Genetic recombination at the buff spore colour locus in Sordaria brevicollis

Summary Asci showing aberrant segregation at the buff spore colour locus in Sordaria brevicollis were selected from crosses between buff mutants and wild type in the presence of closely-linked flanking markers. The frequency of crossing-over associated with aberrant segregations was calculated and corrected to allow for crossovers between the flanking markers incidental to the aberrant segregation. The average frequency of crossing over was found to be related to the class of aberrant ascus studied. 5+:3m and 3+:5m asci showed 16% associated marker recombination while 6+:2m and 2+:6m asci showed 27% recombination. The frequency of tritype and tetratype postmeiotic segregation asci was calculated. Only 3% tetratypes were found and this is thought to indicate a low frequency of symmetric hybrid DNA formation.     

Multiple heterologies increase mitotic double-strand break-induced allelic gene conversion tract lengths in yeast.

Spontaneous and double-strand break (DSB)-induced allelic recombination in yeast was investigated in crosses between ura3 heteroalleles inactivated by an HO site and a +1 frameshift mutation, with flanking markers defining a 3.4-kbp interval. In some crosses, nine additional phenotypically silent RFLP mutations were present at approximately 100-bp intervals. Increasing heterology from 0.2 to 1% in this interval reduced spontaneous, but not DSB-induced, recombination. For DSB-induced events, 75% were continuous tract gene conversions without a crossover in this interval; discontinuous tracts and conversions associated with a crossover each comprised approximately 7% of events, and 10% also converted markers in unbroken alleles. Loss of heterozygosity was seen for all markers centromere distal to the HO site in 50% of products; such loss could reflect gene conversion, break-induced replication, chromosome loss, or G2 crossovers. Using telomere-marked strains we determined that nearly all allelic DSB repair occurs by gene conversion. We further show that most allelic conversion results from mismatch repair of heteroduplex DNA. Interestingly, markers shared between the sparsely and densely marked interval converted at higher rates in the densely marked interval. Thus, the extra markers increased gene conversion tract lengths, which may reflect mismatch repair-induced recombination, or a shift from restoration- to conversion-type repair.     

Dominant enhancer effect of the meiotic mei4 mutant on recombination frequencies restricted to linkage group VI in Podospora anserina.

Summary A mutant which increases second division segregation (SDS) frequency of locus 110 (linkage group VI) was isolated. It was called mei4 because of its meiotic deficiency. The present paper deals with its effect on meiotic recombination when heterozygous. mei4 then only acts on linkage group VI. The SDS frequencies were increased for all markers used, except locus 5 located very close to the centromere. This quasi general enhancement results exclusively in an enlargement of map distance on linkage group VI's proximal part. Crosses involving three mutant genes allowed to check that the distances on the distal part were constant. This is due to a real lack of crossover frequency modification in this region and not to a change of chiasma interference. Among the seven linkage groups of Podospora anserina, group VI exhibits several other particularities concerning meiotic recombination, especially a lower positive chiasma interference and a more regular crossover distribution, suggesting a particular recombination regulation.Laboratoire associé n⁰ 86 du Centre National de la Recherche Scientifique     

Repeat instability at human minisatellites arising from meiotic recombination.

Little is known about the role of meiotic recombination processes such as unequal crossover in driving instability at tandem repeat DNA. Methods have therefore been developed to detect meiotic crossovers within two different GC-rich minisatellite repeat arrays in humans, both in families and in sperm DNA. Both loci normally mutate in the germline by complex conversion-like transfer of repeats between alleles. Analysis shows that inter-allelic unequal crossovers also occur at both loci, although at low frequency, to yield simple recombinant repeat arrays with exchange of flanking markers. Equal crossovers between aligned alleles, resulting in recombinant alleles but without change in repeat copy number, also occur in sperm at a similar frequency to unequal crossovers. Both crossover and conversion show polarity in the repeat array and are co-suppressed in an allele showing unusual germline stability. This provides evidence that minisatellite conversion and crossover arise by a common mechanism, perhaps by alternative processing of a meiotic recombination initiation complex, and implies that minisatellite instability is a by-product of meiotic recombination in repeat DNA. While minisatellite recombination is infrequent, crossover rates indicate that the unstable end of a human minisatellite can act as a recombination warm-spot, even between sequence-heterologous alleles.     

Characterization of recombination in the HLA class II region.

Studies of linkage disequilibrium across the HLA class II region have been useful in predicting where recombination is most likely to occur. The strong associations between genes within the 85-kb region from DQB1 to DRB1 are consistent with low frequency of recombination in this segment of DNA. Conversely, a lack of association between alleles of TAP1 and TAP2 (approximately 15 kb) has been observed, suggesting that recombination occurs here with relatively high frequency. Much of the HLA class II region has now been sequenced, providing the tools to undertake detailed analysis of recombination. Twenty-seven families containing one or two recombinant chromosomes within the 500-kb interval between the DPB1 and DRB1 genes were used to determine patterns of recombination across this region. SSCP analysis and microsatellite typing yielded identification of 127 novel polymorphic markers distributed throughout the class II region, allowing refinement of the site of crossover in 30 class II recombinant chromosomes. The three regions where recombination was observed most frequently are as follows: the 45-kb interval between HLA-DNA and RING3 (11 cases), the 50-kb interval between DQB3 and DQB1 (6 cases), and an 8.8-kb segment of the TAP2 gene (3 cases). Six of the 10 remaining recombinants await further characterization, pending identification of additional informative markers, while four recombinants were localized to other intervals (outliers). Analysis of association between markers flanking HLA-DNA to RING3 (45 kb), as well as TAP1 to TAP2 (15 kb), by use of independent CEPH haplotypes indicated little or no linkage disequilibrium, supporting the familial recombination data. A notable sequence motif located within a region associated with increased rates of recombination consisted of a (TGGA)12 tandem repeat within the TAP2 gene.     

A Recombinational Hotspot at the Triplo-Lethal Locus of Drosophila Melanogaster

In the genome of Drosophila melanogaster there is only one locus, Tpl, that is triplo-lethal; it is also haplo-lethal. Previous work has identified 3 hypomorphic alleles of Tpl which rescue animals carrying a duplication of Tpl, but which are not dominant lethals as null mutations or deficiencies would be. We have found that all three hypomorphic alleles act as site-specific hotspots for recombination when heterozygous with a wild-type homolog. Recombination between the flanking markers ri and Ki is increased 6.5-10.5-fold in the presence of Tpl hypomorphic alleles. The increased recombination was found to occur between Tpl and Ki, while recombination in other adjacent regions is unchanged. The use of isogenic Tpl+ controls, and the use of flanking intervals in the mutant chromosomes allows us to rule out the interchromosomal effect as a cause. We have also observed premeiotic recombination occurring at the Tpl hypomorphic alleles in male heterozygotes. We hypothesize that transposons are responsible for both the hypomorphic phenotype and the high frequency of recombination.     

Recombination Can Initiate and Terminate at a Large Number of Sites within the Rosy Locus of Drosophila Melanogaster

This report presents the results of a recombination experiment designed to question the existence of special sites for the initiation or termination of a recombination heteroduplex within the region of the rosy locus. Intragenic recombination events were monitored between two physically separated rosy mutant alleles ry301 and ry2 utilizing DNA restriction site polymorphisms as genetic markers. Both ry301 and ry2 are known from previous studies to be associated with gene conversion frequencies an order of magnitude lower than single site mutations. The mutations are associated with large, well defined insertions located as internal sites within the locus in prior intragenic mapping studies. On the molecular map, they represent large insertions approximately 2.7 kb apart in the second and third exons, respectively, of the XDH coding region. The present study monitors intragenic recombination in a mutant heterozygous genotype in which DNA homology is disrupted by these large discontinuities, greater than the region of DNA homology and flanking both sides of the locus. If initiation/or termination requires separate sites at either end of the locus, then intragenic recombination within the rosy locus of the heterozygote should be eliminated. Contrary to expectation, significant recombination between these sites is seen.     

Gene Conversion Alone Accounts for More than 90% of Recombination Events at the Am Locus of Neurospora Crassa

We have used closely flanking molecular markers located ~4 kb distal and 6 kb proximal of the am locus to investigate the incidence of crossover events associated with the generation of prototrophic recombinants in a cross heteroallelic am(1) am(6). Ninety-three percent of prototrophs were generated by events that did not recombine the molecular markers, indicating that simple conversion accounts for the formation of most prototrophs and that associated crossovers are much less frequent (~0.07) than estimated previously using more distant flanking markers. This suggests that conversion and crossing over during meiosis may arise from distinct mechanisms or that if, as is widely supposed, conversion and crossing over result from alternate modes of resolution of Holliday junctions then, at least for the am locus of Neurospora, the mode of resolution is strongly biased in favor of retaining the parental association of flanking sequences. Because estimates of the association of conversion and crossing over based on more distant gene markers are similar for yeast and Neurospora (~0.35), our observation may have general significance.     

Identification of an RFLP interval containing Pch2 on chromosome 7AL in wheat.

The gene Pch2 in 'Cappelle Desprez' is one of two genes found in hexaploid wheat known to confer resistance to eyespot disease. This study was conducted to develop an RFLP linkage map of the distal portion of wheat chromosome 7AL, and to locate and identify markers closely associated with Pch2 for use in marker-assisted selection. Ten loci in addition to Pch2 were mapped on chromosome 7AL, using segregation data from 102 homozygous chromosome 7A recombinant substitution lines derived from 'Chinese Spring' x 'Chinese Spring' ('Cappelle Desprez' 7A). The Pch2 locus was bracketed by two RFLP markers, one 11.0 cM distal to Xcdo347 and the other 18.8 cM proximal to Xwg380. The position of Pch2 on chromosome 7AL is similar to that of Pch1 on chromosome 7DL, suggesting that these resistance genes are homoeoloci. Although no single marker was closely linked to Pch2, simultaneous selection of the flanking RFLP markers Xcdo347 and Xwg380 could be used for selecting Pch2, since double recombination occurred in only 3% of the recombinant population. The use of the flanking RFLP markers to select for Pch2, in combination with previously identified Pch1-linked markers, would facilitate the development of cultivars carrying two genes for resistance to eyespot.     

Conjugational Recombination in Escherichia Coli: Genetic Analysis of Recombinant Formation in Hfr X F(-) Crosses

The formation of recombinants during conjugation between Hfr and F(-) strains of Escherichia coli was investigated using unselected markers to monitor integration of Hfr DNA into the circular recipient chromosome. In crosses selecting a marker located ~500 kb from the Hfr origin, 60-70% of the recombinants appeared to inherit the Hfr DNA in a single segment, with the proximal exchange located >300 kb from the selected marker. The proportion of recombinants showing multiple exchanges increased in matings selecting more distal markers located 700-2200 kb from the origin, but they were always in the minority. This effect was associated with decreased linkage of unselected proximal markers. Mutation of recB, or recD plus recJ, in the recipient reduced the efficiency of recombination and shifted the location of the proximal exchange (s) closer to the selected marker. Mutation of recF, recO or recQ produced recombinants in which this exchange tended to be closer to the origin, though the effect observed was rather small. Up to 25% of recombinant colonies in rec(+) crosses showed segregation of both donor and recipient alleles at a proximal unselected locus. Their frequency varied with the distance between the selected and unselected markers and was also related directly to the efficiency of recombination. Mutation of recD increased their number by twofold in certain crosses to a value of 19%, a feature associated with an increase in the survival of linear DNA in the absence of RecBCD exonuclease. Mutation of recN reduced sectored recombinants in these crosses to ~1% in all the strains examined, including recD. A model for conjugational recombination is proposed in which recombinant chromosomes are formed initially by two exchanges that integrate a single piece of duplex Hfr DNA into the recipient chromosome. Additional pairs of exchanges involving the excised recipient DNA, RecBCD enzyme and RecN protein, can subsequently modify the initial product to generate the spectrum of recombinants normally observed.     

Genetic mapping of the Lr20-Pm1 resistance locus reveals suppressed recombination on chromosome arm 7AL in hexaploid wheat.

The Lr20-Sr15-Pm1 resistance locus in hexaploid wheat confers resistance to three different fungal wheat pathogens (leaf rust, stem rust, and powdery mildew). It was previously localized in the distal region of chromosome arm 7AL. As a first step towards the isolation of this complex locus, we performed molecular mapping of the Lr20 and Pm1 genes in three F2 populations. In two populations, a cluster of 8 and 12 markers, respectively, cosegregated with the resistance genes. In a third population based on a cross between a susceptible lr20 mutant and a resistant cultivar, all clustered markers were monomorphic. However, in this population the recombination frequency proximal to the Lr20 gene was up to 60 times higher, indicating that the complete genetic linkage of the clustered markers is not due to a close physical linkage of the probes but is caused by suppressed recombination. This was supported by the analysis of Triticum monococcum BAC clones where no physical linkage between cosegregating probes was observed. Suppressed recombination at the Lr20-Pm1 locus is likely the result of an alien introgression of chromatin from an unidentified wild relative species or is due to chromosomal rearrangements.     

Meiotic recombination and flanking marker exchange at the highly unstable human minisatellite CEB1 (D2S90)

Unequal crossover has long been suspected to play a role in the germline-specific instability of tandem-repeat DNA, but little information exists on the dynamics and processes of unequal exchange. We have therefore characterized new length alleles associated with flanking-marker exchange at the highly unstable human minisatellite CEB1, which mutates in the male germline by a complex process often resulting in the gene conversion-like transfer of repeats between alleles. DNA flanking CEB1 is rich in single-nucleotide polymorphisms (SNPs) and shows extensive haplotype diversity, consistent with elevated recombinational activity near the minisatellite. These SNPs were used to recover mutant CEB1 molecules associated with flanking-marker exchange, directly from sperm DNA. Mutants with both proximal and distal flanking-marker exchange were shown to contribute significantly to CEB1 turnover and suggest that the 5' end of the array is very active in meiotic unequal crossover. Coconversions involving the interallelic transfer of repeats plus immediate flanking DNA were also common, were also polarized at the 5' end of CEB1, and appeared to define a conversion gradient extending from the repeat array into adjacent DNA. Whereas many mutants associated with complete exchange resulted in simple recombinant-repeat arrays that show reciprocity, coconversions were highly gain-biased and were, on average, more complex, with allele rearrangements similar to those seen in the bulk of sperm mutants. This suggests distinct recombination-processing pathways producing, on the one hand, simple crossovers in CEB1 and, on the other hand, complex conversions that sometimes extend into flanking DNA.     

A linkage map of mouse Chromosome 1 using an interspecific cross segregating for the gld autoimmunity mutation

An interspecific backross was used to define a high resolution linkage map of mouse Chromosome (Chr) 1 and to analyze the segregation of the generalized lymphoproliferative disease (gld) mutation. Mice homozygous for gld have multiple features of autoimmune disease. Analysis of up to 428 progeny from the backcross [(C3H/HeJ-gld x Mus spretus)F₁ x C3H/HeJ-gld] established a map that spans 77.6 cM and includes 56 markers distributed over 34 ordered genetic loci. The gld mutation was mapped to a less than 1 cM segment on distal mouse Chr 1 using 357 gld phenotype-positive backcross mice. A second backcross, between the laboratory strains C57BL/6J and SWR/J, was examined to compare recombination frequency between selected markers on mouse Chr 1. Significant differences in crossover frequency were demonstrated between the interspecific backcross and the inbred laboratory cross for the entire interval studied. Sex difference in meiotic crossover frequency was also significant in the laboratory mouse cross. Two linkage groups known to be conserved between segments of mouse Chr 1 and the long arm of human Chrs 1 and 2 where further defined and a new conserved linkage group was identified that includes markers of distal mouse Chr 1 and human Chr 1, bands q32 to q42.     

Evidence for negative interference: clustering of crossovers close to the am locus in Neurospora crassa among am recombinants.

In response to a conflict between two mapping studies in the predicted orientation of the allele map with respect to the centromere, Fincham proposed that recombination events at the Neurospora am locus rarely have an associated crossover. Fincham considered that the elevated levels of crossing over between flanking markers in am recombinants resulted from negative interference, an increased probability of a nearby second event, and on this basis predicted a clustering of crossing over near am in these recombinants. In this article we reevaluate the data from three mapping studies of the am locus and report molecular evidence that shows crossovers to be clustered immediately proximal to am in am recombinants.     

GENETICS OF SORDARIA FIMICOLA. V. ABERRANT SEGREGATION AT THE G LOCUS

Kitani , Y., L. S. Olive , and Arif S. El -Ani . (Columbia U., New York City.) Genetics of Sordaria fimicola. V. Aberrant segregation at the g locus. Amer. Jour. Bot. 49(7): 697–706. Illus. 1962.—Aberrant segregation of the gray-spore color locus in Sordaria fimicola was studied with the aid of closely linked markers. It was found that 6:2 and 5:3 asci occur with about the same frequency, but asci with an excess of wild-type spores occur with a frequency 5.5 times that of asci with an excess of gray spores. Also, the frequency of related crossing over (occurring close to the miscopied loeus and involving the miscopying strand) was much higher than the expected value, and in 5:3 asci it appears to be at least twice that found in 6:2 asci. Nine aberrant 4:4 asci, each with 2 spore pairs heterogeneous for color, were found. These are believed to have resulted from reciprocal double transreplication. The rarest aberrant type was represented by a single 7:1 ascus, which is difficult to explain on the basis of a single meiotic process. Miscopying is discussed with relation to an 8-strand model of paired homologues and the occurrence of localized chromosome pairing during prezygotene DNA synthesis. Several possible explanations for the occurrence of aberrant tetrads are considered. Miscopying has also been found to involve several spore-color loci not previously studied; whereas, several other such mutant loci fail to show evidence of it. One locus (m) shows abnormal segregation of the 6:2 but not the 5:3 type.     

Unequal Exchange and Meiotic Instability of Disease-Resistance Genes in the Rp1 Region of Maize

The Rp1 region of maize was originally characterized as a complex locus which conditions resistance to the fungus Puccinia sorghi, the causal organism in the common rust disease. Some alleles of Rp1 are meiotically unstable, but the mechanism of instability is not known. We have studied the role of recombination in meiotic instability in maize lines homozygous for either Rp1-J or Rp1-G. Test cross progenies derived from a line that was homozygous for Rp1-J, but heterozygous at flanking markers, were screened for susceptible individuals. Five susceptible individuals were derived from 9772 progeny. All five had nonparental combinations of flanking markers; three had one combination of recombinant flanking markers while the other two had the opposite pair. In an identical study with Rp1-G, 20 susceptible seedlings were detected out of 5874 test cross progeny. Nineteen of these were associated with flanking marker exchange, 11 and 8 of each recombinant marker combination. Our results indicate that unequal exchange is the primary mechanism of meiotic instability of Rp1-J and Rp1-G.     

Genetic Evidence That the Meiotic Recombination Hotspot at the His4 Locus of Saccharomyces Cerevisiae Does Not Represent a Site for a Symmetrically Processed Double-Strand Break

In the yeast Saccharomyces cerevisiae, the binding of the Rap1 protein to a site located between the 5' end of the HIS4 gene and the 3' end of BIK1 stimulates meiotic recombination at both flanking loci. By using strains that contain mutations located in HIS4 and BIK1, we found that most recombination events stimulated by the binding of Rap1 involve HIS4 or BIK1, rather than bidirectional events including both loci. The patterns of aberrant segregation indicate that most of the Rap1-stimulated recombination events do not represent the symmetric processing of a double-strand DNA break located at the Rap1-binding site.     

RAD52-Independent Mitotic Gene Conversion in SACCHAROMYCES CEREVISIAE Frequently Results in Chromosomal Loss

We have examined spontaneous, interchromosomal mitotic recombination events between his4 alleles in both Rad+ and rad52 strains of Saccharomyces cerevisiae. In Rad+ strains, 74% of the His+ prototrophs resulted from gene conversion events without exchange of flanking markers. In diploids homozygous for the rad52-1 mutation, the frequency of His+ prototroph formation was less than 5% of the wild-type value, and more than 80% of the gene conversion events were accompanied by an exchange of flanking markers. Most of the rad52 intragenic recombination events arose by gene conversion accompanied by an exchange of flanking markers and not by a simple reciprocal exchange between the his4A and his4C alleles. There were also profound effects on the kinds of recombinant products that were recovered. The most striking effect was that RAD52-independent mitotic recombination frequently results in the loss of one of the two chromosomes participating in the gene conversion event.