Peter J. Roche

Purification and cloning of a corpuscles of Stannius protein from Anguilla australis

The kidneys of teleost fish are associated with tissues containing secretory granules--the corpuscles of Stannius (CS). Electron microscopy indicates that the granules are of a proteinaceous nature and may represent hormones or enzymes of unrecognized physiological and biochemical function. In the present study, two-dimensional gel electrophoresis and electroelution was used to purify the major protein to homogeneity; it is approximately 32,000 Da in the reduced form and glycosylated. From the partial NH2-terminal sequence, a 75-mer oligonucleotide probe was synthesized and used to isolate a cDNA clone from which the complete amino acid sequence of the major CS protein was deduced. Polyclonal antibodies raised against CS homogenates were specific for the CS proteins (confirmed by immunohistochemistry). Hybridization histochemistry was used to confirm the location of the mRNA encoding the isolated protein. Incubation of CS homogenate with eel plasma or ovine renin substrate did not result in any angiotensin-like peptides whereas kidney homogenate did.

Structure and expression of Leydig insulin-like peptide mRNA in the sheep.

We have used PCR to isolate and characterise Leydig insulin-like peptide (Ley I-L) mRNA from sheep ovary. The deduced amino acid sequence of sheep Ley I-L has good homology with the pig and human peptides, having 93% and 77% amino acid identity, respectively. Northern blot analysis revealed abundant expression in both ovary and testis. An examination of ovarian RNA from non-pregnant and pregnant sheep showed that pregnancy did not significantly increase Ley I-L mRNA levels. However mRNA levels did alter depending on whether ovaries contained a corpus luteum. Also ovaries were examined by hybridisation histochemistry to locate the site of expression. Abundant Ley I-L mRNA levels were found in the theca interna cells of the ovary.

Expression of human relaxin genes: characterization of a novel alternatively-spliced human relaxin mRNA species.

Relaxin is a two-chain peptide hormone encoded by two non-allelic genes in humans and great apes, and by a single gene in all other species studied. We have characterized the expression of the human relaxin genes (H1 and H2) in placenta, decidua, prostate and ovary by reverse-transcription/polymerase chain reaction (RT/PCR). H2 relaxin mRNA was detected in the ovary, term placenta, decidua, and prostate gland. In contrast, H1 gene expression was detected only in the prostate gland. In addition to the relaxin PCR product of the predicted size (486 bp), a larger relaxin-specific product (587 bp) was detected in both H1 and H2 amplifications and in amplifications of chimpanzee relaxin from placenta and corpus luteum. Sequencing of human and chimpanzee PCR products, and human relaxin genomic clones, revealed that the larger product arises from an alternatively-spliced relaxin mRNA species incorporating an extra exon. This is the first evidence that the structure of the human and chimpanzee relaxin genes differ from that of other characterized relaxin genes, such as pig and rat. The novel peptide arising from this alternate message would be identical to prorelaxin in the B-chain and part of the C-peptide (extending to the position of the intron) but would differ from prorelaxin in the carboxy-terminal domain. Observation of a similar mRNA species in the chimpanzee suggests that this conserved relaxin-like peptide may have a significant biological role.

Cloning and expression analysis of a cytochrome P-45011β cDNA in sheep

A full length ovine steroid 11 beta-hydroxylase (cytochrome P-450(11 beta)) cDNA clone from a sheep adrenal cortex cDNA library was isolated. Sequence analysis indicates that this cDNA clone resembles bovine P-450(11 beta) cDNA (95% nucleotide sequence homology) more closely than rat P-450(11 beta) cDNA (69% nucleotide sequence homology). Although the levels of nucleotide sequence homology of this cDNA clone to the rat P-450(11 beta) cDNA and the rat P-450aldo cDNA are similar, the putative amino acid sequence shows a closer resemblance to rat P-450aldo protein. Northern blot analysis shows that there are three sizes of transcript and they are expressed throughout the adrenal cortex.

Processing and bioactivity of the corpuscles of Stannius protein of the Australian eel.

The primary structure of the major protein from the Corpuscles of Stannius (CS) of the Australian eel was elucidated from the cDNA sequence and was found to bear close similarity to the N-terminal amino acid sequence of the presumably homologous salmon hormone, teleocalcin (TC). The cDNA sequence predicted a preproprotein of 263 amino acids. Following removal of a 17 amino acid signal peptide, specific monobasic cleavage at an Arg-Phe bond generates the 231 amino acid mature form of the protein. The isolation and sequence determination of the prosequence confirms that the precursor contains a prosegment of 15 residues. Various fragments of the protein have been synthesized chemically and their biological activity assessed. The N-terminal 1–20 fragment of the mature protein inhibits calcium uptake in fingerling trout, the effect being similar, but not equipotent to salmon teleocalcin. Further, infusion of either the N-terminal 1–20 or the 81–94 fragment at 50 μg/h into the renal artery of conscious sheep, caused significant decreases in systemic plasma potassium concentration and in potassium excretion. The 1–20 fragment also gave rise to a small but significant increase in sodium excretion. Infusion of TC at the same rate results in a significant decrease in plasma potassium and phosphate concentration as well as a significant decrease in potassium excretion. Bovine PTH (1–34) at 100 μg/h causes a small decrease in plasma potassium and phosphate and an increase in plasma calcium concentration, and was the only peptide to cause a significant decrease in calcium excretion.

Functional and expression analysis of ovine steroid 11ß-hydroxylase (Cytochrome P45011ß)

In this study, the ovine steroid 11 beta-hydroxylase (P450(11 beta) or CYP11B) cDNA previously reported by us (1) was transfected into COS-7 cells. Using 3H-11-deoxycorticosterone (3H-DOC) as the substrate, and paper partition chromatography for separation of steroid products, the expressed enzyme was shown to catalyse the conversion of DOC to corticosterone (B), 18-hydroxy-11-deoxycorticosterone (18-OH-DOC), 18-hydroxy-corticosterone (18-OH-B), and aldosterone (ALDO). These results suggest that the expressed ovine cDNA exhibited 11 beta-hydroxylase, 18-hydroxylase and aldosterone synthesis activities. The enzymatic activity of the enzyme was further analysed by adding unlabelled steroids to compete with 3H-DOC. The conversion of 3H-DOC to 3H-ALDO was inhibited by the addition of excess DOC, B and 18-OH-DOC, indicating that all these steroids were potential substrates of the enzyme. The results also demonstrated that 18-hydroxylation could occur before 11 beta-hydroxylation with this enzyme. However, the addition of excess cold 18-OH-B had no significant effect on the level of 3H-ALDO that was synthesised. This result could imply that 18-OH-B is not an intermediate involved in the conversion of DOC to aldosterone, or, more likely, the enzyme substrate site is not accessible readily. Our results also indicated that DOC was preferred to 18-OH-DOC as a substrate for the enzyme. We have demonstrated by hybridisation histochemistry using specific oligonucleotide probes that the corresponding P450(11 beta) RNA transcript was present in all zones in the sheep adrenal cortex. In summary, we have shown that the enzyme encoded by the predominant P450(11 beta) cDNA isolated from the sheep adrenocortical cDNA library has all the enzymatic activities to biosynthesise ALDO from DOC. The corresponding transcript of this ovine P450(11 beta) cDNA was located throughout the adrenal cortex and thus the inability of the zonae fasciculata-reticularis to secrete ALDO remains to be understood.

HYBRIDIZATION HISTOCHEMISTRY - LOCATING GENE EXPRESSION

A technique we have called hybridization histochemistry has been developed for the location of specific mRNA populations in specially prepared sections of tissue (Hudson et al 1980; Coghlan et al 1981; Coghlan et al 1984; Hudson et al 1981; Jacobs et al 1983; Coghlan et al 1984). Later the same approach has been used by others to identify specific neurones in the hypothalamus (Gee et al 1983), to study the origin and fate of identified neurones in aphysia (McAllister et al 1983), location of specific genes in Drosophila embryos (McGinnis et al 1984), and enkephalin in the adrenal (Block et al 1984).