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D W Nebert

Publications and source records attributed to D W Nebert.

At least 145 records · Page 8Linked to original sources
PubMed1988

Human debrisoquine 4-hydroxylase (P450IID1): cDNA and deduced amino acid sequence and assignment of the CYP2D locus to chromosome 22.

The enzyme P450db1 (db1) is responsible for the common human defect in drug oxidation known as the "debrisoquine/sparteine polymorphism." Polyclonal antibody against the rat db1 protein was used to screen a human liver lambda gt11 library for the db1 cDNA clone. A cDNA containing the full protein coding sequence was isolated; the deduced NH2-terminal sequence of this cDNA was identical to that derived from direct sequencing of the purified human db1 protein. Comparison of the human db1 with rat db1 revealed 71 and 73% similarities of nucleotides and amino acids, respectively. By use of human-rodent somatic cell hybrids the db1 gene was localized to human chromosome 22 (CYP2D locus).

Amino Acid Sequence↗
PubMed1988

Evolution of the P450 gene superfamily.

A P450 gene nomenclature system has recently been proposed on the basis of divergent evolution of distinct families and subfamilies among eukaryotes and prokaryotes. At present, eleven gene families have been described, eight of which exist in all mammals. The current number of P450 genes in each species is believed to reflect, at least in part, selective advantages as animals and plants have struggled for coexistence during the last 1 billion years. It is likely that P450 genes will also be found in archaebacteria, as well as most or all eubacteria and eukaryotes. Using Wu-Kabat analysis, we propose regions of the P450 protein involved in membrane attachment, flavoprotein-binding, heme-binding, and substrate specificity. The invariant amino acid sequence (F**G***C*G in the heme-binding region) is unique to the fifty P450 proteins characterized to date and is not found in any of the more than 5,430 other proteins presently in the database. The regulatory trans-acting protein factors and their associated upstream cis-acting DNA elements of the orthologous mouse, rat and human P450A1 gene are complex but appear to be highly conserved during the 80 million years since the mammalian radiation.

Amino Acid Sequence↗
PubMed1987

Human P45IA1 upstream regulatory sequences expressing the chloramphenicol acetyltransferase gene. Effect of Ha-MSV enhancer and comparison of transient with stable transformation assays.

Upstream sequences of the human P450IA1 gene were inserted into a promoterless expression vector (pSVO-cat) containing the chloramphenicol acetyltransferase (CAT) gene, with and without the Harvey murine sarcoma virus (Ha-MSV) core enhancer, and either plasmid was transfected into human breast carcinoma MCF-7 and MDA-231 and mouse hepatoma Hepa-1 cell lines. In most instances constitutive and inducible CAT activities in the transient CAT expression assay were similar (within 3-fold) to those in the stable transformation CAT assay (selection of G418-resistant colonies following co-transfection with pSV2-neo). In the case of Ha-MSV-containing constructs stably integrated in the two human breast cancer lines, however, CAT expression was more than two orders of magnitude greater than that transiently expressed in these cells. Since the major difference between these two assays is plasmid copy number, these data suggest the presence of limiting amounts of tissue-specific positive-control enhancer-binding factor(s) in the breast carcinoma cell lines.

Acetyltransferases↗
PubMed1987

Analysis of two benzo[a]pyrene-resistant mutants of the mouse hepatoma Hepa-1 P(1)450 gene via cDNA expression in yeast.

Two benzo[a]pyrene-resistant mutant clones (c1 and c37) of the mouse hepatoma Hepa-1 wild-type (wt) cell line were examined for their lack of P(1)450 [aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH)] activity. From lambda gt11 cDNA libraries, the nearly full-length P(1)450 cDNAs of wt, c1 and c37 were isolated and sequenced. The c1 cDNA was found to have a single mutation leading to premature termination of the protein after Asn-414; a rapidly migrating band corresponding to this truncated protein was found on Western immunoblots. The c37 cDNA was found to have two point mutations, leading to Leu-118----Arg-118 and Arg-245----Pro-245, but otherwise to encode the normal (524-residue) protein; the mature protein was confirmed by Western blot analysis. P(1)450 cDNA from wt, c1 and c37 and chimeric cDNAs between wt and c37 were inserted into the expression vector pAAH5 and expressed in Saccharomyces cerevisiae strain 50.L4. The Leu-118----Arg-118 mutation alone was found to have negligible effect on AHH activity, while the Arg-245----Pro-245 mutation alone leads to a 2- to 3-fold decrease in enzyme activity. The two mutations together totally abrogate AHH activity. The biologic mutant c37 provides the first evidence for the importance of Arg-245, and the complementary function of Leu-118, in normal P(1)450 enzymic function. This alteration in a single amino acid from arginine to proline might block electron flow directly, or change secondary structure of the protein, such that normal monooxygenation of benzo[a]pyrene cannot occur.

Amino Acid Sequence↗
PubMed1987

Localization of UDP glucuronosyltransferase gene(s) on mouse chromosome 5.

The drug-metabolizing UDP glucuronosyltransferases are encoded by genes which constitute a multigene family. One rat gene subfamily codes for at least four constitutive enzyme forms, including those which glucuronidate the androgenic steroids testosterone (UDPGTr-3) and androsterone (UDPGTr-4). In the present study, UDPGTr-3 and UDPGTr-4 cDNAs were used to demonstrate that an homologous subfamily is present in the mouse genome. Using mouse X Chinese hamster somatic cell hybrids, we mapped at least one gene of this UDP glucuronosyltransferase subfamily (UDPGTr-3) to mouse chromosome 5 and suggest the name as the Udpgt-3 locus.

Animals↗
PubMed1987

The P450 gene superfamily: recommended nomenclature.

A nomenclature for the P450 gene superfamily is proposed based on evolution. Recommendations include Roman numerals for distinct gene families, capital letters for subfamilies, and Arabic numerals for individual genes. An updating of this list, which presently includes 65 entries, will be required every 1-2 years. Assignment of orthologous genes is presently uncertain in some cases--between widely diverged species and especially in the P450II family due to the large number of genes. As more is known, it might become necessary to change some gene assignments that are based on our present knowledge.

Alleles↗
PubMed1987

Debrisoquine 4-hydroxylase: characterization of a new P450 gene subfamily, regulation, chromosomal mapping, and molecular analysis of the DA rat polymorphism.

Debrisoquine 4-hydroxylase (P450db1) was purified from rat liver microsomes. Polyclonal antibody was produced and, in conjunction with immunoblots, was used to identify and purify a second immunorelated P450 (P450db2) that does not have debrisoquine hydroxylating activity. The cDNA clones to db1 and db2 were isolated from a lambda gt11 expression library, sequenced, and found to share 78% nucleotide and 73% deduced amino acid similarities. These similarities are evenly dispersed along the sequence except for a region of 190 nucleotides with 99% similarity near the carboxyl terminus of the protein-coding region; this similarity is probably the remnant of a gene conversion event. Both proteins share between 38% and 43% amino acid similarity with P450a, P450b, P450e, P450f, P450PB1, and P450j; these data indicate that P450db1 and P450db2 are members of a separate subfamily within the P450II gene family. Southern blot analysis and preliminary genomic cloning suggest that at least four genes exist in the subfamily, although the present evidence suggests that only db1 and db2 are expressed in rat liver. With the use of 19 mouse X hamster somatic cell hybrids, the db1 and db2 genes were localized to mouse chromosome 15 (P450-2D locus). A polymorphism has been described for debrisoquine metabolism in the DA rat strain, adult females having markedly decreased debrisoquine 4-hydroxylase activity. Our immunoblot analysis and mRNA analysis suggest that debrisoquine 4-hydroxylase deficiency in the female DA rat is not due to a decrease in db1 protein or mRNA. The db1 and db2 proteins are differentially regulated: during development db2 is present at birth while db1 is absent, and db1 increases by 1 week of age; in addition, db1 is slightly induced by phenobarbital, 3-methyl-cholanthrene, and dexamethasone whereas db2 is marginally increased by these latter two agents. These results demonstrate that debrisoquine 4-hydroxylase is a member of a new constitutively expressed P450II sub-family containing two or more genes in the rat and establish that the debrisoquine polymorphism in the DA rat is probably due to a structurally altered db1 protein.

Amino Acid Sequence↗
PubMed1987

Comparison of human mouse P1450 upstream regulatory sequences in liver- and nonliver-derived cell lines.

The foreign chemical tetrachlorodibenzo-p-dioxin (TCDD) is known to interact with the aromatic hydrocarbon receptor and, in turn, activate transcription of the mouse P1450 and P3450 genes. Various lengths of DNA upstream from the human P1450 gene were inserted into the promoterless pSVO-cat prokaryotic expression vector and compared with mouse P1450 upstream sequences similarly treated. The constructs were cotransfected with pSV2-neo into human, mouse, and monkey liver- and nonliver-derived cell lines. After selection in G418, the transformed colonies were treated with control medium, TCDD, or, in some cases, cycloheximide. Pooled transformants were then assayed for chloramphenicol acetyltransferase activity. The data are consistent with the presence of several functional regulatory regions within the upstream DNA: a promoter region, a region that is negatively autoregulated, and a region further upstream that activates transcription and is dependent upon a functional aromatic hydrocarbon receptor. Compared with 1604 base pairs of human P1450 upstream sequences, 1646 base pairs of mouse P1450 upstream sequences exhibit an increased sensitivity to TCDD; this effect was found to require both trans-acting protein factors and cis-acting DNA elements. Our results demonstrate the successful interaction of mouse trans-acting factors with human P1450 upstream sequences and human trans-acting factors with mouse P1450 upstream sequences.

Cell Line↗
PubMed1987

Human P(3)450: cDNA and complete protein sequence, repetitive Alu sequences in the 3' nontranslated region, and localization of gene to chromosome 15.

P(1)450 and P(3)450 are the two members of the dioxin-inducible P450 gene family, one of at least eight families in the entire P450 gene superfamily. The human P(1)450 gene has been previously sequenced. In this report the human liver P(3)450 protein is shown to migrate as a 54-kDa band on NaDodSO4-polyacrylamide gel electrophoresis. The human P(3)450 cDNA (3,064 bp) and complete P(3)450 protein (515 residues; Mr = 58,294) were determined. The human P(3)450 mRNA (3.3 kb) has an unusually long 3' nontranslated region (1,509 bp) primarily due to the inclusion of four copies of Alu repetitive sequences. Comparison of human P(3)450 cDNA with human P(1)450 cDNA and mouse (or rat) P(3)450 cDNA with mouse (or rat) P(1)450 cDNA suggests that an intra-exonic gene conversion event (in the region between 100 and 400 nucleotides from the 5' end of the translated region) most likely occurred between 20 and 80 million years ago. Analysis of 32 human X mouse and 23 human X hamster somatic cell hybrids confirm unequivocally that both P(3)450 and P(1)450 reside on human chromosome 15.

Amino Acid Sequence↗
PubMed1987

Expression of the mouse P(1)450 gene during differentiation without foreign chemical stimulation.

Transcriptional activation of the P(1)450 and P(3)450 genes, mRNA accumulation, and induction of the P(1)450 and P(3)450 enzymes are known to be stimulated by foreign chemicals such as tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene. It has been established that this induction process is dependent on trans-acting factor(s) that include a complex between the environmental chemical inducer and the aromatic hydrocarbon (Ah) receptor. In this report we show that constitutive P(1)450 mRNA but not P(3)450 mRNA is elevated in 7-day-old mouse embryos and following retinoic acid-induced differentiation of F9 embryonal carcinoma cells. In both instances it is shown that P(1)450 gene activation can occur in the absence of a foreign chemical stimulus. These data suggest that the P(1)450 gene may serve an important developmental function during differentiation.

Animals↗
PubMed1986

NAD(P)H:menadione oxidoreductase. Novel purification of enzyme cDNA and complete amino acid sequence, and gene regulation.

NAD(P)H:menadione oxidoreductase induction by polycyclic hydrocarbons is known to be governed by the aromatic hydrocarbon-responsive (Ah) locus. This cytosolic enzyme was isolated from 3-methylcholanthrene-treated rat liver by a rapid two-step procedure with the use of affinity gel purification and fast-protein liquid chromatography. Polyclonal antiserum to menadione reductase was raised in rabbits. On Western (immuno) blot analysis, large increases in this hepatic menadione reductase protein (NMOR1) of 3-methylcholanthrene-treated C57BL/6N but not DBA/2N mice confirmed that induction of this enzyme by 3-methyl-cholanthrene is regulated by the Ah receptor. A cDNA expression library was constructed in lambda gt11 and screened with antiserum. Positive cDNA clones were plaque purified and further characterized by showing enhanced hybridization to 3-methylcholanthrene-induced poly(A+) RNA from rats; the longest cDNA clone (1,501 base pairs) has an open reading frame (bases 75-899) and a nucleotide sequence consistent with a new gene family. On Northern blot analysis, a single 3-methylcholanthrene-inducible rat liver mRNA (approximately 1.6 kilobases) hybridizes to the cDNA probe. On Southern blot analysis a total of 14-16 kilobases of rat genomic DNA fragments hybridize to the cDNA probe, indicating one or a small number of menadione reductase genes in this family. The amino acid sequence (274 residues) and Mr of 30,946 compare well with the size of the rat enzyme (32 kDa) estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence of two internal fragments of the trypsin-digested purified NMOR1 protein is in complete agreement with that deduced from the cDNA nucleotide sequence. This study represents the first cloning and sequencing of a cDNA encoding a Phase II drug-metabolizing enzyme under control of the Ah locus.

Amino Acid Sequence↗
PubMed1986

Methylation differences in the murine P1450 and P3450 genes in wild-type and mutant hepatoma cell culture.

The murine P1450 and P3450 genes and flanking regions contain 14 and 15 Msp I sites (C-C-G-G-), respectively, designated M1 through M14 or M15. These two genes from mouse Hepa-1 wild-type (wt) parent and three mutant cell lines were studied for methylation differences with use of the isoschizomers Msp I and Hpa II. The mutant lines included: c1, having high constitutive P1450 mRNA and believed to carry a mutation in the P1450 structural gene; c2, having negligible levels of Ah receptor; and c4, having a defect in nuclear translocation of the inducer-receptor complex. The P3450 gene was not expressed constitutively or after treatment of these four cell lines with the P1450 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and, correspondingly, the P3450 Msp I sites remained methylated. Treatment of all four cell lines with TCDD did not alter the P1450 methylation pattern, nor was there any evidence of P1450 gene amplification. Treatment of all four lines with 5-azacytidine caused demethylation of the P1450 Msp I sites but did not change the usual P1450 catalytic activity pattern found in each of the lines. The only detectable difference in the P1450 gene among the four lines was hypomethylation of the M9 site in c1 that was not seen in wt, c2 and c4 cells. The M9 site is part of a 9-bp box (5'-C-C-G-G-G-A-C-A-T-3'), located near the beginning of exon 3. It is of interest that the same nine bases are found in intron 2 about 80 bp upstream from the 5' end of exon 3 in the homologous P3450 gene.

Animals↗