Decreased meiotic reciprocal recombination in subtelomeric regions in Saccharomyces cerevisiae.

Reciprocal recombination (chiasma formation) between homologs appears to be essential for promoting chromosome segregation at the first meiotic division. However, chiasmata that form near the ends of chromosomes are much less efficient at promoting segregation. To determine the frequency of reciprocal recombination near the end of a chromosome, genetic markers were inserted at approximately 7 kb intervals within the leftmost 30 kb of chromosome I from Saccharomyces cerevisiae. Analysis of recombination between the markers indicated that meiotic reciprocal recombination rates were much lower than on the rest of the chromosome and that rates increased with distance from the telomere. Thus, S. cerevisiae has evolved a mechanism that minimizes the occurrence of chiasmata that cannot promote meiotic segregation. Low rates of recombination were independent of the SIR2 and SIR3 gene products, suggesting that any mechanism for suppressing recombination was different from transcriptional repression due to a telomere position effect.

The role of centromere alignment in meiosis I segregation of homologous chromosomes in Saccharomyces cerevisiae.

During meiosis, homologous chromosomes pair and then segregate from each other at the first meiotic division. Homologous centromeres appear to be aligned when chromosomes are paired. The role of centromere alignment in meiotic chromosome segregation was investigated in Saccharomyces cerevisiae diploids that contained one intact copy of chromosome I and one copy bisected into two functional centromere-containing fragments. The centromere on one fragment was aligned with the centromere on the intact chromosome while the centromere on the other fragment was either aligned or misaligned. Fragments containing aligned centromeres segregated efficiently from the intact chromosome, while fragments containing misaligned centromeres segregated much less efficiently from the intact chromosome. Less efficient segregation was correlated with crossing over in the region between the misaligned centromeres. Models that suggest that these crossovers impede proper segregation by preventing either a segregation-promoting chromosome alignment on the meiotic spindle or some physical interaction between homologous centromeres are proposed.

Chromosome size-dependent control of meiotic reciprocal recombination in Saccharomyces cerevisiae: the role of crossover interference.

In the yeast Saccharomyces cerevisiae, small chromosomes undergo meiotic reciprocal recombination (crossing over) at rates (centimorgans per kilobases) greater than those of large chromosomes, and recombination rates respond directly to changes in the total size of a chromosomal DNA molecule. This phenomenon, termed chromosome size-dependent control of meiotic reciprocal recombination, has been suggested to be important for ensuring that homologous chromosomes cross over during meiosis. The mechanism of this regulation was investigated by analyzing recombination in identical genetic intervals present on different size chromosomes. The results indicate that chromosome size-dependent control is due to different amounts of crossover interference. Large chromosomes have high levels of interference while small chromosomes have much lower levels of interference. A model for how crossover interference directly responds to chromosome size is presented. In addition, chromosome size-dependent control was shown to lower the frequency of homologous chromosomes that failed to undergo crossovers, suggesting that this control is an integral part of the mechanism for ensuring meiotic crossing over between homologous chromosomes.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: characterization of the 54 kb right terminal CDC15-FLO1-PHO11 region.

Gene density near the ends of Saccharomyces cerevisiae chromosomes is much lower than on the rest of the chromosome. Non-functional gene-fragments are common and a high proportion of the sequences are repeated elsewhere in the genome. This sequence arrangement suggests that the ends of chromosomes play a structural rather than a coding role and may be analogous to the highly repeated heterochromatic DNA of higher organisms. In order to evaluate the function of the ends of S. cerevisiae chromosomes, the rightmost 54-kb of DNA from chromosome I was investigated. The region contains 16 open reading frames (ORFs) and two tRNA genes. Gene-disruption studies indicated that none of these genes are essential for growth on rich or minimal medium, mating or sporulation. In contrast to the central region where 80% of the genes are transcribed when cells are grown on rich medium, only seven ORFs and the two tRNA genes appeared to produce transcripts. Six of the transcribed ORFs were from the centromere-proximal part of the region, leaving the rightmost 35-kb with only a single sequence that is transcribed during vegetative growth. Two genes located 3 and 10-kb from the chromosome I telomere are almost identical to two genes located somewhat further from the chromosome VIII telomere. Surprisingly, the chromosome VIII copies were transcribed while the chromosome I genes were not. These results suggest that the chromosome I genes may be repressed by a natural telomere position effect. The low level of transcription, absence of essential genes as well as the repetitive nature of these sequences are consistent with their having a structural role in chromosome function.

Analysis of a 103 kbp cluster homology region from the left end of Saccharomyces cerevisiae chromosome I.

The DNA sequence and preliminary functional analysis of a 103-kbp section of the left arm of yeast chromosome I is presented. This region, from the left telomere to the LTE1 gene, can be divided into two distinct portions. One portion, the telomeric 29 kbp, has a very low gene density (only five potential genes and 21 kbp of noncoding sequence), does not encode any "functionally important" genes, and is rich in sequences repeated several times within the yeast genome. The other portion, with 37 genes and only 14.5 kbp of noncoding sequence, is gene rich and codes for at least 16 "functionally important" genes. The entire gene-rich portion is apparently duplicated on chromosome XV as an extensive region of partial gene synteney called a cluster homology region. A function can be assigned with varying degrees of precision to 23 of the 42 potential genes in this region; however, the precise function is know for only eight genes. Nineteen genes encode products presently novel to yeast, although five of these have homologs elsewhere in the yeast genome.

Patterns of meiotic double-strand breakage on native and artificial yeast chromosomes.

The preferred positions for meiotic double-strand breakage were mapped on Saccharomyces cerevisiae chromosomes I and VI, and on a number of yeast artificial chromosomes carrying human DNA inserts. Each chromosome had strong and weak double-strand break (DSB) sites. On average one DSB-prone region was detected by pulsed-field gel electrophoresis per 25 kb of DNA, but each chromosome had a unique distribution of DSB sites. There were no preferred meiotic DSB sites near the telomeres. DSB-prone regions were associated with all of the known "hot spots" for meiotic recombination on chromosomes I, III and VI.

The nucleotide sequence of chromosome I from Saccharomyces cerevisiae.

Chromosome I from the yeast Saccharomyces cerevisiae contains a DNA molecule of approximately 231 kbp and is the smallest naturally occurring functional eukaryotic nuclear chromosome so far characterized. The nucleotide sequence of this chromosome has been determined as part of an international collaboration to sequence the entire yeast genome. The chromosome contains 89 open reading frames and 4 tRNA genes. The central 165 kbp of the chromosome resembles other large sequenced regions of the yeast genome in both its high density and distribution of genes. In contrast, the remaining sequences flanking this DNA that comprise the two ends of the chromosome and make up more than 25% of the DNA molecule have a much lower gene density, are largely not transcribed, contain no genes essential for vegetative growth, and contain several apparent pseudogenes and a 15-kbp redundant sequence. These terminally repetitive regions consist of a telomeric repeat called W', flanked by DNA closely related to the yeast FLO1 gene. The low gene density, presence of pseudogenes, and lack of expression are consistent with the idea that these terminal regions represent the yeast equivalent of heterochromatin. The occurrence of such a high proportion of DNA with so little information suggests that its presence gives this chromosome the critical length required for proper function.

LTE1 of Saccharomyces cerevisiae is a 1435 codon open reading frame that has sequence similarities to guanine nucleotide releasing factors.

The DNA sequence of the LTE1 gene on the left arm of chromosome I of Saccharomyces cerevisiae has been determined. The LTE1 open reading frame comprises 4305 bp that can be translated into 1435 amino acid residues. The position of this open reading frame corresponds well to that of a 4.7 kb transcript that has been mapped to this position. The derived amino acid sequence has significant similarities to the amino acid sequence of the guanine nucleotide releasing factor isolated from a rat brain library. The carboxy-terminus of the LTE1 protein also shows similarities to other guanine nucleotide exchange factors of the S. cerevisiae CDC25 family.

Sequencing of chromosome I of Saccharomyces cerevisiae: analysis of the 42 kbp SPO7-CENI-CDC15 region.

Determination of the DNA sequence and preliminary functional analysis of a 42 kbp centromeric section of chromosome I have been completed. The section spans the SPO7-CEN1-CDC15 loci and contains 19 open reading frames (ORFs). They include an apparently inactive Ty1 retrotransposon and eight new ORFs with no known homologs or function. The remaining ten genes have been previously characterized since this part of the yeast genome has been studied in an unusually intensive manner. Our directed sequencing allows a complete ordering of the region.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: analysis of the genes in the FUN38-MAK16-SPO7 region.

Transcribed regions on a 42-kb segment of chromosome I from Saccharomyces cerevisiae were mapped. Polyadenylated transcripts corresponding to eight previously characterized genes (MAK16, LTE1, CCR4, FUN30, FUN31, TPD3, DEP1, and CYS3) and eight new genes were identified. All transcripts were present at one to four copies per cell except for one which was significantly less abundant. This region has been sequenced, and the sizes, locations, and orientations of the transcripts were in nearly perfect agreement with the open reading frames. Disruptions in eight genes identified solely on the basis of a transcribed region, FUN38, FUN25, FUN26, FUN28, FUN30, FUN31, FUN33, and FUN34, indicated that all were nonessential for growth on rich medium at 30 degrees C. Disruption of FUN30, a gene closely related to RAD16 and RAD54, surprisingly resulted in increased resistance to UV irradiation. No additional phenotypes, other than slow growth, were observed for all other mutants. The distribution of essential genes on chromosome I is discussed.

Physical association between nonhomologous chromosomes precedes distributive disjunction in yeast.

During meiosis homologous chromosomes normally pair, undergo reciprocal recombination, and then segregate from each other. Distributive disjunction is the meiotic segregation that is observed in the absence of homologous recombination and can occur for both nonrecombinant homologous chromosomes and completely nonhomologous chromosomes. While the mechanism of distributive disjunction is not known, several models have been presented that either involve or are completely independent of interactions between the segregating chromosomes. In this report, we demonstrate that distributive disjunction in Saccharomyces cerevisiae is preceded by an interaction between nonhomologous chromosomes.

The YAL017 gene on the left arm of chromosome I of Saccharomyces cerevisiae encodes a putative serine/threonine protein kinase.

The DNA sequence of a region between the LTE1 and CYS3 genes on the left arm of chromosome I from Saccharomyces cerevisiae contains an open reading frame (ORF), YAL017, corresponding to the 5.0 kb FUN31 (Function Unknown Now) transcribed region. The predicted protein from this ORF contains 1358 amino acid residues with a molecular weight of 152,531, and an identifiable serine/threonine protein kinase catalytic domain. When compared with other yeast protein kinases, the Yal017p kinase most resembles the SNF1 serine/threonine protein kinase which is involved in regulating sucrose fermentation genes. The Yal017p kinase shows highest amino acid identities with two mammalian carcinoma-related serine/threonine protein kinases; PIM-1, which shows induced expression in T-cell lymphomas; and p78A1, whose expression is lost in human pancreatic carcinomas. Gene disruption of YAL017 indicates that it is non-essential for growth on glucose.

Physical localization of yeast CYS3, a gene whose product resembles the rat gamma-cystathionase and Escherichia coli cystathionine gamma-synthase enzymes.

We have cloned, sequenced and physically mapped the CYS3 gene of Saccharomyces cerevisiae. This gene can complement the cys3-1 allele, and disruptions at this locus lead to cysteine auxotrophy. The predicted CYS3 product is closely related (46% identical) to the rat cystathionine gamma-lyase (Erickson et al., 1990), but differs in lacking cysteine residues. These results provide further evidence that the S288C strain of yeast resembles mammals in synthesizing cysteine solely via a trans-sulfuration pathway. The CYS3 product was found to have strong homology to three other enzymes involved in cysteine metabolism: the Escherichia coli metB and metC products and the S. cerevisiae MET25 gene product. The trans-sulfuration enzymes appear to form a diverged family and carry out related functions from bacteria to mammals.

Sequencing of chromosome I from Saccharomyces cerevisiae: analysis of a 32 kb region between the LTE1 and SPO7 genes.

The DNA sequencing and preliminary functional analysis of a 32 kb section of yeast chromosome I has been completed. This region lies on the left arm of the chromosome between the LTE1 and SPO7 genes and contains 14 open reading frames (ORFs) positioned closely together, with an average spacing of approximately 350 nucleotides between coding regions. Three of these ORFs correspond to previously identified genes, a further three show significant homology with other proteins, while the remaining eight ORFs share no significant homology to genes in the databases.

The CCR4 protein from Saccharomyces cerevisiae contains a leucine-rich repeat region which is required for its control of ADH2 gene expression.

The CCR4 gene from Saccharomyces cerevisiae is required for the transcription of the glucose-repressible alcohol dehydrogenase (ADH2). Mutations in CCR4 also suppress the transcription at the ADH2 and his4-912delta loci caused by defects in the SPT10 (CRE1) and SPT6 (CRE2) genes. The CCR4 gene was mapped to the left arm of chromosome I and cloned by complementation of function using previously isolated segments of chromosome I. DNA sequence analysis of the cloned gene defined CCR4 as a 2511 bp open reading frame that would encode a polypeptide of 837 amino acids. The CCR4 mRNA was found to be 2.8 kb in size and Western analysis identified CCR4 as a 95,000 D protein. Disruption of the CCR4 gene resulted in reduced levels of ADH2 expression under both glucose and ethanol growth conditions and in temperature sensitive growth on nonfermentative medium, phenotypes essentially indistinguishable from previously identified mutations in CCR4. The amino terminus of the CCR4 protein was found to be rich in glutamine residues similar to a number of genes which are required for transcription. More importantly, CCR4 showed similarity to a diverse set of proteins sharing a leucine-rich tandem repeat motif, the presence of which has been implicated in mediating protein-protein interactions. Deletions of several of the five leucine-rich repeats in CCR4 were shown to produce nonfunctional proteins indicating the importance of the repeats to CCR4 activity. This leucine-rich repeat region may mediate the contact CCR4 makes with another factor.

Cloning of chromosome I DNA from Saccharomyces cerevisiae: analysis of the FUN52 gene, whose product has homology to protein kinases.

A gene whose product has homology to protein kinases and is closely related to the Aspergillus nidulans nimA cell-cycle gene was identified on chromosome I of the yeast, Saccharomyces cerevisiae. This gene has been temporarily designated FUN52, where FUN is the acronym for 'function unknown now'. In A. nidulans, nimA is required to enter mitosis. In addition, overexpression of nimA causes premature onset of mitosis and cell cycle arrest. In contrast, S. cerevisiae cells that were either deleted for FUN52 or were overexpressing it had no detectable growth phenotypes. FUN52 proved to be the same as the previously identified KIN3 gene [Jones and Rosamond, Gene 90 (1990) 87-92] that was reported to map on chromosome VI.

Chromosome size-dependent control of meiotic recombination.

Smaller chromosomes have higher rates of meiotic reciprocal recombination (centimorgans per kilobase pair) than larger chromosomes. This report demonstrates that decreasing the size of Saccharomyces cerevisiae chromosomal DNA molecules increases rates of meiotic recombination and increasing chromosome size decreases recombination rates. These results indicate that chromosome size directly affects meiotic reciprocal recombination.

Identification of a Saccharomyces cerevisiae homolog of the SNF2 transcriptional regulator in the DNA sequence of an 8.6 kb region in the LTE1-CYS1 interval on the left arm of chromosome I.

The DNA sequence of an 8.6 kb region of the left arm of chromosome I has been determined. This region, between the LTE1 and CYS1 loci, is approximately 40 kb from the centromere. There are six potential open-reading frames (ORFs), provisionally named YAL001-006 within this fragment of chromosome I. Four of these ORFs can be aligned with previously identified FUN transcripts: FUN28 with YAL006, FUN29 with YAL004, FUN30 with YAL001 and FUN31 with YAL002. The YAL001 ORF shows significant homology to the SNF2 transcriptional regulator. A region of the DNA contains an extensive repeat of the bases C-A-T positioned in the 5' terminus of the YAL004 promoter region.

Distributive disjunction of authentic chromosomes in Saccharomyces cerevisiae.

Distributive disjunction is defined as the first division meiotic segregation of either nonhomologous chromosomes that lack homologs or homologous chromosomes that have not recombined. To determine if chromosomes from the yeast Saccharomyces cerevisiae were capable of distributive disjunction, we constructed a strain that was monosomic for both chromosome I and chromosome III and analyzed the meiotic segregation of the two monosomic chromosomes. In addition, we bisected chromosome I into two functional chromosome fragments, constructed strains that were monosomic for both chromosome fragments and examined meiotic segregation of the chromosome fragments in the monosomic strains. The two nonhomologous chromosomes or chromosome fragments appeared to segregate from each other in approximately 90% of the asci analyzed, indicating that yeast chromosomes were capable of distributive disjunction. We also examined the ability of a small nonhomologous centromere containing plasmid to participate in distributive disjunction with the two nonhomologous monosomic chromosomes. The plasmid appeared to efficiently participate with the two full length chromosomes suggesting that distributive disjunction in yeast is not dependent on chromosome size. Thus, distributive disjunction in S. cerevisiae appears to be different from Drosophila melanogaster where a different sized chromosome is excluded from distributive disjunction when two similar size nonhomologous chromosomes are present.