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G S Roeder

Publications and source records attributed to G S Roeder.

At least 37 records · Page 2Linked to original sources
PubMed1998

Zip2, a meiosis-specific protein required for the initiation of chromosome synapsis.

We describe the identification and characterization of the Saccharomyces cerevisiae ZIP2 gene, which encodes a novel meiosis-specific protein essential for synaptonemal complex formation. In the zip2 mutant, chromosomes are homologously paired but not synapsed. The Zip2 protein localizes to discrete foci on meiotic chromosomes; these foci correspond to sites of convergence between paired homologs that are believed to be sites of synapsis initiation. Localization of Zip2p requires the initiation of meiotic recombination. In a mutant defective in double-strand break repair, Zip2p colocalizes with proteins involved in double-strand break formation and processing. We propose that Zip2p promotes the initiation of chromosome synapsis and that localization of Zip2p to sites of interhomolog recombination ensures synapsis between homologous chromosomes.

Chromosomes, Fungal↗
PubMed1998

Meiotic chromosome morphology and behavior in zip1 mutants of Saccharomyces cerevisiae.

The yeast Zip1 protein (Zip1p) is a component of the central region of the synaptonemal complex (SC). Zip1p is predicted to form a dimer consisting of a coiled-coil domain flanked by globular domains. To analyze the organization of Zip1p within the SC, in-frame deletions of ZIP1 were constructed and analyzed. The results demonstrate that the C terminus but not the N terminus of Zip1p is required for its localization to chromosomes. Deletions in the carboxy half of the predicted coiled-coil region cause decreases in the width of the SC. Based on these results, a model for the organization of Zip1p within the SC is proposed. zip1 deletion mutations were also examined for their effects on sporulation, spore viability, crossing over, and crossover interference. The results demonstrate that the extent of synapsis is positively correlated with the levels of spore viability, crossing over, and crossover interference. In contrast, the role of Zip1p in synapsis is separable from its role in meiotic cell cycle progression. zip1 mutants display interval-specific effects on crossing over.

Antibodies, Fungal↗
PubMed1997

Tam1, a telomere-associated meiotic protein, functions in chromosome synapsis and crossover interference.

The TAM1 gene of Saccharomyces cerevisiae is expressed specifically during meiosis and encodes a protein that localizes to the ends of meiotic chromosomes. In a tam1 null mutant, there is an increase in the frequency of chromosomes that fail to recombine and an associated increase in homolog nondisjunction at meiosis I. The tam1 mutant also displays an increased frequency of precocious separation of sister chromatids and a reduced efficiency of distributive disjunction. The defect in distributive disjunction may be attributable to overloading of the distributive system by the increased number of nonrecombinant chromosomes. Recombination is not impaired in the tam1 mutant, but crossover interference is reduced substantially. In addition, chromosome synapsis is delayed in tam1 strains. The combination of a defect in synapsis and a reduction in interference is consistent with previous studies suggesting a role for the synaptonemal complex in regulating crossover distribution. tam1 is the only known yeast mutant in which the control of crossover distribution is impaired, but the frequency of crossing over is unaffected. We discuss here possibilities for how a telomere-associated protein might function in chromosome synapsis and crossover interference.

Chromosome Mapping↗
PubMed1997

The yeast Red1 protein localizes to the cores of meiotic chromosomes.

Mutants in the meiosis-specific RED1 gene of S. cerevisiae fail to make any synaptonemal complex (SC) or any obvious precursors to the SC. Using antibodies that specifically recognize the Red1 protein, Red1 has been localized along meiotic pachytene chromosomes. Red1 also localizes to the unsynapsed axial elements present in a zip1 mutant, suggesting that Red1 is a component of the lateral elements of mature SCs. Anti-Red1 staining is confined to the cores of meiotic chromosomes and is not associated with the loops of chromatin that lie outside the SC. Analysis of the spo11 mutant demonstrates that Red1 localization does not depend upon meiotic recombination. The localization of Red1 has been compared with two other meiosis-specific components of chromosomes, Hop1 and Zip1; Zip1 serves as a marker for synapsed chromosomes. Double labeling of wild-type meiotic chromosomes with anti-Zip1 and anti-Red1 antibodies demonstrates that Red1 localizes to chromosomes both before and during pachytene. Double labeling with anti-Hop1 and anti-Red1 antibodies reveals that Hop1 protein localizes only in areas that also contain Red1, and studies of Hop1 localization in a red1 null mutant demonstrate that Hop1 localization depends on Red1 function. These observations are consistent with previous genetic studies suggesting that Red1 and Hop1 directly interact. There is little or no Hop1 protein on pachytene chromosomes or in synapsed chromosomal regions.

Cell Nucleolus↗
PubMed1997

A multipurpose transposon system for analyzing protein production, localization, and function in Saccharomyces cerevisiae.

Analysis of the function of a particular gene product typically involves determining the expression profile of the gene, the subcellular location of the protein, and the phenotype of a null strain lacking the protein. Conditional alleles of the gene are often created as an additional tool. We have developed a multifunctional, transposon-based system that simultaneously generates constructs for all the above analyses and is suitable for mutagenesis of any given Saccharomyces cerevisiae gene. Depending on the transposon used, the yeast gene is fused to a coding region for beta-galactosidase or green fluorescent protein. Gene expression can therefore be monitored by chemical or fluorescence assays. The transposons create insertion mutations in the target gene, allowing phenotypic analysis. The transposon can be reduced by cre-lox site-specific recombination to a smaller element that leaves an epitope tag inserted in the encoded protein. In addition to its utility for a variety of immunodetection purposes, the epitope tag element also has the potential to create conditional alleles of the target gene. We demonstrate these features of the transposons by mutagenesis of the SPA2, ARP100, SER1, and BDF1 genes.

DNA Transposable Elements↗
PubMed1995

Sex and the single cell: meiosis in yeast.

Recent studies of Saccharomyces cerevisiae have significantly advanced our understanding of the molecular mechanisms of meiotic chromosome behavior. Structural components of the synaptonemal complex have been identified and studies of mutants defective in synapsis have provided insight into the role of the synaptonemal complex in homolog pairing, genetic recombination, crossover interference, and meiotic chromosome segregation. There is compelling evidence that most or all meiotic recombination events initiate with double-strand breaks. Several intermediates in the double-strand break repair pathway have been characterized and mutants blocked at different steps in the pathway have been identified. With the application of genetic, molecular, cytological, and biochemical methods in a single organism, we can expect an increasingly comprehensive and unified view of the meiotic process.

Chromosomes, Fungal↗
PubMed1995

Roles for two RecA homologs in promoting meiotic chromosome synapsis.

Previous studies have shown that the RAD51 and DMC1 genes of Saccharomyces cerevisiae encode homologs of the Escherichia coli RecA strand exchange enzyme. Results presented here demonstrate that the dmc1 and rad51 mutants undergo nearly complete chromosome synapsis, but synaptonemal complex formation is delayed substantially compared with wild type. In the zip1 mutant, chromosomes are paired homologously, but not synapsed, and the protein backbones (axial elements) of each pair of chromosomes are connected intimately to each other at a few sites referred to herein as axial associations. dmc1 zip1 and rad51 zip1 double mutants assemble axial elements that are not obviously associated, demonstrating that the Dmc1 and Rad51 proteins are required to establish or stabilize axial associations. We propose that axial associations serve to promote meiotic chromosome synapsis and that the absence of these associations accounts for the delayed and inefficient synapsis observed in dmc1 and rad51 strains. During meiosis in haploid yeast, chromosome synapsis takes place between nonhomologous chromosome segments. In a zip1 haploid, axial associations are not apparent, suggesting that these associations depend on interactions between homologous sequences.

Alleles↗
PubMed1995

Zip1-induced changes in synaptonemal complex structure and polycomplex assembly.

The yeast Zip1 protein is a component of the synaptonemal complex (SC), which is an elaborate macromolecular structure found along the lengths of chromosomes during meiosis. Mutations that increase the length of the predicted coiled coil region of the Zip1 protein show that Zip1 influences the width of the SC. Overexpression of the ZIP1 gene results in the formation of two distinct types of higher order structures that are found in the nucleus, but not associated with chromatin. One of these structures resembles the polycomplexes that have been observed in many organisms and are thought to be aggregates of SC components. The second type of structure, which we have termed "networks," does not resemble any previously identified SC-related structure. Assembly of both polycomplexes and networks can occur independently of the Hop1 or Red1 protein, which are thought to be SC components. Our results demonstrate that Zip1 is a structural component of the central region of the SC. More specifically, we speculate that Zip1 is a component of the transverse filaments that lie perpendicular to the long axis of the complex.

Chromosomes, Fungal↗
PubMed1995

The sep1 mutant of Saccharomyces cerevisiae arrests in pachytene and is deficient in meiotic recombination.

Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae promotes homologous pairing of DNA in vitro and sep1 mutants display pleiotropic phenotypes in both vegetative and meiotic cells. In this study, we examined in detail the ability of the sep1 mutant to progress through meiosis I prophase and to undergo meiotic recombination. In meiotic return-to-growth experiments, commitment to meiotic recombination began at the same time in wild type and mutant; however, recombinants accumulated at decreased rates in the mutant. Gene conversion eventually reached nearly wild-type levels, whereas crossing over reached 15-50% of wild type. In an assay of intrachromosomal pop-out recombination, the sep1, dmc1 and rad51 single mutations had only small effects; however, pop-out recombination was virtually eliminated in the sep1 dmc1 and sep1 rad51 double mutants, providing evidence for multiple recombination pathways. Analysis of meiotic recombination intermediates indicates that the sep1 mutant is deficient in meiotic double-strand break repair. In a physical assay, the formation of mature reciprocal recombinants in the sep1 mutant was delayed relative to wild type and ultimately reached only 50% of the wild-type level. Electron microscopic analysis of meiotic nuclear spreads indicates that the sep1 delta mutant arrests in pachytene, with apparently normal synaptonemal complex. This arrest is RAD9-independent. We hypothesize that the Sep1 protein participates directly in meiotic recombination and that other strand exchange enzymes, acting in parallel recombination pathways, are able to substitute partially for the absence of the Sep1 protein.

Cell Cycle Proteins↗
PubMed1995

The yeast MER2 gene is required for chromosome synapsis and the initiation of meiotic recombination.

Mutation of the MER2 gene of Saccharomyces cerevisiae confers meiotic lethality. To gain insight into the function of the Mer2 protein, we have carried out a detailed characterization of the mer2 null mutant. Genetic analysis indicates that mer2 completely eliminates meiotic interchromosomal gene conversion and crossing over. In addition, mer2 abolishes intrachromosomal meiotic recombination, both in the ribosomal DNA array and in an artificial duplication. The results of a physical assay demonstrate that the mer2 mutation prevents the formation of meiosis-specific, double-strand breaks, indicating that the Mer2 protein acts at or before the initiation of meiotic recombination. Electron microscopic analysis reveals that the mer2 mutant makes axial elements, which are precursors to the synaptonemal complex, but homologous chromosomes fail to synapse. Fluorescence in situ hybridization of chromosome-specific DNA probes to spread meiotic chromosomes demonstrates that homolog alignment is also significantly reduced in the mer2 mutant. Although the MER2 gene is transcribed during vegetative growth, deletion or overexpression of the MER2 gene has no apparent effect on mitotic recombination or DNA damage repair. We suggest that the primary defect in the mer2 mutant is in the initiation of meiotic genetic exchange.

Chromosomes, Fungal↗
PubMed1995

Heteroduplex DNA formation and homolog pairing in yeast meiotic mutants.

Previous studies of Saccharomyces cerevisiae have identified several meiosis-specific genes whose products are required for wild-type levels of meiotic recombination and for normal synaptonemal complex (SC) formation. Several of these mutants were examined in a physical assay designed to detect heteroduplex DNA (hDNA) intermediates in meiotic recombination. hDNA was not detected in the rec102, mei4 and hop1 mutants; it was observed at reduced levels in red1, mek1 and mer1 strains and at greater than the wild-type level in zip1. These results indicate that the REC102, MEI4, HOP1, RED1, MEK1 and MER1 gene products act before hDNA formation in the meiotic recombination pathway, whereas ZIP1 acts later. The same mutants assayed for hDNA formation were monitored for meiotic chromosome pairing by in situ hybridization of chromosome-specific DNA probes to spread meiotic nuclei. Homolog pairing occurs at wild-type levels in the zip1 and mek1 mutants, but is substantially reduced in mei4, rec102, hop1, red1 and mer1 strains. Even mutants that fail to recombine or to make any SC or SC precursors undergo a significant amount of meiotic chromosome pairing. The in situ hybridization procedure revealed defects in meiotic chromatin condensation in mer1, red1 and hop1 strains.

Chromosomes, Fungal↗
PubMed1995

Binding of a cell-type-specific RNA splicing factor to its target regulatory sequence.

The transcript of the Saccharomyces cerevisiae MER2 gene is spliced efficiently during meiosis but not during vegetative growth. Efficient splicing of the wild-type MER2 transcript requires the Mer1 protein, which is produced only in meiotic cells. Analysis of deletion and substitution mutations in the MER2 5' exon demonstrates that the unusually large size of this exon plays an important role in splicing regulation. The cis-acting sequences essential for Mer1-dependent splicing of MER2 RNA were determined by the analysis of MER2 deletion mutants and hybrid genes. The 80-base MER2 intron is sufficient for Mer1-dependent splicing in vivo, but sequences in the 5' exon enhance splicing efficiency. The Mer1 protein contains the KH motif found in some RNA-binding proteins, and RNA gel mobility shift assays demonstrate that Mer1 binds specifically to MER2 RNA. Both the transcript derived from the intronless MER2 gene and the transcript consisting only of the intron are able to bind to Mer1 in vitro, but neither has as high affinity for the protein as the intact substrate. RNase T1 footprinting indicates that the Mer1 protein contacts MER2 RNA at several points in the 5' exon and in the intron. Thus, Mer1 interacts directly with a regulatory element in MER2 RNA and promotes splicing.

Base Sequence↗
PubMed1995

Bdf1, a yeast chromosomal protein required for sporulation.

The BDF1 gene of Saccharomyces cerevisiae is required for sporulation. Under starvation conditions, most cells from the bdf1 null mutant fail to undergo one or both meiotic divisions, and there is an absolute defect in spore formation. The Bdf1 protein localizes to the nucleus throughout all stages of the mitotic and meiotic cell cycles. Analysis of spread meiotic nuclei reveals that the Bdf1 protein is localized fairly uniformly along chromosomes, except that it is excluded specifically from the nucleolus. A bdf1 null mutant displays a reduced rate of vegetative growth and sensitivity to a DNA-damaging agent. The BDF1 gene encodes a 77-kDa protein that contains two bromodomains, sequence motifs of unknown function. Separation-of-function alleles suggest that only one of the two bromodomains is required for sporulation, whereas both are required for Bdf1 function in vegetative cells. We propose that the Bdf1 protein is a component of chromatin and that the mitotic and meiotic defects of the bdf1 null mutant result from alterations in chromatin structure.

Alleles↗
PubMed1994

Mutation of a meiosis-specific MutS homolog decreases crossing over but not mismatch correction.

MSH4 is a novel meiosis-specific gene required for wild-type levels of spore viability in S. cerevisiae. The predicted product of the MSH4 gene is homologous to the MutS family of proteins; however, msh4-null mutants have no apparent defect in mismatch repair. msh4 mutant strains display wild-type levels of gene conversion and postmeiotic segregation, but they show a reduction in crossing over and a resultant increase in nondisjunction of homologous chromosomes at meiosis I. Immunofluorescence experiments demonstrate that the Msh4 protein is localized to discrete sites on pachytene chromosomes. We propose that Msh4 interacts with a recombination intermediate to influence its resolution.

Adenosine Triphosphatases↗
PubMed1994

Crossover interference is abolished in the absence of a synaptonemal complex protein.

In the zip1 mutant, meiotic chromosomes fail to synapse, owing to the absence of a structural component of the synaptonemal complex (SC). This mutant has been analyzed for the ability to carry out several functions that have been proposed for the SC. The data presented show that the zip1 mutation does not affect chiasma function and confers only modest defects in meiotic recombination and sister chromatid cohesion. In contrast, crossover interference is completely abolished in the absence of Zip1. These data are the first to establish a molecular link between cytological observations of the SC and the genetic phenomenon of interference.

Chromosomes↗
PubMed1994

Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae.

We have developed a large-scale screen to identify genes expressed at different times during the life cycle of Saccharomyces cerevisiae and to determine the subcellular locations of many of the encoded gene products. Diploid yeast strains containing random lacZ insertions throughout the genome have been constructed by transformation with a mutagenized genomic library. Twenty-eight hundred transformants containing fusion genes expressed during vegetative growth and 55 transformants containing meiotically induced fusion genes have been identified. Based on the frequency of transformed strains producing beta-galactosidase, we estimate that 80-86% of the yeast genome (excluding the rDNA) contains open reading frames expressed in vegetative cells and that there are 93-135 meiotically induced genes. Indirect immunofluorescence analysis of 2373 strains carrying fusion genes expressed in vegetative cells has identified 245 fusion proteins that localize to discrete locations in the cell, including the nucleus, mitochondria, endoplasmic reticulum, cytoplasmic dots, spindle pole body, and microtubules. The DNA sequence adjacent to the lacZ gene has been determined for 91 vegetative fusion genes whose products have been localized and for 43 meiotically induced fusions. Although most fusions represent genes unidentified previously, many correspond to known genes, including some whose expression has not been studied previously and whose products have not been localized. For example, Sec21-beta-gal fusion proteins yield a Golgi-like staining pattern, Ty1-beta-gal fusion proteins localize to cytoplasmic dots, and the meiosis-specific Mek1/Mre4-beta-gal and Spo11-beta-gal fusion proteins reside in the nucleus. The phenotypes in haploid cells have been analyzed for 59 strains containing chromosomal fusion genes expressed during vegetative growth; 9 strains fail to form colonies indicating that the disrupted genes are essential. Fifteen additional strains display slow growth or are impaired for growth on specific media or in the presence of inhibitors. Of 39 meiotically induced fusion genes examined, 14 disruptions confer defects in spore formation or spore viability in homozygous diploids. Our results will allow researchers who identify a yeast gene to determine immediately whether that gene is expressed at a specific time during the life cycle and whether its gene product localizes to a specific subcellular location.

Base Sequence↗
PubMed1994

The yeast med1 mutant undergoes both meiotic homolog nondisjunction and precocious separation of sister chromatids.

A mutant at the yeast MED1 locus was isolated in a screen for sporulation-proficient, meiotic-lethal mutants. Synaptonemal complex formation in the med1 mutant is apparently normal and med1 strains undergo meiotic crossing over at approximately 50% of the wild-type level. The med1 mutant undergoes homolog nondisjunction at meiosis I, presumably as a consequence of the decrease in crossing over. In addition, the mutant undergoes precocious separation of sister chromatids, resulting in chromosome missegregation at both meiotic divisions. We suggest that the med1 mutation perturbs chromosome structure, leading to a reduction in recombination and a defect in sister chromatid cohesion.

Cell Nucleus↗