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A null beta-casein allele (CSN2O) was investigated in Creole and Pyrenean goats producing milk devoid of beta-casein (CSN2). Northern blot analyses of total mammary RNA showed much lower amounts of CSN2 transcripts that were similar in size to the wild-type counterpart. The amount of CSN2O mRNA was roughly 5% of the amount of mRNA obtained at the same age and stage of lactation from CSN2A/A goats. Comparative sequence analyses of full-length CSN2O and CSN2A cDNAs showed that both alleles were of similar size, but allele CSN2O had a one-nucleotide deletion in the 5' end of exon 7, which introduces a premature stop codon. The open reading frame of allele CSN2O encodes a shortened polypeptide of 72 amino acids, compared to 223 amino acids for caprine pre beta-casein A. Comparative analyses of RT-PCR products suggested that alleles CSN2O and CSN2A might also differ in the amount and relative ratio of minor deleted CSN2 transcripts. The lower amount of CSN2O mRNA was associated with the occurrence of the premature stop codon which may mediate a rapid decay of CSN2O mRNA and promote skipping of nucleotide stretches containing premature nonsense triplets.

A 5' truncated caprine (ca) kappa-casein-encoding gene (kappa Cas) was fused to the 3' end of a 3' truncated ca beta Cas. The kappa Cas form comprised the 0.8-kb 3' end of intron 2, the remaining part of the transcription unit containing codons -2 to stop 172, and 0.43 kb of the 3' flanking region. The beta Cas form comprised a 3-kb 5' flanking region and the 5' end of the transcription unit terminating 69 bp downstream from exon 2 which encodes the 15-amino-acid (aa) signal peptide and the first 2 aa of mature beta Cas. The resulting hybrid gene driven by the beta Cas promoter was expressed in all eight lines of transgenic mice investigated, although at different levels. In two lines, the yield of recombinant (re-) kappa Cas was > or = 3 mg/ml of milk. The stage- and mammary tissue-specific expression was similar to that of endogenous beta Cas. The re-kappa Cas differed from its goat milk counterpart by the occurrence of four extra aa at the N-terminal end, indicating that the signal peptidase released the beta Cas signal peptide. According to sedimentation analyses of murine milk containing > or = 3 mg re-kappa Cas/ml, the latter essentially occurred in micelles. Preliminary comparative assays of the behavior of ca alpha s1Cas-kappa Cas and alpha s1Cas-re-kappa Cas mixtures upon incremental addition of Ca2+ showed that re-kappa Cas had the capacity to protect alpha s1Cas against Ca(2+)-induced precipitation in forming stable micelles.(ABSTRACT TRUNCATED AT 250 WORDS)

The 9149-bp transcription unit encoding ovine beta-casein (Cas) and 4636 bp of 5' flanking region were completely sequenced. The gene is composed of nine exons and its overall organization is similar to that of its counterparts from other species. Intron 4, the largest, shares three similar stretches (sizes ranging from 0.1 to 0.3 kb) with the region upstream from the transcription unit. These common sequences are part of short interspersed nuclear elements (SINE) specific to Bovidae (Bov). Intron 4 contains two 274-bp Bov-A2 SINE in opposite orientation, as well as a full-length 569-bp Bov-B SINE. This latter SINE, also present in caprine intron 4, is missing in cattle. This suggests that the amplification of Bov-SINE has continued after the divergence of cattle from sheep and goats, assuming that the presently known sequences are representative of these species.

Bovine alpha s2-casein D (CasD) differs from the common type A by the deletion of a stretch of 9 amino acids (aa) starting at a position not precisely known, either at aa 50, 51, or 52. The sequence of cloned PCR-amplified genomic DNA from three homozygous cows, two unrelated females carrying the CasD allele and one carrying the CasA allele, did not reveal any deletion and showed two identical nucleotide (nt) substitutions in the 1.7-kb region of both CasD alleles encompassing codons 43-75 in the cDNA encoding alpha s2-CasA. This strongly suggests that the deleted bovine alpha s2-CasD arises from skipping the 27-nt exon, now identified as exon VIII, which encodes aa 51-59 of alpha s2-CasA. The G-->T transversion (allele A-->D) affecting the last nt of exon VIII, i.e., the 5' consensus splicing site, might be responsible for the altered splicing of the primary transcript of alpha s2-CasD.

An 18-kb caprine genomic DNA fragment, comprising the beta-casein transcription unit with about 3-kb 5' and 6-kb 3' flanking regions, was microinjected into fertilized one-cell murine eggs. All nine lines of transgenic mice obtained expressed the transgene in their mammary glands, as demonstrated by Northern blot analysis of mRNA in miscellaneous tissues, and qualitative and quantitative analysis of caprine beta-casein in milk, using SDS/PAGE, Western blotting and rocket immunoelectrophoresis. Two lines produced milk containing up to 21-24 mg of the exogenous protein/ml, a yield which is roughly twice that found in goat milk. The yield reached at least 40 mg/ml in some progeny of crossbred G1 transgenic mice. Thus, the investigated gene appears to be a good candidate for making hybrid constructs that might promote an efficient production of valuable foreign proteins in milk.

The two non-allelic forms of alpha s2-casein, occurring in ovine milk, differ by an internal deletion of nine amino acid residues, including both cysteine residues at positions 34 and 42 in the mature chain. Sequencing of several alpha s2-casein cDNA, isolated from the mammary cDNA library of a single lactating ewe, showed three new types which differed from that previously studied. In addition to the expected deletion of codons +34 to +42 affecting 30-40% of mRNA, another structural difference involving an internal stretch of 44 nucleotides in the 5' untranslated region, was found. S1-nuclease protection assays confirmed the existence of several types of the relevant mRNA and sequencing of in-vitro-amplified genomic DNA demonstrated the presence of the 44-nucleotide stretch in the alpha s2-casein transcriptional unit, thus ruling out the possibility of a cloning artefact. The different alpha s2-casein mRNA, four in terms of deletion and two in terms of nucleotide substitutions for a given ewe, can be readily explained by partial exon skipping and allelic differences, respectively. This assumption is well supported by the following observations: 5' and 3' ends of both deleted DNA fragments are similar to those of exons; sequences neighbouring the 44-nucleotide stretch of the genomic DNA perfectly match consensus sequences described for 3' and 5' ends of introns; the rather simple patterns observed on Southern blots of different enzymatic digests of genomic DNA strongly suggest the occurrence of only 1 copy alpha s2-casein gene/haploid genome. During the course of evolution, the alpha s2-casein-encoding gene has undergone many mutations and some of them might have occurred in regions corresponding to consensus splicing regions of the pre-mRNA. Thus, complete skipping of some exons might be responsible for the shorter sizes of rat and mouse alpha s2-casein mRNA. If so, the overall organization of the alpha s2-casein gene in the different species might be more similar than expected from structural comparisons of the cognate mRNA or caseins.

Restriction fragment length polymorphism (RFLP) of ovine casein genes was investigated. Genomic DNA from 56 rams was digested with 10 restriction endonucleases and Southern blots probed with the four ovine casein cDNAs (alpha s1-, beta-, alpha s2- and kappa-Cn). Five enzymes, namely, BglI, PvuII, RsaI, TaqI and HindIII revealed nine different RFLPs. The inheritance of six of these polymorphisms was studied by segregation analysis of gametes in nine rams' families, and each of them could be related to the existence of alleles at the relevant casein locus. A close linkage between the four ovine casein genes was demonstrated since no recombination within the four pairs of loci examined, alpha s1-beta-Cn, alpha s1-kappa-Cn, beta-kappa-Cn and alpha s2-kappa-Cn, was observed in the progeny of double heterozygous rams. The casein genes are thus clustered in the ovine species as in the case of other mammals.

This paper describes the elucidation of the primary structure of the three genetic variants of goat alpha s1-casein, alpha s1-Cn D, E and F, which have been found to be associated with reduced amounts of alpha s1-casein in milk. Variant E has the same electrophoretic mobility as variant B, but differs from the latter by the substitutions of Arg for Lys and of Thr for Ala at positions 100 and 195. A genetically controlled event which does not affect the amino acid sequence of this variant might be responsible for its lower rate of synthesis compared to that of alpha s1-casein B. The deletion of 11 amino acids at positions 59-69 and of 37 amino acids at positions 59-95 in variant B leads to variants D and F. In both cases the deletions, which start at the same position of the polypeptide chain, include the major phosphorylation site of the protein. On the basis of sequence data for casein genes and cDNAs, it was concluded that the deletions occurring in the D and F variants are due to the exclusion of one and several exons, respectively. The observed deletions in the proteins could thus be the consequence of splice site mutations which would induce altered RNA processing and hence reduce the rate of synthesis of the casein.

The extensive polymorphism of caprine alpha s1-casein, which is controlled by at least seven autosomal alleles segregating in a Mendelian fashion, was investigated by RFLP analysis. Genomic DNA from 77 lactating goats, whose genotypes had been previously determined by electrophoretic analysis of milk proteins, was digested with 11 restriction endonucleases and Southern blots were probed with a radiolabelled ovine alpha s1-casein cDNA. Three enzymes, PstI, TaqI and Rsa I, allowed the unambiguous identification of known alleles alpha s1-CnA, E and O and of the allelic pairs [alpha s1-CnD and F] and [alpha s1-CnB and C]. Evidence for a second null allele, termed alpha s1-CnO', and for an additional allele, designated alpha s1-CnF', was provided, which leads to the identification of nine alleles at the alpha s1-Cn locus, in this species. Although only 15 out of the 45 expected genotypes could be fully ascertained, this procedure allows the identification at birth of animals carrying the alpha s1-CnA, B or C alleles associated with a high alpha s1- and whole-casein content.

The amino acid sequence of porcine and human caseinomacropeptides (CMP), the C-terminal glycopeptide released from kappa-casein by chymosin at the initial step of milk coagulation, have been investigated. The complete amino acid sequence of porcine CMP and that of the first 59 amino acid residues of human CMP have been determined. Porcine and human CMPs contain 71 and likely 65 amino acid residues respectively. The extra hexapeptide 38-43 found in porcine CMP arises obviously from the duplication of the DNA fragment coding for the 6 preceding amino acids.

Ovine whole casein contains 2 multiphosphorylated beta-casein components designated as beta 1 and beta 2-caseins. The complete sequence of beta 1-casein and the partial sequence of beta 2-casein have been determined from cyanogen bromide and tryptic digests. The ovine beta 1 and beta 2-caseins have the same polypeptide chain and appear to differ only in that they contain 6 and 5 phosphates respectively. The amino acid composition of ovine beta 1-casein can be written as: Asp4, Asn4, Thr10, ThrP1, Ser9, SerP5, Glu19, Gn21, Pro34, Gly5, Ala4, Val21, Met6, Ile9, Leu22, Tyr3, Phe9, Trp1, Lys12, His5, Arg3. Compared to bovine beta-casein A2, which is made up of 209 residues, ovine beta 1-casein has a deletion of 2 residues (either Pro-179--Tyr-180 or Tyr-180--Pro-181) and 20 largely conservative amino acid substitutions. Although 20% of the substitutions involve proline residues, the proline contents of ovine beta 1 and bovine beta A2-caseins are very similar, around 16%. The average hydrophobicity, calculated according to Bigelow, is 5.51 kJ/residue, which is similar to that calculated for bovine beta-casein A2. The cluster of 4 phosphorylated serine residues and the highly charged nature of the amino terminal region observed for bovine beta-casein are conserved in the ovine beta-caseins. The substitution from Ile-12 (bovine) to Thr-12 (ovine) results in a new phosphorylation site, according to the phosphorylation code proposed for caseins. This site is only partially phosphorylated hence the occurrence of both beta 1 and beta 2-caseins in ovine milk.

An ovine mammary cDNA library has been constructed from total poly(A)+ RNA isolated from the mammary gland of a lactating ewe, using a classical procedure. Blunt-ended double-stranded cDNAs prepared with reverse transcriptase and nuclease S1 were tailed with dCTP, inserted into the dGMP-tailed PstI site of plasmid pBR322 and cloned in E. coli. Five series of homologous clones representing abundant messenger RNAs (strong hybridization with a single-stranded cDNA probe generated from total poly(A)+ RNA) were selected using each time a different predominant cloned ds-cDNA as probe, then identified by positive hybridization-translation of the cognate mRNA and subsequent immunoprecipitation and electrophoresis of the protein. The lengths of alpha s1-, alpha s2-, beta-, kappa-casein and beta-lactoglobulin mRNAs are in the range of 1.2, 1.1, 1.25, 1.0 and 0.85 kb, respectively, as determined by Northern blotting analysis. Five homologous mRNAs of similar sizes were identified in the porcine species by dot blot hybridization and Northern analyses. The nucleotide sequence of alpha s1-casein mRNA was determined by sequencing, according to Maxam and Gilbert, both a 1080 bp long cloned ds-cDNA and a ss-cDNA (268 nucleotides) generated by 5' extension of a 5' terminal truncated radiolabeled fragment (83 bp) of the relevant ds-cDNA, used as primer for reverse transcription. The 3' non coding region (431 nucleotides, excluding the poly(A) tail) represents 70% of the length of the coding region (618 nucleotides) flanked by a 61 nucleotide 5' region. Comparison of sequences of ovine and bovine, rat and guinea-pig alpha s1-casein mRNAs has revealed a greater homology in the 3' and especially 5' non coding regions. In the reading frame, the conserved regions are essentially those corresponding to the signal peptide and phosphopeptide domains. The derived 206 amino acid sequence of ovine pre-alpha s1-casein differs from that of its bovine counterpart (genetic variant B) by 24 amino acid substitutions and a deletion of 8 amino acid residues occurring in the polypeptide chain of the mature protein. Such a variation (84% homology only) in two phylogenetically closely related species indicates a high rate of evolution of alpha s1-casein.

The complete amino acid sequence of Italian water buffalo (Bubalus arnee) caseinomacropeptide, the C-terminal fragment released from kappa-casein by chymosin, has been determined. It contains 64 amino acid residues including one phosphoserine and differs from its bovine (Bos taurus) B counterpart by 10 amino acid substitutions. The sequence of the last 11 amino acid residues of para-kappa-casein is also reported. In relation to the Ala148/Asp substitution which is responsible for the different electrophoretic behaviour of bovine kappa-caseins B and A, water buffalo kappa-casein is homologous to the bovine variant B. It is suggested that a variant Thr136-Ala148 might be the wild type of the Bos genus.

The complete nucleotide sequence of ovine beta-casein mRNA has been determined by sequencing, according to Sanger-Messing, both a recombinant clone isolated from a mammary cDNA pUC 18 library and a single-stranded cDNA generated by reverse transcription from a synthetic 17-mer primer complementary to the 5' part of the mRNA coding frame. The 1088 nucleotide long beta-casein mRNA, excluding the poly(A) tail, contains a coding frame of 669 nucleotides including the stop codon, flanked by 60 and 359 nucleotides in the 5' and 3' untranslated regions, respectively. It arises from the splicing of 9 exons as deduced from gene sequence data. The deduced amino acid sequence differs at 3 positions from that previously determined by direct sequencing of mature beta-casein. Comparison of the ovine, bovine, rat, mouse, and rabbit beta-casein mRNA sequences shows a higher homology in the 3' and 5' untranslated regions. The most conserved regions in the open reading frame are essentially those encoding the signal peptide and the major phosphorylation site.

The fifty of so phosphorylated hydroxyamino acid residues hitherto investigated in caseins from different species have been found to occur in tripeptide sequences -Ser/Thr-X-A- where X represents any amino acid residue and A is an acidic residue. This strongly suggests that phosphorylation of caseins involves basically the stepwise enzymatic recognition of primary and secondary anionic amino acid triplets where the determinants are dicarboxylic residues and phosphoseryl residues, respectively. Studies of genetic variants of bovine caseins have provided clear-cut evidence for the actual occurrence of the former recognition sites. The occurrence of the above tripeptide sequences is a necessary but not a sufficient condition for phosphorylation of caseins to occur. Possible factors of constraint such as different intrinsic properties of both phosphate acceptor residues and acidic determinants, the characteristics of the local environment in terms of overall charge and hydrophilicity, secondary structure and steric hindrance, an insufficient available pool of casein kinase(s)... are discussed. All evidence now available supports the concept that phosphorylation of caseins is a posttranslational event and it is suggested that the process may occur during the transfer of completed polypeptide chains from the smooth endoplasmic reticulum to the Golgi apparatus where most of phosphate incorporation is presumably carried out. This organelle is rich in membrane-bound specific cyclic AMP-independent kinase(s) able in vitro to rephosphorylate specifically although not completely phosphatase-treated caseins and caseinophosphopeptides.

Whole goat kappa-casein was prepared by chromatography of whole casein on hydroxyapatite. Chromatography of whole kappa-casein on DEAE-cellulose separated 5 fractions. All of them were sensitive to chymosin. Their amino acid and carbohydrate composition, phosphate content and molecular weight were determined. Galactose, N-acetylgalactosamine, N-acetyl and N-glycolyl neuraminic acids were identified in whole kappa-casein. It appears that goat kappa-casein, like cow, buffalo and ewe kappa-caseins, is composed of several fractions having identical peptide chains and differing in their carbohydrate contents. The main fraction, devoid of carbohydrate, was treated with chymosin. The para-kappa-casein and caseinomacropeptide were isolated. Their amino acid composition and phosphate content were determined.

The bovine alphas2-, alphas3-, alphas4- and alphas6-caseins [1] were isolated. The 4 proteins had the same amino-acid composition and C-terminal sequence, but different phosphorus contents. From a mixture of these proteins (designated as 'alphas2-complex') and from alphas3-casein a single and identical N-terminal sequence was obtained by Edman degradation. It seems therefore that the 4 proteins have the same peptide chain and only differ in their phosphorus content. For this reason we propose to modify the nomenclature of Annan and Mason [1] and to use in future the single term alphas2 to designate the caseins which have been previously called alphas2,alphas3,alphas4 and alphas6 by these authors. The study of the primary structure of the peptide chain, which has confirmed these results, was undertaken on the S-carboxymethylated alphas2-complex. From a cyanogen bromide digest and from a tryptic hydrolyzate of the alphas2-complex, 5 and 25 peptides were obtained respectively, both sets of peptides accounting for the whole peptide chain. Examination of the tryptic peptides containing methionine combined with the N- and C-terminal sequences of the alphas2-complex and some CNBr peptides, gave the order of the CNBr peptides, H.CN4--CN2--CN5--CN1--CN3.OH, which contain 4, 22, 115, 49 and 17 residues respectively. A partial sequence accounting for half of the peptide chain of the alphas2-complex is given. This peptide chain is likely composed of 207 amino-acid residues.

The amino acid sequence of caprine CMP, the negatively charged C-terminal fragment released by chymosin (rennin EC 3.4.23.4) from goat K-casein at the initial stage of the milk-clotting process, has been investigated. The complete sequence has been determined by analysing chymotryptic and "thermolysin" fragments of the CMP. Caprine CMP contains 66 amino acid residues, 2 being phosphorylated. Asp2, Asn5, Thr11, Ser6, SerP2, Glu7, Gln2, Pro6, Ala9 Val5, Met1, Ile6, Lys3, His1, and the carbohydrate-free polypeptide chain has a molecular weight of 6,998 daltons. The occurrence in caprine CMP of an additional phosphate group, linked to serine 168 in the C-terminal region Thr-Ser168-Thr-Glu170-Val.OH of the polypeptide chain, has given support to the phosphorylation code for caseins that we postulated earlier [28, 27]. According to this hypothesis, a specific phosphoryl kinase may recognize an anionic phosphorylation site corresponding to the tripeptide sequence Thr/Ser-X-Glu, X being any amino acid residue. Since the C-terminal sequence of bovine and caprine CMPs differ by the substitution Ala/Glu170 (caprine), phosphorylation of caprine serine 168 could be explained by the occurrence of the new phosphorylation site Ser168-Thr-Glu170.