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)

Every bulky lesion in DNA can potentially inhibit the Taq DNA polymerase and thereby decrease the amplification produced in the polymerase chain reaction. We investigated the feasibility of using this inhibition to quantify DNA lesions produced by the anticancer drug cisplatin. Products were detected by electrophoresis followed by ethidium bromide staining. Quantitation was obtained by including [32P]dCTP in the amplification reaction and subsequently assessing the incorporated radioactivity. Hamster genomic DNA was platinated in vitro to defined levels and amplified with primers that produce either a 150, 750 or 2,000 base pair fragment. The degree of inhibition of PCR agreed with the predicted level of DNA platination in each size of fragment, suggesting that the polymerase was inhibited by every cisplatin-induced lesion. This method was used to detect cisplatin-induced lesions in the adenine phosphoribosyltransferase gene of CHO cells. Cells were incubated with 0-125 microM cisplatin for 2 h, the DNA was purified and subjected to PCR. A significant decrease in amplification of the 2 kbp fragment was observed in DNA from cells incubated with cisplatin at 75 microM. The degree of inhibition agreed closely with the amount of DNA damage in the overall genome as measured by atomic absorption. No change was detected in amplification of the 150 base fragment which can therefore be used to normalize data for any variations between DNA samples. This assay has the same sensitivity as other methods currently used for the analysis of gene-specific damage. The advantage of this assay is that it obviates the need for specific endonuclease complexes to recognize and cleave DNA adducts as previously required when analyzing damage in specific genomic sequences.
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PMID:A polymerase chain reaction-based method to detect cisplatin adducts in specific genes. 195 80

Using the polymerase chain reaction (PCR) with Taq DNA polymerase, we have amplified a 2.4-kb fragment of genomic DNA containing the adenine phosphoribosyltransferase (APRT) gene from patients with APRT deficiency. Several clones from each patient were sequenced after subcloning the PCR product into M13mp18. Selected regions of the amplified fragment were also sequenced directly. This enabled us to distinguish PCR-induced errors from endogenous mutations and polymorphisms in each clone. 44 PCR errors were found in a total of 57,94 kb of DNA sequenced from 25 clones from 7 patients. All the errors were due to the PCR process and not to subcloning, as shown by sequence analysis of 5 APRT-positive clones isolated from a phage genomic library.
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PMID:Polymerase chain reaction amplification and sequence analysis of human mutant adenine phosphoribosyltransferase genes: the nature and frequency of errors caused by Taq DNA polymerase. 206 30

Six mutations, impairing DNA polymerase of E. coli in combination with the wild type gene for rho factor or ts-mutation rho 15 have been studied in relation to the expression of seven operons having different types of regulation. The expression of genes for glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase is shown to be constitutive and resistant to mutationally altered RNA polymerase and rho factor. The expression of genes for adenine phosphoribosyltransferase and of deo operon is regulated by rho dependent attenuators with attenuation being lifted incomplete medium. Mutation rho 15 decreases the level of enzymes of thr and lac operons independent of mRNA levels of these operons. Mutation rho 15 effect on posttranscriptional level is modified by mutations damaging RNA polymerase. The data obtained suppose RNA polymerase to affect all stages of realization of genetic information, beginning with promoter recognition and RNA synthesis and including the protein synthesis on mRNA.
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PMID:[Effect of mutation changes in RNA-polymerase and transcription termination factor rho on expression of various operons in E. coli]. 302 82