Multiple mRNA species code for two non‐allelic forms of ovine αs2‐casein

Abstract

The two non-allelic forms of as2-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 crs2-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 as2-casein transcriptional unit, thus ruling out the possibility of a cloning artefact.The different a,,-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 as2-casein gene/haploid genome. During the course of evolution, the as2-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 as2-casein mRNA. If so, the overall organization of the as2-casein gene in the different species might be more similar than expected from structural comparisons of the cognate mRNA or caseins.In ruminants, caseins comprise the major fraction of secretory proteins synthesized in mammary epithelial cells. Primary structures of the four bovine caseins (reviewed in [l]) share little similarity, except for the well-conserved multiple phosphorylation sites and signal peptides [2] of the calciumsensitive caseins, asl, as2 and j?. The proposal that they have a common origin [2] was further substantiated by the similarity in organization of the relevant genes in the region spanning the promoter and the 5' end of the transcription unit [3].The striking similarity (40%) between the N-terminal and C-terminal halves of bovine crs2-casein [4] is partially known for the rat [lo] and bovine [l 13 species. It may contain at least nine introns, but sequence data are only available for the region upstream from exon 11.Previous SDS/PAGE studies of ovine as2-casein, synthesized in both cell-free translation systems and mammary gland explants, revealed the occurrence of two polypeptide chains differing by an internal deletion...

Comparative analysis of gene sequence of goat CSN1S1 F and N alleles and characterization of CSN1S1 transcript variants in mammary gland

“…The existence of constitutionally deleted transcripts of exons has been reported in various domestic species, such as goat and sheep by Ferranti et al (1998Ferranti et al ( , 1999, pigs by Alexander et al (1992), horse by Lenasi et al (2003) but also in humans by Martin et al (1996). Multiple forms of transcripts originated by alternative splicing have been also shown for the casein as2 of sheep (Boisnard et al, 1991) and goat (Ramunno et al, 2001). It seems reasonable to hypothesize the existence of forms of transcripts other than those reported in the present study, which have not been detected so far, due to their undetectable amounts.…”

The impact of genetic polymorphisms on the protein composition of ruminant milks

“…Two non-allelic forms produced by different mRNAs [10] have been shown to occur in all the individuals analysed, apparently indicating a stabilised mechanism for the production of protein isoforms of different lengths. Two non-allelic forms of a s2 -casein occurring in ovine milk differ by an internal deletion of nine amino acid residues at positions 34-42 of the a s2 -casein chain.…”

Differential Splicing of Pre‐Messenger RNA Produces Multiple Forms of Mature Caprine αs1‐Casein

“…a,,-casein also exists, in caprine and ovine milks, as two non-allelic forms arising from alternative skipping of exon 6 during the processing of pre-messengers (Boisnard et al, 1991). Thus, complete exon skipping due to mutations occurring within consensus splice site sequences of a,,-, and even (human) p-casein is now well documented Menon et al, 1992;Bouniol et al, 1993;Mohr et al, 1994;Martin and Leroux, 1994).…”