Drora Zenvirth

Sister chromatid‐based DNA repair is mediated by RAD54, not by DMC1 or TID1

In the mitotic cell cycle of the yeast cerevisiae, the sister chromatid is preferred over the homologous chromosome (non‐sister chromatid) as a substrate for DNA double‐strand break repair. However, no genes have yet been shown to be preferentially involved in sister chromatid‐mediated repair. We developed a novel method to identify genes that are required for repair by the sister chromatid, using a haploid strain that can embark on meiosis. We show that the recombinational repair gene RAD54 is required primarily for sister chromatid‐based repair, whereas TID1, a yeast RAD54 homologue, and the meiotic gene DMC1, are dispensable for this type of repair. Our observations suggest that the sister chromatid repair pathway, which involves RAD54, and the homologous chromosome repair pathway, which involves DMC1, can substitute for one another under some circumstances. Deletion of RAD54 in S.cerevisiae results in a phenotype similar to that found in mammalian cells, namely impaired DNA repair and reduced recombination during mitotic growth, with no apparent effect on meiosis. The principal role of RAD54 in sister chromatid‐based repair may also be shared by mammalian and yeast cells.

Multiple sites for double-strand breaks in whole meiotic chromosomes of Saccharomyces cerevisiae

We present a scheme for locating double‐strand breaks (DSBs) in meiotic chromosomes of Saccharomyces cerevisiae, based on the separation of large DNA molecules by pulsed field gel electrophoresis. Using a rad50S mutant, in which DSBs are not processed, we show that DSBs are widely induced in S. cerevisiae chromosomes during meiosis. Some of the DSBs accumulate at certain preferred sites. We present general profiles of DSBs in chromosomes III, V, VI and VII. A map of the 12 preferred sites on chromosome III is presented. At least some of these sites correlate with known ‘hot spots’ for meiotic recombination. The data are discussed in view of current models of meiotic recombination and chromosome segregation.

Four linked genes participate in controlling sporulation efficiency in budding yeast.

Quantitative traits are conditioned by several genetic determinants. Since such genes influence many important complex traits in various organisms, the identification of quantitative trait loci (QTLs) is of major interest, but still encounters serious difficulties. We detected four linked genes within one QTL, which participate in controlling sporulation efficiency in Saccharomyces cerevisiae. Following the identification of single nucleotide polymorphisms by comparing the sequences of 145 genes between the parental strains SK1 and S288c, we analyzed the segregating progeny of the cross between them. Through reciprocal hemizygosity analysis, four genes, RAS2, PMS1, SWS2, and FKH2, located in a region of 60 kilobases on Chromosome 14, were found to be associated with sporulation efficiency. Three of the four “high” sporulation alleles are derived from the “low” sporulating strain. Two of these sporulation-related genes were verified through allele replacements. For RAS2, the causative variation was suggested to be a single nucleotide difference in the upstream region of the gene. This quantitative trait nucleotide accounts for sporulation variability among a set of ten closely related winery yeast strains. Our results provide a detailed view of genetic complexity in one “QTL region” that controls a quantitative trait and reports a single nucleotide polymorphism-trait association in wild strains. Moreover, these findings have implications on QTL identification in higher eukaryotes.

Switching yeast from meiosis to mitosis: double-strand break repair, recombination and synaptonemal complex

When Saccharomyces cerevisiae cells that have begun meiosis are transferred to mitotic growth conditions (‘return‐to‐growth’, RTG), they can complete recombination at high meiotic frequencies, but undergo mitotic cell division and remain diploid. It was not known how meiotic recombination intermediates are repaired following RTG. Using molecular and cytological methods, we investigated whether the usual meiotic apparatus could repair meiotically induced DSBs during RTG, or whether other mechanisms are invoked when the developmental context changes.

An essential role for sodium in the bicarbonate transporting system of the cyanobacterium Anabaena variabilis

The apparent photosynthetic affinity of Anabaena variabilis for extracellular inorganic carbon (Ci) was strikingly increased by Na+. The effect was highly specific for Na+ and was maximal at 40 mM Na+. Na+ supply decreased the apparent K m (Ci) of the Ci transporting system and to a lesser extent increased V max. It did not affect photosynthetic rate expressed as a function of intracellular Ci. We infer an effect of Na+ on the Ci transporting system rather than on the photosynthetic machinery itself. We propose several possible models, including Na+‐H+ antiport for maintenance of intracellular pH during HCO3 uptake, and Na+‐HCO− 3 symport.

Uptake and efflux of inorganic carbon in Dunaliella salina.

The apparent photosynthetic Km (CO2) of air-grown Dunaliella salina is 2 μM as measured both by the filtering centrifugation technique and by O2 electrode. These cells are capable of accumulating inorganic carbon (Cinorg) up to 20 times its concentration in the medium. It is suggested that air-grown Dunaliella cells are able to concentrate CO2 within the cell. Analysis of the efflux of Cinorg from cells previously loaded with H14CO3-demonstrated the existence of an internal pool which has an half-time of depletion of 2.5–7 min depending on the conditions of the experiment. This finding indicates that the internal Cinorg pool is not readily exchangeable with the external medium. Furthermore, the influence of the presence or absence of unlabelled Cinorg in the medium during the efflux experiment on the half-time observed indicate that efflux of Cinorg is not a simple diffusion process but is rather carrier-mediated.

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.

Modulation of the transcription regulatory program in yeast cells committed to sporulation.

BackgroundMeiosis in budding yeast is coupled to the process of sporulation, where the four haploid nuclei are packaged into a gamete. This differentiation process is characterized by a point of transition, termed commitment, when it becomes independent of the environment. Not much is known about the mechanisms underlying commitment, but it is often assumed that positive feedback loops stabilize the underlying gene-expression cascade.ResultsWe describe the gene-expression program of committed cells. Sporulating cells were transferred back to growth medium at different stages of the process, and their transcription response was characterized. Most sporulation-induced genes were immediately downregulated upon transfer, even in committed cells that continued to sporulate. Focusing on the metabolic-related transcription response, we observed that pre-committed cells, as well as mature spores, responded to the transfer to growth medium in essentially the same way that vegetative cells responded to glucose. In contrast, committed cells elicited a dramatically different response.ConclusionOur results suggest that cells ensure commitment to sporulation not by stabilizing the process, but by modulating their gene-expression program in an active manner. This unique transcriptional program may optimize sporulation in an environment-specific manner.

Spore germination in Saccharomyces cerevisiae: global gene expression patterns and cell cycle landmarks

BackgroundSpore germination in the yeast Saccharomyces cerevisiae is a process in which non-dividing haploid spores re-enter the mitotic cell cycle and resume vegetative growth. To study the signals and pathways underlying spore germination we examined the global changes in gene expression and followed cell-cycle and germination markers during this process.ResultsWe find that the germination process can be divided into two distinct stages. During the first stage, the induced spores respond only to glucose. The transcription program during this stage recapitulates the general transcription response of yeast cells to glucose. Only during the second phase are the cells able to sense and respond to other nutritional components in the environment. Components of the mitotic machinery are involved in spore germination but in a distinct pattern. In contrast to the mitotic cell cycle, growth-related events during germination are not coordinated with nuclear events and are separately regulated. Thus, genes that are co-induced during G1/S of the mitotic cell cycle, the dynamics of the septin Cdc10 and the kinetics of accumulation of the cyclin Clb2 all exhibit distinct patterns of regulation during spore germination, which allow the separation of cell growth from nuclear events.ConclusionTaken together, genome-wide expression profiling enables us to follow the progression of spore germination, thus dividing this process into two major stages, and to identify germination-specific regulation of components of the mitotic cell cycle machinery.

Meiotic double-strand breaks in Schizosaccharomyces pombe

Abstract Meiotic DNA double-strand breaks (DSBs) are associated with recombination hot spots in the yeast Saccharomyces cerevisiae and are believed to initiate the process of recombination. Until now, meiosis-induced breaks have not been shown to occur regularly in other organisms. Here we show, by pulsed-field gel electrophoresis of DNA, that meiotic DSBs occur transiently in all three chromosomes of the fission yeast Schizosaccharomyces pombe. In a repair defective mutant, carrying a mutation in the RecA homolog gene rhp51, meiotic DSBs accumulate. In contrast to expectation from the genetic map of S. pombe, however, many chromosomal DNA molecules remain unbroken during meiosis.