Steroid hormones generally mediate their effects by interacting with specific receptors which then bind to defined DNA sequences in the regulatory regions of target genes to activate expression (see Gronmeyer (1992) and references therein). However, the post-transcriptional regulation of gene expression by steroid hormones is also well documented (see Nielsen & Shapiro 1990). Indeed steroid hormones were amongst the first agents to be demonstrated to play a role in mRNA stabilization (Palmiter & Carey 1974). For example, glucocorticoid hormones have been shown to enhance the stability of growth hormone mRNA (Paek & Axel 1987), testosterone has been reported to induce changes in the poly(A) tail length of the mRNA encoding cystatin-related protein (Vercaeren et al. 1992) and testosterone and/or oestrogen induce changes in the poly(A) tail length of the vasopressin mRNA (Carter & Murphy 1993) associated with changes in mRNA accumulation. However, it is still unclear how steroids mediate these
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J. C. Pascall and K. D. Brown
The 5′-flanking sequence of the mouse epidermal growth factor gene has been isolated from a mouse genomic DNA library. S1 nuclease mapping indicated that the transcription start sites used in the submaxillary gland and the kidney are identical. Computer-aided sequence comparisons have indicated regions of the gene which may be involved in hormonal regulation.
J C Pascall, M A Surani, S C Barton, T J Vaughan, and K D Brown
A transgenic mouse line (EGF/Tag) has been established in which expression of SV40 T-antigen is directed by a 5·5 kb fragment of the 5′-flanking region of the mouse epidermal growth factor (EGF) gene. Of the two principal sites of EGF expression in mice, submaxillary gland and kidney, T-antigen mRNA and protein were detected in the former but not in the latter tissue of the EGF/Tag animals. T-antigen expression in the submaxillary gland was restricted to the EGF-producing cells of the granular convoluted tubules, and the oncoprotein induced hyperplasia of these cells. T-antigen levels were markedly higher in the submaxillary glands of male compared with female transgenic mice, suggesting that expression of the transgene was androgen-regulated, like the endogenous EGF gene. These results indicate that the 5·5 kb fragment upstream of the mouse EGF gene contains the DNA enhancer elements required for hormonally regulated expression in the submaxillary gland. Since the hyperplastic submaxillary glands of the EGF/Tag mice continue to synthesize EGF, these glands provide a tissue source from which it may prove possible to establish EGF-secreting cell lines for further in vitro studies of the mechanisms regulating expression of the EGF gene.
J. C. Pascall, D. S. C. Jones, S. M. Doel, J. M. Clements, M. Hunter, T. Fallon, M. Edwards, and K. D. Brown
A portion of the pig epidermal growth factor (EGF) gene has been isolated and characterized. The nucleotide sequencies of exons 20 and 21, which encode the EGF region of the precursor protein, show 85% similarity with the human EGF gene sequence. In addition, conservation of the intron—exon boundaries between the two species was generally observed. Although the pig exon 21 appeared to lack a single nucleotide at its 5′ end relative to the human gene, sequences obtained by direct amplification of the genomic DNA around the 5′ end of this exon using the polymerase chain reaction, and from a pig EGF cDNA recombinant isolated from a kidney library, indicated that the deletion was probably a cloning artifact. Comparison of the predicted amino acid sequence of pig EGF with that of EGF from other species, as well as with several other polypeptides which bind to the EGF receptor, indicated conservation of Gly18, Tyr37, Gly39 and Arg41 in addition to all six cysteine residues and Leu47, which are known to be critical for biological activity. A synthetic gene encoding the predicted amino acid sequence of pig EGF was expressed in yeast. The recombinant polypeptide was shown to compete with 125I-labelled mouse EGF for binding to cells and to stimulate DNA synthesis in quiescent monolayers of Swiss 3T3 cells.