The Tn3 β-Lactamase Gene Acts as a Hotspot for Meiotic Recombination in Yeast

Although genetic distances are often assumed to be proportional to physical distances, chromosomal regions with unusually high (hotspots) or low (coldspots) levels of meiotic recombination have been described in a number of genetic systems. In general, the DNA sequences responsible for these effects have not been determined. We report that the 5' region of the beta-lactamase (ampR) gene of the bacterial transposon Tn3 is a hotspot for meiotic recombination when inserted into the chromosomes of the yeast Saccharomyces cerevisiae. When these sequences are homozygous, both crossing over and gene conversion are locally stimulated. The 5' end of the beta-lactamase gene is about 100-fold "hotter" for crossovers than an average yeast DNA sequence.     

Chromosome size-dependent control of meiotic reciprocal recombination in Saccharomyces cerevisiae: the role of crossover interference.

In the yeast Saccharomyces cerevisiae, small chromosomes undergo meiotic reciprocal recombination (crossing over) at rates (centimorgans per kilobases) greater than those of large chromosomes, and recombination rates respond directly to changes in the total size of a chromosomal DNA molecule. This phenomenon, termed chromosome size-dependent control of meiotic reciprocal recombination, has been suggested to be important for ensuring that homologous chromosomes cross over during meiosis. The mechanism of this regulation was investigated by analyzing recombination in identical genetic intervals present on different size chromosomes. The results indicate that chromosome size-dependent control is due to different amounts of crossover interference. Large chromosomes have high levels of interference while small chromosomes have much lower levels of interference. A model for how crossover interference directly responds to chromosome size is presented. In addition, chromosome size-dependent control was shown to lower the frequency of homologous chromosomes that failed to undergo crossovers, suggesting that this control is an integral part of the mechanism for ensuring meiotic crossing over between homologous chromosomes.     

The coupling of crossing over and homologous synapsis in maize inversions

Because fresh initiations of synapsis must occur for homologous synapsis of internal heterozygously inverted chromosome segments, attention has been directed at homologous synapsis and crossing over in overlapping paracentric inversions in the long arm of chromosome 1 of maize. In an earlier study with a relatively short inversion (where double crossovers within the inversion were rare), a recombination nodule (RN) was generally found at pachytene in reverse paired (homologously synapsed) inverted regions. Crossover frequency within the inversion, which could be independently estimated from analysis of bridge and fragment frequency at anaphase I and II, closely corresponded to crossover frequency estimated from observed RN frequency in pachytene inversion loops. These findings were consistent with the interpretation that establishment of homologous synapsis in this case is generally coupled to crossing over. This coupling suggests that there is very early commitment to the form of resolution of recombination intermediates that results in reciprocal recombination events instead of conversion only or other noncrossover events. This study examines another, larger paracentric inversion in the long arm of chromosome 1 that completely overlaps the first inversion. It is sufficiently longer than the first inversion that double crossover events are found within it with substantial frequency and interference considerations are feasible. This study confers additional insight into the interrelationships of synapsis and crossing over and the probable sequence in which the various involved processes usually occur. It raises the strong possibility that crossovers can be initiated during the alignment phase that precedes synapsis.     

Meiosis in Asynaptic Yeast

The Saccharomyces cerevisiae red1 mutant fails to assemble synaptonemal complex during meiotic prophase. This mutant displays locus-specific reductions in interchromosomal gene conversion and a moderate reduction in crossing over. The occurrence of a significant amount of meiotically induced recombination in the red1 mutant indicates that the synaptonemal complex is not absolutely required for meiotic exchange. The RED1 gene product is required for intrachromosomal recombination in some assays but not others. Chromosomes that have undergone reciprocal exchange nevertheless nondisjoin in red1 mutants, indicating that crossovers are not sufficient for disjunction. Epistasis studies reveal that HOP1 is epistatic to RED1, and that RED1 acts in an independent pathway from MER1. A model for the function of the RED1 gene product in chromosome synapsis is discussed.     

Induction of Intrachromosomal Recombination in Yeast by Inhibition of Thymidylate Biosynthesis

The biosynthesis of thymidylate in the yeast Saccharomyces cerevisiae can be inhibited by antifolate drugs. We have found that antifolate treatment enhances the formation of leucine prototrophs in a haploid strain of yeast carrying, on the same chromosome, two different mutant leu2 alleles separated by Escherichia coli plasmid sequences. That this effect is a consequence of thymine nucleotide depletion was verified by the finding that provision of exogenous thymidylate eliminates the increased production of Leu+ colonies. DNA hybridization analysis revealed that recombination, including reciprocal exchange, gene conversion and unequal sister-chromatid crossing over, between the duplicated genes gave rise to the induced Leu+ segregants. Although gene conversion unaccompanied by crossing over was responsible for the major fraction of leucine prototrophs, events involving reciprocal exchange exhibited the largest increase in frequency. These data show that recombination is induced between directly repeated DNA sequences under conditions of thymine nucleotide depletion. In addition, the results of this and previous studies are consistent with the possibility that inhibition of thymidylate biosynthesis in yeast may create a metabolic condition that provokes all forms of mitotic recombination.     

Nonhomologous Synapsis and Reduced Crossing over in a Heterozygous Paracentric Inversion in Saccharomyces Cerevisiae

Homologous chromosome synapsis (``homosynapsis') and crossing over are well-conserved aspects of meiotic chromosome behavior. The long-standing assumption that these two processes are causally related has been challenged recently by observations in Saccharomyces cerevisiae of significant levels of crossing over (1) between small sequences at nonhomologous locations and (2) in mutants where synapsis is abnormal or absent. In order to avoid problems of local sequence effects and of mutation pleiotropy, we have perturbed synapsis by making a set of isogenic strains that are heterozygous and homozygous for a large chromosomal paracentric inversion covering a well marked genetic interval and then measured recombination. We find that reciprocal recombination in the marked interval in heterozygotes is reduced variably across the interval, on average to ~55% of that in the homozygotes, and that positive interference still modulates crossing over. Cytologically, stable synapsis across the interval is apparently heterologous rather than homologous, consistent with the interpretation that stable homosynapsis is required to initiate or consummate a large fraction of the crossing over observed in wild-type strains. When crossing over does occur in heterozygotes, dicentric and acentric chromosomes are formed and can be visualized and quantitated on blots though not demonstrated in viable spores. We find that there is no loss of dicentric chromosomes during the two meiotic divisions and that the acentric chromosome is recovered at only 1/3 to 1/2 of the expected level.     

Crossing over is rarely associated with mitotic intragenic recombination in Schizosaccharomyces pombe

Chromosomal rearrangements can result from crossing over during ectopic homologous recombination between dispersed repetitive DNA. We have previously shown that meiotic ectopic recombination between artificially dispersed ade6 heteroalleles in the fission yeast Schizosaccharomyces pombe frequently results in chromosomal rearrangements. The same recombination substrates have been studied in mitotic recombination. Ectopic recombination rates in haploids were approximately 1-4 x 10(-6) recombinants per cell generation, similar to allelic recombination rates in diploids. In contrast, ectopic recombination rates in heterozygous diploids were 2.5-70 times lower than allelic recombination or ectopic recombination in haploids. These results suggest that diploid-specific factors inhibit ectopic recombination. Very few crossovers occurred in ade6 mitotic recombination, either allelic or ectopic. Allelic intragenic recombination was associated with 2% crossing over, and ectopic recombination between multiple different pairing partners showed 1-7% crossing over. These results contrast sharply with the 35-65% crossovers associated with meiotic ade6 recombination and suggest either differential control of resolution of recombination intermediates or alternative pathways of recombination in mitosis and meiosis.     

Intrachromosomal Recombination in Saccharomyces cerevisiae : Reciprocal Exchange in an Inverted Repeat and Associated Gene Conversion

Intrachromosomal gene conversion has not shown a strong association with reciprocal exchanges. However, reciprocal exchanges do occur between intrachromosomal repeats. To understand the relationship between reciprocal exchange and gene conversion in repeated sequences the recombination behavior of an inverted repeat was studied. We have found that in one orientation a single copy of the kanr gene of the bacterial transposon Tn903 flanked by part of the inverted repeats IS903 does not give G418 resistance in Saccharomyces cerevisiae. A reciprocal exchange in the IS903 repeats inverts the kanr gene, which then gives G418 resistance in a single copy. Using this as a selection for intrachromosomal reciprocal exchange we have introduced multiple restriction site heterologies into the IS903 repeats and examined the crossover products for associated gene conversions. Approximately 50% of crossovers, both in mitosis and meiosis, were associated with a gene conversion. This suggests that these crossovers result from an intermediate that gives a gene conversion in 50% of the events, that is, both reciprocal exchange and gene conversion between repeated sequences have a common origin. The data are most consistent with a heteroduplex mismatch repair mechanism.     

Homology Search and Choice of Homologous Partner during Mitotic Recombination

Homologous recombination is an important DNA repair mechanism in vegetative cells. During the repair of double-strand breaks, genetic information is transferred between the interacting DNA sequences (gene conversion). This event is often accompanied by a reciprocal exchange between the homologous molecules, resulting in crossing over. The repair of DNA damage by homologous recombination with repeated sequences dispersed throughout the genome might result in chromosomal aberrations or in the inactivation of genes. It is therefore important to understand how the suitable homologous partner for recombination is chosen. We have developed a system in the yeast Saccharomyces cerevisiae that can monitor the fate of a chromosomal double-strand break without the need to select for recombinants. The broken chromosome is efficiently repaired by recombination with one of two potential partners located elsewhere in the genome. One of the partners has homology to the broken ends of the chromosome, whereas the other is homologous to sequences distant from the break. Surprisingly, a large proportion of the repair is carried out by recombination involving the sequences distant from the broken ends. This repair is very efficient, despite the fact that it requires the processing of a large chromosomal region flanking the break. Our results imply that the homology search involves extensive regions of the broken chromosome and is not carried out exclusively by sequences adjacent to the double-strand break. We show that the mechanism that governs the choice of homologous partners is affected by the length and sequence divergence of the interacting partners, as well as by mutations in the mismatch repair genes. We present a model to explain how the suitable homologous partner is chosen during recombinational repair. The model provides a mechanism that may guard the integrity of the genome by preventing recombination between dispersed repeated sequences.     

A Fine-Structure Map of Spontaneous Mitotic Crossovers in the Yeast Saccharomyces cerevisiae

Homologous recombination is an important mechanism for the repair of DNA damage in mitotically dividing cells. Mitotic crossovers between homologues with heterozygous alleles can produce two homozygous daughter cells (loss of heterozygosity), whereas crossovers between repeated genes on non-homologous chromosomes can result in translocations. Using a genetic system that allows selection of daughter cells that contain the reciprocal products of mitotic crossing over, we mapped crossovers and gene conversion events at a resolution of about 4 kb in a 120-kb region of chromosome V of Saccharomyces cerevisiae. The gene conversion tracts associated with mitotic crossovers are much longer (averaging about 12 kb) than the conversion tracts associated with meiotic recombination and are non-randomly distributed along the chromosome. In addition, about 40% of the conversion events have patterns of marker segregation that are most simply explained as reflecting the repair of a chromosome that was broken in G1 of the cell cycle.     

A yeast centromere acts in cis to inhibit meiotic gene conversion of adjacent sequences

The centromere of chromosome III (CEN3) of yeast has been examined for its ability to inhibit meiotic recombination in adjacent sequences. The effect of the centromere was investigated when it was adjacent to both of the recombining sequences (homozygous) or adjacent to only one of the two recombining DNA segments (hemizygous). When homozygous, CEN3 exerts a bidirectional repression of crossing over and a strong inhibition of gene conversion. This suggests that CEN3 reduces the frequency of crossing over by interfering with the initiation of proximal recombination events. When hemizygous, CEN3 impairs the ability of adjacent sequences to act as the recipient of genetic information during gene conversion. These results support the idea that the initiating event in yeast meiotic recombination involves the recipient molecule.     

Effects of Homology, Size and Exchange on the Meiotic Segregation of Model Chromosomes in Saccharomyces Cerevisiae

In most eukaryotic organisms, chiasmata, the connections formed between homologous chromosomes as a consequence of crossing over, are important for ensuring that the homologues move away from each other at meiosis I. Some organisms have the capacity to partition the rare homologues that have failed to experience reciprocal recombination. The yeast Saccharomyces cerevisiae is able to correctly partition achiasmate homologues with low fidelity by a mechanism that is largely unknown. It is possible to test which parameters affect the ability of achiasmate chromosomes to segregate by constructing strains that will have three achiasmate chromosomes at the time of meiosis. The meiotic partitioning of these chromosomes can be monitored to determine which ones segregate away from each other at meiosis I. This approach was used to test the influence of homologous yeast DNA sequences, recombination intiation sites, chromosome size and crossing over on the meiotic segregation of the model chromosomes. Chromosome size had no effect on achiasmate segregation. The influence of homologous yeast sequences on the segregation of noncrossover model chromosomes was negligible. In meioses in which two of the three model chromosomes experienced a crossover, they nearly always disjoined at meiosis I.     

Gene conversion and crossing over along the 405-kb left arm of Saccharomyces cerevisiae chromosome VII

Gene conversions and crossing over were analyzed along 10 intervals in a 405-kb region comprising nearly all of the left arm of chromosome VII in Saccharomyces cerevisiae. Crossover interference was detected in all intervals as measured by a reduced number of nonparental ditypes. We have evaluated interference between crossovers in adjacent intervals by methods that retain the information contained in tetrads as opposed to single segregants. Interference was seen between intervals when the distance in the region adjacent to a crossover was < approximately 35 cM (90 kb). At the met13 locus, which exhibits approximately 9% gene conversions, those gene conversions accompanied by crossing over exerted interference in exchanges in an adjacent interval, whereas met13 gene conversions without an accompanying exchange did not show interference. The pattern of exchanges along this chromosome arm can be represented by a counting model in which there are three nonexchange events between adjacent exchanges; however, maximum-likelihood analysis suggests that approximately 8-12% of the crossovers on chromosome VII arise by a separate, noninterfering mechanism.     

Branching out: meiotic recombination and its regulation

Homologous recombination is a dynamic process by which DNA sequences and strands are exchanged. In meiosis, the reciprocal DNA recombination events called crossovers are central to the generation of genetic diversity in gametes and are required for homolog segregation in most organisms. Recent studies have shed light on how meiotic crossovers and other recombination products form, how their position and number are regulated and how the DNA molecules undergoing recombination are chosen. These studies indicate that the long-dominant, unifying model of recombination proposed by Szostak et al. applies, with modification, only to a subset of recombination events. Instead, crossover formation and its control involve multiple pathways, with considerable variation among model organisms. These observations force us to 'branch out' in our thinking about meiotic recombination.     

Saturation mapping of a gene-rich recombination hot spot region in wheat

Physical mapping of wheat chromosomes has revealed small chromosome segments of high gene density and frequent recombination interspersed with relatively large regions of low gene density and infrequent recombination. We constructed a detailed genetic and physical map of one highly recombinant region on the long arm of chromosome 5B. This distally located region accounts for 4% of the physical size of the long arm and at least 30% of the recombination along the entire chromosome. Multiple crossovers occurred within this region, and the degree of recombination is at least 11-fold greater than the genomic average. Characteristics of the region such as gene order and frequency of recombination appear to be conserved throughout the evolution of the Triticeae. The region is more prone to chromosome breakage by gametocidal gene action than gene-poor regions, and evidence for genomic instability was implied by loss of gene collinearity for six loci among the homeologous regions. These data suggest that a unique level of chromatin organization exists within gene-rich recombination hot spots. The many agronomically important genes in this region should be accessible by positional cloning.     

Effects of Mutagen-Sensitive Mus Mutations on Spontaneous Mitotic Recombination in Aspergillus

Methyl methane-sulfonate (MMS)-sensitive, radiation-induced mutants of Aspergillus were shown to define nine new DNA repair genes, musK to musS. To test mus mutations for effects on mitotic recombination, intergenic crossing over was assayed between color markers and their centromeres, and intragenic recombination between two distinguishable adE alleles. Of eight mutants analyzed, four showed significant deviations from mus+ controls in both tests. Two mutations, musK and musL, reduced recombination, while musN and musQ caused increases. In contrast, musO diploids produced significantly higher levels only for intragenic recombination. Effects were relatively small, but averages between hypo- and hyperrec mus differed 15-20-fold. In musL diploids, most of the rare color segregants resulted from mitotic malsegregation rather than intergenic crossing over. This indicates that the musL gene product is required for recombination and that DNA lesions lead to chromosome loss when it is deficient. In addition, analysis of the genotypes of intragenic (ad+) recombinants showed that the musL mutation specifically reduced single allele conversion but increased complex conversion types (especially recombinants homozygous for ad+). Similar analysis revealed differences between the effects of two hyperrec mutations; musN apparently caused high levels solely of mitotic crossing over, while musQ increased various conversion types but not reciprocal crossovers. These results suggest that mitotic gene conversion and crossing over, while generally associated, are affected differentially in some of the mus strains of Aspergillus nidulans.     

Studies on crossover-specific mutants and the distribution of crossing over in Drosophila females

In Drosophila females, the majority of recombination events do not become crossovers and those that do occur are nonrandomly distributed. Furthermore, a group of Drosophila mutants specifically reduce crossing over, suggesting that crossovers depend on different gene products than noncrossovers. In mei-218 mutants, crossing over is reduced by approximately 90% while noncrossovers and the initiation of recombination remain unchanged. Importantly, the residual crossovers have a more random distribution than wild-type. It has been proposed that mei-218 has a role in establishing the crossover distribution by determining which recombination sites become crossovers. Surprisingly, a diverse group of genes, including those required for double strand break (DSB) formation or repair, have an effect on crossover distribution. Not all of these mutants, however, have a crossover-specific defect like mei-218 and it is not understood why some crossover-defective mutants alter the distribution of crossovers. Intragenic recombination experiments suggest that mei-218 is required for a molecular transition of the recombination intermediate late in the DSB repair pathway. We propose that the changes in crossover distribution in some crossover-defective mutants are a secondary consequence of the crossover reductions. This may be the activation of a regulatory system that ensures at least one crossover per chromosome, and which compensates for an absence of crossovers by attempting to generate them at random locations.     

Molecular analysis of a hybrid gene encoding human glycophorin variant Miltenberger V-like molecule

The genomic structure of a human glycophorin variant, Miltenberger class V-like molecule (MiV*), was examined. Southern blot analysis of total genomic DNA revealed that the 5' half of the MiV* gene derived from glycophorin A (GPA) gene whereas the 3' half derived from glycophorin B (GPB) gene. This structure is reciprocal to another glycophorin variant, Sta, which has a GPB-GPA hybrid structure. The genomic sequences around the crossing-over point were amplified by polymerase chain reaction, and the sequences were determined. Comparison of the nucleotide sequences of the GPA, GPB, and MiV* genes indicates that the crossing-over point is located in the region around the 3' end of intron 3 of the GPA gene. This place is different from the crossing-over point for Sta, which was found to be highly homologous to that for haptoglobin-related genes. However, the nucleotide sequences within the presumptive crossing-over point for the MiV* gene were found to be homologous in a reverse orientation to the crossing-over point proposed for haptoglobin-related genes. These results suggest strongly that homologous recombination through unequal crossing over can be facilitated by specific genomic elements such as those in common for formation of MiV*, Sta, and haptoglobin-related genes. The present study also localized the gene of the third glycophorin, GPE, at chromosome 4, q31.1 band, the same locus as for the GPA and GPB genes. The results indicate that GPE was not involved in generating MiV* or Sta hybrid gene despite the fact that it is localized adjacent to the GPA and GPB genes.     

Meiotic Recombination on Artificial Chromosomes in Yeast

We have examined the meiotic recombination characteristics of artificial chromosomes in Saccharomyces cerevisiae. Our experiments were carried out using minichromosome derivatives of yeast chromosome III and yeast artificial chromosomes composed primarily of bacteriophage lambda DNA. Tetrad analysis revealed that the artificial chromosomes exhibit very low levels of meiotic recombination. However, when a 12.5-kbp fragment from yeast chromosome VIII was inserted into the right arm of the artificial chromosome, recombination within that arm mimicked the recombination characteristics of the fragment in its natural context including the ability of crossovers to ensure meiotic disjunction. Both crossing over and gene conversion (within the ARG4 gene contained within the fragment) were measured in the experiments. Similarly, a 55-kbp region from chromosome III carried on a minichromosome showed crossover behavior indistinguishable from that seen when it is carried on chromosome III. We discuss the notion that, in yeast, meiotic recombination behavior is determined locally by small chromosomal regions that function free of the influence of the chromosome as a whole.     

Meiotic Crossing over between Nonhomologous Chromosomes Affects Chromosome Segregation in Yeast

Meiotic recombination between artificial repeats positioned on nonhomologous chromosomes occurs efficiently in the yeast Saccharomyces cerevisiae. Both gene conversion and crossover events have been observed, with crossovers yielding reciprocal translocations. In the current study, 5.5-kb ura3 repeats positioned on chromosomes V and XV were used to examine the effect of ectopic recombination on meiotic chromosome segregation. Ura(+) random spores were selected and gene conversion vs. crossover events were distinguished by Southern blot analysis. Approximately 15% of the crossover events between chromosomes V and XV were associated with missegregation of one of these chromosomes. The missegregation was manifest as hyperploid spores containing either both translocations plus a normal chromosome, or both normal chromosomes plus one of the translocations. In those cases where it could be analyzed, missegregation occurred at the first meiotic division. These data are discussed in terms of a model in which ectopic crossovers compete efficiently with normal allelic crossovers in directing meiotic chromosome segregation.