Recombination at his-3 in Neurospora declines exponentially with distance from the initiator,cog

By deletion of 1.8 kb of sequence between cog(L) and his-3 and replacement with sequences of different lengths, we have generated a set of Neurospora strains in which the distance between cog(L) and the site at which recombination is selected varies from 1.7 to nearly 6 kb. Each of the manipulated strains includes cog(L), a highly active recombination hotspot, and rec-2, thus allowing high-frequency recombination. In addition, each is a his-3 mutant, either K26 or K480. The frequency of His(+) recombinants in progeny of these crosses is inversely proportional to the distance between his-3 and cog. Specifically, there is a linear relationship between log(10) (recombination frequency) and the distance in base pairs, indicating that as distance decreases, the rate of interallelic recombination increases exponentially. An exponential relationship between distance separating markers and the chance of co-conversion has been found in both Drosophila and fission yeast, indicating that the extension of recombination events may be a stochastic process in most organisms. On the basis of these and additional data presented in this article, we conclude that recombination is initiated at cog(L) in >17% of meioses, that most conversion tracts are very short, and that few extend >14 kb.     

Recombination events in Neurospora crassa may cross a translocation breakpoint by a template-switching mechanism

To assist investigation of the effect of sequence heterology on recombination in Neurospora crassa, we inserted the Herpes simplex thymidine kinase gene (TK) as an unselected marker on linkage group I, giving a gene order of Cen-his-3-TK-cog-lpl. We show here that in crosses heterozygous for TK, conversion of a his-3 allele on one homolog is accompanied by transfer of the heterologous sequence between cog and his-3 from the other homolog, indicating that recombination is initiated centromere-distal of TK. We have identified a 10-nucleotide motif in the cog region that, although unlikely to be sufficient for hotspot activity, is required for high-frequency recombination and, because conversion of silent sequence markers declines on either side, may be the recombination initiation site. Additionally, we have mapped conversion tracts in His(+) progeny of a translocation heterozygote, in which the translocation breakpoint separates cog from the 5' end of his-3. We present molecular evidence of recombination on both sides of the breakpoint. Because recombination is initiated close to cog and the event must therefore cross the translocation breakpoint, we suggest that template switching occurs in some recombination events, with repair synthesis alternating between use of the homolog and the initiating chromatid as template.     

Use of fluorescent protein to analyse recombination at three loci in Neurospora crassa

We have inserted a histone H1-GFP fusion gene adjacent to three loci on different chromosomes of Neurospora crassa and made mating pairs in which a wild type version of GFP is crossed to one with a mutation in the 5' end of GFP. The loci are his-3, am and his-5, chosen because recombination mechanisms appear to differ between his-3 and am, and because crossing over adjacent to his-5, like his-3, is regulated by rec-2. At his-3, the frequencies of crossing over between GFP and the centromere and of conversion of 5'GFP to GFP(+) are comparable to those obtained by classical recombination assays, as is the effect of rec-2 on these frequencies, suggesting that our system does not alter the process of recombination. At each locus we have obtained sufficient data, on both gene conversion and crossing over, to be able to assess the effect of deletion of any gene involved in recombination. In addition, crosses between a GFP(+) strain and one with normal sequence at all three loci have been used to measure the interval to the centromere and to show that GFP experiences gene conversion with this system. Since any gene expressed in meiosis is silenced in Neurospora if hemizygous, any of our GFP(+) strains can be used as a quick screen to determine if a gene deleted by the Neurospora Genome Project is involved in crossing over or gene conversion.     

Diversification of exogenous genes in vivo in Neurospora

We have adapted the meiotic recombination hotspot cog of Neurospora crassa for shuffling exogenous DNA, providing a means of generating novel genes in situ from sequences introduced into chromosomes. Genes to be diversified are inserted between the his-3 locus and cog. Diversification crosses are heterozygous both for alleles of the exogenous DNA and for auxotrophic alleles of his-3. Progeny selected for ability to grow without histidine supplementation are enriched for exchange events within the exogenous DNA. Exchange events initiated by cog can propagate past DNA sequences mismatched for more than 370 bp and complete exchanges in patches of matched sequence as short as 24 bp, parameters that make the system suited for use in the directed evolution of genes for protein engineering. Here we demonstrate the system by shuffling human immunoglobulin kappa chain genes and also endoglucanase genes derived from different species of fungi.     

Alleles of the hotspot cog are codominant in effect on recombination in the his-3 region of Neurospora

There are two naturally occurring functional alleles of the recombination hotspot cog, which is located 3.5 kb from the his-3 locus of Neurospora crassa. The presence of the cog+ allele in a cross significantly increases recombination in the his-3 region compared to a cross homozygous for the cog allele. Data obtained shortly after discovery of cog+ suggested that it was fully dominant to cog. However, a dominant cog+ conflicts with observations of hotspots in Saccharomyces cerevisiae and Schizosaccharomyces pombe, in which recombination is initiated independently of homolog interactions, and suggests recombination mechanisms may differ in Neurospora and yeast. We present evidence that cog alleles are codominant in effect on both allelic recombination in his-3 and crossing over between loci flanking his-3. In addition, we show that genetic background variation has at least a twofold effect on allelic recombination. We speculate that variation in genetic background, together with the complexities of recombination in crosses bearing close mutant alleles, accounts for the previous conclusion that cog+ is dominant to cog.     

Occurrence in wild strains of Neurospora crassa of genes controlling genetic recombination.

Each of the main laboratory wild stocks of N. crassa carries one of two alleles at the rec-1 and rec-2 loci and one of three at the rec-3 locus. The constitutions of the stocks are given in Fig. 1. Some of those conserved are evidently not the originals. The third rec-3 gene (rec-3L), found in Lindegren A, controls recombination at the am-1 locus to a level between that of rec-3+ and rec-3, the relative levels being 1 : 8 : 25. At the his-2 locus rec-3L is indistinguishable from rec-3+ in its level of control. This proves that there are minor differences between the control (con) genes, near to am-1 and his-2, which recognize products of rec-3 genes. Further, this is the first clear evidence, though indirect, that the binding sites for products of rec genes are situated in the chromosome regions where recombination is modulated.     

Targeting vectors for gene diversification by meiotic recombination in Neurospora crassa.

We have constructed a pair of vectors, pDV2 and pDV3, that enable targeted insertion of exogenous DNA into Linkage Group I of Neurospora crassa at the his-3 locus. Transplaced sequences are inserted between his-3 and the cog(L) recombination hot spot and include his-3 mutations that allow meiotic recombination initiated by cog(L) to be monitored. Selection of correctly placed transforming DNA is based on complementation between different his-3 alleles borne by the plasmids and transformation hosts. The system allows investigation of the effect of any given sequence on recombination as well as diversification of sets of related sequences in vivo for directed evolution of genes.     

Mutation of msh-2 in Neurospora crassa does not reduce the incidence of recombinants with multiple patches of donor chromosome sequence

The Neurospora homologue msh-2 of the Escherichia coli mismatch repair gene mutS was mutated by repeat-induced point mutation (RIP) of a 1.9-kb duplication covering 1661bp of the coding sequence and 302 bp 5' of the gene. msh-2(RIP-LK1) exhibited a mutator phenotype conferring a 17-fold increase in the frequency of spontaneous mitotic reversion of his-3 allele K458. In msh-2(RIP-LK1) homozygotes, recombination frequency at the his-3 locus increased up to 2.9-fold over that in msh-2(+) diploids. Progeny of crosses homozygous msh-2(RIP-LK1), like those from crosses homozygous msh-2(+) frequently had multiple patches of donor chromosome sequence, suggesting that patchiness in msh-2(+) crosses is not explained by incomplete repair of heteroduplex DNA by MSH-2. These findings are consistent with data from the analysis of events in a Neurospora translocation heterozygote that suggested multiple patches of donor chromosome sequence arising during recombination reflect multiple template switches during DNA repair synthesis.     

The control of allelic recombination at histidine loci in Neurospora crassa

The gene rec-1⁺ which reduces allelic recombination at the his-1 locus by a factor of between 15 and 30 has no effect upon allelic recombination at the his-2, his-3, his-5, his-6 and his-7 loci. Other genes controlling recombination at two of these loci, namely rec-x at his-2 and rec-w at his-3, have been found. There is a strong possibility that rec-x may be identical with rec-3, so far known to regulate recombination only at the am-1 locus. It is probable that the stocks used all carry a rec⁺ gene which regulates recombination at the his-6 locus, since all prototroph frequencies are low, but no regulatory gene active at the his-5 and his-7 loci.     

Genetic factors affecting allelic recombination at the histidine-3 locus ofNeurospora crassa

Summary In addition to the generec-4, other genetic factors affect the frequency of allelic recombination in thehis-3 locus. One dominant factor, designated asrec-6 ⁺, in association withrec-4 ⁺ causes greater reduction in prototrophic frequency than obtained withrec-4 ⁺ alone. The action ofrec 6 ⁺ in crosses recessive homozygous forrec-4 is not established at the present. The effect ofrec-6 ⁺ is recognised only with onehis-3 allele but not with another. Interaction ofrec-4 ⁺ orrec-4 with other genetic factors can give approximately ten fold variation in the prototrophic frequencies obtained with a pair of alleles. It is suggested that the control of the rate of mutations during meiosis might be one of the roles of the recombination genes.     

Genetic Analysis of the Reciprocal Translocation T2(I;VIII) of Aspergillus Using the Technique of Mitotic Mapping in Homozygous Translocation Diploids

A UV-induced sulphite-requiring mutant (sD50) consistently shows mitotic linkage to groups I and VIII in haploids from heterozygous mapping diploids. This linkage was found to be due to a reciprocal translocation T2(I;VIII) which could not be separated from the sulphite requirement in about 100 tested progeny from heterozygous crosses, and both may well have been induced by the same mutational event. T2(I;VIII) is the first case of a reciprocal translocation in Aspergillus which showed meiotic linkages between markers of different linkage groups, and, in addition, involved chromosome arms containing markers suitable for complete mapping by the technique of mitotic recombination in homozygous translocation diploids.-Using various selective markers, haploid segregants and diploid crossovers of all possible types were isolated from homozygous translocation diploids. (1) Haploid segregants showed new linkage relationships in T/T diploids: all available markers of VIII now segregated as a group with the majority of the markers of I, except for the markers of the left tip of I. These formed a separate linkage group and are presumably translocated to VIII. (2) Diploid mitotic crossovers confirmed this information and showed that the orientation of the translocated segments was unchanged. These findings conclusively demonstrate that T2(I;VIII) is a reciprocal translocation due to an exchange of the left tip of group I with the long right arm of group VIII.-Since the position of the break on VIIIR was found to be at sD50 this marker could be used to map the break on IL by meiotic recombination in heterozygous crosses. In addition, such crosses showed reduced recombination around the breaks, so that it was possible to sequence markers which normally are barely linked.     

Recombination block in the Spore killer region of Neurospora

Spore killers Sk-2K and Sk-3K are chromosomal meiotic drive factors in Neurospora. In heterozygous crosses, ascospores not containing the Spore killer die. Sk-2K and Sk-3K, which differ in killing specificity, were found to be associated with suppression of recombination in a centromere-spanning region of linkage group III, and investigation of that recombination block is reported here. The block covers a region that is normally 30 to 40 map units long. A locus (r(Sk-2)) conferring resistance to Sk-2K maps to the left end of the recombination block. Recombination is normal in r(Sk-2) X Sk sensitive but blocked in Sk-2K X r(Sk-2); so the block does not depend upon killing. By selective plating, SkK stocks carrying genetic markers within the block were obtained at frequencies on the order of 10(-5) or 10(-6). Since this tight block is far beyond what has been observed for genetic reduction of recombination, a structural basis is assumed. No evidence of chromosome rearrangement was obtained. Crosses homozygous for Sk-2K show normal crossing-over and map order for the flanking markers cum and his-7 and three included markers (acr-7, acr-2, and leu-1). Results would be consistent with a divergence of sequence great enough to interfere with homologous pairing.     

Expansions and contractions of the genetic map relative to the physical map of yeast chromosome III.

To examine the relationship between genetic and physical chromosome maps, we constructed a diploid strain of the yeast Saccharomyces cerevisiae heterozygous for 12 restriction site mutations within a 23-kilobase (5-centimorgan) interval of chromosome III. Crossovers were not uniformly distributed along the chromosome, one interval containing significantly more and one interval significantly fewer crossovers than expected. One-third of these crossovers occurred within 6 kilobases of the centromere. Approximately half of the exchanges were associated with gene conversion events. The minimum length of gene conversion tracts varied from 4 base pairs to more than 12 kilobases, and these tracts were nonuniformly distributed along the chromosome. We conclude that the chromosomal sequence or structure has a dramatic effect on meiotic recombination.     

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.     

Meiotic Recombination in Neurospora crassa Proceeds by Two Pathways with Extensive Holliday Junction Migration

Analysis of thousands of Δmsh-2 octads using our fluorescent recombination system indicates that, as in other filamentous fungi, symmetric heteroduplex is common in the his-3 region of Neurospora crassa. Symmetric heteroduplex arises from Holliday junction migration, and we suggest this mechanism explains the high frequency of His⁺ spores in heteroallelic crosses in which recombination is initiated cis to the his-3 allele further from the initiator, cog⁺. In contrast, when recombination is initiated cis to the his-3 allele closer to cog⁺, His⁺ spores are mainly a result of synthesis-dependent strand annealing, yielding asymmetric heteroduplex. Loss of Msh-2 function increases measures of allelic recombination in both his-3 and the fluorescent marker gene, indicating that mismatches in asymmetric heteroduplex, as in Saccharomyces cerevisiae, tend to be repaired in the direction of restoration. Furthermore, the presence of substantial numbers of conversion octads in crosses lacking Msh-2 function suggests that the disjunction pathway described in S. cerevisiae is also active in Neurospora, adding to evidence for a universal model for meiotic recombination.     

Meiotic transmission of Drosophila pseudoobscura chromosomal arrangements

Drosophila pseudoobscura harbors a rich gene arrangement polymorphism on the third chromosome generated by a series of overlapping paracentric inversions. The arrangements suppress recombination in heterokaryotypic individuals, which allows for the selective maintenance of coadapted gene complexes. Previous mapping experiments used to determine the degree to which recombination is suppressed in gene arrangement heterozygotes produced non-recombinant progeny in non-Mendelian ratios. The deviations from Mendelian expectations could be the result of viability differences between wild and mutant chromosomes, meiotic drive because of achiasmate pairing of homologues in heterokaryotypic females during meiosis, or a combination of both mechanisms. The possibility that the frequencies of the chromosomal arrangements in natural populations are affected by mechanisms other than adaptive selection led us to consider these hypotheses. We performed reciprocal crosses involving both heterozygous males and females to determine if the frequency of the non-recombinant progeny deviates significantly from Mendelian expectations and if the frequencies deviate between reciprocal crosses. We failed to observe non-Mendelian ratios in multiple crosses, and the frequency of the non-recombinant classes differed in only one of five pairs of reciprocal crosses despite sufficient power to detect these differences in all crosses. Our results indicate that deviations from Mendelian expectations in recombination experiments involving the D. pseudoobscura inversion system are most likely due to fitness differences of gene arrangement karyotypes in different environments.     

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.     

The Relationships among Transmission Frequency, Male Recombination and Progeny Production in DROSOPHILA MELANOGASTER

The T-007 second chromosome line, which was originally isolated in 1970 from a natural population of Drosophila melanogaster at Harlingen, south Texas, has previously been shown to be associated with several unusual genetic phenomena. In the present study, two characteristics, distorted transmission frequency and male recombination, were analyzed in relation to the progeny production of T-007 heterozygous individuals. The following points were established: (1) Distorted transmission frequency in the T-007 heterozygous male was mainly due to "elimination" of T-007 chromosomes among the progeny, while no such elimination occurred for the normal partner chromosome. (2) Transmission frequency and progeny production of the T-007 heterozygous females were normal, or at least almost normal. (3) The frequency of male recombination increased with an increasing degree of distortion. This was due to an increased number of recombinants produced per male and to a decreased number of progeny receiving the T-007 chromosome.     

The meiotic recombination hot spot ura4A in Schizosaccharomyces pombe

The meiotic recombination hot spot ura4A (formerly ura4-aim) of Schizosaccharomyces pombe was observed at the insertion of the ura4+ gene 15 kb centromere-proximal to ade6 on chromosome III. Crosses heterozygous for the insertion showed frequent conversion at the heterology with preferential loss of the insertion. This report concerns the characterization of 12 spontaneous ura4A mutants. A gradient of conversion ranging from 18% at the 5' end to 6% at the 3' end was detected. A novel phenomenon also was discovered: a mating-type-related bias of conversion. The allele entering with the h+ parent acts preferentially as the acceptor for conversion (ratio of 3:2). Tetrad analysis of two-factor crosses showed that heteroduplex DNA is predominantly asymmetrical, enters from the 5' end, and more often than not covers the entire gene. Restoration repair of markers at the 5' end was inferred. Random spore analyses of two-factor crosses and normalization of prototroph-recombinant frequencies to physical distance led to the demonstration of map expansion: Crosses involving distant markers yielded recombinant frequencies higher than the sum of the frequencies measured in the subintervals. Finally, marker effects on recombination were defined for two of the ura4A mutations.