Guinea‐pig casein A cDNA

Abstract

The nucleotide sequence (1036 bases) of guinea-pig casein A mRNA has been determined. Two cDNA recombinant plasmids contained a total of 993 base pairs, including part of the 5' noncoding region, and the complete coding and 3' noncoding region. The remaining 5' noncoding sequence was obtained by primer extension.The deduced 223-amino-acid-coding sequence of guinea-pig pre-casein A exhibited 30 % homology with bovine uS2 casein, the most striking similarities being in the locations of potential phosphorylation sites.During lactation, the expression of the milk protein genes of the epithelial cells of the mammary gland is modulated by a combination of peptide and steroid hormones [I]. An attractive feature of this system is that differential regulation of the a-lactalbumin and casein genes is apparent during the onset of lactation [2 -41, making it a particularly suitable system for studying the hormonal control of a family of genes.In the course of our studies using the lactating guinea-pig mammary gland as a model system, we have cloned and characterised cDNA copies of the three major guinea-pig casein mRNAs and cr-lactalbumin mRNA [5]. We have recently reported the sequence of the latter [6]. We now report the complete nucleotide sequence of one of the casein cDNAs, that encoding guinea-pig casein A (using our previous nomenclature based on the relative mobility of the protein on polyacrylamide gels [7]). This was a necessary prerequisite for our current studies on the genomic organisation, expression and hormonal regulation of the milk protein genes. Examination of the amino acid sequence of this casein, deduced from the nucleotide sequence, reveals significant homology with bovine a;, casein. This is most apparent in terms of the locations of potential phosphorylation sites and sequences of the signal peptide, implicating a conservation of these functionally important domains. MATERIALS AND METHODS Muter iulsRestriction endonucleases BstNI, Fnu4HI and RsaI were purchased from Bio-Labs through CP Labs Ltd (Bishop's Stortford, Herts, UK); all other restriction enzymes, T4 DNA polymerase and T4 polynucleotide kinase were from Bethesda Research Labs (Cambridge UK). AMV reverse transcriptase (batch G-91180) was provided by. Dr. J. W. Beard (Life Sciences Inc., St Petersburg, FL, USA). Recombinant plasmidsThe construction and characterisation of pgpN33, a recombinant plasmid containing a cDNA copy of guinea-pig casein A mRNA inserted into the EcoRI site of pAT153 using homopolymeric dA . dT tails, has been described previously [5]. A second recombinant pgpKG8, showing extensive overlap with the inserted sequence of pgpN33 but possessing an additional sequence at one end, was isolated from a similar library obtained by insertion of lactating guinea-pig mammary gland double-stranded cDNA, prepared as described by Land et al.[S], into the PstI site of pAT153 using dG . dC homopolymer tails. Chemical DNA sequencing of recombinant plasmidsProcedures for the preparation of restriction fragments, 5' and 3' end labelling, ...

Physiology and Biochemistry of Lactation

“…With the aid of these techniques attention has also widened to include caseins and whey proteins of the cow Brew, 1972;Ribadeau-Dumas et al, 1972;Grosclaude et al, 1973;Brignon et al, 1977), sheep Oolles et al, 1974;Richardson and Mercier, 1979), goat (MacGillivray et al, 1979;Preaux et al, 1979), deer (McDougall and Stewart, 1976), horse (Visser et al, 1982), rabbit (Hopp and Woods, 1979;Dayal et al, 1982), guinea pig (Hall et al, 1984a(Hall et al, , 1984b, rat (Brown et al, 1977;Hirose et al, 1981;Blackburn et al, 1982;Qasba and Safaya, 1984), mouse (Kinkade et al, 1976;Hennighausen et al, 1982a,b), human (Findlay and Brew, 1972;Metz-Boutigue et al, 1984) and other species. References to j3-lactoglobulin biosynthesis in various species may be found in Godovac-Zimmerman and Braunitzer (1987).…”

Evolution of the casein multigene family: conserved sequences in the 5′ flanking and exon regions

“…As an initial attempt to elucidate the mechanism of coordinate regulation of the casein genes, the structure and organization of the casein mRNAs and the corresponding genomic sequences encoding these mRNAs have been studied. E0 I RL Press Umited, Oxford, England. Extensive nucleotide sequence analysis of a number of casein eRNAs from rat (5,6), mouse (7), guiniea pig (8,9), and cow (10), has revealed a considerable divergence among the casein sequences, except for three regions which are highly conserved. These include the 5' non-coding, signal peptide and casein kinase phosphorylation sequences (6).…”

Localization of two interchain disulfide bridges in dimers of bovine αs2‐casein

“…In human, the protein is not present at all (Yamauchi et al, 1981) and in mouse and rat the part of the polypeptide chain containing the cysteines is deleted (Henninghausen et al, 1982;Hobbs and Rosen, 1982). In sheep and guinea pig the two cysteines are present in the corresponding positions but it is not known whether aS2-casein in these species forms dimers (Boisnard and Petrissant, 1985;Hall et al, 1984). Hence, an important functional role for the cysteine residues is not indicated by comparative sequence studies.…”