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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Efficiencies of insertion and extension at a single site-directed abasic lesion, X, were measured while varying 5'- and 3'-template bases adjacent to X. The preference for insertion was found to be A > G > T approximately C, with the "upstream" (3'-neighboring) template base perturbing insertion efficiencies by an order of magnitude or more. Efficiencies of synthesis past the abasic lesion depended strongly on the "downstream" (5'-neighboring) template base and on the properties of the polymerase. HIV-1 RT favored "direct" extension of X.A > X.G > X.T > X.C, by addition of the next correct nucleotide. However, it was found that X.C, least favored for direct extension, was most favored for "misalignment" extension, occurring when the DNA structure in the vicinity of the lesion collapsed to realign a primer 3'-C terminus opposite a downstream template G site. Polymerase properties have an important role in copying abasic lesions. Drosophila DNA polymerase alpha, HIV-1, and AMV reverse transcriptases had "little" difficulty inserting opposite abasic lesions, with efficiencies comparable to misinsertions opposite normal template bases. However, AMV RT did not extent past the lesion using direct or misalignment mechanisms. Wild-type and mutant T4 DNA polymerases were used to show that although exonucleolytic proofreading inhibits lesion bypass, the presence of a highly active proofreading exonuclease is not sufficient to prevent bypass.
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PMID:Nucleotide insertion and primer extension at abasic template sites in different sequence contexts. 809 71

The ability of thermostable DNA polymerases to mediate template-dependent DNA synthesis in the presence of phenol has been examined as monitored by amplification of a specific Borrelia burgdorferi rRNA sequence. Tth DNA polymerase displayed the unique property of maintaining both DNA- and RNA-dependent DNA polymerase activities in the presence of 2%-5% (vol/vol) of phenol-saturated PBS buffer. Tth DNA polymerase mediated reverse transcriptase activity was unaffected by phenol-saturated phosphate-buffered saline concentrations as high as 15% (vol/vol). By contrast, Taq DNA Polymerase was inactive under these conditions. The ability to function in the presence of phenol can greatly simplify reverse transcriptase, PCR and reverse transcription-PCR protocols since the phenol-saturated aqueous phase of a phenol partition can be added directly to the reaction mixtures. The simplicity of the procedures described should have applicability to a broad range of basic research, clinical and forensic applications.
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PMID:A distinctive property of Tth DNA polymerase: enzymatic amplification in the presence of phenol. 813 48

Polymerase chain reaction (PCR) was used to identify Rickettsia prowazekii, the etiologic agent of epidemic typhus. For the PCR, Thermus thermophilus thermostable DNA polymerase was applied with buffer containing a relatively low Mg2+ concentration (1.5-2 mM with dNTP's at 250 microM each). A primer pair used to amplify a 448-base-pair (bp) fragment of R. prowazekii genome was synthesized on the basis of the DNA sequence of gene rpa14/16, coding for a precursor of the mature polypeptides of molecular weight (Mr) 14,000 and/or 16,000 (16kD) from R. prowazekii strain E. For determining the specificity of the primer pair, purified genomic DNAs of 16 rickettsial and 10 other bacterial strains were used.
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PMID:Identification of Rickettsia prowazekii using the polymerase chain reaction. 815 68

Human herpesvirus 6 (HHV-6) is a recently described T-cell pathogen whose medical relevance and molecular biology are just beginning to be addressed. As a first look at the regulation of viral genes, control of the HHV-6 DNA polymerase promoter was examined. Polymerase gene transcription in HHV-6-infected cells was found to initiate from a single site located 115 bases upstream of the translation start codon. A polymerase promoter-chloramphenicol acetyltransferase reporter gene construct failed to be expressed in uninfected T cells but was highly active in HHV-6-infected cells. Mutational data indicated that the polymerase promoter is TATA-less. Mutational analysis also revealed that the major upstream promoter regulatory element required for transcriptional activity in HHV-6-infected cells is a palindromic ATF/CREB transcription factor binding site. The significance of this site for promoter induction was further demonstrated by the fact that the polymerase ATF/CREB element, when appended to a heterologous basal promoter, is highly responsive to HHV-6 infection. Two protein complexes were found to bind in a specific manner to the ATF/CREB motif in both uninfected and HHV-6-infected T-cell nuclear extracts. Site-specific mutation of the ATF/CREB site resulted in loss of protein binding as well as loss of promoter activity in HHV-6-infected cells.
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PMID:An ATF/CREB site is the major regulatory element in the human herpesvirus 6 DNA polymerase promoter. 815 67

This replication method, which was introduced in 1985, has been used to find and identify microorganisms in the environment, among others in samples of soil, sediments and waters. A gene or a DNA fragment specific to a microorganism is replicated in vitro by a chain reaction catalyzed by DNA polymerase (PCR: Polymerase Chain Reaction) and analyzed by electrophoretic procedures. At the moment in most legislations bacteriological criteria for drinking water depend on E. coli and other bacteria referring to fecal contamination (fecal coliforms and enterococci). Absence of these bacteria does not necessarily exclude contamination of water with protozoa or virus. Detection of the latter by common methods is difficult and time-consuming. Application of PCR to these purposes is interesting. During the last years several protocols have been developed such as methods for the detection of E. coli, bacteria referring to fecal contamination, pathogens like Legionella pneumophila as well as Salmonella and Shigella, enterovirus and protozoa i.e. Giardia. Compared to the traditional methods an obvious advantage of the new methods lies in their velocity, sensitivity and specificity. This review introduces to several different applications of PCR. Although this method is still restricted to specialized laboratories at the moment, it will gain importance as a complement to traditional methods for the detection of pathogenic microorganisms in water as soon as simple tests will be available.
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PMID:[The use of PCR for detecting pathogenic microorganisms in water]. 820 34

A meiotic DNA polymerase that is present at a high level of activity in meiotic cells of a basidiomycete, Coprinus cinereus, was purified to near homogeneity using synthetic RNA homopolymer [poly(C)] cellulose column chromatography. This report presents the first extensive purification and characterization of any eukaryotic DNA polymerase having a role in meiosis. This enzyme is a single polypeptide with a molecular mass of 65,000. Activity in this enzyme requires magnesium ions and occurs at an optimal pH of 7.5. It is strongly inhibited by dideoxythymidine triphosphate but is relatively insensitive to aphidicolin and N-ethylmaleimide and can use poly(C)/oligo(dG)12-18 as a template-primer. Polymerase activity can be found only in cells at meiotic prophase, even though the enzyme has been identified in somatic cells in an inactive state using immunoblot analysis. Its distinctive distribution makes possible a genetic and biochemical analysis of functional role of a meiotic DNA polymerase in meiotic recombination, repair and synthesis.
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PMID:A meiotic DNA polymerase from Coprinus cinereus: further purification and characterization. 830 25

Two human glioma cell lines (U87MG and U373MG) were evaluated for their thermal enhancement of radiation sensitivity and its correlation to the degree of inactivation of DNA polymerase alpha and beta. The data showed that hyperthermia increased radiation sensitivity in a time- and temperature-dependent manner. The differential heat sensitivity of the two cell lines was reflected in the degree of polymerase inactivation. Polymerase inactivation was also dependent on time and temperature and was greater for polymerase beta than alpha. The degree of polymerase inactivation correlated well with the thermal enhancement ratio (TER) calculated at the 1.0% survival level. This correlation was poor for the TER at the 50% survival level. The correlations were better for polymerase beta than alpha. The small differences in thermal sensitivity between the two cell lines primarily at 41 and 42 degrees C could not be explained by correlation between polymerase inactivation and TER. Incubation between hyperthermia and irradiation resulted in recovery of polymerase activity and loss of radiosensitization. Levels of polymerase beta after hyperthermia may be used to predict thermal enhancement of radiosensitivity for low survival levels, but possibly not in the shoulder region of the radiation survival curve. Small cell line-dependent differences in thermal sensitivity may not be resolved in these comparisons.
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PMID:A comparison of the enhancement of radiation sensitivity and DNA polymerase inactivation by hyperthermia in human glioma cells. 831 26

Pneumocystis carinii pneumonia (PCP) is a leading cause of death among AIDS patients in the United States. Our analysis of P. carinii protein-coding genes has revealed a significant A + T codon bias. Polymerase chain reaction (PCR) was utilized to isolate and identify the genes encoding calmodulin, beta-tubulin, DNA polymerase II, and RNA polymerases I, II and III from P. carinii. Primer pairs were designed to incorporate P. carinii codon preference to known conserved protein regions from other organisms. This strategy should be useful for a large variety of P. carinii genes and assist in the comprehensive analysis of the genomic structure of this important pathogen.
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PMID:Isolation and identification of six Pneumocystis carinii genes utilizing codon bias. 832 3

Polymerase chain reaction (PCR) involves alternate denaturing and re-annealing of DNA in test samples in the presence of appropriate oligonucleotide primers complementary to opposite strands of the target DNA together with a heat-stable DNA polymerase, Mg2+ and the four nucleotide triphosphates. DNA target segments can be 'amplified' ten-millionfold by 25-35 such cycles. Even greater amplification (approximately 10(12)-fold) with enhanced specificity can be obtained by a second set of amplification cycles using a further pair of 'nested' primers sited within the DNA sequence defined by the original primers. PCR can be applied to the study of the whole range of transfusion-transmitted infections, both plasma and cell associated; RNA viruses can be analyzed if a DNA copy is made from the viral RNA by treatment with reverse transcriptase. In a transfusion context, the retroviruses (HIV-1, HIV-2, HTLV-I, HTLV-II), HCV and HBV have been the viruses most intensively subjected to PCR analysis. The advantages of PCR in this context include its ability to detect virus during the 'window period' or seronegative stages of infections and its value as a marker for viraemia and for the detection of viruses in products made from large pools of plasma. True immunity may also be differentiated from persistent infection in the presence of antibody. Similarly, PCR can overcome problems of diagnosis of acute infection caused by the presence of passively transferred antibody. Detailed strain differentiation is also possible by PCR, in conjunction with sequencing or with the aid of restriction endonucleases.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Polymerase chain reaction and transfusion microbiology. 838 93

Polymerase chain reaction was used to detect herpes simplex virus (HSV) specific deoxyribonucleic acid (DNA) sequences in acute and convalescent cerebrospinal fluid (CSF) and brain tissue of a 78-year-old man and in CSF of a neonate who died of complications owing to herpes simplex virus encephalitis (HSVE). Polymerase chain reaction (PCR) was carried out for 35 cycles with a set of primers that bracketed a 92 base pair segment unique to the HSV DNA polymerase gene. Amplified DNA was electrophoresed on 3 percent agarose gel, blotted onto a nylon membrane, and probed with 32p-labeled oligonucleotide internal to the primers. The HSV specific DNA sequences were detected in the specimens from both patients. No HSV specific DNA was detected in CSFs from 20 patients with suspected Lyme disease or neurosyphilis. Polymerase chain reaction is a rapid and noninvasive technique for the diagnosis of HSVE.
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PMID:Use of polymerase chain reaction for laboratory diagnosis of herpes simplex virus encephalitis. 839 76


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