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PubMed2000

A family of cAMP-response-element-related DNA sequences with meiotic recombination hotspot activity in Schizosaccharomyces pombe.

The heptamer sequence ATGACGT is essential for activity of the M26 meiotic recombination hotspot in the ade6 gene of Schizosaccharomyces pombe. Hotspot activity is associated with binding of the heterodimeric transcription factor Atf1.Pcr1 to M26. We have found that the sequences (C/T/G) TGACGT also bound Atf1.Pcr1 and acted as meiotic hotspots, but unlike M26 they must be followed by A or C for Atf1.Pcr1 binding and hotspot activity. The basis of the hotspot activity of CTGACGTA (ade6-3013) appears to be identical to that of M26: hotspot activity of both sequences was abolished in cells mutant for atf1, pcr1, spc1, or wis1 and was undetectable in mitotic recombination and in meiotic recombination when located on a plasmid. Both hotspot sequences were sites of micrococcal nuclease hypersensitivity in meiotic chromatin, suggesting that they create an open chromatin structure during meiosis at the site of the hotspots. The newly identified hotspot sequences (C/T/G)TGACGT(A/C) and M26 are closely related to the cAMP response element (CRE) consensus sequence for binding of cAMP-responsive transcription factors such as Atf1.Pcr1, suggesting a link between transcription and meiotic recombination. These results significantly expand the list of identified sequences with meiotic recombination hotspot activity in S. pombe from a single sequence to a family of CRE-related sequences.

Base Sequence↗
PubMed2004

Fission yeast global repressors regulate the specificity of chromatin alteration in response to distinct environmental stresses.

The specific induction of genes in response to distinct environmental stress is vital for all eukaryotes. To study the mechanisms that result in selective gene responses, we examined the role of the fission yeast Tup1 family repressors in chromatin regulation. We found that chromatin structure around a cAMP-responsive element (CRE)-like sequence in ade6-M26 that is bound by Atf1.Pcr1 transcriptional activation was altered in response to osmotic stress but not to heat and oxidative stresses. Such chromatin structure alteration occurred later than the Atf1 phosphorylation but correlated well with stress-induced transcriptional activation at ade6-M26. This chromatin structure alteration required components for the stress-activated protein kinase (SAPK) cascade and both subunits of the M26-binding CREB/ATF-type protein Atf1.Pcr1. Cation stress and glucose starvation selectively caused chromatin structure alteration around CRE-like sequences in cta3(+) and fbp1(+) promoters, respectively, in correlation with transcriptional activation. However, the tup11Delta tup12Delta double deletion mutants lost the selectivity of stress responses of chromatin structure and transcriptional regulation of cta3(+) and fbp1(+). These data indicate that the Tup1-like repressors regulate the chromatin structure to ensure the specificity of gene activation in response to particular stresses. Such a role for these proteins may serve as a paradigm for the regulation of stress response in higher eukaryotes.

Activating Transcription Factor 1↗
PubMed2005

Differential activation of M26-containing meiotic recombination hot spots in Schizosaccharomyces pombe.

Certain genomic loci, termed hot spots, are predisposed to undergo genetic recombination during meiosis at higher levels relative to the rest of the genome. The factors that specify hot-spot potential are not well understood. The M26 hot spot of Schizosaccharomyces pombe is dependent on certain trans activators and a specific nucleotide sequence, which can function as a hot spot in a position- and orientation-independent fashion within ade6. In this report we demonstrate that a linear element (LE) component, Rec10, has a function that is required for activation of some, but not all, M26-containing hot spots and from this we propose that, with respect to hot-spot activity, there are three classes of M26-containing sequences. We demonstrate that the localized sequence context in which the M26 heptamer is embedded is a major factor governing whether or not this Rec10 function is required for full hot-spot activation. Furthermore, we show that the rec10-144 mutant, which is defective in full activation of ade6-M26, but proficient for activation of other M26-containing hot spots, is also defective in the formation of LEs, suggesting an intimate link between higher-order chromatin structure and local influences on hot-spot activation.

Alleles↗
PubMed2000

[Genetic control of purine biosynthesis in Pichia methanolica yeast. The ADE5 (PUR4) gene, controlling 5'-phosphoribosylformyl glycineamide synthetase].

By comparing published and experimental data on spontaneous mutability of early genes controlling biosynthesis of purine nucleotides (BPN) in different yeast species in the system "from red to white," it was shown that the PUR4 gene encoding 5'-phosphoribosylformyl glycinamidine synthetase (FGAM-synthetase) (EC 6.3.5.3) is the most mutable gene in yeast Saccharomyces cerevisiae (the ADE6 gene), Schizosaccharomyces pombe (the ade3 gene), and Pichia methanolica (the ADE5 gene). This correlates with a considerably large size of the FGAM-synthetase polypeptide, as compared to the products of other genes belonging to this group. Study of characteristics of spontaneous mutations in early BPN genes of P. methanolica demonstrated that the vast majority of unstable ade5sU alleles (mutations with a high reversion frequency ranging from 0.2 x 10(-6) to 2 x 10(-6)) appeared solely among mutants for the ADE5 gene. Based on these results, it was assumed that there are two independent mechanisms responsible for reversions of spontaneous mutations in this gene. The DNA sequence that can compensate for the P. methanolica ade5 mutation and probably is the structural P-ADE5 gene, was cloned from a genomic library of P. methanolica by the ade6 mutation complementation in the recipient S. cerevisiae strain.

Carbon-Nitrogen Ligases with Glutamine as Amide-N-↗
PubMed1996

Isolation and characterization of a cisplatin-resistant strain of Schizosaccharomyces pombe.

A cis-diamminedichloroplatinum [DDP (cisplatin)]-resistant population of Schizosaccharomyces pombe was developed through chronic exposure of the 972 h- strain to increasing concentrations of the drug. The resulting cells, designated wtr2, were 5.25-fold resistant to DDP, are resistance was retained by clone isolated from this population in the absence of drug for > or = 5 months. After backcrossing and isolation of a single clone, random spore analysis gave a segregation ratio close to 1:1 for DDP resistance and sensitivity. Tetrad analysis confirmed a mendelian 2:2 segregation, suggesting that a single nuclear gene was responsible for the DDP-resistant phenotype. Stable diploids obtained from the mating of a resistant spore carrying the ade6-216 marker with the mei2-102-ade6-210 meiosis-deficient mutant remained resistant, indicating that the resistant phenotype was expressed dominantly. There was no difference between the accumulation of the DDP analog [3H]dichloro(ethylenediamine)-platinum(II) into whole cells derived from the sensitive and the resistant spores obtained from the last backcross. The resistant clones from a single tetrad did not have an increased level of glutathione and were collaterally sensitive to cadmium and arsenite. We conclude that in S. pombe, a stable and dominant DDP-resistant phenotype can be mediated by a single allele, that the phenotype is not accompanied by cross-resistance to cadmium or arsenite, and that the mechanism is not associated with a significant alteration in glutathione level or DDP uptake.

Alleles↗
PubMed1998

[New phenotypic manifestation of the ad2 mutation in Saccharomyces cerevisiae yeast--the inability to grow on a synthetic medium with glycerol and hypoxanthine].

The ADE2 gene of Saccharomyces cerevisiae yeast encodes aminoimidazole ribonucleotide-carboxylase (AIR-carboxylase), an enzyme catalyzing the sixth stage of purine nucleotide biosynthesis. Strains bearing the ade2 mutation are able to grow on a glucose-containing synthetic medium with the addition of adenine or hypoxanthine, which under the action of the cellular phosphoribosyltransferases are converted into adenosine monophosphate and inosine monophosphate, respectively. Our studies showed that ade2 mutants were unable to grow on a synthetic medium with glycerol and hypoxanthine. This newly described feature is not constitutively manifested, because some strains can contain suppressor mutations which restore the ability to grow on a synthetic medium with glycerol and hypoxanthine. The ade4, ade5, ade8, ade6, and ade7 mutations were found to suppress the phenotypic manifestation of the ade2 mutations via inactivation of enzymes catalyzing the first, second, third, fourth and fifth stages of purine biosynthesis, while the ade1 mutation, which inactivates enzyme of the seventh stage, lacks suppressive activity. Strains with single adenine mutations, ade4, ade5, ade8, ade6, ade7, or ade1 grow on glycerol- and hypoxanthine-containing media. Our data suggest that the new property of the ade2 mutations could be associated with the accumulation of the AIR-carbole-ribonucleotide. A mutation resulting in the requirement for serine on the medium with glycerol, but not glucose, is described.

Carboxy-Lyases↗
PubMed1996

Mutational analysis of the gene for Schizosaccharomyces pombe RNase MRP RNA, mrp1, using plasmid shuffle by counterselection on canavanine.

Reverse genetics in fission yeast is hindered by the lack of a versatile established plasmid shuffle system. In order to screen efficiently and accurately through plasmid-borne mutations in the essential gene for the RNA component of RNase MRP, mrp1, we have developed a system for plasmid shuffling in fission yeast using counterselection on canavanine. The system takes advantage of the ability of the Saccharomyces cerevisiae CAN1 gene to complement a Schizosaccharomyces pombe can1-1 mutation. Two general use plasmids were constructed that allow directional cloning and initial selection for histidine before counterselection by canavanine. The strain constructed for plasmid shuffling carries auxotrophic markers for ade6, leul, ura4 and his3 along with the can1-1 mutation. Using this system we examined several partial deletions and point mutations in conserved nucleotides of Schizosaccharomyces pombe RNase MRP RNA for their ability to complement a chromosomal deletion of the mrp1 gene. The degree of background canavanine resistance as well as plasmid-plasmid recombination encountered in these experiments was sufficiently low to suggest that the system we have set up for counterselection by canavanine in fission yeast using multicopy plasmids will be widely useful.

Base Sequence↗
PubMed1997

Type II myosin involved in cytokinesis in the fission yeast, Schizosaccharomyces pombe.

We have cloned an unique gene encoding the heavy chain of a type II myosin in the fission yeast, Schizosaccharomyces pombe. The myo2+ gene encodes a protein of 1526 amino acids with a predicted molecular weight of 177 kDa and containing consensus binding motifs for both essential and regulatory light chains. The S. pombe myo2+ head domain is 45% identical to myosin IIs from Saccharomyces cerevisiae and Homo sapiens and 40% identical to Drosophila melanogaster Structurally, myo2+ most closely resembles budding yeast MYO1, the tails of both myosin IIs containing a number of proline residues that are predicted to substantially disrupt the ability of these myosins to form coiled coils. The myo2+ gene is located on chromosome III, 8.3 map units from ade6+. Deletion of approximately 70% of the coding sequence of myo2+ is lethal but myo2delta spores can acquire a suppressor mutation that allows them to form viable microcolonies consisting of filaments of branched cells with aberrant septa. Overexpression of myo2+ results in the inhibition of cytokinesis; cells become elongated and multinucleate and fail to assemble a functional cytokinetic actin ring and are either aseptate or form aberrant septa. These results suggest that a contractile actin-myosin based cytokinetic mechanism appeared early in the evolution of eukaryotic cells and further emphasise the utility of fission yeast as a model organism in which to study the molecular and cellular basis of cytokinesis.

Amino Acid Sequence↗
PubMed2000

Sequence analysis of two cosmids from Schizosaccharomyces pombe chromosome III.

We report the complete sequence of two cosmids, SPCC895 (38457 bp insert, EMBL Accession No. AL035247) and SPCC1322 (42068 bp insert, EMBL Accession No. AL035259), localized on chromosome III of the Schizosaccharomyces pombe genome. Fourteen Coding DNA sequences (CDSs) were identified in SPCC895 and 17 in SPCC1322. Two known genes were found in each cosmid: map2 and gms1 on SPCC895, encoding the mating type P-factor precursor and an UDP-galactose transporter, respectively, and bub1 and ade6 in SPCC1322, encoding a protein kinase and a phosphoribosylaminoimidazole carboxylase, respectively. The fission yeast K RNA gene has been localized to SPCC895. Three ribosomal proteins have been predicted among these two cosmids. Nine CDSs similar to known proteins were found on SPCC895, and seven on SPCC1322. They include putative genes for an uridylate kinase, a proteasome catalytic component, an ion transporter, a checkpoint protein, a translation initiation protein, a SNARE complex protein, a protein involved in cytoskeletal organization, a spindle pole body-associating protein, pre-mRNA splicing factor RNA helicase, a 3'-5' exonuclease for RNA 3' ss-tail, an UTP-glucose-1-phosphate uridylyltransferase, a leukotriene A(4) hydrolase, a member of the RanBP7-importin beta-Cse1p superfamily, a Ca(++)-calmodulin-dependent serine/threonine protein kinase and a prohibitin antiproliferative protein. One CDS is predicted to be an integral membrane protein. One CDS from SPCC895 is similar to a CDS of unknown function from Saccharomyces cerevisiae and three from SPCC1322 are similar to CDSs of unknown function from Candida albicans, S. cerevisiae and Sz. pombe, respectively. Finally, one CDS of SPCC895 and three of SPCC1322 correspond to orphan genes.

Amino Acid Sequence↗
PubMed1992

Species specific protein--DNA interactions may determine the chromatin units of genes in S.cerevisiae and in S.pombe.

Yeast genes, such as URA3, are chromatin units characterized by positioned nucleosomes and flanking nuclease sensitive regions (NSRs). To investigate the structural determinants at the chromatin level in vivo, the URA3 gene was dissected into three parts (U5', Umid and U3'), and the chromatin structures of the individual parts were analysed after insertion into minichromosomes and after chromatin assembly in vivo in Saccharomyces cerevisiae. While nucleosome positions were altered on Umid, the 5'-end and the 3'-end of URA3 maintained their native structures (a positioned nucleosome and a NSR each) independent of the site or orientation of insertion. This suggests that the chromatin unit of the native URA3 gene is dominated by strong protein boundaries at the 5'- and 3'-ends. In an alternative approach, we investigated whether nucleosome positions or NSRs were maintained when the whole URA3 gene was placed on a shuttle vector and assembled into chromatin by Schizosaccharomyces pombe providing different proteins, but the same nucleosomal spacing. In a complementary exchange experiment, the ade6 gene of S.pombe was shuttled to S.cerevisiae. In spite of a general conservation of histone proteins and nucleosome core structures, neither nucleosome positions nor NSRs were maintained in the heterologous background. The results demonstrate that chromatin structures are species specific and that the structural boundaries of yeast genes may be dominated by strong species specific protein-DNA interactions.

Chromatin↗
PubMed2005

Construction and characterization of a series of vectors for Schizosaccharomyces pombe.

A set of vectors was created to allow cloning and expression studies in Schizosaccharomyces pombe. These vectors had a uniform backbone with an efficient Sz. pombe ARS, ARS3002, but different selectable markers--his3+, leu1+, ade6+ and ura4+. The vectors functioned efficiently as autonomously replicating plasmids that could also be converted into integrating vectors. The ura4+-containing vector was used to construct a Sz. pombe genomic library.

Gene Library↗
PubMed2006

Growth arrest and chromosome instability in aneuploid yeast.

Aneuploid generation and stability are biologically important. In the present study, we investigated fission yeast aneuploids, focusing on the process through which aneuploidy is resolved into stable euploidy. The viability and growth patterns of aneuploid spores were greatly influenced by culture conditions, including nutrition and temperature. Germ tube formation and DNA synthesis in a major portion of aneuploids were greatly delayed or arrested. Observation of individual spores and their growth profiles revealed that a certain type(s) of aneuploid resolved its aneuploidy into normal euploids through anomalous cell divisions, which in many cases produced dead cells. Another type of aneuploid, disomy of chromosome 3, the only maintainable aneuploid between n and 2n, showed a peculiar cell division arrest phenotype under a certain growth condition. Microcolonies that formed from this type of aneuploid often contained a population of cells that became incompetent for cell division. This cell division arrest was not due to a nutritional limitation. During this peculiar process of colony formation, stable haploids or diploids were frequently produced. All other types of aneuploids are usually inviable, at least under our experimental conditions. To examine the aneuploid issue more systematically, we constructed a system to select for disomy of chromosome 1 or 2 using intragenic complementation of ade6-M210 and -M216 alleles. This genetic selection system revealed that fission yeast aneuploids can be stabilized through structural chromosome changes, including partial duplication and circular mini-chromosomes.

Aneuploidy↗
PubMed1994

Functional expression of human poly(ADP-ribose) polymerase in Schizosaccharomyces pombe results in mitotic delay at G1, increased mutation rate, and sensitization to radiation.

The activity of poly(ADP-ribose) polymerase (PADPRP), a chromatin-associated enzyme present in most eukaryotic cells, is stimulated by DNA strand breaks, suggesting a role for the enzyme in the cellular response to DNA damage. However, the primary function of PADPRP remains unknown. We have selected Schizosaccharomyces pombe as a simple eukaryotic system in which to study PADPRP function because this fission yeast shares with mammalian cells important cellular features possibly associated with poly-(ADP-ribos)ylation pathways. We investigated the existence of an endogenous yeast PADPRP by DNA and RNA hybridization to mammalian probes under low-stringency conditions and by PADPRP activity assays. Our data indicate that fission yeasts are naturally devoid of PADPRP. We therefore isolated S. pombe strains expressing PADPRP by transformation with a human full-length PADPRP cDNA under the control of the SV40 early promoter. The human PADPRP construct was transcribed and translated in S. pombe, generating a major transcript of the same size (3.7 kb) as that detected in mammalian cells and a 113-kDa polypeptide, identical in size to the native human PADPRP protein. Yeast recombinant PADPRP was enzymatically active and was recognized by antibodies to human PADPRP. S. pombe cells expressing PADPRP (SPT strains) showed a stable phenotype that was characterized by: (i) cell cycle retardation as a result of a specific delay at the G1 phase, (ii) decreased cell viability in stationary cultures, (iii) enhanced rates of spontaneous and radiation-induced ade6-ade7 mutations, and (iv) increased sensitivity to radiation. SPT strains may prove efficient tools with which to investigate PADPRP functions in eukaryotic cells.

Dose-Response Relationship, Radiation↗
PubMed1993

Molecular and genetic characterization of SPT4, a gene important for transcription initiation in Saccharomyces cerevisiae.

Mutations in the SPT4 gene of Saccharomyces cerevisiae were isolated as suppressors of delta insertion mutations that interfere with adjacent gene transcription. Recent genetic evidence indicates that the SPT4 protein functions with two other proteins, SPT5 and SPT6, in some aspect of transcription initiation. In this work we have characterized the SPT4 gene and we demonstrate that spt4 mutations, like spt5 and spt6 mutations, cause changes in transcription. Using the cloned SPT4 gene, spt4 null mutations were constructed; in contrast to spt5 and spt6 null mutants, which are inviable, spt4 null mutants are viable and have an Spt- phenotype. The DNA sequence of the SPT4 gene predicts a protein product of 102 amino acids that contains four cysteine residues positioned similarly to those of zinc binding proteins. Mutational analysis suggests that at least some of these cysteines are essential for SPT4 function. Genetic mapping showed that SPT4 is a previously unidentified gene that maps to chromosome VII, between ADE6 and CLY8.

Base Sequence↗
PubMed1993

A study of integrative transformation in Schizosaccharomyces pombe.

Using the one-step gene disruption technique, we studied the effect of various parameters on the disruption frequency (percentage of homologous integrants) and transformation efficiency (number of transformants per microgram of input DNA) of integrative transformation in Schizosaccharomyces pombe. We used suc1 as the target gene for disruption and ura4 as the selectable marker. Our results are as follows. 1) Use of the strong adh1 promoter to drive the expression of ura4 did not affect the disruption frequency but modestly increased the transformation efficiency. 2) The transformation method had a profound effect, with the lithium acetate method yielding both a 10-fold higher disruption frequency compared to the protoplast method and a 5- to 10-fold higher transformation efficiency. 3) The presence of increasing amounts of non-homologous sequences at the ends of the transforming DNA decreased the disruption frequency by up to 5-fold but had no effect on the transformation efficiency. We also describe the use of the sup3-5 allele in an ade6-704 genetic background to discriminate between the products of homologous versus non-homologous integration, thereby promoting the identification of rare homologous integrants.

Alleles↗
PubMedDate not supplied

Physical mapping of the Schizosaccharomyces pombe histone genes.

The histone-encoding genes in Schizosaccharomyces pombe were physically mapped by hybridisation to filters containing cosmid and P1 genomic libraries. The H2A.2 gene and the H2A.1-H2B.1 gene pair mapped between the ade6 and rikI genes on chromosome III. The three H4-H3 gene pairs were mapped to three different regions by a H4.1 probe. Southern analysis of clones from each region revealed the positions of the three H4-H3 gene pairs. H4.1-H3.1 was localised to chromosome I between the mei2 and rad1 genes; H4.2-H3.2 mapped between rad3 and cdc2 on chromosome II; H4.3-H3.3 was localised to a region between the nuc1 and puc1 genes on chromosome II.

Blotting, Southern↗
PubMed1991

Synchronized meiosis and recombination in fission yeast: observations with pat1-114 diploid cells.

The mutation pat1-114 has been used to synchronize meiosis in the fission yeast Schizosaccharomyces pombe. We have investigated several aspects of such synchronized meiotic cultures. In both pat1-114 and pat1+ diploids, meiotic landmark events are initiated at the same time after meiosis induction, but synchrony is much more pronounced in the pat1-114-driven meiosis. Commitment to recombination and to meiosis have been timed at 2 h after meiotic induction. Due to a seven-fold reduction of intragenic recombination frequency in the ade6 region of pat1-114 diploids, physical analysis of recombination has not been possible. We have distinguished three factors that influence intragenic recombination frequencies: temperature, azygotic versus zygotic meiosis, and the nature of the pat1 allele. Differences and similarities in the timing of meiotic landmarks in S. cerevisiae and S. pombe are discussed.

Cell Cycle↗
PubMed1985

Direct selection of mutants influencing gene conversion in the yeast Schizosaccharomyces pombe.

In Schizosaccharomyces pombe, a suppressor-active mutation at the anticodon site of the tRNASerUCA gene sup3 leads to opal (UGA)-specific suppression. Second-site mutations (rX) in sup3 inactivate the suppressor. The sup3-UGA, rX double mutants are genetically unstable in meiotic selfings, due to the intergenic transfer of information between sup3 and the unlinked genes sup9 and sup12 (Hofer et al. 1979; Munz and Leupold 1981; Munz et al. 1982). These three genes have considerable sequence homology over about 200 base pairs (Hottinger et al. 1982). Mutants showing a decrease or an increase of the meiotic instability at sup3 have been selected. One mutation (rec3-8) increases both the genetic instability and the frequency of intragenic recombination in sup3 by one order of magnitude. It has no effect on the stability of the nonsense alleles arg1-230 (UAA), ade6-704 and ural1-61 (UGA) or on the frequency of crossing-over between sup3 and the closely linked gene cdc8. The existence of a common genetic control over intragenic recombination and genetic instability at sup3 provides a direct way of selecting for rec mutants in homothallic haploid strains of S. pombe carrying a suppressor-inactive allele of sup3. It also supports the hypothesis that the instability of mutant alleles of this gene is due to chromosome mispairing at meiosis allowing sup3 to pair with sup9 or sup12 and then to undergo recombination by gene conversion restoring the suppressor-active allele sup3-UGA from the suppressor-inactive allele sup3-UGA, rX.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗