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ABSTRACT

Reverse transcription-PCR was used to clone the coding region of the donkey (Equus asinus) glycoprotein hormone α-subunit transcript from pituitary gland RNA. The donkey α-subunit sequence demonstrated considerable identity with the horse (97% at the nucleotide level), confirming the very close evolutionary linkage between these two species. The predicted amino acid sequence revealed that the donkey α-subunit has the same unusual C-terminus as the horse α-subunit, when compared with all other mammalian α-subunits, including a Tyr-His transposition between positions 87 and 93 and Ile instead of Ser as the C-terminal residue. Since recent evidence indicates important involvement of this region of the α-subunit in receptor binding, these findings provide a possible partial explanation for the unique biological properties of the equine gonadotrophins.

ABSTRACT

A 514 bp cDNA transcript coding for 78% of horse (Equus caballus) GH has been cloned and sequenced. The deduced amino acid sequence corresponded precisely to that previously obtained by protein sequencing and, in addition, provided new sequence information for the signal peptide. The missing 3′ fragment of the cDNA was reconstructed using synthetic oligonucleotides and site-specific directed mutagenesis. The complete cDNA sequence was then inserted into an expression vector (PIN-III-lpp^p−5) which utilizes a bacterial signal peptide to secrete the expressed product into the periplasmic space of Escherichia coli. Western blot analysis of cell lysates and periplasmic fractions prepared from cells harbouring this construct revealed significant quantities of immunoreactive GH and indicated that the bacterial signal peptide was successfully cleaved from the fusion protein on secretion. Recombinant-derived horse GH, recovered by osmotic shock from the periplasm, was active in a heterologous radioimmunoassay and a horse liver radioreceptor assay and resulted in a recovery of 0·5–2 mg GH/l cell culture. An apparent limitation on the secretion rate of horse GH in E. coli, possibly involving a block to translocation across the cytoplasmic membrane, prevented higher levels of expression being obtained.

ABSTRACT

A 246 bp cDNA clone representing the C-terminal region of the donkey (Equus asinus) chorionic gonadotrophin (CG)-β subunit was isolated from a placental library. The transcript contained the 3′ untranslated region and 42% of the CG-β subunit coding region (amino acid residues 85–146 of the mature peptide). Comparison of the deduced donkey amino acid sequence with the published horse CG-β subunit protein sequence (where they overlapped) revealed an overall homology of 61%. However, most of the differences were in the C-terminal extension, which is thought not to be important for gonadotrophic activity, and appeared to be due to two nucleotide insertions in the donkey sequence (compared with a deduced horse nucleotide sequence) leading to a reading-frame shift. Amino acid homology in the disulphide 'core' region was 81%. Some of the differences in this region were in the 'determinant loop' (residues 93–100) and these are interpreted in relation to the observed biological activities of horse and donkey CG.

The deduced amino acid sequence of the donkey cDNA indicated that it was larger than the majority of gonadotrophin-β subunits due to a C-terminal extension. Primate and horse CG (and horse LH) β subunits have analogous C-terminal extensions. The extension in the donkey subunit is 25 amino acid residues in length, compared with 28 in the horse and 24 in man. Comparisons with other available subunit DNA sequences indicated that, like the human CG-β gene, the donkey gene probably evolved from an ancestral LH-like β gene, following nucleotide deletions that allowed readthrough into previously untranslated DNA. Furthermore, both the human and donkey CG-β genes make use of the original LH polyadenylation sequence AAUAAA for translational termination and polyadenylation. We conclude that the C-terminal extension arose independently in equids and primates but through similar mechanisms.

ABSTRACT

The polymerase chain reaction (PCR) was used to generate a 131 bp cDNA encoding part of the sheep endometrial oxytocin receptor. The nucleotide sequence of this cDNA was 93.8% identical to the human oxytocin receptor sequence in this region. When used to probe Northern blots of sheep endometrial RNA the PCR product identified a 6.7 kb mRNA which appeared and disappeared during the oestrous cycle in parallel with the oxytocin receptor molecule as measured by ligand binding. The sheep endometrial receptor mRNA was significantly larger than the human myometrial mRNA (4.7 kb). It is suggested that the cloned cDNA described here is an appropriate probe for use where it is required to measure sheep oxytocin receptor mRNA.

ABSTRACT

A bovine trophoblast interferon (IFN-τ) gene promoter sequence (− 450 to +26 bp relative to the transcription start site) led to expression of reporter gene (CAT) constructs transfected into L929 (murine fibroblast) or JAR (human choriocarcinoma) cells. Expression depended on the presence of an exogenous (SV40) enhancer. Poly(I)(C) activated endogenous IFN production in L929 and JAR cells but had no consistent effect on CAT expression. Similar results were obtained in L929 cells with inactivated Newcastle disease virus. There was no 'priming' effect of exogenous Type I IFN. Deletion mutants revealed sites exerting negative control on expression between −338 and −247 bp, and between −150 and −71 bp; these regions contained sequences resembling previously identified negative regulatory domains. In the absence of viral inducibility it is proposed that negative regulation contributes towards the stringent control of expression characteristic of IFN-τ genes.

ABSTRACT

A sheep endometrial oxytocin receptor (OTR) cDNA (1·5 kb) was isolated from a λ-ZAP library using a reverse transcription-PCR product probe generated from oestrous endometrial mRNA. The sheep OTR cDNA shared an overall similarity of 82% with human OTR cDNA, 85% with pig OTR cDNA and 76% with rat OTR cDNA. The encoded receptor was a 391 amino acid polypeptide 94% similar to human OTR, 94% similar to pig OTR and 93% similar to rat OTR. The sheep OTR contained two additional amino acids compared with human OTR which were located in the highly GC-rich third intracytoplasmic loop. This region is thought to be associated with G protein coupling and signal transduction. Expression of the cDNA in Cos-7 cells and measurement of oxytocin-induced phosphoinositide turnover confirmed that it coded for a functional product. The affinity of the expressed receptor was comparable with that observed for the in vivo receptor.

ABSTRACT

Genomic and cDNA clones for bovine trophoblast interferon (IFN) have been isolated by probing a bovine genomic library and a bovine embryonic (day-18 post coitus) cDNA library respectively with the ovine trophoblast IFN cDNA. The two DNA sequences were identical; sequence analysis demonstrated 80% identity between the amino acid sequence of bovine trophoblast IFN and ovine trophoblast IFN, and 70% identity with a previously identified bovine IFN-α2. Southern blotting of bovine genomic DNA indicated the presence of a minimum of three trophoblast IFN genes. Primer extension analysis identified the transcription start site in the 5′ flanking region of the bovine IFN gene. Computer-aided analysis of the 5′ flanking sequence demonstrated a similarity with that of bovine IFN-α2 and the existence of a possible viral induction sequence.

ABSTRACT

Expression of the RNA coding for the ACTH—β-lipotrophin precursor, pro-opiomelanocortin (POMC), has been demonstrated in five human small-cell lung cancer (SCLC) cell lines. Using Northern and slot-blot hybridization analysis of RNA and a bovine POMC cDNA as probe, the processed POMC RNA from SCLC cells was found to be approximately 1350 nucleotides in length, which is larger than that found in the normal human pituitary. Expression of the POMC gene was confirmed by measurement of ACTH precursors secreted by the cells, using a novel two-site immunoradiometric assay based on monoclonal antibodies, which directly quantifies both POMC and pro-ACTH but does not recognize ACTH. Levels of POMC in medium accumulated throughout the growth of the cells, in contrast to POMC RNA which showed a relatively constant level of expression. We conclude that human SCLC cell lines are valuable models for studying the aberrant expression and regulation of the human POMC gene.

ABSTRACT

The PCR technique and highly degenerate oligonucleotide primers were used to amplify a 282 bp fragment of the horse (Equus caballus) epidermal growth factor (EGF) cDNA. The clone corresponded to 94 amino acids of the EGF precursor molecule. The deduced amino acid sequence of the 53 residue EGF mitogenic peptide within the precursor sequence showed 60–70% identity with five other published EGF sequences. The PCR cDNA fragment hybridized to a 4·9 kb transcript in horse kidney and endometrial RNA which was of a similar size to the mature EGF transcript found in other mammalian species.

The horse cDNA clone was used in Northern blots to monitor EGF expression in the endometrium of pregnant mares up to day 83 of gestation (term=330–340 days). The level of expression increased from day 33 and showed a further dramatic increase between days 35 and 45, which coincides with the onset of implantation and placentation in this species. Levels remained elevated up to day 83. The horse DNA sequence was used to design sense and antisense oligonucleotide probes (45-mers) for in situ hybridization studies. The antisense probe showed specific hybridization to the glandular, but not lumenal, epithelial cells of the endometrium and there was no signal in fetal membranes. The in situ hybridization signal increased between days 35 and 45 to a similar degree to that observed in the Northern blot analysis. This dramatic increase in EGF expression in the glandular epithelium of the mare's endometrium during pregnancy may provide a mitogenic stimulus to the endometrium and/or trophoblast to facilitate placental differentiation and attachment. Alternatively, the precursor could be involved in the endometrial gland secretory process which is necessary to produce uterine milk for fetal sustenance.

The PCR cloning methods used in this study should be generally applicable to the cloning of EGF cDNAs from other species.

ABSTRACT

A cloned cDNA has been isolated by probing a sheep blastocyst cDNA library using a synthetic oligonucleotide representing the N-terminal amino acid sequence of the antiluteolytic protein, ovine trophoblast protein-1. Sequence analysis of the cDNA confirms the 70% homology between the antiluteolysin and the interferon-α family of proteins; however, the sequence reported here differs at several points from previously reported amino acid and cDNA sequences for the antiluteolysin. In-vitro translation of day-16 poly(A)⁺ RNA indicated that antiluteolysin mRNA is a major constituent of total mRNA at this stage of blastocyst development, and Northern blotting confirmed that antiluteolysin mRNA production occurred between days 13 and 22 after oestrus. This is consistent with the stage at which embryonic extracts are antiluteolytic on administration in vivo. These and other data confirm that the ovine trophoblast antiluteolysin is an interferon, and suggest that at least five isoforms of this protein may exist.