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M Keaveney, M G Parker, and F Gannon

ABSTRACT

A well-conserved feature of the steroid receptor gene family is the presence of an exceptionally long 3′ untranslated region (UTR). Analysis of this sequence from the human oestrogen receptor (hER) gene showed the presence of a number of AT-rich regions that included thirteen repeats of the ATTTA motif, an element known to have a destabilizing effect in other systems. In the region 3′ of the gene there were a further eight copies of this pentamer. Also located in this sequence were two members of the Alu repetitive family in inverse orientation and in a tandem arrangement. Transfection experiments in which the 3′ UTR and 3′ flanking sequence were included in chloramphenicol acetyltransferase expression vectors revealed a large destabilization effect with several different fragments. This inherent instability appears to be determined by the primary nucleotide sequence but may act in conjunction with other factors. This posttranscriptional regulatory mechanism may contribute to the control of the level of the hER mRNA.

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P Sourdaine, M G Parker, J Telford, and W R Miller

ABSTRACT

Aromatase activity may be detected in most, but not all, breast cancers, and in certain tumours there appears to be decreased sensitivity to the aromatase inhibitor 4-hydroxyandrostenedione (4-OHA). The aims of the present study were to measure aromatase activity, and its sensitivity to 4-OHA, in breast tumours, and to examine the CYP19 gene encoding the aromatase cytochrome P450 (P450arom) for the presence of mutations.

In vitro aromatase activity and sensitivity to 4-OHA were measured by determining the conversion of tritiated testosterone to tritiated oestradiol in breast tumour tissue in the absence and presence of 4-OHA (10 nm). Genomic DNA was extracted from five tumours: one showing no detectable aromatase activity and four displaying evidence of aromatase activity (two sensitive and two insensitive to 4-OHA). Subsequent PCR-single-strand conformation polymorphism analysis revealed a variation in the mobility of single-stranded DNA for exons III, VII and X, corresponding, as shown by direct sequencing of PCR products, to common polymorphism of the aromatase gene. This study does not provide evidence for mutation in the coding exons of the P450arom gene which would account for either the absence of aromatase activity or its changed sensitivity to 4-OHA in breast cancers.

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S.G. Hillier, P.T.K. Saunders, R. White, and M.G. Parker

ABSTRACT

Oestrogen receptor mRNA expression in mouse ovaries was analysed by Northern blotting of total RNA using 32P-labelled RNA probes complementary to different functional domains of the oestrogen receptor. The ~6·5 kb mouse oestrogen receptor mRNA transcript was present in immature and adult ovaries at extremely low abundance compared with uterus and oviduct. Using a probe complementary to the steroid-binding domain of the oestrogen receptor (probe EF), a novel RNA transcript of ~1·5 kb was also found in the ovaries but was absent from uterus and oviduct. The melting temperature of the hybrid produced by the ~1·5 kb transcript with probe EF was ~10°C lower than that produced by authentic oestrogen receptor mRNA, which demonstrates incomplete sequence homology between the two transcripts and indicates that the ~1·5 kb RNA is not a truncated form of oestrogen receptor mRNA. Furthermore, the ~1·5 kb RNA lacks the DNA-binding domain found in the oestrogen receptor. The ~1·5 kb RNA, but not oestrogen receptor mRNA, was enriched in total RNA from isolated granulosa cells compared with residual ovarian tissue. The encoded product of this novel oestrogen receptor-related RNA could be a steroid-binding protein involved in oestrogen action in the ovaries.

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M Wong, M S Ramayya, G P Chrousos, P H Driggers, and K L Parker

ABSTRACT

The orphan nuclear receptor steroidogenic factor 1 (SF-1) plays key roles in endocrine development and function. Initially identified as a positive regulator of the cytochrome P450 steroid hydroxylases, analyses of knockout mice deficient in SF-1 revealed that SF-1 is essential for adrenal and gonadal development, pituitary gonadotropin expression and formation of the ventromedial hypothalamic nucleus. Although more limited in scope, analyses of SF-1 in humans similarly have suggested that SF-1 is important for differentiated function in adrenocortical and gonadotrope adenomas. In the hope of extending our understanding of SF-1 function by identifying possible roles of SF-1 in clinical endocrine disorders, we isolated the FTZ-F1 gene encoding human SF-1 and mapped it to chromosome 9q33. In this report, we characterize the sequence and structural organization of the human cDNA and gene encoding SF-1, providing new insights into comparative aspects of SF-1 structure that will facilitate efforts to study the role of this transcription factor in human endocrine disorders.

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K J Parker, P M Jones, C H Hunton, S J Persaud, C G Taylor, and S L Howell

ABSTRACT

The liberation of arachidonic acid (AA), by phospholipase A2 (PLA2), is the rate-limiting step in a number of cell signalling pathways. In the pancreatic β-cell, AA itself is thought to participate in the regulation of insulin secretion. Recently a Ca2+-sensitive, AA-selective cytosolic PLA2 (type IV cPLA2) has been isolated from the human monocyte U937 cell line. Although the DNA sequence of this enzyme implies a molecular weight of 85 kDa, the protein migrates with a molecular weight of 100-110 kDa on SDS-PAGE. In many cell types, cPLA2s which are reactive towards antibodies raised against the type IV cPLA2 have been shown to hydrolyse AA from membrane glycerophospholipids. Using a polyclonal antibody raised against a recombinant form of type IV cPLA2, we have detected an immunoreactive protein with a molecular weight of 93·5 kDa in rat islets of Langerhans. Furthermore, we have detected similar immunoreactive proteins in insulin-secreting β-cell lines and have shown co-expression of type IV cPLA2 immunoreactivity and insulin immunoreactivity in rat pancreatic β-cells. Under non-stimulatory conditions the 93·5 kDa immunoreactive protein detected in rat islets of Langerhans was located predominantly in the cytosolic fraction. We have shown that immunoprecipitation of the rat immunoreactive protein from rat islet homogenates significantly decreases the total dithiothreitol/β-mercaptoethanol-insensitive PLA2 activity by 56·4±7% This provides further evidence that the immunoreactive rat protein is a type IV cPLA2 and is responsible for a large component of the PLA2 activity in rat islets of Langerhans. It is possible that, in the rat β-cell, type IV cPLA2 couples the increase in intracellular Ca2+, brought about by insulin secretagogues, to the liberation of AA and the subsequent release of insulin.