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• W2074418097 abstract "A rapid amplification of cDNA ends method, using degenerate oligonucleotides based upon the N-terminal amino acid sequence of human hepatic deoxyribonuclease II (DNase II), allowed a novel cDNA encoding DNase II to be constructed from thyroid gland RNA. The composite nucleotide sequence (1593 bases) included an open reading frame of 1080 bases, which encoded a single polypeptide of 360 amino acids (signal peptide, 16; propeptide, 91; mature protein, 253). Although the sequence of DNase II showed no significant homology to other mammalian proteins, its cDNA structural organization resembled those of the lysosomal cathepsin families. The two parts of the cDNA corresponding to the propeptide and the mature protein were expressed in Escherichia coli, and the recombinant polypeptides thus obtained were strongly stained with an anti-DNase II antibody on Western blotting. DNase II is ubiquitously expressed in human tissues, and the DNase II gene (DNASE2) was assigned to chromosome 19. A rapid amplification of cDNA ends method, using degenerate oligonucleotides based upon the N-terminal amino acid sequence of human hepatic deoxyribonuclease II (DNase II), allowed a novel cDNA encoding DNase II to be constructed from thyroid gland RNA. The composite nucleotide sequence (1593 bases) included an open reading frame of 1080 bases, which encoded a single polypeptide of 360 amino acids (signal peptide, 16; propeptide, 91; mature protein, 253). Although the sequence of DNase II showed no significant homology to other mammalian proteins, its cDNA structural organization resembled those of the lysosomal cathepsin families. The two parts of the cDNA corresponding to the propeptide and the mature protein were expressed in Escherichia coli, and the recombinant polypeptides thus obtained were strongly stained with an anti-DNase II antibody on Western blotting. DNase II is ubiquitously expressed in human tissues, and the DNase II gene (DNASE2) was assigned to chromosome 19. Deoxyribonuclease II (DNase II 1The abbreviations used are: DNase II, deoxyribonuclease II; aa, amino acid(s); AUAP, abridged universal amplification primer; bp, base pair(s); IPTG, isopropyl-1-thio-β-d-galactopyranoside; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; SRED, single radial enzyme diffusion; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase. , EC 3.1.22.1) is one of two distinct types of DNase present in mammalian tissues and body fluids. In the absence of metal ions, it hydrolyzes DNA to 3′-phosphoryl oligonucleotides under acidic conditions and has therefore been designated “acid DNase” (1Bernardi G. Boyer P. 3rd Ed. The Enzymes. 4. Academic Press, New York1971: 271-287Google Scholar). DNase II was isolated directly from lysosomes in the porcine spleen (2Liao T.-H. Liao W.-H. Chang H.-C. Lu K.-S. Biochim. Biophys. Acta. 1989; 1007: 15-22Crossref PubMed Scopus (37) Google Scholar) and from rat and monkey livers (3Dulaney J.T. Touster O. J. Biol. Chem. 1972; 247: 1424-1432Abstract Full Text PDF PubMed Google Scholar, 4Oosthuizen M.M.J. Myburgh J.A. Schabort J.C. Int. J. Biochem. 1984; 16: 1207-1215Crossref PubMed Scopus (4) Google Scholar). DNase II activity is known to occur in various mammalian tissues (5Allfrey V. Mirsky A.E. J. Gen. Physiol. 1952; 36: 227-241Crossref PubMed Scopus (73) Google Scholar, 6Cordonnier C. Bernardi G. Can. J. Biochem. 1968; 46: 989-995Crossref PubMed Scopus (42) Google Scholar, 7Yasuda T. Nadano D. Awazu S. Kishi K. Biochim. Biophys. Acta. 1992; 1119: 185-193Crossref PubMed Scopus (76) Google Scholar, 8Takeshita H. Yasuda T. Nadano D. Tenjo E. Sawazaki K. Iida R. Kishi K. Int. J. Biochem. 1994; 26: 1025-1031Crossref PubMed Scopus (29) Google Scholar). Although the enzymological properties of DNase II from different tissues and species are very similar, inconsistencies in the chemical properties of these enzymes (for example, with regard to molecular mass or subunit structure) have been recognized. Bernardi (1Bernardi G. Boyer P. 3rd Ed. The Enzymes. 4. Academic Press, New York1971: 271-287Google Scholar) suggested that the hog splenic DNase II may have a dimeric structure composed of two similarly sized subunits, while the bovine hepatic enzyme appeared to consist of one 27-kDa polypeptide chain (9Lesca P. J. Biol. Chem. 1976; 251: 116-123Abstract Full Text PDF PubMed Google Scholar). However, the porcine hepatic and splenic DNase IIs have been demonstrated to consist of two non-identical subunits (2Liao T.-H. Liao W.-H. Chang H.-C. Lu K.-S. Biochim. Biophys. Acta. 1989; 1007: 15-22Crossref PubMed Scopus (37) Google Scholar, 10Liao T.-H. J. Biol. Chem. 1985; 260: 10708-10713Abstract Full Text PDF PubMed Google Scholar). One of the hallmarks of apoptosis or programmed cell death is the enzymatic internucleosomal cleavage of chromatin to yield an electrophoretic ladder pattern of DNA fragments. Several molecules that might be responsible for this endonucleolytic activity have been characterized from or detected in various sources (11Peitsch M.C. Mannherz H.G. Tschopp J. Trends Cell Biol. 1994; 4: 37-41Abstract Full Text PDF PubMed Scopus (155) Google Scholar). Barry and Eastman (12Barry M.A. Eastman A. Arch. Biochem. Biophys. 1993; 300: 440-450Crossref PubMed Scopus (433) Google Scholar) demonstrated that a nuclear DNase II can mediate internucleosomal DNA digestion during apoptosis in Chinese hamster ovary cells following intracellular acidification. Furthermore, Torriglia et al. (13Torriglia A. Chaudun E. Chany-Fournier F. Jeanny J.-C. Courtois Y. Counis M.-F. J. Biol. Chem. 1995; 270: 28579-28585Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) suggested the specific involvement of DNase II in physiological nuclear degradation during lens fiber cell differentiation in chick embryos. However, although information on the cDNA encoding DNase II would allow the detailed elucidation of both the inherent structural properties of the enzyme and the intrinsic involvement of DNase II in apoptosis, no cDNA has as yet been cloned from any mammal. In contrast to DNase I (EC 3.1.21.1), which has been studied extensively with regard to biochemical and human genetic aspects (14Kishi K. Yasuda T. Ikehara Y. Sawazaki K. Sato W. Iida R. Am. J. Hum. Genet. 1990; 47: 121-126PubMed Google Scholar, 15Yasuda T. Awazu S. Sato W. Iida R. Tanaka Y. Kishi K. J. Biochem. (Tokyo). 1990; 108: 393-398Crossref PubMed Scopus (84) Google Scholar, 16Yasuda T. Kishi K. Yanagawa Y. Yoshida A. Ann. Hum. Genet. 1995; 59: 1-15Crossref PubMed Scopus (71) Google Scholar, 17Yasuda T. Nadano D. Takeshita H. Tenjo E. Sawazaki K. Ootani M. Kishi K. FEBS Lett. 1995; 359: 211-214Crossref PubMed Scopus (25) Google Scholar), DNase II has been less well characterized, particularly in human systems. Hitherto, DNase II has been only partially purified from gastric mucosa, uterine cervix (18Yamanaka M. Tsubota Y. Anai M. Ishimatsu K. Okumura M. Katsuki S. Takagi Y. J. Biol. Chem. 1974; 249: 3884-3889Abstract Full Text PDF PubMed Google Scholar), urine (7Yasuda T. Nadano D. Awazu S. Kishi K. Biochim. Biophys. Acta. 1992; 1119: 185-193Crossref PubMed Scopus (76) Google Scholar, 19Murai K. Yamanaka M. Akagi K. Anai M. J. Biochem. (Tokyo). 1980; 87: 1097-1103PubMed Google Scholar), and lymphoblasts (20Harosh I. Binninger D.M. Harris P.V. Mezzina M. Boyd J.B. Eur. J. Biochem. 1991; 202: 479-484Crossref PubMed Scopus (37) Google Scholar). We previously developed a sensitive and specific method for the quantitative detection of DNase II activity in human tissues and body fluids (7Yasuda T. Nadano D. Awazu S. Kishi K. Biochim. Biophys. Acta. 1992; 1119: 185-193Crossref PubMed Scopus (76) Google Scholar) and were consequently able to discover that human urinary and leukocyte DNase II shows genetic polymorphism with respect to its activity levels (21Yasuda T. Nadano D. Sawazaki K. Kishi K. Ann. Hum. Genet. 1992; 56: 1-10Crossref PubMed Scopus (35) Google Scholar). The distribution of DNase II activity displays clear-cut bimodality in the Japanese population, and population and family studies found the activity levels to be controlled by two alleles, the dominant DNASE2*H and the recessiveDNASE2*L, which are present at a single autosomal locus. These result in high and low activity, respectively. Differences in DNase II activity between various strains of mice have also been suggested to be determined by a single autosomal locus (22Koizumi T. Exp. Anim. 1995; 44: 323-327Crossref PubMed Scopus (1) Google Scholar). Thus, it has been confirmed that DNase II is one of the limited number of enzymes that exhibits genetic polymorphism in its activity levels. However, it remains to be determined whether the genetic control over activity levels is due to variation in the structural or regulatory gene for DNase II. Clarification of the molecular basis for the genetic polymorphism of DNase II requires the structure of a cDNA for DNase II to be determined and analyzed. In the present study, we purified the DNase II from human liver and determined its N-terminal amino acid (aa) sequence. This was used as the the basis of a polymerase chain reaction (PCR)-based cloning strategy for the construction of a cDNA coding for human DNase II. We report herein the molecular cloning process and the complete nucleotide sequence of DNase II cDNA. We also describe the bacterial expression of this cDNA and the chromosomal assignment of the gene coding for DNase II (DNASE2). Finally, we present evidence that DNase II is ubiquitously expressed in human tissues. This is the first report of the cloning and characterization of a cDNA for mammalian DNase II. Superscript II RNase H− reverse transcriptase (RT), RNase H, Taq DNA polymerase, a dNTP mixture, 5′- and 3′-rapid amplification of cDNA ends (RACE) systems, and an oligo(dT) primer were obtained from Life Technologies, Inc. All other chemicals used were of reagent grade or the purest grade available commercially. All the oligonucleotide primers used in this study were synthesized and purchased from Life Technologies, Inc. An antibody against human DNase II was produced by injecting a mixture of human urinary DNase II, purified essentially as described previously (7Yasuda T. Nadano D. Awazu S. Kishi K. Biochim. Biophys. Acta. 1992; 1119: 185-193Crossref PubMed Scopus (76) Google Scholar), and Freund's complete adjuvant into a Japanese white rabbit. The antibody reacted strongly with the enzyme on SDS-polyacrylamide gel electrophoresis (PAGE). Furthermore, the enzymatic activity of both the purified enzyme and human urine was completely inhibited by the antibody. The antibody showed no reactivity toward human DNase I. A specific antibody against human DNase I was prepared according to a previously described method (15Yasuda T. Awazu S. Sato W. Iida R. Tanaka Y. Kishi K. J. Biochem. (Tokyo). 1990; 108: 393-398Crossref PubMed Scopus (84) Google Scholar). Seventeen different kinds of human tissue sample were obtained from eight individuals (between 18 and 82 years of age) who had died from traumatic shock or loss of blood with no pathological changes. The samples were collected within about 20 h of death and stored at −80 °C until use. DNase II activity was determined by a single radial enzyme diffusion (SRED) method as described previously (7Yasuda T. Nadano D. Awazu S. Kishi K. Biochim. Biophys. Acta. 1992; 1119: 185-193Crossref PubMed Scopus (76) Google Scholar, 21Yasuda T. Nadano D. Sawazaki K. Kishi K. Ann. Hum. Genet. 1992; 56: 1-10Crossref PubMed Scopus (35) Google Scholar). One unit of DNase II activity was defined as an increase of 1.0 in the absorbance at 260 nm. Levels of DNase II activity in each tissue sample were determined as follows (23Yasuda T. Takeshita H. Nakajima T. Hosomi O. Nakashima Y. Kishi K. Biochem. J. 1997; 325: 465-473Crossref PubMed Scopus (46) Google Scholar). The human tissues were cut into small pieces, washed with cold saline to remove excess blood, homogenized using an Ultra-turrax (IKA-WERK, Staufen, Germany) in 1–2 ml 50 mm Tris (pH 7.5) containing 1.0 mmphenylmethylsulfonyl fluoride (Sigma), then centrifuged at 15,000 × g for 20 min. The resulting supernatants were used in the subsequent analysis. A whole liver (ca. 1200 g) was obtained at autopsy from a 57-year-old woman 15 h after death due to loss of blood and was stored at −80 °C until use. DNase II was purified from the liver according to a modification of a previously described method (7Yasuda T. Nadano D. Awazu S. Kishi K. Biochim. Biophys. Acta. 1992; 1119: 185-193Crossref PubMed Scopus (76) Google Scholar). Details of the purification will be published elsewhere. The purified DNase II was subjected to SDS-PAGE in 12.5% gel according to the method of Laemmli (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). After the electrophoretic run, the protein was transferred onto an Immobilon-PSQ membrane (Millipore, Bedford, MA) by electroblotting. The portion of the membrane carrying the enzyme was directly subjected to automatic Edman degradation using a protein sequencer (model 477A; Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Total RNA was separately extracted from the thyroid gland and spleen of a 48-year-old man, obtained at autopsy 12 h after death due to loss of blood, by the acid guanidinium isothiocyanate-phenol-chloroform extraction method (25Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). First, the DNA fragment corresponding to the 3′-end region of the human DNase II cDNA was obtained by the 3′-RACE method (26Frohman M.A. Dush M.K. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8998-9002Crossref PubMed Scopus (4341) Google Scholar) using a 3′-RACE system. The synthesis of the first strand of the total RNA was performed using the RT reaction as follows. After denaturation at 70 °C for 10 min, total RNA (about 2 μg) was incubated with 200 units of Superscript II at 42 °C for 50 min in a reaction mixture (20 μl) comprising 20 mm Tris (pH 8.4), 50 mmKCl, 2.5 mm MgCl2, 0.5 mm each dNTP, 10 mm dithiothreitol, and 500 nm adaptor primer (Life Technologies, Inc.), 5′-GGCGACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3′. After heating at 70 °C for 10 min, the reaction mixture was further incubated with 2 units of RNase H at 37 °C for 20 min. The material obtained was used for nested PCR amplification (27Newton C.R. Graham A. PCR. Alden Press Ltd, Oxford1994: 27-38Google Scholar) with two partially overlapped degenerate primers, based on the N-terminal aa sequence of human DNase II (primer-1, 5′-GA(T/C)CA(T/C)GA(T/C)GGIGGITT(T/C)TGG-3′; primer-2, 5′-GGIGGITT(T/C)TGG(T/C)TIGTICA-3′). A 2-μl aliquot of the material was subjected to the first PCR amplification using a Minicycler (model PTC-150, MJ Research, Watertown, MA). The PCR reaction mixture (50 μl) comprised 20 mm Tris (pH 8.3), 50 mm KCl, 1.5 mm MgCl2, 0.01 mmdithiothreitol, 0.2 mm each dNTP, 2.5 units TaqDNA polymerase, 4 μm gene-specific primer-1, and 4 μm abridged universal amplification primer (AUAP; Life Technologies, Inc.), 5′-GGCCACGCTCGACTAGTAC-3′. After denaturation at 94 °C for 3 min, amplification was carried out for 35 cycles, each of which involved denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min and extension at 72 °C for 2 min, followed by a further 10-min extension at 72 °C. Subsequently, a 2-μl aliquot of the first PCR product was subjected to a second PCR amplification using a set of AUAP primer and the downstream gene-specific primer-2 under the same amplification conditions as those used for the first PCR. These 3′-RACE products were directly subcloned into TA cloning vector pCR II (Invitrogen). The plasmid DNA from 40 independent clones was isolated and sequenced. Next, the 5′-end region of the cDNA was likewise amplified by the RACE method using a 5′-RACE system. The 5′-gene-specific primers used for this RACE were based on the nucleotide sequence data obtained in this study (primer-3, 5′-GTCAGCTGCTTGCCCATCTTCG-3′; primer-4, 5′-CTATGAGGCCAACTGTATGCAGC-3′; and primer-5, 5′-AGCAGGGTCTGCCCGTAGGTAC-3′). The total RNA (about 1 μg) from the human thyroid gland and spleen was reverse-transcribed using primer-3 priming and Superscript II; then the first strand products were isolated using a QIAquickTM PCR Purification kit (QIAGEN, Chatsworth, CA). After poly(C)-tailing of the first strand products according to the manufacturer's instructions, the first PCR was carried out using the 5′-gene-specific primer-5 and an anchor primer (Life Technologies, Inc.), 5′-CUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3′ under the same amplification conditions, except that the annealing temperature used was 57 °C. Subsequently, a 2-μl aliquot of the first PCR product was subjected to the second PCR amplification using the upstream 5′-gene-specific primer-4 and AUAP. The 5′-RACE products obtained were subcloned into the pCR II vector (Invitrogen) and sequenced. Nucleotide sequences were determined by the dideoxy chain-termination method using a Dye Terminator Cycle sequencing kit (FS, Applied Biosystems). The sequencing run was performed on a Genetic Analyzer (model 310, Applied Biosystems). All DNA sequences were confirmed by reading both DNA strands. Total RNA was extracted from 17 different kinds of human tissue obtained from a 25-year-old woman at autopsy, 15 h after death due to loss of blood, and from leukocytes (25Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). A 1-μg sample of each RNA was reverse-transcribed using oligo(dT) priming. A 1.5-μl aliquot of each sample was subjected to PCR analysis. A set of two primers, 6 (5′-TCTTCCATGCGTGGGCACAC-3′) and 7 (5′-GATCTTATAAGCTCTGCTGG-3′), corresponding to the N- and C-terminal portions of mature human DNase II protein, were used. The DNase II gene transcript was amplified by PCR as described above, except that the annealing temperature was 60 °C, and the amplified products were subjected to electrophoresis on 2% (w/v) agarose gel. The resulting bands were visualized by ethidium bromide staining. A 782-bp DNA fragment containing the complete coding sequence for mature human DNase II protein was obtained by RT-PCR amplification of total RNA derived from the same thyroid gland sample using a set of two primers, DN2-N1 (5′-AAAAGGATCCTCTTCCATGCGTGGGCACA-3′) and DN2-C1 (5′-CCCCCAGCTCTTAGATCTTATAAGCTCTGCTGG-3′), in which BamHI and SacI sites were incorporated. After reverse transcription with oligo(dT) priming, the subsequent PCR reaction was carried out for 35 cycles of denaturation (94 °C, 1 min), annealing (60 °C, 1 min), and extension (72 °C, 2 min). After digestion with BamHI and SacI (all the endonucleases were purchased from New England Biolabs, Beverly, MA), the fragment was cloned into the pQE-30 expression vector (QIAGEN). The resulting construct, designated pQE-30/mature protein, was transformed intoE. coli SG13009 (pREP4). In addition, a 293-bp DNA fragment containing the complete coding sequence for the DNase II propeptide was amplified by RT-PCR of the same total RNA with a set of two primers, DN2-N2 (5′-AAAAGGATCCCTGACCTGCTACGGGGACTC-3′) and DN2-C2 (5′-CCCCAAGCTTGTCCTGAGCCTTGCTGGGTTG-3′), in which BamHI and HindIII sites were also incorporated, respectively. After digestion, the fragment was likewise cloned into the pQE-30 expression vector. The construct, termed pQE-30/propeptide, was transformed into E. coli M15 (pREP4). After the transformed cells had been grown according to the supplier's instructions, isopropyl-1-thio-β-d-galactopyranoside (IPTG) was added to a final concentration of 2 mm, and the incubation was continued for another 4 h. The cells were harvested by centrifugation, resuspended in 0.1 m sodium phosphate (pH 8.0) containing 0.01 m Tris and 8 m urea, then lysed by gently vortexing. Both the expressed proteins derived from these constructs contained six consecutive His residues as an affinity tag and were isolated using Ni-nitrilo-triacetic acid resin (QIAGEN) according to the manufacturer's instructions. Both of the recombinant proteins obtained and the native enzyme isolated from human liver were subjected to SDS-PAGE and detected by immunoblotting using anti-human DNase II antibody (28Yasuda T. Nadano D. Takeshita H. Kishi K. Biochem. J. 1993; 296: 617-625Crossref PubMed Scopus (26) Google Scholar). Genomic DNAs extracted from a panel of 20 different cloned human × rodent (hamster or mouse) hybrid cell lines were obtained from BIOS Laboratories (New Haven, CT). A set of two primers, 5 and 6, was used to amplify the human DNase II-specific DNA fragment from a panel of somatic cell hybrids. About 50 ng of DNA was subjected to PCR under the same amplification conditions as above, except that the annealing temperature used was 60 °C. The products were separated on 1.5% (w/v) agarose gel and visualized by ethidium bromide staining. Using human DNA as a template, a 1.7-kilobase (kb) DNA fragment was specifically amplified by these primers. Upon Southern blotting, the human DNase II cDNA probe hybridized well with the fragment. Otherwise, these primers failed to demonstrate any specific products in either hamster or mouse DNAs. Therefore, the presence of the amplified 1.7-kb fragment upon PCR with these primers allowed unequivocal identification of the human component. No systematic examination of the tissue distribution of DNase II has so far been carried out in humans or other mammals. Under optimal assay conditions established on the basis of the catalytic properties of purified human urinary and hepatic DNase II, DNase II activity was determined in 17 different tissue extracts by the SRED method (7Yasuda T. Nadano D. Awazu S. Kishi K. Biochim. Biophys. Acta. 1992; 1119: 185-193Crossref PubMed Scopus (76) Google Scholar). As shown in Table I, the adrenal gland, thyroid gland, lymph nodes, and pituitary gland showed high activity. In contrast to mammalian DNase I (23Yasuda T. Takeshita H. Nakajima T. Hosomi O. Nakashima Y. Kishi K. Biochem. J. 1997; 325: 465-473Crossref PubMed Scopus (46) Google Scholar, 29Nadano D. Yasuda T. Kishi K. Clin. Chem. 1993; 39: 448-452Crossref PubMed Scopus (205) Google Scholar, 30Takeshita H. Yasuda T. Nakajima T. Hosomi O. Nakashima Y. Kishi K. Biochem. Mol. Biol. Int. 1997; 42: 65-75PubMed Google Scholar, 31Yasuda T. Sawazaki K. Nadano D. Takeshita H. Nakanaga M. Kishi K Clin. Chim. Acta. 1993; 218: 5-16Crossref PubMed Scopus (34) Google Scholar, 32Yasuda T. Takeshita H. Sawazaki K. Nadano D. Iida R. Miyahara S. Kishi K. J. Forensic Sci. 1996; 41: 862-864Crossref PubMed Google Scholar), all the tissue samples examined exhibited a significant level of activity. The activities detected in these tissues were completely abolished by anti-human DNase II antibody but not by anti-human DNase I. From these findings, it seems reasonable to conclude that the activity detected by the SRED method was indeed derived from DNase II. The tissue activity distribution and the amount of tissue available meant that the liver was the most suitable organ for the preparation of human DNase II.Table IDistribution of DNase II in human tissuesTissueDNase II activityDNase II gene transcript1-b+ = amplification of a 762-bp DNase II-specific fragment by RT-PCR using primers 6 and 7. NT, not tested.No. of samplesRangeActivity1-aValues are means ± standard deviations of triplicate determinations for each tissue, derived from different individuals. ND, not determined.units/g wet weightCerebrum81.0–7.52.7 ± 1.9+Cerebellum81.6–4.92.4 ± 1.0+Pituitary gland712.0–35.024.0 ± 7.3+Submaxillary gland74.4–7.75.3 ± 1.4+Thyroid gland612.0–62.030.0 ± 16.0+Parotid gland52.3–7.54.2 ± 1.8+ThymusND+Heart81.9–7.04.2 ± 2.0+Lung87.1–21.012.0 ± 5.1+Stomach83.0–11.06.7 ± 2.0+Liver68.8–30.020.0 ± 5.8+Pancreas75.2–10.09.5 ± 3.6+Kidney83.3–21.09.5 ± 6.0+Adrenal gland66.2–11034.0 ± 36.0+Spleen86.7–18.012.0 ± 4.2+Small intestine82.3–8.64.7 ± 2.0+Large intestine81.7–8.64.4 ± 1.4+Lymph nodes59.2–78.028.0 ± 22.0NT1-a Values are means ± standard deviations of triplicate determinations for each tissue, derived from different individuals. ND, not determined.1-b + = amplification of a 762-bp DNase II-specific fragment by RT-PCR using primers 6 and 7. NT, not tested. Open table in a new tab With regard to the aa sequence of mammalian DNase II, only one description of the aa sequence around an essential histidine residue of the porcine DNase II has so far been reported (33Oshima R.G. Price P.A. J. Biol. Chem. 1973; 248: 7522-7526Abstract Full Text PDF PubMed Google Scholar). In this study, about 20 μg of human DNase II was obtained from a whole liver, and its catalytic and immunological properties were found to closely resemble those of human urinary DNase II (7Yasuda T. Nadano D. Awazu S. Kishi K. Biochim. Biophys. Acta. 1992; 1119: 185-193Crossref PubMed Scopus (76) Google Scholar). When the isolated enzyme was subjected to SDS-PAGE followed by protein staining, a nearly single band was observed at a position corresponding to approximately 32 kDa on the gel. Edman degradation allowed the N-terminal aa sequence of this protein to be identified up to the 30th residue as follows: Ser-Ser-Met-Arg-Gly-His-Thr-Lys-Gly-Val-Leu-Leu-Leu-Asp-His-Asp-Gly-Gly-Phe-Trp-Leu-Val-His-Ser-Val-Pro-Asn-Phe-Pro-Pro. Comparison of this aa sequence with those in the SWISS-PROT protein sequence data base showed no significant homologies. Due to its high DNase II activity (as shown in Table I) and its wide availability among the human tissue samples collected, the thyroid gland was selected for the preparation of total RNA. Total RNA extracted from a thyroid gland sample was separately amplified by the 3′- and 5′-RACE methods (26Frohman M.A. Dush M.K. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8998-9002Crossref PubMed Scopus (4341) Google Scholar) to construct a cDNA encoding human DNase II. Two partially overlapped degenerate oligonucleotides, primer-1 and -2, corresponding to the aa sequence from Asp-14 to Trp-20 and from Gly-17 to His-23, respectively, were prepared for nested PCR amplification. Following 3′-RACE of the total thyroid RNA with the gene-specific primer-1, PCR amplification was performed using the downstream primer-2. After cloning into pCR II vector, 40 independent clones were selected from the 3′-RACE cDNA library and sequenced to screen for a clone that would insert a cDNA encoding DNase II. Five clones (DN2–3R) with an insert 1.2 kb long had a nucleotide sequence that completely corresponded to the aa sequence from Ser-24 to Pro-30. Next, the 5′-end region of the cDNA was amplified by 5′-RACE. After reverse transcription of the thyroid RNA with primer-3 priming, nested PCR amplification using primer-5 followed by the upstream primer-4 yielded a unique 0.55-kb fragment. After subcloning, the clone (DN2–5R) in which the corresponding RACE product was inserted was selected and sequenced. The first ATG codon was found at position 73 of the sequence. Sequence analysis of each RACE product showed the composite sequence (1593 bp) to include an open reading frame of 1080 bp, along with portions of the 5′-untranslated (72 bp) and 3′-untranslated (441 bp) regions (Fig. 1). The open reading frame started at position 73 with an ATG initiation codon and ended with a TAA stop codon at position 1153. The sequence flanking the ATG codon at position 73 was compatible with the consensus sequence for an initiator sequence (34Kozak M. J. Mol. Biol. 1987; 196: 947-950Crossref PubMed Scopus (996) Google Scholar). The 3′-untranslated region was followed by a short poly(A) tail. A putative polyadenylation signal (ATTAAA) was located 21 bp upstream of the poly(A) tail. The 3′-untranslated region of DNase II cDNA had a longer span than that of DNase I cDNA. In humans, this is about 150 bp (16Yasuda T. Kishi K. Yanagawa Y. Yoshida A. Ann. Hum. Genet. 1995; 59: 1-15Crossref PubMed Scopus (71) Google Scholar), in rabbits, 147 bp (23Yasuda T. Takeshita H. Nakajima T. Hosomi O. Nakashima Y. Kishi K. Biochem. J. 1997; 325: 465-473Crossref PubMed Scopus (46) Google Scholar), and in the mouse, 163 bp (30Takeshita H. Yasuda T. Nakajima T. Hosomi O. Nakashima Y. Kishi K. Biochem. Mol. Biol. Int. 1997; 42: 65-75PubMed Google Scholar). It has been reported (35Faust P.L. Kornfeld S. Chirgwin J.M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4910-4914Crossref PubMed Scopus (263) Google Scholar) that a long 3′-untranslated region is a common feature of lysosomal protease mRNAs. Also, the nucleotide sequence of the open reading frame of the cDNA derived from the thyroid gland was completely consistent with that obtained from the spleen of the same individual. The aa sequence predicted by nucleotide analysis is shown in Fig. 2. Assuming that DNase II translation starts at the first ATG, the open reading frame coded for a protein of 360 aa, in which the presence of a stretch of hydrophobic aa close to the initial Met strongly suggested the presence of a signal peptide, which is also present in lysosomal enzymes. The predicted signal peptide exhibited the common characteristics found among signal peptides: hydrophobic aa clusters in the interior (Leu-4 to Val-21) and a residue with a short side chain for cleavage by the signal peptidase (Ala-15). Human cathepsins B and D have been predicted to be cleaved after the Ala-X-Ala sequence (35Faust P.L. Kornfeld S. Chirgwin J.M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4910-4914Crossref PubMed Scopus (263) Google Scholar, 36Chan S.J. Segundo B.S. McCormick M.B. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7721-7725Crossref PubMed Scopus (229) Google Scholar). Considering the “(−3, −1) rule” (37von Heijne G. Nucleic A" @default.
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