Strong association between polymorphisms in an intronic microsatellite and in the coding sequence of the BoLA-DRB3 gene: implications for microsatellite stability and PCR-based DRB3 typing.

A highly polymorphic microsatellite in the bovine DRB3 gene was characterized by polymerase chain reaction (PCR) analysis and DNA sequencing. A very strong association between expressed DRB3 polymorphism and microsatellite alleles was revealed by PCR analysis of genomic DNA from 116 animals representing three breeds of cattle. The results indicated a low frequency of microsatellite length mutations as the association was consistent over breeds. The DRB3 microsatellite may be utilized in a PCR-based typing method of bovine class II alleles. The microsatellite polymorphism did not distinguish all known DRB3 alleles, but it was shown that this method may be complemented by the use of allele-specific PCR based on the extensive polymorphism in the DRB3 exon 2. The DNA sequences of seven microsatellite alleles, associated with different class II haplotypes, were determined. The DRB3 microsatellite is composed of three repeat motifs, a stretch of at least 10 uninterrupted (TG)n dinucleotides, a long but interrupted stretch of (GA)n dinucleotides, and a few (CAGA)n tetranucleotides. There were pronounced sequence differences between alleles and the results indicated that the evolution of this microsatellite has involved length mutations of the dinucleotide repeats as well as point mutations causing interruptions in the dinucleotide repeats.