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1.
Human dioxin-inducible cytosolic NAD(P)H:menadione oxidoreductase. cDNA sequence and localization of gene to chromosome 16 总被引:9,自引:0,他引:9
A K Jaiswal O W McBride M Adesnik D W Nebert 《The Journal of biological chemistry》1988,263(27):13572-13578
NAD(P)H:menadione oxidoreductase (NMOR1) is a flavoprotein that catalyzes the two-electron reduction of various redox dyes and quinones. It has been proposed that this enzyme may have a protective effect against cancer caused by quinones and their metabolic precursors. We show that tetrachlorodibenzo-p-dioxin (TCDD) treatment of the human hepatoblastoma cell line Hep-G2 produces a 5-fold induction of NMOR activity. Several overlapping human NMOR1 cDNAs were isolated from a human liver lambda gt 11 expression library, and their composite sequence corresponds to an mRNA of 2448 nucleotides containing a continuous open reading frame encoding a protein of 274 residues (molecular weight, 30,880). The corresponding human NMOR1 mRNA has an unusually long 3'-untranslated region (1679 base pairs) with four potential polyadenylation signals (I-IV) at positions 986, 1460, 1838, and 2419 and a single copy of human Alu repetitive sequence between polyadenylation sites II and III. Southern blot analysis of human genomic DNA suggests the presence of a single NMOR1 gene approximately 10 kilobases (KB) in length. The use of three of the aforementioned polyadenylation signals is likely to account for the three different species (2.7, 1.7, and 1.2 kb) of mRNA hybridizing to NMOR1 cDNA in Hep-G2 cells. Indeed several partial cDNA clones were isolated that corresponded to the mRNA derived by use of the proximal polyadenylation signal. Interestingly, the longest (2.7 kb) mRNA species was induced severalfold by TCDD, whereas the other two mRNAs (1.7 and 1.2 kb) were induced to a much lesser extent by TCDD treatment. The human NMOR1 cDNA and protein are 83 and 85% similar to rat liver cytosolic NMOR1 cDNA and protein, respectively. Southern analysis of DNA from 54 human x mouse and 39 human x hamster somatic cell hybrids shows that the NMOR1 gene resides on human chromosome 16. 相似文献
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Y Inoue Y Kawasaki T Harada K Hatakeyama H Kagamiyama 《The Journal of biological chemistry》1991,266(31):20791-20796
6-Pyruvoyl-tetrahydropterin synthase, which catalyzes the second step in the biosynthesis of tetrahydrobiopterin, was purified approximately 18,000-fold to apparent homogeneity from rat liver. The molecular mass of the native enzyme was estimated to be 83 kDa by gel filtration. The enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 17 kDa. Up to 24 residues of the NH2-terminal sequence were determined by Edman degradation, which released a single amino acid at each step. These results indicate that the enzyme consists of identical subunits. The purified enzyme was digested with lysyl endopeptidase or V8 protease, and 11 peptide fragments were isolated. On the basis of the sequences of these peptides, oligonucleotides were synthesized and used to screen a rat liver cDNA library, and one cDNA clone was isolated. The complete nucleotide sequence of the 1176-base pair cDNA was then determined. The deduced amino acid sequence contained 144 amino acid residues, but a NH2-terminal four-amino acid sequence was not found in the purified protein. Therefore, the mature protein consists of 140 amino acids. A single mRNA band of 1.3 kilobases was obtained by RNA blot analysis of rat liver. The predicted amino acid sequence of 6-pyruvoyl-tetrahydropterin synthase was compared with the Protein Sequence Database of the National Biomedical Research Foundation, revealing significant local similarity to large T antigens from the polyomavirus family. 相似文献
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NAD(P)H:quinone oxidoreductases (NQOs) are flavoproteins that catalyze the oxidation of NADH or NADPH by various quinones and oxidation-reduction dyes. We have previously described a complementary DNA that encodes a dioxin-inducible cytosolic form of human NAD(P)H:quinone oxidoreductase (NQO1). In the present report we describe the nucleotide sequence and deduced amino acid sequence for a cDNA clone that is likely to encode a second form of NAD(P)H:quinone oxidoreductase (NQO2) which was isolated by screening a human liver cDNA library by hybridization with a NQO1 cDNA probe. The NQO2 cDNA is 976 nucleotides long and encodes a protein of 231 amino acids (Mr = 25,956). The human NQO2 cDNA and protein are 54% and 49% similar to human liver cytosolic NQO1 cDNA and protein, respectively. COS1 cells transfected with NQO2 cDNA showed a 5-7-fold increase in NAD(P)H:quinone oxidoreductase activity as compared to nontransfected cells when either 2,6-dichlorophenolindophenol or menadione was used as substrate. Western blot analysis of the expressed NQO1 and NQO2 cDNA proteins showed cross-reactivity with rat NQO1 antiserum, indicating that NQO1 and NQO2 proteins are immunologically related. Northern blot analysis shows the presence of one NQO2 mRNA of 1.2 kb in control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated human hepatoblastoma Hep-G2 cells and that TCDD treatment does not lead to enhanced levels of NQO2 mRNA as it does for NQO1 mRNA. Southern blot analysis of human genomic DNA suggests the presence of a single gene approximately 14-17 kb in length. The NQO2 gene locus is highly polymorphic as indicated by several restriction fragment length polymorphisms detected with five different restriction enzymes.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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M Tanaka S Ohno S Adachi S Nakajin M Shinoda Y Nagahama 《The Journal of biological chemistry》1992,267(19):13451-13455
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Czirják G Burkhart WA Moyer MB Antal J Shears SB Enyedi P 《Biochimica et biophysica acta》1999,1444(3):326-336
A rat cytoplasmic aminopeptidase P was purified from liver cytosol with a procedure including an affinity elution step with 3 microM inositol 1,3,4-trisphosphate. Proteolytic fragments were generated, sequenced and the enzyme was cloned from a rat liver cDNA library. The structure shows high (87.8% and 95.5%, respectively) sequence identity at the nucleotide and amino acid levels with the previously described human putative cytoplasmic aminopeptidase P. The cloned rat enzyme was functionally expressed in Escherichia coli and also in COS-1 cells. Western blot analysis, using an antibody generated against the recombinant protein, and Northern blot hybridization showed ubiquitous expression of the protein in different tissues with the highest expression level in the testis. 相似文献
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cDNA encoding type B subunit of rat phosphoglycerate mutase: its isolation and nucleotide sequence. 总被引:1,自引:0,他引:1
K Uchida 《Archives of biochemistry and biophysics》1991,288(2):558-561
A cDNA encoding the nonmuscle-specific (type B) subunit of phosphoglycerate mutase (PGAM-B) was isolated and characterized. A cDNA probe, synthesized by the polymerase chain reaction (PCR) from rat liver cell mRNA using mixed primers specific to the amino acid sequence of human PGAM-B, was used to screen a rat liver cell cDNA library. The identity of the cDNA was confirmed by amino acid sequence data for 24 peptides obtained by digesting the purified protein with three different endopeptidases. The coding region encoded a polypeptide composed of 253 amino acid (plus the initiator Met). RNA blot analysis showed a single mRNA species of 1.7 kilobases in rat liver cell. The deduced amino acid sequence of rat PGAM-B was identical to that of human PGAM-B except for only one substitution at position 251 near the carboxyl terminus (valine for the rat and alanine for the human). 相似文献
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Recently two reports [J. A. Robertson et al. (1986) J. Biol. Chem. 261, 15794-15799 and R. M. Bayney et al. (1987) J. Biol. Chem. 262, 572-575] have appeared concerning the nucleotide sequence of quinone reductase cDNA clones. Although the cDNA clones are virtually identical, they diverge in the 5' region that encodes the NH2 terminus of the protein. In order to clarify the sequence of this region, we have isolated quinone reductase clones from a rat genomic library using a cDNA clone, pDTD55, isolated and characterized by our laboratory. We have determined the sequence of exons 1 and 2 of the structural gene by double-stranded sequencing using oligonucleotide primers. The sequence of exons 1 and 2 of the quinone reductase structural gene along with our previous nucleotide sequence analysis of pDTD55 as well as conventional amino acid sequence analysis of the purified protein indicates that quinone reductase is composed of 274 amino acids with a molecular weight of 30,946. These data agree with the published sequence of lambda NMOR1 reported by Robertson et al. 相似文献
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Ke-Nan Qin Maria I. New K.-C. Cheng 《The Journal of steroid biochemistry and molecular biology》1993,46(6)
Rat liver 3α-hydroxysteroid dehydrogenase cDNA was previously cloned by us. In this study, we used the rat cDNA as the probe to screen a human liver lambda gt11 cDNA library. A total of four different cDNAs were identified and sequenced. The sequence of one of the cDNAs is identical to that of the human chlordecone reductase cDNA except that our clone contains a much longer 5′-coding sequence than previously reported. The other three cDNAs display high degrees of sequence homology to those of both rat 3α-hydroxysteroid dehydrogenase and human chlordecone reductase. Because 3α-hydroxysteroid dehydrogenase and human chlordecone reductase belong to the aldo-keto reductase superfamily, we named these human clones HAKRa to HAKRd. Northern blot analysis showed that the liver expresses the highest levels of all four clones. Expression of all four clones was also detected in the brain, kidney, lung, and testis, whereas the placenta expressed only the messenger RNA for HAKRb. Genomic blot analysis using HAKRb as the probe detected multiple DNA fragments hybridized to the probe and a high degree of restriction fragment length polymorphism, suggesting the complexity of this supergene family. 相似文献
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cDNA and deduced amino acid sequence of a mouse DNA repair enzyme (APEX nuclease) with significant homology to Escherichia coli exonuclease III 总被引:11,自引:0,他引:11
S Seki K Akiyama S Watanabe M Hatsushika S Ikeda K Tsutsui 《The Journal of biological chemistry》1991,266(31):20797-20802
We purified a mouse DNA repair enzyme having apurinic/apyrimidinic endonuclease, DNA 3'-phosphatase, 3'-5'-exonuclease and DNA 3' repair diesterase activities, and designated the enzyme as APEX nuclease. A cDNA clone for the enzyme was isolated from a mouse spleen cDNA library using probes of degenerate oligonucleotides deduced from the N-terminal amino acid sequence of the enzyme. The complete nucleotide sequence of the cDNA (1.3 kilobases) was determined. Northern hybridization using this cDNA showed that the size of its mRNA is about 1.5 kilobases. The complete amino acid sequence for the enzyme predicted from the nucleotide sequence of the cDNA (APEX nuclease cDNA) indicates that the enzyme consists of 316 amino acids with a calculated molecular weight of 35,400. The predicted sequence contains the partial amino acid sequences determined by a protein sequencer from the purified enzyme. The coding sequence of APEX nuclease was cloned into pUC18 SmaI and HindIII sites in the control frame of the lacZ promoter. The construct was introduced into BW2001 (xth-11, nfo-2) strain cells of Escherichia coli. The transformed cells expressed a 36.4-kDa polypeptide (the 316 amino acid sequence of APEX nuclease headed by the N-terminal decapeptide of beta-galactosidase) and were less sensitive to methyl methanesulfonate than the parent cells. The fusion product showed priming activity for DNA polymerase on bleomycin-damaged DNA and acid-depurinated DNA. The deduced amino acid sequence of mouse APEX nuclease exhibits a significant homology to those of exonuclease III of E. coli and ExoA protein of Streptococcus pneumoniae and an intensive homology with that of bovine AP endonuclease 1. 相似文献
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Molecular cloning of complementary deoxyribonucleic acid for an androgen-regulated epididymal protein: sequence homology with metalloproteins 总被引:5,自引:0,他引:5
N J Charest D R Joseph E M Wilson F S French 《Molecular endocrinology (Baltimore, Md.)》1988,2(10):999-1004
Acidic epididymal glycoprotein (AEG) is a 31,000 molecular weight secretory protein of the rat epididymis. Screening of a rat epididymal cDNA library with affinity-purified AEG antiserum yielded cDNA for AEG. Identity of the clones was verified by comparison of amino acid sequence of the purified protein with the sequence derived from the nucleotide sequence of the cDNA isolates. Two classes of AEG cDNA, approximately 1500 base pairs (bp) and 950 bp in length, differed by 538 bp in the 3'-untranslated region and by four single nucleotide mismatches, one of which was in the coding region. Northern blot hybridization of epididymal RNA revealed two species of AEG mRNA, corresponding in length to each type of cDNA. Analysis of RNA from individual animals provided evidence that the two mRNA species are the products of allelic genes. In vivo studies demonstrated that the level of total AEG mRNA is regulated by androgen. Amino acid sequence homology of AEG with metal-binding domains of several proteins suggests that AEG is a metalloprotein. 相似文献
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Complementary DNA clones encoding 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. The sizes of the cDNA inserts ranged from 1.3-2.3 kilobases. Sequence analysis indicated that variation in the DNA size was due to heterogeneity in the length of 3' noncoding sequences. A full-length cDNA clone of 1286 basepairs contained an open reading frame encoding a protein of 322 amino acids with an estimated mol wt of 37 kDa. When expressed in E. coli, the encoded protein migrated to the same position on sodium dodecyl sulfate-polyacrylamide gels as the enzyme purified from rat liver cytosols. The protein expressed in bacteria was highly active in androsterone reduction in the presence of NAD as cofactor, and this activity was inhibited by indomethacin, a potent inhibitor of 3 alpha HSD. The predicted amino acid sequence of 3 alpha HSD was related to sequences of several other enzymes, including bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase, and frog lens epsilon-crystalline, suggesting that these proteins belong to the same gene family. 相似文献
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Characterization of a cDNA for human glutathione-insulin transhydrogenase (protein-disulfide isomerase/oxidoreductase) 总被引:1,自引:0,他引:1
A human liver cDNA expression library in lambda-phage gt11 was screened with monoclonal antibodies to rat liver protein-disulfide isomerase/oxidoreductase (EC 5.3.4.1/1.8.4.2), also known as glutathione-insulin transhydrogenase (GIT). The nucleotide sequence of the largest cDNA insert (hgit-1) was determined. It contained approx. 1500 basepairs, representing an estimated 65% of the glutathione-insulin transhydrogenase message. The amino-acid sequence deduced from this cDNA insert contains a 7-amino-acid long polypeptide determined by sequencing the active-site fragment isolated from the rat GIT protein. A comparison of the nucleotide sequence of hgit-1 and a previously reported nucleotide sequence of rat glutathione-insulin transhydrogenase cDNA shows that the human hgit-1 clone corresponds to the middle of the transhydrogenase message at amino-acid residue number 275 of the rat protein, and codes for 206 amino-acid residues, including one of the two active-site regions of glutathione-insulin transhydrogenase, a stop codon (TAA), a long 3'-noncoding region of over 800 bases, a polyadenylation signal (AATAA), and a 29 base poly(A) tail. There exists high homology between the human and rat enzymes (94% in the overall amino-acid sequence, with 100% in the active site region and 81% in the nucleotide sequence within the coding portion of hgit-1). As with the rat enzyme, the human enzyme shows some identity with another dithiol-disulfide-exchange protein, Escherichia coli thioredoxin. Like rat cDNA, the human hgit-1 cDNA hybridized to rat mRNA of 2500 bases on a Northern blot. The relative quantitative abundance of GIT mRNA in nine rat tissues studied using hgit-1 as a hybridization probe was found to be in the same order as previously found with the rat cDNA. Thus, the above studies indicate that glutathione-insulin transhydrogenase is a highly conserved protein and that the human hgit-1 cDNA is suitable for use as a probe for further studies on gene regulation of this enzyme. 相似文献
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Molecular cloning of cDNA for acetyl-coenzyme A carboxylase 总被引:4,自引:0,他引:4
D H Bai M E Pape F López-Casillas X C Luo J E Dixon K H Kim 《The Journal of biological chemistry》1986,261(26):12395-12399
Poly(A)+ RNA from lactating rat mammary glands was size-fractionated to enrich the relative amount of acetyl-CoA carboxylase mRNA. The enriched mRNA was used to generate a lambda gt11 cDNA library. Initial screening with polyclonal antiserum to acetyl-CoA carboxylase produced three positive clones. Western blot analysis revealed that two clones, lambda DH3 and lambda KH18, synthesized 165,000-dalton proteins that were recognized by antibodies to acetyl-CoA carboxylase and beta-galactosidase, indicating that acetyl-CoA carboxylase/beta-galactosidase fusion proteins were produced. Competition experiments with purified acetyl-CoA carboxylase further demonstrated that the fusion proteins contained acetyl-CoA carboxylase protein segments. Antibodies which are specific to the fusion proteins were isolated. These antibodies cross-reacted only with acetyl-CoA carboxylase in a preparation of partially purified acetyl-CoA carboxylase. In addition, the antibodies immunoprecipitated enzyme activity from a crude liver homogenate. Northern blot analysis of total RNA revealed two RNA species: one 10 kilobases and the other 3.0 kilobases. The levels of these RNA species increased when starved animals were fed a fat-free diet, indicating that they are coordinately regulated. 相似文献