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R G Pestell, V E Hammond and R J Crawford

ABSTRACT

DNA elements governing transcription of the ovine cytochrome P-450 side-chain cleavage (CYP11A1) gene were investigated. Three overlapping genomic clones for the ovine CYP11A1 gene were isolated and characterized. The transcriptional start site was located 51 nucleotides upstream from the initiating methionine. Gene transfer experiments were conducted in murine adrenocortical Y1 cells and human choriocarcinoma JEG-3 cells using chloramphenicol acetyltransferase reporter gene constructs containing promoter fragments from −2700 to −177 bp.

The results demonstrate that DNA elements sufficient to convey a basal level of expression and cyclic AMP (cAMP) responsiveness lie within 177 bp of the transcriptional start, although the possibility that additional regulatory elements reside outside this 177 bp has not been excluded. The ovine 5′ flanking sequence demonstrated 92% homology with the bovine sequence, extending over the entire fragment. In contrast, only four significant regions of conservation between the ovine, murine, rat and human CYP11A1 promoters were found. These regions are positioned within 200 bp upstream of the transcriptional start site.

DNase 1 footprinting was performed to identify DNA elements able to bind nuclear proteins. Primary adrenocortical and placental tissues from sheep were used as the source of nuclear extracts to detect DNA-protein interactions relevant to CYP11A1 gene expression in vivo. Five regions of protection were detected in the first −634 bp of the ovine CYP11A1 promoter. Three of these elements corresponded to the regions which are well-conserved between species. The other two elements resembled activating protein-1 (AP-1) and AP-4 sites and overlapping AP-2/Sp1 sites, and are conserved in the bovine gene but not in other species.

Nuclear protein extracts from adrenals of sheep with different serum ACTH levels (i.e. ACTH-treated, dexamethasone-treated and untreated sheep) protected similar regions of the ovine CYP11A1 promoter fragment. Similarly, the regions protected did not differ when nuclear protein from JEG-3 cells treated with cAMP was compared with that of untreated JEG-3 cells. These results suggest that induction of CYP11A1 gene transcription by ACTH in the ovine adrenal and by cAMP in JEG-3 cells in culture is not mediated by changes in binding of the proteins that interact directly with these footprinted elements.

The elements footprinted by extracts from primary ovine tissue lie within the 177 bp sufficient for cAMP-regulated expression. The correspondence of these elements either to regions conserved between species or to known consensus binding sites suggests that these sequences are cis elements involved in regulating transcription of the ovine CYP11A1 gene in vivo.

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J M Gunnersen, P J Roche, G W Tregear and R J Crawford

ABSTRACT

Relaxin is a peptide hormone which is produced in human reproductive tissues including the ovary and prostate gland. Little is known of the molecular events regulating relaxin gene transcription. We have studied this question using gene transfer of relaxin promoter/reporter gene constructs into a relaxin-expressing cell line. A number of human cell lines expressed relaxin as detected by reverse transcription-PCR. In one of these lines, the prostate adenocarcinoma cell line LNCaP.FGC, relaxin mRNA was also detected by Northern blot analysis. The DNA sequences of the proximal 5′-flanking regions (∼900 nucleotides) of the two human relaxin genes, HI and H2, were determined. Deletion constructs containing portions of the 5′-flanking regions of HI and H2 linked to the bacterial chloramphenicol acetyl transferase reporter gene were prepared. The expression of the reporter gene constructs was analysed in the LNCaP.FGC cell line and the results of these transient transfection assays have led to the identification of positive and negative regulatory regions within the 5′-flanking DNA. A difference in activity of the H1 and H2 gene promoters in this prostate cell line was observed, with the H2 promoter being more active. This situation may mimic that occurring in vivo since the relaxin secreted from the prostate gland into seminal fluid is the product of the H2 gene.

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G. P. Aldred, P. Fu, R. J. Crawford and R. T. Fernley

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.

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R. J. Crawford, V. E. Hammond, P. J. Roche, P. D. Johnston and G. W. Tregear

ABSTRACT

The gene encoding rhesus monkey relaxin has been investigated. A cDNA library was prepared using corpus luteal RNA from a pregnant rhesus monkey, cDNA clones encoding relaxin were isolated and the nucleotide sequence was determined. The amino acid sequence of rhesus monkey preprorelaxin, predicted from the cDNA, demonstrates that the sequence has not been strongly conserved when compared with that of man, although features characteristic of the relaxin molecule have been maintained. This structural information will allow production of rhesus monkey relaxin, leading to studies investigating the bioactivity of relaxin in a homologous primate system. Southern blot analysis indicated that there is only one relaxin gene in the rhesus monkey and baboon genomes. In this respect these primate genomes are different from the human genome which contains two relaxin genes.