Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The catalytic reaction of renin, an aspartyl proteinase, with angiotensinogen is the rate-limiting step fo the renin-angiotensin system involved in the maintenance of blood pressure and electrolyte balance in mammals. We have characterized species-specific expression of the hepatic renin gene by RNase protection experiment, primer extension analysis, and promoter assay using an in vitro DNA transfection. RNase protection experiments revealed that the renin gene is expressed in rat liver, but neither in mouse nor in human. Primer extension analysis identified the putative promoter region of the rat renin gene, which contains TATAAAA sequence, a canonical regulatory DNA element. In order to test whether the upstream region of the renin gene with respect to the putative transcription initiation site is a functional promoter, we have examined the ability of the 5'-flanking sequences of the rat renin gene as well as the human and mouse genes to activate expression of a reporter gene containing the bacterial chloramphenicol acetyltransferase (CAT)-coding sequences, by transient transfection assays. In transfected HepG2 cells, a hepatoma cell line, only the rat renin promoter was capable of driving the CAT gene expression. These results suggested that the rat-specific renin gene expression in the liver could be primarily determined by its promoter specificity.
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PMID:Species-specific expression of the hepatic renin gene. 820 34

We used pregnant and pseudopregnant Wistar-Imamichi rats, prepared at a breeding farm, for production of transgenic rats. The donor and recipient rats were transported to our facility for ova manipulation and embryo implantation, respectively. As a foreign gene, 1.9 kb of a hybrid gene was constructed with the rat renin promoter and chloramphenicol acetyltransferase-coding gene. Fertilized oocytes were collected from the donor and the foreign DNA was microinjected into the pronuclei of 539 oocytes by the method of Hochi. The manipulated oocytes were cultured to the 2-cell stage. One hundred seventeen 2-cell embryos were implanted in the recipient rats. Of 67 newborns, 55 rats grew up to be 3-week-old weanlings. Genomic DNA was extracted from the tail of these weanlings and examined by polymerase chain reaction analysis. Six transgenic rats were found to have been generated by the present method. In this way, transgenic rats were produced by an efficient combination of breeding farm utilization and laboratory research.
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PMID:Production of transgenic rats using pregnant and pseudopregnant rats prepared at a breeding farm. 835 70

Identification of regulatory elements in the human renin gene promoter has been hindered by the lack of suitable renin-expressing cell lines. In this paper, the SV40 viral enhancer is coupled to a human renin promoter/chloramphenicol acetyltransferase (CAT) reporter gene construct, to determine whether promoter regulatory elements can be identified in the context of enhanced transcription in the normally non-renin-expressing HeLa cell line. The present results indicate that the SV40 enhancer can overcome tissue-specificity of the human renin promoter, and confer correctly initiated transcriptional activity to HeLa cells. Analysis of a series of 5'-end deletions of the human renin promoter linked to the CAT gene and SV40 enhancer identified negative regulatory elements between positions-275 and -225, and between-142 and -102 of the human renin promoter, as well as a positive regulatory element between -225 and -142. Thus, studies of renin promoter regulatory elements need not be limited to renin-expressing cells, but can be performed in non-renin-expressing cells with the addition of an enhancer. This strategy can be generally applicable to the study of tissue-specific gene regulation in cases where there are no specific cell lines available.
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PMID:Deletional analysis of the human renin promoter: transcriptional activation by the SV40 enhancer enables identification of promoter regulatory elements in non-renin-expressing cells. 839 Sep 24

Angiotensin I-converting enzyme (ACE) is a key enzyme in the regulation of systemic blood pressure and plays a major role in the renin-angiotensin and bradykinin-kinin systems, at the luminal surface of the vascular endothelia. To identify the promoter region, the transcription regulatory elements and the cell specificity of the ACE gene, five successive DNA deletions of the 5' upstream region (-1214, -754, -472, -343, -132 bp relative to the start site of transcription) were isolated and fused in sense and antisense orientations to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene in the promoterless plasmid pBLCAT3. Promoter activities were measured in transient transfection assays using three different cell lines from rabbit endothelium (RE), human embryocarcinoma (Tera-1) and hepatocarcinoma cells (HepG2). All five fragments of the ACE promoter region directed expression of the CAT gene when transfected into the endothelial and the embryocarcinoma cells, which contain endogenous ACE mRNA and express ACE activity. In contrast only minimal levels of promoter activity were obtained on transfection into hepatocarcinoma cells in which endogenous ACE mRNA and ACE activity were not detected. Transfection of RE and Tera-1 cells demonstrated that promoter activity was defined by the length of the ACE promoter sequence inserted into the construct. The 132 bases located upstream from the transcription start site were sufficient to confer ACE promoter activity, whereas the sequences upstream from -472 bp and between -343 bp and -132 bp were responsible for a decrease of promoter activity. Furthermore, the minimal 132 bp of the ACE promoter contains elements which direct cell-specific CAT expression. In addition, the DNA transfection study in the presence of dexamethasone suggested that the potential glucocorticoid regulatory elements, located in the sequence of the ACE promoter, are not functional.
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PMID:Functional analysis of the human somatic angiotensin I-converting enzyme gene promoter. 839 96

Renin, a key enzyme controlling blood pressure, is mainly synthesized in kidney. To characterize the rat renin promoter, we have constructed a reporter gene containing the 238-bp putative regulatory region linked to a bacterial chloramphenicol acetyltransferase (CAT), and analyzed its promoter activity by in vitro transfection and introduction of the CAT fusion gene into germline of mice. CAT activity was detected in transfected embryonic kidney-derived 293 cells, but not in HeLa cells derived from cervical carcinoma, showing that the putative promoter region of the rat renin gene directs transcription in a cell type-dependent manner. To examine whether the sequence was sufficient to regulate the expression of the CAT chimeric gene in mice, we generated seven transgenic mice carrying the reporter construct. Unexpectedly, the transgene was not expressed in any of the independent transgenic mice examined. These results suggest one possibility that an additional control region may be required for efficient expression of the rat renin promoter in developing mice.
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PMID:Rat renin promoter activity in cultured cells and transgenic mice. 839 29

Although transfection of renin gene into adult liver resulted in increased blood pressure (BP) for 1 week, sustained transgene expression must be considered to produce a continuous hypertensive animal. We hypothesized that gene transfer into neonatal rats would result in long-term transgene expression, given with highly replicating hepatocytes in neonates. Initially, chloramphenicol acetyltransferase (CAT) vector was transfected into the liver of 1-day-old rats. Immunohistochemical staining showed positive staining of CAT throughout the liver. Therefore, we transfected renin vector to study biological effects. At 2, but not 4 and 8, weeks, a significant increase in plasma angiotensin II concentration was observed in rats transfected with renin vector. Expression of renin mRNA in the liver transfected with renin vector could be detected at least up to 6 weeks, while no significant changes in BP were observed. These results demonstrated that in vivo gene transfer into the neonatal liver resulted in sustained transgene expression, suggesting the potential use of in vivo gene transfer as a tool to produce a novel model.
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PMID:Sustained transgene expression by transfection of renin gene into liver of neonates. 869 31

Mouse As4.1 cells, obtained after transgene-targeted oncogenesis to induce neoplasia in renal renin-expressing cells, express high levels of renin mRNA from the endogenous Ren-1(c) gene. We have used these cells to characterize the role of the Ren-1(c) proximal promoter (+6 to -117) in the regulation of renin gene transcription. It was found that 4.1 kilobases (kb) of Ren-1(c) 5'-flanking sequence, in combination with the proximal promoter, are required for strong activation (approximately 2 orders of magnitude over the basal level of the promoter alone) of the chloramphenicol acetyltransferase reporter in transfection assays. Within the 4.1-kb fragment, a 241-base pair region was identified that retains full activity in an orientation-independent manner in combination with the promoter. The resulting transcripts initiate at the normal renin start site. Electrophoretic mobility shift assays identified a sequence at approximately position -60 in the promoter region that binds nuclear proteins specific for renin-expressing As4.1 cells. Mutations in this sequence, which disrupt binding of nuclear protein(s), completely abolish activation of transcription by the 4. 1-kb fragment. Activation of transcription by the 241-base pair enhancer was still observed, although it was diminished in magnitude (60-fold over the mutated promoter alone). We present a model derived from the current data that suggests that regulation of renin expression is achieved through cooperation of transcription factors binding at the proximal promoter element and a distal enhancer element to abrogate or override the effects of an intervening negative regulatory region.
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PMID:Role of proximal promoter elements in regulation of renin gene transcription. 879 16

The As4.1 cell line was established from a mouse kidney tumor by transgene-targeted tumorogenesis. These cells express high levels of renin mRNA from their endogenous renin gene and release approximately eightfold-more prorenin than active renin in culture. Levels of renin mRNA in As4.1 cells are decreased in a dose-dependent manner by the addition of physiological concentrations of cytokine interleukin-1 to the media. Stability of renin mRNA and initial rates of release of active renin and prorenin were not significantly altered by interleukin-1. In contrast, transcription initiated from a construct that consisted of 4.1 kilobases of renin 5' flanking sequence fused to a reporter gene (chloramphenicol acetyltransferase) was markedly inhibited by interleukin-1. On the basis of our findings, we conclude that downregulation of renin synthesis caused by interleukin-1 occurs primarily at the level of transcription and that DNA sequence or sequences mediating that effect are positioned within 4.1 kilobases upstream of the renin gene. The physiological relevance of this regulation is related to the events that occur during septic shock, characterized by hypotension, cardiovascular collapse, multiple organ failure, and high mortality. Unexpectedly, hypotension associated with septic shock does not lead to activation of the renin-angiotensin system. The hypotension in septicemia is believed to be mediated by the combined action of many modulators including cytokines, and data presented here suggest direct involvement of interleukin-1 in this process.
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PMID:Downregulation of renin gene expression by interleukin-1. 926 Sep 85


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