Â Â S. Dõaz², G. Giovambattista, F. N. Dulout and P. Peral-Garcõa
Â Â Centro de Investigaciones en Genetica Basica y Aplicada (CIGEBA), Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, La Plata, Argentina
Genetic variation in the equine leucocyte antigen-DRB (ELA-DRB) second exonwas investigated using polymerase chain reaction (PCR) ampli®cation, restriction fragment length polymorphism (RFLP) of PCR products (PCR-RFLP) and deoxyribonucleic acid (DNA) sequencing. Eight distinct PCR-RFLP patterns could be identi®ed in the studied Argentine Creole (AC) horses. The number of observed patterns per individual ranged from four to six, thus con®rming the presence of multiple DRBcopies in AC horses. Three PCR-RFLP alleles and three new sequences were identi®ed. The estimated rates of synonymous and non-synonymous substitutions among ELA-DRB exon 2 sequences were higher within the antigen recognition site (ABS) than on the non-ABS. Phylogenetic analysis showed that the nucleotide sequences clustered in two main groups, while some sequences were not included in eithergroup. Finally, the identi®cation of the number of alleles per animal, the phylogenetic and segregation analyses allowed us to explain the number of ELA-DRB loci. However, it was not possible to identify speci®c alleles with speci®c loci. Keywords Argentine Creole horses, ELA-DRB, MHC, PCR-RFLP, polymorphism.
Class II major histocompatibility complex (MHC) products are highlypolymorphic cell-surface molecules involved in the initiation of the immune response to foreign antigens. These molecules are heterodimers constituted by a and b chains encoded by closely linked A and B genes. The polymorphic sites of the class II genes are mainly located in exon 2, which codes for the ®rst extracellular domain or the
Address for correspondence Â Â Â P. Peral-Garcõa, Centro deInvestigaciones en Genetica Basica y Aplicada (CIGEBA), Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, 60 y 118 C.C. 296, C.P. B1900AVW, La Plata, Argentina. E-mail: email@example.com *The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers AF144564, AF170067 andAY026311. ² Â Â Fellow from the Comision Nacional de Investigaciones Cientõ®cas y Â Tecnicas (CONICET). Accepted for publication 26 June 2001
antigen binding site (ABS). Most class II genes show large genetic variation among and within species in both the number of loci and alleles (Bontrop et al. 1999; Lewin et al. 1999). There appear to be multiple DRB loci in horses. Fraser & Bailey (1996)proposed the existence of as many as three DRB copies in domestic horses, Equus caballus. However, Hedrick et al. (1999) proposed the presence of only two DRB genes in E. przewalski. To date, 11 equine leukocyte antigenDRB (ELA-DRB) exon 2 sequences have been reported for E. caballus (Gustavsson & Andersson 1994; Fraser & Bailey 1996) and six additional ones for E. przewalski (Hedrick et al. 1999).Considering the similarity between alleles described to date and the absence of a speci®c typing method for ELA-DRB genes, it has been dif®cult to assign sequences to speci®c loci in domestic horses. In this study we analysed the polymorphism of ELA-DRB genes in Argentine Creole (AC) horses using restriction fragment length polymorphism of polymerase chain reaction products (PCR-RFLP) anddeoxyribonucleic acid (DNA) sequencing methods, in order to identify additional
Ó 2001 International Society for Animal Genetics, Animal Genetics, 32, 257±263
Dõaz, Giovambattista, Dulout, Peral-Garcõa Â Â
ELA-DRB sequences in domestic horses. The number of alleles per animal, phylogenetic and segregation analyses allowed us to address two fundamental questions: the number of ELA-DRB loci...