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, ...

The entire guinea-pig alpha-lactalbumin gene was isolated from a genomic DNA library constructed in the bacteriophage lambda L47. The complete nucleotide sequence of the gene and its immediate 5' and 3' flanking sequences were determined and compared with those of the human and rat alpha-lactalbumin genes. This demonstrates that the size, organization and sequence of the exons is highly conserved between species, and reveals the presence of the highly conserved potential regulatory 'milk box' consensus sequence [RGAAGRAAA(N)TGGACAGAAATCAA(CG)TTTCTA] between positions -140 and -110 relative to the transcriptional start point. A guinea-pig LINE repeat sequence was also present in the 5' flanking region between positions -520 and -1195. Transfection of the alpha-lactalbumin gene cloned in a bovine papilloma virus vector into the mouse C127 and human MCF-7 mammary tumour cell-lines gave rise to stable but seemingly constitutive expression of alpha-lactalbumin. Expression was from the correct transcriptional start point, resulting in the accumulation of correctly processed mRNA and the secretion of alpha-lactalbumin into the culture medium.

Nucleotide sequence analysis of cloned guinea-pig casein B cDNA sequences has identified two casein B variants related to the bovine and rat alpha s1 caseins. Amino acid homology was largely confined to the known bovine or predicted rat phosphorylation sites and within the 'signal' precursor sequence. Comparison of the deduced nucleotide sequence of the guinea-pig and rat alpha s1 casein mRNA species showed greater sequence conservation in the non-coding than in the coding regions, suggesting a functional and possibly regulatory role for the non-coding regions of casein mRNA. The results provide insight into the evolution of the casein genes, and raise questions as to the role of conserved nucleotide sequences within the non-coding regions of mRNA species.