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)

The relationship between changes in mRNA abundance and enzyme activity was determined for three genes over a span of nearly 3 h during amino acid production in Corynebacterium glutamicum. Gene expression changes during C. glutamicum fermentations were examined by complementary DNA (cDNA) microarrays and by a second method for quantitating RNA levels, competitive reverse transcriptase-PCR (RT-PCR). The results obtained independently by both methods were compared and found to be in agreement, thus validating the quantitative potential of DNA microarrays for gene expression profiling. Evidence of a disparity between mRNA abundance and enzyme activity is presented and supports our belief that it is difficult to generally predict protein activity from quantitative transcriptome data. Homoserine dehydrogenase, threonine dehydratase, and homoserine kinase are enzymes involved in the biosynthesis of l-isoleucine and other aspartate-derived amino acids in C. glutamicum. Our data suggest that different underlying regulatory mechanisms may be connected with the expression of the genes encoding each of these three enzymes. Indeed, whereas in one case the increases in enzyme activity exceeded those in the corresponding mRNA abundance, in another case large increases in the levels of gene expression were not congruent with changes in enzyme activity.
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PMID:Disparity between changes in mRNA abundance and enzyme activity in Corynebacterium glutamicum: implications for DNA microarray analysis. 1265 16

The VP2 hypervariable region of P97/302 local infectious bursal disease virus (IBDV) isolate was amplified by the reverse transcriptase (RT) nested polymerase chain reaction (PCR) and cloned. This region of P97/302 local isolate was sequenced and compared with eight other reported IBDV sequences. The result showed that P97/302 IBDV was most identical to the reported very virulent IBDV strains because it has amino acid substitutions at positions 222, 256, 294, and 299, which encode alanine, isoleucine, isoleucine, and serine, respectively. This region can be digested with restriction enzymes of Taq1, Sty1, Ssp1 but not with Sac1. The P97/302 isolate was then used for the optimization of RT nested PCR enzyme-linked immunosorbent assay (ELISA). The RT nested PCR ELISA was able to detect 10(-4) dilution of the infected bursa homogenates and was 10 times more sensitive when compared with the agarose gel detection method. The RT nested PCR ELISA can detect up to 0.48 ng of the PCR product. The specificity of this nested PCR ELISA was also high (100%).
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PMID:Sequence analysis of Malaysian infectious bursal disease virus isolate and the use of reverse transcriptase nested polymerase chain reaction enzyme-linked immunosorbent assay for the detection of VP2 hypervariable region. 1271 71

Short interspersed nuclear elements (SINEs) are dispersed repetitive DNA sequences that are major components of all mammalian genomes. They have been described in almost all lineages of Euarchontoglires (rodents, rabbits, primates, flying lemurs, and tree shrews), except in flying lemurs. Most SINE family members are composed of three distinct regions: a 5' tRNA-related region, a tRNA-unrelated region, and a short tandem repeat at the 3' end that is AT-rich. The newly discovered SINE family in Cynocephalus deviates from this common structure. All 30 SINE loci analyzed in this family lack a tRNA-unrelated region and are composed exclusively of tRNA-related elements. Therefore, this novel SINE structure, described for the first time in mammalian genomes, was designated as t-SINE. The t-SINE family exhibits a high copy number and is specific to flying lemurs. Three major t-SINE subfamilies could be distinguished on the basis of characteristic nucleotides, deletions, insertions, and duplications. These sequence-specific characteristics within subfamilies and sub-subfamilies reveal that they are derived copies of distinct progenitors. We present evolutionary relationships between subfamilies and compare relationships between the subfamilies and the isoleucine tRNA gene. t-SINE amplification occurred through multiple sources and is supposedly mobilized via the L1-encoded reverse transcriptase-dependent retrotranspositional mechanism in trans.
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PMID:Unique mammalian tRNA-derived repetitive elements in dermopterans: the t-SINE family and its retrotransposition through multiple sources. 1288 66

The recombinant protein RTL1Tc, encoded by the non-LTR (long terminal repeat) retrotransposon L1Tc from Trypanosoma cruzi, has been shown to have reverse transcriptase (RT) activity using poly(rA)/oligo(dT) and poly(rC)/oligo(dG) homopolymers as template/primers. The optimal RT activity was detected at a concentration of 5 mM Mg2+, pH 8 and between 28 and 37% degrees C. Site-directed mutagenesis in the RT catalytic site proved that substitution of aspartic acid 313 for isoleucine (RT D313IL1Tc) practically abolishes the RT activity of the RTL1Tc protein. RT-polymerase chain reaction assays revealed that the RTL1Tc protein has the ability to use both homologous and heterologous RNA templates. Also, it is shown that the RTL1Tc protein is capable of synthesizing complementary DNA molecules by consecutive switching of the oligo molecule, which the protein uses as a template. This template switching may be involved in the retroelement integration process.
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PMID:Characterization of reverse transcriptase activity of the L1Tc retroelement from Trypanosoma cruzi. 1468 92

Methionine at position 184 of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) was changed to valine, isoleucine, threonine, or alanine in an HIV-1-based vector. The vectors were analyzed for replication capacity and for resistance to the nucleoside analog 2',3'-dideoxy-3'thiacytidine (3TC) using a single-cycle assay. Viruses containing the valine or isoleucine mutations were highly resistant to 3TC and replicated almost as well as the wild-type virus. The virus containing the threonine mutation was resistant to 3TC, but replicated about 30% as well as the wild-type. The alanine mutation conferred partial resistance to 3TC, but replicated poorly. The amounts of viral DNA synthesized decreased in 3TC-treated cells when the cells were infected with wild-type virus and the M184A mutant. The effect of these mutations on the generation of the ends of the linear viral DNA was determined using the sequence of the 2-LTR circle junctions. The M184T mutation increased the proportion of 2-LTR circle junctions containing a tRNA insertion, suggesting that the mutation affected the RNase H activity of RT.
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PMID:Mutations at position 184 of human immunodeficiency virus type-1 reverse transcriptase affect virus titer and viral DNA synthesis. 1506 12

Focal epilepsies in young patients are frequently associated with differentiated glioneuronal tumours. Dysplastic neurones represent a characteristic neuropathological feature of gangliogliomas, the most common entity encountered in this group. Here, we have analysed two major components of the reelin pathway involved in neuronal migration and cortical development, that is, p35 and disabled-1 (dab1), in gangliogliomas. Genomic structures of human dab1 and p35 were identified 'in silico' using the HTGS databank, NCBI BLAST 2.1. DNA sequence analysis was carried out in gangliogliomas obtained from 29 epilepsy patients vs. peripheral blood DNA from non-affected control individuals (n = 100). Gene expression of dab1 and p35 was determined by real-time RT-PCR (reverse transcriptase polymerase chain reaction) in gangliogliomas (n = 14) vs. non-neoplastic central nervous system tissue (n = 20). The human dab1 gene contains 13 coding exons and is located on chromosome 1p31-32. A single coding exon constitutes the human p35 gene, which is located on chromosome 17q11.2. A novel homologueous genomic region on chromosome 2 has to be taken into account for future studies on p35. One ganglioglioma patient showed a unique polymorphism in the p35 gene. The single base exchange (C to A) at nucleotide 904 of the p35 cDNA (GenBank X80343, start ATG, codon 302) results in a leucine-isoleucine amino acid substitution. No mutations of the dab1 and p35 genes in gangliogliomas were observed. However, significantly lower levels of dab1 and p35 gene transcripts were detected in gangliogliomas compared to controls (dab1 28.24%, t-test P < 0.001; p35 21.28%, t-test P < 0.001, in gangliogliomas vs. controls). Our data suggest that mutational events of dab1 and p35 are not involved in the molecular pathogenesis of gangliogliomas. A potential functional role of these developmentally regulated genes for the formation of epileptogenic glioneuronal lesions remains to be elucidated.
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PMID:The reelin pathway components disabled-1 and p35 in gangliogliomas--a mutation and expression analysis. 1517 76

Chronic hepatitis B virus (HBV) infection can cause severe liver disease, including cirrhosis and hepatocellular carcinoma. Lamivudine is a relatively recent alternative to alpha interferon for the treatment of HBV infection, but unfortunately, resistance to lamivudine commonly develops during monotherapy. Lamivudine-resistant HBV mutants display specific mutations in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the viral polymerase (reverse transcriptase [rt]), which is the catalytic site of the enzyme, i.e., methionine 204 to isoleucine (rtM204I) or valine (rtM204V). The latter mutation is often accompanied by a compensatory leucine-to-methionine change at codon 180 (rtL180M). In the present study, a novel sequencing method, pyrosequencing, was applied to the detection of lamivudine resistance mutations and was compared with direct Sanger sequencing. The new pyrosequencing method had advantages in terms of throughput. Experiments with mixtures of wild-type and resistant viruses indicated that pyrosequencing can detect minor sequence variants in heterogeneous virus populations. The new pyrosequencing method was evaluated with a small number of patient samples, and the results showed that the method could be a useful tool for the detection of lamivudine resistance in the clinical setting.
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PMID:Pyrosequencing for detection of lamivudine-resistant hepatitis B virus. 1547 42

A successful transition of Listeria monocytogenes from the extracellular to the intracellular environment requires a precise adaptation response to conditions encountered in the host milieu. Although many key steps in the intracellular lifestyle of this gram-positive pathogen are well characterized, our knowledge about the factors required for cytosolic proliferation is still rather limited. We used DNA microarray and real-time reverse transcriptase PCR analyses to investigate the transcriptional profile of intracellular L. monocytogenes following epithelial cell infection. Approximately 19% of the genes were differentially expressed by at least 1.6-fold relative to their level of transcription when grown in brain heart infusion medium, including genes encoding transporter proteins essential for the uptake of carbon and nitrogen sources, factors involved in anabolic pathways, stress proteins, transcriptional regulators, and proteins of unknown function. To validate the biological relevance of the intracellular gene expression profile, a random mutant library of L. monocytogenes was constructed by insertion-duplication mutagenesis and screened for intracellular-growth-deficient strains. By interfacing the results of both approaches, we provide evidence that L. monocytogenes can use alternative carbon sources like phosphorylated glucose and glycerol and nitrogen sources like ethanolamine during replication in epithelial cells and that the pentose phosphate cycle, but not glycolysis, is the predominant pathway of sugar metabolism in the host environment. Additionally, we show that the synthesis of arginine, isoleucine, leucine, and valine, as well as a species-specific phosphoenolpyruvate-dependent phosphotransferase system, play a major role in the intracellular growth of L. monocytogenes.
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PMID:Identification of Listeria monocytogenes genes contributing to intracellular replication by expression profiling and mutant screening. 1638 46

Baseline HIV-1 resistance data are important for resistance monitoring purposes especially in regions initiating large-scale antiretroviral treatment programs. We examined 40 protease and 35 reverse transcriptase amino acid sequences of HIV-1 subtype C from drug inexperienced patients from rural settings in South Africa for resistance mutations. Samples were collected between 2001 and 2004 prior to the availability of antiretrovirals through public health institutions. Ninety-five percent of patients had no major mutations in the protease gene, although substitutions M46L (2.5%) and G73S (2.5%), which according to the Stanford Genotypic Resistance Interpretation Algorithm are considered major mutations, were detected. In addition, a high prevalence of minor mutations was observed in the protease, with at least three minor resistance-associated mutations in 37% of the isolates. An isoleucine insertion at codon 37 was detected in one sequence. Most of the RT sequences were wild-type, although V118I (8.5%) and Y318F (5.7%) associated with resistance to lamivudine and nevirapine, respectively, were observed. Our data suggest that major resistance mutations among the drug-inexperienced population in South Africa may be rare, and routine resistance testing before the initiation of therapy in this initial stage of the treatment program may not be necessary.
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PMID:Resistance mutational analysis of HIV type 1 subtype C among rural South African drug-naive patients prior to large-scale availability of antiretrovirals. 1720 75

Long-term lamivudine (LMV) treatment of chronic hepatitis B almost inevitably engenders viral resistance. Mutations that result in the replacement of the methionine at position 204 of the deoxynucleoside triphosphate-binding site of the hepatitis B virus (HBV) reverse transcriptase (rt) by isoleucine, valine, or (rarely) serine (rtM204I/V/S) confer high-level resistance to LMV but reduce replication efficiency. The subsequent selection or coselection of secondary mutations that partially restore replication efficiency is common and may influence drug resistance. Genotyping has shown that LMV treatment can select for HBV rtL80V/I mutants, but their prevalence and phenotype have not been documented. Analysis of a large sequence database revealed that rtL80V/I occurred almost exclusively in association with LMV resistance, and 85% of these isolates encoded rtL80I. Coselection of rtL80V/I occurred in 46% of isolates in which LMV resistance was attributable to rtM204I but only 9% of those in which resistance was attributable to rtM204V. Moreover, rtL80V/I did not occur in HBV genotype A isolates but occurred at similar frequencies in genotype B, C, and D isolates. In vitro phenotyping showed that although the rtL80I mutant by itself replicated less efficiently and was hypersensitive to LMV compared to the replication efficiency and sensitivity of its wild-type parent, the presence of rtL80I enhanced the replication efficiency of rt204I/V mutants without significantly affecting LMV resistance. Molecular modeling revealed that rt80 does not interact directly with the enzyme's substrates. Collectively, these results suggest that coselection of rtL80V/I and rtM204I/V occurs because the former compensates for the loss of replication efficiency associated with the acquisition of LMV resistance, particularly in the case of rtM204I.
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PMID:The L80I substitution in the reverse transcriptase domain of the hepatitis B virus polymerase is associated with lamivudine resistance and enhanced viral replication in vitro. 1802 22


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