Cloning of rabbit prolactin cDNA and prolactin gene expression in the rabbit mammary gland.

cDNA clones coding for rabbit prolactin were isolated from a pituitary library using a rat prolactin RNA probe. One cDNA contained 873 bases including the entire coding sequence of rabbit prolactin, its signal peptide and the 5' and 3' untranslated regions of 44 and 145 nucleotides respectively. The deduced amino acid sequence of the cloned prolactin cDNA presented a 93-78% identity with mink, porcine and human prolactins. The prolactin gene transcription was investigated by RT-PCR analysis in several organs of midlactating New Zealand White rabbits. The ectopic transcription of the prolactin gene was examined in more detail in the mammary gland. A strong PCR signal was detected in the mammary gland of virgin does and was also observed during pregnancy and at the beginning of lactation. This PCR signal was very weak in mid-lactating and absent in post-weaning mammary gland.

Cloning and structural analysis of four genes encoding interferon-omega in rabbit.

By using an ovine interferon-tau (IFN-tau) cDNA probe, four recombinant phages were isolated from a rabbit genomic library and sequenced from nucleotides -450 to 1,300 relative to the CAP site. Each of the four rabbit genes contains an open reading frame of 595 nucleotides and code for proteins that exhibit structural characteristics of the interferon-omega (IFN-omega) family. They display more than 98% identity in their coding regions. The deduced amino acid sequences share > 96% sequence similarity. In contrast, the 5' and 3' noncoding regions have diverged considerably (approximately 50% identity). Amino acid comparisons of rabbit IFN-omega with IFN-omega of other species reveal the highest degree of identity with human (72%), followed by porcine (68%) IFN-omega. Rabbit IFN-omega displays only 57% sequence similarity with ovine IFN-tau. The coding regions of the four genes subcloned in a cytomegalovirus eukaryotic expression vector and transfected in monkey COS-7 cells direct the production of proteins that protect bovine and rabbit cells against vesicular stomatitis virus infection, thus demonstrating that these genes encode fully active IFN proteins. The expression of these genes was studied in Sendai-induced rabbit leukocytes. A single band of poly(A)+RNA hybridized with a rabbit IFN-omega probe under stringent conditions, whereas no IFN-omega transcript was detected with RNA isolated from uninduced leukocytes. Southern blot analysis suggest the existence of at least eight IFN-omega genes or pseudogenes in the rabbit genome.

Multiple mRNA species code for two non-allelic forms of ovine alpha s2-casein.

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.

Cloning and structural analysis of two distinct families of ovine interferon-alpha genes encoding functional class II and trophoblast (oTP) alpha-interferons.

Ovine trophoblast protein (oTP) is a polypeptide secreted by ovine trophectoderm from day 11 to 21, which plays a key role in maternal recognition of pregnancy. Structural analyses established that oTP shares extensive homology with class II alpha-interferon (IFN-alpha II) subfamily. Previous screening of an ovine genomic DNA library probed with an oTP cDNA incidently resulted in the isolation of a functional IFN-alpha II gene and two relevant pseudogenes, as shown by sequence analysis and study of expression in eukaryotic COS cells. The expected oTP gene together with a cognate pseudogene was successfully isolated from the series of clones selected from another genomic library probed with the oTP cDNA, using two specific oligonucleotides, each one complementary to a region of oTP cDNA with little homology with the IFN-alpha II gene and related pseudogenes. Southern blotting of ovine genomic DNA indicated the existence of at least five trophoblast IFN-alpha genes or pseudogenes. Nucleotide sequence comparisons showed that the oTP gene exhibits a higher homology (90%) with bovine trophoblast IFN gene (Stewart et al. (1990) J. Mol. Endocrinol. 4, 275-282) than with oIFN-alpha II gene (70%), thus providing evidence that embryonic IFNs constitute a distinct subfamily of IFN-alpha s.

Complete sequence of ovine alpha s2-casein messenger RNA.

The primary structure of mRNA coding for ovine alpha s2 casein has been determined by chemical sequencing of three cDNA clones and the primer extension products of the longest one. The mRNA was 1,024 nucleotides long, excluding the poly(A) tail. The length of the 5' noncoding, coding and 3' noncoding regions was 53, 669 and 302 nucleotides, respectively. A comparison of the nucleotide sequence of ovine alpha s2-casein and guinea-pig casein A mRNAs revealed an extensive homology in the 5' and 3' noncoding regions. The deduced amino acid sequence of ovine alpha s2-casein was compared with its bovine and guinea-pig counterparts. Moreover, an heterogeneity was evidenced in the mRNA population of the alpha s2-casein.

Purification and properties of the major tRNAHis from sheep liver.

The major isoacceptor of tRNAHis from sheep liver was purified. Two different methods were described which both took advantage of the presence of the hypermodified Q nucleotide in this tRNA. In the first procedure, CNBr treatment of tRNA which was previously enriched in tRNAHis greatly increased the efficiency of the subsequent chromatographic steps. The tRNAHis obtained by this technique showed a specific activity of 1,690 picomoles of histidine per A260 unit. In the second one, a twenty-fold enrichment in tRNAHis was obtained by fractionation of crude tRNA on acetylated DBAE-cellulose columns. The nucleotide composition of the tRNA obtained by the CNBr procedure was determined. No thymine ribotide could be detected. When this tRNA was submitted to periodate oxidation and tritium borohydride reduction, a radioactive label was introduced in this molecule which was assumed to be located in the Q nucleotide.

Correlation between the presence of tRNA His GUG and the erythropoietic function in foetal sheep liver.

Histidyl-tRNAs from foetal and adult sheep liver were compared to their reticulocyte counterparts. The combination of various techniques revealed the existence of two histidyl-tRNA species in reticulocytes, one of which was not retained on acetylated DBAE-cellulose columns and was guanylatable. Three histidyl-tRNA isoacceptors were identified in foetal liver. Two of these species were not adsorbed on acetylated DBAE-cellulose but only one was found to be guanylatable. An identical chromatographic behaviour on RPC-5 columns was observed for guanylated histidyl-tRNAs from both origins. These results suggest the occurrence of a GUG anticodon in these guanine-accepting tRNAs. In foetal liver the amount of guanylatable histidyl-tRNA was estimated to be 7% of the total tRNA population. This observation is in agreement with the erythropoietic function of liver during the foetal life.

Biochemical research on oogenesis. Nucleotide sequence of initiator tRNA from oocytes and from somatic cells of Xenopus laevis.

The nucleotide sequence of initiator tRNA, tRNAfMet, from vitellogenic oocytes of Xenopus laevis was determined. The sequence was deduced from analysis of all T1 and pancreatic oligonucleotides and comparison with the sequence of initiator tRNA from other animal species. At least 80% of all initiator tRNA molecules from oocytes have the same nucleotide sequence. This means that most and probably all initiator tRNA genes which are active in oocytes are identical to one another. No structural difference was observed between liver and oocyte initiator tRNAs. Initiator tRNA from X. laevis has the same nucleotide sequence as initiator tRNA from several species of mammals. The genes coding for this RNA have therefore remained unchanged in the mammalian and amphibian lines for at least 300000000 years.