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)

Under certain conditions, T4 gene 32 protein is known to increase the efficiency of different enzymes, such as Taq DNA polymerase, reverse transcriptase, and telomerase. In this study, we compared the efficiency of the SMART PCR cDNA synthesis kit with and without the T4 gene 32 protein. The use of this cDNA synthesis procedure, in combination with T4 gene 32 protein, increases the yield of RT-PCR products from approximately 90% to 150%. This effect is even observed for long mRNA templates and low concentrations of total RNA (25 ng). Therefore, we suggest the addition of T4 gene 32 protein in the RT-PCR mixture to increase the efficiency of cDNA synthesis, particularly in cases when low amounts of tissue are used.
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PMID:Increased yield of PCR products by addition of T4 gene 32 protein to the SMART PCR cDNA synthesis system. 1146 24

To date the majority of sequencing technologies have been based on use of gel plates. In this study sequencing by capillary electrophoresis for HIV-1 genotyping on the CEQ 2000 sequencer (Beckman Coulter Inc.) has been investigated and compared to an 'in house' protocol on the Prism-377 sequencer (Applied Biosystems) and to the HIV-1 TruGene kit (Visible Genetics Inc.), two gel plate-based systems. Plasma from 20 HAART-treated patients with virological failure were analyzed for protease (PR) and reverse transcriptase (RT) genes. A total of 520 RT codons (26/patient) and 360 PR codons (18/patient) related to antiretroviral drug resistance were evaluated. The overall agreement between CEQ 2000 and Prism-377 results was 100% for the RT and PR primary and secondary mutations. The overall agreement between CEQ 2000 and TruGene was 100% for primary and > or =97% for secondary mutations. Discrepant results would have never led to errors in genotype interpretation. Performances for a 24 patients/week/one technician genotyping throughput were analyzed. For Prism-377, TruGene and CEQ 2000, manual processing required 5, 4 and 2,5 days, sequence data analysis needed additional 3, 1 and 2 days and cost/patient was approximately 49, 214 and 39 $, respectively. The CEQ 2000 sequencer offers a reliable alternative for fast and cost effective HIV drug resistance analysis.
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PMID:Comparison of capillary electrophoresis sequencing with the new CEQ 2000 DNA Analysis System to conventional gel based systems for HIV drug resistance analysis. 1154 79

Real-time PCR is an accurate method that can be used for the quantification of specific DNA molecules. Here we provide a protocol for SYBR Green I in real-time PCR applications using plastic reaction tubes. We report that SYBR Green I is alkali labile and once degraded inhibits the PCR. In our optimized protocol, diluted aliquots of SYBR Green I remain stable for at least two weeks. We also evaluated different cDNA synthesis protocols for the quantification of multiple genes from the same cDNA preparation. The best result was obtained with cDNAs synthesized by OmniScript reverse transcriptase from 2.5 microg total RNA using oligo d(T)18 primers. The cDNA reactions could be diluted 1:25, allowing the quantification of up to 125 different medium expressed genes of Arabidopsis. Extension times ranged between 20 and 40 bp/s for accurate quantification of PCR products up to approximately 400 bp in the Rotor-Gene 2000 system. Using our optimized real-time PCR protocol, the reproducibility and amplification efficiency was high and comparable to a commercially available SYBR Green I kit. Furthermore, the sensitivity allowed us to quantify 10-20 copies of mRNA and dsDNA. Thus, the protocol eliminates the need for expensive pre-made kits.
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PMID:Evaluation of a homemade SYBR green I reaction mixture for real-time PCR quantification of gene expression. 1196 1

An observational study assessed the longitudinal use of a new line probe assay for the detection of protease mutations. Probe assays for detection of reverse transcriptase (Inno-LiPA HIV-1 RT; Innogenetics) and protease (prototype kit Inno-LiPA HIV Protease; Innogenetics) mutations gave results for 177 of 199 sequential samples collected over 2 years from 26 patients failing two nucleoside reverse transcriptase inhibitors and one protease inhibitor (first line: indinavir, n = 6; ritonavir, n = 10; and saquinavir, n = 10). Results were compared to recombinant virus protease inhibitor susceptibility data (n = 87) and to clinical and virological data. Combinations of protease mutations (M46I, G48V, I54V, V82A or -F, I84V, and L90M) predicted phenotypic resistance to the protease inhibitor and to nelfinavir. The sum of protease mutations was associated with virological and clinical outcomes from 6 and 3 months on, respectively. Moreover, a poorer clinical outcome was linked to the sum of reverse transcriptase mutations. In conclusion, despite the limited number of patients studied and the restricted number of codons investigated, probe assay-based genotyping correlates with phenotypic drug resistance and predicts new Centers for Disease Control and Prevention stage B and C clinical events and virological outcome. Line probe assays provide additional prognostic information and should be prospectively investigated for their potential for treatment monitoring.
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PMID:Longitudinal use of a line probe assay for human immunodeficiency virus type 1 protease predicts phenotypic resistance and clinical progression in patients failing highly active antiretroviral therapy. 1201 10

Alterations in gene expression represent key events in carcinogenesis. We have studied HPV-induced cervical carcinogenesis, using an HPV-33-positive cell line (UT-DEC-1) established from a low-grade vaginal dysplasia (VAIN-I). Early-passage cells contained HPV-33 in episomal form, but these were superseded at later passages by cells carrying only integrated virus. To gain insight into the biologic significance of HPV integration, we compared the level of gene expression in normal vaginal keratinocytes, early-passage and late-passage UT-DEC-1 cells, using cDNA microarrays. Total RNA was isolated from cells by CsCl-gradient centrifugation, reverse-transcribed with MMLV reverse transcriptase and labeled with alpha-(32)P ATP. A cDNA microarray expression profile analysis was performed with Clontech's Human Cancer 1.2 cDNA expression array kit. The 16 upregulated genes (cut-off 2-fold), identified by comparing both cell types to control keratinocytes, appeared to support cell-cycle progression or to be functional in mitosis. These included, e.g., MCM4 DNA replication licensing factor, cdc2p34 and chromatin assembly factor 1 p48 subunit. Downregulated genes (44 altogether) interfered with apoptosis and cell adhesion, including the apoptosis-inducing genes FRAP, Bik and caspase-9 precursor. The most significant differences between the late and early passages (29 and 46 constantly up- and downregulated genes without any fluctuation) were overexpression of the transcription factors E2F5 with its dimerization partner DP1, NF-kappa B and serine/threonine kinases and underexpression of enzymes of the MAPK pathway. Acquisition of a selective growth advantage after viral integration might be explained by a major shift from a MAPK pathway to cell-cycle dysregulation (G(2)/M).
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PMID:Transcriptional profiling of a human papillomavirus 33-positive squamous epithelial cell line which acquired a selective growth advantage after viral integration. 1211 47

Thrombin appears to underlie myometrial contractions in response to intrauterine bleeding. In a similar fashion, thrombin generated within the uterus in the absence of active bleeding could also produce contractions. These studies sought to determine whether functionally active prothrombin is expressed in the pregnant and nonpregnant rat uterus. Uteri were obtained from proestrus/estrus and timed-pregnant Sprague-Dawley rats. Western blots were performed using antithrombin antibodies. Immunohistochemical studies were performed using the same antibodies along with the Vector Elite ABC kit. Qualitative reverse transcriptase-polymerase chain reaction studies were performed using rat prothrombin-specific oligonucleotide primers. In vitro uterine contraction studies were performed using Taipan snake venom (an exogenous prothrombinase) and components of the plasma prothrombinase complex (Factors Xa and V) with and without pretreatment with thrombin inhibitors (heparin or hirudin). The Western blots demonstrated prothrombin peptides in myometrial tissue from estrus and pregnant rats. The immunohistochemical studies confirmed prothrombin peptides in both the circular and longitudinal myometrium, along with the endometrium. The reverse transcriptase-polymerase chain reaction studies demonstrated prothrombin mRNA in the endometrium and placenta, but not in the myometrial smooth muscle. The Taipan snake venom stimulated a significant increase in contractions, which were suppressed by pretreatment with heparin and hirudin. The Factor Xa and V complex also significantly stimulated uterine contractions, which were likewise inhibited by hirudin. These studies provide evidence supporting the expression of functionally active prothrombin in the pregnant and nonpregnant rat uterus. Based on the presence of its mRNA, prothrombin appears to be synthesized in the endometrium and placenta; in contrast, the myometrial smooth-muscle cells appear to sequester preformed prothrombin. These results support the hypothesis that intrauterine thrombin could play an autocrine/paracrine role in the regulation of contractile activity.
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PMID:Intrauterine expression of prothrombin in the sprague-dawley rat. 1238 11

Drug resistance and poor virological responses are associated with well-characterized mutations in the viral reading frames that encode the proteins that are targeted by currently available antiretroviral drugs. An integrated system was developed that includes target gene amplification, DNA sequencing chemistry (TRUGENE HIV-1 Genotyping Kit), and hardware and interpretative software (the OpenGene DNA Sequencing System) for detection of mutations in the human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase sequences. The integrated system incorporates reverse transcription-PCR from extracted HIV-1 RNA, a coupled amplification and sequencing step (CLIP), polyacrylamide gel electrophoresis, semiautomated analysis of data, and generation of an interpretative report. To assess the accuracy and robustness of the assay system, 270 coded plasma specimens derived from nine patients were sent to six laboratories for blinded analysis. All specimens contained HIV-1 subtype B viruses. Results of 270 independent assays were compared to "gold standard" consensus sequences of the virus populations determined by sequence analysis of 16 to 20 clones of viral DNA amplicons derived from two independent PCRs using primers not used in the kit. The accuracy of the integrated system for nucleotide base identification was 98.7%, and the accuracy for codon identification at 54 sites associated with drug resistance was 97.6%. In a separate analysis of plasma spiked with infectious molecular clones, the assay reproducibly detected all 72 different drug resistance mutations that were evaluated. There were no significant differences in accuracy between laboratories, between technologists, between kit lots, or between days. This integrated assay system for the detection of HIV-1 drug resistance mutations has a high degree of accuracy and reproducibility in several laboratories.
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PMID:Accuracy of the TRUGENE HIV-1 genotyping kit. 1268 49

The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System are designed to sequence the protease (PR)- and reverse transcriptase (RT)-coding regions of human immunodeficiency virus type 1 (HIV-1) pol. Studies were undertaken to determine the accuracy of this assay system in detecting resistance-associated mutations and to determine the effects of RNA extraction methods, anticoagulants, specimen handling, and potentially interfering substances. Samples were plasma obtained from HIV-infected subjects or seronegative plasma to which viruses derived from wild-type and mutant infectious molecular clones (IMC) of HIV-1 were added. Extraction methods tested included standard and UltraSensitive AMPLICOR HIV-1 MONITOR, QIAGEN viral RNA extraction mini kit, and QIAGEN Ultra HIV extraction kit, and NASBA manual HIV-1 quantitative NucliSens. Sequence data from test sites were compared to a "gold standard" reference sequence to determine the percent agreement. Comparisons between test and reference sequences at the nucleotide level showed 97.5 to 100% agreement. Similar results were obtained regardless of extraction method, regardless of use of EDTA or acid citrate dextrose as anticoagulant, and despite the presence of triglycerides, bilirubin, hemoglobin, antiretroviral drugs, HIV-2, hepatitis C virus (HCV), HBV, cytomegalovirus, human T-cell leukemia virus type 1 (HTLV-1), or HTLV-2. Samples with HIV-1 RNA titers of >or=1,000 copies/ml gave consistent results. The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System consistently generate highly accurate sequence data when tested with IMC-derived HIV and patient samples.
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PMID:Performance characteristics of the TRUGENE HIV-1 Genotyping Kit and the Opengene DNA Sequencing System. 1268 50

In this study, the synthesis of some benzoxazoles and their analogues were described and their antiviral activities were studied together with the previously synthesized 2,5,6-trisubstituted benzoxazole, benzothiazole, benzimidazole and oxazolo(4,5-b)pyridine derivatives. The reverse transcriptase (RT) inhibitory activity of these compounds was determined using a commercial kit and assay system which utilizes the scintillation proximity assay principle. The results are concentration at which the compound inhibits RT activity by 50%). The compounds inhibited the in vitro binding of thymidine to the RT enzyme exhibiting IC50 values between 6.3 x 10(5) mumol/l-0.34 mumol/l and their activities were compared to some standard drugs such as 3'-azido-2',3'-dideoxythymidine triphosphate and dideoxythymidine triphosphate.
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PMID:Synthesis and HIV-1 reverse transcriptase inhibitor activity of some 2,5,6-substituted benzoxazole, benzimidazole, benzothiazole and oxazolo(4,5-b)pyridine derivatives. 1278 23

Telomerase is an RNA-dependent DNA polymerase that synthesizes TTAGGG telomeric DNA onto chromosome ends to compensate for sequence loss during DNA replication. It has been detected in 85-90% of all primary human cancers, implicating that the telomerase seems to be reactivated in tumors and that such activity may play a role in the tumorigenic process. The purpose of this study was to evaluate telomerase activity, human telomerase RNA (hTR), and telomerase reverse transcriptase (TERT) in stomach cancer and to determine their potential relationships to clinicopathologic parameters. Frozen and corresponding methacarn-fixed paraffin-embedded tissue samples were obtained from 51 patients with gastric adenocarcinoma and analyzed for telomerase activity by using a TRAPeze ELISA kit. Tissue sections of all the samples were further investigated for hTR and TERT by in situ hybridization and a sensitive immunohistochemical technique, respectively. Telomerase activity was detected in 37 (73%) tumors. Telomerase positivity from methacarn-fixed paraffin blocks was found to be 35% of that from frozen tissues. hTR was overexpressed in 46 (90%) samples: 33/37 (89%) with and 13/14 (93%) without telomerase activation. Expression of TERT was demonstrated in 40 (78%) cases: 30/37 (81%) with and 10/14 (71%) without telomerase. Telomerase activity correlated well with depth of invasion (P =.037) and tumor differentiation (P =.022), whereas hTR significantly correlated with nodal metastasis (P=.047) and tumor size (P=.023). These data suggest that reactivated telomerase may play a significant role in the tumorigenesis of gastric cancer and may reflect, along with enhanced hTR, the malignant potential of the tumor. It is noteworthy that methacarn-fixed tissue cannot as yet substitute for the frozen section in the TRAP assay.
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PMID:Expression of telomerase activity, human telomerase RNA, and telomerase reverse transcriptase in gastric adenocarcinomas. 1286 Oct 67


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