Alec J. Jeffreys

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.

THE PIGMAP CONSORTIUM LINKAGE MAP OF THE PIG (SUS SCROFA).

A linkage map of the porcine genome has been developed by segregation analysis of 239 genetic markers. Eighty-one of these markers correspond to known genes. Linkage groups have been assigned to all 18 autosomes plus the X Chromosome (Chr). As 69 of the markers on the linkage map have also been mapped physically (by others), there is significant integration of linkage and physical map data. Six informative markers failed to show linkage to these maps. As in other species, the genetic map of the heterogametic sex (male) was significantly shorter (∼16.5 Morgans) than the genetic map of the homogametic sex (female) (∼21.5 Morgans). The sex-averaged genetic map of the pig was estimated to be ∼18 Morgans in length. Mapping information for 61 Type I loci (genes) enhances the contribution of the pig gene map to comparative gene mapping. Because the linkage map incorporates both highly polymorphic Type II loci, predominantly microsatellites, and Type I loci, it will be useful both for large experiments to map quantitative trait loci and for the subsequent isolation of trait genes following a comparative and candidate gene approach.

Characterization of a panel of highly variable minisatellites cloned from human DNA

Five of the most variable loci detected in human DNA by hybridization with DNA fingerprint probes have been cloned and characterized. Each locus consists of a tandem‐repetitive minisatellite, with repeat units ranging in length from 9 to 45 base pairs depending on the locus. All of these cloned minisatellites act as locus‐specific hybridization probes, and detect extremely variable Mendelian loci with heterozygosities ranging from 90 to 99%. These five hypervariable loci, together with a previously‐isolated minisatellite designated pΛg3, are dispersed over four autosomes (chromosomes 1, 5, 7 and 12). Syntenic pairs on chromosomes 1 and 7 show no detectable pair‐wise linkage, and thus these hypervariable loci show no evidence of clustering within the genome and should provide valuable markers for mapping inherited disease. The locus‐specific minisatellites act as very sensitive hybridization probes, and can be pooled to detect several hypervariable loci simultaneously. The applications of these probes in individual identification, paternity testing and analysis of cell chimaerism are discussed, and are illustrated by an analysis of forensic specimens from two victims who had been sexually assaulted and murdered.

Repeat unit sequence variation in minisatellites: A novel source of DNA polymorphism for studying variation and mutation by single molecule analysis

Variation in internal minisatellite structure can be analyzed by mapping variant repeat units within amplified alleles. A system capable of distinguishing greater than 10(70) allelic states at the human hypervariable locus D1S8 has been developed. Population surveys of internal allelic structure indicate that D1S8 alleles evolve rapidly along haploid chromosome lineages. Internal mapping of deletion mutant alleles physically selected from genomic DNA provides further evidence that germline and somatic mutations altering the number of allelic repeat units seldom if ever arise by unequal exchange between alleles. The existence of low level germline mosaicism for new mutants further indicates that many germline mutation events are premeiotic. Physical selection of new mutants also allows minisatellite mutation rates to be estimated directly in human DNA.

DNA sequence variants in the Gγ-, Aγ-, δ- and β-globin genes of man

DNA prepared from 60 unrelated individuals was cleaved with one of eight different restriction endonucleases and the resulting DNA fragments were separated by agarose gel electrophoresis. DNA fragments containing G gamma-, A gamma-, delta- or beta-globin genes were detected by Southern blot hybridization, using as probe either a 32P-labeled cloned DNA copy of rabbit beta-globin messenger RNA or labeled human beta- and G gamma- globin cDNA plasmids. Three types of variant restriction enzyme patterns of globin DNA fragments were detected in otherwise normal individuals. One variant pattern, found in only one person, was caused by an additional restriction endonuclease Pst I cleavage site in the center of the delta- globin gene intervening sequence; the subject was heterozygous for the presence of this cleavage site and was shown to have inherited it from her mother. Another variant pattern resulted from the appearance of an endonuclease Hind III cleavage site in the intervening sequence of the A gamma-globin gene; this variant is polymorphic, with a gene frequency for the presence of the intragenic Hind III site of 0.23. This Hind III cleavage site polymorphism is also found in the G gamma-globin gene intervening sequence and thus the polymorphism itself appears to be duplicated over the pair of gamma-globin loci. These variants can be used to derive an approximate estimate of the total number of different DNA sequence variants in man.

Where the crossovers are: recombination distributions in mammals

Until recently, recombination studies in humans and mice had identified only a few anecdotal examples of crossover hot spots. Recently, the pace of discovery has accelerated. In every genomic segment that has been examined at sufficiently high resolution, recombination events have a punctate recombination distribution: they are clustered within small (1–2-kb) regions that are surrounded by large stretches of recombinationally suppressed DNA. Here, we review progress in understanding the distribution of mammalian recombination events, tie mammalian results together with informative studies in budding yeast and discuss the consequences of these findings for genome diversity and evolution.

Clustering of hypervariable minisatellites in the proterminal regions of human autosomes

Six of the human minisatellites detected by DNA fingerprint probes have been localized by in situ hybridization to human metaphase chromosomes. These hypervariable loci are not dispersed at random in the human genome, but show preferential, though not exclusive, localization to terminal G-bands of human autosomes. Two of the proterminal minisatellites are very closely linked to other variable loci. Sequence analysis of one of these additional minisatellites suggests that the two linked minisatellites arose by independent amplification of different repeat units. The proterminal regions of human autosomes may therefore be rich in minisatellites, analogous to the pseudoautosomal terminal pairing region of human sex chromosomes that is similarly abundant in hypervariable minisatellites.

DNA fingerprinting : approaches and applications

Molecular Genetics of Hypervariable DNA.- Principles and recent advances in human DNA fingerprinting.- Generation of variability at VNTR loci in human DNA.- Human VNTR sequences in porcine HTF-islands.- Oligonucleotide fingerprinting using simple repeat motifs: a convenient, ubiquitously applicable method to detect hypervariability for multiple purposes.- DNA fingerprinting of the human intestinal parasite Giardia intestinalis with hypervariable minisatellite sequences.- Human variable number of tandem repeat probes as a source of polymorphic markers in experimental animals.- DNA Fingerprinting: the utilization of minisatellite probes to detect a somatic mutation in the Proteus syndrome.- Genetic variability of satellite sequence in the dipteran Musca domestica.- Population Genetics and Evolutionary Biology.- Analysis of population genetic structure by DNA fingerprinting.- Population genetics of hypervariable loci: analysis of PCR based VNTR polymorphism within a population.- Population genetic data determined for five different single locus minisatellite probes.- Multilocus and single locus minisatellite analysis in population biological studies.- Helpers-at-the-nest in European Bee-eaters (Merops apiaster): a genetic analysis.- Cloning, characterization and evolution of Indian peafowl Pavo christatus minisatellite loci.- Use of sex-linked minisatellite fragments to investigate genetic differentiation and migration of North American populations of the peregrine falcon (Falco peregrinus).- Economically-important Animals and Plants.- Hypervariable DNA markers and their applications in the chicken.- Cloning of hypervariable minisatellite and simple sequence microsatellite repeats for DNA fingerprinting of important aquacultural species of salmonids and tilapias.- Genetic factors accountable for line-specific DNA fingerprint bands in quail.- Identification of markers associated with quantitative trait loci in chickens by DNA fingerprinting.- Two dimensional DNA-fingerprinting in animals.- Applications of DNA fingerprinting in plant breeding.- Oligonucleotide fingerprinting in plants and fungi.- The isolation and characterisation of plant sequences homologous to human hypervariable minisatellites.- Implementation of DNA Typing.- DNA fingerprinting: a biotechnology in business.- DNA fingerprinting: its applications in forensic case work.- Tracking the violent criminal offender through DNA typing profiles - a national database system concept.- The quality control of cell banks using DNA fingerprinting.- Detection of amplified VNTR alleles by direct chemiluminescence: application to the genetic identification of biological samples in forensic cases.- Genetic typing using automated electrophoresis and fluorescence detection.

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.

Uniparental paternal disomy in Angelman's syndrome

Angelmans syndrome and Prader-Willi syndrome are both causes of mental retardation with recognisable, but quite different, clinical phenotypes. Both are associated with deletions of chromosome 15q11-13, of maternal origin in Angelmans and paternal in Prader-Willi. Prader-Willi can arise by inheritance of two chromosomes 15 from the mother and none from the father (uniparental maternal disomy). In 2 patients with Angelmans syndrome we found evidence of uniparental paternal disomy. The phenotypic effects of maternal and paternal disomy of chromosome 15 are very different and inheritance of two normal 15s from one parent does not lead to normal development--strong evidence in man for genomic imprinting, in which the same gene has different effects dependent upon its parental origin.