Structural analysis of 5'-flanking regions of rat, mouse and human renin genes reveals the presence of a transposable-like element in the two mouse genes.

The two renin genes of the mouse (Ren1 and Ren2) are expressed at different levels in the submaxillary gland (SMG). In contrast to mice, there is no detectable renin gene expression in the rat SMG. To determine the molecular basis for these different levels of renin expression, we have compared the 5'-flanking regions of the rat and mouse genes. The sequence of mouse, but not rat, genes reveals the presence in Ren1 and Ren2 of a large insertion, probably a new class of transposable elements. A second, apparently unrelated shorter insertion is present only in Ren2. Otherwise, the mouse and rat 5'-flanking sequences are well conserved and resemble the corresponding region of the human Ren gene, indicating that the insertions occurred after the separation of the rat and mouse species but before the duplication of the mouse Ren gene. We suggest that these structural differences may have a role in the differential expression of the Ren genes in mice and other animals.

Thyroxine and testosterone transcriptionally regulate renin gene expression in the submaxillary gland of normal and transgenic mice carrying extra copies of the Ren2 gene.

Expression of the mouse renin genes (Ren1 and Ren2) in the submaxillary gland of female mice has been analyzed following administration of thyroxine (T4) or dihydrotestosterone (DHT). Both hormones appear to act independently on mRNA accumulation which increases about 5 fold over basal level. In vitro transcription assays in isolated nuclei demonstrate that both hormones act at the transcriptional level. The effects of DHT and T4 were also analyzed in transgenic mice obtained by microinjection of the Ren2 gene. We show that T4 is as efficient as DHT in promoting renin mRNA accumulation in these transgenic animals, in spite of their low basal level of Ren2 mRNA. Structural comparison of the Ren1 and Ren2 promoters with those of other genes regulated by T4 shows the conservation of two discrete regions.

Two isoforms of the kidney androgen-regulated protein are encoded by two alleles of a single gene in OF1 mice.

Two cDNA clones coding for two forms of the mouse kidney androgen-regulated protein (KAP) distinguished by their electrophoretic mobilities on SDS gel electrophoresis have been isolated from libraries prepared from strains of mice having one (BALB/c) or two (OF1) forms of the KAP protein. The corresponding mRNAs have identical sizes, as well as identical sequences in their 5' non-translated regions. The size difference observed between the two proteins is due to two point mutations in the coding region of the KAP mRNA, leading to two amino-acid changes one of which resulted in the substitution of a glycine for a glutamic acid. As shown by in vitro transcription/translation experiments, these two amino-acid differences are responsible for the shift in the apparent molecular weight of the protein on SDS gels. Both forms of the protein are more abundant in males than in females. In vitro translation of kidney RNAs isolated from six different strains and species of mice revealed the presence of other forms of the KAP protein, characterized by small variations of their molecular weights. Southern blot analysis data are consistent with the presence of only one kap gene in the mouse genome. A restriction fragment length polymorphism has been observed, which does not correlate with the protein polymorphism, indicating the presence of another allele in the OF1 mouse genome.

Extra-renal transcription of the renin genes in multiple tissues of mice and rats.

Expression of the mouse renin genes (Ren-1 and Ren-2) and of the unique rat renin gene was determined in several extra-renal tissues of mice and rats by primer-directed enzymatic amplification of cDNAs. In addition to the adrenal glands, testis, and ovaries, renin transcripts are detected in the liver, whole brain, and hypothalamus and, at lower levels, in spleen, thymus, lung, and prostate. Expression of the rat renin gene correlates with that of the mouse Ren-1 gene with the notable exception of the submaxillary gland where renin transcripts are found only in mice. The levels of renin transcripts in the liver of females from both species are higher than in males. In mice, the relative levels of Ren-1 and Ren-2 transcripts vary widely in different tissues. These results support the hypothesis of a local renin-angiotensin system in multiple extra-renal sites and imply the existence of complex mechanisms of regulation of the renin gene, previously thought to be expressed in a tissue-specific manner.

High level of accumulation of a mRNA coding for a precursor-like protein in the submaxillary gland of male rats.

NaDodSO4/PAGE analysis of in vitro translation products of rat submaxillary gland (SMG) mRNAs has revealed an important sexual dimorphism. Moreover, most of the rat male-specific major translation products differ in size from those translated from male mouse SMG mRNAs. To characterize proteins accumulated in the rat SMG under androgen control, a cDNA library was constructed. Here we report the nucleotide sequence of a 0.7-kilobase mRNA that is 1000-3000 times more abundant in male rats than in female rats. The predicted corresponding protein, SMR1, has a molecular weight of 16,000 and contains a signal peptide for secretion and potential signals for glycosylation. An interesting feature of SMR1 is the presence, in a hydrophilic region, of the tetrapeptide Gln-His-Asn-Pro surrounded by two pairs of basic residues that represent potential cleavage sites for maturation enzymes. In rats, the tissue distribution of the SMR1 mRNA is restricted to the SMG and the prostate. Only very low amounts of SMR1 mRNA can be detected in the SMG of male or female mice. Southern blot analysis indicates the presence of three genes in rats but only one in mice. Hypotheses on the physiological role of SMR1-derived peptides in male rats are discussed.

Regulated expression of the Ren-2 gene in transgenic mice derived from parental strains carrying only the Ren-1 gene.

The Ren-2 gene encoding the mouse submaxillary gland (SMG) renin was microinjected into the pronuclei of fertilized eggs from mice carrying only the Ren-1 gene. In addition to the whole transcription unit, the injected DNA contained 2.5 and 3 kb of upstream and downstream flanking sequences, respectively. Three independent transgenic mice lines were obtained; two of them had integrated one copy of the Ren-2 gene, the last one had integrated five and eleven copies at two independent sites. Independently of the number of Ren-2 copies integrated, the pattern of Ren-2 gene expression in all the transgenic mice was identical to that observed in wild-type animals in which Ren-1 and Ren-2 are closely linked on chromosome 1. In particular, the exogenous Ren-2 gene was only transcribed in the kidney and in the SMG. In the kidney, Ren-1 and Ren-2 mRNAs were present at a comparable level, whereas in the SMG Ren-2 mRNA was at least 100-fold more abundant than Ren-1 mRNA. Moreover, Ren-2 expression in the SMG was positively regulated by androgens. Only one difference between transgenic mice and wild-type mice carrying the Ren-2 gene has been observed: the basal level of Ren-2 transcription in the SMG of transgenic females was lower than in two-gene strain females. Androgen treatment of transgenic females induced SMG renin mRNA to a level identical to that of transgenic males. This suggests that the basal level of SMG renin mRNA is dependent upon cis-acting elements which are not present in the microinjected fragment.

Renin-promoter SV40 large T-antigen transgenes induce tumors irrespective of normal cellular expression of renin genes.

Chimeric genes containing the 5'-flanking regions of the mouse renin genes, Ren1 and Ren2, associated with the early region of the simian virus 40 (SV40) were constructed. The two recombinant genes which contain, respectively, 0.45- and 2.5-kb the Ren1 and Ren2 5'-flanking sequences, named Ren1Tag and Ren2Tag, were microinjected into fertilized eggs. Tumors arose after a latency of 5-9 months in mouse lines harboring these hybrid genes except for one, in which a different and earlier pathology was observed (peripheral neuropathies). Most of the pathologies developed by these transgenic mice reflect the tumorigenic spectrum of the SV40 early region gene (choroid plexus, kidney, intestinal tumors, and peripheral neuropathies). None of these tumors arose from renin-producing cells nor produced renin. As suggested by the tumor pathology, the expression of the SV40 large T-antigen did not follow the normal expression of the Ren1 and the Ren2 genes since SV40 large T-antigen mRNA was found in tissues which normally do not express renin.