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A J Klar

Publications and source records attributed to A J Klar.

At least 55 records · Page 3Linked to original sources
PubMed1990

Regulation of fission yeast mating-type interconversion by chromosome imprinting.

Mating types of the fission yeast Schizosaccharomyces pombe interchange nonrandomly in a cell lineage so that only one cell among four granddaughters of a cell ever switches, and the sister of the newly switched cell switches efficiently in consecutive cell divisions, thereby producing chains of recurrent switching. The programme of cellular differentiation is mediated by inheritance of parental DNA chains at the mating type locus (mat1) by progeny cells. This review summarizes recent results suggesting that two types of imprinting events at the mat1 locus are required to generate the specific pattern of switching in a cell lineage. One of those is a site- and strand- specific event that is required before the mat1 locus can be cleaved in vivo. The other is a double-stranded break at mat1 that is healed by gene conversion in the progeny cells resulting in switching the mat1 locus.

Cell Differentiation↗
PubMed1987

The mother-daughter mating type switching asymmetry of budding yeast is not conferred by the segregation of parental HO gene DNA strands.

The Saccharomyces cerevisiae HO gene, which encodes a site-specific endonuclease, is transcribed in the parent (mother) cell but not in the daughter (bud) cell. Consequently mother cells can switch their mating type whereas bud cells cannot. Whether or not the different capabilities of these cells are due to a nonrandom segregation of parental HO gene DNA strands to progeny cells is tested here by assaying the pattern of switching in cells where the HO gene is inverted in the chromosome. In an inverted HO strain, as is the case with the normal HO arrangement strains, only the mother cells were found to switch. Thus, the possibility of asymmetric segregation of parental HO gene DNA strands does not regulate the mother-daughter asymmetry of switching.

Cell Cycle↗
PubMed1986

Initiation of meiotic recombination by double-strand DNA breaks in S. pombe.

Mitotic gene conversion and reciprocal recombination have recently been shown to be efficiently initiated by double-strand DNA breaks (DSBs) in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. We tested whether DSBs could also initiate meiotic recombination at the mat1 locus in S. pombe. The mat1 switching-mechanism-generated DSB found in mitotically growing cells can be repaired without mat1 switching, since strains deleted for both donor loci (mat2-P and mat3-M) have the break but do not produce inviable cells. A (mat1-P X mat1-M) cross produced a high frequency (20%) of 3:1 gene conversions of mat1 in meiotic tetrads. Gene conversion events were associated with the recombination of flanking markers. Strains lacking the DSB failed to convert. Thus, the DSB at mat1 promotes efficient meiotic recombination in fission yeast.

DNA↗
PubMed1986

Double-strand breaks can initiate meiotic recombination in S. cerevisiae.

We investigated the effects of double-strand breaks on meiotic recombination in yeast. A double-strand break was introduced at the MATa locus by sporulation of a MAT alpha inc/MATa diploid under inducing conditions for the HO-encoded endonuclease; 14% of the resulting tetrads had undergone 4 alpha:0a conversion. Conversion at MAT was associated with co-conversion of a closely linked marker and an increased recombination frequency for flanking markers. We also studied the sporulation products of a diploid heterozygous at the HIS4 locus for an insertion of a 100 bp fragment of MATa containing the HO endonuclease cut site. Under inducing conditions, a significant number of tetrads were formed that had undergone gene conversions in favor of the HIS4+ allele. Although double-strand breaks can initiate meiotic recombination in yeast, the data suggest that they do not normally do so.

DNA↗
PubMed1986

Cloning and characterization of four SIR genes of Saccharomyces cerevisiae.

Mating type in the yeast Saccharomyces cerevisiae is determined by the MAT (a or alpha) locus. HML and HMR, which usually contain copies of alpha and a mating type information, respectively, serve as donors in mating type interconversion and are under negative transcriptional control. Four trans-acting SIR (silent information regulator) loci are required for repression of transcription. A defect in any SIR gene results in expression of both HML and HMR. The four SIR genes were isolated from a genomic library by complementation of sir mutations in vivo. DNA blot analysis suggests that the four SIR genes share no sequence homology. RNA blots indicate that SIR2, SIR3, and SIR4 each encode one transcript and that SIR1 encodes two transcripts. Null mutations, made by replacement of the normal genomic allele with deletion-insertion mutations created in the cloned SIR genes, have a Sir- phenotype and are viable. Using the cloned genes, we showed that SIR3 at a high copy number is able to suppress mutations of SIR4. RNA blot analysis suggests that this suppression is not due to transcriptional regulation of SIR3 by SIR4; nor does any SIR4 gene transcriptionally regulate another SIR gene. Interestingly, a truncated SIR4 gene disrupts regulation of the silent mating type loci. We propose that interaction of at least the SIR3 and SIR4 gene products is involved in regulation of the silent mating type genes.

Cloning, Molecular↗
PubMed1985

Map positions of yeast genes SIR1, SIR3 and SIR4.

The HML and HMR loci in the yeast Saccharomyces cerevisiae each contain a complete copy of mating-type information. HML and HMR normally are transcriptionally inactive due to four unlinked genes, known as MAR or SIR or CMT. The map position of MAR1 (SIR2) has been reported previously; it is located on the left arm of chromosome IV, 27 cM from the centromere. Using conventional meiotic and mitotic mapping combined with recombinant DNA techniques, we have mapped three other SIR genes. SIR1 maps near the telomere of the right arm of chromosome XI; SIR3 (MAR2) maps to the right arm of chromosome XII, 31 cM distal to URA4; and SIR4 maps to the right arm of chromosome IV, 16 cM proximal to LYS4.

Alleles↗
PubMed1985

SUM1, an apparent positive regulator of the cryptic mating-type loci in Saccharomyces cerevisiae.

The mating-type information residing at the HML and HMR loci in Saccharomyces cerevisiae is kept unexpressed by the action of at least four MAR (or SIR) loci. To determine possible interactions between the MAR/SIR gene products and to find new regulatory loci, we sought extragenic suppressors of the mar1-1 mutation. A strain with the genotype HMLa MAT alpha HMRa mar1-1 is unable to mate because of the simultaneous expression of a and alpha information. A mutant of this strain was isolated that exhibits an alpha phenotype and, therefore, presumably fails to express the HML and HMR loci. We designate the new locus SUM1 (suppressor of mar). The mutation is recessive, centromere unlinked and does not correspond to the MAT, HML, HMR, SIR1, MAR1, MAR2 (SIR3) or SIR4 loci. The sum1 mutation affects expression of both a and alpha information at the HM loci. Suppression by sum1-1 is neither allele specific nor locus specific as it suppresses a deletion mutation of the MAR1 locus and mutations in SIR3 and SIR4. The sum1-1 mutation has no discernible phenotype in a Mar+ strain. We propose that the MAR/SIR gene products negatively regulate the SUM1 locus, the gene product of which is necessary for expression of the HM loci.

Crosses, Genetic↗
PubMed1984

Regulation of mating-type information in yeast. Negative control requiring sequences both 5' and 3' to the regulated region.

The genome of the yeast Saccharomyces cerevisiae contains three complete copies of the genetic information governing cell mating type. Normally, only the information in one of the copies (the MAT locus) is expressed; the other two copies (HML and HMR) are repressed and serve as donors of mating-type sequences that can be transposed to MAT in cells capable of switching mating type. We have mutagenized the silent HMR locus and have found that the repression of this locus requires two sites, one lying on each side of the mating-type sequences at HMR. The regulatory sites are positioned outside of the sequences that are included in the pair of divergent transcripts coded for by HMR, and lie about 1000 base-pairs to either side of the central promoter region of the locus. Deletion of one of the regulatory sites results phenotypically in complete loss of repression, whereas deletion of the other site gives only partial loss of control. Both of the sites are associated with an autonomous replication activity, though the relationship between this activity and the process of repression is unclear.

Base Sequence↗
PubMed1984

Rearrangements of the transposable mating-type cassettes of fission yeast.

The fission yeast, Schizosaccharomyces pombe, switches mating type every few cell divisions. Switching is controlled by the genes of the mating-type locus, which consists of three components, mat1, mat2-P and mat3-M, each separated by approximately 15 kb. Copy transposition of P (Plus) or M (Minus) information from mat2-P or mat3-M into the expression locus mat1 mediates cell type switching. The mating-type locus undergoes events at high frequency (10(-2)-10(-6)) which stabilize one or other mating type. These events are shown to be rearrangements which result in either deletion or insertion of DNA between cassettes.

Ascomycota↗
PubMed1984

Genes required for initiation and resolution steps of mating-type switching in fission yeast.

The fission yeast Schizosaccharomyces pombe switches mating type by transposition of a copy of DNA derived from either of the two storage cassettes, mat2 -P and mat3 -M, into the expression locus, mat1 . The recombinational event of switching is initiated by a double-stranded DNA break present in approximately 20% of the molecules at mat1 . Fifty-three mutants defective in switching of mating type have been isolated previously, and each has been assigned to 1 of 10 linkage groups. One group consists of cis-acting mutations at mat1 , which reduce the amount of the DNA double-strand cut. The remaining nine groups are mutations in genes that are unlinked to the mating-type locus and are studied here. Three ( swi1 , -3, -7) are required for formation of the double-strand cut, whereas the others are not. Mutants of three genes ( swi4 , -8, -9) undergo high-frequency rearrangement of the mating-type locus indicative of errors of resolution of recombinational intermediates. The remaining three ( swi2 , -5, -6) have normal levels of cut, do not make errors of resolution, and possibly are required either for efficient utilization of the cut or determining the directionality of switching. The data suggest that the switching process can be dissected into genetically distinguishable steps.

Ascomycota↗
PubMed1984

Involvement of double-strand chromosomal breaks for mating-type switching in Saccharomyces cerevisiae.

The yeast S. cerevisiae switches a and alpha cell types by a transposition mechanism that replaces genetic information residing at the mating-type locus (MAT) with information copied from either of the two donor loci, HML and HMR. The donor HML and HMR loci contain the same genetic information as the MATa and MAT alpha alleles, yet they do not switch. Additionally, Strathern et al. (1982) have described an in vivo double-strand DNA break found at subgenomic levels (approximately 2% of MAT DNA) within the MAT locus but not within HML and HMR. We have examined the role of this double-strand DNA break in the switching process. Cell lineage studies show that strains containing deletions of the donor HML and HMR loci produce lethal progeny in the exact pattern described for MAT switching in standard strains. Our interpretation is that the double-strand MAT break in the deletion strains cannot be repaired because of the lack of the donor loci, resulting in cell death. We suggest that the double-strand DNA break is an initiating event for switching and that this event is lethal in the absence of the donor loci. MAT mutants isolated as survivors from this "pedigree of death" define a site required for switching where the double-strand break occurs. We have also examined marl mutant strains in which the donor loci are expressed and observed to switch (Klar et al. 1981a). The double-strand DNA cut appears at the HM loci in these strains. Thus, there is a strong correlation between the presence or absence of the double-strand break at each cassette and its ability or inability to switch as observed at the single cell level.

Alleles↗
PubMed1983

A site-specific endonuclease essential for mating-type switching in Saccharomyces cerevisiae.

We have detected two site-specific endonucleases in strains of Saccharomyces cerevisiae. One endonuclease, which we call YZ endo, is present only in yeast strains that are undergoing mating-type interconversion. The site at which YZ endo cleaves corresponds to the in vivo double-strand break occurring at the mating-type locus in yeast undergoing mating-type interconversion. YZ endo generates a site-specific double-strand break having 4-base 3' extensions terminating in 3' hydroxyl groups. The site of cleavage occurs in the Z1 region near the YZ junction of the mating-type locus. Mutant mating-type loci known to decrease the frequency of mating-type interconversion are correspondingly poor substrates for YZ endo in vitro. In vitro analysis of a number of such altered recognition sites has delimited the sequences required for cleavage. The molecular genetics of mating-type interconversion is discussed in the context of this endonucleolytic activity. The second endonuclease, which we refer to as Sce II, is present in all strains of S. cerevisiae we have examined. The cleavage site of Sce II has been determined and proves to be unrelated to the cleavage site of YZ endo.

Base Sequence↗
PubMed1983

Efficient production of a ring derivative of chromosome III by the mating-type switching mechanism in Saccharomyces cerevisiae.

The mating-type switches in the yeast Saccharomyces cerevisiae occur by unidirectional transposition of replicas of unexpressed genetic information, residing at HML or HMR, into the mating-type locus (MAT). The source loci, HML and HMR, remain unchanged. Interestingly, when the HM cassettes are expressed, as in marl strains, the HML and HMR cassettes can also efficiently switch, apparently by obtaining genetic information from either of the other two cassettes (Klar et al., Cell 25:517-524, 1981). We have isolated a novel chromosome III rearrangement in heterothallic (marl ho) strains, which is also produced efficiently in marl HO cells, presumably the consequence of a recombination event between HML and HMR. The fusion results in the loss of sequences which are located distal to HML and to HMR and produces a ring derivative of chromosome III. Cells containing such a ring chromosome are viable as haploids; apparently, no essential loci are located distal to the HM loci. The fusion cassette behaves as a standard HM locus with respect to both regulation by the MAR/SIR control and its role in switching MAT.

Base Sequence↗
PubMed1982

Directionality of yeast mating-type interconversion.

The mating-type a and alpha alleles of the yeast Saccharomyces cerevisiae interconvert by a transposition-substitution reaction where replicas of the silent mating loci, at HML and HMR, are transmitted to the expressed mating-type locus (MAT). HML is on the left arm and HMR on the right arm, while MAT is in the middle of chromosome III. Cells with the genotype HML alpha HMRa switch mating type efficiently at a frequency of about 86%. Since well over 50% of the cells switch, it is thought that switches do not occur randomly, but are directed to occur to the opposite mating-type allele. In contrast, we report that strains possessing the reverse HMLa HMR alpha arrangement switch (phenotype) inefficiently at a maximum of about 6%. The basis for this apparent reduced frequency of switching is that these strains preferentially yield futile homologous MAT locus switches--that is, MATa to MATa and MAT alpha to MAT alpha--and consequently, most of these events are undetected. We used genetically marked HM loci to demonstrate that alpha cells preferentially choose HMR as donor and a cells preferentially choose HML as donor, irrespective of the genetic content of the silent loci. Because of this feature, HML alpha HMRa strains generate predominantly heterologous while HMLa HMR alpha strains produce predominantly homologous MAT switches. The control for directionality of switching therefore is not at the level of transposing heterologous mating-type information, but only at the level of choosing HML versus HMR as the donor. In strains where the preferred donor locus is deleted, the inefficient donor becomes capable of donating efficiently. Thus the preference seems to be mediated by competition between the HM loci for donating information to MAT.

Alleles↗
PubMed1982

Homothallic switching of yeast mating type cassettes is initiated by a double-stranded cut in the MAT locus.

A double-stranded DNA cut has been observed in the mating type (MAT) locus of the yeast Saccharomyces cerevisiae in cultures undergoing homothallic cassette switching. Cutting is observed in exponentially growing cells of genotype HO HML alpha MAT alpha HMR alpha or HO HMLa MATa HMRa, which switch continuously, but not in a/alpha HO/HO diploid strains, in which homothallic switching is known to be shut off. Stationary phase cultures do not exhibit the cut. Although this site-specific cut occurs in a sequence (Z1) common to the silent HML and HMR cassettes and to MAT, only the Z1 sequence at the MAT locus is cut. The cut at MAT occurs in the absence of the HML and HMR donor cassettes, suggesting that cutting initiates the switching process. An assay for switching on hybrid plasmids containing mata- cassettes has been devised, and deletion mapping has shown that the cut site is required for efficient switching. Thus a double-stranded cut at the MAT locus appears to initiate cassette transposition-substitution and defines MAT as the recipient in this process.

Chromosome Mapping↗
PubMed1981

Regulation of transcription in expressed and unexpressed mating type cassettes of yeast.

The genes that control the a, alpha and a/alpha cell types in Saccharomyces are carried on transposable elements known as a and alpha cassettes which reside at three different chromosomal loci. Examination of the transcripts by R-looping and filter hybridization indicates that each cassette is capable of producing two divergent transcripts. Cassettes at the MAT locus are transcribed constitutively. Transcription of cassettes at HML and HMR is prevented by trans-acting negative regulators.

DNA Transposable Elements↗