Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Twelve cytochrome P450 cDNA fragments were cloned from Drosophila melanogaster by reverse transcriptase/PCR (RT/PCR) using degenerate oligonucleotide primers. The corresponding genes belong to several subfamilies of the CYP4 and CYP9 P450 families. Only two of these genes, Cyp4dl and Cyp4d2, have previously been described. In situ hybridization of each of the cDNA fragments showed two clusters of genes; one near the tip of the X chromosome and the other on the left arm of chromosome 2. Interestingly the latter cluster comprises widely divergent genes belonging both to the CYP9 and CYP4 families and also to the CYP6 family (Cyp6a2). Putative allelic variants of several of the genes were found in different insecticide-resistant and -susceptible strains (Hikone R, Haag 79 and Oregon R). The identification of these genes and alleles will allow us to clarify the involvement of P450s in xenobiotic metabolism and will facilitate a genetic analysis of P450 functions in insects.
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PMID:Cytochrome P450 gene clusters in Drosophila melanogaster. 867 71

The cytochrome P450 2C24 gene is characterized by the capability to generate, in rat kidney, a transcript containing exons 2 and 4 spliced at correct sites but having the donor site of exon 4 directly joined to the acceptor site of exon 2 (exon scrambling). By reverse transcriptase-PCR analysis, it is now shown that the only exons present in the scrambled transcript are exons 2, 3, and 4 and that this molecule lacks a poly(A)+ tail. Furthermore, the use of PCR primers in both orientations of either exon 2 or exon 4 revealed that the orders of the exons in the scrambled transcript are 2-3-4-2 and 4-2-3-4, respectively. These results, combined with the observation that P450 2C24 is a single-copy gene, with no duplication of the exon 2 to exon 4 segment, suggest that the scrambled transcript has properties consistent with that of a circular molecule. In line with this is the observation of an increased resistance of the transcript to phosphodiesterase I, a 3'-exonuclease. Moreover, an alternatively processed cytochrome P450 2C24 mRNA, lacking the three scrambled exons and having exon 1 directly joined to exon 5, has been identified in kidney and liver, tissues that express the scrambled transcript. This complete identity of the exons that are absent in the alternatively processed mRNA but present in the scrambled transcript is interpreted as indicative of the possibility that exon scrambling and exon skipping might be interrelated phenomena. It is therefore proposed that alternative pre-mRNA processing has the potential to generate not only mRNAs lacking one or more exons but also circular RNA molecules.
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PMID:Circular RNAs from transcripts of the rat cytochrome P450 2C24 gene: correlation with exon skipping. 869 51

A method was developed using reverse transcriptase-polymerase chain reaction (RT-PCR) to selectively detect and qualitatively determine the levels of mRNA expression of the major isoenzymes of cytochrome P450 (P450 1A1, 1A2, 2B1/2, 2C11, 2E1, 3A1, 3A2, and 4A1) and fatty acyl-CoA oxidase (FACO) in the rat. Total liver RNA was isolated from male Sprague-Dawley rats treated with various inducers of cytochrome P450 (P450) and analyzed for the presence and relative quantities of each P450 isoenzyme mRNA using this technique. The specificity of the oligonucleotide primers used in the detection of each P450 mRNA was tested and confirmed through the simultaneous analysis of liver microsomal protein preparations for the presence of constitutive or inducible P450 apoprotein and enzyme activities using western immunoblotting and specific enzyme activity measures, respectively. This method of P450 expression analysis is proven to be highly specific and readily applicable for the assessment of P450 enzyme induction and down-regulation in the rat during routine toxicology studies when expression of the gene product is regulated by transcriptional activation and/or mRNA stabilization.
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PMID:Analysis of rat cytochrome P450 isoenzyme expression using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). 876 76

Human bronchial epithelial cells (BEC), a primary defense against inhaled materials, are the progenitor cells for bronchogenic carcinomas and have important metabolic capabilities. We used reverse transcriptase-polymerase chain reaction (RT-PCR) to identify xenobiotic metabolism enzymes expressed in primary BEC and alveolar macrophages (AM) of non-smoking volunteers. Cytochromes P450 (CYP) 1A1, 1B1, 2B7, 2E1, and 4B1 and microsomal epoxide hydrolase (mEH) were expressed in BEC but not AM. CYP2F1 was expressed in BEC, but it was expressed at barely detectable levels or not at all in AM. NADPH oxidoreductase (NADPH OR), microsomal glutathione transferase (GST 12), glutathione transferase mu, phenol sulfotransferase (PST), thermolabile phenol sulfotransferase (TL PST), and the clara cell-specific gene, CC10 were expressed in both BEC and AM. CYP3A4 and glucuronosyl transferases-1 and 2 were not expressed in either BEC or AM. In contrast to primary BEC, of the genes evaluated, the immortalized human bronchial epithelial cell line BEP2D constitutively expressed only CYP1A1, CYP2E1, NADPH OR, glucuronosyl transferase 1, GST 12, GST mu, PST, TL PST, and CC10. The loss of xenobiotic metabolism enzyme gene expression in the BEP2D cell line may result from either reduced exposure to inducing agents, or loss of differentiative characteristics in culture. It is clear from the data comparing BEC and AM that there are important intertissue differences in expression of xenobiotic metabolism enzymes.
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PMID:Xenobiotic metabolism enzyme gene expression in human bronchial epithelial and alveolar macrophage cells. 884 77

In pregnant mares, eCG stimulates luteal androgen and estrogen production, increasing plasma concentrations 2- to 3-fold. To study how these changes are regulated, we examined the expression of mRNA for the steroidogenic enzymes 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), cytochrome P450 17 alpha-hydroxylase/17,20-lyase (P450 17 alpha), and cytochrome P450 aromatase (P450arom) in equine primary corpora lutea using Northern blot analyses. Three equine specific cDNAs were generated by reverse transcriptase polymerase chain reaction. When compared to human, bovine, and rat sequences, the nucleotide identities were 82%, 84%, and 76%, respectively, for 3 beta-HSD cDNA (843 base pairs [bp]); 79%, 80% and 66% for P450(17) alpha cDNA (541 bp); and 80%, 83% and 75% for P450arom cDNA (289 bp). The P450(17) alpha cDNA sequence demonstrated 99.6% nucleotide identity with the previously published sequence for equine testicular P450(17) alpha. Luteal tissue samples were collected at three times: diestrus (Days 8-10), early pregnancy before the onset of eCG secretion (Days 29-35), and early pregnancy after the onset of eCG secretion (Days 42-45). Although no significant changes were observed in 3 beta-HSD expression, P450(17) alpha and P450arom demonstrated stage-specific transcriptional regulation. Steady-state levels of P450(17) alpha mRNA were similar during diestrus and early pregnancy before the onset of eCG secretion but increased significantly after the onset of eCG secretion. Cytochrome P450arom mRNA levels decreased significantly after the onset of eCG secretion. Steady-state levels of P450arom mRNA were highest in luteal tissue collected during pregnancy before the onset of eCG secretion and intermediate during diestrus. Secretion of eCG appears to increase luteal estrogen synthesis by a transcriptional up-regulation of P450(17) alpha expression. These data suggest that availability of aromatizable androgens may be rate-limiting in luteal estrogen synthesis before the onset of eCG secretion.
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PMID:Differential transcription of steroidogenic enzymes in the equine primary corpus luteum during diestrus and early pregnancy. 909 61

The molecular basis of metabolic resistance to pyrethroids in Helicoverpa armigera is currently under debate. Substantial indirect evidence supports a role for both esterase- and cytochrome-P450-mediated metabolism. However, the relative roles played by these two mechanisms in field-based resistance is uncertain. Our understanding of the importance of P450-mediated metabolism is hindered by the paucity of cloned genes from this species, and the corresponding absence of data on rates of insecticide metabolism by functionally expressed P450s. To facilitate P450 gene isolation from H. armigera we used degenerate primers in the reverse transcriptase-polymerase chain reaction (RT-PCR) to clone P450 gene fragments from the RNA of a pyrethroid-resistant strain. Here we report the isolation of eight new P450 genes: seven from the CYP4 family and one CYP9. One of these genes, CYP4G8, is two-fold over-expressed in the resistant strain, whereas the other CYP4s showed either similar or undetectable levels of expression. CYP9A3 appears to be a homolog of the putatively resistance-associated CYP9A1 of Heliothis virescens. However, no difference in expression between the H. armigera strains was detected. CYP6B2, a gene previously reported to be over-expressed in a different pyrethroid-resistant strain of H. armigera, also revealed non-detectable levels of expression in both strains. These observations suggest that different P450s may be over-expressed in different resistant strains, and emphasize that recombinant expression will be necessary in order to define precisely their individual substrate specificities and ability to metabolize pyrethroids. The gene fragments described here represent an important first step in this direction.
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PMID:Cytochrome P450 genes from Helicoverpa armigera: expression in a pyrethroid-susceptible and -resistant strain. 930 92

Hepatic enzyme induction is often evoked as the cause for a variety of effects in animal studies, e.g. hepatic hypertrophy and secondary thyroid neoplasms in rodents. In clinical practice, enzyme induction often enhances drug clearance and may result in reduced efficacy. For example, carbamazepine or rifampin treatment induces P450 3A in humans, and as a result, dramatically reduces the efficacy of midazolam or cyclosporine. Due to species differences in substrate specificity and the regulation of various drug-metabolizing enzymes, assessing enzyme induction in human tissues is a desirable goal. Since induction often occurs as a result of increased synthesis of mRNA coding for a particular enzyme, induction may be quantified by measuring specific mRNA levels. We describe an approach to quantifying mRNA levels using reverse transcriptase-polymerase chain reaction (RT-PCR). This approach makes use of either radiolabeled PCR primers or fluorimetric quantification of product and does not require the synthesis of a competitor RNA or DNA molecule. Thus, Cyp2B1/2 mRNA can be shown to be induced 17-fold in the H4IIE rat hepatoma cell line. Likewise, Cyp3A and Cyp2A6 mRNAs can be shown to be induced in primary human hepatocytes cultured on collagen-coated plates and treated with rifampin for 72 h. By contrast, mRNA levels for Cyp1A1 and Cyp2E1 were not increased by rifampin treatment. This report demonstrates the potential of this approach for examining a number of mRNAs from drug-treated cultured cells, as a means of assessing metabolic enzyme induction.
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PMID:Quantitative reverse transcriptase/PCR assay for the measurement of induction in cultured hepatocytes. 940 49

We developed a quantitative competitive reverse transcriptase-polymerase chain reaction (QC RT-PCR) assay to measure mRNA levels of seven human cytochrome P450 (P450, CYP) genes and microsomal epoxide hydrolase (EH) simultaneously. This assay employs an exogenous recombinant RNA (rcRNA) molecule as an internal standard that shares PCR primer and hybridization probe sequences with CYP1A1, CYP1A2, CYP2A6/7, CYP2D6, CYP2E1, CYP2F1, CYP3A4/5/7, and EH mRNA. Because each rcRNA molecule contains several primer sequences, an entire battery of genes that exhibit differential responsiveness to various classes of xenobiotics may be measured simultaneously from one population of cDNA molecules. In this study, we demonstrated the precision and power of the assay using small amounts of human liver total RNA. We also report for the first time quantitative profiles of P450 and EH mRNA abundance in eight human livers. Cytochrome P450 2E1 mRNA maintained the highest abundance (average 6.67 x 10(7) molecules/microg of total RNA) and least variation (13 fold) in all livers examined. Cytochrome P450 1A2, CYP2A6/7, CYP2D6, CYP3A4/5, and EH mRNAs were approximately one order of magnitude less abundant than CYP2E1 transcripts, with CYP2D6 levels exhibiting the greatest variation (220 fold) between individuals. This QC RT-PCR assay should prove valuable for measuring basal and induced mRNAs in different cell types in vitro, as well as in biomonitoring applications where individuals are exposed or hypersusceptible to certain xenobiotic-initiated toxicities.
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PMID:Quantification of multiple human cytochrome P450 mRNA molecules using competitive reverse transcriptase-PCR. 953 3

The cytochromes P450 are a large family of haemoproteins which have a major role in the oxidative metabolism of a wide range of xenobiotics and some endogenous compounds. In this study the presence of individual members of the CYP1, CYP2 and CYP3 P450 families has been investigated by reverse transcriptase polymerase chain reaction in different regions of normal human brain consisting of frontal and temporal cortices, mid brain, cerebellum, pons and medulla. All the P450s were identified in specific regions of brain with CYP1A1 and CYP2C being the most frequently expressed forms of P450. Sequencing identified the CYP2C PCR product as CYP2C8. This study indicates that individual P450 mRNAs are present in human brain and are found in specific brain regions. The distribution of individual P450s in different regions of human brain is likely to be highly important in determining the response of the brain to toxic foreign compounds.
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PMID:Regional distribution of individual forms of cytochrome P450 mRNA in normal adult human brain. 958 55

The presence of P450 in a murine macrophage cell line, RAW264.7, was investigated to clarify the biological role and regulation of P450. Microsomes of RAW264.7 cells were isolated and subjected to immunoblotting with anti-rat CYP2A1, 2B1, and 4A2 antibodies. The microsomes gave staining bands with all these antibodies, suggesting the presence of mouse Cyp2a, 2b, and 4a isoforms in RAW264.7. RAW264. 7 cells were treated with typical inducers of P450 (phenobarbital, clofibrate, beta-naphthoflavone and 3-methylcholanthrene). None of these chemicals induced these P450s. Stimulation of RAW264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (INF-gamma) which increase inducible nitric oxide synthase (iNOS) and cytokines in cells decreased Cyp4a protein but not Cyp2a and 2b proteins. To identify P450 isoforms in RAW264.7, we used polymerase chain reaction (PCR) primers for mouse Cyp2a4, 2a12, 2b9/10, 4a10, and 4a12. Total RNA was isolated from these cells and converted to cDNA by reverse transcriptase. PCR was done with these primers and the amplified nucleotides were analyzed by a DNA sequencer. Only Cyp2b9/10 and 4a12 primers gave clear bands, although all primers gave clear bands from liver total RNA. Nucleotide sequences of these products amplified by PCR were identical with Cyp2b9 and 4a12. These findings indicate that Cyp2b9 and 4a12 were present in a macrophage cell line, RAW264.7, and the regulation of P450 by inducers and cytokine differed from that in liver.
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PMID:P450 isoforms in a murine macrophage cell line, RAW264.7, and changes in the levels of P450 isoforms by treatment of cells with lipopolysaccharide and interferon-gamma. 963 May 46


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