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)

Hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4) catalyzes the methylation of acetylserotonin to complete the synthesis of melatonin in the pineal and retina. A complete 1728 nucleotide cDNA encoding rat pineal HIOMT was isolated, characterized, and used to evaluate day/night levels of HIOMT mRNA. As previously reported for HIOMT enzyme activity, HIOMT mRNA levels were also greater in the pineal than in the retina. Northern blot analysis and in situ hybridization were useful for detection of HIOMT mRNA in the pineal but not the retina, whereas the reverse transcriptase-polymerase chain reaction or RNase protection assay revealed transcripts for HIOMT both in the pineal and retina. Investigating HIOMT mRNA levels in rat pineal and retina at 6 time-points throughout a 24 h period revealed higher levels of HIOMT message during darkness. The daily fluctuation in HIOMT mRNA persisted in constant darkness, verifying an endogenous circadian rhythm both in the pineal and retina. In mammalian pineals, sympathetic innervation, synthesizing norepinephrine that activates beta (beta) adrenergic receptors, entrain several circadian bodily functions through the synthesis and release of melatonin. A single injection of the beta-adrenergic agonist, isoproterenol, induced a dramatic increase of HIOMT mRNA levels in the light-adapted pineal, in vivo. Moreover, a single injection of the beta-adrenergic antagonist, propranolol, prevented the nocturnal increase of pineal HIOMT mRNA. Using a combination of methods, it has been shown that the level of HIOMT mRNA fluctuates daily in both the pineal gland and retina. This day/night rhythm can be modulated either by beta receptor agonists or antagonists when applied appropriately during the circadian cycle, suggesting that the mRNA changes in HIOMT may be controlled at the transcriptional level.
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PMID:Circadian regulation of hydroxyindole-O-methyltransferase mRNA levels in rat pineal and retina. 893 Mar 56

The high frequency of incorporation of non-complementary nucleotides by HIV-1 reverse transcriptase is likely to be a major factor in the exceptionally rapid accumulation of viral mutations during the course of AIDS infections. To investigate whether this high level of infidelity is also associated with the incorporation of nucleotide analogs, we analyzed O6-methyldeoxyguanosine triphosphate and compared the incorporation of this analog by HIV-1 reverse transcriptase to that catalyzed by other DNA synthesizing enzymes. Our results indicate that O6-methyldeoxyguanosine triphosphate serves as a substrate for DNA synthesized in vitro by HIV-1 RT on both DNA and RNA templates. The product DNA contains the modified purine; it is sensitive to the repair enzyme, O6-methylguanine methyltransferase, which specifically reacts with DNA containing methylated guanines at the O6 position. Using a forward mutation assay we demonstrated that the nucleotide analog incorporated by HIV-1 RT is mutagenic. The mutations produced are single-base substitutions opposite template thymidines and result in A:T --> G:C transitions. The incorporation of a mutagenic nucleotide by HIV-1 RT highlights the possibility of increasing the rate of mutagenesis of HIV by the use of nucleotides that form non-complementary base pairs at high frequency.
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PMID:Mutagenesis by human immunodeficiency virus reverse transcriptase: incorporation of O6-methyldeoxyguanosine triphosphate. 906 14

We describe a new primer design strategy for PCR amplification of unknown targets that are related to multiply-aligned protein sequences. Each primer consists of a short 3' degenerate core region and a longer 5' consensus clamp region. Only 3-4 highly conserved amino acid residues are necessary for design of the core, which is stabilized by the clamp during annealing to template molecules. During later rounds of amplification, the non-degenerate clamp permits stable annealing to product molecules. We demonstrate the practical utility of this hybrid primer method by detection of diverse reverse transcriptase-like genes in a human genome, and by detection of C5DNA methyltransferase homologs in various plant DNAs. In each case, amplified products were sufficiently pure to be cloned without gel fractionation. This COnsensus-DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) strategy has been implemented as a computer program that is accessible over the World Wide Web (http://blocks.fhcrc.org/codehop.html) and is directly linked from the BlockMaker multiple sequence alignment site for hybrid primer prediction beginning with a set of related protein sequences.
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PMID:Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. 951 32

During the pathogenesis of human hepatocellular carcinoma (HCC), the CpG island encompassing the pi-class glutathione S-transferase gene (GSTP1) becomes hypermethylated. Repression of transcription accompanying CpG island hypermethylation has been proposed to be mediated by methyl-CpG binding domain (MBD) proteins. We report here that inhibition of transcription from hypermethylated GSTP1 promoters in Hep3B HCC cells, which fail to express GSTP1 mRNA or GSTP1 polypeptides, appears to be mediated by MBD2. Treatment of Hep3B cells with 5-azadeoxycytidine (5-aza-dC), a methyltransferase inhibitor, activated GSTP1 expression, whereas treatment with trichostatin A, a histone deacetylase inhibitor, had little effect. To more precisely assess the contribution of the pattern of GSTP1 CpG island methylation on GSTP1 mRNA expression, Hep3B cells were treated for 72 h with 5-aza-dC and then subjected to limiting dilution cloning. Bisulfite sequencing was used to map the methylation patterns of the GSTP1 promoter region in GSTP1-expressing and -non-expressing clones. In the clone that expressed GSTP1 mRNA determined by Northern blot analysis and quantitative reverse transcriptase (RT)-PCR, widespread demethylation of at least one GSTP1 allele was evident. Chromatin immunoprecipitation experiments revealed the presence of MBD2, but not Sp1, at the GSTP1 promoter in Hep3B cells. In contrast, Sp1 was detected at the GSTP1 promoter in a GSTP1-expressing Hep3B 5-aza-dC subclone. To test whether MBD2 might be responsible for the inhibition of GSTP1 transcription from hypermethylated GSTP1 promoters, siRNAs were used to reduce MBD2 polypeptide levels in Hep3B cells. SssI-catalyzed methylation of GSTP1 promoter sequences resulted in diminished luciferase reporter activity after transfection into Hep3B cells. However, when hypermethylated GSTP1 promoter sequences were transfected into Hep3B cells that had been treated with siRNA-targeting MBD2 mRNA, no repression of luciferase reporter expression was evident. These findings implicate MBD2 in the repression of GSTP1 expression associated with GSTP1 CpG island hypermethylation in HCC cells.
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PMID:Methyl-CpG binding domain protein 2 represses transcription from hypermethylated pi-class glutathione S-transferase gene promoters in hepatocellular carcinoma cells. 1196 Sep 94

Methylation of bacterial DNA can regulate microbial growth and virulence. Expression of hpyIM, a conserved methyltransferase of the gastric pathogen Helicobacter pylori, was quantitated in gastric biopsy specimens from 41 H. pylori-infected patients and during growth in vitro, by quantitative reverse transcriptase-polymerase chain reaction and/or RNA slot-blot analysis, to determine whether levels of transcription were associated with pathologic outcome, as based on both severity of gastritis and inflammatory cytokine levels, or were regulated by bacterial growth phase. The effects that hpyIM inactivation has on bacterial morphology were determined by electron microscopy. Expression of hpyIM varied dramatically within colonized gastric tissue, and levels were not related to either colonization density, severity of inflammation, mucosal IL-8 concentrations, or clinical disease. In vitro, hpyIM expression was higher during log-phase growth and was required for normal bacterial morphology, suggesting that hpyIM expression may be growth-phase regulated within the gastric niche.
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PMID:Characterization of expression of a functionally conserved Helicobacter pylori methyltransferase-encoding gene within inflamed mucosa and during in vitro growth. 1235 74

Beta-alanine (Ala) betaine, an osmoprotectant suitable under saline and hypoxic environments, is found in most members of the halophytic plant family Plumbaginaceae. In Limonium latifolium (Plumbaginaceae), it is synthesized via methylation of beta-Ala by the action of a trifunctional S-adenosyl L-methionine (Ado-Met): beta-Ala N-methyltransferase (NMTase). Peptide sequences from purified beta-Ala NMTase were used to design primers for reverse transcriptase-PCR, and several cDNA clones were isolated. The 5' end of the cDNA was cloned using a 5'-rapid amplification of cDNA ends protocol. A 500-bp cDNA was used as a probe to screen a lambda-gt10 L. latifolium leaf cDNA library. Partial cDNA clones represented two groups, NMTase A and NMTase B, differing only in their 3'-untranslated regions. The full-length NMTase A cDNA was 1,414 bp and included a 1128-bp open reading frame and a 119-bp 5'-untranslated region. The deduced amino acid sequence of 375 residues had motifs known to be involved in the binding of Ado-Met. The NMTase mRNA was expressed in L. latifolium leaves but was absent in Limonium sinuatum, a member of the genus that lacks the synthetic pathway for beta-Ala betaine. NMTase mRNA expression was high in young and mature leaves and was enhanced by light. NMTase cDNA was expressed in yeast (Saccharomyces cerevisiae) under the control of a galactose-inducible promoter. Protein extracts of galactose-induced recombinant yeast had Ado-Met-specific NMTase activities that were highly specific to beta-Ala, N-methyl beta-Ala, and N,N-dimethyl beta-Ala as methyl acceptors. NMTase activities were not detectable in comparable protein extracts of yeast, transformed with vector control. The NMTase protein sequence shared homology with plant caffeic acid O-methyltransferases and related enzymes. Phylogenetic analyses suggested that beta-Ala NMTase represents a novel family of N-methyltransferases that are evolutionarily related to O-methyltransferases.
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PMID:beta-alanine N-methyltransferase of Limonium latifolium. cDNA cloning and functional expression of a novel N-methyltransferase implicated in the synthesis of the osmoprotectant beta-alanine betaine. 1285 43

Modified nucleosides in tRNAs play important roles in tRNA structure, biosynthesis and function, and serve as crucial determinants of bacterial growth and virulence. In the yeast Saccharomyces cerevisiae, mutants defective in N1-methylation of a highly conserved adenosine (A58) in the TPsiC loop of initiator tRNA are non-viable. The yeast m1A58 methyltransferase is a heterotetramer consisting of two different polypeptide chains, Gcd14p and Gcd10p. Interestingly, while m1A58 is not found in most eubacteria, the mycobacterial tRNAs have m1A58. Here, we report on the cloning, overexpression, purification and biochemical characterization of the Rv2118c gene-encoded protein (Rv2118p) from Mycobacterium tuberculosis, which is homologous to yeast Gcd14p. We show that Rv2118c codes for a protein of approximately 31 kDa. Activity assays, modified base analysis and primer extension experiments using reverse transcriptase reveal that Rv2118p is an S-adenosyl-l-methionine-dependent methyltransferase which carries out m1A58 modification in tRNAs, both in vivo and in vitro. Remarkably, when expressed in Escherichia coli, the enzyme methylates the endogenous E.coli initiator tRNA essentially quantitatively. Furthermore, unlike its eukaryotic counterpart, which is a heterotetramer, the mycobacterial enzyme is a homotetramer. Also, the presence of rT modification at position 54, which was found to inhibit the Tetrahymena pyriformis enzyme, does not affect the activity of Rv2118p. Thus, the mycobacterial m1A58 tRNA methyltransferase possesses distinct biochemical properties. We discuss aspects of the biological relevance of Rv2118p in M.tuberculosis, and its potential use as a drug target to control the growth of mycobacteria.
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PMID:Mycobacterium tuberculosis Rv2118c codes for a single-component homotetrameric m1A58 tRNA methyltransferase. 1496 Jul 15

Imprinted genes are expressed from a single allele due to differential methylation of maternal or paternal alleles during gametogenesis. Dnmt3L (DNA cytosine-5-methyltransferase 3 like), a member of de novo methyltransferase Dnmt3 protein family, is a regulator of maternal imprinting. In the present study, we have characterized the promoter region of the mouse Dnmt3L gene. Transient transfection assays performed with 5'-deletion promoter constructs indicated a minimal promoter area within 440 bp upstream from the translational start site. Longer promoter constructs showed decreased activity, suggesting the presence of repressor elements within the upstream sequences. According to electrophoretic mobility-shift assays and mutation analysis, the minimal promoter region contained four functional binding sites for the Sp1 (specificity protein 1) family of transcription factors, Sp1 and Sp3. In vitro methylation of Dnmt3L promoter constructs decreased the transcriptional activity significantly, demonstrating down-regulation by cytosine methylation. This was supported by the results from bisulphite sequencing and real-time quantitative reverse transcriptase-PCR analysis of different mouse cell lines and tissues. In testis and embryonic stem cells showing strong Dnmt3L expression, all CpG sites studied were fully unmethylated, whereas non-expressive cell lines and tissues with lesser Dnmt3L expression showed complete or diverse CpG methylation levels. Treatment of Dnmt3L non-expressive cell lines with deacetylase inhibitor trichostatin A and methyltransferase inhibitor 5-aza-2'-deoxycytidine induced the expression of Dnmt3L mRNA. Furthermore, we show that the repressional effect of longer promoter fragments was also relieved by these inhibitors, altogether indicating an epigenetic control for Dnmt3L gene regulation.
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PMID:Epigenetic modifications affect Dnmt3L expression. 1501 37

DNA methylation and epigenetic inactivation of the O6-methylguanine methyltransferase (MGMT) gene induces MGMT deficiency, reducing the tumor cell's DNA repair capacity and increasing its susceptibility to alkylating chemotherapeutic agents. Consequently, adult patients whose tumors are deficient in MGMT have better outcomes with alkylator chemotherapy, and MGMT methylation has been proposed as a screening marker of deficient tumors. In order to test the feasibility of this approach for medulloblastoma, a common brain tumor in children, we determined the methylation status, mRNA expression pattern, and protein expression of MGMT in a panel of clinical specimens. Methylation-specific polymerase chain reaction analysis revealed methylation of MGMT in 28 of 37 tumor samples. Quantitative real-time reverse transcriptase-polymerase chain reaction showed a range of expression of MGMT mRNA varying more than 20-fold. However, there was no correlation found between MGMT methylation and mRNA expression. Immunohistochemistry demonstrated that all tumors were immunoreactive for MGMT in the nucleus of the medulloblastoma cells in a heterogeneous pattern. The intercell variability of MGMT complement explained the discordance between methylation and expression. Therefore, MGMT methylation as determined by methylation-specific polymerase chain reaction cannot be used as a marker for MGMT deficiency in medulloblastoma. Further, these findings support the use of pharmacological MGMT depletion as a rational approach for intensification of alkylator chemotherapy in the treatment of medulloblastoma.
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PMID:Intercellular heterogeneity of expression of the MGMT DNA repair gene in pediatric medulloblastoma. 1527 12

The gene product of cfr from Staphylococcus sciuri confers resistance to chloramphenicol, florfenicol and clindamycin in Staphylococcus spp. and Escherichia coli. Cfr is not similar to any other known chloramphenicol resistance determinant. Comparative investigation of E. coli with and without a plasmid-encoded Cfr showed a decreased drug binding to ribosomes in the presence of Cfr. As chloramphenicol/florfenicol and clindamycin have partly overlapping drug binding sites on the ribosome, the most likely explanation is that Cfr modifies the RNA in the drug binding site. This hypothesis was supported by drug footprinting data that showed both a decreased drug binding and an enhanced reverse transcriptase stop at position 2504, which corresponds to a modification at position A2503 at the drug binding site. A 45 n long RNA fragment containing the appropriate region was isolated and MALDI-TOF mass spectrometry in combination with tandem mass spectrometry showed an additional methylation at position A2503. Moreover, reduced methylation was detected at nucleotide C2498. The results show that Cfr is an RNA methyltransferase that targets nucleotide A2503 and inhibits ribose methylation at nucleotide C2498, thereby causing resistance to chloramphenicol, florfenicol and clindamycin.
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PMID:A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: methylation of 23S ribosomal RNA at A2503. 1609 Oct 44


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