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PubMed1986

Identification and characterization of four new GCD genes in Saccharomyces cerevisiae.

Mutant strains, resistant against the amino acid analogues 5-methyltryptophan, 5-fluorotryptophan and canavanine were isolated, starting with a trp2 leaky auxotrophic strain. Of 10 such strains, only four turned out to be of the "general control derepressed" (gcd) mutant type. Three other isolates were shown to be defective in the general amino acid permease system, while the remaining three strains displayed low spore viability and were not further investigated. Complementation tests amongst the four new gcd-mutant strains, including strain RH558 gcd2-1 isolated earlier, yielded five complementation groups: GCD2, GCD3, GCD4, GCD5, and GCD6. All mutant strains showed a dual phenotype, which was not separable by wild type backcrosses: "constitutive derepression" and "slow growth". Epistasis of all gcd mutations over gcn1-1, gcn2-1 and gcn3-1 was found with respect to both phenotypes, except for gcd5-1, which was lethal in these combinations. On the other hand gcn4-101 was found to be epistatic over all gcd mutations, but only with respect to the "constitutive derepression" phenotype, and not to "slow growth"; again the combination with gcd5-1 was lethal. Mutation gcd2-1 was mapped on chromosome VII, 50 cM from leu1 and 22 cM from ade6. A new model is discussed, in which GCD-genes are involved in the amino acid uptake into the vacuoles.

Amino Acids↗
PubMed1977

Protoplast fusion of Schizosaccharomyces pombe Auxotrophic mutants of identical mating-type.

Protoplasts of methionine- and lysine-requiring h- mutants isolated from the L972 h- strain of Schizosaccharomyces pombe were fused. The protoplasts were obtained from the cells with enzymes produced by Trichoderma viride. When a mixture of the protoplasts was treated with 30% PEG 4000 solution containing 10 mM CaCl2, cell fusion and complementation was attained with a frequency of 0.17%. Both fusion partners were recovered among the spores after crossing of the fusion products with the strain M210 ade6 h+. Cytological and haploidization examinations showed that the fusion cells are not heterokaryons, and that the increased amount of genetic material is situated in one nucleus.

Ascomycota↗
PubMed1994

Hotspots of homologous recombination.

Homologous recombination occurs at higher than average frequency at and near hotspots. Hotspots are special nucleotide sequences recognized by proteins that promote, directly or indirectly, a rate limiting step of recombination. This review focuses on two well-studied examples, the Chi sites of the bacterium Escherichia coli and the M26 site of the fission yeast Schizosaccharomyces pombe. Chi, 5' G-C-T-G-G-T-G-G 3', is recognized by the RecBCD enzyme, which nicks the DNA near Chi and produces a 3'-ended single-stranded DNA 'tail'; this tail is a potent substrate for homologous pairing by RecA and single-stranded DNA binding proteins. M26, 5' A-T-G-A-C-G-T 3', is recognized by a heterodimeric protein and stimulates, by an as-yet-unknown mechanism, meiotic recombination at and near the ade6 gene. Additional hotspots in bacteria, fungi, and mammals enhance recombination directly or indirectly via a variety of mechanisms. Although hotspots are widespread among organisms, the biological role of their localized enhancement of recombination remains a matter of speculation.

Binding Sites↗
PubMed2006

Cloning the Schizosaccharomyces pombe lys2+ gene and construction of new molecular genetic tools.

Molecular genetic analyses in Schizosaccharomyces pombe rely on selectable markers that are used in cloning vectors or to mark targeted gene deletions and other integrated constructs. In this study, we used genetic mapping data and genomic sequence information to predict the identity of the S. pombe lys2(+) gene, which is homologous to Saccharomyces cerevisiae LYS4(+). We confirmed this prediction, showing that the cloned SPAC343.16 gene can complement a lys2-97 mutant allele, and constructed the lys2(+)-based cloning vector pRH3. In addition, we deleted the S. pombe his7(+) gene with a lys2(+) -marked polymerase chain reaction (PCR) product and the S. pombe lys2(+) gene with a his7(+)-marked PCR product. Strains carrying these deletions of lys2(+) or his7(+) serve as relatively efficient hosts for the deletion of the ade6(+) gene by lys2(+)-- or his7(+)--marked PCR products when compared with hosts carrying lys2 or his7 point mutations. Therefore, these studies provide plasmids and strains allowing the use of lys2(+) as a selectable marker, along with improved strains for the use of his7(+) to mark gene deletions.

Alleles↗
PubMed1997

Tolerance of low pH in Schizosaccharomyces pombe requires a functioning pub1 ubiquitin ligase.

A strain of Schizosaccharomyces pombe carrying a disrupted Na+/H+ antiporter gene (sod2::sup3-5), in addition to the common auxotrophic mutations, ade6-216, ura4-D18 and leu1-32, is highly sensitive to media adjusted to pH 6.9. Reversion analysis of this strain yielded a group of revertants capable of growth at pH 6.9. Two of the revertants elongated and failed to form colonies at pH 3.5. Genetic characterization of one of the pH-sensitive elongated strains, J227, showed the presence of two independently segregating mutations. One, pub1 (protein ubiquitin ligase 1), has recently been reported as an E3 protein ubiquitin ligase involved in cdc25 turnover. The second has been named elp3-1 (elongated at low pH). Genetic dissection of the original strain revealed that poor growth at high pH was due to the presence of the auxotrophic markers, suggesting a possible inhibitory effect of high pH on the function of permeases responsible for uptake of the necessary nutrients. Suppression of the high pH sensitivity required the presence of both the pub1-1 and elp3-1 mutations. While the pub1-1 mutation reduced the capacity of cells to tolerate relatively moderate concentrations of LiCl (3 mM) in liquid culture, it was capable of partially suppressing the extreme Li+ sensitivity caused by the sod2 disruption. Under these conditions, the growth of pub1-1 sod2::ura4 double mutant cells was improved over that of either pub1-1 or sod2::ura4 cells. The elp3-1 mutation had no effect on the Li+ tolerance in either wild-type or sod2::ura4 backgrounds. pub1-1 cells are elongated and incapable of colony formation at pH 3.5. In contrast, elp3-1 cells are elongated at pH 3.5 and pH 5.5 (the normal pH of minimal medium) but can form colonies under both conditions. J227 cells are significantly longer than either single mutant at pH 3.5 and do not form colonies but are visually similar to elp3-1 cells at pH 5.5. Complementation cloning in the J227 background yielded a genomic clone of pub1, allowing us to define the intron-exon structure of the gene. Sequences with high homology to the predicted amino acid sequence of pub1 have been identified in Saccharomyces cerevisiae (RSP5/NPI1), human (hRPF1), mouse (mNedd4), and rat (rNedd4). Based on the nature of our mutant selection, the pH-sensitive phenotype of the strains selected, and the known involvement of RSP5/ NPI1 in membrane permease turnover in S. cerevisiae, we hypothesize a role for pub1, either directly or indirectly, in regulating membrane transport processes. This is further supported by the broad range of effects that the pub1-1 mutation exerts on overall performance of cells at high and low external pH, and in the presence of toxic levels of Li+.

Amino Acid Sequence↗
PubMed1998

Alteration of the largest subunit of RNA polymerase II and its effect on chromosome stability in Schizosaccharomyces pombe.

A mutation in the RNA polymerase II largest subunit (RpII LS) that is related to abnormal induction of sister chromatid exchange has previously been described the CHO-K1 cell mutant tsTM4. To elucidate the molecular basis of this effect we introduced the mutation into the homologous site in the Schizosaccharomyces pombe rpbl gene, which encodes RpII LS. Since the tsTM4 mutant exhibited a decrease in the rate of DNA synthesis in cells arrested in S phase at the nonpermissive temperature, we focussed on the study of growth, the cell cycle, and chromosome stability at various temperatures. First, we examined the effects of the mutation on haploid yeast cells. The mutant showed slower growth than the wild type, but cell growth was not arrested at the nonpermissive temperature. When growing cells were shifted to the nonpermissive temperature, an accumulation of cells in G1 and/or G0 was observed. Tetrad analysis suggested that these phenotypes were associated with the mutation. In diploid cells, chromosome instability was detected by loss of intragenic complementation between two alleles of the ade6 gene. An abnormal fraction of cells containing an intermediate DNA content was also observed by FACS analysis. The accumulation of this fraction may reflect the fact that a large number of cells are in S phase or have an abnormal DNA content as a result of chromosome instability. These observations demonstrate that the S. pombe rpb1 mutant exhibits a phenotype very similar to that of the CHO-K1 cell mutant tsTM4.

Amino Acid Sequence↗
PubMed1990

High levels of chromosome instability in polyploids of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was used to study the genetic consequences of polyploidy in a unicellular organism. Isogenic diploid (2N), triploid (3N) and tetraploid (4N) strains with a genetically marked chromosome VII (cyh2-leu1-CEN7-ade6) were constructed and were used to follow the loss of one, two or three chromosome VII's during mitosis. We found that as ploidy increased, the frequency of loss of a single chromosome VII increased: Loss of one copy of chromosome VII occurred at a rate nearly 30-fold higher in triploids and approximately 1000-fold higher in tetraploids than in the diploid. Loss of two or three copies occurred at an even greater frequency. These findings suggest either that aneuploidy (3N-1, 3N-2, 4N-1, 4N-2, 4N-3) increases genome instability or that multiple chromosome loss events occur at high frequency. Polyploidy appears to dramatically increase chromosome loss, presumably due to the inability of the cell to undergo proper chromosome segregation. The biological significance and possible causes for the instability of polyploidy in unicellular organisms such as yeast are discussed.

Chromosomes, Fungal↗
PubMed1994

Position effect variegation at fission yeast centromeres.

Chromatin structure at Schizosaccharomyces pombe centromeres is unusual. The insertion of the ura4 gene within these centromeres resulted in genetically identical cells mosaic for its expression. Placement of the ade6 gene within cen1 or cen3 resulted in red-white sectored colonies, demonstrating the instability of gene expression. The occurrence of pink colonies implied that intermediate levels of repression were established. Repression of both genes within centromeres was temperature sensitive. The chromatin structure of the ura4 gene at centromeres was altered, suggesting that the unusual chromatin encroaches into the gene and inhibits normal expression. These repressive effects at S. pombe centromeres resemble the classical phenomenon of position effect variegation imposed by Drosophila heterochromatin on nearby genes. However, since the epigenetic states can be set at intermediate levels of expression, a purely euchromatin-heterochromatin dichotomy does not apply. A model for the epigenetic regulation of genes placed within S. pombe centromeres is presented.

Base Sequence↗
PubMed1990

The ADE2 gene from Saccharomyces cerevisiae: sequence and new vectors.

We have determined the sequence of a DNA fragment encoding the ADE2 gene from Saccharomyces cerevisiae. A DNA fragment of 2241 bp capable of complementing ade2 mutations was modified so it is available as a single BglII fragment for use in yeast vectors or for gene disruptions. The minimal fragment codes for a putative protein which is highly similar to the protein encoded by the ADE6 gene from Schizosaccharomyces pombe and to the proteins encoded by the purEK operon of Escherichia coli.

Amino Acid Sequence↗
PubMed2004

Roles of histone acetylation and chromatin remodeling factor in a meiotic recombination hotspot.

Histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling factors (ADCRs) are involved in selective gene regulation via modulation of local chromatin configuration. Activation of the recombination hotspot ade6-M26 of Schizosaccharomyces pombe is mediated by a cAMP responsive element (CRE)-like sequence, M26, and a heterodimeric ATF/CREB transcription factor, Atf1.Pcr1. Chromatin remodeling occurs meiotically around M26. We examined the roles of HATs and ADCRs in chromatin remodeling around M26. Histones H3 and H4 around M26 were hyperacetylated in an M26- and Atf1-dependent manner early in meiosis. SpGcn5, the S. pombe homolog of Gcn5p, was required for the majority of histone H3 acetylation around M26 in vivo. Deletion of gcn5+ caused a significant delay in chromatin remodeling but only partial reduction of M26 meiotic recombination frequency. The snf22+ (a Swi2/Snf2-ADCR homologue) deletion and snf22+ gcn5+ double deletion abolished chromatin remodeling and significant reduction of meiotic recombination around M26. These results suggest that HATs and ADCRs cooperatively alter local chromatin structure, as in selective transcription activation, to activate meiotic recombination at M26 in a site-specific manner.

Acetylation↗
PubMed1997

Region-specific meiotic recombination in Schizosaccharomyces pombe: the rec11 gene.

Mutations in the rec11 gene of Schizosaccharomyces pombe reduce meiotic recombinant frequencies by as much as a factor of 300 on chromosome III but less than a factor of 4 in the intervals tested on chromosomes I and II. To gain insight into the function of this region- (or chromosome-) specific activator of recombination, we have cloned and sequenced the rec11 gene. Meiotic crosses with rec11 disruption mutations placed the rec11 gene 6 cM from ade6 on chromosome III. Transcripts of rec11 accumulated transiently at 2-3 h after induction of melosis in a pat1-114 (Ts) mutant. Reverse transcriptase/polymerase chain reaction (RT-PCR) analysis of these transcripts revealed eight introns. The spliced RNA is predicted to encode a polypeptide of 923 amino acids with only very limited homology to reported proteins. The transient accumulation of rec11 transcripts and the phenotype of rec11 mutations suggest that the novel rec11 gene product acts early in meiosis to activate recombination preferentially on chromosome III.

Base Sequence↗
PubMed2006

Hsk1 kinase is required for induction of meiotic dsDNA breaks without involving checkpoint kinases in fission yeast.

Cdc7 kinase, conserved through evolution, is known to be essential for mitotic DNA replication. The role of Cdc7 in meiotic recombination was suggested in Saccharomyces cerevisiae, but its precise role has not been addressed. Here, we report that Hsk1, the Cdc7-related kinase in Schizosaccharomyces pombe, plays a crucial role during meiosis. In a hsk1 temperature-sensitive strain (hsk1-89), meiosis is arrested with one nucleus state before meiosis I in most of the cells and meiotic recombination frequency is reduced by one order of magnitude, whereas premeiotic DNA replication is delayed but is apparently completed. Strikingly, formation of meiotic dsDNA breaks (DSBs) are largely impaired in the mutant, and Hsk1 kinase activity is essential for these processes. Deletion of all three checkpoint kinases, namely Cds1, Chk1, and Mek1, does not restore DSB formation, meiosis, or Cdc2 activation, which is suppressed in hsk1-89, suggesting that these aberrations are not caused by known checkpoint pathways but that Hsk1 may regulate DSB formation and meiosis. Whereas transcriptional induction of some rec genes and horsetail movement are normal, chromatin remodeling at ade6-M26, a recombination hotspot, which is prerequisite for subsequent DSB formation at this locus, is not observed in hsk1-89. These results indicate unique and essential roles of Hsk1 kinase in the initiation of meiotic recombination and meiosis.

Bromodeoxyuridine↗
PubMed1992

Chromosomal context dependence of a eukaryotic recombinational hot spot.

The single base-pair mutation M26 in the ade6 gene of the fission yeast Schizosaccharomyces pombe creates a hot spot for meiotic homologous recombination. When DNA fragments containing M26 and up to 3.0 kilobases of surrounding DNA were moved to the ura4 gene or to a multicopy plasmid, M26 had no detectable hot spot activity. Our results indicate that nucleotide sequences at least 1 kilobase away from M26 are required for M26 hot spot activity and suggest that, as for transcriptional promoters, a second site or proper chromatin structure is required for activation of this eukaryotic recombinational hot spot. We discuss the implications of these results for studies of other meiotic recombinational hot spots and for gene targeting.

Chromosomes, Fungal↗
PubMed1985

Rearrangement of the genetic map of chromosome VII of Saccharomyces cerevisiae.

The genetic map of the right arm of chromosome VII of Saccharomyces cerevisiae includes markers on a distal segment for which meiotic linkage to the centromere-proximal marker cly8 has not previously been demonstrated. According to the currently accepted map, SUF4 is the most distal marker on the right arm. We have shown by tetrad analysis that SUF4 is linked to cly8 and ade6. The genetic distance between SUF4 and cly8 is 29 cM. These data indicate that the genetic map of the right arm of chromosome VII should be revised by inverting the orientation of the distal segment so that SUF4 is located near cly8, and SUC1 and MAL1 are the most distal markers. With this revision, all of the polymeric fermentation markers that have been mapped are located at the ends of chromosomes.

Chromosome Mapping↗
PubMed1986

Suppressors of the ras2 mutation of Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains two members of the ras gene family. Strains with disruptions of the RAS2 gene fail to grow efficiently on nonfermentable carbon sources. This growth defect can be suppressed by extragenic mutations called sra. We have isolated 79 independent suppressor mutations, 68 of which have been assigned to one of five loci. Eleven additional dominant mutations have not been assigned to a specific locus. Some sra1 and SRA4 and all SRA3 mutations were RAS independent, allowing growth of yeast cells that lack a functional RAS gene. Mutations in sra1, SRA3, SRA4 and sra6 are linked to his6, ino1, met3 and ade6, respectively. Some sra mutants have pleiotropic phenotypes that affect glycogen accumulation, sporulation, viability, respiratory capacity and suppression of two cell-division-cycle mutations, cdc25 and cdc35. The proposed functions of many of the suppressor genes are consistent with the model in which RAS activates adenylate cyclase.

Adenylyl Cyclases↗
PubMed1998

The M26 hotspot of Schizosaccharomyces pombe stimulates meiotic ectopic recombination and chromosomal rearrangements.

Homologous recombination is increased during meiosis between DNA sequences at the same chromosomal position (allelic recombination) and at different chromosomal positions (ectopic recombination). Recombination hotspots are important elements in controlling meiotic allelic recombination. We have used artificially dispersed copies of the ade6 gene in Schizosaccharomyces pombe to study hotspot activity in meiotic ectopic recombination. Ectopic recombination was reduced 10-1000-fold relative to allelic recombination, and was similar to the low frequency of ectopic recombination between naturally repeated sequences in S. pombe. The M26 hotspot was active in ectopic recombination in some, but not all, integration sites, with the same pattern of activity and inactivity in ectopic and allelic recombination. Crossing over in ectopic recombination, resulting in chromosomal rearrangements, was associated with 35-60% of recombination events and was stimulated 12-fold by M26. These results suggest overlap in the mechanisms of ectopic and allelic recombination and indicate that hotspots can stimulate chromosomal rearrangements.

Alleles↗
PubMed1999

Localization and properties of a silencing element near the mat3-M mating-type cassette of Schizosaccharomyces pombe.

Transcription is repressed in a segment of Schizosaccharomyces pombe chromosome II that encompasses the mat2-P and mat3-M mating-type cassettes. Chromosomal deletion analysis revealed the presence of a repressor element within 500 bp of mat3-M. This element acted in synergy with the trans-acting factors Swi6, Clr1, Clr2, Clr3, and Clr4 and had several properties characteristic of silencers: it did not display promoter specificity, being able to silence not only the M mating-type genes but also the S. pombe ura4 and ade6 genes placed on the centromere-distal side of the mat3-M cassette; it could repress a gene when placed further than 2.6 kb from the promoter and it acted in both orientations, although with different efficiencies, the natural orientation repressing more stringently than the reverse. Following deletion of this element, two semistable states of expression of the mat3-M region were observed and these two states could interconvert. The deletion did not affect gene expression in the vicinity of the mat2-P cassette, 11 kb away from mat3-M. Conversely, deleting 1.5 kb on the centromere-proximal side of the mat2-P cassette, which was previously shown to partially derepress transcription around mat2-P, had no effect on gene expression near mat3-M. A double deletion removing the mat2-P and mat3-M repressor elements had the same effect as the single deletions on their respective cassettes when assayed in cells of the M mating type. These observations allow us to refine a model proposing that redundant pathways silence the mating type region of S. pombe.

Base Sequence↗
PubMed1999

Position effect variegation at the mating-type locus of fission yeast: a cis-acting element inhibits covariegated expression of genes in the silent and expressed domains.

Schizosaccharomyces pombe switches its mating type by transposing a copy of unexpressed genes from the respective mat2 or mat3 cassettes to mat1. The donor cassettes are located in a silent domain that is separated from the expressed mat1 cassette by the L region. We monitored the expression of ade6 from sites in the L region and examined the relationship between the expression state at these sites and at sites within the silent domain. Results indicate that: (1) the silent domain extends into the L region, but repression is gradually alleviated with increasing distance from mat2, and overexpression of swi6 enhances PEV in the L region; (2) a transcriptionally active chromatin state, associated with reporter gene expression in the L region, spreads toward the silent domain; (3) a cis-acting element, located at the junction between the L region and mat2-P, ensures repression in the silent domain, regardless of the expression state in the L region; and (4) repression in mat1-P cells is less stringently controlled than in mat1-M cells. We discuss the functional organization of the mat region and genetic elements that ensure separation between repressed and derepressed domains.

Adenine↗