Proc. Nati. Acad. Sci. USA Vol. 81, pp. 5999-6003, October 1984 Biochemistry Structure of the human renin gene (hypertension/aspartyl proteinase/nucleotide sequence/splice junction) HITOSHI MIYAZAKI*, AKIYOSHI FUKAMIZU*, SHIGEHISA HIROSE*, TAKASHI HAYASHI*, HITOSHI HORI*, HIROAKI OHKUBOt, SHIGETADA NAKANISHIt, AND KAZUO MURAKAMI** *Institute of Applied Biochemistry, University of Tsukuba, Ibaraki 305, Japan; and tInstitute for Immunology, Kyoto University Faculty of Medicine, Kyoto 606, Japan Communicated by Leroy Hood, June 27, 1984 ABSTRACT The human renin gene was isolated from a between the intron-exon organization of the gene and the Charon 4A human genomic library and characterized. The tertiary structure of the protein. gene spans about 11.7 kilobases and consists of 10 exons and 9 introns that map at points that could be variable surface loops MATERIALS AND METHODS of the enzyme. The complete coding regions, the 5'- and 3'- Materials. All restriction enzymes were obtained from flanking regions, and the exon-intron boundaries were se- either New England Biolabs or Takara Shuzo (Kyoto, Ja- quenced. The active site aspartyl residues Asp-38 and Asp-226 pan). Escherichia coli alkaline phosphatase and T4 DNA li- are encoded by the third and eighth exons, respectively. The gase were from Takara Shuzo. [_y-32P]ATP (>5000 Ci/mmol; extra three amino acids (Asp-165, Ser-166, Glu-167) that are 1 Ci = 37 GBq) and [a-32P]dCTP (=3000 Ci/mmol) were not present in mouse renin are encoded by the separate sixth from Amersham. exon, an exon as small as 9 nucleotides. The positions of the Screening. A human genomic library, prepared from partial introns are in remarkable agreement with those in the human Alu I and Hae III digestion and ligated into the EcoRI arms pepsin gene, supporting the view that the genes coding for of the X vector Charon 4A, was kindly provided by T. aspartyl proteinases have arisen as the result of duplication of Maniatis (4). A portion of the amplified library plated on E. a common ancestral gene. As in most eukaryotic genes, the coli LE 392 was screened, by the method of Benton and putative T-A-T-A and C-A-A-T sequences, which may play a Davis (5), for sequences that hybridize with cDNA probes role in the initiation of gene transcription, are found in the [Pst I-Hpa II and Hpa II-Rsa I fragments of pHRn 321 (3)] vicinity of -29 and -51 nucleotides of the cap site. Further labeled with 32P by nick-translation to a specific activity of 2 upstream, at nucleotides -456 to -451, is located the x 108 cpm/,ug. Hybridization was conducted at 65°C for hexanucleotide T-G-T-T-C-T, which has recently been sug- 12-24 hr and the filters were sequentially washed several gested as a binding site for the glucocorticoid receptor. In the times in (i) 0.3 M NaCl/0.03 M sodium citrate/0.1% 3'-flanking region, there is the conserved hexanucleotide se- at quence A-A-T-A-A-A, thought to be necessary for polyade- NaDodSO4 room temperature, (ii) 0.3 M NaCI/0.03 M nylylation. Blot-hybridization analyses of the isolated gene sodium citrate/0.1% NaDodSO4 at 65°C, (iii) 15 mM NaClI1.5 done and the total cellular DNA after digestion with restriction mM sodium citrate/0.1% NaDodSO4 at 65°C, and (iv) 0.3 M enzymes revealed that human renin is encoded by a single NaCl/0.03 M sodium citrate at room temperature and ex- gene. posed to Fuji x-ray film RX-50 with a DuPont Cronex Light- ning Plus screen. Positive clones were picked and rescreened Renin is an aspartyl proteinase synthesized mainly in the at low plaque density to achieve high purity. Preparation of juxtaglomerular cells of the kidney, which are located in the phage DNA was carried out as described (6). afferent arterioles immediately proximal to the glomeruli (1). All cloning experiments were performed in accordance When the blood flow through the kidney becomes in- with the guidelines for research involving recombinant DNA adequate, renin is secreted into the circulation where it acts molecules issued by the Ministry of Education, Science and on a plasma protein to produce the decapeptide angiotensin Culture of Japan. I. Under the influence of the converting enzyme, the angio- Subcloning. DNA fragments from the positive phage clones tensin I derived from the larger parent protein angiotensin- were digested with EcoRI and the four fragments spanning ogen, in turn, becomes angiotensin II, which has potent the entire length of the gene were isolated by electrophoresis vasoconstrictor and aldosterone secretion-stimulating activi- through 0.7% agarose gels. The purified DNA fragments ties. Renin is, therefore, the key enzyme of the renin- were subcloned into the EcoRI site of plasmid pBR322 since angiotensin-aldosterone cascade aimed at increasing blood larger quantities of the EcoRI fragments were required for pressure and extracellular fluid volume (2). sequence determination. Transformations of E. coli HB101 Recently we (3) have reported the isolation and sequence were performed as described by Morrison (7). analysis of a human renin cDNA clone. From the cDNA Mapping and Sequencing. DNA samples were digested sequence, the human renin precursor, preprorenin, was with various restriction enzymes under conditions specified shown to be composed of 406 amino acids. In the present by the supplier and separated by electrophoresis on 0.7% and study, we used the human renin cDNA to isolate the cor- 2.5% agarose or 5% polyacrylamide gels. Ethidium bromide- responding gene sequence from a human genomic library. stained gels were photographed under UV light and DNA Analysis of the organization and structure of the renin gene was transferred to nitrocellulose filters in 0.9 M NaCl/0.09 M revealed that it exists in a single copy, spans roughly 11.7 sodium citrate as described by Southern (8). Hybridization kilobases (kb), and consists of 10 exons. We also examined was carried out at 65°C for 12 hr with nick-translated cDNA the intron-exon junctions to see if there is a correlation probes as described above, and filters were autoradiographed at - 70°C with intensifier screens. The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: kb, kilobase(s). in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5999 Downloaded by guest on September 25, 2021 6000 Biochemistry: Miyazaki et al. Proc. Natl. Acad. Sci. USA 81 (1984) Appropriate restriction fragments were isolated from poly- represents the original structure of the human renin gene that acrylamide gels and sequenced according to Maxam and is present as a single copy in the chromosome. Gilbert (9) by using the 5' end-labeling method. The se- Organization of the Cloned Gene. From the results of single quence strategy used is shown in Fig. 1C. and double digestion experiments using the subcloned frag- ments, a partial restriction map of the human renin gene was RESULTS constructed (Fig. 1B). The restriction fragments containing Isolation ofa Genomic Clone for Human Renin and Subclon- an exonic sequence and its surrounding regions, identified by ing of Its EcoRI Fragments. A human genomic library con- blot hybridization with a nick-translated [32P]cDNA probe, structed in the X phage Charon 4A (4) was screened for the were subjected to nucleotide sequence analysis to determine renin gene by using a nick-translated cDNA insert as a the exon-intron junctions. The subcloned 0.8-kb fragment hybridization probe. After screening 400,000 recombinant that did not hybridize with the cDNA probe was also se- phages, 7 positive clones were identified. They were plaque- quenced to cover the entire coding sequence. As shown in purified and their DNA was isolated for restriction endonu- Figs. 1 and 2, human renin gene consists of 10 exons and 9 clease characterization. Preliminary restriction enzyme introns. Central parts ofthe introns were not sequenced. The analysis of these clones indicated that they covered similar exon sequences are identical with those of the corresponding regions of about 12 kb of the human genome. Two clones, region of the cDNA (3). In accordance with the general G-T- designated XHRn 42 and XHRn 72, were further analyzed and A-G rule, all of the introns begin with the dinucleotide G-T shown to have three EcoRI sites (Fig. lA). The orientation of and end with A-G. More extensive consensus sequences the gene was determined by using the 5' and 3' specific have been proposed for the RNA splice sites by analyzing a cDNA probes generated by digesting the human renin cDNA great number of eukaryotic genes that are transcribed by at a unique Hpa II site. Four EcoRI fragments (the 5'-most RNA polymerase II (12, 13). The donor (C-A-G/G-T-A-A-T- 7.8-kb, the 3'-most 5.7-kb, and the overlapping 3.4-kb and G-T) and the acceptor [QC)11-N-T-A-G/G] consensus se- 0.8-kb fragments shown in Fig. 1A), which together comprise quences are reasonably well matched by the intron-exon 17.7 kb of the human genome, were subcloned for the sub- junctions of the renin gene (Fig. 2). sequent sequence analysis. When compared to mouse renin, human renin contains Soubrier et al. (10) have shown that the renin gene exists three extra amino acids (Asp-165, Ser-166, Glu-167; the in a single copy in the human genome. To confirm this and to numbering system begins from Leu-1 of mature renin) at the establish the absence of major sequence rearrangement of middle of its polypeptide chain.