Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Zidovudine (azidothymidine (AZT)) inhibits human immunodeficiency virus replication, prolongs survival, and delays progression of acquired immune deficiency syndrome. We determined AZT-induced molecular and ultrastructural changes in the rat heart. Rats (3 per group) were given drinking water with or without AZT (0.2 to 1.0 mg/ml; 29 to 102 mg/kg/day). After 21, 35, or 49 days, hearts were glutaraldehyde-fixed by abdominal aortic perfusion, processed, and examined by transmission electron microscopy. In parallel, myocardial RNA was extracted from hearts (AZT dose: 1 mg/ml; 35 days) and subjected to Northern analysis using cDNA probes for: alpha c-actin, troponin C, mitochondrial creatine kinase and malate dehydrogenase, a portion of the mitochondrial genome containing cytochrome b coding region (pMM26), and glyceraldehyde-3-phosphate dehydrogenase. Results showed marked and widespread cardiac mitochondrial swelling with fractured and disrupted cristae after 35 days of 1 mg/ml AZT. After a 14-day recovery, these ultrastructural defects did not reverse. Changes were not present in myocardium after 21 days of AZT nor after 35 days of lower dose AZT (0.2 mg/ml). Mitochondrial cytochrome b mRNA expression was depressed in AZT-treated rat hearts (35 days; 1 mg/ml AZT). mRNAs encoding glyceraldehyde-3-phosphate dehydrogenase, alpha c-actin, troponin C, mitochondrial creatine kinase, malate dehydrogenase, and mitochondrial ribosomal RNAs remained unchanged. AZT disrupts cardiac mitochondrial ultrastructure and expression of mitochondrial cytochrome b mRNA in a dose- and time-dependent fashion. The mechanism of AZT cardiotoxicity may relate to inhibition of mitochondrial DNA replication (at the level of DNA polymerase gamma) as postulated by others.
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PMID:Mitochondrial ultrastructural and molecular changes induced by zidovudine in rat hearts. 171 47

The antigenic structure of glyceraldehyde-3-phosphate dehydrogenase of the most representative Enterobacteriaceae species were compared with an antiserum to Escherichia coli glyceraldehyde-3-phosphate dehydrogenase. The results of the immunodiffusion experiments were confirmed and specified by micro-complement fixation studies. They demonstrated a total immunological identity between the E. coli enzyme and the enzymes of Alcalescens-dispar and the Shigella species, a marked relatedness of the Salmonella species enzyme and a more or less significant relation of the enzymes of the other Enterobacteriaceae species. Moreover, the micro-complement fixation had the same sensitivity and a better selectivity than the DNA/DNA hybridizations. The results show that, like DNA polymerase, this enzyme has evolved more slowly than the other enzymes studied at this time.
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PMID:[Immunological study of glyceraldehyde-3-phosphate dehydrogenase in Enterobacteriaceae; taxonomic value]. 640 65

Fifteen samples from 11 patients suffering from chronic lymphocytic leukaemia (CLL; 5 untreated, 6 chemotherapeutically treated) were analysed for their individual gene expression of the multidrug resistance (MDR) associated genes encoding mdr1/P-glycoprotein, mrp, and topoisomerase II alpha/beta-isoenzymes by a complementary DNA polymerase chain reaction (cDNA-PCR) approach. The expression of glyceraldehyde-3-phosphate dehydrogenase (gapdh) served as standard. Thereby, we generally found high mdr1- and mrp-, but low topoisomerase II alpha-mRNA levels. While mdr1 levels of the CLL samples were mostly found to be in the range of values measured in the T-lymphoblastoid, P-glycoprotein MDR cell line CCRF VCR 100, mrp levels were usually found to be 2-4-fold higher compared therewith. This might represent a multifactorial MDR in CLL. In contrast to the low or even absent topoisomerase II alpha gene expression, however, the expression of the topoisomerase II beta gene was generally high in the CLL lymphocytes exceeding the value observed in the cell line CCRF VCR 100 up to 5-fold. mdr1 gene expression correlated significantly with mrp gene expression in samples from patients having received chemotherapy (rs = 0.5833, P < 0.05, n = 10). In two patients the follow-up analysis revealed combined increases in mdr1- and mrp-gene expression levels in the course of the disease.
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PMID:High mdr1- and mrp-, but low topoisomerase II alpha-gene expression in B-cell chronic lymphocytic leukaemias. 795 50

Synthesis of radiolabeled DNA probes via polymerase chain reaction (PCR) is a convenient alternative to the more conventional methods of random primer-labeling and nick translation. PCR requires less template and allows the synthesis of radiolabeled probes from specific sequences contained within cloning vectors and genomic DNA. Under nucleotide imbalance conditions where the concentration of the radiolabeled nucleotide was 0.825 microM and the other dNTPs were each > 25 microM, amplification by Taq DNA polymerase was inhibited. Reducing the concentrations of the unlabeled dNTPs resulted in greater yields of amplification product with maximal yield obtained when the concentration of three unlabeled nucleotides was two- to eightfold higher than that of the limiting labeled nucleotide. When we utilized this amplification method for synthesis of an 800-bp glyceraldehyde-3-phosphate (GAPDH) dehydrogenase probe, 87% of the added [32P]dCTP was incorporated into amplification product. Application of this method for synthesis of high specific activity probes ( > 4 x 10(9) cpm/micrograms) up to 2.6 kb in length is demonstrated and utility of the 800-bp GAPDH probe for hybridization to Northern blots for detection of GAPDH mRNA is presented.
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PMID:Nucleotide imbalance and polymerase chain reaction: effects on DNA amplification and synthesis of high specific activity radiolabeled DNA probes. 798 88

Inhibition of DNA polymerase gamma-function mediated by 3'-azido-3'-deoxythymidine (AZT) has been proposed to cause a myopathy by reducing mitochondrial DNA (mtDNA) replication. Repeated bouts of exercise stimulate an increase in mtDNA replication, mitochondrial content, and mitochondrial volume fraction. Therefore, adaptation of rat skeletal muscle [tibialis anterior (TA)] mitochondria exposed to AZT (1 mg/ml for 35 days) and then to electrical stimulation for 8 h/day (7, 14, 21 days) with continued AZT treatment was examined. Fourteen and 21 days of stimulation increased TA cytochrome oxidase (CO) activity, mtDNA, and CO subunit III and VIc mRNA levels in both groups. The TA CO activity and CO III mRNA increases after 14 and 21 days of stimulation were diminished in AZT-treated rats. TA glyceraldehyde-3-phosphate dehydrogenase was reduced in normal rats after chronic stimulation but was unchanged in AZT-treated rats. Chronic stimulation increased the mitochondrial volume fraction by 80 and 40% in normal and AZT-treated rats, respectively. These results indicate diminution, but not complete inhibition, of mitochondrial adaptation by AZT-treated skeletal muscle in response to stimulation.
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PMID:Skeletal muscle mitochondria from AZT-treated rats have a diminished response to chronic electrical stimulation. 882 81

We have developed real-time PCR systems to quantitate feline cytokine gene expression. The method is based on the cleavage of fluorescent dye-labelled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by a Sequence Detection System. The feline-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation of complementary DNA versus genomic DNA amplification products. Quantitative analysis of cytokine cDNA concentrations was performed in comparison to feline GAPDH. Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an amplification control and allowed for correction of variations in the efficiencies of RNA extraction and reverse transcription. GAPDH mRNAs were readily detectable in cDNAs prepared from unstimulated feline peripheral blood mononuclear cells (PBMCs) and from frozen cell pellets, while cytokines (Interleukin (IL)-4, IL-10, IL-12 p35, IL-12 p40, IFNgamma, IL-16) were expressed at variable amounts. IFNgamma transcription was found to be upregulated in stimulated PBMCs and feline cell lines. The synthesis of cDNA and the performance of the PCR in separate tubes proved to be of superior sensitivity compared to a single-tube based system. The assays described are highly reproducible, require no post-PCR manipulation of the amplicons and permit the analysis of several hundred PCR reactions per day. With this method it is possible to detect and quantify cytokine mRNA expression reliably in small amounts of cells even after storage of samples for at least 5 years.
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PMID:Quantitative real-time PCR for the measurement of feline cytokine mRNA. 1058 8

Here we present a novel methodology to quantitate bovine cytokines and growth factors contributing to immunity against bacterial infections of the mammary gland in cattle. Real-time TaqMan PCR systems were developed to overcome limitations of conventional quantitative PCR methods. The TaqMan method is based on the cleavage of fluorescent dye-labeled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by an automated spectrophotometer integrated in a sequence detection system (Applied Biosystems, Foster City, CA). The bovine-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation between complementary DNA (cDNA) and genomic DNA (gDNA) amplification products. Quantitative analysis of cytokine cDNA was performed in comparison to bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an amplification control and allowed for correction of variations in different numbers of cells in the starting material, in the efficiencies of RNA extraction and reverse transcription. With this method, high-throughput analysis of large numbers of samples was possible within a short time. In addition, decreasing the numbers of working steps shortened the time for analysis and increased accuracy. Profiles of cytokines (interleukin (IL)-2, IL-6, IL-8, IL-12 p40, TNF-alpha, IFN-gamma) and granulocyte-macrophage colony stimulating factor (GM-CSF) were established in normal lactating cattle. Differences of cytokine profiles obtained with the real-time TaqMan PCR system and conventional methods are discussed.
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PMID:Quantitation of bovine cytokine mRNA in milk cells of healthy cattle by real-time TaqMan polymerase chain reaction. 1113 25

Single nucleotide instability (SNI), an increase in spontaneous point mutation rates (MRs) without involvement of microsatellite instability, is present in rat mammary carcinoma cell lines and human breast cancer cell lines. A:T to C:G transversions, which are generally rare, were frequently observed in two rat mammary carcinoma cell lines and in their primary carcinomas, and were considered to be related to the molecular mechanism of SNI. In this study, two known molecular mechanisms that cause increases of A:T to C:G transversions, inactivation of the MutT mammalian homologue (Mth1) gene and overexpression of the DNA polymerase k (Pol k) gene, were analyzed in two rat mammary carcinoma cell lines and 11 rat primary carcinomas. PCR-SSCP analysis revealed no mutations in the entire Mth1 coding region. Quantitative real-time RT-PCR analysis showed that Mth1 mRNA expression was slightly, but significantly, increased in the primary carcinomas (P = 0.001 using GAPDH for normalization, and P = 0.002 using histone H4, t-test), contrary to our expectation, and was decreased to 1 / 2 in the cell lines. The expression of Pol k, which is known to be error-prone with frequent A:T to C:G transversions, was rather decreased in the cell lines and primary carcinomas. Inactivation of Mth1 and overexpression of Pol k were unlikely to have caused SNI in the two rat mammary carcinoma cell lines with a high frequency of A:T to C:G transversions, and searching for other unknown molecular mechanisms is important.
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PMID:The absence of Mth1 inactivation and DNA polymerase kappa overexpression in rat mammary carcinomas with frequent A:T to C:G transversions. 1203 45

To examine the expression of laminin 5 genes (LAMA3, LAMB3, and LAMC2) encoding the three polypeptide chains alpha3, beta3, and gamma2, respectively, in human keratinocytes, we developed novel quantitative polymerase chain reaction (PCR) methods utilizing Thermus aquaticus DNA polymerase, specific primers, and fluorescein-labeled probes with the ABI PRISM 7700 sequence detector system. Gene expression levels of LAMA3, LAMB3, and LAMC2 and glyceraldehyde-3-phosphate dehydrogenase were quantitated reproducibly and sensitively in the range from 1 x 10(2) to 1 x 10(8) gene copies. Basal gene expression level of LAMB3 was about one-tenth of that of LAMA3 or LAMC2 in human keratinocytes, although there was no clear difference among immunoprecipitated protein levels of alpha3, beta3, and gamma2 synthesized in radio-labeled keratinocytes. Human serum augmented gene expressions of LAMA3, LAMB3, and LAMC2 in human keratinocytes to almost the same extent, and this was associated with an increase of the laminin 5 protein content measured by a specific sandwich enzyme-linked immunosorbent assay. These results demonstrate that the absolute mRNA levels generated from the laminin 5 genes do not determine the translated protein levels of the laminin 5 chains in keratinocytes, and indicate that the expression of the laminin 5 genes may be controlled by common regulation mechanisms.
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PMID:Quantitative analysis of laminin 5 gene expression in human keratinocytes. 1585 26

Synthetic DNA templates were compared with authentic cDNA templates as standards for the real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The single-stranded DNA template used here targeted the multidrug resistant transporter P-glycoprotein/MDR1. The double-stranded DNA template, targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was synthesized using an exonuclease-free large fragment E. coli DNA polymerase I. The human colon carcinoma cell line Caco-2 and human duodenum biopsies were used to prepare the authentic cDNA templates. The standard lines were comparable for the synthetic DNA templates and authentic cDNA templates. Long-term cryopreservation at -80 degrees C resulted in the destabilization of the synthetic single-stranded DNA template compared with the authentic cDNA templates in the case of MDR1, whereas for GAPDH, the stability of the synthetic double-stranded DNA template was comparable with that of the authentic cDNA templates. Even for the synthetic DNA templates, repetitive freeze-thawing resulted in destabilization, especially at lower concentrations, and degradation products might have interfered with the RT-PCR's efficiency. The synthetic DNA templates are better than the authentic cDNA templates, but more than 5 cycles of repetitive freeze-thawing should be avoided.
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PMID:Comparison of synthetic DNA templates with authentic cDNA templates in terms of quantification by real-time quantitative reverse transcription polymerase chain reaction. 1650 61


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