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)

Utilizing the reverse transcriptase-linked polymerase chain reaction, we analyzed the capacity of three groups of rat conceptal tissues to express cytochrome P4501A1 (CYP1A1) mRNA during the dysmorphogenesis-sensitive stage of organogenesis. The visceral yolk sac, ectoplacental cone and embryo proper each were investigated on day 12 of gestation with and without prior exposure in utero to 3-methylcholanthrene as inducing agent. With two sets of discriminating oligonucleotide primers, definitive, reproducible signals were detectable only in tissues from 3-methylcholanthrene preexposed conceptuses. Signals of highest intensity were observed with visceral yolk sac tissues and signals of lowest intensity were observed with tissues of the embryo per se. Specificities of the amplified cDNAs were verified using Southern blotting with hybridization to an internal oligonucleotide probe. The results indicate that organogenesis-stage conceptual tissues of the rat will express CYP1A1 mRNA in response to environmental transregulating agents.
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PMID:Organogenesis-stage cytochrome P450 isoforms: utilization of PCR for detection of CYP1A1 mRNA in rat conceptal tissues. 206 63

Plastid (pt) and mitochondrial (mt) DNA were isolated from the unicellular green alga KS3/2, which is presumed to be a species of the genera Ankistrodesmus or Monoraphidium. The DNA species are characterized by their different densities (pt, 1.685 g/ml; mt, 1.695 g/ml), individual restriction patterns, and their respective sizes of 130 and 47 kb. Using an intronic sequence from fungal mitochondria as a hybridization probe, intron sequences from mtDNA and from ptDNA were identified. DNA sequencing of the cloned ptDNA intron revealed that the plastid gene for subunit IV of the cytochrome b6/f petD complex is interrupted by a group II intron of unusual length (3533 bp). This was confirmed by transcript analysis. The intron encodes an open reading frame (ORF) which shows significant homology with reverse transcriptase genes from various genetic elements. This discovery is unique for plastomes and indicates that introns, retrotransposons, insertion elements and retroviruses may have a common evolutionary origin.
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PMID:The intron of a plastid gene from a green alga contains an open reading frame for a reverse transcriptase-like enzyme. 247 55

The use of synthetic oligodeoxyribonucleotides as tools for the isolation, characterization and mutagenesis of eukaryote genes has played a major role in the molecular definition of the CYC1 locus of Saccharomyces cerevisiae, which is the structural gene for the apoprotein of iso-1-cytochrome c. Thus, the possibility of using a synthetic oligodeoxyribonucleotide as a probe to identify and monitor the isolation of a specific gene was first established by model studies which defined the melting temperatures (TmS) for duplexes of oligonucleotides of different lengths and base compositions. This led to the isolation of the CYC1 locus using a synthetic 13 nucleotide probe. A more convenient strategy for determination of DNA sequences by the Sanger method was provided by using synthetic oligodeoxyribonucleotides as primers with denatured double-strand plasmid DNA as template. By this means, the sequence of the CYC1 locus was determined by "walking" along the gene without isolating restriction fragments of the DNA or separating DNA strands. Synthetic oligodeoxyribonucleotides, used as primers for reverse transcriptase with mRNA as template, were also used to precisely define the 5'- and 3'-ends of the iso-1-cytochrome c mRNA. Yet another application of synthetic oligodeoxyribonucleotides is their use as specific mutagens after in vitro incorporation into double-stranded DNA. In the case of iso-1-cytochrome c, this mutagenic strategy is being used to define the role of conserved amino-acids in cytochrome function.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthetic oligodeoxyribonucleotides as tools in molecular genetics: the characterization of the CYC1 (iso-1-cytochrome c encoding) locus of Saccharomyces cerevisiae. 300 92

Cytochrome P-450 mRNA has been partially purified from membrane-bound polysomes of the livers of phenobarbital-treated rats by SDS-phenol-chloroform extraction, followed by poly(U)-Sepharose chromatography and by centrifugation through a sucrose density gradient. Cytochrome P-450 mRNA activity was detected near 18S in the sucrose density gradient, accounting for approximately 5% of total mRNA activity on the basis of [3H]leucine incorporation in an in vitro translation system of wheat germ. Complementary DNA (cDNA) which had been synthesized on the partially purified mRNA by AMV reverse transcriptase was inserted into the Pst I site of pBR 322. After bacterial transformation, and in situ colony hybridization using [32P]cDNA as a probe, a colony carrying cytochrome P-50 cDNA sequence was identified by a hybridization-arrested translation assay. Sequence complementarity of the inserted DNA sequence to cytochrome P-450 mRNA was further confirmed by a positive hybridization-translation assay. The mRNA isolated from the partially purified mRNA preparation by hybridizing it with the recombinant DNA (III-8-10) showed enriched synthesis of a protein product whose apparent molecular weight was consistent with that of cytochrome P-450, and which was immunoprecipitable with anti-cytochrome P-450 antibody.
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PMID:Construction and identification of a hybrid plasmid containing DNA sequence complementary to phenobarbital-inducible cytochrome P-450 messenger RNA from rat liver. 616 9

Poly(A)+ RNA was isolated from membrane-bound polysomes of the livers of 3-methylcholanthrene (MC)-treated rats, and was partially purified by sucrose density gradient centrifugation. The mRNA was translated in an in vitro rabbit reticulocyte lysate system, and assayed for the synthesis of MC-inducible forms of cytochrome P-450 (cytochrome P-450MC) using anti-cytochrome P-450c antibody which reacted with two types of cytochrome P-450MC, P-450c, and P-450d. The mRNA activity for cytochrome P-450MC was located at around 18S, accounting for approximately 5% of total mRNA activity. The double-stranded complementary DNA which had been synthesized from the partially purified mRNA by avian myeloblastosis virus reverse transcriptase and DNA polymerase I (Klenow enzyme) was cloned in Escherichia coli X1776, using plasmid pBR322 as a cloning vector. After differential colony hybridization using [32P]cDNA's synthesized from mRNA preparation of MC-treated or untreated rat liver as a probe, a clone (3-9-1) carrying cytochrome P-450MC cDNA sequence was identified by a positive hybridization-translation assay. The specific mRNA hybridized with plasmid 3-9-1 DNA showed an enriched synthesis of a protein with apparent molecular weight of 56,000 daltons, which was immunoprecipitable with anti-P-450c antibody. In RNA blot analysis with MC-, polychlorinated biphenyls (PCB)-, and phenobarbital (PB)-induced mRNA as well as uninduced mRNA, a longer cDNA (P-34) which had been isolated by hybridization with the insertion of clone 3-9-1, and the previously isolated PB-inducible cytochrome P-450b cDNA (Fujii-Kuriyama et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2793-2797) hybridized with mRNA preparations in an inducer-specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Molecular cloning of a complementary DNA to 3-methylcholanthrene-inducible cytochrome P-450 mRNA from rat liver. 631 66

Poly(A)+-RNA obtained from the livers of 3-methylcholanthrene (3MC)-treated rats was translated into cytochrome P-450c in a cell-free reticulocyte system. In this translational system, no precursor cytochrome P-450c was observed. The mRNA responsible for the synthesis of this cytochrome was isolated by immunoprecipitation of liver polyribosomes obtained at 15 hr after 3MC treatment, and a cDNA was constructed by the reverse transcriptase reaction. The cDNA was further purified by hybridizing at a high R0t (product of RNA concentration and incubation time) to poly(A)+-RNA isolated from control rat liver, and the nonhybridized, single-stranded cDNA was isolated by hydroxylapatite chromatography. This cDNAp-450c was employed in hybridization reactions with poly(A)+-RNA isolated from the livers of rats treated with 3MC for various times. These studies indicated a maximal induction of mRNAp-450c at about 15 hr after 3MC injection, although levels of this mRNA were significantly increased by 7 hr. The mRNAp-450c concentration had diminished by 24 hr but remained higher than control levels for at least 48 hr. These studies establish an effect of 3MC upon the accumulation of mRNAp-450c in rat liver.
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PMID:Administration of 3-methylcholanthrene to rats increases the specific hybridizable mRNA coding for cytochrome P-450c. 694 71

A sensitive assay of multiple mRNAs by reverse transcriptase-polymerase chain reaction was adopted to study the hormonally regulated expression of steroidogenic enzymes in primary rat granulosa cells in culture. As little as 15-60 ng total RNA prepared from cultured cells were reverse transcribed in the presence of pd(T)6, and polymerase chain reaction was conducted in the presence of specific oligonucleotide pairs designed to identify cDNAs of steroidogenic enzymes. In combination with Northern blot analysis of cholesterol side-chain cleavage cytochrome P450 (P450scc) message, it is shown that a novel protein kinase inhibitor, tyrphostin AG18, arrests the FSH-induced accumulation of P450scc mRNA. This inhibition is dose dependent (IC50, 15 microM) and reversible. The addition of 80 microM AG18 to cells containing high levels of P450scc mRNA caused a rapid decline of the cytochrome message (t 1/2, 5 h), similar to the effect of 30 micrograms/ml alpha-amanitin. However, concomitant addition of the two drugs did not accelerate the mRNA degradation process, suggesting that AG18 does not affect message stabilization. Tyrphostin AG18 did not affect mRNA species that are not FSH inducible, such as the ribosomal protein L19, or the constitutively expressed low levels of steroid 5 alpha-reductase mRNA. Moreover, even the extremely high levels of P450scc mRNA in granulosa-lutein cells, being cAMP independent and terminally differentiated a few hours after LH surge, were not affected by the addition of AG18 in culture. In contrast, two additional key and FSH-inducible steroidogenic enzymes, i.e. aromatase cytochrome P450 and 3 beta-hydroxysteroid dehydrogenase-I, were inhibited by AG18 at their mRNA levels. These results suggest that an as yet undetermined tyrosine kinase pathway is involved in the cAMP-dependent signal transduction pathway of FSH action, so that the presence of AG18 does not allow FSH induction of gene expression to occur.
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PMID:Tyrosine kinase inhibitor AG18 arrests follicle-stimulating hormone-induced granulosa cell differentiation: use of reverse transcriptase-polymerase chain reaction assay for multiple messenger ribonucleic acids. 751 96

Neurosteroids are steroids that are synthesized de novo in the brain and include some classical (adrenal and gonadal steroids) and some unique brain-specific steroids. Neurosteroids are thought to mediate their action through ion gated channel receptors such as gamma-aminobutyric acid(A) and N-methyl-D-aspartate rather than through classical nuclear steroid hormone receptors. Some enzymes involved in neurosteroidogenesis have been identified as those found in steroidogenic tissues, and some may be unique to the brain. We previously demonstrated that the messenger RNAs (mRNA) for the cholesterol side-chain cleavage enzyme, cytochrome P450scc, and one form of 11 beta-hydroxylase, cytochrome P450c11 beta, are regionally expressed in the adult rat brain. However, cytochrome P450c17, which has 17-hydroxylase and 17,20-lyase activity and is thought to be required for the synthesis of dehydroepiandrosterone, was not detected in any region of the rat brain, even though dehydroepiandrosterone is one of the most abundant neuroactive steroids. We now demonstrate that P450c17 is expressed in the nervous system of the developing rodent embryo. By ribonuclease protection assays, P450c17 mRNA was found in the trunk but not in the head of rat embryos but reverse transcriptase-polymerase chain reaction analysis showed expression of P450c17 mRNA in the head of E15.5 to E19.5 rat embryos. Immunocytochemically detectable P450c17 protein was expressed in the nervous system as early as embryonic day E10.5 in the mouse, mainly in tissue derived from the neural crest. Neuronal cell bodies as well as fibers staining for P450c17 were observed in the central and peripheral nervous systems. The sites of P450c17 expression in the peripheral nervous system suggest it may be involved in a wide variety of sensory-motor functions. In the central nervous system, cell bodies expressing P450c17 are found in the hind brain, in mesencephalic nuclei, and in a region in the location of the locus coeruleus, but in cells distinct from those expressing the dopamine-beta-hydroxylase. Furthermore, its particular location and temporal expression in axons reaching the cortical areas suggest it is a marker for the axonal growth in this region, and that its neurosteroid product may be a signal for targeting cortical axons during embryogenesis.
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PMID:Steroidogenic enzyme P450c17 is expressed in the embryonic central nervous system. 758 60

Drug metabolizing enzymes, particularly those involved in the metabolism of carcinogenic chemicals, were characterized in cultured human keratinocytes. Using immunoblotting experiments, we analysed the expression of phase I enzymes, cytochrome P4501A1 (CYP1A1) and NADPH reductase, and phase II enzymes, phenol UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) isoform pi, in the presence of either classical inducers (i.e. 3-methylcholanthrene, dimethylbenz[a]anthracene, phenobarbital, and clofibrate) or all-trans retinoic acid (RA). This study has shown that the expression of CYP1A1 and UGT is concomitantly induced by 3-methylcholanthrene, dimethylbenz[a]anthracene, and RA, and that of NADPH reductase is only enhanced by phenobarbital and RA. In contrast, the expression of GST pi was not affected by the inducers. Using the reverse transcriptase-polymerase chain reaction, we have demonstrated that the effects of 3-methylcholanthrene, dimethylbenz[a]anthracene and RA on CYP1A1 expression correlate with an increase of CYP1A1 mRNA level. Our results indicate that, with the exception of clofibrate, xenobiotics and RA differentially modulate the expression of drug metabolizing enzymes.
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PMID:Constitutive and inducible expression of drug metabolizing enzymes in cultured human keratinocytes. 775 27

Investigations with chemical inhibitors and with inhibitory antibodies specific for cytochrome P4501A-catalyzed ethoxyresorufin (ethoxyphenoxazone) O-deethylation and 2-acetylaminofluorene (N-2-fluorenylacetamide) ring hydroxylation indicated that cytochrome(s) P450 of the 1A subfamily was functionally expressed in human embryonic hepatic tissues at very early stages (days 50-60) of gestation. Lack of detectable capacity of hepatic microsomal enzymes to catalyze either N-hydroxylation of 2-acetylaminofluorene or O-demethylation of methoxyresorufin indicated that functional cytochrome P4501A2 is expressed minimally or negligibly in human embryonic hepatic tissues. By contrast, profound inhibition of the ring hydroxylation of 2-acetylaminofluorene and of the O-deethylation of ethoxyresorufin by 7,8-benzoflavone as well as by anti-cytochrome P4501A1 antibodies indicated the presence of significant levels of functional cytochrome P4501A1 in hepatic microsomes of human embryos. Using the reverse transcriptase-linked polymerase chain reaction with specific oligonucleotide primers, we also detected significant expression of cytochrome P4501A1 mRNA in human embryonic livers. Polymerase chain reaction amplification, cloning and sequencing of the corresponding cDNA provided evidence that the cytochrome P4501A1 mRNA expressed in human embryonic tissues was identical to that expressed in adult human tissues. The results of the study have important implications in terms of the embryotoxic effects of chemicals that are known to be substrates, inhibitors or inducers of cytochrome P4501A1 and to which pregnant women are exposed.
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PMID:Expression of functional cytochrome P4501A1 in human embryonic hepatic tissues during organogenesis. 788 87


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