1. The bovine ti-casein gene has been isolated as a series of overlapping I clones and shown to consist of five exons distributed over a total length of approximately 13 kb. Most of the mature protein-coding sequence is contained in a single large exon.2. Approximately 65% of the gene has been sequenced together with portions of the 5'-and 3'-flanking sequences. The immediate 5'-flanking sequence contains several motifs which are characteristic of upstream regions including a TATA box, a CAAT box, a sequence similar to that recognized by transcription factor AP-1 and a purine-rich sequence resembling that found upstream in all other lactoprotein genes. Other possible regulatory sequences are found upstream of exon 4.3. The organization of the K-casein gene, together with its upstream sequence, confirms previous conclusions that it is unrelated to the calcium-sensitive-casein gene familiy to which it is linked. Evidence is presented which supports a previous suggestion that ic-casein and the fibrinogens are evolutionarily related.4. Intron sequences contain several examples of the A family of the artiodactyl Alu-like repeated sequences, together with a single example of a C-family sequence. The remainders of the introns of the rc-casein gene, compared with the repeat elements and exons, are A + T-rich.5. Among the I clones isolated, representatives were found of the A and B genetic variants which can be distinguished by restriction-enzyme analysis. Several other examples of polymorphisms in the non-coding region were found.The caseins are the predominant proteins in the milk of most species. As well as being the main source of amino acids for the suckling infant, the caseins serve to raise the calcium and phosphate concentrations in milk to levels well in excess of the solubility product of calcium phosphate by forming loosely ordered aggregates, termed micelles, which sequester calcium phosphate. Bone formation in the young animal thus depends on the ability of casein to transport appropriate quantities of calcium phosphate in milk.In bovine milk more than 20 individual components can be resolved when whole casein is analyzed electrophoretically under dissociating conditions [I]. These result from posttranslational modification and genetic variation of four primary translation products which correspond to asl-, clS2-, pand k--caseins. The xsl-, xs2-and p-caseins are insoluble in the presence of calcium ion at the concentrations at which it occurs in milk and these caseins are referred to as the calciumsensitive caseins. In contrast, K-casein is insensitive to the presence of calcium and is referred to as the micelle stabilizer because it is essential for the formation of stable casein micelles [2]. Following ingestion, the caseins are immobilized in the stomach as a result of clot formation. This occurs when chymosin (rennin) or pepsin specifically cleaves a single PheMet bond in K-casein to form insoluble para-K-casein (105 residues) and a soluble macropeptide containing the C-termind 64 amino acid residues of ...

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The rat alpha- and bovine alpha s1-casein genes have been isolated and their 5' sequences determined. The rat alpha-, beta-, gamma- and bovine alpha s1-casein genes contain similar 5' exon arrangements in which the 5' noncoding, signal peptide and casein kinase phosphorylation sequences are each encoded by separate exons. These findings support the hypothesis that during evolution, the family of casein genes arose by a process involving exon recruitment followed by intragenic and intergenic duplication of a primordial gene. Several highly conserved regions in the first 200 base pairs of the 5' flanking DNA have been identified. Additional sequence homology extending up to 550 base pairs upstream of the CAP site has been found between the rat alpha- and bovine alpha s1-casein sequences. Unexpectedly, the 5' flanking promoter regions are conserved to a greater extent than both the entire mature coding and intron regions of these genes. These conserved 5' flanking sequences may contain potential cis regulatory elements which are responsible for the coordinate expression of the functionally-related casein genes during mammary gland development.

SUMMARYThe effect of varying the casein composition of artificial micelle milk on rennet coagulation time and syneresis was examined in order to determine whether either of these processes is dependent on the concentration of particular casein components. It was found that the levels of κ-and β-caseins had a significant effect on coagulation, whereas syneresis was only affected by the level of β-casein. Partial dephosphorylation of preformed micelles or the incorporation of dephosphorylated or partly dephosphorylated β-casein into artificial micelle milk was found to have an adverse effect on both coagulation and syneresis. It was concluded that the phosphate groups of casein, particularly those of β-casein, are directly involved in the micelle-micelle interactions which occur during coagulation and syneresis.

Introduction Methodology Casein cDNA sequences The

The effect of preheating skim milk and artificial micelle milk on curd syneresis was studied. The inhibition of syneresis caused by heat was dependent on the presence of /?-lactoglobulin (/?-lg) and to a lesser extent a-lactalbumin. The degree of inhibition increased with increasing amounts of added /?-lg and preheating temperature. This agrees with the hypothesis that the detrimental effect of preheating on syneresis is due to complex formation between /?-lg and /c-casein. This complex appeared to be mediated via thiol-disulphide exchange and its formation appeared to interfere with the micelle-micelle interactions responsible for syneresis.

Beta-lactoglobulin (P-lg) is the major whey protein found in bovine milk and its protein structure is known (1). A role for ,6-lg in the transport of retinol to the infant has been suggested (2). Here we report the full length cDNA sequence of the ,-lg A variant. Although isolation of cDNAs for this protein (3) and partial sequences for this cDNA have recently been reported (4-5), they differ significantly from the sequence reported here. The sequence of Jamieson et al.

1. A preparation which contains protein kinase activity capable of rephosphorylating dephosphorylated @,I and P-caseins has been prepared from lactating bovine mammary gland. This activity is localised in the Golgi fraction and in this respect is similar to that obtained previously from rat tissue. It differs from the rat preparation in being unable to rephosphorylate dephosphorylated x-casein.2. Progressive dephosphorylation of asl and P-caseins increases the rate of their rephosphorylation in the presence both of Ca2+ and of Mg2+. For 8-casein significant non-specific phosphorylation occurs in the presence of Mg2+ while in the presence of Ca2+ the level of phosphorylation is very low.3. Comparison of the products of the action of this kinase on partially dephosphorylated aSl and B-caseins indicates that seryl residues grouped with pre-existing phosphoseryl residues are phosphorylated preferentially in the presence of Mg2 +, while the phosphorylation of isolated single seryl residues is promoted by Ca2+.4. Digests of aSl-casein, prepared by treatment with cyanogen bromide, have been fractionated and the three major peptides, separated. After dephosphorylation two of these, which between them contain all of the phosphate of aS1-casein, are rephosphorylated at sites occupied in the native protein by phosphoseryl residues, i. e., at positions 46, 48, 75 and 115 of the a,l-casein molecule. Evidence is presented which shows that this specificity of action also operates when intact aSl-casein is used as phosphate acceptor. It is concluded that the enzyme responsible for specific phosphorylation acts through recognition of an amino acid sequence and does not require the provision of a specific structural conformation in the protein substrate.Determination of the sequences of amino acid residues occurring in the three major components of bovine casein, @,I, P and x-caseins, has defined accurately the locations of the phosphate residues in these proteins [1,2]. Altogether some 14 seryl residues are phosphorylated and a prominent feature of the primary structures of the a ,~ and p-caseins is a region of homology which includes a cluster of four phosphoseryl residues. In aSl-casein this extends over residues 63 -70 thus : Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser-(P)-Glu-Glu, a sequence which is repeated in residues 14-21 of /?-casein with the substitution of leucine for isoleucine. In addition to these a,l-casein contains four phosphoseryl residues located at positions 46, 48, 75 and 11 5 and P-casein contains one located at position 35. %-Casein contains only one phosphoseryl residue. It has been shown by Turkington and Topper [3] that in mouse mammary glands at least a significant proportion of the phosphate of casein is incorporated after polypeptide synthesis rather than by incorporation of phosphorylated amino acids, and more recently Bingham and coworkers [4,5] have detected in the Golgi apparatus of lactating rat mammary gland a protein kinase for which dephosphorylated bovine caseins were found to act as better phosphat...