PubMed Health⌕ Search

Biomedical subjects

A J Klar

Publications and source records attributed to A J Klar.

At least 37 records · Page 2Linked to original sources
PubMed1996

Chromosomal inheritance of epigenetic states in fission yeast during mitosis and meiosis.

Inheritance of the active and inactive states of gene expression by individual cells is crucial for development. In fission yeast, mating-type region consists of three loci called mat1, mat2, and mat3. Transcriptionally silent mat2 and mat3 loci are separated by a 15 kb interval, designated the K-region, and serve as donors of information for transcriptionally active mat1 interconversion. In a strain carrying replacement of 7.5 kb of the K-region with the ura4 gene, we discovered that ura4 silencing and efficiency of mating-type switching were covariegated and were regulated by an epigenetic mechanism. Genetic analyses demonstrated that epigenetic states were remarkably stable not only in mitosis but also in meiosis and were linked to the mating-type region. This study indicates that different epigenetic states are heritable forms of chromatin organization at the mat region.

Blotting, Southern↗
PubMed1994

A model for specification of the left-right axis in vertebrates.

The mechanisms that underlie the formation of the left-right embryonic axis in vertebrates are not known. The programmed pattern of cell-type change in fission yeast results from the inheritance of specific chromatids of the parental chromosome. Here, I address how such a model may explain left-right specification of the viscera in mice. The model proposes that DNA replication produces different chromatids, and that these specific chromatids of both homologs are nonrandomly segregated to daughter cells to specify the left-right axis of the embryo. Such a model presents a simple explanation of the interesting phenotype of the newly discovered insertional mutation inv in mice, which causes reversal of the left-right axis, proposing that it is caused by a chromosomal inversion.

Animals↗
PubMed1994

Three additional linkage groups that repress transcription and meiotic recombination in the mating-type region of Schizosaccharomyces pombe.

The mating-type genes of Schizosaccharomyces pombe are found at three locations in the same chromosomal region. These genes are in an active configuration at the mat1 locus and in an inactive configuration at the mat2 and mat3 loci. The mechanism that represses transcription of mat2 and mat3 also inactivates other promoters introduced nearby and is accompanied by a block to meiotic recombination in the mat2-mat3 interval, suggesting that this mechanism involves a particular chromatin structure. We present evidence that the transcription and recombination blocks require three newly defined trans-acting loci, clr2, clr3 and clr4, in addition to the previously identified clr1, rik1 and swi6 loci. We also investigated the role of mat2 cis-acting sequences in silencing. Four cis-acting elements that repress mat2 in a plasmid context were previously identified. Deletion of two of these elements proved to have little effect in a chromosomal context. However, when combined with mutations in trans-acting genes, deletion of the same two elements greatly enhanced mat2 expression. The observed cumulative effects suggest a redundancy in the silencing mechanism.

Gene Deletion↗
PubMed1994

Sap1, a protein that binds to sequences required for mating-type switching, is essential for viability in Schizosaccharomyces pombe.

The pattern of mating-type switching in cell pedigrees of the fission yeast Schizosaccharomyces pombe is dictated by the inheritance of specific DNA chains at the mating-type locus (mat1). The recombination event essential for switching is initiated by a site-specific double-strand break at mat1. The switch-activating protein, Sap1, binds in vitro to a mat1 cis-acting site that was shown earlier to be essential for efficient mating-type switching. We isolated the sap1 gene by using oligonucleotides corresponding to the amino acid sequence of purified Sap1 protein. The sequence of that gene predicted a 30-kDa protein with no significant homology to other canonical DNA-binding protein motifs. To facilitate its biochemical characterization, Sap1 was expressed in Escherichia coli. The protein expressed in bacteria displayed the same DNA-binding specificities as the protein purified from S. pombe. Interestingly, analysis of a sap1 null mutation showed that the gene is essential for growth even in a strain in which mating-type switching is prohibited because of a defect in generation of the double-strand break. Thus, the sap1 gene product implicated in mating-type switching is shown to be essential for cell viability.

Amino Acid Sequence↗
PubMed1993

DNA polymerase-alpha is essential for mating-type switching in fission yeast.

In the fission yeast Schizosaccharomyces pombe, the double-stranded chromosomal break (DSB) at the mating-type locus (mat1) initiates recombination during mating-type switching. A constant DSB level is maintained throughout the cell-cycle. In the strand-segregation model for mating-type switching, it was postulated that if the DSB is generated during or soon after mat1 replication, one of the chromatids could be repaired and switched during replication in the next cell cycle, while the other chromatid inherits the break. Here we report a molecular characterization of swi7, one of the genes required for DSB formation. Surprisingly, a gene complementing the swi7 mutation maps to chromosome I and encodes S. pombe DNA polymerase-alpha. Disruption of this gene is lethal in both switching and non-switching strains, as expected. S. pombe DNA polymerase-alpha must therefore play a role in generating the DSB at mat1, suggesting that DSB formation is coupled with DNA replication.

Amino Acid Sequence↗
PubMed1993

Lineage-dependent mating-type transposition in fission and budding yeast.

The basis of cellular differentiation is perhaps best understood in the yeast mating-type switching system. The yeast cell produces daughter cells that differ from each other or from their parent cell via developmentally regulated genomic rearrangements. Recent experiments on cell-type determination in fission yeast have revealed that this process is determined by the inheritance of specific parental chromosome strands by the progeny cells.

DNA Transposable Elements↗
PubMed1993

Directionality of fission yeast mating-type interconversion is controlled by the location of the donor loci.

Cells of homothallic strains of Schizosaccharomyces pombe efficiently switch between two mating types called P and M. The phenotypic switches are due to conversion of the expressed mating-type locus (mat1) by two closely linked silent loci, mat2-P and mat3-M, that contain unexpressed information for the P and M mating types, respectively. In this process, switching-competent cells switch to the opposite mating type in 72-90% of the cell divisions. Hence, mat2-P is a preferred donor of information to mat1 in M cells, whereas mat3-M is a preferred donor in P cells. We investigated the reason for the donor preference by constructing a strain in which the genetic contents of the donor loci were swapped. We found that switching to the opposite mating type was very inefficient in that strain. This shows that the location of the silent cassettes in the chromosome, rather than their content, is the deciding factor for recognition of the donor for each cell type. We propose a model in which switching is achieved by regulating accessibility of the donor loci, perhaps by changing the chromatin structure in the mating-type region, thus promoting an intrachromosomal folding of mat2 or mat3 onto mat1 in a cell type-specific fashion. We also present evidence for the involvement of the Swi6 and Swi6-mod trans-acting factors in the donor-choice mechanism. We suggest that these factors participate in forming the proposed folded structure.

Base Sequence↗
PubMed1993

The mechanism of fission yeast mating-type interconversion: evidence for two types of epigenetically inherited chromosomal imprinted events.

Mating types of the fission yeast S. pombe interchange such 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 developmental program is known to be mediated through specific parental chromosomal DNA-strand inheritance at the mating-type locus (mat1). A heritable DNA strand- and sequence-specific imprinting event was previously postulated to be required to cleave the chromosome at mat1 to initiate recombination required for switching. The pedigree analysis presented here demonstrates that swi1, swi3, and swi7 genes are required for the DNA cleavage step directly, and not for the imprinting function, since mutations in these genes reduce the efficiency of initial (i.e., switching of one in four granddaughters) and recurrent switching equally. However, when the mat1 gene sequence is placed adjacent to the indigenous mat1 locus, apparently it is imprinted inefficiently, since recurrent switches of the inserted locus occur nearly five times more often than the initial switches. This "runaway switching" is the first evidence formally demonstrating both the existence of and the requirement for an imprinted mat1 DNA, making it cleavable and consequently swichable. Second, the double-stranded break constitutes another imprinted event as it is evidenced to be inherited from the parental cell.

Chromosomes, Fungal↗
PubMed1992

Developmental choices in mating-type interconversion in fission yeast.

Fission yeast cells follow a specific pattern of mating (cell) type switching in single cell pedigrees. Asymmetric cell divisions producing sisters of different developmental fates result from inheritance of specific parental DNA strands according to the classical model of semiconservative replication and segregation.

Gene Expression Regulation, Fungal↗
PubMed1992

The clr1 locus regulates the expression of the cryptic mating-type loci of fission yeast.

The mat2-P and mat3-M loci of fission yeast contain respectively the plus (P) and minus (M) mating-type information in a transcriptionally silent state. That information is transposed from the mat2 or mat3 donor locus via recombination into the expressed mating-type locus (mat1) resulting in switching of the cellular mating type. We have identified a gene, named clr1 (for cryptic loci regulator), whose mutations allow expression of the mat2 and mat3 loci. clr1 mutants undergo aberrant haploid meiosis, indicative of transcription of the silent genes. Production of mRNA from mat3 is detectable in clr1 mutants. Furthermore, the ura4 gene inserted near mat3, weakly expressed in wild-type cells, is derepressed in clr1 mutants. The clr1 mutations also permit meiotic recombination in the 15-kb mat2-mat3 interval, where recombination is normally inhibited. The clr1 locus is in the right arm of chromosome II. We suggest that clr1 regulates silencing of the mat2 and mat3 loci, and participates in establishing the "cold spot" for recombination by organizing the chromatin structure of the mating-type region.

Chromatin↗
PubMed1992

Active genes in budding yeast display enhanced in vivo accessibility to foreign DNA methylases: a novel in vivo probe for chromatin structure of yeast.

Unlike higher eukaryotes, where an inverse correlation has been generally observed between gene expression and methylation of CpG sites, the budding yeast Saccharomyces cerevisiae lacks DNA methylation. Gene regulatory mechanisms can function independently of DNA methylation in yeast, and yeast strains expressing foreign DNA methylases that modify adenine and CpG residues have been found to be viable. We have used such strains to determine whether the transcriptional status of genes can influence the level of their DNA methylation in vivo. Several genes were tested, for example, GAL1, -7, and -10, PHO5, HMRa and HML alpha, and STE2 and STE3. Surprisingly, we found that all the genes displayed severalfold more methylation in the expressed state as compared to the repressed state. This procedure serves as a novel in vivo probe for the chromatin structure of yeast and potentially for higher eukaryotes.

Blotting, Southern↗
PubMed1991

A novel switch-activating site (SAS1) and its cognate binding factor (SAP1) required for efficient mat1 switching in Schizosaccharomyces pombe.

The pattern of parental DNA strand inheritance at the mating type locus (mat1) determines the pattern of mat1 switching in a cell lineage by regulating the formation of the site-specific double-stranded break (DSB) required for mating type interconversion in Schizosaccharomyces pombe. To study the molecular basis of this programmable cell type change, we conducted structural and functional analyses of the DNA sequence flanking the DSB at mat1. We have identified and characterized a DNA-binding activity that interacts with a specific sequence located 140 bp from the DSB site. Deletion analysis of DNA sequences located distal to mat1 cassette revealed the presence of at least two switch-activating sites (SAS1 and SAS2), both of which are required for generating an efficient level of DSBs and consequently, for efficient switching. We found that SAS1 overlaps with the target site of the DNA-binding activity called SAP1 (for switch-activating protein). Point mutations generated in the SAS1 element that adversely affect binding of SAP1 protein in vitro were found to reduce the efficiency of switching in vivo, suggesting the requirement of SAP1 for switching. Pedigree analysis revealed that SAS1 is equally required for initial switching (one switch in four grand-daughters of a cell) and for consecutive switching (where the sister of a recently switched cell switches again), indicating that the two developmentally asymmetric cell divisions required to generate a particular pattern of switching share the same molecular control mechanism.

Base Sequence↗
PubMed1991

The mechanism of fission yeast mating type interconversion: seal/replicate/cleave model of replication across the double-stranded break site at mat1.

The interconversion of cell type in the fission yeast, Schizosaccharomyces pombe, is initiated by a double-stranded break (DSB) found at the mating type locus (mat1). A heritable site- and strand-specific DNA "imprinting" event at mat1 was recently hypothesized to be required to make the mat1 locus cleavable, and the DSB was suggested to be produced one generation before the actual switching event. It is known that only one cell among four granddaughters of a cell ever switches, and the sister of the recently switched cell switches efficiently in consecutive cell divisions. The feature of consecutive switching creates a major difficulty of having to replicate chromosomes possessing the DSB. The mat1 cis-acting leaky mutation, called smt-s, reduces the level of the DSB required for switching and is shown here to be a 27-bp deletion located 50 bp away from the cut site. Determination of the pattern and frequency of switching of the mutant allele by cell lineage studies has allowed us to conclude the following: (1) the chromosome with the DSB is sealed and replicated, then one of the specific chromatids is cleaved again to generate switching-competent cells in consecutive cell divisions and (2) the smt-s mutation affects DNA cleavage and not the hypothesized DNA imprinting step.

Alleles↗
PubMed1991

swi6, a gene required for mating-type switching, prohibits meiotic recombination in the mat2-mat3 "cold spot" of fission yeast.

Mitotic interconversion of the mating-type locus (mat1) of the fission yeast Schizosaccharomyces pombe is initiated by a double-strand break at mat1. The mat2 and mat3 loci act as nonrandom donors of genetic information for mat1 switching such that switches occur primarily (or only) to the opposite mat1 allele. Location of the mat1 "hot spot" for transposition should be contrasted with the "cold spot" of meiotic recombination located within the adjoining mat2-mat3 interval. That is, meiotic interchromosomal recombination in mat2, mat3 and the intervening 15-kilobase region does not occur at all. swi2 and swi6 switching-deficient mutants possess the normal level of double-strand break at mat1, yet they fail to switch efficiently. By testing for meiotic recombination in the cold spot, we found the usual lack of recombination in a swi2 mutant but a significant level of recombination in a swi6 mutant. Therefore, the swi6 gene function is required to keep the donor loci inert for interchromosomal recombination. This finding, combined with the additional result that switching primarily occurs intrachromosomally, suggests that the donor loci are made accessible for switching by folding them onto mat1, thus causing the cold spot of recombination.

Genes, Fungal↗
PubMed1990

The developmental fate of fission yeast cells is determined by the pattern of inheritance of parental and grandparental DNA strands.

A key feature for development consists of producing sister cells that differ in their potential for cellular differentiation. Following two cell divisions, a haploid Schizosaccharomyces pombe cell produces one cell in four 'granddaughters' with a changed mating cell type, implying nonequivalence of sister cells in each of two consecutive cell divisions. The observed pattern of switching is analogous to the mammalian 'stem cell' lineage by which a cell produces one daughter like itself while the other daughter is advanced in its developmental program. It is tested here whether sisters differ because of unequal distribution of cytoplasmic and/or nuclear components to them or due to inheriting a specific parental DNA chain at the mating type locus. Only the DNA strand-segregation model predicts that those cells engineered to contain an inverted tandem duplication of the mating type locus should produce equivalent sisters. Consequently, two 'cousins' in four related granddaughter cells should switch. The results verified the prediction, thus establishing that all cells otherwise fully possess the potential to switch. Therefore, the program of cell type change in S.pombe cell lineages is determined by the pattern of DNA strand inheritance at the mating type locus. A specific DNA sequence present at the mating type locus is postulated to be the cause of developmental asymmetry between sister cells. A general model for cellular differentiation is proposed in which the act of DNA replication itself is hypothesized to produce developmentally nonequivalent sister genomes.

DNA, Fungal↗
PubMed1990

Extragenic suppressors of mar2(sir3) mutations in Saccharomyces cerevisiae.

The silent mating-type genes (HML and HMR) of Saccharomyces cerevisiae are kept under negative transcriptional control by four trans-acting MAR (or SIR) loci. We have isolated extragenic suppressors of the mar2-1 mutation which, based on genetic complementation tests, define two additional loci involved in regulating the expression of HML and HMR. A strain with the genotype HMLa MAT alpha HMRa mar2-1 is sterile due to the simultaneous expression of a and alpha information. Two mutants exhibiting an alpha phenotype (which may result from the restoration of MAR/SIR repression) were isolated and genetically characterized. The mutations in these strains: (1) are recessive, (2) are capable of suppressing a mar2-deletion mutation, (3) are unlinked to MAT, (4) complement one another as well as the previously identified sum1-1 mutation, and (5) are not new alleles of the known MAR/SIR loci. We designate these new regulatory loci SUM2 and SUM3 (suppressor of mar). Unlike the sum1-1 mutation, suppression by sum2-1 and sum3-1 is mar2-locus specific. Both sum2-1 and sum3-1 affect the expression of a information at the HM loci. Transcript analysis shows a significant reduction in HMLa and HMRa gene transcription in mar2-1 sum2-1 and mar2-1 sum3-1 cells. Furthermore, we have found genetic evidence to suggest that mar2-1 sum2-1 cells exhibit only partial expression of silent alpha information. We conclude that the SUM2 and SUM3 gene products are required for expression of the HM loci and act downstream of the MAR2 (SIR3) gene function. Possible mechanisms for the action of the SUM gene products are discussed.

Alleles↗
PubMed1990

The sum1-1 mutation affects silent mating-type gene transcription in Saccharomyces cerevisiae.

The silent mating-type genes (HML and HMR) of Saccharomyces cerevisiae are kept under negative transcriptional control by the trans-acting products of the four MAR/SIR loci. MAR/SIR gene mutations result in the simultaneous derepression of HML and HMR gene expression. The sum1-1 mutation was previously identified as an extragenic suppressor of mutations in MAR1 (SIR2) and MAR2 (SIR3). As assayed genetically, sum1-1 is capable of restoring repression of silent mating-type information in cells containing mar1 or mar2 null mutations. We show here that the mating-type phenotype associated with sum1-1 results from a dramatic reduction in the steady-state level of HML and HMR gene transcripts. At the same time, the sum1-1 mutation has no significant effect on the level of each of the four MAR/SIR mRNAs.

Blotting, Northern↗