The Α-S2-casein gene (CASAS2) has been mapped to the homoeologous cattle, sheep, and goat chromosomes 4 and to the long arm of a bovine chromosome translocation, t(4;8), using nonradioactive in situ hybridization and simultaneous fluorescent R-banding. The t(4;8) has been characterized by GTG-, GBG-, and RBG-banding and by silver staining of nucleolus organizer regions. These results confirm the previous (ISCNDA, 1989) localization of the casein gene group to chromosome 4 of cattle, sheep, and goats. We propose that the discrepancy between our results and earlier assignments of the casein gene group to chromosome 6 of cattle and sheep can be explained by the fact that the chromosome identified as No. 6 in the Reading Conference (1976) report was renamed chromosome 4 in the ISCNDA (1989) standardized karyotype of both species.
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...
The function of osteoclasts is to digest the calcified bone matrix. Osteoclasts, together with myotubes, are among the rare examples of multinucleated cells found in higher vertebrates, resulting from the fusion of mononucleated progenitors belonging to the monocyte/macrophage lineage. So far, no information is available about function and transcriptional activity of multiple nuclei in osteoclasts. We have used a run-on technique to visualize RNA synthesis in individual nucleus. We provide the first evidence that nuclei of resorbing osteoclasts, isolated from chick embryo long bones, or differentiated in vitro from murine spleen cells in presence of RANKL and macrophage-colony stimulating factor, are all transcriptionally active. Nevertheless, if transcriptional activity is the same for all the nuclei within a cell, its level varies between osteoclasts: osteoclasts with highly active nuclei are always associated with resorption pits. We found that global transcription activity of resorbing osteoclasts seeded on calcified matrix is down-regulated after 5-h treatment with calcitonin, which transiently blocks resorption. This effect is reversible because calcitonin removal led to nuclear transcription activation. These results indicate a strong correlation between transcription and resorption. Finally, our data indicate that the resorption pit surface is linearly related to the nuclei number per osteoclast, strongly suggesting that functional advantage of osteoclast multinucleation is to improve resorption efficiency.
SummaryTwo alleles, A and B, were previously described at the goat αs2‐casein locus. Isoelectric focusing allowed us to subdivide the former one in two new alleles, called A and C. Although αs2‐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 αs2‐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’.
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