THE HINGE DELETION ALLELIC VARIANT OF PORCINE IGA RESULTS FROM A MUTATION AT THE SPLICE ACCEPTOR SITE IN THE FIRST C-ALPHA INTRON

Recently published genomic and cDNA sequences for porcine IgA suggeste d that the splice acceptor site in the C alpha 1-C alpha 2 intron was an AA rather than an AG dinucleotide. This possibility was tested in a n in vitro HeLa cell splicing system using an RNA substrate correspond ing to the genomic DNA with the putative AA splice site. Data indicate d that splicing occurred at a cryptic AG site 12 nucleotides into the C alpha 2 domain rather than at the AA site. The possibility that swin e B cells could use either site was tested by preparing the cDNAs from 13 different samples representing nine animals and amplifying the seg ment from the first C alpha 1 nucleotide to nucleotide 532 in C alpha 2 (genomic DNA numbering system). Analysis on a 6% polyacrylamide sequ encing gel revealed two polynucleotide products in most samples that d iffered by the expected 12 nucleotides; suggesting that swine could us e both splice sites. Sequence analysis confirmed that the shorter form was spliced at the downstream site and the larger form at the apparen t upstream AA site. However, when the genomic DNA from an animal expre ssing only the longer polynucleotide was cloned and sequenced, the ups tream splice acceptor site was AG not AA. Thus the data suggested that porcine IgA occurred in two allelic forms, designated IgA(a) and IgA( b), which differ by an apparent G to A mutation in the last nucleotide of intron 1 resulting in a short-hinged (two amino acids, IgA(b)) var iant, in which the downstream cryptic splice site is used, as well as a ''normal-hinged'' (six amino acids, IgA(a)) variant. Evidence that I gA(a) and IgA(b) are allelic was confirmed by genotypic analyses of pr ogeny from matings of IgA(a)/IgA(b) heterozygotes. Evidence that both transcripts are functional was confirmed by showing that serum IgA lev els were similar in animals homozygous for each variant.