Physical Lengths of Meiotic and Mitotic Gene Conversion Tracts in Saccharomyces Cerevisiae

Physical lengths of gene conversion tracts for meiotic and mitotic conversions were examined, using the same diploid yeast strain in all experiments. This strain is heterozygous for a mutation in the URA3 gene as well as closely linked restriction site markers. In cells that had a gene conversion event at the URA3 locus, it was determined by Southern analysis which of the flanking heterozygous restriction sites had co-converted. It was found that mitotic conversion tracts were longer on the average than meiotic tracts. About half of the tracts generated by spontaneous mitotic gene conversion included heterozygous markers 4.2 kb apart; none of the meiotic conversions included these markers. Stimulation of mitotic gene conversion by ultraviolet light or methylmethanesulfonate had no obvious effect on the size or distribution of the tracts. Almost all conversion tracts were continuous.     

Genetic and Physical Analysis of Double-Strand Break Repair and Recombination in Saccharomyces Cerevisiae

We have investigated HO endonuclease-induced double-strand break (DSB) recombination and repair in a LACZ duplication plasmid in yeast. A 117-bp MATa fragment, embedded in one copy of LACZ, served as a site for initiation of a DSB when HO endonuclease was expressed. The DSB could be repaired using wild-type sequences located on a second, promoterless, copy of LACZ on the same plasmid. In contrast to normal mating-type switching, crossing-over associated with gene conversion occurred at least 50% of the time. The proportion of conversion events accompanied by exchange was greater when the two copies of LACZ were in direct orientation (80%), than when inverted (50%). In addition, the fraction of plasmids lost was significantly greater in the inverted orientation. The kinetics of appearance of intermediates and final products were also monitored. The repair of the DSB is slow, requiring at least an hour from the detection of the HO-cut fragments to completion of repair. Surprisingly, the appearance of the two reciprocal products of crossing over did not occur with the same kinetics. For example, when the two LACZ sequences were in the direct orientation, the HO-induced formation of a large circular deletion product was not accompanied by the appearance of a small circular reciprocal product. We suggest that these differences may reflect two kinetically separable processes, one involving only one cut end and the other resulting from the concerted participation of both ends of the DSB.     

Genetic and Physical Mapping of Telomeres in the Rice Blast Fungus, Magnaporthe Grisea

Telomeric restriction fragments were genetically mapped to a previously described linkage map of Magnaporthe grisea, using RFLPs identified by a synthetic probe, (TTAGGG)(3). Frequent rearrangement of telomeric sequences was observed in progeny isolates creating a potential for misinterpretation of data. Therefore a consensus segregation data set was used to minimize mapping errors. Twelve of the 14 telomeres were found to be genetically linked to existing RFLP markers. Second-dimensional electrophoresis of restricted chromosomes confirmed these linkage assignments and revealed the chromosomal location of the two unlinked telomeres. We were thus able to assign all 14 M. grisea telomeres to their respective chromosome ends. The Achilles' cleavage (AC) technique was employed to determine that chromosome 1 markers 11 and CH5-120H were ~1.8 Mb and 1.28 Mb, respectively, from their nearest telomeres. RecA-AC was also used to determine that unlinked telomere 6 was ~530 kb from marker CH5-176H in strain 2539 and 580 kb in Guy11. These experiments indicated that large portions of some chromosome ends are unrepresented by genetic markers and provided estimates of the relationship of genetic to physical distance in these regions of the genome.     

Genes Required for Vacuolar Acidity in Saccharomyces Cerevisiae

Mutations that cause loss of acidity in the vacuole (lysosome) of Saccharomyces cerevisiae were identified by screening colonies labeled with the fluorescent, pH-sensitive, vacuolar labeling agent, 6-carboxyfluorescein. Thirty nine vacuolar pH (Vph-) mutants were identified. Four of these contained mutant alleles of the previously described PEP3, PEP5, PEP6 and PEP7 genes. The remaining mutants defined eight complementation groups of vph mutations. No alleles of the VAT2 or TFP1 genes (known to encode subunits of the vacuolar H(+)-ATPase) were identified in the Vph- screen. Strains bearing mutations in any of six of the VPH genes failed to grow on medium buffered at neutral pH; otherwise, none of the vph mutations caused notable growth inhibition on standard yeast media. Expression of the vacuolar protease, carboxypeptidase Y, was defective in strains bearing vph4 mutations but was apparently normal in strains bearing any of the other vph mutations. Defects in vacuolar morphology at the light microscope level were evident in all Vph- mutants. Strains that contained representative mutant alleles of the 17 previously described PEP genes were assayed for vacuolar pH; mutations in seven of the PEP genes (including PEP3, PEP5, PEP6 and PEP7) caused loss of vacuolar acidity.     

Multiple Pathways for Homologous Recombination in Saccharomyces Cerevisiae

The genes in the RAD52 epistasis group of Saccharomyces cerevisiae are necessary for most mitotic and meiotic recombination events. Using an intrachromosomal inverted-repeat assay, we previously demonstrated that mitotic recombination of this substrate is dependent upon the RAD52 gene. In the present study the requirement for other genes in this epistasis group for recombination of inverted repeats has been analyzed, and double and triple mutant strains were examined for their epistatic relationships. The majority of recombination events are mediated by a RAD51-dependent pathway, where the RAD54, RAD55 and RAD57 genes function downstream of RAD51. Cells mutated in RAD55 or RAD57 as well as double mutants are cold-sensitive for inverted-repeat recombination, whereas a rad51 rad55 rad57 triple mutant is not. The RAD1 gene is not required for inverted-repeat recombination but is able to process spontaneous DNA lesions to produce recombinant products in the absence of RAD51. Furthermore, there is still considerably more recombination in rad1 rad51 mutants than in rad52 mutants, indicating the presence of another, as yet unidentified, recombination pathway.     

Mechanisms of Gene Conversion in Saccharomyces Cerevisiae

In red-white sectored colonies of Saccharomyces cerevisiae, derived from mitotic cells grown to stationary phase and irradiated with a light dose of x-rays, all of the segregational products of gene conversion and crossing over can be ascertained. Approximately 80% of convertants are induced in G1, the remaining 20% in G2. Crossing over, in the amount of 20%, is found among G1 convertants but most of the crossovers are delayed until G2. About 20% of all sectored colonies had more than one genotype in one or the other sector, thus confirming the hypothesis that conversion also occurs in G2. The principal primary event in G2 conversion is a single DNA heteroduplex. It is suggested that the close contact that this implies carries over to G2 when crossing over and a second round of conversion occurs.     

``break Copy'''' Duplication: A Model for Chromosome Fragment Formation in Saccharomyces Cerevisiae

Introduction of a chromosome fragmentation vector (CFV) into the budding yeast Saccharomyces cerevisiae results in a targeted homologous recombination event that yields an independently segregating chromosome fragment (CF) and an alteration in the strain's karyotype. Fragmentation with an acentric CFV directed in a centromere-proximal orientation generates a CF that contains all sequences proximal to the targeting segment and results in loss of the endogenous targeted chromosome to yield a 2N-1+CF karyotype. In contrast, fragmentation with a centric CFV directed in a centromere-distal orientation generates a CF that contains all sequences distal to the targeting segment and retention of the endogenous targeted chromosome to yield a 2N+CF karyotype. We have termed this phenomenon ``break copy' duplication. Using yeast strains in which the centromere had been transposed to a new location, it was demonstrated that the centromere inhibited break copy duplication. These data suggest that CF formation is the product of an unscheduled DNA replication event initiated by the free end of the CFV and is analogous to a ``half' double-strand break gap-repair reaction. We suggest that break copy duplication may have evolved as a mechanism for maintenance of ploidy following DNA breakage.     

A Test of the Double-Strand Break Repair Model for Meiotic Recombination in Saccharomyces Cerevisiae

We tested predictions of the double-strand break repair (DSBR) model for meiotic recombination by examining the segregation patterns of small palindromic insertions, which frequently escape mismatch repair when in heteroduplex DNA. The palindromes flanked a well characterized DSB site at the ARG4 locus. The ``canonical'''' DSBR model, in which only 5'' ends are degraded and resolution of the four-stranded intermediate is by Holliday junction resolvase, predicts that hDNA will frequently occur on both participating chromatids in a single event. Tetrads reflecting this configuration of hDNA were rare. In addition, a class of tetrads not predicted by the canonical DSBR model was identified. This class represented events that produced hDNA in a ``trans'''' configuration, on opposite strands of the same duplex on the two sides of the DSB site. Whereas most classes of convertant tetrads had typical frequencies of associated crossovers, tetrads with trans hDNA were parental for flanking markers. Modified versions of the DSBR model, including one that uses a topoisomerase to resolve the canonical DSBR intermediate, are supported by these data.     

A Role for Rev3 in Mutagenesis during Double-Strand Break Repair in Saccharomyces Cerevisiae

Recombinational repair of double-strand breaks (DSBs), traditionally believed to be an error-free DNA repair pathway, was recently shown to increase the frequency of mutations in a nearby interval. The reversion rate of trp1 alleles (either nonsense or frameshift mutations) near an HO-endonuclease cleavage site is increased at least 100-fold among cells that have experienced an HO-mediated DSB. We report here that in strains deleted for rev3 this DSB-associated reversion of a nonsense mutation was greatly decreased. Thus REV3, which encodes a subunit of the translesion DNA polymerase &, was responsible for the majority of these base substitution errors near a DSB. However, rev3 strains showed no decrease in HO-stimulated recombination, implying that another DNA polymerase also functioned in recombinational repair of a DSB. Reversion of trp1 frameshift alleles near a DSB was not reduced in rev3 strains, indicating that another polymerase could act during DSB repair to make these frameshift errors. Analysis of spontaneous reversion in haploid strains suggested that Rev3p had a greater role in making point mutations than in frameshift mutations.     

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.     

Analysis of a Circular Derivative of Saccharomyces Cerevisiae Chromosome III: A Physical Map and Identification and Location of Ars Elements

DNA was isolated from a circular derivative of chromosome III to prepare a library of recombinant plasmids enriched in chromosome III sequences. An ordered set of recombinant plasmids and bacteriophages carrying the contiguous 210-kilobase region of chromosome III between the HML and MAT loci was identified, and a complete restriction map was prepared with BamHI and EcoRI. Using the high frequency transformation assay and extensive subcloning, 13 ARS elements were mapped in the cloned region. Comparison of the physical maps of chromosome III from three strains revealed that the chromosomes differ in the number and positions of Ty elements and also show restriction site polymorphisms. A comparison of the physical map with the genetic map shows that meiotic recombination rates vary at least tenfold along the length of the chromosome.     

Distributive Disjunction of Authentic Chromosomes in Saccharomyces Cerevisiae

Distributive disjunction is defined as the first division meiotic segregation of either nonhomologous chromosomes that lack homologs or homologous chromosomes that have not recombined. To determine if chromosomes from the yeast Saccharomyces cerevisiae were capable of distributive disjunction, we constructed a strain that was monosomic for both chromosome I and chromosome III and analyzed the meiotic segregation of the two monosomic chromosomes. In addition, we bisected chromosome I into two functional chromosome fragments, constructed strains that were monosomic for both chromosome fragments and examined meiotic segregation of the chromosome fragments in the monosomic strains. The two nonhomologous chromosomes or chromosome fragments appeared to segregate from each other in approximately 90% of the asci analyzed, indicating that yeast chromosomes were capable of distributive disjunction. We also examined the ability of a small nonhomologous centromere containing plasmid to participate in distributive disjunction with the two nonhomologous monosomic chromosomes. The plasmid appeared to efficiently participate with the two full length chromosomes suggesting that distributive disjunction in yeast is not dependent on chromosome size. Thus, distributive disjunction in S. cerevisiae appears to be different from Drosophila melanogaster where a different sized chromosome is excluded from distributive disjunction when two similar size nonhomologous chromosomes are present.     

Nonsense Mutations in Essential Genes of Saccharomyces Cerevisiae

A new method for isolating nonsense mutations in essential yeast genes has been used to develop a collection of 115 ochre mutations that define 94 complementation groups. The mutants are isolated in a genetic background that includes an ochre suppressor on a metastable plasmid and a suppressible colony-color marker on a chromosome. When the parental strain is plated on a rich medium, the colonies display a pattern of red, plasmid-free sectors on a white background. Mutants containing an ochre mutation in any essential yeast gene give rise to nonsectoring, white colonies, since cell growth is dependent on the presence of the plasmid-borne suppressor. Analysis of the data suggests that mutations are being recovered from a pool of approximately 250 genes.     

Isolation of Com1, a New Gene Required to Complete Meiotic Double-Strand Break-Induced Recombination in Saccharomyces Cerevisiae

We have designed a screen to isolate mutants defective during a specific part of meiotic prophase I of the yeast Saccharomyces cerevisiae. Genes required for the repair of meiotic double-strand breaks or for the separation of recombined chromosomes are targets of this mutant hunt. The specificity is achieved by selecting for mutants that produce viable spores when recombination and reductional segregation are prevented by mutations in SPO11 and SPO13 genes, but fail to yield viable spores during a normal Rec(+) meiosis. We have identified and characterized a mutation com1-1, which blocks processing of meiotic double-strand breaks and which interferes with synaptonemal complex formation, homologous pairing and, as a consequence, spore viability after induction of meiotic recombination. The COM1/SAE2 gene was cloned by complementation, and the deletion mutant has a phenotype similar to com1-1. com1/sae2 mutants closely resemble the phenotype of rad50S, as assayed by phase-contrast microscopy for spore formation, physical and genetic analysis of recombination, fluorescence in situ hybridization to quantify homologous pairing and immunofluorescence and electron microscopy to determine the capability to synapse axial elements.     

An Examination of Adaptive Reversion in Saccharomyces Cerevisiae

Reversion to Lys+ prototrophy in a haploid yeast strain containing a defined lys2 frameshift mutation has been examined. When cells were plated on synthetic complete medium lacking only lysine, the numbers of Lys+ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number. An examination of the distribution of the numbers of early appearing Lys+ colonies from independent cultures suggests that the mutations to prototrophy occurred randomly during nonselective growth. In contrast, an examination of the distribution of late appearing Lys+ colonies indicates that the underlying reversion events occurred after selective plating. No accumulation of Lys+ revertants occurred when cells were starved for tryptophan, leucine or both lysine and tryptophan prior to plating selectively for Lys+ revertants. These results indicate that mutations accumulate more frequently when they confer a selective advantage, and are thus consistent with the occurrence of adaptive mutations in yeast.     

Fitness Effects of Ty Transposition in Saccharomyces Cerevisiae

It has been suggested that the primary evolutionary role of transposable elements is negative and parasitic. Alternatively, the target specificity and gene regulatory capabilities of many transposable elements raise the possibility that transposable element-induced mutations are more likely to be adaptively favorable than other types of mutations. Populations of Saccharomyces cerevisiae containing large amounts of variation for Ty1 genomic insertions were constructed, and the effects of Ty1 copy number on two components of fitness, yield and growth rate were determined. Although mean stationary phase density decreased with increased Ty1 copy number, the variance and range increased. The distributions of stationary phase densities indicate that many Ty1 insertions have negative effects on fitness, but also that some may have positive effects. To test directly for adaptively favorable Ty1 insertions, populations containing large amounts of variability for Ty1 copy number were grown in continuous culture. After 98-112 generations the frequency of clones containing zero Ty1 elements had decreased to approximately 0.0, and specific Ty1-containing clone families had predominated. Considering that most of the genetic variation in the populations was due to Ty1 transposition, and that Ty1 insertions had, on average, a negative effect on fitness, we conclude that Ty1 transposition events were directly responsible for the production of adaptive mutations in the clones that predominated in the populations.     

Meiotic Chromosome Pairing in Triploid and Tetraploid Saccharomyces Cerevisiae

Meiotic chromosome pairing in isogenic triploid and tetraploid strains of yeast and the consequences of polyploidy on meiotic chromosome segregation are studied. Synaptonemal complex formation at pachytene was found to be different in the triploid and in the tetraploid. In the triploid, triple-synapsis, that is, the connection of three homologues at a given site, is common. It can even extend all the way along the chromosomes. In the tetraploid, homologous chromosomes mostly come in pairs of synapsed bivalents. Multiple synapsis, that is, synapsis of more than two homologues in one and the same region, was virtually absent in the tetraploid. About five quadrivalents per cell occurred due to the switching of pairing partners. From the frequency of pairing partner switches it can be deduced that in most chromosomes synapsis is initiated primarily at one end, occasionally at both ends and rarely at an additional intercalary position. In contrast to a considerably reduced spore viability (~40%) in the triploid, spore viability is only mildly affected in the tetraploid. The good spore viability is presumably due to the low frequency of quadrivalents and to the highly regular 2:2 segregation of the few quadrivalents that do occur. Occasionally, however, quadrivalents appear to be subject to 3:1 nondisjunction that leads to spore death in the second generation.     

Distance-Independence of Mitotic Intrachromosomal Recombination in Saccharomyces Cerevisiae

Many genetic studies have shown that the frequency of homologous recombination depends largely on the distance in which recombination can occur. We have studied the effect of varying the length of duplicated sequences on the frequency of mitotic intrachromosomal recombination in Saccharomyces cerevisiae. We find that the frequency of recombination resulting in the loss of one of the repeats and the intervening sequences reaches a plateau when the repeats are short. In addition, the frequency of recombination to correct a point mutation contained in one of these repeats is not proportional to the size of the duplication but rather depends dramatically on the location of the mutation within the repeated sequences. However, the frequency of mitotic interchromosomal reciprocal recombination is dependent on the distance separating the markers. The difference in the response of intrachromosomal and interchromosomal mitotic recombination to increasing lengths of homology may indicate there are different rate-limiting steps for recombination in these two cases. These findings have important implications for the maintenance and evolution of duplicated sequences.