Celia A. May

Intense and highly localized gene conversion activity in human meiotic crossover hot spots

Meiotic gene conversion has an important role in allele diversification and in the homogenization of gene and other repeat DNA sequence families, sometimes with pathological consequences. But little is known about the dynamics of gene conversion in humans and its relationship to meiotic crossover. We therefore developed screening and selection methods to characterize sperm conversions in two meiotic crossover hot spots in the major histocompatibility complex (MHC) and one in the sex chromosomal pseudoautosomal pairing region PAR1 (ref. 9). All three hot spots are active in gene conversion and crossover. Conversion tracts are short and define a steep bidirectional gradient centered at the peak of crossover activity, consistent with crossovers and conversions being produced by the same recombination-initiating events. These initiations seem to be spread over a narrow zone, rather than occurring at a single site, and seem preferentially to yield conversions rather than crossovers. Crossover breakpoints are more broadly diffused than conversion breakpoints, suggesting either differences between conversion and crossover processing after initiation or the existence of a quality control checkpoint at which short interactions between homologous chromosomes are preferentially aborted as conversions.

PRDM9 variation strongly influences recombination hot-spot activity and meiotic instability in humans.

PRDM9 has recently been identified as a likely trans regulator of meiotic recombination hot spots in humans and mice. PRDM9 contains a zinc finger array that, in humans, can recognize a short sequence motif associated with hot spots, with binding to this motif possibly triggering hot-spot activity via chromatin remodeling. We now report that human genetic variation at the PRDM9 locus has a strong effect on sperm hot-spot activity, even at hot spots lacking the sequence motif. Subtle changes within the zinc finger array can create hot-spot nonactivating or enhancing variants and can even trigger the appearance of a new hot spot, suggesting that PRDM9 is a major global regulator of hot spots in humans. Variation at the PRDM9 locus also influences aspects of genome instability—specifically, a megabase-scale rearrangement underlying two genomic disorders as well as minisatellite instability—implicating PRDM9 as a risk factor for some pathological genome rearrangements.

Human minisatellites, repeat DNA instability and meiotic recombination.

Minisatellites include some of the most variable loci in the human genome and are superb for dissecting processes of tandem repeat DNA instability. Single DNA molecule analysis has revealed different mutation processes operating in the soma and germline. Low‐level somatic instability results in simple intra‐allelic rearrangements. In contrast, high frequency germline instability involves complex gene conversions and is therefore recombinational in nature, almost certainly occurring at meiosis. To determine whether true meiotic crossovers occur at human minisatellites, we have used polymorphisms near the repeat array to recover recombinant DNA molecules directly from sperm DNA. Analysis of minisatellite MS32 has revealed an intense and highly localised meiotic crossover hotspot centred upstream of the array, the first example of a human hotspot defined at the molecular level. This hotspot extends into the beginning of the repeat array, resulting in unequal and equal crossovers. Array crossovers occur much less frequently than array conversions but appear to arise by a common process, most likely by alternative processing of a recombination initiation complex. The location of MS32 at the boundary of a recombination hotspot suggests that this locus has evolved as a by‐product of localised meiotic recombination activity, and that minisatellites might in general mark recombinationally proficient hotspots or hot domains in the genome. Finally, sperm crossover analysis makes it possible to explore the molecular rules that govern human meiotic recombination, and to detect phenomena such as meiotic drive that could provide a possible connection between recombination and DNA sequence diversity itself.

Minisatellite mutation frequency in human sperm following radiotherapy

Screening pedigrees for inherited minisatellite length changes provides an efficient means of monitoring repeat DNA instability but has given rise to apparently contradictory results regarding the effects of radiation on the human germline. To explore this further in individuals with known radiation doses and to potentially gain information on the timing of mutation induction, we have used an extremely sensitive single molecule approach to quantify the frequencies of mutation at the hypervariable minisatellites B6.7 and CEB1 in the sperm of three seminoma patients following hemipelvic radiotherapy. Scattered radiation doses to the testicles were monitored and pre-treatment sperm DNA was compared with sperm derived from irradiated pre-meiotic, meiotic and post-meiotic cells. We show no evidence for mutation induction in any of the patients and discuss this finding in the context of previous population studies using minisatellites as reporter systems, one of which provided evidence for radiation-induced germline mutation.

Analysis of meiotic recombination products from human sperm.

Traditional methods for surveying meiotic recombination in humans are limited to pedigree and linkage disequilibrium analyses. We have developed assays that allow the direct detection of crossover and gene conversion molecules in batches of sperm DNA. To date, we have characterized 26 recombination hotspots by allele-specific PCR and selectively amplified recombinant DNA molecules from these regions. These analyses have revealed that meiotic crossover hotspots in humans are highly localized and flanked by DNA segments where recombination is suppressed. The centers of crossover hotspots are also active in noncrossover recombination, displaying short conversion tracts.

Transmission Distortion Affecting Human Noncrossover but Not Crossover Recombination: A Hidden Source of Meiotic Drive

Meiotic recombination ensures the correct segregation of homologous chromosomes during gamete formation and contributes to DNA diversity through both large-scale reciprocal crossovers and very localised gene conversion events, also known as noncrossovers. Considerable progress has been made in understanding factors such as PRDM9 and SNP variants that influence the initiation of recombination at human hotspots but very little is known about factors acting downstream. To address this, we simultaneously analysed both types of recombinant molecule in sperm DNA at six highly active hotspots, and looked for disparity in the transmission of allelic variants indicative of any cis-acting influences. At two of the hotspots we identified a novel form of biased transmission that was exclusive to the noncrossover class of recombinant, and which presumably arises through differences between crossovers and noncrossovers in heteroduplex formation and biased mismatch repair. This form of biased gene conversion is not predicted to influence hotspot activity as previously noted for SNPs that affect recombination initiation, but does constitute a powerful and previously undetected source of recombination-driven meiotic drive that by extrapolation may affect thousands of recombination hotspots throughout the human genome. Intriguingly, at both of the hotspots described here, this drive favours strong (G/C) over weak (A/T) base pairs as might be predicted from the well-established correlations between high GC content and recombination activity in mammalian genomes.

A major recombination hotspot in the XqYq pseudoautosomal region gives new insight into processing of human gene conversion events

Recombination plays a fundamental role in meiosis. Non-exchange gene conversion (non-crossover, NCO) may facilitate homologue pairing, while reciprocal crossover (CO) physically connects homologues so they orientate appropriately on the meiotic spindle. In males, X-Y homologous pairing and exchange occurs within the two pseudoautosomal regions (PARs) together comprising <5% of the human sex chromosomes. Successful meiosis depends on an obligatory CO within PAR1, while the nature and role of exchange within PAR2 is unclear. Here, we describe the identification and characterization of a typical ~1 kb wide recombination hotspot within PAR2. We find that both COs and NCOs are strongly modulated in trans by the presumed chromatin remodelling protein PRDM9, and in cis by a single nucleotide polymorphism (SNP) located at the hotspot centre that appears to influence recombination initiation and which causes biased gene conversion in SNP heterozygotes. This, the largest survey to date of human NCOs reveals for the first time substantial inter-individual variation in the NCO:CO ratio. Although the extent of biased transmission at the central marker in COs is similar across men, it is highly variable among NCO recombinants. This suggests that cis-effects are mediated not only through recombination initiation frequencies varying between haplotypes but also through subsequent processing, with the potential to significantly intensify meiotic drive of hotspot-suppressing alleles. The NCO:CO ratio and extent of transmission distortion among NCOs appear to be inter-related, suggesting the existence of two NCO pathways in humans.

Charomid cloning vectors meet the pedipalpal chelae: single‐locus minisatellite DNA probes for paternity assignment in the harlequin beetle‐riding pseudoscorpion

We describe the first application of the charomid‐cloning method for developing single‐locus minisatellite DNA probes in a terrestrial arthropod. From a genomic library of the neotropical pseudoscorpion, Cordylochernes scorpioides, we have isolated two probes with heterozygosities exceeding 95%. These probes yielded single‐locus patterns after only low stringency washing and in the absence of genomic competitor DNA. Analysis of three pedigrees indicated germline stability and showed no evidence of linkage between the loci. Patterns of allelic transmission generally conformed closely to Mendelian expectations but large offspring numbers did enable detection of one example of significant bias in allele inheritance. Two test cases are presented to illustrate the clarity and power with which these probes can establish paternity: (i) a female mated to three unrelated males, and (ii) a female mated to two of her brothers. In both cases, a single probe could be used to assign the paternity of all offspring.

Polymorphic Sex-Specific Sequences in Birds of Prey

A red kite (Milvus milvus) clone, which cross-hybridizes to a human minisatellite, reveals complex polymorphic patterns in members of the Accipitridae, a family of 217 species of birds of prey, which includes the kites, hawks and eagles. The total absence of hybridization to males implies that the sequence is located on the W chromosome, and stable inheritance from mother to daughter suggests that the patterns evolve as haploid matrilines. This has allowed not only the development of a simple and rapid sexing technique but may also provide a means of monitoring matriarchal success and dispersion. As an example, a survey of 36 continental European red kite nests revealed 24 different haplotypes, whereas only 2 were found among 53 Welsh nests. The results show the effect of a dramatic bottleneck in Wales and may provide evidence of recent introgression from the continent.

Single-locus profiling reveals loss of variation in inbred populations of the red kite (Milvus milvus)

The genetic analysis of inbred populations has been facilitated by the development of Variable Number Tandem Repeat (VNTR) probes. We describe here the first use of a synthetic, concatenated simple sequence probe to detect a single VNTR locus and its use in differentiating populations of the red kite, one of Europe’s most threatened birds of prey. Numbering 11000-13000 pairs, this species is patchily distributed, comprising several populations, some of which have recently suffered major bottlenecks. By using the (CCAT)n probe we have examined variation within and among four of these. The levels of heterozygosity and allelic diversity reflect each population’s history but also reveal substructuring within the indigenous British population in Wales. Here a group of peripheral territories, which are amongst the most successful in the population, exhibit significantly lower heterozygosity than the main population, and appear to receive little genetic input from it.