G. Shirleen Roeder

Large-scale analysis of the yeast genome by transposon tagging and gene disruption

Economical methods by which gene function may be analysed on a genomic scale are relatively scarce. To fill this need, we have developed a transposon-tagging strategy for the genome-wide analysis of disruption phenotypes, gene expression and protein localization, and have applied this method to the large-scale analysis of gene function in the budding yeast Saccharomyces cerevisiae. Here we present the largest collection of defined yeast mutants ever generated within a single genetic background—a collection of over 11,000 strains, each carrying a transposon inserted within a region of the genome expressed during vegetative growth and/or sporulation. These insertions affect nearly 2,000 annotated genes, representing about one-third of the 6,200 predicted genes in the yeast genome. We have used this collection to determine disruption phenotypes for nearly 8,000 strains using 20 different growth conditions; the resulting data sets were clustered to identify groups of functionally related genes. We have also identified over 300 previously non-annotated open reading frames and analysed by indirect immunofluorescence over 1,300 transposon-tagged proteins. In total, our study encompasses over 260,000 data points, constituting the largest functional analysis of the yeast genome ever undertaken.

ZIP1 is a synaptonemal complex protein required for meiotic chromosome synapsis

ZIP1 is a novel meiosis-specific gene required for chromosome synapsis and cell cycle progression in S. cerevisiae. zip1 strains undergo homologous chromosome pairing, but are defective in synaptonemal complex (SC) formation. The zip1 mutation confers a uniform arrest in meiosis prior to the first division. zip1 strains display nearly wild-type levels of commitment to meiotic recombination; however, mature reciprocal recombinants are not formed until cells are released from meiotic arrest by return to growth medium. DNA sequence analysis of ZIP1 reveals structural homology to a number of proteins containing coiled coils. Immunofluorescence experiments using anti-ZIP1 antibodies demonstrate that the ZIP1 protein localizes to synapsed meiotic chromosomes but not to unsynapsed axial elements. Taken together, these data suggest that ZIP1 is a component of the central region of the SC. We propose a model in which ZIP1 acts as a molecular zipper to bring homologous chromosomes in close apposition.

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.

Recombination-stimulating sequences in yeast ribosomal DNA correspond to sequences regulating transcription by RNA polymerase I

A DNA sequence (HOT1) from the repeated ribosomal RNA gene cluster of Saccharomyces cerevisiae can stimulate genetic exchange when inserted at novel locations in the yeast genome. Localization of the sequences required for HOT1 activity demonstrates that two noncontiguous fragments of DNA are required for the stimulation of recombination. One of these fragments contains the transcription initiation site for the major 35S ribosomal RNA precursor. The other contains an enhancer of RNA polymerase I transcription. We suggest that transcription by RNA polymerase I initiating in the inserted rDNA and proceeding through the adjacent sequences is responsible for the stimulation of exchange. Consistent with this interpretation, insertion of the putative termination site for RNA polymerase I transcription between HOT1 and the adjacent recombining DNA abolishes the recombination stimulation. Transcription through both copies of the homologous recombining sequences appears to be necessary for enhanced exchange.

Pch2 Links Chromatin Silencing to Meiotic Checkpoint Control

The PCH2 gene of Saccharomyces cerevisiae is required for the meiotic checkpoint that prevents chromosome segregation when recombination and chromosome synapsis are defective. Mutation of PCH2 relieves the checkpoint-induced pachytene arrest of the zip1, zip2, and dmc1 mutants, resulting in chromosome missegregation and low spore viability. Most of the Pch2 protein localizes to the nucleolus, where it represses meiotic interhomolog recombination in the ribosomal DNA, apparently by excluding the meiosis-specific Hop1 protein. Nucleolar localization of Pch2 depends on the silencing factor Sir2, and mutation of SIR2 also bypasses the zip1 pachytene arrest. Under certain circumstances, Sir3-dependent localization of Pch2 to telomeres also provides checkpoint function. These unexpected findings link the nucleolus, chromatin silencing, and the pachytene checkpoint.

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.

Cis-acting, recombination-stimulating activity in a fragment of the ribosomal DNA of S. cerevisiae.

Special mechanisms for stimulating recombination among the nearly identical repeat units of certain multigene families may exist in order to maintain their sequence homogeneity. We have found evidence for such a recombination-stimulating activity in the tandemly repeated ribosomal RNA genes of yeast. A fragment of the yeast ribosomal DNA (rDNA), containing the 5S gene, nontranscribed spacer DNA, and part of the 25S gene, causes a localized stimulation of recombination when inserted at novel locations in the yeast genome. The rDNA fragment stimulates both interchromosomal and intrachromosomal mitotic recombination but not meiotic recombination. To stimulate mitotic recombination, the fragment must act on both copies of the recombining gene. Furthermore, the rDNA fragment stimulates exchange only when inserted with the 5S gene proximal to, and the 25S gene distal to, the recombining alleles.

Imposition of Crossover Interference through the Nonrandom Distribution of Synapsis Initiation Complexes

Meiotic crossovers (COs) are nonrandomly distributed along chromosomes such that two COs seldom occur close together, a phenomenon known as CO interference. We have used genetic and cytological methods to investigate interference mechanisms in budding yeast. Assembly of the synaptonemal complex (SC) initiates at a few sites along each chromosome, triggered by a complex of proteins (including Zip2 and Zip3) called the synapsis initiation complex (SIC). We found that SICs, like COs, display interference, supporting the hypothesis that COs occur at synapsis initiation sites. Unexpectedly, we found that SICs show interference in mutants in which CO interference is abolished; one explanation is that these same mutations eliminate the subset of COs that normally occur at SICs. Since SICs are assembled in advance of SC and they are properly positioned even in the absence of SC formation, these data clearly demonstrate an aspect of interference that is independent of synapsis.

The Meiosis-Specific Hop2 Protein of S. cerevisiae Ensures Synapsis between Homologous Chromosomes

The hop2 mutant of S. cerevisiae displays a novel phenotype: meiotic chromosomes form nearly wild-type amounts of synaptonemal complex, but most chromosomes are engaged in synapsis with nonhomologous partners. The meiosis-specific Hop2 protein localizes to chromosomes prior to and during synapsis and in the absence of the double-strand breaks that initiate recombination. hop2 strains sustain a wild-type level of meiotic double-strand breaks, but these breaks remain unrepaired. The hop2 mutant arrests at the pachytene stage of meiotic prophase with the RecA-like protein Dmc1 located at numerous sites along synapsed chromosomes. We propose that the Hop2 protein functions to prevent synapsis between nonhomologous chromosomes.

Meiosis-specific RNA splicing in yeast

Previous studies have suggested that the differentiated state of meiosis in yeast is regulated primarily at the transcriptional level. This study reports a case of posttranscriptional regulation of a gene whose product is essential for meiosis. The MER2 gene is transcribed in mitosis as well as meiosis; however, the transcript is spliced efficiently to generate a functional gene product only in meiosis. Meiotic levels of splicing depend on the MER1 gene product, which is also essential for meiosis and which is produced only in meiotic cells. Therefore, at least one of the functions of the MER1 protein is to mediate splicing of the MER2 transcript. Genetic data suggest that the MER1 gene may also be responsible for splicing the transcript of at least one other gene.