Generation of Superoxide and In Vitro Inactivation of Viruses by Aldopentoses

Ren-1 renin is synthesized in the kidney of every mouse. Ren-2 renin has been observed in the submandibular gland (SMG) of male mice carrying two renin genes. However, it is not known if Ren-2 renin is in the kidney and blood of the two-renin gene mice. In this study, a direct ELISA for Ren-2 renin (SMG renin) was established by a sandwich method. This ELISA could measure the Ren-2 active renin in the range from 1 to 100ng and distinguish Ren-2 active renin from not only Ren-1 renin but also Ren-2 prorenin. By a combination of this assay system and conventional methods, the pro-form as well as the active form of Ren-2 renin was found in the kidney and plasma of male AKR mice carrying two-renin genes.     

Kidney and submaxillary gland renins are encoded by two non-allelic genes in Swiss mice.

Two distinct phenotypic groups of inbred strains of mice, with different amounts of submaxillary gland (SMG) renin have been described. We have previously shown that strains with high levels of SMG renin, such as Swiss or AKR mice, have two renin genes, Rn1 and Rn2, per haploid genome, while strains with low levels of SMG, such as BALB/c or C57Bl/6, have only one renin gene. We now report the molecular cloning of cDNA copies of Swiss mouse kidney renin mRNA and present nucleotide sequence data of the recombinant clones. Comparison of these sequences with the sequence of Swiss mouse SMG renin mRNA we have previously reported, demonstrates that Swiss mice express the two non-allelic genes, Rn1 and Rn2.     

Evolution and Variation of Renin Genes in Mice

Inbred strains of mice carry Ren-1, a gene encoding the thermostable Renin-1 isozyme. Ren-1 is expressed at relatively low levels in mouse submandibular gland and kidney. Some strains also carry Ren-2, a gene encoding the thermolabile Renin-2 isozyme. Ren-2 is expressed at high levels in the mouse submandibular gland and at very low levels, if at all, in the kidney. Ren-1 and Ren-2 are closely linked on mouse chromosome 1, show extensive homology in coding and noncoding regions and provide a model for studying the regulation of gene expression. An investigation of renin genes and enzymatic activity in wild-derived mice identified several restriction site polymorphisms as well as putative variants in renin gene expression and protein structure. The number of renin genes carried by different subpopulations of wild-derived mice is consistent with the occurrence of a gene duplication event prior to the divergence of M. spretus (2.75–5.5 million yr ago). This conclusion is in agreement with a prior estimate based upon comparative sequence analysis of Ren-1 and Ren-2 from inbred laboratory mice.     

Molecular mechanism of tissue-specific regulation of mouse renin gene expression by cAMP. Identification of an inhibitory protein that binds nuclear transcriptional factor.

Renin gene expression in the mouse kidney and submandibular gland (SMG) are differentially regulated by cAMP. In this study, we examined the potential molecular mechanism responsible for this tissue-specific regulation. 32P end-labeled synthetic oligonucleotide containing mouse renin cAMP-responsive element (CRE) was incubated with kidney nuclear extracts from either control or cAMP-treated mice and analyzed by gel mobility shift assay. Our results demonstrated that cAMP induced a nuclear protein which complexed with the CRE oligonucleotide in a specific manner. This nuclear protein-DNA binding was competed effectively by the oligonucleotide containing human chorionic gonadotropin alpha-subunit CRE but not by the mouse renin DNA fragment from which the CRE was deleted by site-directed mutagenesis. In contrast, no DNA-protein complex formation could be detected when this [32P]CRE oligonucleotide was incubated with the SMG nuclear extract from control or cAMP-treated mice. However, CRE-binding protein complex formation was demonstrated in the SMG nuclear extract when the incubation was performed in the presence of 0.8% sodium deoxycholate and 1.2% Nonidet P-40, detergents that dissociate protein-protein complexes. Furthermore, in the absence of deoxycholate, we observed that SMG nuclear extract attenuated the binding of the kidney CRE-binding protein to mouse renin CRE in a dose-dependent manner and this inhibitory effect of SMG nuclear extract disappeared in the presence of sodium deoxycholate. This inhibitory nuclear protein in SMG is specific for CRE-binding protein since it does not affect nuclear protein binding to synthetic DNA oligonucleotides of human collagenase AP-1 and human metallothionein AP-2. Our data further suggest that inhibitory nuclear protein is present in lower quantities in other extrarenal tissues, i.e. testes, liver, brain, heart, but is not detectable in the kidney. Taken together, these results suggest that the SMG and certain extrarenal tissues contain nuclear trans-acting factor(s) that interact with CRE-binding protein, thereby interfering with its binding to mouse renin CRE. The presence of this inhibitory protein in the mouse SMG nucleus may contribute to the tissue-specific regulation of the renin gene expression by cAMP.     

Physical Characterization of Genetic Rearrangements at the Mouse Renin Loci

Many inbred strains of mice have a single locus encoding renin, Ren-1, whereas other inbred strains have two tandemly linked loci, Ren-1 and Ren-2. Each of these renin genes in inbred mice exhibits a unique pattern of tissue-specific expression. As a prerequisite to understanding the structural basis for the expression differences, we have physically characterized the sequence organization of this chromosomal region in both types of strains. Pulsed field gel electrophoresis was initially used to compare the long-range structure of this region in C57BL/6 (Ren-1) and DBA/2 (Ren-1 + Ren-2) mice. The structure in both inbred strains is extremely similar, except for an additional 30 kb containing Ren-2 in DBA/2 mice. The boundaries of the extra 30-kb segment were sequenced and compared to homologous sequences flanking the Ren-1 alleles. This analysis identified the precise recombination site, and also the presence of a large insertion, between the renin loci in DBA/2. The renin gene duplication apparently resulted from recombination between sequences sharing little homology, suggesting that nonhomologous chromosomal breakage and rejoining may have been involved mechanistically in the event.     

Close physical linkage of the murine Ren-1 and Ren-2 loci

In addition to the Ren-1 gene common to all mice, some inbred strains carry a second copy of the renin structural gene, Ren-2. These two loci are tightly linked genetically on mouse chromosome one. We have used pulsed field gel electrophoresis (PFGE) to study the physical arrangement of the two renin genes in the inbred strain DBA/2. PFGE mapping permitted the construction of a restriction map of the Ren loci spanning roughly 120 Kb. The results indicate that the genes are transcribed in the same relative direction, that Ren-2 lies upstream relative to Ren-1, and that the respective coding sequences are separated by approximately 20 Kb.     

The mouse Rn locus: S allele of the renin regulator gene results from a single structural gene duplication.

Inbred strains of mice have been divided into two distinct phenotypic groups having different levels of renin activity regulated by androgen in the submaxillary gland (SMG). Strains carrying the Rnrs allele of the renin gene regulator, located on chromosome 1, have a high level of renin activity; strains carrying the Rnrb allele have a low level of renin activity. The level of SMG renin activity correlates with the level of renin mRNA. We have analyzed, by Southern blot hybridization, the organization of renin genes in both strains. Strains carrying the Rnrb allele, such as BALB/c or C57 Bl/6, or CH3 mice, have one renin structural gene per haploid genome, while those having the Rnrs allele, such as AKR or Swiss mice, have two renin genes. We have also identified renin genes in mice belonging to different biochemical groups: Mus spretus has one renin gene while M. vrania and M. musculus brevirostris have two renin genes.     

N-linked glycosylation affects the processing of mouse submaxillary gland prorenin in transfected AtT20 cells

Most mouse inbred strains carry two renin genes, Ren-1 and Ren-2, Renin-2, the product of the Ren-2 gene, is highly expressed in the submaxillary gland. It is a renin isoenzyme 96% similar to kidney renin-1, but unglycosylated. In order to investigate if glycosylation of prorenin affects its processing and/or secretion we have introduced two potential N-linked glycosylation sites into preprorenin-2 cDNA using site-directed mutagenesis. Expression plasmids were derived from wild-type and mutant renin-2 cDNA and were transfected into AtT20 cells. Both transfected cells, expressing glycosylated or unglycosylated forms, secreted prorenin and renin by the constitutive and regulated pathways, respectively. Prorenin was correctly processed to active renin but the second maturation site was not cleaved in AtT20 cells. The comparison of glycosylated and unglycosylated renin expression showed a diminished secretion of glycosylated active renin. Prevention of glycosylation with tunicamycin resulted in an improved secretion of active renin. Moreover, the efficiency of the trypsin activation in vitro was reduced for glycosylated prorenin and it was restored when the activation was performed on mutant renin secreted from tunicamycin-treated cells. It is proposed that the bulky carbohydrates attached to prorenin constitute a steric hindrance to proteolysis by maturation enzymes.     

Molecular cloning of two distinct renin genes from the DBA/2 mouse.

We report the molecular cloning of cDNA copies of DBA/2 mouse submaxillary gland (SMG) renin mRNA, which were used to probe Southern transfers of mouse genomic DNA. The results suggested either that there is a single renin gene containing a large intron in that part of the gene corresponding to the probe, or that there are two distinct renin genes. We have shown that the latter is the case by cloning and isolating two similar but distinct renin genes from DBA/2 mouse DNA. Restriction maps of the regions containing the two renin genes are presented, together with nucleotide sequence data locating a complete exon coding for amino acids 268-315 of mouse SMG renin.     

A DNA polymorphism,consistent with gene duplication,correlates with high renin levels in the mouse submaxillary gland

The renin regulatory locus (Rnr) is a genetic element governing mouse submaxillary gland (SMG) renin levels. A 45,000 dalton polypeptide detectable after in vitro translation of mouse SMG mRNA has been identified by genetic and physical criteria as SMG renin. A cDNA recombinant clone specific for SMG renin has been isolated and used to demonstrate that the previously described genetic regulation of SMG renin levels is manifest at the level of renin mRNA concentration. The renin cDNA clone has also been used in Southern blot analyses to study the organization of homologous DNA sequences in strains carrying different alleles at the Rnr locus. Restriction digest patterns of high renin strains (Rnr^s) are characteristically distinct from patterns observed for low renin strains (Rnr^b) and are suggestive of a structural gene duplication at the chromosome 1 locus in high renin strains. However, gene dosage cannot account for the increased levels in high renin strains, since SMG renin levels in Rnr^s and those in Rnr^b may differ up to 100-fold.     

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.     

Molecular cloning and nucleotide sequence of a human renin cDNA fragment.

We have studied human renin messenger RNA by hybridization with the mouse submaxillary gland (SMG) renin cDNA probe. The human kidney messenger RNA is about 1.6 kilobase (kb) long, similarly to the mouse SMG renin mRNA. A kidney renin cDNA clone of 1.1 kb length was obtained. A comparison of nucleotide sequences of mouse and human cDNA clones reveals conservation of residues involved in catalytic mechanisms and a potential glycosylation site. The human renin molecular probe allowed us to study renin expression in human chorionic tissue. The chorionic and kidney renin messenger RNAs are similar in length. The Southern blot analysis reveals the presence of a single renin gene in human DNA.