David L. Neil

Complex gene conversion events in germline mutation at human minisatellites

Mutation at the human minisatellites MS32, MS205 and MS31A has been investigated by characterizing mutant alleles in pedigrees and in the case of MS32 by direct analysis of mutant molecules in single sperm. Most mutations at all three loci are polar, involving the preferential gain of a few repeat units at one end of the tandem repeat array. Incoming repeats can be derived from the same allele or the homologous chromosome, though they are frequently rearranged during mutation. Lack of exchange of flanking markers suggests the involvement of complex conversion–like events in the generation of mutant alleles. At MS32, high frequency mutation processes in sperm appear to be largely germline specific and to occur at a constant rate irrespective of allele size. Together with mutational polarity, this implies that germline instability is controlled by elements outside the tandem repeat array.

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.

Minisatellite variant repeat mapping: Application to DNA typing and mutation analysis

Most DNA typing systems assay allele length variation at tandemly repeated loci such as minisatellites and microsatellites. Allele length measurements are approximate, which impedes the use of such loci in forensic analysis and in studies of allelic variability at hypervariable loci. We now review progress in the development of alternative DNA typing systems based on allelic variation in the interspersion patterns of variant repeat units along minisatellite alleles. Minisatellite variant repeat mapping by PCR (MVR-PCR) not only provides a powerful new digital approach to DNA typing, but also for the first time allows investigation of the true level of allelic variability at minisatellite loci and of the mutational mechanisms that generate ultravariability.

Minisatellite mutation and recombination

A prominent feature of the human genome, and many other higher eukaryotic genomes, is the abundance of repeated sequences. These multi-copy sequences may be dispersed around the genome, like the Alu and L1 elements (Schmid and Jelinek,1982; Singer and Skowronski,1985), or, by contrast, found in tandemly repeated arrays. These tandem arrays fall into a wide variety of size classes: the major alphoid satellite sequences, for example (Waye and Willard,1986), which exist in chromosome-specific subclasses, between them account for about 5% of the human genome. The blocks of alphoid satellite are composed of tandem arrays up to 5Mb in length (Willard,1991), and appear to correspond to the functional part of human centromeres (Willard,1990). At the other end of the scale, “microsatellites”, short arrays (generally up to 60bp) of dinucleotide repeats, are extremely abundant, and widely dispersed in the genome (Weber and May,1989; Litt and Luty,1989; Weber,1990). Our work, and this review, concern tandem arrays of intermediate size, the “minisatellite” loci (Jeffreys et al.,1985a, Jeffreys et al.,1985b). These arrays have a total length usually in the range 0.5-30kb, composed of short repeated units. The repeat units at the loci we have studied in detail range between 8 and 90bp in length.