Cloning and Sequencing of a Complementary Deoxyribonucleic Acid Coding for a Bovine αs1-Casein A from Mammary Tissue of a Homozygous B Variant Cow

Abstract

A cDNA clone for bovine αs1-casein variant A was isolated from a mammary gland cDNA library using a synthetic degenerate oligonucleotide probe. The largest Pst I insert containing an EcoR I site was sequenced. It contained 1090 base pairs, 47 in the 5′ noncoding region, 603 in the coding region and 440 in the 3′ noncoding region. The nucleotide sequence was compared with three published cDNA sequences for αs1-casein variant B. The most obvious difference was the absence of the 39 bases encoding the 13 amino acids that are present in die B variant but absent from the A variant. In addition, five other single base positions differed within individual codons among the four sequences at the third base for each codon, but this did not change the amino acids encoded. There were, however, a number of differences found in the 3′ noncoding region. The isolated cDNA was subjected to site-directed mutagenesis to replace a Val-Ile dipeptide with Phe-Phe to increase the chymosin sensitivity of the protein. When the milk proteins from mammary gland tissue extracts were typed, the αs1-casein A gene product was not detected.