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.

Organization of the yeast Zip1 protein within the central region of the synaptonemal complex.

The yeast Zip1 protein is a component of the central region of the synaptonemal complex (SC). Zip1 is predicted to form an alpha-helical coiled coil, flanked by globular domains at the NH(2) and COOH termini. Immunogold labeling with domain-specific anti-Zip1 antibodies demonstrates that the NH(2)-terminal domain of Zip1 is located in the middle of the central region of the SC, whereas the COOH-terminal domain is embedded in the lateral elements of the complex. Previous studies have shown that overproduction of Zip1 results in the assembly of two types of aggregates, polycomplexes and networks, that are unassociated with chromatin. Our epitope mapping data indicate that the organization of Zip1 within polycomplexes is similar to that of the SC, whereas the organization of Zip1 within networks is fundamentally different. Zip1 protein purified from bacteria assembles into dimers in vitro, and electron microscopic analysis demonstrates that the two monomers within a dimer are arranged in parallel and in register. Together, these results suggest that two Zip1 dimers, lying head-to-head, span the width of the SC.

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.

The origins of gene instability in yeast.

Two unstable mutations at the his4 locus of yeast are due to the insertion of the transposable elements Ty912 and Ty917 into the his4 regulatory region. The two transposons are related, one being derived from the other by a substitution of 4000 base pairs of DNA. Element Ty912 includes identical terminal repeats, whereas the terminal repeats of Ty917 are not identical. Transposition of Ty912 or Ty917 generates 5-base-pair duplications of the target DNA at either end of the element. Expression and reversion of a his4 gene containing Ty912 or Ty917 is controlled by three unlinked regulatory genes. The properties of these regulatory genes are similar to those described for the controlling elements in maize.

Chromosome synapsis and genetic recombination: their roles in meiotic chromosome segregation.

Chromosome synapsis and genetic recombination ensure the faithful segregation of chromosomes at meiosis I by establishing physical connections between homologs. Recent observations suggest that recombination may also play a role in the homology search process that precedes synapsis.

The pachytene checkpoint.

The pachytene checkpoint prevents meiotic nuclear division in cells that fail to complete meiotic recombination and chromosome synapsis. This control mechanism prevents chromosome missegregation that would lead to the production of aneuploid gametes. The pachytene checkpoint requires a subset of proteins that function in the mitotic DNA damage checkpoint. In budding yeast, the pachytene checkpoint also requires meiosis-specific chromosomal proteins and, unexpectedly, proteins concentrated in the nucleolus. Progress has been made in identifying components of the cell-cycle machinery that are impacted by the checkpoint.

MER1, a yeast gene required for chromosome pairing and genetic recombination, is induced in meiosis.

The yeast MER1 gene is required for the production of viable meiotic products and for meiotic recombination. Cytological analysis of chromosome spreads from a mer1 mutant indicates that the MER1 gene product is also required for normal chromosome pairing. mer1 strains make axial elements, precursors to the synaptonemal complex; however, the chromosomes in most nuclei do not become fully synapsed. The DNA sequence of the MER1 coding region was determined; the MER1 open reading frame encodes a 270-amino-acid protein with a molecular mass of 31.1 kilodaltons. The MER1 protein shows limited sequence similarity to calmodulin. Expression of the MER1 gene was examined by RNA blot hybridization analysis and through the construction and analysis of mer1::lacZ fusion genes. Expression of the MER1 gene is meiotically induced and required the IME1 gene product. Thus, expression of the MER1 gene early in meiosis is required for proper chromosome pairing and meiotic recombination.

Control of yeast gene expression by transposable elements: maximum expression requires a functional Ty activator sequence and a defective Ty promoter.

Integration of a transposable element adjacent to a gene frequently results in an alteration in expression of the nearby gene. The purpose of our experiments was to identify cis-acting sequences within a yeast transposon (Ty) that are important for expression of the adjacent gene. The role of these sequences in Ty transcription was also analyzed in order to examine the relationship between Ty and adjacent gene expression. Three naturally occurring Ty elements located at the HIS4 locus were examined. These Ty elements differed by multiple sequence changes and had different effects on HIS4 expression. To determine which sequences were important to Ty and HIS4 expression, Ty::lacZ and Ty::HIS4::lacZ fusion genes were constructed and analyzed. Results of these experiments indicated that a sequence element is present in the Ty epsilon region that is necessary for HIS4 expression but which has only a modest effect on Ty transcription. Additionally, a mutation in the Ty promoter region decreased Ty transcription and increased HIS4 expression. The opposite effects of this mutation on Ty and adjacent gene expression were probably caused by promoter competition.

A unique pathway of double-strand break repair operates in tandemly repeated genes.

The RAD52 gene product of the yeast Saccharomyces cerevisiae is required for most spontaneous recombination and almost all double-strand break (DSB) repair. In contrast to recombination elsewhere in the genome, recombination in the ribosomal DNA (rDNA) array is RAD52 independent. To determine the fate of a DSB in the rDNA gene array, a cut site for the HO endonuclease was inserted into the rDNA in a strain containing an inducible HO gene. DSBs were efficiently repaired at this site, even in the absence of the RAD52 gene product. Efficient RAD52-independent DSB repair was also observed at another tandem gene array, CUP1, consisting of 18 repeat units. However, in a smaller CUP1 array, consisting of only three units, most DSBs (ca. 80%) were not repaired and resulted in cell death. All RAD52-independent DSB repair events examined resulted in the loss of one or more repeat units. We propose a model for DSB repair in repeated sequences involving the generation of single-stranded tails followed by reannealing.

Transcription by RNA polymerase I stimulates mitotic recombination in Saccharomyces cerevisiae.

The recombination-stimulating sequence HOT1 is derived from the ribosomal DNA array of Saccharomyces cerevisiae and corresponds to sequences that promote transcription by RNA polymerase I. When inserted at a chromosomal location outside the ribosomal DNA array, HOT1 stimulates mitotic recombination in the adjacent sequences. To investigate the relationship between transcription and recombination, transcription promoted by HOT1 was directly examined. These studies demonstrated that transcription starts at the RNA polymerase I initiation site in HOT1 and proceeds through the chromosomal sequences in which recombination is enhanced. Linker insertion mutations in HOT1 were generated and assayed for recombination stimulation and for promoter function; this analysis demonstrated that the same sequences are required for both activities. These results indicate that the ability of HOT1 to enhance recombination is related to, and most likely dependent on, its ability to promote transcription.

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.

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.

Splicing of the meiosis-specific HOP2 transcript utilizes a unique 5' splice site.

The Saccharomyces cerevisiae HOP2 gene is required to prevent formation of synaptonemal complex between nonhomologous chromosomes during meiosis. The HOP2 gene is expressed specifically in meiotic cells, with the transcript reaching maximum abundance early in meiotic prophase. The HOP2 coding region is interrupted by an intron located near the 5' end of the gene. This intron contains a nonconsensus 5' splice site (GUUAAGU) that differs from the consensus 5' splice signal (GUAPyGU) by the insertion of a nucleotide and by a single nucleotide substitution. Bases flanking the HOP2 5' splice site have the potential to pair with sequences in U1 small nuclear RNA, and mutations disrupting this pairing reduce splicing efficiency. HOP2 pre-mRNA is spliced efficiently in the absence of the Mer1 and Nam8 proteins, which are required for splicing the transcripts of two other meiosis-specific genes.

Isolation and characterization of the SPT2 gene, a negative regulator of Ty-controlled yeast gene expression.

The his4-917 mutation of Saccharomyces cerevisiae results from the insertion of the Ty element Ty917 into the regulatory region of the HIS4 gene and renders the cell His-. The hist4-912 delta mutant, which carries a solo delta in the 5'-noncoding region of HIS4, is His+ at 37 degrees C but His- at 23 degrees C. Both these mutations interfere with HIS4 expression at the transcriptional level. The His- phenotype of both insertion mutations is suppressed by mutations at the SPT2 locus. The product of the wild-type SPT2 gene apparently represses HIS4 transcription in these mutant strains; this repression is relieved when the SPT2 gene is destroyed by mutation. The repression of transcription by SPT2 presumably results from an interaction between the SPT2+ gene product and Ty or delta sequences. In this paper, we report the cloning and DNA sequence analysis of the wild-type SPT2 gene and show that the gene is capable of encoding a protein of 333 amino acids in length. In addition, we show that a dominant mutation of the SPT2 gene results from the generation of an ochre codon which is presumed to lead to a shortened SPT2 gene product.

Intrachromosomal movement of genetically marked Saccharomyces cerevisiae transposons by gene conversion.

In this paper, we describe the movement of a genetically marked Saccharomyces cerevisiae transposon. Ty912(URA3), to new sites in the S. cerevisiae genome. Ty912 is an element present at the HIS4 locus in the his4-912 mutant. To detect movement of Ty912, this element has been genetically marked with the S. cerevisiae URA3 gene. Movement of Ty912(URA3) occurs by recombination between the marked element and homologous Ty elements elsewhere in the S. cerevisiae genome. Ty912(URA3) recombines most often with elements near the HIS4 locus on chromosome III, less often with Ty elements elsewhere on chromosome III, and least often with Ty elements on other chromosomes. These recombination events result in changes in the number of Ty elements present in the cell and in duplications and deletions of unique sequence DNA.

Bypass of a meiotic checkpoint by overproduction of meiotic chromosomal proteins.

The Saccharomyces cerevisiae zip1 mutant, which exhibits defects in synaptonemal complex formation and meiotic recombination, triggers a checkpoint that causes cells to arrest at the pachytene stage of meiotic prophase. Overproduction of either the meiotic chromosomal protein Red1 or the meiotic kinase Mek1 bypasses this checkpoint, allowing zip1 cells to sporulate. Red1 or Mek1 overproduction also promotes sporulation of other mutants (zip2, dmc1, hop2) that undergo checkpoint-mediated arrest at pachytene. In addition, Red1 overproduction antagonizes interhomolog interactions in the zip1 mutant, substantially decreasing double-strand break formation, meiotic recombination, and homologous chromosome pairing. Mek1 overproduction, in contrast, suppresses checkpoint-induced arrest without significantly decreasing meiotic recombination. Cooverproduction of Red1 and Mek1 fails to bypass the checkpoint; moreover, overproduction of the meiotic chromosomal protein Hop1 blocks the Red1 and Mek1 overproduction phenotypes. These results suggest that meiotic chromosomal proteins function in the signaling of meiotic prophase defects and that the correct stoichiometry of Red1, Mek1, and Hop1 is needed to achieve checkpoint-mediated cell cycle arrest at pachytene.

MEI4, a meiosis-specific yeast gene required for chromosome synapsis.

The MEI4 gene product is required for meiotic induction of recombination and viable spore production in the yeast Saccharomyces cerevisiae. DNA sequence analysis shows that the MEI4 gene encodes a 450-amino-acid protein bearing no homology to any previously identified protein. The MEI4 coding region is interrupted by a small intron located near the 5' end of the gene. Efficient splicing of the MEI4 transcript is not dependent on the MER1 protein, which is required for splicing the transcript of another meiotic gene, MER2. Expression of a mei4::lacZ fusion gene is meiosis-specific and depends on both heterozygosity at the mating-type locus and nutrient limitation. Northern (RNA) blot hybridization analysis suggests that MEI4 gene expression is regulated at the level of transcription. A functional MEI4 gene is not required for meiotic induction of transcription of the MER1, MER2, MEK1, RED1, SPO11, or RAD50 gene. Cytological analysis of mei4 mutant strains during meiotic prophase demonstrates that the chromosomes form long axial elements that fail to undergo synapsis. The meiosis II division is delayed in mei4 strains.

Role for the silencing protein Dot1 in meiotic checkpoint control.

During the meiotic cell cycle, a surveillance mechanism called the "pachytene checkpoint" ensures proper chromosome segregation by preventing meiotic progression when recombination and chromosome synapsis are defective. The silencing protein Dot1 (also known as Pch1) is required for checkpoint-mediated pachytene arrest of the zip1 and dmc1 mutants of Saccharomyces cerevisiae. In the absence of DOT1, the zip1 and dmc1 mutants inappropriately progress through meiosis, generating inviable meiotic products. Other components of the pachytene checkpoint include the nucleolar protein Pch2 and the heterochromatin component Sir2. In dot1, disruption of the checkpoint correlates with the loss of concentration of Pch2 and Sir2 in the nucleolus. In addition to its checkpoint function, Dot1 blocks the repair of meiotic double-strand breaks by a Rad54-dependent pathway of recombination between sister chromatids. In vegetative cells, mutation of DOT1 results in delocalization of Sir3 from telomeres, accounting for the impaired telomeric silencing in dot1.

MEI4, a yeast gene required for meiotic recombination.

Mutants at the MEI4 locus were detected in a search for mutants defective in meiotic gene conversion. mei4 mutants exhibit decreased sporulation and produce inviable spores. The spore inviability phenotype is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The MEI4 gene has been cloned from a yeast genomic library by complementation of the recombination defect and has been mapped to chromosome V near gln3. Strains carrying a deletion/insertion mutation of the MEI4 gene display no meiotically induced gene conversion but normal mitotic conversion frequencies. Both meiotic interchromosomal and intrachromosomal crossing over are completely abolished in mei4 strains. The mei4 mutation is able to rescue the spore-inviability phenotype of spo13 and 52 strains (i.e., mei4 spo13 rad52 mutants produce viable spores), indicating that MEI4 acts before RAD52 in the meiotic recombination pathway.

A chromosome containing HOT1 preferentially receives information during mitotic interchromosomal gene conversion.

The recombination-stimulating sequence, HOT1, is derived from yeast ribosomal DNA and corresponds to the sequences required for promotion of transcription by RNA polymerase I. The effect of HOT1 on mitotic interchromosomal gene conversion was examined in diploid strains carrying his4 heteroalleles. When HOT1 is inserted adjacent to both copies of HIS4, the frequency of His+ recombinants is increased approximately 10-fold. When HOT1 is present on only one of the two homologs, recombination is enhanced and the his4 gene on the HOT1-containing chromosome is preferentially converted. In both pairs of his4 heteroalleles examined, HOT1 stimulates conversion of the his4 mutation which is further from the site of HOT1 insertion more than it stimulates conversion of the HOT1-proximal his4 allele. Compared to recombinants isolated from control strains that lack HOT1, HOT1-promoted His+ recombinants are more often homozygous for sequences distal to HIS4. The preferential conversion of sequences on the HOT1-containing chromosome is consistent with the double-strand-gap repair model of recombination and suggests that HOT1-promoted gene conversion initiates with a double-strand break in HOT1-adjacent sequences.