Cloning and sequence analysis of cdna for rat angiotensinogen (angiotensin/recombinant DNA/DNA sequence/blood pressure)

Transcription

1 Proc. Natl Acad. Sci. USA Vol. 80, pp , April 1983 Biochemistry Cloning and sequence analysis of cdna for rat angiotensinogen (angiotensin/recombinant DNA/DNA sequence/blood pressure) HIROAKI OHKUBO*, RYoICHIRO KAGEYAMA*, MAYUMI UJIHARA*, TADAAKI HIROSEt, SEICHI INAYAMAt, AND SHIGETADA NAKANISHI* *Institute for Immunology, Kyoto University Faculty of Medicine, Kyoto 606, Japan; and tpharmaceutical Institute, Keio University School of Medicine, Tokyo 160, Japan Communicated by Joseph L. Goldstein, January 20, 1983 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 ), thus releasing the decapeptide angiotensin I. The latter is then cleaved by angiotensin converting enzyme (dipeptidyl carboxypeptidase, peptidyldipeptide hydrolase, EC ), 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 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact responsible for the regulation of angiotensinogen production have not been elucidated. Recently, angiotensinogen has been purified to homogeneity from several species and the estimated molecular weights of the protein molecules of rat, human, and hog angiotensinogens have been reported to be about 50,000 (4, 5). Direct sequence determination of amino-terminal portions of rat and human angiotensinogens has shown that angiotensin I decapeptide is located at the amino-terminal part of the precursor molecule (6, 7). However, neither the primary structure of a large remaining portion of the precursor molecule nor its possible biological function have been elucidated. To study the regulatory mechanisms involved in the production of angiotensinogen and to reveal its entire primary structure, we have constructed an angiotensinogen cdna clone from rat liver mrnas. Here, we present the nucleotide sequence of the cloned cdna which allows us to predict the entire coding sequence for rat angiotensinogen together with the structure of the 3' untranslated region and most of the 5' untranslated region. MATERIALS AND METHODS Primer Extension Reaction. As glucocorticoids have been reported to stimulate the synthesis of rat liver angiotensinogen (4, 8), total liver RNA was prepared as described (9) from adult male Wistar rats that were administered dexamethasone (10,ug/ ml of drinking water) for 4 days. Poly(A)-containing RNA was obtained by twice subjecting the total RNA extracted to oligo(dt)- cellulose chromatography (10). The primer extension reaction was carried out with 50 ng of a mixture of 24 synthetic oligodeoxyribonucleotides labeled with 32P at the 5' end (3 X 106 cpm/pmol), 150 Ag of poly(a)-containing RNA as template, and 525 units of avian myeloblastosis virus reverse transcriptase in a total volume of 150,ul as described (11). The mixture of oligodeoxyribonucleotides was synthesized by the modified triester method (12). The cdna synthesized was fractionated in a 7% polyacrylamide/7 M urea gel as described (13). Cloning Procedure. A cdna library was constructed by the method of Okayama and Berg (14) using 20,ug of poly(a)-containing RNA and 5,ug of the vector/primer DNA. Escherichia coli HB101 or X1776, transformed and selected for ampicillinresistance as described (15), was first screened by hybridization (16) at 36 C (17) with a mixture of 24 synthetic oligodeoxyribonucleotides described in Results. The hybridization-positive clones obtained were then screened by hybridization at 50 C (16) with a radioactive angiotensin-specific cdna probe. All of the cloning procedures were conducted in accordance with the guidelines for research involving recombinant DNA molecules issued by the Ministry of Education, Science, and Culture of Japan. Analytical Procedures. Procedures for restriction endonuclease digestion and 5'-end labeling of DNA were as described

2 Biochemistry: Ohkubo et al. (18). DNA sequence analysis was- carried out by the procedure of Maxam and Gilbert (13). RNA blot hybridization analysis was carried out according to the procedure of Alwine et al (19); poly(a)-containing RNA isolated from livers of dexamethasonetreated rats was denatured with 1 M glyoxal/50% dimethyl sulfoxide (20), electrophoresed on a 1.5% agarose gel, and transferred to diazobenzyloxymethyl~paper. The hybridization probe was labeled by nick-translation (21). Proc. Natl. Acad. Sci. USA 80 (1983) 2197 RESULTS AND DISCUSSION Isolation and Characterization of a cdna Clone. Our approach for the isolation of a cloned DNA sequence specific for angiotensinogen involved chemical synthesis of two mixtures of A_ G A 24 oligodeoxyribonucleotides each, 5' T-G- -A-A- -G-G- -T- G C G A A_ A_ A A G-G-A-T 3' (oligo 1) and 5' T-G- -A-A- -G-G- -T-G-G-A-T 3' T G T G T (oligo 2), as shown in Fig.. la. These tetradecamers represent all possible.complementary sequences corresponding to the pentapeptide sequence Ile-His-Pro-Phe-His of rat angiotensin I (excluding the third nucleotide residue of the ninth histidine codon for angiotensin I). The oligodeoxyribonucleotide mixtures were used as primers to initiate reverse transcription of rat liver mrna. The oligo 1 and oligo 2 primers were labeled with 32P at the 5' end and single-stranded cdnas were extended from these primers with reverse transcriptase. The cdnas synthesized were analyzed by urea/polyacrylamide gel electrophoresis (Fig. lb). Similar electrophoretic patterns were observed between the cdnas extended from the oligo 1 primer and those from the oligo 2 primer, except that a band with a mobility corresponding to a size of approximately 160 nucleotides was missing in the oligo 2-primed cdnas. To identify the primed cdna that contained an angiotensin-specific nucleotide sequence, two major transcripts with mobilities corresponding to sizes of approximately 160 and 250 nucleotides were isolated and their sequences were determined by the procedure of Maxam and Gilbert (13). A sequence of 12 nucleotide residues immediately upstream from the primer sequence in the cdna of approximately 160 nucleotides but not in that of approximately 250 nucleotides coincided precisely with the anticoding sequence corresponding to the amino-terminal amino acid sequence Asp-Arg-Val-Tyr of angiotensin I (data not shown). Thus, the cdna of approximately 160 nucleotides represents a specific reverse transcription of angiotensinogen mrna. Because the radioactivity of the angiotensin-specific cdna thus obtained did not seem to be enough for direct use as a hybridization probe to screen the rat liver cdna bank, cdna clones that carry DNA sequences complementary to one of the oligo 1 tetradecamer sequences were first selected by hybridization with the 32P-labeled oligo 1 mixture. In this way, 17 hybridization-positive clones were isolated from about 70,000 transformants derived from the rat liver cdna bank. When these 17 hybridization-positive clones were rescreened with the angiotensin-specific cdna probe, one clone, pragl6, was found to hybridize positively. Clone pragl6 carried a cdna insert of about 1,900 nucleotide residues, including the poly(da)poly(dt) and poly(dg). poly(dc) tails. Restriction endonuclease analysis (Fig. 2) of the cloned cdna indicated that all, the restriction sites observed in the nucleotide sequence determined for the primer-extended approximately 160-nucleotide cdna were located in the 5'-terminal portion of the cloned cdna with direct one-to-one correspondence. In agreement with this result, nucleotide sea angiotensin I amino acid sequence Ile His Pro Phe His mrna U 5'-AUC CAU CCC UUU CAU-3' A G A A G A oligo 1 3'-TAG GTG GGC AAG GT -5' T oligo 2 3'-TAG GTG GGT AAG b A Or~~~~~~~~~~~~~~~~~~~~~~~~~~~ALg 281- ff3 194 Origin B T GT -5' FIG. 1. (a) Synthetic oligodeoxyribonucleotides used for priming reverse transcription of rat liver mrna and for probing cloned cdna for rat angiotensinogen. All possible coding sequences and the corresponding tetradecamers synthesized as two pools (designated oligo 1 and oligo 2) are given for the carboxyl-terminal pentapeptide sequence of rat angiotensin I (fifth to ninth residue of angiotensin I). (b) Urea/ polyacrylamide gel electrophoresis of cdnas primed by the mixture of synthetic oligodeoxyribonucleotides. cdna was.synthesized with rat liver poly(a)-containing RNA using two kinds of oligodeoxyribonucleotide mixtures as primers and analyzed on a 7% polyacrylamide/7 M urea gel. An autoradiogram is shown: lane A, oligo 1-primed cdnas; lane B, oligo 2-primed cdnas. The sizes of the primed cdna species given in nucleotides on the right side of the autoradiogram were estimated by comparison with the 5'-end labeled Hae m cleavage products of 4X174 DNA, the sizes of which are given in nucleotides on the left side of the autoradiogram. quence analysis of the cloned cdna in both directions from one of these restriction sites, Acc I (nucleotide residue 81), showed that the nucleotide sequence surrounding the Acc I site corresponded exactly to the amino-terminal sequence of 17 amino acid residues, including angiotensin I, reported for rat angiotensinogen (6). Nucleotide Sequence of mrna Codingfor.Angiotensinogen Precursor and Assignment of Protein Sequence. Clone pragl6 was subjected to nucleotide sequefice analysis according to the strategy indicated in Fig. 2. The nucleotide sequence was determined on both strands of the cdna for all but 53 and 16 residues corresponding to the 5' and 3' ends of the mrna, respectively; for these regions, sequence determination on both strands was technically difficult, but the sequence data were reliable. The primary structure of the mrna coding for the angiotensinogen precursor (preangiotensinogen) was deduced from the 1,762-nucleotide sequence determined (Fig. 3). Angiotensin I is encoded by nucleotide residues The 5'-terminal sequence of the mrna deduced from the inserted cdna se-

3 2198 Biochemistry: Ohkubo et al. Proc. Natl. Acad. Sci. USA 80 (1983) -I I Y 1 0 n ax *4Ncm)) OD (. O cm _ ) _ =I _IK-Lt 1I11 111z CI 0OD It to '1 = Zo Ib O tou)n o-z IC) z =Z = V ar((4 UOQ~~I -) Nl 4 o4 aq- v) if) OD O00) W dov ) to T (D OD 4 _3_) _ > 4 b > 4 - < l 0I -* a- <~~l4 -l l * -l- of b -I - l a He * - I - FIG. 2. Strategy for determining the sequence of the cdna insert in clone pragl6. The map displays only the relevant restriction endonuclease sites, which are identified by numbers indicating the 5'-terminal nucleotide generated by cleavage (for the nucleotide numbers, see Fig. 3). The sequence corresponding to the coding region is indicated by the open box; the closed box indicates the coding region for angiotensin I. The sequence used as a hybridization probe for screening a cdna bank and for priming the reverse transcription is indicated by the line directly beneath the closed box; the wavy line represents the primer-extended cdna. The poly(da)poly(dt) and poly(dg)-poly(dc) tails are not included in the map. The direction and extent of sequence determinations are shown by horizontal arrows; the sites of 5'-end labeling are indicated by short vertical lines on the arrows. quence extended to 159 nucleotides upstream from the nucleotide residue corresponding to the 5' end of the oligodeoxyribonucleotides used for primer extension. This size is closely similar to that of the angiotensin-specific cdna extended from the oligo 1 primer (approximately 160 nucleotides long) (Fig. lb). This indicates that the cdna insert in clone pragl6 contains almost all of the sequence corresponding to the 5'-terminal sequence of the mrna. On the basis of this finding, the translational initiation site was assigned to the methionine codon AUG at position 1-3, which is the first AUG triplet within the mrna sequence deduced. This assignment was further supported by the presence of an in-frame nonsense codon UAG (residues -60 to -58) located upstream from the putative initiation methionine. The first 24 amino acid residues starting with the initiation methionine and proceeding to the amino acid sequence of angiotensin include a large number of hydrophobic amino acids (16 nonpolar residues) and terminate in a residue with a small neutral side chain, glycine. This characteristic structure would represent the signal peptide similar to that of other secretory proteins (22). The reading frame shown in Fig. 3 is the only one without multiple termination codons and consists of 1,4 nucleotide residues encoding 477 amino acid residues, which are followed by the termination codon, UGA. Removal of the putative signal peptide by cleavage between the glycine at position 24 and the first amino acid (aspartate-25) of angiotensin would result in a mature angiotensinogen of 453 amino acid residues, with a calculated molecular weight of 49,548. This value agrees well with the molecular weight of 50,000 for the carbohydrate-free angiotensinogen, which was calculated from the glycosylated form of the purified protein (Mr = 56,400) (5). Relative to this, the amino acid sequence shown in Fig. 3 was found to possess three potential glycosylation sites conforming to the canonical Asn-X- Ser (or threonine) sequence (23) at amino acid positions 47-49, , and The amino acid composition deduced from the nucleotide sequence agrees with the composition reported from amino acid analysis of purified rat angiotensinogen (5) (Table 1). All of these results support the authenticity of the amino acid sequence deduced from the cloned cdna sequence. The 3' untranslated region of the mrna is 270 nucleotides long [excluding the poly(a) tract]. It has been suggested that the hexanucleotide A-A-U-A-A-A found nucleotides upstream from the polyadenylylation site in most eukaryotic mrnas serves as a signal for polyadenylylation (24). However, instead of this common sequence, 10 consecutive adenosine residues (nos. 1,671-1,680) were found at the corresponding region of rat preangiotensinogen mrna, although the sequence A-A-U- A-A-A (residues 1,572-1,577) appeared 125 nucleotides upstream from the polyadenylylation site. To correlate the length of rat preangiotensinogen mrna with the size of the cdna insert, rat liver poly(a)-containing RNA was analyzed by the blot hybridization technique. One band was observed corresponding to a length for the mrna of approximately 1,850 nucleotides (Fig. 4). Assuming a length for the poly(a) tail of approximately 90 nucleotides, the size of the mrna estimated is consistent with that of the inserted cdna sequence in clone pragl6. Thus, the consecutive adenosine residues may be involved in polyadenylylation after transcription, although the nucleotide sequence deduced from a single cdna clone must be viewed cautiously because of the possibility of errors occurring during in vitro cdna synthesis or DNA cloning. It should be noted that preangiotensinogen exhibits a molecular organization similar to that of the arginine vasopressin/ neurophysin II precursor, in that the biologically active peptide (angiotensin or vasopressin) in both precursor molecules is preceded directly by a signal peptide and followed by a long carboxyl-terminal sequence (25). Moreover, as with the neurophysin II sequence, the carboxyl-terminal region of angiotensinogen contains two internally homologous sequences located between residues and residues ; 6 out of 11 amino acid residues, including a cysteine residue, are present at equivalent positions. Neurophysin II has a carrier function for arginine vasopressin after cleavage from its precursor protein (26). Thus, it would be interesting to investigate whether FIG. 3 (on next page). Primary structure of rat preangiotensinogen mrna. The nucleotide sequence of the mrna was deduced from that of the cdna insert in clone pragl6. Nucleotide residues are numbered in the 5' to 3' direction, beginning with the first residue of the AUG triplet codingfor the initiation methionine. The nucleotides on the 5' side of residue 1 are indicated by negative numbers. The predicted amino acid sequence of preangiotensinogen is shown above the nucleotide sequence. The amino acid residues are numbered beginning with the initiation methionine. The angiotensin I sequence is boxed with a solid line. The internally homologous amino acid sequences described in the text are underlined.

4 Biochemistry: Ohkubo et al. Proc. Natl. Acad. Sci. USA 80 (1983) Met Thr Pro Thr Gly Ala Gly Leu Lys Ala Thr Ile Phe 5'----AUAGCUGUGCUUGUCUGGGCUGGAGCUAAAGGACACACAGAAGCMGUCCACAGAUCCGUG AUG ACU CCC ACG GGG GCA GGC CUG AAG GCC ACC AUC UUC ii Cys Ile Leu Thr Trp Val Ser Leu Thr Ala Gly Asp Arg Val Tyr Ile His Pro Phe His Leu Leu Tyr Tyr Ser Lys Ser Thr Cys Ala UGC AUC CUG ACC UGG GUC AGC CUG ACA GCU GGG GAC CGC GUA UAC AUC CAC CCC UUU CAU CUC CUC UAC UAC AGC MG AGC ACC UGC GCC Gln Leu Glu Asn Pro Ser Val Glu Thr Leu Pro Glu Pro Thr Phe Glu Pro Val Pro Ile Gln Ala Lys Thr Ser Pro Val Asp Glu Lys CAG CUG GAG AAC CCC AGU GUG GAG ACG CUC CCA GAG CCA ACC UUU GAG CCU GUG CCC AUU CAG GCC AAG ACC UCC CCC GUG GAU GAG MG Thr Leu Arg Asp Lys Leu Val Leu Ala Thr Glu Lys Leu Glu Ala Glu Asp Arg Gln Arg Ala Ala Gln Val Ala Met Ile Ala Asn Phe ACC CUG CGA GAU MG CUC GUG CUG GCC ACU GAG MG CUA GAG GCU GAG GAU CGG CAG CGA GGU GCC CAG GUC GCG AUG AUU GCC AAC UUC Met Gly Phe Arg Met Tyr Lys Met Leu Ser Glu Ala Arg Gly Val Ala Ser Gly Ala Val Leu Ser Pro Pro Ala Leu Phe Gly Thr Leu AUG GGU UUC CGC AUG UAC MG AUG CUG AGU GAG GCA AGA GGU GUA GCC AGU GGG GCC GUC CUC UCU CCA CCG GCC CUC UUU GGC ACC CUG Val Ser Phe Tyr Leu Gly Ser Leu Asp Pro Thr Ala Ser Gln Leu Gln Val Leu Leu Gly Val Pro Val Lys Glu Gly Asp Cys Thr Ser GUC UCU UUC UAC CUU GGA UCG UUG GAU CCC ACG GCC AGC CAG CUG CAG GUG CUG CUG GGC GUC CCU GUG AAG GAG GGA GAC UGC ACC UCC Arg Leu Asp Gly His Lys Val Leu Thr Ala Leu Gln Ala Val Gln Gly Leu Leu Val Thr Gln Gly Gly Ser Ser Ser Gln Thr Pro Leu CGG CUG GAC GGA CAU MG GUC cug ACU GCC CUG CAG GCU GUU CAG GGC UUG CUG GUC ACC CAG GGU GGA AGC AGC AGC CAG ACA CCC CUG Leu Gln Ser Thr Val Val Gly Leu Phe Thr Ala Pro Gly Leu Arg Leu Lys Gln Pro Phe Val Glu Ser Leu Gly Pro Phe Thr Pro Ala CUA CAG UCC ACC GUG GUG GGC CUC UUC ACU GCC CCA GGC UUG CGC CUA MA CAG CCA UUU GUU GAG AGC UUG GGU CCC UUC ACC CCC GCC Ile Phe Pro Arg Ser Leu Asp Leu Ser Thr Asp Pro Val Leu Ala Ala Gln Lys Ile Asn Arg Phe Val Gln Ala Val Thr Gly Trp Lys AUC UUC CCU CGC UCU CUG GAC UUA UCC ACU GAC CCA GUU CUU GCU GCC CAG MA AUC MC AGG UUU GUG CAG GCU GUG ACA GGG UGG AAG Met Asn Leu Pro Leu Glu Gly Val Ser Thr Asp Ser Thr Leu Phe Phe Asn Thr Tyr Val His Phe Gln Gly Lys Met Arg Gly Phe Ser AUG AAC UUG CCA CUA GAG GGG GUC AGC ACG GAC AGC ACC CUA UUU UUC MC ACC UAC GUU CAC UUC CAA GGG MG AUG AGA GGC UUC UCC Gln Leu Thr Gly Leu His Glu Phe Trp Val Asp Asn Ser Thr Ser Val Ser Val Pro Met Leu Ser Gly Thr Gly Asn Phe Gln His Trp CAG CUG ACU GGG CUC CAU GAG UUC UGG GUG GAC MC AGC ACC UCA GUG UCU GUG CCC AUG CUC UCG GGC ACU GGC AAC UUC CAG CAC UGG Ser Asp Ala Gln Asn Asn Phe Ser Val Thr Arg Val Pro Leu Gly Glu Ser Val Thr Leu Leu Leu Ile Gln Pro Gln CY5 Ala Ser Asp AGU GAC GCC CAG MC MC UUC UCC GUG ACA CGC GUG CCC CUG GGU GAG AGU GUC ACC CUG CUG CUG AUC CAG CCC CAG UGC GCC UCA GAU Leu Asp Arg Val Glu Val Leu Val Phe Gln His Asp Phe Leu Thr Trp Ile Lys Asn Pro Pro Pro Arg Ala Ile Arg Leu Thr Leu Pro CUG GAC AGG GUG GAG GUC CUC GUC UUC CAG CAC GAC UUC CUG ACU UGG AUA AAG AAC CCG CCU CCU CGG GCC AUC CGU CUG ACC CUG CCG oo Gln Leu Glu Ile Arg Gly Ser Tyr Asn Leu Gln Asp Leu Leu Ala Gln Ala Lys Leu Ser Thr Leu Leu Gly Ala Glu Ala Asn Leu Gly CAG CUG GM AUU CGG GGA UCC UAC MC CUG CAG GAC CUG CUG GCU CAG GCC MG CUG UCU A-CC CUU UUG GGU GCU GAG GCA AMU CUG GGC Lys Met Gly Asp Thr Asn Pro Arg Val Gly Glu Val Leu Asn Ser Ile Leu Leu Glu Leu Gln Ala Gly Glu Glu Glu Gln Pro Thr Glu AAG AUG GGU GAC ACC MGCCCC CGA GUG GGA GAG GUU CUC AAC AGC AUC CUC CUU GM CUC CM GCA GGC GAG GAG GAG CAG CCC ACA GAG Ser Ala Gln Gln Pro Gly Ser Pro Glu Val Leu Asp Val Thr Leu Ser Ser Pro Phe Leu Phe Ala Ile Tyr Glu Arg Asp Ser Gly Ala UCU GCC CAG CAG CCU GGC UCA CCC GAG GUG CUG GAC GUG ACC CUG AGC AGU CCG UUC CUG UUC GCC AUC UAC GAG CGG GAC UCA GGU GCG Leu His Phe Leu Gly Arg Val Asp Asn Pro Gln Asn Val Val CUG CAC UUU CUG GGC AGA GUG GAU MC CCC CM MU GUG GUG UGA UGCCUCCUGUGUAGCCAUGGAGACAAGGCCAGCGUCAGAGAGCUAUCCUGGGCAAAAU CAGUGCCUUCAaCCCUGCUUCCSGLIAtUCCUUCCAGCAAGGCAGAGGCCGUCUCCUUGGAGAUGGCGCUAACUGAGAAUAAUGAUGAGCAGCAGCCUCCUGGGGUGUGGGUUUGUU ! UGGACACUGGGGUGAGAGCCAGGAGCUGGCACUCUGUAUAGGAGGACUGCCAUCCUGGAAMAAMAUGGACCAMAACMUGUUUGUG ' ioo FIG. 3. (Legend appears at the bottom of the preceding page.)

5 2200 Biochemistry: Ohkubo et al. Table 1. Comparison of amino acid compositions of rat angiotensinogen Predicted from Amino acid nucleotide sequence Reported* Aspartic acid 20 Asparagine Threonine 30 Serine Glutamic acid 25 Glutamine Proline Glycine 45 Alanine 33 Valine Methionine 7 Isoleucine Leucine Tyrosine 8 8 Phenylalanine Lysine Histidine Arginine Cysteine ND Tryptophan ND Total Predicted numbers of amino acid were calculated by assuming that angiotensinogen is formed by removal of the amino-terminal signal peptide of 24 amino acid residues. ND, not determined. * Data taken from Hilgenfeldt et al. (5). the long carboxyl-terminal region of angiotensinogen has some biological role after the release of angiotensin I. We have recently cloned specific cdna sequences for both Origin 28S 23S 18S 16S FIG. 4. Size determination of the mrna coding for rat preangiotensinogen. A 9-,tg sample of rat liver poly(a)-containing RNA was analyzed. The hybridization probe used was the 1,097-base-pair Acc I fragment (residues 81-1,177) derived from clone pragl6. The size markers used were rat and Escherichia coli rrna. Proc. Natl. Acad. Sci. USA 80 (1983) low molecular weight and high molecular weight kininogens from bovine liver mrna sequences (18). Both kininogens are precursor proteins of bradykinin, which is released by limited proteolysis of kininogens by kallikreins and causes hypotension (references cited in ref. 18). Thus, angiotensin and bradykinin exert opposite effects on the regulation of blood pressure. Because neither the molecular basis responsible for angiotensinogen biosynthesis nor that for kininogen biosynthesis are well understood, the cloned cdna for angiotensinogen together with those for the kininogens may provide the tools necessary for detailed study of the regulation of blood pressure. This investigation was supported in part by research grants from the Ministry of Education, Science, and Culture of Japan; the Institute of Physical and Chemical Research; and the Mitsubishi Foundation. 1. Reid, I. A., Morris, B. J. & Ganong, W. F. (1978) Annu. Rev. Physiol. 40, Skeggs, L. T., Dorer, F. E., Levine, M., Lentz, K. E. & Kahn, J. R. (1980) in The Renin-Angiotensin System, eds. Johnson, J. A. & Anderson, R. R. (Plenum, New York), pp Ramsay, D. J. (1979) Neuroscience 4, Tewksbury, D. A. (1981) in Biochemical Regulation of Blood Pressure, ed. Soffer, R. L. (Wiley, New York), pp Hilgenfeldt, U., Blandfort, N. & Hackenthal, E. (1980) in Enzymatic Release of Vasoactive Peptides, eds. Gross, F. & Vogel, G. (Raven, New York), pp Bouhnik, J., Clauser, E., Strosberg, D., Frenoy, J.-P., Menard, J. & Corvol, P. (1981) Biochemistry 20, Tewksbury, D. A., Dart, R. A. & Travis, J. (1981) Biochem. Biophys. Res. Commun. 99, Weigand, K., Wernze, H. & Falge, C. (1977) Biochem. Biophys. Res. Commun. 75, Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. (1979) Biochemistry 18, Aviv, H. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, Agarwal, K. L., Brunstedt, J. & Noyes, B. E. (1981)J. Biol. Chem. 256, Hirose, T., Crea, R. & Itakura, K. (1978) Tetrahedron Lett. 28, Maxam,. A. M. & Gilbert, W. (1980) Methods Enzymol. 65, Okayama, H. & Berg, P. (1982) Mol. Cell. Biol. 2, Morrison, D. A. (1979) Methods Enzymol. 68, Hanahan, D. & Meselson, M. (1980) Gene 10, Wallace, R. B., Shaffer, J., Murphy, R. F., Bonner, J., Hirose, T. & Itakura, K. (1979) Nucleic Acids Res. 6, Nawa, H., Kitamura, N., Hirose, T., Asai, M., Inayama, S. & Nakanishi, S. (1983) Proc. Natl. Acad. Sci. USA 80, Alwine, J. C., Kemp, D. J. & Stark, G. R. (1977) Proc. Natl. Acad. Sci. USA 74, McMaster, G. K. & Carmichael, G. G. (1977) Proc. Natl. Acad. Sci. USA 74, Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977) J. Mol. Biol. 113, Blobel, G. & Dobberstein, B. (1975) J. Cell Biol. 67, Marshall, R. D. (1974) Biochem. Soc. Symp. 40, Proudfoot, N. J. & Brownlee, G. G. (1976) Nature (London) 263, Land, H., Schfitz, G., Schmale, H. & Richter, D. (1982) Nature (London) 295, Brownstein, M. J., Russell, J. T. & Gainer, H. (1980) Science 207,