In many organisms, a synaptonemal complex (SC) intimately connects each pair of homologous chromosomes during much of the first meiotic prophase and is thought to play a role in regulating recombination. In the yeast Saccharomyces cerevisiae, the central element of each SC contains Zip1, a protein orthologous to mammalian SYCP1. To study the dynamics of SCs in living meiotic cells, a functional ZIP1::GFP fusion was introduced into yeast and analyzed by fluorescence video microscopy. During pachytene, SCs exhibited dramatic and continuous movement throughout the nucleus, traversing relatively large distances while twisting, folding, and unfolding. Chromosomal movements were accompanied by changes in the shape of the nucleus, and all movements were reversibly inhibited by the actin antagonist Latrunculin B. Normal movement required the NDJ1 gene, which encodes a meiosisspecific telomere protein needed for the attachment of telomeres to the nuclear periphery and for normal kinetics of recombination and meiosis. These results show that SC movements involve telomere attachment to the nuclear periphery and are actindependent and suggest these movements could facilitate completion of meiotic recombination.actin ͉ meiosis ͉ recombination ͉ synaptonemal complex ͉ yeast S porulation of diploid cells in the ascomycete Saccharomyces cerevisiae is accompanied by a typical meiotic cell cycle that culminates with the production of four haploid ascospores. Chromosomes pair, undergo recombination, and then segregate from each other in two successive divisions. Reciprocal recombination between homologous chromosomes takes place during prophase of the first meiotic division (prophase I) and is essential for proper segregation. In S. cerevisiae, prophase I can be divided into leptotene-, zygotene-, pachytene-, and diplotene-like substages defined by the state of chromosome pairing and condensation. During leptotene, chromosomes organize on proteinaceous axial elements, and double-strand breaks (DSBs) begin to appear in DNA, initiating recombination. At the same time, perinuclear telomeres begin to cluster near the spindle pole body (the yeast centrosome), pushing chromosomes into a bouquetlike configuration at the leptotene/zygotene transition. At zygotene, the axial elements pair or synapse at the sites of DSBs, and tripartite proteinaceous structures called synaptonemal complexes (SCs) begin to form between homologous chromosomes (1-3). At pachytene, the SCs have matured by a zipper-like mechanism into ribbon-like structures that are embedded at each end in the nuclear envelope and intimately connect each pair of homologues from end to end (3-5). Mature SCs are present for at least 1 h in strain SK1 and longer in other strains, so that pachytene accounts for a relatively large part of prophase I (6-8). In S. cerevisiae, DNA strand invasion and Holliday junction recombination intermediates are observed throughout zygotene and pachytene (9-12), suggesting that recombination is completed during pachytene, presumably when chromosomes...

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.

Analysis of cloned sequences for yeast histone genes H2A and H2B reveals that there are only two copies of this pair of genes within the haploid yeast genome. Within each copy, the genes for H2A and H2B are separated by approximately 700 bp of spacer DNA. The two copies are separated from one another in the yeast genome by a minimum distance of 35-60 kb. Sequence homology between the two copies is restricted to the genes for H2A and H2B; the spacer DNA between the genes is nonhomologous. In both copies, the genes for H2A and H2B are divergently transcribed. In addition, both plasmids code for other nonhistone proteins. Sequences coding for histones H3 and H4 have not been detected in the immediate vicinity of the genes for H2A and H2B.

Chromosome I from the yeast Saccharomyces cerevisiae contains a DNA molecule of -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 yeast Saccharomyces cerevisiae has been the focus of intensive study as a model eukaryote. As part of this effort, an international program is under way to determine the nucleotide sequence of the 16 chromosomes that constitute its 13.5-Mbp nuclear genome. This endeavor will provide both a complete eukaryotic gene set and a reference set of experimentally amenable genes for comparison with those of other organisms. Currently, four yeast chromosomes have been sequenced (1-4); all have a high gene density, and a majority of the genes found are newly sequenced and of unknown function. Chromosome I is the smallest S. cerevisiae chromosome. It contains a DNA molecule that is only 231 kbp, making it the smallest known fully functional nuclear chromosome. This chromosome has been studied intensively, and mutants are available for a large number of its genes (5-7). Here we report the nucleotide sequence of chromosome I and describe several unusual features of its gene organization and chromosome structure as well as many newly discovered genes.** MATERIALS AND METHODS DNA Sources. Four sources of chromosome I DNA, all from S288C-derived yeast strains, were used to generate the tem-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.plates for DNA sequencing. These were the library of Riles et aL (8), a cosmid from the collection of Dujon (9), chromosome walking (10), and PCR amplified fragments of genomic DNA. DNA fragments, except those generated by PCR which were used directly, were subcloned into the Bluescript KS(+) plasmid from Stratagene prior to sequencing. All DNA sequencing was performed using double-stranded DNA templates.DNA Sequencing. Two methods were used for sequencing DNA templates: manual sequencing and machine-based sequencing with an Applied Biosystems sequencing machine (model 373A). Our manual sequencing used unidirectional nested deletions and was carried out as described (11, 12). For machine-based sequencing, three sets of templates were used: unidirectional nested deletions, PCR amplified chromosomal DNA, and, for the region spanning YAL062 to CDC24, cosmid DNA was shotgun cloned into Bluescript KS(+). In summary, the procedure for the Applied Biosystems machine (model 373A) used dye-labeled dideoxynucleotide terminators and a cycle sequencing kit (Prism Ready reaction dye terminator kit; Perkin-Elmer) and the protocol provided by the supplier. This method allowed us to process all four sequencing reactions in a single reaction tube. The cycle amplification reactions were performed with a Perkin-Elmer ...

The MAL6 locus is one of five closely related unlinked loci, any one of which is sufficient for fermentation of maltose in Saccharomyces. Previous genetic analysis indicated that this locus is defined by two complementation groups, MALp and MALg. MALp reportedly is a regulatory gene required for inducible synthesis of the two enzymatic functions needed for fermentation: maltose permease and maltase. We have investigated the physical and genetic structure of the MAL6 locus, which has been isolated on a recombinant DNA plasmid. One subclone of the region, pDF-1, was found to encode a single transcribed region and to contain the MALp gene. A second subclone, pl, was shown to contain the MALg function but surprisingly had not one but two maltose-inducible transcripts. Subclones having only one of these transcribed regions lacked MALg activity. The three transcribed regions have been named MAL61 and MAL62, which correspond to MALg, and MAL63, which corresponds to MALp. This clustered arrangement of a regulatory gene adjacent to the sequences it controls has not previously been described in eukaryotes and is reminiscent of bacterial operons except that the messenger RNA molecules are not polycistronic.

Improved methods for the formation and stabilization of R-loops for visualization in the electron microscope are presented. The two complementary strands of a duplex DNA are photochemically crosslinked once every 1 to 3 kb using 4, 5', 8 trimethylpsoralen. R-loops are then formed by incubation with RNA in 70% formamide at a temperature above the DNA melting temperature. Finally, the R-loops are stabilized by modifying the free single strand of DNA with glyoxal, thus minimizing the displacement of the hybridized RNA by branch migration. In this manner R-loops can be formed and visualized at a high frequency irrespective of the base composition of the nucleic acid of interest.

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Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYKI genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pykl-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYKI genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYKI genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYKI, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYKI were only -7.5 kb apart, although the genetic map distance between them is -13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to -3 kb, the region between CDC24 and PYKI appears to undergo meiotic recombination at an unusually high frequency.