Cloning of cDNA Sequence Coding for Bovine α_s1-Casein

Abstract

Double-stranded cDNAs were synthesized using total polyA-containing mRNAs isolated from the mammary gland of a lactating cow as templates. Escherichia coli χ1776 was transformed with the hybrid DNA of bacterial plasmid pBR322 and cDNA constructed by the dG-dC tailing method. Plasmids containing cDNA inserts at the Pst I restri tion sites were selected by resistance to tetracycline and by sensitivity to ampicillin. Those containing milk protein cDNA sequences were selected by colony hybridization using radioactive (³²P) single-stranded cDNAs as probes. Recombinant plasmids were isolated from those clones showing intense signals on the auto-radiograph, and grouped into roughly six types by restriction endonuclease cleavage patterns of the cDNA sequences. Partial nucleotide sequences of two types of clones were determined by the method of Maxam and Gilbert. The amino acid sequence predicted from the nucleotide sequence of one clone coincided with that of α_s1-casein, a major component of bovine milk protein. The cloned cDNA sequence is 900 nucleotides in length, but may lack a DNA sequence coding the N-terminal region as well as the signal sequence of α_s1-casein.