Residual recombination in Neurospora crassa spo11 deletion homozygotes occurs during meiosis.

Spo11 is considered responsible for initiation of meiotic recombination in higher organisms, but previous analysis using spo11 (RIP) mutants suggests that the his-3 region of Neurospora crassa experiences spo11-independent recombination. However, despite possessing several stop codons, it is conceivable that the mutants are not completely null. Also, since lack of spo11 interferes with chromosomal pairing and proper segregation at Meiosis I, spores can be partially diploid for a period after meiosis. Thus, it is possible that the recombination observed could be an abnormal event, occurring during the period of aneuploidy rather than during meiosis. To test the former hypothesis, we generated spo11 deletion homozygotes. Using crosses heteroallelic for his-3 mutations, we showed that His(+) progeny are generated in spo11 deletion homozygotes at a frequency at least as high as in wild type and, as in the spo11 (RIP) mutants, local crossing over is not reduced. To test the latter hypothesis, we utilised mutations in either end of a histone H1-GFP fusion gene, inserted between the recombination hotspot cog and his-3, in which GFP(+) spores arise as a result of recombination in a cross between the two GFP alleles. In a control cross homozygous for spo11 (+), the frequency at which GFP(+) spores arise is comparable to the frequency of His(+) spores and glowing nuclei first appear during prophase, prior to metaphase I, as expected for a product of meiotic recombination. Similarly in spo11 deletion homozygotes, GFP(+) spores arise at high frequency and glowing nuclei are first seen before metaphase, indicating that allelic recombination occurs during meiosis in the absence of spo11. We have therefore shown that spo11 is not essential for either his-3 allelic recombination or crossing over in the vicinity of his-3, and that spo11-independent allelic recombination is meiotic, indicating that there is a spo11-independent mechanism for initiation of recombination in Neurospora.

A crossover hotspot near his-3 in Neurospora crassa is a preferential recombination termination site.

During analysis of 148 unselected Neurospora crassa octads, an above average rate of crossing over was detected within a 360-base region near the 3' end of his-3, suggesting a hotspot for crossing over about 1.8 kb away from the recombination initiation site within cog. Homozygous deletion of the 360-base region increases exchanges in his-3 and on the far side of his-3 from cog, with the heterozygote showing an intermediate increase. We conclude that recombination events initiated at cog terminate within the 360-base sequence more often than in other sections of the cog-his-3 interval and, since some of these terminations will be resolved as crossovers, a cluster of crossovers at this location is the outcome. Removal of this termination site increases the chance that an event will reach his-3, resulting in recombination within the gene, or extend past it to yield a crossover on the other side of his-3. The deleted sequence has substantial predicted secondary structure, including a complex predicted stem-loop, suggesting that DNA secondary structure may be responsible for the termination.

High density analysis of randomly selected Neurospora octads reveals conversion associated with crossovers located between cog and his-3.

We analysed 148 octads from a Neurospora cross maximised for sequence heterology in the his-3 region and detected non-Mendelian segregation at his-3, cot-1 and lys-4 loci. This was in all cases 6:2 or 2:6, with no evidence of post-meiotic segregation (PMS) in these genes. High density snp analysis was used to place crossovers between his-3 and the centromere-distal marker ad-3, and sequencing to refine the location of crossovers between his-3 and the recombination hotspot cog. Crossovers appeared to have a non-random distribution, falling close to his-3 or more than 40 kb distal, and all those in which the location was determined were flanked by sequences showing gene conversion and/or PMS amongst the polymorphisms. This octad study confirms the validity of assumptions made during random spore analyses and suggests that recombination hotspots at cot-1 and lys-4 may, unlike the relatively cold recombination initiator at the am locus, be high frequency recombinators similar to cog.

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.

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.

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.

Structure and function analysis of the calcium-related gene spray in Neurospora crassa.

The spray gene was cloned, and wildtype and mutant alleles were sequenced. Spray(+) has a 3452-bp open reading frame plus seven introns. The spray mutant had a T --> G transversion close to the carboxyl end, creating a stop codon (TGA). The sequence shows no match to genes of known function, but the carboxyl end shows seven transmembrane domains and matches putative membrane proteins of yeast. The most abundant transcript detected was 4.4 kb in size. Repeat-induced point mutagenesis produced the mutant spray phenotype. Electrophysiological analysis showed that ion fluxes in the spray plasma membrane are normal; furthermore, whereas the spray mutant was known to have no organelle-based calcium fluorescence, the cytosol shows a tip-high calcium gradient. The spray mutant is sensitive to calcineurin inhibitors. The results suggest that the SPRAY protein is located in an organellar membrane, regulating the distribution of Ca(2+) via calcineurin.

Recombinational landscape across a 650-kb contig on the right arm of linkage group V in Neurospora crassa.

We have established a 650-kb walk spanning the sp to ure-1 interval on Neurospora crassa linkage group V. Loci covered by the walk are, from proximal to distal, sp; rec-2; ure-2; DNA polymerase delta; am; gul-1; ace-5; and ure-1. We have found extensive DNA polymorphism in this region and used this to examine the recombinational landscape. Crossovers are not evenly distributed over the region covered by the contig.

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.

Analysis of conversion tracts associated with recombination events at the am locus of Neurospora crassa.

In cross B163, heteroallelic am1 am6 and heterozygous for both conventional genetic flanking markers and closer molecular markers, we previously found that the majority on flanker exchanges were remote from events that generated prototrophic recombinants. We report here that natural polymorphisms distinguishing the parents of cross B163 also include sequences within and closely flanking am. Segregation of these markers in B163 prototrophs confirms that the majority of meiotic recombination events at am resulted from gene conversion. Conversion of am6, the distal allele, is more frequent than conversion of am1. Twelve percent of tracts in am6 convertants were discontinuous while 30% of continuous tracts converting am6 extend less than 741 bp.

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 approximately 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 am1 am6. 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 (approximately 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 (approximately 0.35), our observation may have general significance.

The orientation of gene maps by recombination of flanking markers for the am locus of Neurospora crassa.

Fincham (1967), Smyth (1973 b) and Rambosek and Kinsey (1983) have each generated fine-structure maps of the am gene of Neurospora crassa. Each map had a consistent linear order of alleles but the assignment of an orientation with respect to other linkage group-V loci differed. Fincham found the end marked by the am6 allele to be at the distal end of the locus, Smyth found am6 to be at the proximal end while the data of Rambosek and Kinsey did not suggest an orientation. Smyth s orientation has been adopted as the standard, but not unreservedly. We have aligned the genetic and physical maps of the am gene, showing that am6 is at the distal end, supporting Fincham's orientation. However, we suggest that an assumption used to orient fine structure genetic maps is flawed and that the conflicting orientation between these three studies follows from the different choice of flanking markers.

The effect of rec-2 on repeat-induced point-mutation (RIP) and recombination events that excise DNA sequence duplications at the his-3 locus in Neurospora crassa.

In Neurospora crassa, duplicated DNA suffers both extensive repeat-induced point-mutation (RIP) and also excision by recombination events during the dikaryotic phase of the life cycle that precedes karyogamy and meiosis (reviewed by Selker 1990). This paper describes experiments designed to test the effect of rec-2, a gene known to modulate the local level of meiotic recombination at his-3, on RIP and the excision of tandem duplications. Duplications carrying his-3 sequences and a marker, hygr, that confers hygromycin resistance were constructed by targeted transformation. RIP and excisive recombination were assessed from the progeny of crosses heterozygous for a duplication and having different combinations of rec-2 alleles. In the presence of rec-2+, excision of hygr was reduced to about half of that in crosses homozygous for rec-2. In contrast, rec-2+ had little influence on the frequency of duplications that escaped RIP. Thus, in addition to reducing recombination between his-3 alleles during meiosis, rec-2+ also influences recombination events that lead to the excision of duplications carrying his-3. However, RIP may be independent.

The initiation site for recombination cog is at the 3' end of the his-3 gene in Neurospora crassa.

Recombination at his-3 in Neurospora crassa is thought to be initiated through a site designated cog which lies in the his-3 to ad-3 interval of linkage group I. Fragments of the his-3 gene were used to transform various his-3 mutant alleles to prototrophy in order to link the genetic map to the nucleotide sequence. It was established that cog is at the 3' end of his-3 and is therefore not the his-3 promoter. This suggests that cog may be dissimilar to a number of yeast recombinators which are associated with promoters of transcription.