RESUMO
Previous studies on the fourth inversion of the t complex, In17(4), suggest that loci near the center of this inversion have been subjected to segmental recombination during the past 1-2 million years. We have used a combination of PCR-based restriction site (PBR) analysis and DNA sequencing to perform a high-resolution analysis of a 2-million base pair (Mbp) segment in the middle of In17(4). We examined 21 restriction sites that are polymorphic between t haplotypes and their wild-type homologs, over nine distinct loci. In addition, we examined several other polymorphic sites through DNA sequence analysis of two of these nine loci. We analyzed several haplotypes in this way, including the "complete" t haplotypes tw2, t0, tw32, tw71, and tw75. We show that only tw32 is a true "complete" t haplotype; the remaining four t haplotypes have segments of wild-type DNA ranging from less than 100 bp to 2 Mbp. The sizes of these wild-type DNA segments are consistent with their being generated by gene-conversion events. The 2-Mbp segment is located in a region that may contain the t-complex distorter gene Tcd2. One of the nine loci examined in this study is Fgd2, a gene that has been proposed to encode Tcd2. Sequencing and PBR data show that at least a portion of the Fgd2 gene has been converted to the wild-type within tw71 and tw75mice.
Assuntos
Haplótipos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Recombinação Genética , Animais , Sequência de Bases , Cromossomos de Mamíferos , Genoma , Fatores de Troca do Nucleotídeo Guanina/genética , Masculino , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Região do Complexo-t do GenomaRESUMO
Microsatellite markers are useful tools for understanding the evolutionary history of discrete segments of the mammalian genome. We used the microsatellite marker D17Mit23 to study the portion of the mouse genome known as the t complex, a naturally occurring variant of Chromosome 17. We identified an allelic variant of D17Mit23, which is shared by two forms of the t complex, the t haplotypes t(w2) and t(Lub2). Polymerase chain reaction (PCR) analysis of DNA samples from mice that were heterozygous for either t haplotype resulted in gel patterns with prominent bands of higher molecular weight in addition to the bona-fide D17Mit23 alleles. The appearance of these higher molecular weight bands, although consistent with heteroduplex formation, was not diminished through the use of reconditioning PCR. We used a modified form of asymmetric PCR, called "unilateral PCR," to show that the higher molecular weight bands are heteroduplexes and to identify their constituent strands. Certain microsatellite motifs may be especially prone to the production of prominent heteroduplex products, and this may lead to the erroneous genotyping of DNA samples.
Assuntos
Repetições de Microssatélites/genética , Ácidos Nucleicos Heteroduplexes/genética , Reação em Cadeia da Polimerase/métodos , Alelos , Animais , DNA/genética , Feminino , Haplótipos/genética , Masculino , Camundongos , Dados de Sequência Molecular , Peso MolecularRESUMO
To examine genetic exchange between t haplotypes and their wild-type homologs, four previously identified mouse Chromosome (Chr) 17 variants termed mosaic haplotypes were analyzed in detail. Three of these haplotypes-one from a Mus musculus population in Bulgaria, one from a Mus domesticus population in Chile, and one from a M. domesticus population in Germany-display properties indicative of the t complex. All four haplotypes are exceptional because they are characterized by the presence of a few wild-type DNA markers in the distal inversion [In(17)4] of a t haplotype chromosome: thus, they are classified as mosaic t haplotypes. The mosaic pattern for each haplotype is distinct, however. We compared the mosaic haplotypes with each other, and with several well-characterized laboratory t haplotypes, by analyzing several DNA markers in the In(17)4 region of the t complex, where all of the mosaicism occurs. We used a combination of high-resolution restriction mapping, DNA sequencing, and analysis of new DNA markers to classify the haplotypes. This analysis shows that segmental exchange, either by gene conversion or double crossing-over, has occurred at molecular markers in the vicinity of a gene, Dnahc8, that is a candidate for the t complex distorter locus Tcd2. While it is unclear whether segmental exchanges have included the Tcd2 gene, it is apparent that several independent recombination events have occurred in In(17)4 during the recent evolution of t haplotypes.
