Plant & Animal Genome VI Conference

P11

SEARCHING AND IDENTIFICATION OF GENOMIC MUTATIONS IN AND AROUND THE AMINOPEPTIDASE N GENE REVEALS HIGHLY VARIABLE NON-CODING REGIONS

1. Department of Animal Science, 225 Kildee Hall, Iowa State University, Ames, Iowa 50011
2. Dept. of Animal Science, 225C Kildee Hall, Iowa State University, Ames, IA 50011

Aminopeptidase N (AMPEPN) has been identified as the porcine receptor for Transmissible Gastroenteritis Virus (TGEV) which causes diarrhea with high mortality in young pigs. We have conducted initial research to find genetic differences associated with the AMPEPN gene in swine. We first identified 20 putative exons based on porcine AMPEPN cDNA sequence and used polymerase chain reaction (PCR) to specifically amplify regions of interest and to search for sequence polymorphisms in both introns and exons. Twenty-six pairs of primers were designed to cover the entire genomic region including the 5' and 3' untranscribed regions. Confirmed PCR product sequences demonstrated that we are able to correctly amplify 16 entire exons and partially amplify 4 exons including neighboring introns. Based on the confirmed PCR product sequences, primers were selected to specifically amplify individual exons/introns and to compare sequence differences among Meishan, Minzhu, Duroc, Hampshire, Landrace and Yorkshire pigs. Preliminary data show that 17 point mutations have been identified. Among these 17 mutations, 8 mutations has been verified with PCR-RFLP, 5 with rare restriction enzymes are yet to be verified and 4 are without restriction sites. Three exonic polymorphisms are silent mutations. We have also identified four sequencing errors in the published cDNA data. The most variable non-coding region of the gene resides in 5' untranscribed region and the most variable coding sequence is in exon 1. The mutations identified provide tools for genomic analysis of the gene. (Partial financial support provided by BRDC is appreciated).