A single nucleotide deletion resulting in a premature stop codon is associated with marked reduction of transcripts from a goat beta-casein null allele.

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.

High-level, stage- and mammary-tissue-specific expression of a caprine kappa-casein-encoding minigene driven by a beta-casein promoter in transgenic mice.

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)

Biochemical and genetic analysis of variant C of caprine alpha s2-casein (Capra hircus).

Two alleles, A and B, were previously described at the goat alpha s2-casein locus. Isoelectric focusing allowed us to subdivide the former one in two new alleles, called A and C. Although alpha s2-casein C cannot actually be distinguished from its A counterpart by starch or polyacrylamide gel electrophoresis, it differs from the previous allele by a single substitution Lys (A)/Ile (C) at position 167, which was confirmed at the nucleotide level. The frequencies of the three alpha s2-casein alleles A, B and C were estimated to be 0.85, 0.04 and 0.11 in the French dairy breeds 'Alpine' and 'Saanen'.

Bovine alpha s2-casein D is generated by exon VIII skipping.

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.

High expression of the caprine beta-casein gene in transgenic mice.

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.

Sequence of the goat alpha-lactalbumin-encoding gene: comparison with the bovine gene and evidence of related sequences in the goat genome.

The isolation and characterization of a genomic clone encoding goat alpha-lactalbumin (alpha LA) are described, and its sequence was aligned with that of its bovine counterpart. The 5'-flanking regions are highly homologous, but two small insertions were found in the goat sequence. One of these insertions fills the gap found in the so-called 'milk-box' consensus sequence of the bovine gene. Southern analysis indicates the presence of several alpha LA-related sequences in the goat genome, as previously observed in cow and sheep, which suggests that the relevant duplication event(s) occurred before divergence of these ruminant species.