David B. Kaback

Chromosome mobility during meiotic prophase in Saccharomyces cerevisiae.

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 meiosis-specific 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 actin-dependent and suggest these movements could facilitate completion of meiotic recombination.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome.

Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 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 pyk1-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 PYK1 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 PYK1 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, PYK1, 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 PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.

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

The preferred positions for meiotic double-strand breakage were mapped onSaccharomyces 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.

Meiotic Recombination at the Ends of Chromosomes in Saccharomyces Cerevisiae

Meiotic reciprocal recombination (crossing over) was examined in the outermost 60–80 kb of almost all Saccharomyces cerevisiae chromosomes. These sequences included both repetitive gene-poor subtelomeric heterochromatin-like regions and their adjacent unique gene-rich euchromatin-like regions. Subtelomeric sequences underwent very little crossing over, exhibiting approximately two- to threefold fewer crossovers per kilobase of DNA than the genomic average. Surprisingly, the adjacent euchromatic regions underwent crossing over at twice the average genomic rate and contained at least nine new recombination “hot spots.” These results prompted an analysis of existing genetic mapping data, which showed that meiotic reciprocal recombination rates were on average greater near chromosome ends exclusive of the subtelomeres. Thus, the distribution of crossovers in S. cerevisiae appears to resemble that found in several higher eukaryotes where the outermost chromosomal regions show increased crossing over.

In Vivo Analysis of Synaptonemal Complex Formation During Yeast Meiosis

During meiotic prophase a synaptonemal complex (SC) forms between each pair of homologous chromosomes and is believed to be involved in regulating recombination. Studies on SCs usually destroy nuclear architecture, making it impossible to examine the relationship of these structures to the rest of the nucleus. In Saccharomyces cerevisiae the meiosis-specific Zip1 protein is found throughout the entire length of each SC. To analyze the formation and structure of SCs in living cells, a functional ZIP1::GFP fusion was constructed and introduced into yeast. The ZIP1::GFP fusion produced fluorescent SCs and rescued the spore lethality phenotype of zip1 mutants. Optical sectioning and fluorescence deconvolution light microscopy revealed that, at zygotene, SC assembly was initiated at foci that appeared uniformly distributed throughout the nuclear volume. At early pachytene, the full-length SCs were more likely to be localized to the nuclear periphery while at later stages the SCs appeared to redistribute throughout the nuclear volume. These results suggest that SCs undergo dramatic rearrangements during meiotic prophase and that pachytene can be divided into two morphologically distinct substages: pachytene A, when SCs are perinuclear, and pachytene B, when SCs are uniformly distributed throughout the nucleus. ZIP1::GFP also facilitated the enrichment of fluorescent SC and the identification of meiosis-specific proteins by MALDI-TOF mass spectroscopy.

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.

Telomeric silencing of an open reading frame in Saccharomyces cerevisiae.

The endmost chromosome I ORF is silenced by a natural telomere position effect. YAR073W/IMD1 was found to be transcribed at much higher levels in sir3 mutants and when its adjacent telomere was removed from it. These results suggest that telomeres play a role in silencing actual genes.

Meiotic segregation of circular plasmid-minichromosomes from intact chromosomes in Saccharomyces cerevisiae

SummaryDistributive disjunction is defined by first meiotic division segregation of either two nonhomologous chromosomes that lack homologous pairing partners, or of two homologous chromosomes that have failed to undergo crossing-over. In the yeast Saccharomyces cerevisiae, plasmid minichromosomes, synthetic linear chromosomes and a fragment of a real chromosome have been observed to segregate from nonhomologous DNA species at the first meiotic divisions. Suggesting that this organism may have a distributive mechanism for chromosome segregation. However, it is not known whether intact chromosomes also participate in a distributive process. To determine whether intact, full length, S. cerevisiae chromosomes could segregate from nonhomologous chromosomal species, the meiotic behavior of an unpaired intact copy of chromosome I has been analyzed with respect to several centromere-containing circular plasmid minichromosomes. Strains monosomic or trisomic for chromosome I were transformed with centromere plasmids containing either homologous or nonhomologous inserts, sporulated, and analyzed genetically both for the presence of plasmid and for the number of copies of chromosome 1. Each plasmid segregated from an intact unpaired copy of chromosome I at the first meiotic division in a significant majority (63–93%) of the asci examined. These results suggest that intact chromosomes from S. cerevisiae are capable of distributive disjunction.

A selfish DNA element engages a meiosis-specific motor and telomeres for germ-line propagation

The yeast 2 micron plasmid engages a meiosis-specific motor that orchestrates telomere-led chromosome movements for its telomere-associated segregation during meiosis I.

A simple method for isolating disomic strains of Saccharomyces cerevisiae

A simple method to select disomic (N + 1) strains that should be applicable for almost any chromosome in Saccharomyces cerevisiae is presented. A diploid heterozygous for a KanMX knock‐out mutation in an essential gene is sporulated and viable geneticin (G418)‐resistant colonies selected. Disomic products of a missegregation or non‐disjunction event containing a copy of both the wild‐type essential gene and its complementary KanMX knock‐out allele make up most of the viable colonies. This method has been used to isolate disomic haploids for a variety of chromosomes. It is appropriately named MARV (for missegregation‐associated restoration of viability) and is easily adaptable to virtually any strain. Copyright