Recombination and the Progression of Meiosis in Saccharomyces Cerevisiae

Recombination is an essential part of meiosis; in almost all organisms, including Saccharomyces cerevisiae, proper chromosome segregation and the viability of meiotic products is dependent upon normal levels of recombination. In this article we examine the kinetics of the meiotic divisions in four mutants defective in the initiation of recombination. We find that mutations in any of three Early Exchange genes (REC104, REC114 or REC102) confer a phenotype in which the reductional division occurs earlier than in an isogenic wild-type diploid. We also present data confirming previous reports that strains with a mutation in the Early Exchange gene MEI4 undergo the first division at about the same time as wild-type cells. The rec104 mutation is epistatic to the mei4 mutation for the timing of the first division. These observations suggest a possible relationship between the initiation of recombination and the timing of the reductional division. These data also allow these four Early Exchange genes examined to be distinguished in terms of their role in coordinating recombination with the reductional division.     

The REC41 gene of Saccharomyces cerevisiae: isolation and genetic analysis

Recombination-deficient strains have been proven useful for the understanding of the genetic control of homologous recombination. As the genetic screens used to isolate recombination-deficient (rec(-)) yeast mutants have not been saturated, we sought to develop a simple colony color assay to identify mutants with low or elevated rates of recombination. Using this system we isolated a collection of rec(-) mutants. We report the characterization of the REC41 gene identified in this way. REC41 is required for normal levels of interplasmid recombination and gamma-ray induced mitotic interchromosomal recombination. The rec41-1 mutant failed to grow at 37 degrees C. Microscopic analysis of plated cells showed that 45-50% of them did not form visible colonies at permissive temperature. Haploid cells of the rec41 mutant show the same gamma-ray sensitivity as wild type ones. However, the diploid rec41 mutant shows gamma-ray sensitivity which is comparable with heterozygous REC41/rec41-1 diploid cells. This fact indicates semidominance of the rec41-1 mutation. Diploid strains homozygous for the rec41 rad52 mutations had the same gamma-ray sensitivity as single rad52 diploids and exhibited dramatically decreased growth rate. The expression of the HO gene does not lead to inviability of rec41 cells. The rec41 mutation has an effect on meiosis, likely meiotic recombination, even in the heterozygous state. We cloned the REC41 gene. Sequence analysis revealed that the REC41 gene is encoded by ORF YDR245w. Earlier, this ORF was attributed to MNN10, BED1, SLC2, CAX5 genes. Two multicopy plasmids with suppressers of the rec41-1 mutation (pm21 and pm32) were isolated. The deletion analysis showed that only DNA fragments with the CDC43 and HAC1 genes can partially complement the rec41-1 mutation.     

Procedures used in the induction of mitotic recombination and mutation in the yeast Saccharomyces cerevisiae.

Techniques are described for the use of various yeast strains to detect the induction of (1) mitotic crossing-over, (2) mitotic gene conversion, (3) forward mutation and (4) reverse mutation. The technique for the detection of mitotic crossing over is based on a diploid that carries two different alleles of the gene locus ade2. These alleles differ in their extent of colony pigmentation engendered on low-adenine media, and they complement each other to the effect that the diploid is white. Mitotic crossing over results in the formation of twin-sectored colonies with a red and a pink sector. The technique for the detection of mitotic gene conversion is based on the use of a heteroallelic diploid carrying two non-complementing alleles that cause a nutritional requirement. Mitotic gene conversion leads to the restoration of intact and dominant wild-type alleles that alleviate the nutritional requirement so that convertant cells can be selected on a minimal medium. The forward mutation technique is based on the use of a haploid strain with a defect in the ade2-gene locus which causes the formation of red colonies. Induction of forward mutation in a number of other loci prevents the accumulation of this red pigment so that induction of mutation can be detected by the formation of pink and white colonies. The reverse mutation technique is based on the restoration or compensation of a mutational defect causing a growth requirement. Mutants can be selected for on a minimal medium.     

Positional cloning and characterization of mouse mei8, a disrupted allelle of the meiotic cohesin Rec8

A novel mutation, mei8, was isolated in a forward genetic screen for infertility mutations induced by chemical mutagenesis of ES cells. Homozygous mutant mice are sterile. Mutant females exhibit ovarian dysgenesis and lack ovarian follicles at reproductive maturity. Affected males have small testes due to arrest of spermatogenesis during meiotic prophase I. Genetic mapping and positional cloning of mei8 led to the identification of a mutation in Rec8, a homolog of the yeast meiosis-specific cohesin gene REC8. Analysis of meiosis in Rec8(mei8)/Rec8(mei8) spermatocytes showed that, while initiation of recombination and synapsis occurs, REC8 is required for the completion and/or maintenance of synapsis, cohesion of sister chromatids, and the formation of chiasmata, as it is in other organisms. However, unlike yeast and Caenorhabditis elegans, localization of REC8 on meiotic chromosomes is not required for the assembly of axial elements.     

Testing of some permitted food colours for the induction of gene conversion in diploid yeast.

12 permitted food colours in use were screened for geno-toxicity. Mitotic gene conversion in Saccharomyces cerevisiae was used as the end-point. Each food colour was tested in stationary-phase as well as log-phase cells but without microsomal activation. These food colours did not cause any increase in mitotic gene conversion in diploid yeast BZ 34.     

The PSO3 gene is involved in error-prone intragenic recombinational DNA repair in Saccharomyces cerevisiae

Summary The induction of gene conversion and mitotic crossing-over by photoaddition of psoralens, 254 nm ultraviolet radiation, and nitrogen mustards was determined in diploid cells homozygous for the pso3-1 mutation and in the corresponding wild type of Saccharomyces cerevisiae. For these different agents, the frequency of non-reciprocal events (conversion) is reduced in the pso3-1 mutant compared to the wild type. In contrast, the frequency of reciprocal events (crossing-over) is increased at a range of doses. These observations, together with the block in induced mutagenesis for both reverse and forward mutations previously reported for the pso3-1 mutant, suggest that the PS03 gene product plays a role in mismatch repair of short patch regions. The block in gene conversion in the pso3 homozygous diploid leads, in the case of nitrogen mustards, to specific repair intermediates which are lethal to the cells.     

Enhancing effects of cytosine arabinoside on ethyl methanesulfonate-induced 6-thioguanine resistance mutations in Chinese hamster V79 cells

We studied the synergistic enhancement effects of two chemicals which are different in their mechanism of action on DNA in cells. The test chemicals used were ethyl methanesulfonate (EMS) as an alkylating agent and cytosine arabinoside (Ara-C) as an analogue of cytidine. For determination of mutagenesis we measured the induction of resistance to 6-thioguanine (6-TG) in Chinese hamster V79 cells. EMS had a strong mutagenic effect on V79 cells, but for Ara-C the results were less clear. In this study, Ara-C had no detectable effect in inducing mutation up to a concentration of 5 X 10(-4) M. The mutation frequency of combined treatment with EMS and Ara-C was significantly higher than that obtained with EMS alone. These results indicate that Ara-C had an enhancing effect on mutations induced by EMS.     

The modification of induced genetic change in yeast by an amino acid analogue

Summary Treatment of diploid yeast cultures with the amino acid analogue, para-fluorophenylalanine (PFPA), at concentrations which caused inhibition of growth, resulted in up to 5 fold increases in the frequency of mitotic gene conversion at two different heteroallelic loci. With haploid yeast cultures, growth in PFPA increased the rate of forward mutation to canavanine resistance by at least 2 fold.Growth of diploids in PFPA prior to exposure to the deaminating agent nitrous acid, the cross-linking agent mitomycin C, the alkylating chemical ethylmethanesulphonate (EMS) and UV light resulted in significant changes in the potency of these diverse mutagens to induce intragenic recombination. For all four mutagens, increased frequencies of gene convertants/viable cell were observed in those cultures which had been exposed to the amino acid analogue prior to mutagen treatment. In haploid WT yeast cells, amino acid analogue incorporation resulted in an enhanced frequency of UV induced forward mutation to canavanine resistance whilst in a DNA repair deficient rad 6 mutant this interaction between UV and PFPA was abolished.The results have been interpreted on the basis of incorporation of the analogue into enzymes involved with DNA replication with a consequent loss of fidelity of such enzymes and increased errors in base incorporation.     

Evaluation and re-evaluation of genetic radiation hazards in man. I. Interspecific comparison of estimates of mutation rates.

A detailed presentation is made of the experimental data from the various systems used by Abrahamson et al. [2] to conclude that the per locus per rad (low LET) radiation-induced forward mutation rates in organisms, whose DNA content varies by a factor of about 1000, is proportional to genome size. Additional information pertinent in this context is also reviewed. It is emphasized that the mutation rates cited by Abrahamson et al. [2], although considered as pertaining to mutations at specific loci, actually derive from a broad variety of genetic end-points. It is argued that an initial (if not sufficient) condition for sound inter-specific mutation rate comparisions, covering a wide range of organisms and detecting systems of various sensitivities, requires a reasonalbly consistent biological definition of a specific locus mutation, namely, a transmissible intra-locus change. Granting the differences between systems in their resolving power to detect intragenic change, the data cited in this paper do not support the existence of a simple proportionality between radiotion-induced intra-locus mutation rate and genome size for the different species reviewed here. Furthermore, in Drosophila melanogaster, where individual salivary gland chromosome bands (that can differ greatly in DNA content) are usually associated with individual loci or at least distinct complementation groups, radiation-induced intra-locus mutation rates are not correlated with apparent differences in the DNA content of bands. This result is incompatible with the notion that most of the DNA in a band represents a radiation-mutable target capable of eliciting the kind of mutation observed in mutation rate experiments. All these considerations argue against the validity of the hypothesis of Abrahamson et al. [2] and their generalization that, for the evaluation of genetic radiation hazards in man, we can now "extrapolate from mutation rates obtained in lower organisms to man with greater confidence" on the basis of DNA content (italics are ours).     

The Rec102 Mutant of Yeast Is Defective in Meiotic Recombination and Chromosome Synapsis

A mutation at the REC102 locus was identified in a screen for yeast mutants that produce inviable spores. rec102 spore lethality is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The rec102 mutation completely eliminates meiotically induced gene conversion and crossing over but has no effect on mitotic recombination frequencies. Cytological studies indicate that the rec102 mutant makes axial elements (precursors to the synaptonemal complex), but homologous chromosomes fail to synapse. In addition, meiotic chromosome segregation is significantly delayed in rec102 strains. Studies of double and triple mutants indicate that the REC102 protein acts before the RAD52 gene product in the meiotic recombination pathway. The REC102 gene was cloned based on complementation of the mutant defect and the gene was mapped to chromosome XII between CDC25 and STE11.     

Potential Germline Competition in Animals and Its Evolutionary Implications

Mutation, mitotic crossing over and mitotic gene conversion can create genetic diversity in otherwise uniform diploid cell lineages. In the germline this diversification may result in competition between diploid germline phenotypes, with subsequent biases in the frequency of alleles transmitted to the offspring. Sperm competition is a well documented feature of many higher organisms and a model is developed to quantify this process. Competition, and hence selection, can also occur by differential survival of diploid lineages before meiosis. It is concluded that under certain circumstances germline selection is an efficient means of eliminating unfavorable alleles from the population. This does not require differences in adult fertility or viability which is the usual mechanism cited as causing changes in gene frequency in a population. It is proposed that such competition may play a role in maintaining the efficiency of basic metabolic pathways.     

Pulsed-field gel electrophoresis indicates genotypic heterogeneity among Campylobacter upsaliensis strains

Abstract A non-flocculent strain of Saccharomyces cerevisiae was selected after EMS mutation of a flocculent and heterozygous FLO1 locus diploid. The analysis of 25 asci from this diploid showed in all cases segregation 0F:4NF, thus confirming that it was probably affected in the desired gene. After sporulation and dissection of asci, three haploid strains were chosen, which were altered in the locus FLO1 . Crossing these three strains with two other ones having markers for ADE1 and pho11::LEU2 , we could map the mutation at ca. 4.3 cM and ca. 37.7 cM from the PHO11 and ADE1 loci respectively.     

Integrating genomics,bioinformatics, and classical genetics to study the effects of recombination on genome evolution

This study presents compelling evidence that recombination significantly increases the silent GC content of a genome in a selectively neutral manner, resulting in a highly significant positive correlation between recombination and "GC3s" in the yeast Saccharomyces cerevisiae. Neither selection nor mutation can explain this relationship. A highly significant GC-biased mismatch repair system is documented for the first time in any member of the Kingdom Fungi. Much of the variation in the GC3s within yeast appears to result from GC-biased gene conversion. Evidence suggests that GC-biased mismatch repair exists in numerous organisms spanning six kingdoms. This transkingdom GC mismatch repair bias may have evolved in response to a ubiquitous AT mutational bias. A significant positive correlation between recombination and GC content is found in many of these same organisms, suggesting that the processes influencing the evolution of the yeast genome may be a general phenomenon. Nonrecombining regions of the genome and nonrecombining genomes would not be subject to this type of molecular drive. It is suggested that the low GC content characteristic of many nonrecombining genomes may be the result of three processes (1) a prevailing AT mutational bias, (2) random fixation of the most common types of mutation, and (3) the absence of the GC-biased gene conversion which, in recombining organisms, permits the reversal of the most common types of mutation. A model is proposed to explain the observation that introns, intergenic regions, and pseudogenes typically have lower GC content than the silent sites of corresponding open reading frames. This model is based on the observation that the greater the heterology between two sequences, the less likely it is that recombination will occur between them. According to this "Constraint" hypothesis, the formation and propagation of heteroduplex DNA is expected to occur, on average, more frequently within conserved coding and regulatory regions of the genome. In organisms possessing GC-biased mismatch repair, this would enhance the GC content of these regions through biased gene conversion. These findings have a number of important implications for the way we view genome evolution and suggest a new model for the evolution of sex.     

Characterization of an in vitro micronucleus assay with Syrian hamster embryo fibroblasts

The use of Syrian hamster embryo cells for assessing genotoxicity provides the unique opportunity to determine 5 different end-points (gene mutations, DNA-strand breaks, aneuploidy, DNA repair (unscheduled DNA synthesis, UDS) and neoplastic transformation) in the one cell system. This approach allows direct comparisons of results produced under identical conditions of dose at target, metabolism and bioavailability. We report here on the characterization of an additional end-point in the same cell system: the formation of micronuclei indicating chromosomal changes induced by chemicals. For a preliminary validation of this new test system we have investigated 14 carcinogens and 3 non-carcinogenic structural analogues in order to evaluate the significance of micronucleus induction for carcinogenic properties. All tested carcinogens induced micronuclei in a dose-dependent manner; all non-carcinogens yielded negative results. Correlations between the formation of micronuclei and the Ames test, induction of UDS, cell transformation and the in vivo bone marrow micronucleus test are demonstrated.     

Estimating the contribution of mutation, recombination and gene conversion in the generation of haplotypic diversity

Recombination occurs through both homologous crossing over and homologous gene conversion during meiosis. The contribution of recombination relative to mutation is expected to be dramatically reduced in inbreeding organisms. We report coalescent-based estimates of the recombination parameter (rho) relative to estimates of the mutation parameter (theta) for 18 genes from the highly self-fertilizing grass, wild barley, Hordeum vulgare ssp. spontaneum. Estimates of rho/theta are much greater than expected, with a mean rho/theta approximately 1.5, similar to estimates from outcrossing species. We also estimate rho with and without the contribution of gene conversion. Genotyping errors can mimic the effect of gene conversion, upwardly biasing estimates of the role of conversion. Thus we report a novel method for identifying genotyping errors in nucleotide sequence data sets. We show that there is evidence for gene conversion in many large nucleotide sequence data sets including our data that have been purged of all detectable sequencing errors and in data sets from Drosophila melanogaster, D. simulans, and Zea mays. In total, 13 of 27 loci show evidence of gene conversion. For these loci, gene conversion is estimated to contribute an average of twice as much as crossing over to total recombination.     

Radiation equivalent of genotoxic chemicals: Validation in cultured mammalian cell lines

Published data on mutations induced by ionizing radiation and 6 monofunctional alkylating agents, namely EMS, MMS, ENNG, MNNG, ENU and MNU, in different cell lines (Chinese hamster ovary, Chinese hamster lung V79, mouse lymphoma L5178 and human cells) were analysed so that radiation-equivalent chemical (REC) values could be calculated.REC values thus obtained for a given alkylating agent with different cell lines fall within a narrow range suggesting its validation in cultured mammalian cell systems including human.     

Characterization of REC104, a gene required for early meiotic recombination in the yeast Saccharomyces cerevisiae.

The REC104 gene was initially defined by mutations that rescued the inviability of a rad52 spo 13 haploid strain in meiosis. We have observed that rec104 mutant strains undergo essentially no induction of meiotic gene conversion, and we have not been able to detect any meiotic crossing over in such strains. The REC104 gene has no apparent role in mitosis, since mutations have no observable effect on growth, mitotic recombination, or DNA repair. The DNA sequence of REC104 reveals that it is a previously unknown gene with a coding region of 549-bp, and genetic mapping has localized the gene to chromosome VIII near FUR1. Expression of the REC104 gene is induced in meiosis, and it appears that the gene is not transcribed in mitotic cells. Possible roles for the REC104 gene product in meiosis are discussed.     

Colorimetric broth microdilution method for the antifungal screening of plant extracts against yeasts

Screening plant extracts for antifungal activity is increasing due to demand for new antifungal agents, but the testing methods present many challenges. Standard broth microdilution methods for antifungal susceptibility testing of available antifungal agents are available now, but these methods are optimised for single compounds instead of crude plant extracts. In this study we evaluated the standard NCCLS method as well as a modification which uses spectrophotometric determination of the end-points with a plate reader. We also evaluated another standard method, the EUCAST method, which is a similar microdilution assay to the NCCLS method, but uses a larger inoculum size and a higher glucose concentration in the medium as well as spectrophotometric end-point determination. The results showed that all three methods had some drawbacks for testing plant extracts and thus we modified the NCCLS broth microdilution method by including a colorimetric indicator-resazurin for end-point determination. This modified method showed good reproducibility and clear-cut end-point, plus the end-point determination needed no instruments. It enabled us to evaluate the activity of a selection of extracts from six Combretaceous plants against three Candida spp. and thus provided pharmacological evidence for some traditional uses of these plants while assisting the identification of the active ingredients.     

Genetic effects of 5-azacytidine in Saccharomyces cerevisiae

The base analog 5-azacytidine induced a variety of genetic and epigenetic effects in different organisms. It was tested in two diploid strains of the yeast Saccharomyces cerevisiae to study the induction of point mutation, mitotic reciprocal crossing-over, mitotic gene conversion (strain D7) and mitotic aneuploidy (strain D61.M). It was used on cells growing in its presence for 4-5 generations. There was a strong induction of both types of mitotic recombination and point mutation. However, there was no induction of mitotic chromosomal malsegregation under the same conditions.     

A yeast strain for simultaneous detection of induced mitotic crossing over,mitotic gene conversion and reverse mutation

A diploid yeast strain is described which can be used to study induction of mitotic crossing over, mitotic gene conversion and reverse mutation.Mitotic crossing over can be detected visually as pink and red twin sectored colonies which are due to the formation of homozygous cells of the genotype ade240/ade240 (deep red) and ade-2-119/ade2-119 (pink) from the originally heteroallelic condition ade2-40/ade2-119 which forms white colonies.Mitotic gene conversion is monitored by the appearance of tryptophan non-requiring colonies on selective media. The alleles involved are tryp5-12 and trp5-27 derived from the widely used strain D4.Mutation induction can be followed by the appearance of isoleucine non-requiring colonies on selective media. D7 is homoallelic ilv1-92/ilv1-92. The isoleucine requirement caused by ilv1-92 can be alleviated by true reverse mutation and allele non-specific suppressor mutation.The effects of ethyl methanesulfonate (EMS), nitrous acid, ultraviolet light and hycanthone methanesulfonate were studied with D7 stationary phase cells. Mitotic crossing over as monitored by red/pink twin sectored colonies was almost equally frequent among normal and convertant cells. This showed again that mitotic recombination is not due to the presence fo a few cells committed to meiosis in an otherwise mitotic cell population.The dose-response curves for induction of mitotic gene conversion and reversion of the isoleucine requirement were exponential. In contrast to this, the dose-response curve for induction of twin sectored red and pink colonies reached a plateau at doses giving about 30% cell killing. This could partly be due to lethal segregation in the progeny of treated cells.None of the agents tested would induce only one type of mitotic recombination, gene conversion or crossing over. There was, however, some mutagen specificity in the induction of isoleucine prototrophs.