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

A DNA technique for in situ hybridization developed by Kumar & Collins (1994) for use on polytene chromosomes of adult Anopheles mosquitoes (Diptera: Culicidae) was modified for use with Simulium larval salivary gland chromosomes (Diptera: Simuliidae). Cloned fragments of several Simulium genes (coding for aspartate amino transferase, cytochrome P450 and DNA polymerase) were successfully mapped physically by assigning specific band locations in Simulim sanctipauli V. & D. This represents the first attempt at locating genes beyond the resolution of linkage to inversions in any blackfly species.
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PMID:DNA in situ hybridization on polytene chromosomes of Simulium sanctipauli at loci relevant to insecticide resistance. 1087 68

The formation of DNA adducts in human HepG2 cells and human hepatocytes exposed to 14C-labelled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was examined using Accelerator Mass Spectrometry (AMS). PhIP generated DNA adducts in a linear dose-dependent manner between 100 pM and 20 micro M. Co-treatment with the dietary isothiocyanate, sulforaphane (SFN, 1-10 micro M), or the flavonoid, quercetin (5-20 micro M), significantly reduced the level of PhIP-DNA adducts in a dose-dependent manner. The degree of protection was dependent on PhIP concentration, i.e. after 100 pM PhIP exposure, SFN or quercetin reduced adduct levels to below the limit of detection (0.15 amol PhIP/ micro g DNA) but at higher PhIP exposure (10 nM and 1 micro M), the protection was 60 and 10%, respectively. The involvement of phase I, phase II and DNA repair enzymes in this protection against PhIP-DNA adduct formation was investigated using real-time RT-PCR and enzyme activity assays. In intact HepG2 cells, quercetin inhibited cytochrome P450 (CYP)1A2, the main phase I enzyme responsible for PhIP bioactivation. In contrast, SFN induced phase II detoxification enzymes, UDP-glucuronosyltransferase 1A1 and glutathione S-transferase A1 mRNA expression. SFN and quercetin showed no effect on DNA repair, neither in terms of the level of PhIP-DNA adducts, when cells were treated with phytochemicals after the carcinogen exposure, nor the regulation of mRNA expression of two DNA repair enzymes, apurinic endonuclease and DNA polymerase beta. This study indicates that dietary isothiocyanates and flavonoids modulate phase I and phase II enzyme expression, hence increasing the rate of detoxification of the dietary carcinogen PhIP in human HepG2 cells but do not affect the rate of PhIP-DNA adduct repair. The formation of PhIP-DNA adducts in human hepatocytes was also dose-dependent with PhIP-concentration and the levels of protection by SFN or quercetin were up to 60% after 10 nM PhIP treatment, but showed large inter-individual variation with no observed protection in some individuals.
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PMID:Sulforaphane and quercetin modulate PhIP-DNA adduct formation in human HepG2 cells and hepatocytes. 1294 46

Primer extension reaction (PEXT) is the most widely used approach to genotyping of single nucleotide polymorphisms (SNP). It is based on the high accuracy of nucleotide incorporation by the DNA polymerase. We propose a dual-analyte bio/chemiluminometric method for the simultaneous detection of the PEXT reaction products of the normal and mutant allele in a high sample-throughput format. PCR-amplified DNA fragments that span the SNP of interest are subjected to two PEXT reactions using normal and mutant primers in the presence of digoxigenin-dUTP and biotin-dUTP. Both primers contain a d(A)30 segment at the 5'-end but differ in the final nucleotide at the 3'-end. Under optimized conditions only the primer that is perfectly complementary with the interrogated DNA will be extended by DNA polymerase and lead to a digoxigenin- or biotin-labeled product. The products of the PEXT reactions are mixed, denatured, and captured in microtiter wells through hybridization with immobilized oligo(dT) strands. Detection is performed by adding a mixture of antidigoxigenin-alkaline phosphatase (ALP) conjugate and a streptavidin-aequorin conjugate. The flash-type bioluminescent reaction of aequorin is triggered by the addition of Ca2+. ALP is then measured by adding the appropriate chemiluminogenic substrate. The method was evaluated by genotyping two SNPs of the human mannose-binding lectin gene (MBL2) and one SNP of the cytochrome P450 gene CYP2D6. Patient genotypes showed 100% concordance with direct DNA sequencing data.
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PMID:Genotyping of single nucleotide polymorphisms by primer extension reaction and a dual-analyte bio/chemiluminometric assay. 1755 27

This laboratory investigation was carried out at the Faculty of Sciences, Mahidol University, Thailand during October 2007 to May 2009. The objectives of this study include: the search for heterologous expression of the cytochrome P450 CYP6AA3 enzyme of the Anopheles minimus mosquitoes in relation to Malaria disease and to provide some information on molecular mechanism of insects' pyrethroid resistance. The polymerase chain reaction aided by the Pfu DNA polymerase and some specific generated primers were used to modify the CYP6AA3 gene. The PCR product was ligated with a predigested pET-3a at the NdeI and BamHI restriction sites. The modified CYP6AA3 enzyme was expressed in the Escherichia coli BL21 (DE3) pLysS in order to achieve a high amount of soluble form of its expression. The results showed that the use of the isopropyl-beta-D-thiogalactopyranoside (IPTG) and incubation together with ferric chloride and delta-aminolevulinic acid did not increase any soluble form of the CYP6AA3 enzyme. A significant amount of soluble enzyme was produced upon the replacement of the 30 N-terminal residues with a short peptide where it gave Ldelta30CYP6AA3 protein and after purification process was taken place, it yielded up to 10.64 mg 10 L(-1) or approximately 1 mg L(-1) of the homogenous Ldelta30CYP6AA3. When this purified Ldelta30CYP6AA3 protein was used in a metabolizing process with the cypermethrin, deltamethrin and permethrin substrates, it gave their apparent Km values for cypermethrin and deltamethrin of 12.5 and 23.5 microM, respectively. The heterologous expression carried out with the use of the E. coli gave a high amount of soluble CYP6AA3 enzyme of the An. minimus mosquitoes hence the modified technique being used was successfully achieved.
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PMID:An expression of an insect membrane-bound cytochrome P450 CYP6AA3 in the Escherichia coli in relation to insecticide resistance in a malarial vector. 2193 50