Differential Mismatch Repair Can Explain the Disproportionalities between Physical Distances and Recombination Frequencies of Cyc1 Mutations in Yeast

Recombination rates have been examined in two-point crosses of various defined cyc1 mutations that cause the loss or nonfunction of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. Recombinants arising by three different means were investigated, including X-ray induced mitotic recombination, spontaneous mitotic recombination, and meiotic recombination. Heteroallelic diploid strains were derived by crossing cyc1 mutants containing a series of alterations at or near the same site to cyc1 mutants containing alterations at various distances. Marked disproportionalities between physical distances and recombination frequencies were observed with certain cyc1 mutations, indicating that certain mismatched bases can significantly affect recombination. The marker effects were more pronounced when the two mutational sites of the heteroalleles were within about 20 base pairs, but separated by at least 4 base pairs. Two alleles, cyc1-163 and cyc1-166, which arose by G.C----C.G transversions at nucleotide positions 3 and 194, respectively, gave rise to especially high rates of recombination. Other mutations having different substitutions at the same nucleotide positions were not associated with abnormally high recombination frequencies. We suggest that these marker effects are due to the lack of repair of either G/G or C/C mismatched base pairs, while the other mismatched base pair of the heteroallele undergoes substantial repair. Furthermore, we suggest that diminished recombination frequencies are due to the concomitant repair of both mismatches within the same DNA tract.     

Role of DNA Sequences in Genetic Recombination in the Iso-1-Cytochrome c Gene of Yeast. II. Comparison of Mutants Altered at the Same and Nearby Base Pairs

X-ray-induced mitotic recombination rates and spontaneous meiotic recombination rates have been determined in two-point crosses of various defined cyc1 mutants of the yeast Saccharomyces cerevisiae. All but one of the 17 cyc1 mutants chosen for this study contained either the addition, deletion or substitution of single base-pairs located within a defined segment of the gene that corresponds to the 11 amino acid residues at the amino terminus of iso-1-cytochrome c; approximately half of these mutants had alterations of the AUG initiation codon, some at the same base pair. Up to 66-fold differences in X-ray-induced recombination rates were observed when the same cyc1 mutant was crossed to cyc1 mutants having different alterations in the AUG initiation codon; over a ten-fold difference was observed in series of homologous crosses involving mutants with different changes at the same base-pair. Recombination rates that were associated with specific cyc1 mutants co-segregated with the particular alleles following meiosis, and comparable recombination patterns were also observed for independently isolated, identical mutations. With the mutants used in this study, the frequencies of meiotic recombination did not differ as markedly, suggesting a dissimilar dependence on specific DNA sequences for these two modes of recombination. These disproportionalities of recombination rates suggest that the nature of the mismatched bases influences the recombination process, but not in a way that can be simply interpreted.     

Comparative Recombination Distances among Zea Mays L. Inbreds, Wide Crosses and Interspecific Hybrids

Recombination distances and linkage heterogeneity were compared among a wide range of maize inbreds, wide crosses and maize X teosinte hybrids. Twelve maize and four teosinte races were backcrossed to stocks fixed for rare marker alleles on chromosome arm 1L. Recombination fraction estimates were higher for exotic germplasm than for either U.S. maize or maize X teosinte crosses. Serrano, Tuxpeno and a US-adapted inbred line of tropical origin, NC300, exhibited enhanced recombination. Three of the four maize X teosinte hybrids had little or no recombination between two loci. The observed recombination ``shrinkage' resulted from an apparent inversion in the vicinity of the Amp1 locus. Average recombination distances among common marker loci for composite maps were highly variable, even when map construction was restricted to maize germplasm of similar origins.     

Marker Effects in the Genetic Transduction of Tryptophan Mutants of E. COLI

Recombination frequencies have been determined in crosses involving 28 mutant strains for 20 of which the site of the alteration is known from studies of amino-acid substitutions in the protein products. Three of these mutants showed especially high frequencies of recombination when crossed to other single mutants or when crossed to a strain carrying two alterations at opposite ends of the trpA gene. There is no obvious molecular explanation of the high recombination of these three mutants. They include one missense mutant, one amber and one ochre. The low-frequency recombination mutants include all these same classes as well as frameshift mutants. There is nothing unique about the intragenic location of the high-recombination mutants; in each case there is at least one low-recombination mutant in the same codon.-Crosses involving mutants which were isolated in an altered wild type have shown that the behavior of a high-recombination mutant does not result from its molecular configuration alone, but from its combination with the homologous wild-type sequence from the other parent.-Several lines of evidence indicate that recombination in this system frequently involves closely-spaced double exchanges (about 40 codons apart).     

Genetic Order of the Galactose Structural Genes in Saccharomyces cerevisiae

The galactose structural genes of Saccharomyces cerevisiae were ordered by determining the genotypes of mitotic and meiotic recombinants from crosses heterozygous for the three genes. The most probable order is centromere-gal7-gal10-gal1. Nonreciprocal recombination was more frequent than reciprocal exchange, and both mitotic and meiotic co-conversions involving mutant sites in all three genes were observed.     

Genetic studies of the lac repressor. III. Additional correlation of mutational sites with specific amino acid residues

The complete results of the analysis of over 5300 independently derived nonsense mutations in the lacI gene are presented. These have been mapped and divided into specific sites. A total of 105 nonsense mutations derived from 90 different codons can be distinguished, of which several are the result of tandem double base changes induced by ultraviolet light. With the aid of results determined in a preceding paper (Miller et al., 1977), the majority of these mutations have been assigned to points in the gene coding for specific residues in the lac repressor. This allows a detailed correlation of the physical and genetic map.Recombination studies have been carried out using mutations at known sites. For crosses involving mutations separated by less than 30 nucleotides (the main object of this study), a significant lack of agreement between distance and recombination frequency has been found.     

Influence of genetic background and heterozygosity on meiotic recombination in Arabidopsis thaliana.

Plant breeding relies on genetic variability generated by meiotic recombination. Control of recombination frequencies is not yet possible, but would significantly extend the options for plant-breeding strategies. A prerequisite would be variability of recombination frequencies. In this study, 15 transgenic kanamycin (KR) and hygromycin (HR) resistance gene insertions mapping to the five Arabidopsis thaliana chromosomes were used as genetic markers. Recombination frequencies were determined from the frequencies of resistance phenotypes within populations segregating for linked KR and HR markers. Recombination frequencies of marker pairs were compared among these four ecotypes, among F1s in both reciprocal forms derived from these ecotypes, and between F1s and their parent lines. On average, the recombination frequencies in F1 crosses were substantially higher (up to 2-fold) than in the homozygous parental ecotypes. A strong negative correlation between genetic similarities of ecotypes and recombination frequencies was detected for two adjacent marker pairs located on the long arm of chromosome 3, but not for marker pairs in other genomic regions. Our results suggest that heterozygosity influences recombination in plant breeding, and cannot be ignored in genetic mapping of genomes.     

Comparisons of recombination frequencies in hybrids involving telocentric and bibrachial wheat chromosomes

Telosomic stocks have been extensively used to map genes to chromosome arms and to determine gene-to-centromere genetic distances. It has been suggested that if a chromosome arm is present as a telosome, recombination frequencies will be drastically reduced in the centromeric region. However, previous studies have not considered the bias in recombination estimates due to selection against aneuploid gametes produced by failure of pairing at the first meiotic division. Formulas are derived here for adjusting recombination estimates for this bias. Adjusted recombination frequencies between markers located on both sides of the centromeres are analyzed in three different pairs of wheat (Triticum aestivum) isogenic segregating populations involving bibrachial and telocentric chromosomes. Recombination frequencies estimated from crosses involving telocentric chromosomes were not significantly different from recombination frequencies estimated from isogenic crosses involving bibrachial chromosomes. The implications of the present findings for karyotype evolution, and specifically for Robertsonian fissions and fusions, are discussed. Received: 10 March 1999 / Accepted: 17 June 1999     

Exploring the pathways of homologous recombination.

There has been significant progress in elucidating the mechanisms by which meiotic and mitotic recombination occur. Double-strand breaks in particular have been the object of attention in studies on meiotic gene conversion, site-specific mitotic recombination, the repair of transposon excision and the transformation of cells with linearized DNA. A combination of genetic analysis and physical studies of molecular recombination intermediates have established that double-strand breaks can occur by two different mechanisms.     

The Impact of Recombination Hotspots on Genome Evolution of a Fungal Plant Pathogen

Recombination has an impact on genome evolution by maintaining chromosomal integrity, affecting the efficacy of selection, and increasing genetic variability in populations. Recombination rates are a key determinant of the coevolutionary dynamics between hosts and their pathogens. Historic recombination events created devastating new pathogens, but the impact of ongoing recombination in sexual pathogens is poorly understood. Many fungal pathogens of plants undergo regular sexual cycles, and sex is considered to be a major factor contributing to virulence. We generated a recombination map at kilobase-scale resolution for the haploid plant pathogenic fungus Zymoseptoria tritici. To account for intraspecific variation in recombination rates, we constructed genetic maps from two independent crosses. We localized a total of 10,287 crossover events in 441 progeny and found that recombination rates were highly heterogeneous within and among chromosomes. Recombination rates on large chromosomes were inversely correlated with chromosome length. Short accessory chromosomes often lacked evidence for crossovers between parental chromosomes. Recombination was concentrated in narrow hotspots that were preferentially located close to telomeres. Hotspots were only partially conserved between the two crosses, suggesting that hotspots are short-lived and may vary according to genomic background. Genes located in hotspot regions were enriched in genes encoding secreted proteins. Population resequencing showed that chromosomal regions with high recombination rates were strongly correlated with regions of low linkage disequilibrium. Hence, genes in pathogen recombination hotspots are likely to evolve faster in natural populations and may represent a greater threat to the host.     

Meiosis in NEUROSPORA CRASSA. II. Genetic and Cytological Characterization of Three Meiotic Mutants

Three recessive meiotic mutants, asc(DL95), asc(DL243) and asc(DL879), were detected by the abortion of many of their ascospores and were analyzed using both cytological and genetic methods. Even though asc(DL95), asc (DL243) and the previously studied meiotic mutant, mei-1 (Smith 1975; Lu and Galeazzi (1978), complement one another in crosses, they apparently do not recombine (DeLange and Griffiths (1980). Thus, they may represent alleles of the same gene or comprise a gene cluster. Ascospore abortion in these mutants is caused by abnormal disjunction of meiotic chromosomes. In crosses homozygous for asc(DL95), asc(DL879) or mei-1, both pairing of homologs and meiotic recombination frequencies are reduced. In each case, this primary defect is followed by the formation of univalents at metaphase I and their irregular segregation. The mutant asc(DL243) has a defect in ascus formation, and later in disjunction during the second meiotic and post-meiotic divisions. The first-acting defect before or during karyogamy results in the abortion of most cells. Some cells manage to proceed past this block. During the second meiotic division, most chromosomes of the few resulting asci are attached to only one of the two spindle-pole bodies. Disjunction at the post-meiotic division is also highly irregular. This mutant appears to be defective in the attachment of one spindle-pole body to a set of chromosomes. The defect may involve either a centromere-associated product or a spindle-pole body.     

Genetic control of recombination partner preference in yeast meiosis. Isolation and characterization of mutants elevated for meiotic unequal sister-chromatid recombination.

Meiotic exchange occurs preferentially between homologous chromatids, in contrast to mitotic recombination, which occurs primarily between sister chromatids. To identify functions that direct meiotic recombination events to homologues, we screened for mutants exhibiting an increase in meiotic unequal sister-chromatid recombination (SCR). The msc (meiotic sister-chromatid recombination) mutants were quantified in spo13 meiosis with respect to meiotic unequal SCR frequency, disome segregation pattern, sporulation frequency, and spore viability. Analysis of the msc mutants according to these criteria defines three classes. Mutants with a class I phenotype identified new alleles of the meiosis-specific genes RED1 and MEK1, the DNA damage checkpoint genes RAD24 and MEC3, and a previously unknown gene, MSC6. The genes RED1, MEK1, RAD24, RAD17, and MEC1 are required for meiotic prophase arrest induced by a dmc1 mutation, which defines a meiotic recombination checkpoint. Meiotic unequal SCR was also elevated in a rad17 mutant. Our observation that meiotic unequal SCR is elevated in meiotic recombination checkpoint mutants suggests that, in addition to their proposed monitoring function, these checkpoint genes function to direct meiotic recombination events to homologues. The mutants in class II, including a dmc1 mutant, confer a dominant meiotic lethal phenotype in diploid SPO13 meiosis in our strain background, and they identify alleles of UBR1, INP52, BUD3, PET122, ELA1, and MSC1-MSC3. These results suggest that DMC1 functions to bias the repair of meiosis-specific double-strand breaks to homologues. We hypothesize that the genes identified by the class II mutants function in or are regulators of the DMC1-promoted interhomologue recombination pathway. Class III mutants may be elevated for rates of both SCR and homologue exchange.     

High-resolution mapping and recombination interval analysis of mouse Chromosome 17

Analysis of homologous recombination in eukaryotes has shown that some meiotic crossing-over occurs preferentially at specific genomic sites of limited physical distance called recombinational hotspots. In the mouse, recombinational hotspots have only been defined in the major histocompatibility complex (MHC) on chromosome (Chr) 17. In an attempt to examine whether hotspots are unique to the MHC or are present throughout the genome, high-resolution linkage maps of Chr 17 based on five backcrosses involving different inbred strains have been generated. These maps separate many markers that were previously shown at the same map position and allow a detailed analysis of recombination patterns across Chr 17. Corresponding recombination intervals in these maps have been compared for the identification of intervals with very little or no recombination in certain genetic crosses and considerable recombination in other genetic crosses. This approach has been termed Recombination Interval Analysis. Possible haplotype-dependent non-MHC hotspots, as well as previously identified MHC hotspots, have been detected by interval analysis. Received: 1 December 1997/ Accepted: 27 February 1998     

Genetic mapping of mitochondrial markers by recombinational analysis in Chlamydomonas reinhardtii

The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies. In crosses, the meiotic products only inherit the mitochondrial genome of the mating type minus (paternal) parent. In contrast mitotic zygotes transmit maternal and paternal mitochondrial DNA copies to their diploid progeny and recombinational events between molecules of both origins frequently occur. Six mitochondrial mutants unable to grow in the dark (dk^? mutants) were crossed in various combinations and the percentages of wild-type dk⁺ recombinants were determined in mitotic zygotes when all progeny cells had become homoplasmic for the mitochondrial genome. In crosses between strains mutated in the COB (apocytochrome ) gene and strains mutated in the COX1 (subunit 1 of cytochrome oxidase) gene, the frequency of recombination was 13.7% (± 3.2%). The corresponding physical distance between the mutation sites was 4.3 kb. In crosses between strains carrying mutations separated by about 20 bp, a recombinational frequency of 0.04% (± 0.02%) was found. Two other mutants not yet characterized at the molecular level were also used for recombinational studies. From these data, a linear genetic map of the mitochondrial genome could be drawn. This map is consistent with the positions of the mutation sites on the mitochondrial DNA molecule and thereby validates the method used to generate the map. The frequency of recombination per physical distance unit (3.2% ± 0.7% per kilobase) is compared with those obtained for other organellar genomes in yeasts and Chlamydomonas.     

Direction of chromosome rearrangements in Saccharomyces cerevisiae by use of his3 recombinational substrates.

We used the his3 recombinational substrates (his3 fragments) to direct large interchromosomal (translocations) and intrachromosomal (deletions and tandem duplications) rearrangements in the yeast Saccharomyces cerevisiae. In strains completely deleted for the wild-type HIS3 gene, his3 fragments, one containing a deletion of 5' amino acid coding sequences and the other containing a deletion of 3' amino acid coding sequences, were first placed at preselected sites by homologous recombination. His+ revertants that arose via spontaneous mitotic recombination between the two his3 fragments were selected. This strategy was used to direct rearrangements in both RAD52+ and rad52 mutant strains. Translocations occurred in the RAD52+ genetic background and were characterized by orthogonal field alternating gel electrophoresis of yeast chromosomal DNA and by standard genetic techniques. An unexpected translocation was also identified in which HIS3 sequences were amplified. Two types of tandem duplications of the GAL(7, 10, 1) locus were also directed, and one type was not observed in rad52 mutants. Recombination mechanisms are discussed to account for these differences.     

Genetic Modification of Recombination Rate in TRIBOLIUM CASTANEUM

Asymmetrical responses were obtained in a replicated study of 15 generations of two-way selection for recombination rate between the ruby (rb) and jet (j) loci in Tribolium castaneum. Recombination rates in the two replicate high lines increased from an average of 0.22 in the base populations to an average of 0.42 at generation 15. Recombination rate pooled over the 15 generations of selection in each low line was significantly less than the control but there was no clear downward trend in response to selection for decreased recombination rate. The realized heritabilities were 0.16 +/- 0.03 and 0.17 +/- 0.02 in the two high lines, and were not significantly different from zero in the two low lines. Selection was based on crossing over in cis females only; however, rates measured in cis males after 12 generations showed the same response patterns as female rates. Similar response patterns were also determined for recombination measured in trans males and females at generation 18 following three generations of relaxed selection. The distribution of recombination rates measured in backcross beetles [(H X L) X H and (H X L) X L] at generation 12 indicated polygenic control with those genes decreasing recombination rate being dominant. Detailed analysis of recombination rates in F1's produced by interline crosses at generation 15 confirmed the directional dominance findings. Under a polygenic model of recombination modifiers in which low recombination is dominant to high, average recombination rates will increase as inbreeding progresses, thus providing a mechanism for the production of new gene combinations in small populations.     

Genetic control of recombination processes in Aspergillus nidulans. I. Effect of uvs-mutations on the frequency of spontaneous and ultraviolet ray-induced intergenic recombination]

Effect of 3 uvs mutations (uvs 12, 19 and 25) on recombination processes in Aspergillus nidulans is studied. All the mutations are found either to affect the fertility of carp bodies and germination ability of askospores, or result in complete inability of heterokaryons to form cleistocarpia. Two mutations change the frequency of spontaneous meitotic crossing-over at pro-paba region of the chromosome I and do not affect the rate of mitotic recombination at w-centromeric region of the chromosome II: uvs 12 mutation increases, and uvs 19 mutation decreases the frequency of meiotic recombination. One mutation (uvs 25) decreases the rate of spontaneous mitotic crossing-over. All uvs mutations decrease the frequency of VU light induced mitotic recombination at w-centromeric region of the chromosome II. The data obtained, together with earlier reported characteristics of uvs mutants, suggest that recombination mechanisms in yeast participate in reparation processes more actively than in prokariotes. Different effects of the same uvs mutations on spontaneous frequency of meiotic and mitotic crossing-over draw to the conclusion that genetic control and molecular mechanisms of these processes in A. nidulans are not identical.     

Recombination in Relation to Ultraviolet Sensitivity in CHLAMYDOMONAS REINHARDI

A mutant strain of Chlamydomonas reinhardi which is UV sensitive as a result of a single-gene chromosomal mutation has also been found to exhibit reduced recombination. In crosses homozygous for the mutant allele, a reduction in recombination frequency was demonstrated in two different linkage groups and in three different genetic backgrounds. Thus a single mutation affecting UV sensitivity also has a possible effect on recombination. Such a mutant could be analogous to the rec(-) mutants discovered in E. coli and as such be useful in the study of recombination mechanisms. Three additional UV-sensitive isolates were tested. Recombination was not altered in these mutant strains.     

The effects of RAD52 epistasis group genes on various types of spontaneous mitotic recombination in Saccharomyces cerevisiae

The role of RAD52 epistasis group genes on spontaneous mitotic recombination was examined using three different types of spontaneous mitotic recombination in Saccharomyces cerevisiae. The spontaneous recombination between homologous sequences in a plasmid and a chromosome was essentially unaffected by null mutations in any of the RAD52 epistasis group genes. Recombination between genes in separate autonomously replicating plasmids was reduced 833-fold in a rad52 null mutant, but only 2- to at most 20-fold in rad50, 51, 54, 55, 57 null mutants. Recombination between tandemly repeated heteroalleles in an autonomously replicating plasmid was reduced almost 100-fold in a rad52 null mutant, but is either unaffected or slightly increased in rad50, 51, 54, 55, 57 null mutants. The finding that RAD50, 51, 54, 55, 57 are dispensable or marginally involved in these spontaneous recombinations suggests further that spontaneous mitotic recombination in S. cerevisiae might be processed by other than RAD52 epistasis group.     

Genetic control of sex-dependent meiotic recombination in the major histocompatibility complex of the mouse.

Meiotic recombination within the proximal region of the major histocompatibility complex (MHC) of the mouse is not random but occurs in clusters at certain restricted sites, so-called recombinational hotspots. The wm7 haplotype of the MHC, derived from the wild mouse, enhances recombination specifically during female meiosis within a fragment of 1.3 kb of DNA located between the A beta 3 and A beta 2 genes in genetic crosses with laboratory haplotypes. Previous studies revealed no significant strain differences in nucleotide sequences around the hotspot, irrespective of the ability of the strain to enhance the recombination. It appeared that a distant genetic element might, therefore, control the rate of recombination. In the present study, original recombinants whose breakpoints were defined by direct sequencing of PCR-amplified DNAs were tested for the rate of secondary recombination in the crosses with laboratory strains in order to determine the location of such a genetic element. The results clearly demonstrated that the chromosomal segment proximal to the hotspot is essential for enhancement of recombination. Moreover, the male recombination is suppressed by a segment distal to the hotspot.