Cloning and sequence analysis of cDNA for rat angiotensinogen.

Abstract

A mixture of tetradecamer oligodeoxyribonucleotides complementary to the codons specifying the carboxylterminal sequence, Ile-His-Pro-Phe-His, of angiotensin was chemically synthesized as two pools and used for the isolation of a cDNA clone specific for angiotensinogen from a cDNA bank of rat liver mRNA sequences. The two pools (oligo 1 and oligo 2), each containing 24 oligodeoxyribonucleotides, were first used as primers to initiate reverse transcription of rat liver mRNA. One of the pools (oligo 1) was found to prime a specific 32P-labeled cDNA of approximately 160 nucleotides that contained the anticoding sequence corresponding exactly to the amino acid sequence of rat angiotensin. This cDNA, in turn, was used to rescreen cDNA clones that were isolated by initially selecting the rat liver cDNA bank by hybridization with the oligo 1 mixture. One clone thus obtained, designated pRagl6, was subjected to nucleotide sequence analysis and verified to contain a nearly full-length cDNA sequence coding for rat angiotensinogen precursor. The deduced amino acid sequence indicates that the precursor molecule consists of angiotensinogen of 453 amino acid residues and a putative signal peptide of 24 amino acid residues. The predicted molecular weight and amino acid composition of angiotensinogen agree well with those determined by using the purified protein. An angiotensin moiety is located at the amino-terminal part of angiotensinogen, preceded directly by the signal peptide and followed by a large carboxyl-terminal sequence that contains two internally homologous sequences and three potential glycosylation sites.Angiotensinogen (renin substrate), a glycoprotein mainly synthesized by the liver and secreted into the circulating blood, is cleaved by the enzyme renin (EC 3.4.99.19), thus releasing the decapeptide angiotensin I. The latter is then cleaved by angiotensin converting enzyme (dipeptidyl carboxypeptidase, peptidyldipeptide hydrolase, EC 3.4.15.1), forming the octapeptide angiotensin II. Angiotensin II is the principal biologically active peptide that causes arteriolar vasoconstriction and stimulates aldosterone secretion from adrenal cortex (reviewed in refs. 1 and 2). Angiotensin formation has also been shown to occur in the central nervous system, where angiotensin appears to be involved in causing thirst and in control of the secretion of vasopressin and corticotropin (ACTH) (3). Through these actions, the renin-angiotensin system plays an important role in the control of blood pressure and hydromineral balance. Evidence is accumulating that the level of angiotensinogen in the circulation is as important as that of renin in determining the rate of formation of angiotensin and therefore the activity of the renin-angiotensin system (1). Angiotensinogen production is regulated by several factors such as glucocorticoids, estrogens, and angiotensin II (1, 4). However, the mechanisms responsible for the regulation of angiotensinogen production have not been elucidated.Recently, angiotensinogen has been purif...

Insulin-Mediated Growth in Aortic Smooth Muscle and the Vascular Renin-Angiotensin System

“…Insulin treatment (1000 U/mL) increased angiotensinogen mRNA expression by 25-fold (arbitrary density units, PϽ0.05), whereas it had no effect on renin or AT 1 -R mRNA expression (Figure 1, top and bottom). Northern blot analysis for insulin-stimulated angiotensinogen mRNA production in VSMCs showed a similar several-fold increase in angiotensinogen with insulin, with its signal set at approximately 18S 28 (data not shown).…”

“…Total RNA was quantified by absorbance at 260 nm and stored at Ϫ80°C until use. Messenger RNAs of angiotensinogen, 28 renin, 29 AT 1 -R, 30,31 and GAPDH were quantified in the total RNA extract by RNA protection assay with […”

“…Blots were prehybridized at 45°C for 4 hours in buffer including 5ϫ SSPE, 50% formamide, 5ϫ Denhardt's solution, 50 g/mL denatured salmon sperm DNA, 0.5% SDS, and 1% PEG and then hybridized in the same buffer with 1 to 2ϫ10 6 cpm of probe at 45°C for 20 hours. Probes were synthesized to a complete rat angiotensinogen cDNA, 28 pRang-6, or to complete rat renin cDNA, 29 pRen-31, which had been cloned in pGEM-4 (Promega). A KpnI/EcoRI cDNA fragment that encodes for rat AT 1A -R mRNA was kindly provided by Dr Jeffrey K. Harrison (University of Florida, Gainesville, Fla).…”

Reactive oxygen species blockade and action of insulin on expression of angiotensinogen gene in proximal tubular cells

“…After denaturation at 94 C for 3 min, angiotensinogen, renin and -actin cDNAs were co-amplified in the same tube under the following conditions: 94 C for 20 s, 60 C for 20 s, and 72 C for 40 s. After 30 cycles, PCRs were further extended at 72 C for 5 min. The sense and antisense rat angiotensinogen primers used were 5 -CCT CGC TCT CTG GAC TTA TC-3 and 5 -CAG ACA CTG AGG TGC TGT TG-3 , corresponding to nucleotide sequences N+676 to N+695 and N+882 to N+901 of rat angiotensinogen cDNA (Ohkubo et al 1983) respectively. The sense and antisense rat renin primers were 5 -CTG CCA CCT TGT TGT GTG AG-3 and 5 -CCA GTA TGC ACA GGT CAT CG-3 , corresponding to the nucleotide sequences N+1033 to N+1052 and N+1277 to N+1296 of rat renin cDNA (Burnham et al 1997) respectively.…”

Computer selection of oligonucleotide probes from amino acid sequences for use in gene library screening

“…Inclusion of presynthesized trimers has the effect that the MDT mode calculates the actual number of mRNA sequences which could code for the amino acid sequence of the probe. Consequently, this calculation mode will be of greatest value to users who wish to synthesize each of the possible mRNA sequences separately for independent use (9).…”