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Cloning of cDNAs Encoding Porcine and Human DNase II
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
247, 864–869 (1998)
RC988839
Cloning of cDNAs Encoding Porcine and Human DNase II1 Daisuke Shiokawa* and Sei-ichi Tanuma*,†,2 *Department of Biochemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, 12 Funagawara-cho, Ichigaya, Shinjuku-ku, Tokyo 162, Japan; and †Research Institute for Bioscience, Science University of Tokyo, 2669 Yamazaki, Noda, Chiba 278, Japan
Received May 21, 1998
We report the molecular cloning of cDNAs encoding porcine and human DNase II and the genomic structure of the human DNase II gene. The full length cDNAs for porcine and human DNase II were isolated by polymerase chain reaction on the basis of amino acid sequences determined for the tryptic peptides of porcine liver DNase II. The porcine and human cDNAs contain 1095 and 1083 bp open reading frames, respectively, and encode 364 and 360 amino acid proteins with calculated molecular masses of 40157 and 39555, respectively. The amino acid sequencing of purified porcine DNase II reveals two N-termini with corresponding sequences present within the same open reading frame, suggesting proteolytic processing for the covalently bonded subunit structure of DNase II. Northern blot analysis demonstrated that a single transcript of 2.0 kb mRNA coding for DNase II is ubiquitously expressed in human tissues. A database search revealed that the human genomic sequence of chromosome 19p13.2 contains the DNase II gene. Characterization of the genomic sequence showed that the DNase II gene consists of six exons separated by five introns whose splice acceptor/donor sites agree with the GT/AG rule. q 1998 Academic Press
Deoxyribonuclease II (DNase II) is a well-characterized acid endonuclease that catalyzes the hydrolysis of DNA into 3*-phosphoryl oligonucleotides in the absence 1 The nucleotide sequences reported in this paper have been submitted to the GenBank/EMBL/DDBJ data bank with Accession Numbers AF060221 (porcine DNase II) and AF060222 (human DNase II). 2 To whom correspondence should be addressed. Fax: 81-3-32683045. E-mail: [email protected]. Abbreviations used: CRTC, calreticulin; EKLF, erythroid cell-specific transcription factor of the Kruppel zinc finger family; FARS, phenylalanine tRNA synthetase; GCDH, glutaryl-CoA dehydrogenase; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; PTH, phenylthiohydantoin; PVDF, polyvinylidene difluoride.
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of divalent metal ions (1). DNase II is a ubiquitous enzyme whose activity is found in wide variety of animal tissues; its subcellular localization has been assigned mainly to lysosomes (2, 3). Although the physiological significance of DNase II is not yet fully understood, it is assumed to play important roles in DNA catabolism and DNA fragmentation during apoptosis (1, 4-6). Apoptosis is an active suicidal process in which cells die in a genetically controlled manner. Apoptosis plays important roles under a variety of physiological circumstances in multicellular organisms (7, 8). Cleavage of chromosomal DNA into nucleosomal units is the biochemical feature that characterizes apoptotic cell death (9, 10). At present, several divalent cation-dependent endonucleases, including DNase I, Nuc-18, DNase g, and CAD, have been suggested as candidates for the apoptotic endonuclease (11-17). DNase II has also been proposed to be involved in specific cases of apoptosis (4-6). Therefore, an ‘‘old’’ enzyme, DNase II, is again becoming the focus of research. Knowledge about DNase II, including its purification and physical and enzymatic properties, is expanded, but the major problem in its study, that is, the identification of its cDNA or gene, remains to be solved. We describe here the cDNA cloning of porcine and human DNase II. The tissue distribution of the mRNA and the genomic structure of the human DNase II gene are also reported. MATERIALS AND METHODS Purification of DNase II from porcine liver. All operations were performed at 0–47C unless otherwise indicated. The crude enzyme fraction was obtained from 200 g of porcine liver according to the procedure of Bernardi et al. (18). DNase II was purified from the preparation as previously reported by Liao (19) with some modifications. Step 1: S Cartridge HPLC. The crude enzyme fraction was dialyzed against 20 mM acetate-NaOH (pH 4.7) and subjected to Econo-Pac S HPLC (Bio-Rad) in a cartridge equilibrated with 20 mM acetate-
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NaOH (pH 4.7). The proteins retained by the S Cartridge were eluted with a linear gradient of 0 - 1 M NaCl in 50 mM acetate-NaOH (pH 4.7) at a flow rate of 1 ml/min. Step 2: Blue5PW HPLC. The active fractions were pooled and subjected to blue5PW HPLC (Tosoh) on a column equilibrated with buffer N. Proteins were eluted with a linear gradient of 0 - 1 M NaCl in 50 mM acetate-NaOH (pH 4.7) at a flow rate of 0.5 ml/min. Step 3: Hydroxylapatite HPLC. The pooled active fraction was loaded onto a PD10 column (Pharmacia) equilibrated with 10 mM phosphate-NaOH (pH 6.8), and then subjected to Econo-Pac hydroxylapatite HPLC (Bio-Rad) on a column equilibrated with the same buffer. Proteins were eluted with a linear gradient of 10 - 400 mM sodium phosphate (pH 6.8) at a flow rate of 0.5 ml/min. Step 4: Superdex HR75 gel filtration HPLC. The active fractions were concentrated to 800 ml with Ultrafree C3-LCC (Millipore) and then subjected to gel filtration HPLC on a Superdex HR75 column (Pharmacia) equilibrated with 0.5 M NaCl in 20 mM Mes-NaOH (pH 5.6). Elution was carried out with the same buffer at a flow rate of 0.5 ml/min. Step 5: SP5PW HPLC. The enzyme fractions were pooled and desalted on a PD10 column and subjected to SP5PW HPLC (Tosoh) in a column equilibrated with 20 mM Mes-NaOH (pH 5.6). The proteins were eluted with a linear gradient of 0 - 1.0 M NaCl in 20 mM MesNaOH (pH 5.6) at a flow rate of 0.3 ml/min. The active fractions were pooled and stored at 0207C. This enzyme preparation was used for amino acid sequencing. Enzyme assay. DNase II activity was detected based on its ability to digest the plasmid DNA. In this assay, 1 ml of a 100-fold diluted aliquot of a column fraction was added to 10 ml of reaction buffer [50 mM acetate-NaOH (pH 4.7) and 50 ng/ml pBluescript KS/ (Stratagene)]. The enzyme reaction was carried out by incubation at 377C for 5 min and terminated by extraction with phenol/CHCl3. The resulting mixture was analyzed by 1% agarose gel electrophoresis. Amino acid sequencing. Aliquots of the final enzyme preparation (containing approximately 150 pmol of DNase II) were subjected to 10% SDS-PAGE. After separation, the proteins were electrotransferred onto PVDF membranes (Immobilone-PSQ) in transfer buffer [10 mM Caps-NaOH (pH 11.0), 10% methanol] at 200 mA for 60 min. The 35 kDa DNase II band was visualized by Ponceau S staining (0.1% Ponceau S, 1% acetic acid) and the band was cut out. The protein retained on the membrane strip was subjected to trypsin digestion. The peptide fragments released from the membrane were separated by an ABI 173A reverse-phase HPLC system (Applied Biosystems) and sequenced using a Procise cLC protein sequencer (Applied Biosystems). For the determination of the N terminal sequence, the membrane strip prepared as described above was subjected directly to amino acid sequencing. cDNA clonings of porcine and human DNase II. Based on the partial amino acid sequences of porcine DNase II, degenerated oligonucleotide primers were designed for the PCR amplification of a partial cDNA fragment of porcine liver cDNA. The amplification by PCR with sense {5*-ACKCHTAYCCNATGGTKTAYGAYTA-3*} and antisense {5*-GCRAAMGAKTGRAANGANGCMCC-3*} primers was carried out for 30 cycles in a Perkin Elmer Thermal Cycler using EX Taq polymerase (Takara); each cycle consisted of denaturation at 947C for 30 s, annealing at 557C for 30 s, and extension at 727C for 1 min. The sense and antisense primers corresponded to the amino acid sequences TYPMVYDY and GASFQSFA, respectively, and yielded a product of 158 bp. The PCR product was subcloned into pBluescript KS/ (Stratagene) and the nucleotide sequences were determined on both strands by cycle sequencing using a 7-deaza Thermo Sequenase kit (Amersham) and DSQ1000 DNA sequencer (Shimadzu). Two oligonucleotide primers, sense (GSP2/pII; 5*-TAGTCAAGGGCCATCATGTTCTCC-3*) and antisense (GSP1/pII; 5*TTGATGTGAGTGTTACACTGCTGTTCC-3*), were generated from the partial cDNA sequence and used to clone the full length cDNA for porcine DNase II by rapid amplification of cDNA ends (RACE)
reactions. RACE reactions were performed using a Marathon cDNA amplification kit (Clontech) according to the manufacture’s protocol. In 5* and 3* RACE reactions, adaptor-ligated cDNA was generated from porcine liver polyA(/) RNA and PCR amplifications were carried out using gene-specific primers GSP1/PII and GSP2/PII, respectively, and a linker primer (AP1; 5*-CCATCCTAATACGACTCACTATAGGGC-3*) by 35 cycles using EX Taq polymerase (Takara); each cycle consisted of denaturation at 947C for 30 s, annealing and extension at 727C for 90 s. The resulting PCR product was subcloned into pBluescript KS/ (Stratagene) and sequenced as described above. To minimize the possibility that artificial mutations were introduced during PCR amplification, we used EX Taq DNA polymerase throughout the experiments and at least ten independent clones were sequenced to confirm the cDNA sequences. The Expressed Sequence Tags (EST) subdivision of the NCBI GenBank database was searched with the deduced amino acid sequence of porcine DNase II using the tblastn program. As a result, we identified human EST clones (GenBank AA224257, AA419060, and AA419087) potentially coding for DNase II. On the basis of their nucleotide sequences, oligo-nucleotide primers, sense (5*-CCATGGCCCTGACCTGCTACGGGGAC-3*) and antisense (5*-CCATGGTTAGATCTTATAAGCTCTGCTGGGC-3*), were designed for the PCR amplification of a partial cDNA fragment of human DNase II from a Marathon-Ready human placenta cDNA (Clontech). Nco I sites flanking the coding sequences are bold faced. Amplification by PCR was carried out for 35 cycles using EX Taq polymerase (Takara); each cycle consisted of denaturation at 947C for 30 s, annealing at 607C for 30 s, and extension at 727C for 1 min. The PCR product was subcloned into pBluescript KS/ (Stratagene) and the nucleotide sequence was determined on both strands. This plasmid, named pBSIIhIIM, was used to design the following PCR primers and as a probe in Northern blot analysis. Two oligonucleotide primers, sense (GSP2/hII; 5*-TGAATCGGAACCAGGGAGAGGAGC-3*) and antisense (GSP1/hII; 5*-GCATGGAAGAGTCCTGAGCCTTGC-3*), were generated from the cDNA sequence, and the full length cDNA for human DNase II was cloned by the RACE method as described above. Northern blot analysis. Human Multiple Choice Northern lots #1 and #6 (Ori Gene) were hybridized with a 32P-labeled DNase II cDNA fragment, prepared from the plasmid pBSIIhIIM by Nco I digestion, in hybridization buffer (51 SSPE, 51 Denhardt’s solution, 50% formamide, 0.1% SDS, 100 mg/ml heat-denatured salmon sperm DNA) overnight at 427C. The resulting filters were washed for 30 min at 377C in 11 SSC containing 0.1% SDS, then for 30 min at 507C in 0.11 SSC containing 0.1% SDS, and then exposed to X-ray film for 24 h at 0807C using intensifying screens.
RESULTS AND DISCUSSION Purification and Amino Acid Sequencing of Porcine DNase II The purification of porcine liver DNase II was carried out by sequential column chromatographies on S cartridge, blue5PW, hydroxylapatite, Superdex HR75, and SP5PW. The purity of the final enzyme preparation was analyzed by SDS-PAGE. Result indicating the homogenous purification of DNase II is shown in Fig. 1A. Based on its mobility on SDS gel, the molecular mass of DNase II is estimated to be 35 kDa. By repetition of the purification, we obtained approximately 10 mg of pure DNase II protein. This enzyme preparation was used for the following amino acid sequencing of the protein. Aliquots of the purified DNase II were subjected to
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FIG. 1. Purification and amino acid sequencing of porcine DNase II. (A) Aliquots of the final enzyme preparation obtained by SP5PW HPLC were analyzed by 12% SDS-PAGE. Protein bands were visualized by silver staining. The band representing DNase II is marked by the arrow head. (B) Peptide map of DNase II digested with trypsin. Tryptic peptides generated by in situ digestion were separated by reverse-phase HPLC as described under Materials and Methods. The amino acid sequences of the peptides were analyzed as described under Materials and Methods, and the results are summarized in the right panel. X indicates an amino acid residue that could not be identified.
SDS-PAGE and transferred onto PVDF membranes. The 35 kDa DNase II band visualized by Ponceu S staining was cut out and subjected to in situ trypsin digestion. The elution profile of the tryptic peptides of DNase II on reverse-phase HPLC is shown in Fig. 1B. The peptides eluted in each peak were collected and subjected to amino acid sequencing. As a result, we determined the amino acid sequences of five tryptic peptides TP-1 Ç TP-5 (Fig. 1B right panel). Using these sequences, we tried to clone a cDNA for porcine DNase II. Isolation and Characterization of Porcine and Human DNase II cDNAs The complete nucleotide sequence of the porcine cDNA is illustrated in Fig. 2A. Sequence analysis reveals an open reading frame of 1095 bp encoding 364 amino acids including six potential N-glycosylation sites (Asp-X-Thr/Ser) with a calculated molecular mass of 40157. A partial amino acid sequence, ATEDHSKW, was previously determined for a proteolitic peptide of porcine DNase II, and the 5th histidine of this peptide is thought to be the active site for the catalytic action of DNase II (19). In this clone, this sequence is found in at amino acids 293 (Ala) to 300 (Trp) (Fig. 2A, double underline). This provides convincing evidence that the cDNA really encodes the porcine DNase II protein. We next attempted to clone a cDNA for human DNase II. A homology search of the EST subdivision of the NCBI GenBank database was performed using the predicted amino acid sequence of porcine DNase II as a query. As a result, we identified some human EST sequences showing high homology to porcine DNase II at the amino acid level. On the basis of the EST sequences, we cloned the full length cDNA for human DNase II by the PCR method. The human cDNA has an open reading frame of 1083
base pairs encoding 360 amino acids including four potential N-glycosylation sites with a calculated molecular mass of 39555 (Fig. 2B). Comparison of the deduced amino acid sequences of porcine and human DNase II reveals the proteins share 73.4 % identity (Fig. 3A). During the preparation of this manuscript, the isolation of a cDNA encoding human DNase II has been reported (20). Although the 1083 bp open reading frames are the same, the polyadenylation site is different from that determined here for our sequence. This indicates the existence of multiple polyadenylation sites in the human DNase II mRNA. A database search reveals that DNase II shows no apparent homology to any previously reported cDNAs or proteins, suggesting that DNase II is a novel class of endonuclease unrelated to enzymes or proteins with known functions. As illustrated by the Kyte-Doolittle hydrophobicity plots (Fig. 3), both predicted porcine and human proteins have N-terminal hydrophobic domains that potentially act as signal sequences for extracellular secretion. This is consistent with the previous observation that a portion of the DNase II is found in extracellular body fluids (2, 21, 22) and suggests that DNase II is first synthesized as a precursor protein and next converted to the mature enzyme by removal of the N-terminal signal sequence. To examine this possibility, we determined the N-terminal sequence of the purified porcine DNase II. Interestingly, two amino acid peaks appeared in every PTH-amino acid-releasing cycle. By comparison with the deduced protein sequence of porcine DNase II, these two peaks were found to derive from two individual sequences, NT-1 and NT-2 (Table 1), corresponding to amino acids 17 (Leu) Ç 34 (Lys) and 108 (Ser) Ç 132 (Val), respectively (Fig. 2A). This suggests that the mature enzyme is generated from its precursor protein by removal of the N-terminal signal peptide and further cleaved into two covalently linked
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FIG. 2. Characterization of the cDNAs for porcine and human DNase II. The complete sequences of the porcine (A) and human (B) DNase II cDNAs and their translation products are illustrated. The predicted amino acid sequences are shown from the first ATG codons in the open reading frames. In-frame stop codons upstream of the first ATGs are indicated in bold letters. Nucleotide and amino acid numbers are shown at the left. Putative polyadenylation signals are outlined. Potential N-linked glycosylation sites are marked by asterisks. The amino acid sequences corresponding to the tryptic peptides (Fig. 1B, right) and N-terminal sequences (Table 1) are indicated by underlining with the peptide numbers (B). The amino acid sequence corresponding to that previously determined for porcine DNase II (19) is double underlined (B).
polypeptides. In a previous report, Liao described the subunit structure of porcine DNase II; the 45 kDa holoenzyme is composed of two non-covalently associated 35 kDa (a) and 10 kDa (b) subunits (19). Whether the a and b subunits are identical to our two covalently linked polypeptides [20 (Leu) Ç 109 (His) (10 kDa) and 110 (Ser) Ç 364 (Asp) (28 kDa)] remains to be determined. It is of note that the sum of the molecular masses of these two polypeptides (38 kDa) is still higher than those determined by SDS-PAGE (35 kDa) (Fig.
1A). This suggests that further processing is required for the maturation of DNase II. The exact subunit structure of the mature DNase II protein remains undetermined and requires further studies. Tissue Distribution of DNase II mRNA The tissue distribution of the DNase II mRNA was determined by Northern blot analysis. The blots of polyA(/) RNAs prepared from various human tissues
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FIG. 4. Tissue distribution of DNase II mRNA. DNase II mRNA expression in adult human tissues was analyzed by Northern blot. The identities of the RNAs are indicated at the top of each lane. The position of the 2.1 kb DNase II mRNA is indicated by the arrowhead.
Genomic Organization of the Human DNase II Gene
FIG. 3. (A) Comparison of the deduced amino acid sequences of porcine and human DNase II. A minimum number of gaps (shown by dashes) were introduced to give maximum homology. Shadowed areas indicate amino acid identity between the proteins. Processing sites determined by porcine DNase II are marked by vertical arrows. (B) Hydropathy profiles of porcine and human DNase II. The hydropathy indexes of the deduced amino acid sequences of porcine (upper panel) and human (lower panel) DNase II were calculated according to the method of Kyte–Doolittle (24).
We obtained a human genomic sequence (GenBank AD000092) containing the DNase II gene by searching the GenBank database using the human DNase II cDNA sequence as a query. This genomic sequence covers a 111 kb region of human chromosome 19q13.2. As illustrated in Fig. 5A, six open reading frames are included within this gene-rich region. Comparison of the genomic sequence with the DNase II cDNA sequence reveals that one of the putative open reading frames encodes the DNase II protein and that the DNase II gene consists of six exons separated by five introns (Fig. 5B). The size of the individual introns ranges between 0.1 and 2.0 kb (Table 2). The entire locus spans approximately 6 kb. The sequences of all exon/intron junctions are in agreement with the GT/
were hybridized with a 32P-labeled DNase II cDNA fragment under high stringency conditions. As shown in Fig. 4, the expression of a single transcript of 2.1 kb DNase II mRNA was observed in all tissues tested. The ubiquitous expression of DNase II suggests a general function that is not specific for a particular tissue or cell type. FIG. 5. Schematic diagram of the human DNase II gene in chromosome 19. (A) Schematic illustration of the AD000092 human chromosome 19 genomic sequence. The DNase II gene is indicated by the shadowed box and other genes by open boxes. The identities of the genes are indicated at the top of each box. (B) Genomic organization of the DNase II gene. Exons are shown as boxes. The open and filled boxes indicate untranslated and coding regions, respectively. Exon numbers are indicated at the bottom of each exon.
TABLE 1 N-terminal
Amino acid sequences
NT-1 NT-2
LTXYGDSGQPVDWFVXYK SXNRGHTKXVLLXDQEGXFWLIHSV
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TABLE 2 Exon (size)
5π-Splice donor (cDNA position)
1 (179 bp)
GCCTGTAGACTGgtgagtgcgcag (179)
137 bp
tccgacctccagGTTCGTGGTCTA (180)
2 (181 bp)
AACACCAGCCAGgtgaagggggcc (360)
91 bp
atccgcccgcagCTCGCCTTCCTG (361)
3 (79 bp)
GGCACACGAAGGgtgaggcctgga (439)
1966 bp
cttttggtccagGTGTCCTGCTCC (440)
4 (165 bp)
TCTCGAAGATGGgtaagtcgagtc (604)
90 bp
cttgtcctccagGCAAGCAGCTGA (605)
5 (198 bp)
AATTTGGAGATGgtgagtctcaag (802)
2018 bp
catcctttgcagACCTGTACTCCG (803)
Intron size
3π-Splice acceptor (cDNA position)
6 (1153 bp)
AG rule for splice donor and acceptor sites in eukaryotic genes. The chromosomal localization of the human DNase II gene has been mapped to chromosome 19 (20, 23). This is consistent with our results that human DNase II gene is found in the genomic sequence of chromosome 19q13.2. In this study, we cloned the full length cDNAs for porcine and human DNase II on the basis of sequence information obtained by amino acid sequencing of porcine liver DNase II. Furthermore, we investigated the tissue distribution and genomic organization of the human DNase II gene. Our data provide important insights into the physiological roles of DNase II in various cellular activities.
6. 7. 8. 9. 10. 11.
12. 13. 14.
ACKNOWLEDGMENTS
15. We thank A. Kaneko, M. Tanaka, and Y.Nakabayashi for their excellent technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education.
16. 17.
REFERENCES
18.
1. Bernardi, G. (1971) in The Enzymes (Boyer, P. D., Ed.) 3rd ed., Vol. 4, pp. 271–287 Academic Press, New York. 2. Yasuda, T., Nadano, D., Awazu, S., and Kishi, K. (1992) Biochim. Biophys. Acta 1119, 185–193 3. Liao, T. H., Liao, W. C., Chang, H. C., and Lu, K. S. (1989) Biochim. Biophys. Acta 1007, 15–22 4. Barry M. A., and Eastman, A. (1993) Arch. Biochem. Biophys. 300, 440–450. 5. Torrigla, A., Chaudun, E., Chany-Fournier, F., Jeanny, J. C.,
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Courtois, Y., and Counis, M. F. (1995) J. Biol. Chem. 270, 28579– 28585. Gottlieb, R. A., Nordberg, J., Skowronski, E., and Babior, B. M. (1996) Proc. Natl. Acad. Sci. USA 93, 654–658. Kerr, J. F. R., Wyllie, A. H., and Currie, A. R. (1972) Br. J Cancer 26, 239–257. Wyllie, A. H. (1995) Curr.Opin. Genet. Dev. 5, 97–104. Wyllie, A. H. (1980) Nature 284, 555–556. Arends, M. J., Morris, R. G., and Wyllie, A. H. (1990) Am. J. Pathol. 136, 593–601. Peitsch, M. C., Polzar, B., Stepham, H., Crompton, T., MacDonald, H. R., Mannherz, H. G., and Tshopp, J. (1993) EMBO J. 12, 371–377. Gaido, M. L., and Cidlowski, J. A. (1991) J. Biol. Chem. 266, 18580–18585. Shiokawa, D., Ohyama, H., Yamada, T., Takahashi, K., and Tanuma, S. (1994) Eur. J. Biochem. 226, 23–30. Shiokawa, D., Ohyama, H., Yamada, T., and Tanuma, S. (1997) Biochem. J. 326, 675–681. Shiokawa, D., Iwamatsu, A., and Tanuma, S. (1997) Arch. Biochem. Biophys. 346, 15–20. Shiokawa, D., and Tanuma, S. (1998) Biochem. J. in press. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., and Nagata, S. (1998) Nature 391, 43–50. Bernardi, G., Bernardi, A., and Chersi, A. (1966) Biochim. Biophys. Acta 129, 1–11. Liao, T. H. (1985) J. Biol. Chem. 260, 10708–10713. Yasuda, T., Takashita, H., Ikeda, R., Nakajima, T., Hosumi, O., Nakashima, Y., and Kishi, K. (1998) J. Biol. Chem. 273, 2610– 2616. Murai, K., Yamanaka, M., Akagi, K., and Anai, M. (1980) J. Biochem. 87, 1097–1103. Koizumi, T. (1995) Exp. Anim. 44, 169–171. Brook, J. D., Shaw, D. J., Meredith, L., Bruns, G. A. P., and Harper, P. S. (1984) Hum. Genet. 68, 282–285. Kite, J., and Doolittle, R. F. (1982) J. Mol. Biol. 175, 105–132.
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RC988839
Cloning of cDNAs Encoding Porcine and Human DNase II1 Daisuke Shiokawa* and Sei-ichi Tanuma*,†,2 *Department of Biochemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, 12 Funagawara-cho, Ichigaya, Shinjuku-ku, Tokyo 162, Japan; and †Research Institute for Bioscience, Science University of Tokyo, 2669 Yamazaki, Noda, Chiba 278, Japan
Received May 21, 1998
We report the molecular cloning of cDNAs encoding porcine and human DNase II and the genomic structure of the human DNase II gene. The full length cDNAs for porcine and human DNase II were isolated by polymerase chain reaction on the basis of amino acid sequences determined for the tryptic peptides of porcine liver DNase II. The porcine and human cDNAs contain 1095 and 1083 bp open reading frames, respectively, and encode 364 and 360 amino acid proteins with calculated molecular masses of 40157 and 39555, respectively. The amino acid sequencing of purified porcine DNase II reveals two N-termini with corresponding sequences present within the same open reading frame, suggesting proteolytic processing for the covalently bonded subunit structure of DNase II. Northern blot analysis demonstrated that a single transcript of 2.0 kb mRNA coding for DNase II is ubiquitously expressed in human tissues. A database search revealed that the human genomic sequence of chromosome 19p13.2 contains the DNase II gene. Characterization of the genomic sequence showed that the DNase II gene consists of six exons separated by five introns whose splice acceptor/donor sites agree with the GT/AG rule. q 1998 Academic Press
Deoxyribonuclease II (DNase II) is a well-characterized acid endonuclease that catalyzes the hydrolysis of DNA into 3*-phosphoryl oligonucleotides in the absence 1 The nucleotide sequences reported in this paper have been submitted to the GenBank/EMBL/DDBJ data bank with Accession Numbers AF060221 (porcine DNase II) and AF060222 (human DNase II). 2 To whom correspondence should be addressed. Fax: 81-3-32683045. E-mail: [email protected]. Abbreviations used: CRTC, calreticulin; EKLF, erythroid cell-specific transcription factor of the Kruppel zinc finger family; FARS, phenylalanine tRNA synthetase; GCDH, glutaryl-CoA dehydrogenase; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; PTH, phenylthiohydantoin; PVDF, polyvinylidene difluoride.
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of divalent metal ions (1). DNase II is a ubiquitous enzyme whose activity is found in wide variety of animal tissues; its subcellular localization has been assigned mainly to lysosomes (2, 3). Although the physiological significance of DNase II is not yet fully understood, it is assumed to play important roles in DNA catabolism and DNA fragmentation during apoptosis (1, 4-6). Apoptosis is an active suicidal process in which cells die in a genetically controlled manner. Apoptosis plays important roles under a variety of physiological circumstances in multicellular organisms (7, 8). Cleavage of chromosomal DNA into nucleosomal units is the biochemical feature that characterizes apoptotic cell death (9, 10). At present, several divalent cation-dependent endonucleases, including DNase I, Nuc-18, DNase g, and CAD, have been suggested as candidates for the apoptotic endonuclease (11-17). DNase II has also been proposed to be involved in specific cases of apoptosis (4-6). Therefore, an ‘‘old’’ enzyme, DNase II, is again becoming the focus of research. Knowledge about DNase II, including its purification and physical and enzymatic properties, is expanded, but the major problem in its study, that is, the identification of its cDNA or gene, remains to be solved. We describe here the cDNA cloning of porcine and human DNase II. The tissue distribution of the mRNA and the genomic structure of the human DNase II gene are also reported. MATERIALS AND METHODS Purification of DNase II from porcine liver. All operations were performed at 0–47C unless otherwise indicated. The crude enzyme fraction was obtained from 200 g of porcine liver according to the procedure of Bernardi et al. (18). DNase II was purified from the preparation as previously reported by Liao (19) with some modifications. Step 1: S Cartridge HPLC. The crude enzyme fraction was dialyzed against 20 mM acetate-NaOH (pH 4.7) and subjected to Econo-Pac S HPLC (Bio-Rad) in a cartridge equilibrated with 20 mM acetate-
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NaOH (pH 4.7). The proteins retained by the S Cartridge were eluted with a linear gradient of 0 - 1 M NaCl in 50 mM acetate-NaOH (pH 4.7) at a flow rate of 1 ml/min. Step 2: Blue5PW HPLC. The active fractions were pooled and subjected to blue5PW HPLC (Tosoh) on a column equilibrated with buffer N. Proteins were eluted with a linear gradient of 0 - 1 M NaCl in 50 mM acetate-NaOH (pH 4.7) at a flow rate of 0.5 ml/min. Step 3: Hydroxylapatite HPLC. The pooled active fraction was loaded onto a PD10 column (Pharmacia) equilibrated with 10 mM phosphate-NaOH (pH 6.8), and then subjected to Econo-Pac hydroxylapatite HPLC (Bio-Rad) on a column equilibrated with the same buffer. Proteins were eluted with a linear gradient of 10 - 400 mM sodium phosphate (pH 6.8) at a flow rate of 0.5 ml/min. Step 4: Superdex HR75 gel filtration HPLC. The active fractions were concentrated to 800 ml with Ultrafree C3-LCC (Millipore) and then subjected to gel filtration HPLC on a Superdex HR75 column (Pharmacia) equilibrated with 0.5 M NaCl in 20 mM Mes-NaOH (pH 5.6). Elution was carried out with the same buffer at a flow rate of 0.5 ml/min. Step 5: SP5PW HPLC. The enzyme fractions were pooled and desalted on a PD10 column and subjected to SP5PW HPLC (Tosoh) in a column equilibrated with 20 mM Mes-NaOH (pH 5.6). The proteins were eluted with a linear gradient of 0 - 1.0 M NaCl in 20 mM MesNaOH (pH 5.6) at a flow rate of 0.3 ml/min. The active fractions were pooled and stored at 0207C. This enzyme preparation was used for amino acid sequencing. Enzyme assay. DNase II activity was detected based on its ability to digest the plasmid DNA. In this assay, 1 ml of a 100-fold diluted aliquot of a column fraction was added to 10 ml of reaction buffer [50 mM acetate-NaOH (pH 4.7) and 50 ng/ml pBluescript KS/ (Stratagene)]. The enzyme reaction was carried out by incubation at 377C for 5 min and terminated by extraction with phenol/CHCl3. The resulting mixture was analyzed by 1% agarose gel electrophoresis. Amino acid sequencing. Aliquots of the final enzyme preparation (containing approximately 150 pmol of DNase II) were subjected to 10% SDS-PAGE. After separation, the proteins were electrotransferred onto PVDF membranes (Immobilone-PSQ) in transfer buffer [10 mM Caps-NaOH (pH 11.0), 10% methanol] at 200 mA for 60 min. The 35 kDa DNase II band was visualized by Ponceau S staining (0.1% Ponceau S, 1% acetic acid) and the band was cut out. The protein retained on the membrane strip was subjected to trypsin digestion. The peptide fragments released from the membrane were separated by an ABI 173A reverse-phase HPLC system (Applied Biosystems) and sequenced using a Procise cLC protein sequencer (Applied Biosystems). For the determination of the N terminal sequence, the membrane strip prepared as described above was subjected directly to amino acid sequencing. cDNA clonings of porcine and human DNase II. Based on the partial amino acid sequences of porcine DNase II, degenerated oligonucleotide primers were designed for the PCR amplification of a partial cDNA fragment of porcine liver cDNA. The amplification by PCR with sense {5*-ACKCHTAYCCNATGGTKTAYGAYTA-3*} and antisense {5*-GCRAAMGAKTGRAANGANGCMCC-3*} primers was carried out for 30 cycles in a Perkin Elmer Thermal Cycler using EX Taq polymerase (Takara); each cycle consisted of denaturation at 947C for 30 s, annealing at 557C for 30 s, and extension at 727C for 1 min. The sense and antisense primers corresponded to the amino acid sequences TYPMVYDY and GASFQSFA, respectively, and yielded a product of 158 bp. The PCR product was subcloned into pBluescript KS/ (Stratagene) and the nucleotide sequences were determined on both strands by cycle sequencing using a 7-deaza Thermo Sequenase kit (Amersham) and DSQ1000 DNA sequencer (Shimadzu). Two oligonucleotide primers, sense (GSP2/pII; 5*-TAGTCAAGGGCCATCATGTTCTCC-3*) and antisense (GSP1/pII; 5*TTGATGTGAGTGTTACACTGCTGTTCC-3*), were generated from the partial cDNA sequence and used to clone the full length cDNA for porcine DNase II by rapid amplification of cDNA ends (RACE)
reactions. RACE reactions were performed using a Marathon cDNA amplification kit (Clontech) according to the manufacture’s protocol. In 5* and 3* RACE reactions, adaptor-ligated cDNA was generated from porcine liver polyA(/) RNA and PCR amplifications were carried out using gene-specific primers GSP1/PII and GSP2/PII, respectively, and a linker primer (AP1; 5*-CCATCCTAATACGACTCACTATAGGGC-3*) by 35 cycles using EX Taq polymerase (Takara); each cycle consisted of denaturation at 947C for 30 s, annealing and extension at 727C for 90 s. The resulting PCR product was subcloned into pBluescript KS/ (Stratagene) and sequenced as described above. To minimize the possibility that artificial mutations were introduced during PCR amplification, we used EX Taq DNA polymerase throughout the experiments and at least ten independent clones were sequenced to confirm the cDNA sequences. The Expressed Sequence Tags (EST) subdivision of the NCBI GenBank database was searched with the deduced amino acid sequence of porcine DNase II using the tblastn program. As a result, we identified human EST clones (GenBank AA224257, AA419060, and AA419087) potentially coding for DNase II. On the basis of their nucleotide sequences, oligo-nucleotide primers, sense (5*-CCATGGCCCTGACCTGCTACGGGGAC-3*) and antisense (5*-CCATGGTTAGATCTTATAAGCTCTGCTGGGC-3*), were designed for the PCR amplification of a partial cDNA fragment of human DNase II from a Marathon-Ready human placenta cDNA (Clontech). Nco I sites flanking the coding sequences are bold faced. Amplification by PCR was carried out for 35 cycles using EX Taq polymerase (Takara); each cycle consisted of denaturation at 947C for 30 s, annealing at 607C for 30 s, and extension at 727C for 1 min. The PCR product was subcloned into pBluescript KS/ (Stratagene) and the nucleotide sequence was determined on both strands. This plasmid, named pBSIIhIIM, was used to design the following PCR primers and as a probe in Northern blot analysis. Two oligonucleotide primers, sense (GSP2/hII; 5*-TGAATCGGAACCAGGGAGAGGAGC-3*) and antisense (GSP1/hII; 5*-GCATGGAAGAGTCCTGAGCCTTGC-3*), were generated from the cDNA sequence, and the full length cDNA for human DNase II was cloned by the RACE method as described above. Northern blot analysis. Human Multiple Choice Northern lots #1 and #6 (Ori Gene) were hybridized with a 32P-labeled DNase II cDNA fragment, prepared from the plasmid pBSIIhIIM by Nco I digestion, in hybridization buffer (51 SSPE, 51 Denhardt’s solution, 50% formamide, 0.1% SDS, 100 mg/ml heat-denatured salmon sperm DNA) overnight at 427C. The resulting filters were washed for 30 min at 377C in 11 SSC containing 0.1% SDS, then for 30 min at 507C in 0.11 SSC containing 0.1% SDS, and then exposed to X-ray film for 24 h at 0807C using intensifying screens.
RESULTS AND DISCUSSION Purification and Amino Acid Sequencing of Porcine DNase II The purification of porcine liver DNase II was carried out by sequential column chromatographies on S cartridge, blue5PW, hydroxylapatite, Superdex HR75, and SP5PW. The purity of the final enzyme preparation was analyzed by SDS-PAGE. Result indicating the homogenous purification of DNase II is shown in Fig. 1A. Based on its mobility on SDS gel, the molecular mass of DNase II is estimated to be 35 kDa. By repetition of the purification, we obtained approximately 10 mg of pure DNase II protein. This enzyme preparation was used for the following amino acid sequencing of the protein. Aliquots of the purified DNase II were subjected to
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FIG. 1. Purification and amino acid sequencing of porcine DNase II. (A) Aliquots of the final enzyme preparation obtained by SP5PW HPLC were analyzed by 12% SDS-PAGE. Protein bands were visualized by silver staining. The band representing DNase II is marked by the arrow head. (B) Peptide map of DNase II digested with trypsin. Tryptic peptides generated by in situ digestion were separated by reverse-phase HPLC as described under Materials and Methods. The amino acid sequences of the peptides were analyzed as described under Materials and Methods, and the results are summarized in the right panel. X indicates an amino acid residue that could not be identified.
SDS-PAGE and transferred onto PVDF membranes. The 35 kDa DNase II band visualized by Ponceu S staining was cut out and subjected to in situ trypsin digestion. The elution profile of the tryptic peptides of DNase II on reverse-phase HPLC is shown in Fig. 1B. The peptides eluted in each peak were collected and subjected to amino acid sequencing. As a result, we determined the amino acid sequences of five tryptic peptides TP-1 Ç TP-5 (Fig. 1B right panel). Using these sequences, we tried to clone a cDNA for porcine DNase II. Isolation and Characterization of Porcine and Human DNase II cDNAs The complete nucleotide sequence of the porcine cDNA is illustrated in Fig. 2A. Sequence analysis reveals an open reading frame of 1095 bp encoding 364 amino acids including six potential N-glycosylation sites (Asp-X-Thr/Ser) with a calculated molecular mass of 40157. A partial amino acid sequence, ATEDHSKW, was previously determined for a proteolitic peptide of porcine DNase II, and the 5th histidine of this peptide is thought to be the active site for the catalytic action of DNase II (19). In this clone, this sequence is found in at amino acids 293 (Ala) to 300 (Trp) (Fig. 2A, double underline). This provides convincing evidence that the cDNA really encodes the porcine DNase II protein. We next attempted to clone a cDNA for human DNase II. A homology search of the EST subdivision of the NCBI GenBank database was performed using the predicted amino acid sequence of porcine DNase II as a query. As a result, we identified some human EST sequences showing high homology to porcine DNase II at the amino acid level. On the basis of the EST sequences, we cloned the full length cDNA for human DNase II by the PCR method. The human cDNA has an open reading frame of 1083
base pairs encoding 360 amino acids including four potential N-glycosylation sites with a calculated molecular mass of 39555 (Fig. 2B). Comparison of the deduced amino acid sequences of porcine and human DNase II reveals the proteins share 73.4 % identity (Fig. 3A). During the preparation of this manuscript, the isolation of a cDNA encoding human DNase II has been reported (20). Although the 1083 bp open reading frames are the same, the polyadenylation site is different from that determined here for our sequence. This indicates the existence of multiple polyadenylation sites in the human DNase II mRNA. A database search reveals that DNase II shows no apparent homology to any previously reported cDNAs or proteins, suggesting that DNase II is a novel class of endonuclease unrelated to enzymes or proteins with known functions. As illustrated by the Kyte-Doolittle hydrophobicity plots (Fig. 3), both predicted porcine and human proteins have N-terminal hydrophobic domains that potentially act as signal sequences for extracellular secretion. This is consistent with the previous observation that a portion of the DNase II is found in extracellular body fluids (2, 21, 22) and suggests that DNase II is first synthesized as a precursor protein and next converted to the mature enzyme by removal of the N-terminal signal sequence. To examine this possibility, we determined the N-terminal sequence of the purified porcine DNase II. Interestingly, two amino acid peaks appeared in every PTH-amino acid-releasing cycle. By comparison with the deduced protein sequence of porcine DNase II, these two peaks were found to derive from two individual sequences, NT-1 and NT-2 (Table 1), corresponding to amino acids 17 (Leu) Ç 34 (Lys) and 108 (Ser) Ç 132 (Val), respectively (Fig. 2A). This suggests that the mature enzyme is generated from its precursor protein by removal of the N-terminal signal peptide and further cleaved into two covalently linked
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FIG. 2. Characterization of the cDNAs for porcine and human DNase II. The complete sequences of the porcine (A) and human (B) DNase II cDNAs and their translation products are illustrated. The predicted amino acid sequences are shown from the first ATG codons in the open reading frames. In-frame stop codons upstream of the first ATGs are indicated in bold letters. Nucleotide and amino acid numbers are shown at the left. Putative polyadenylation signals are outlined. Potential N-linked glycosylation sites are marked by asterisks. The amino acid sequences corresponding to the tryptic peptides (Fig. 1B, right) and N-terminal sequences (Table 1) are indicated by underlining with the peptide numbers (B). The amino acid sequence corresponding to that previously determined for porcine DNase II (19) is double underlined (B).
polypeptides. In a previous report, Liao described the subunit structure of porcine DNase II; the 45 kDa holoenzyme is composed of two non-covalently associated 35 kDa (a) and 10 kDa (b) subunits (19). Whether the a and b subunits are identical to our two covalently linked polypeptides [20 (Leu) Ç 109 (His) (10 kDa) and 110 (Ser) Ç 364 (Asp) (28 kDa)] remains to be determined. It is of note that the sum of the molecular masses of these two polypeptides (38 kDa) is still higher than those determined by SDS-PAGE (35 kDa) (Fig.
1A). This suggests that further processing is required for the maturation of DNase II. The exact subunit structure of the mature DNase II protein remains undetermined and requires further studies. Tissue Distribution of DNase II mRNA The tissue distribution of the DNase II mRNA was determined by Northern blot analysis. The blots of polyA(/) RNAs prepared from various human tissues
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FIG. 4. Tissue distribution of DNase II mRNA. DNase II mRNA expression in adult human tissues was analyzed by Northern blot. The identities of the RNAs are indicated at the top of each lane. The position of the 2.1 kb DNase II mRNA is indicated by the arrowhead.
Genomic Organization of the Human DNase II Gene
FIG. 3. (A) Comparison of the deduced amino acid sequences of porcine and human DNase II. A minimum number of gaps (shown by dashes) were introduced to give maximum homology. Shadowed areas indicate amino acid identity between the proteins. Processing sites determined by porcine DNase II are marked by vertical arrows. (B) Hydropathy profiles of porcine and human DNase II. The hydropathy indexes of the deduced amino acid sequences of porcine (upper panel) and human (lower panel) DNase II were calculated according to the method of Kyte–Doolittle (24).
We obtained a human genomic sequence (GenBank AD000092) containing the DNase II gene by searching the GenBank database using the human DNase II cDNA sequence as a query. This genomic sequence covers a 111 kb region of human chromosome 19q13.2. As illustrated in Fig. 5A, six open reading frames are included within this gene-rich region. Comparison of the genomic sequence with the DNase II cDNA sequence reveals that one of the putative open reading frames encodes the DNase II protein and that the DNase II gene consists of six exons separated by five introns (Fig. 5B). The size of the individual introns ranges between 0.1 and 2.0 kb (Table 2). The entire locus spans approximately 6 kb. The sequences of all exon/intron junctions are in agreement with the GT/
were hybridized with a 32P-labeled DNase II cDNA fragment under high stringency conditions. As shown in Fig. 4, the expression of a single transcript of 2.1 kb DNase II mRNA was observed in all tissues tested. The ubiquitous expression of DNase II suggests a general function that is not specific for a particular tissue or cell type. FIG. 5. Schematic diagram of the human DNase II gene in chromosome 19. (A) Schematic illustration of the AD000092 human chromosome 19 genomic sequence. The DNase II gene is indicated by the shadowed box and other genes by open boxes. The identities of the genes are indicated at the top of each box. (B) Genomic organization of the DNase II gene. Exons are shown as boxes. The open and filled boxes indicate untranslated and coding regions, respectively. Exon numbers are indicated at the bottom of each exon.
TABLE 1 N-terminal
Amino acid sequences
NT-1 NT-2
LTXYGDSGQPVDWFVXYK SXNRGHTKXVLLXDQEGXFWLIHSV
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TABLE 2 Exon (size)
5π-Splice donor (cDNA position)
1 (179 bp)
GCCTGTAGACTGgtgagtgcgcag (179)
137 bp
tccgacctccagGTTCGTGGTCTA (180)
2 (181 bp)
AACACCAGCCAGgtgaagggggcc (360)
91 bp
atccgcccgcagCTCGCCTTCCTG (361)
3 (79 bp)
GGCACACGAAGGgtgaggcctgga (439)
1966 bp
cttttggtccagGTGTCCTGCTCC (440)
4 (165 bp)
TCTCGAAGATGGgtaagtcgagtc (604)
90 bp
cttgtcctccagGCAAGCAGCTGA (605)
5 (198 bp)
AATTTGGAGATGgtgagtctcaag (802)
2018 bp
catcctttgcagACCTGTACTCCG (803)
Intron size
3π-Splice acceptor (cDNA position)
6 (1153 bp)
AG rule for splice donor and acceptor sites in eukaryotic genes. The chromosomal localization of the human DNase II gene has been mapped to chromosome 19 (20, 23). This is consistent with our results that human DNase II gene is found in the genomic sequence of chromosome 19q13.2. In this study, we cloned the full length cDNAs for porcine and human DNase II on the basis of sequence information obtained by amino acid sequencing of porcine liver DNase II. Furthermore, we investigated the tissue distribution and genomic organization of the human DNase II gene. Our data provide important insights into the physiological roles of DNase II in various cellular activities.
6. 7. 8. 9. 10. 11.
12. 13. 14.
ACKNOWLEDGMENTS
15. We thank A. Kaneko, M. Tanaka, and Y.Nakabayashi for their excellent technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education.
16. 17.
REFERENCES
18.
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