase 2), and spindle extension (phase 3). Sister centromere separation (anaphase A) rapidly occurred at the end of phase 2. The centromere showed dynamic movements throughout phase 2 as it moved back and forth and was transiently split in two before its separation, suggesting that the centromere was positioned in a bioriented manner toward the poles at metaphase. Microtubule-associating Dis1 was required for the occurrence of constant spindle length and centromere movement in phase 2. Normal transition from phase 2 to 3 needed DNA topoisomerase II and Cut1 but not Cut14. The duration of each phase was highly dependent on temperature.

Oscillatory nuclear movement in fission yeast meiotic prophase is driven by astral microtubules, as revealed by continuous observation of chromosomes and microtubules in living cells.

Using a computerized fluorescence microscope system to observe fluorescently stained cellular structures in vivo, we have examined the dynamics of chromosomes and microtubules during the process of meiosis in the fission yeast Schizosaccharomyces pombe. Fission yeast meiotic prophase is characterized by a distinctive type of nuclear movement that is led by telomeres clustered at the spindle-pole body (the centrosome-equivalent structure in fungi): the nucleus oscillates back and forth along the cell axis, moving continuously between the two ends of the cell for some hours prior to the meiotic divisions. To obtain a dynamic view of this oscillatory nuclear movement in meiotic prophase, we visualized microtubules and chromosomes in living cells using jellyfish green fluorescent protein fused with alpha-tubulin and a DNA-specific fluorescent dye, Hoechst 33342, respectively. Continuous observation of chromosomes and microtubules in these cells demonstrated that the oscillatory nuclear movement is mediated by dynamic reorganization of astral microtubules originating from the spindle-pole body. During each half-oscillatory period, the microtubules extending rearward from the leading edge of the nucleus elongate to drive the nucleus to one end of the cell. When the nucleus reversed direction, its motion during the second half of the oscillation was not driven by the same microtubules that drove its motion during the first half, but rather by newly assembled microtubules. Reversible inhibition of nuclear movement by an inhibitor of microtubule polymerization, thiabendazole, confirmed the involvement of astral microtubules in oscillatory nuclear movement. The speed of the movement fluctuated within a range 0 to 15 micron/minute, with an average of about 5 microm/minute. We propose a model in which the oscillatory nuclear movement is mediated by dynamic instability and selective stabilization of astral microtubules.

A novel fission yeast gene, kms1+, is required for the formation of meiotic prophase-specific nuclear architecture.

In the meiotic prophase nucleus of the fission yeast Schizosaccharomyces pombe, chromosomes are arranged in an oriented manner: telomeres cluster in close proximity to the spindle pole body (SPB), while centromeres form another cluster at some distance from the SPB. We have isolated a mutant, kms1, in which the structure of the meiotic prophase nucleus appears to be distorted. Using specific probes to localize the SPB and telomeres, multiple signals were observed in the mutant nuclei, in contrast to the case in wild-type. Genetic analysis showed that in the mutant, meiotic recombination frequency was reduced to about one-quarter of the wild-type level and meiotic segregation was impaired. This phenotype strongly suggests that the telomere-led rearrangement of chromosomal distribution that normally occurs in the fission yeast meiotic nucleus is an important prerequisite for the efficient pairing of homologous chromosomes. The kms1 mutant was also impaired in karyogamy, suggesting that the kms1+ gene is involved in SPB function. However, the kms1+ gene is dispensable for mitotic growth. The predicted amino acid sequence of the gene product shows no significant similarity to known proteins.

Phosphorylation of RNA-binding protein controls cell cycle switch from mitotic to meiotic in fission yeast.

Meiosis generates haploid gametes from diploid cells and is an almost universal feature of eukaryotic organisms. But little is known about how the switch from mitotic to meiotic cell cycles is molecularly controlled. In the fission yeast Schizosaccharomyces pombe, inactivation of the protein kinase Pat1(Ran1) upon nutrient deprivation triggers entry into the meiotic cell cycle. Here we show that the RNA-binding protein Mei2 is a substrate of Pat1 kinase and that dephosphorylation of Mei2 is sufficient to switch cells from the mitotic cell cycle into meiosis. Mei2 is localized mainly in the cytoplasm of proliferating cells but is seen as a single spot close to the microtubule organizing centre in prophase nuclei during meiosis. Our results, and others from a metazoan, emphasize the crucial role of RNA-binding proteins in the initiation and execution of meiosis.

Meiotic nuclear reorganization: switching the position of centromeres and telomeres in the fission yeast Schizosaccharomyces pombe.

In fission yeast meiotic prophase, telomeres are clustered near the spindle pole body (SPB; a centrosome-equivalent structure in fungi) and take the leading position in chromosome movement, while centromeres are separated from the SPB. This telomere position contrasts with mitotic nuclear organization, in which centromeres remain clustered near the SPB and lead chromosome movement. Thus, nuclear reorganization switching the position of centromeres and telomeres must take place upon entering meiosis. In this report, we analyze the nuclear location of centromeres and telomeres in genetically well-characterized meiotic mutant strains. An intermediate structure for telomere-centromere switching was observed in haploid cells induced to undergo meiosis by synthetic mating pheromone; fluorescence in situ hybridization revealed that in these cells, both telomeres and centromeres were clustered near the SPB. Further analyses in a series of mutants showed that telomere-centromere switching takes place in two steps; first, association of telomeres with the SPB and, second, dissociation of centromeres from the SPB. The first step can take place in the haploid state in response to mating pheromone, but the second step does not take place in haploid cells and probably depends on conjugation-related events. In addition, a linear minichromosome was also co-localized with authentic telomeres instead of centromeres, suggesting that telomere clustering plays a role in organizing chromosomes within a meiotic prophase nucleus.

Telomere-led premeiotic chromosome movement in fission yeast.

The movement of chromosomes that precedes meiosis was observed in living cells of fission yeast by fluorescence microscopy. Further analysis by in situ hybridization revealed that the telomeres remain clustered at the leading end of premeiotic chromosome movement, unlike mitotic chromosome movement in which the centromere leads. Once meiotic chromosome segregation starts, however, centromeres resume the leading position in chromosome movement, as they do in mitosis. Although the movement of the telomere first has not been observed before, the clustering of telomeres is reminiscent of the bouquet structure of meiotic-prophase chromosomes observed in higher eukaryotes, which suggests that telomeres perform specific functions required for premeiotic chromosomal events generally in eukaryotes.

A low copy number central sequence with strict symmetry and unusual chromatin structure in fission yeast centromere.

Fission yeast centromeres vary in size but are organized in a similar fashion. Each consists of two distinct domains, namely, the approximately 15-kilobase (kb) central region (cnt+imr), containing chromosome-specific low copy number sequences, and 20- to 100-kb outer surrounding sequences (otr) with highly repetitive motifs common to all centromeres. The central region consists of an inner asymmetric sequence flanked by inverted repeats that exhibit strict identity with each other. Nucleotide changes in the left repeat are always accompanied with the same changes in the right. The chromatin structure of the central region is unusual. A nucleosomal nuclease digestion pattern formed on unstable plasmids but not on stable chromosome. DNase I hypersensitive sites correlate with the location of tRNA genes in the central region. Autonomously replicating sequences are also present in the central region. The behavior of truncated minichromosomes suggested that the central region is essential, but not sufficient, to confer transmission stability. A portion of the outer repetitive region is also required. A larger outer region is necessary to ensure correct meiotic behavior. Fluorescence in situ hybridization identified individual cens. In the interphase, they cluster near the nuclear periphery. The central sequence (cnt+imr) may play a role in positioning individual chromosomes within the nucleus, whereas the outer regions (otr) may interact with each other to form the higher-order complex structure.

A large number of tRNA genes are symmetrically located in fission yeast centromeres.

We report here that the fission yeast centromere regions in the three chromosomes contain no less than 36 symmetrically arranged tRNA-coding sequences, and many of them are located within the inner inverted regions that are thought to be essential for the centromere function. There are 11 different species of tRNA-coding sequences, and four of them are identical to those previously known in this organism. This high-density distribution of tRNA genes in the centromere regions is surprising, as the fission yeast centromeres were thought to form transcriptionally inactive structures.

Characterization of Schizosaccharomyces pombe minichromosome deletion derivatives and a functional allocation of their centromere.

A 530 kb long Schizosaccharomyces pombe linear minichromosome, Ch16, containing a centric region of chromosome III, has previously been made. In the present study, we constructed a number of deletions in the right and/or left arms of Ch16, and compared their structure and behaviour with Ch16. The functional centromere, cen3, is allocated within a 120 kb long region which is covered by the shortest derivative, Ch10, and is comprised mostly of centromeric repeating sequences. The shortest minichromosome is stable in mitosis and the copy number control is apparently precise. In monosomic meiosis it segregates normally. In disomic meioses, however, the frequency of non-disjunction is very high, suggesting that it may not form a pair. The mitotic loss rate of one of the left-arm deletions, ChR32, which lacks a part of the centromeric repeating sequence, is the highest of all the deletions. This deletion also exhibits the highest precocious sister chromatid separation in meiosis I, suggesting that sister chromatid association might become weakened in ChR32. Our results indicate that the proper meiotic segregation of S.pombe minichromosomes is dependent upon the formation of a bivalent. S.pombe may not have the 'distributive segregation' found with Saccharomyces cerevisiae minichromosomes.

Composite motifs and repeat symmetry in S. pombe centromeres: direct analysis by integration of NotI restriction sites.

S. pombe centromeres are large and complex. We introduced a method that enables us to characterize directly centromere DNAs. Genomic DNA fragments containing cen1, cen2, or cen3, respectively, are made by cleaving NotI sites integrated on target sites and are partially restricted for long-range mapping in PFG electrophoresis. The 40 kb long cen1 consists of two inverted approximately 10 kb motifs, each containing centromeric elements dg and dh, flanked by a central region. In cen2, three motifs are arranged in inverted and direct orientations with flanking domains, making up the approximately 70 kb long repetitious region. In cen3, approximately 15 copies of dg-dh constitute a region longer than 100 kb. A set of inverted motifs with an approximately 15 kb central region might be a prototype for the S. pombe centromeres. The motifs appear to play a role in chromosome stability and segregation. Their action may be additive, and the mutual directions of dg and dh inside a motif may not be essential for function.

Construction of a Not I restriction map of the fission yeast Schizosaccharomyces pombe genome.

Pulsed field gel electrophoresis and large DNA technology were used to construct a Not I restriction map of the entire genome of the fission yeast Schizosaccharomyces pombe. There are 14 detectable Not I sites in S. pombe 972h: 9 sites on chromosome I and 5 sites on chromosome II, while no Not I sites were found on chromosome III. The 17 fragments (including intact chromosome III) generated by Not I digestion were resolved by PFG electrophoresis. These fragments ranged in size from 4.5 kb to approximately 3.5 Mb. Various strategies were applied in determining, efficiently, the order of the fragments on the chromosomes. The genomic size measured by adding all the fragments together is about 14 Mb and the sizes of the three chromosomes are I, 5.7 Mb, II, 4.6 to 4.7 Mb, and III, 3.5 Mb. These are generally somewhat smaller than estimated previously.