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ABSTRACT

The primary structure of the sheep renin precursor has been determined from its cDNA sequence. A library of cDNA clones was constructed from adrenalectomized sheep kidney poly(A)⁺ RNA and screened for sheep renin sequences with a cloned mouse renin cDNA probe. Of the 300 000 clones generated, 24 were hybridization positive and the nucleotide sequences of two of the longest clones were determined. These clones coded for the mature sheep renin protein and the 3′-untranslated sequence but did not extend to the amino-terminal region of preprorenin. Clones corresponding to the 5′ region of renin mRNA were generated by the polymerase chain reaction and their nucleotide sequences determined. The sheep renin precursor consists of 400 amino acids with a putative leader sequence of 14 amino acids and a putative 45 or 53 amino acid prosegment. The mature sheep renin protein has a 73% sequence identity with human renin. Northern analysis demonstrated the presence of renin mRNA in the kidney but not in other tissues in the sheep. While sodium depletion of sheep caused a rise in renin mRNA in the kidney, adrenalectomy also led to a large increase in renal renin mRNA. Southern analysis of genomic DNA suggests that there is only one gene coding for renin in the sheep.

ABSTRACT

The angiotensinogen gene encodes the precursor protein for the potent vasoconstrictor angiotensin II. Although the gene is expressed in several tissues, the liver is the major source of circulating protein. In previous in-vivo studies we have found that a minigene containing 750 bp of 5′-flanking sequence is transcribed in a manner which largely parallels the expression of the endogenous gene. In this report, we characterized conserved elements in the promoter region, in order to determine their role in the transcription of the angiotensinogen gene. Constructs fused to the chloramphenicol acetyl transferase (CAT) reporter gene were transfected into hepatocarcinoma Hep G2 cells as well as into non-hepatic cell lines. We found that 5′-deletion mutant constructs, containing sequences from + 25 to −90 bp and −321 to −750 bp, were each able to activate transcription. These constructs contain the TATA box and core promoter sequences, including an Spl-binding site, and two glucocorticoid responsive elements respectively. In the non-hepatic cell lines, HeLa and Jeg-3, we found that the constructs were transcribed at a much lower rate when compared with the expression of a plasmid containing the Rous sarcoma virus long terminal repeat fused to the CAT gene. Constructs which included sequence 5′ to −244 were oestrogen inducible. An element which is conserved between rodent and human angiotensinogen promoters is contained within a sequence which is oestrogen responsive, while another binds the liver-enriched transcriptional activator hepatocyte nuclear factor 1. However, the role of this transactivator in the transcription of angiotensinogen remains uncertain.

ABSTRACT

Secretion of parathyroid hormone-related protein (PTHrP) by sheep fetal parathyroid glands is reported to be an important factor in the maintenance of a placental calcium pump. The aim of the present study was to determine whether the developing rat parathyroid glands express PTHrP or parathyroid hormone (PTH), or both. Hybridisation histochemistry was used to detect transcription of PTHrP and PTH in serial paraffin sections through the 12·5- and 13·5-day rat embryo parathyroid anlage, as well as in sections through the 17·5-day embryonic and adult parathyroid glands. Results show strong expression of PTH in the 13·5-day embryonic parathyroid anlage, as well as in the parathyroid gland of the 17·5-day embryo and adult. Transcription of the PTHrP gene was not detected.

The more sensitive technique of reverse transcription PCR was then performed. The pharyngeal region of 11·5-, 12·5- and 13·5-day rat embryos was dissected out and, at each stage, RNA was extracted from these tissues, as well as pooled tissues from the rest of the embryo. RNA that had been extracted from adult thyroid/parathyroid tissue was also tested. After reverse transcription, the resulting cDNAs were amplified by PCR (50 cycles) using specific PTH and PTHrP primers. The results show an abundance of PTH mRNA, specific to the pharyngeal region of the 13·5-day embryo, as well as to adult thyroid/parathyroid tissue. PTHrP expression was detected at very low levels in both parathyroid and extraparathyroid tissues. The presence of immunoreactive PTHrP and immunoreactive PTH in the pharyngeal region and rest of the body of 12·5- and 13·5-day rat embryos was assessed by specific RIAs. Whilst immunoreactive PTHrP was not detected in any of the tissues assayed, immunoreactive PTH was detected only in the pharyngeal region of the 13·5-day embryo. This confirms the results obtained from the gene expression studies.

We conclude then that, in the developing rat embryo, PTH rather than PTHrP is more likely to play a role in calcium regulation. This is in contrast with the reported situation in the sheep, and suggests that fundamental species differences in fetal calcium regulation exist in mammals.