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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytochrome P450 (CYP) 2C9 catalyses the metabolism of a wide range of drugs. Previous studies have shown the differences in the amino acid composition among CYP2C9 variants at Cys144/Arg, Tyr358/Cys, Leu359/Ile, and Gly417/Asp. PCR-endonuclease digestion methods have been developed to detect these four possible polymorphisms. The T416-->C mutation in exon 3 of CYP2C9 (Cys144-->Arg) creates an Ava II site. In the 135 subjects we tested, all leukocyte DNA samples showed a complete Ava II digestion indicating homozygous C416 (Arg144). A Tyr358-->Cys mutation will create a Nsi I site at codon 1057-1063 in exon 7. In 40 subjects tested, all samples showed negative results. DNA sequencing on a few samples showed Tyr358Ile359. A mismatched PCR primer pair was then designed to detect codon C1061-->A (Leu359-->Ile) mutation. In 115 subjects tested, 111 samples showed a complete Nsi I digestion (Ile359) and four samples showed heterozygous results. Another mismatched PCR primer pair was used to confirm the C1061 codon in heterozygous subjects. The four heterozygous subjects showed partial digestion with endonuclease Kpn I, which confirmed the heterozygous Ile/Leu at amino acid 359. The G1236-->A mutation in exon 8 of CYP2C9 (Gly417-->Asp) creates a Hph I site. In all 46 subjects, homozygous G1236 (Gly417) was found. Most Chinese subjects actually have Arg144 Tyr358 Ile359 Gly417 in CYP2C9 as previously reported human-2. Furthermore, we found an A-->T (+12 position in intron 2) mutation in our CYP2C9 sequencing process. The mutation creates a NIa III site.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Detection of CYP2C9 polymorphism based on the polymerase chain reaction in Chinese. 1196 81

The expression of Lewis fucosyltransferase (FT) mRNA was examined in gastric mucosa from two Lewis-positive [Le(+)] and two Lewis-negative [Le(-)] individuals. Northern blot analysis demonstrated that levels of mRNA were similar in both Le(+) and Le(-) gastric mucosa. We isolated the protein-coding region of the Lewis FT cDNA from Le(+) and Le(-) gastric mucosa by polymerase chain reaction (PCR) amplification. The sequence of cDNA from the Le(-) gastric mucosa shows two single-base substitutions of G for T at position 59 and of A for G at position 508 from the A of the initiation codon of cDNA. These substitutions may be the cause of changes in two amino acid residues, Arg for Leu at position 20 and Ser for Gly at position 170 from the N-terminal. To determine whether either or both of these base substitutions is responsible for the Le(-) gene, we constructed chimera cDNAs and expressed them in COS cells. Those COS cells transfected with a chimera cDNA containing a mutation of the 508th nucleotide did not express Lewis antigen, whereas those cells transfected with a chimera cDNA containing the 59th nucleotide mutation expressed Lewis antigen, indicating that a single-base change from G to A at position 508 is responsible for the Le(-) gene. The G to A transition at position 508 created a new site for PvuII endonuclease. The digestion by PvuII endonuclease of PCR products between the 386th and 612th nucleotides of Lewis FT cDNA from one of the Le(-) individuals proved to be homozygous for the PvuII site. However, the other Le(-) individual was heterozygous for the PvuII site, suggesting the presence of other Le(-) allele(s). Thus, we isolated one of the silent Lewis genes (le).
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PMID:Analysis of Lewis fucosyltransferase genes from the human gastric mucosa of Lewis-positive and -negative individuals. 821 40

Tap-1 and Tap-2 genes code for a heterodimeric peptide transporter required for the normal maturation and surface expression of class I molecules. Polymorphic variants of these MHC encoded genes occur in rats and humans. After failing to amplify a 3' polymerase chain reaction (PCR) product from thymic and splenic cDNA of the nonobese nondiabetic (NON) strain, we considered it possible that Tap-1 polymorphism was present, since cDNA from CBA/J, C57BL/6, BALB/c, and NOD (nonobese diabetic) mice all yielded Tap-1 3' products. Overlapping PCR fragments spanning the highly conserved ATP-binding cassette (ABC) were generated for purposes of restriction endonuclease analysis, studies of IFN-gamma regulation, and sequencing. To avoid amplifying other members of the transporter family, we used a gel-purified 1670-bp Tap-1 PCR "long product" as template for nested PCR. Sequencing revealed three polymorphic alleles. The most divergent was for the NON strain and involved two non-conserved amino acid substitutions (Arg-->Cys397 and Leu-->Arg491) and three silent mutations. NON mice show an abnormal pattern of class I (Kb) expression and a sizeable reduction in the percentage of CD8+ cells in the blood and thymus. In F2 segregants, the low CD8 phenotype mapped to the MHC. Tap-1 genes of NON and C57BL/6 mice were equally sensitive to up-regulation by IFN-gamma. We conclude that the mouse Tap-1 transporter gene, like the Tap-2 of the rat and the Tap-1 and Tap-2 of the human, is polymorphic. The extensive variation and specific codon changes of Tap-1 in the NON mouse raise the possibility that this gene is the MHC locus responsible for altering the intrathymic development of CD8+ T cells.
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PMID:Polymorphism in the mouse Tap-1 gene. Association with abnormal CD8+ T cell development in the nonobese nondiabetic mouse. 822 29

Nitric oxide is a free radical (NO) formed biologically through the oxidation of L-arginine by nitric oxide synthases. NO is produced transiently in mammalian cells for intercellular signaling and in copious quantities to cause cytostasis and cytotoxicity. In the latter situation, NO is a deliberate cytotoxic product of activated macrophages, along with other reactive oxygen species such as hydrogen peroxide (H2O2) and superoxide (O2-). Escherichia coli has a complex set of responses to H2O2 and O2- that involves approximately 80 inducible proteins; we wondered whether these bacteria might induce analogous defenses against nitric oxide. We show here that a multigene system controlled by the redox-sensitive transcriptional regulator SoxR is activated by NO in vivo. This induction confers bacterial resistance to activated murine macrophages with kinetics that parallel the production of NO by these cells. Elimination of specific SoxR-regulated genes diminishes the resistance of these bacteria to the cytotoxic macrophages. The required functions include manganese-containing superoxide dismutase, endonuclease IV (a DNA-repair enzyme for oxidative damage), and micF, an antisense regulator of the outer membrane porin OmpF. These results demonstrate that SoxR is a sensor for cellular exposure to NO, and that the soxRS response system may contribute to bacterial virulence.
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PMID:Activation by nitric oxide of an oxidative-stress response that defends Escherichia coli against activated macrophages. 823 47

In the fibrinogen molecule, a total of seven sites have been tentatively identified as polymorphic; however, disagreements about these sites have been observed among the various protein and DNA sequence data published. To allow examination of the potential polymorphic sites at the DNA level, human genomic DNA samples were prepared from 110 unrelated, healthy individuals. Either allele-specific polymerase chain reaction (ASPCR) amplification or PCR amplification followed by restriction endonuclease digestion was used to detect the presence of possible polymorphisms. Two polymorphic sites were confirmed, one at A alpha 312 (Thr/Ala) by RsaI restriction analysis, and a second at B beta 448 (Arg/Lys) by MnlI restriction analysis. Mendelian inheritance of both polymorphisms was demonstrated and allele frequencies were estimated as 0.76/0.24 and 0.85/0.15 for the A alpha 312 and B beta 448 sites, respectively. The sites at A alpha 47, A alpha 296, B beta 162, B beta 296, and gamma 88 showed no evidence of variation in any of our samples. The amino acid polymorphisms at A alpha 312 and B beta 448 reflect conservative residue changes with unknown effects on fibrinogen structure or function. An additional, previously unrecognized DNA sequence variant was detected in a single individual in the second intron of the A alpha chain using HinfI restriction analysis.
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PMID:Human fibrinogen polymorphic site analysis by restriction endonuclease digestion and allele-specific polymerase chain reaction amplification: identification of polymorphisms at positions A alpha 312 and B beta 448. 840 Feb 61

The gene I protein (pI) of the f1 filamentous bacteriophage is a non-capsid protein that is required for the assembly of the bacteriophage. It spans the Escherichia coli inner membrane once with its amino terminus in the cytoplasm and its carboxyl-terminal portion in the periplasm. The presence of moderate amounts of this protein in the membrane results in rapid inhibition of cell growth, probably from a loss of membrane potential. Previous observations defined a 55-amino-acid sequence within pI required for its membrane insertion which includes a 20-residue hydrophobic stretch preceded by a 13-residue positively charged amphiphilic helix. To define the minimal sequence required for membrane translocation and for growth inhibition, a deletion analysis was performed on a tripartite fusion construct containing the 55-residue pI sequence flanked upstream by the amino-terminal portion of EcoRI endonuclease and downstream by the enzymatic portion of alkaline phosphatase. Only the 20-residue hydrophobic stretch immediately preceded by 1 arginine residue is required for membrane insertion of the fusion proteins. This region also sufficed to inhibit cell growth provided it contained protein domains exposed in both the cytoplasm and periplasm. It was not possible to separate the domains required for membrane insertion and cell growth inhibition. No requirement for the positively charged amphiphilic helix was detected either for membrane insertion or growth inhibition, suggesting that it plays a role in phage assembly and not membrane insertion.
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PMID:The membrane domain of a bacteriophage assembly protein. Membrane insertion and growth inhibition. 844 12

The frequency in human genomic DNA of three human interferon (IFN) alpha genes which differ from each other by a single base substitution was examined in 11 normal individuals. The polymerase chain reaction (PCR) was used to amplify the coding sequence for the Hu-IFN-alpha 2, Hu-IFN-alpha A, and Hu-IFN-alpha 2(Arg) sequences from genomic DNA. The PCR products were then cloned and individual clones were sequenced. PCR products were also analyzed by restriction endonuclease analysis for the IFN-alpha 2 and IFN-alpha A genes by use of a HinfI site which is eliminated by the substitution of an A for a G in IFN-alpha A. The IFN-alpha A gene which was cloned from the myeloblastoid cell line KG-1 was not observed in any of the 201 clones sequenced from normal individuals or in the Namalwa cell line. It was detected in KG-1 cell genomic DNA where it represented 49% of the clones sequenced. Similarly the IFN-alpha 2(Arg) gene was not detected in normal individuals or in the KG-1 cell line but only in the lymphoblastoid cell line from which it was cloned. In Namalwa cells the IFN-alpha 2(Arg) sequence represented 35% of the clones sequenced while the IFN-alpha 2 sequence comprised 59%. Therefore, both the IFN-alpha A and IFN-alpha 2(Arg) sequences represent alleles of the IFN-alpha 2 gene.
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PMID:Human interferon-alpha A, -alpha 2, and -alpha 2(Arg) genes in genomic DNA. 850 97

Three novel point mutations were detected in the glucocerebrosidase gene of three unrelated Gaucher's disease patients by direct sequencing of PCR products. The first is a C to G change at position 4263 in the genomic sequence (exon 7) which results in a proline to arginine change at position 266 in the mature enzyme (P266R). The second is a G to C change at position 5276 in the genomic sequence (exon 8) which results in an aspartic acid to histidine change at position 315 (D315H). The third is a C to A change at position 5286 in the genomic sequence (exon 8) which results in an alanine to aspartic acid change at position 318 (A318D). The first mutation destroys an AvaII restriction endonuclease site, the second creates a BspMI site and the third creates a BamH I site.
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PMID:Three unrelated Gaucher's disease patients with three novel point mutations in the glucocerebrosidase gene (P266R, D315H and A318D). 854 70

Animal studies and cell culture experiments demonstrated that posttranscriptional editing of the transcript of the GluR-2 gene, resulting in substitution of an arginine for glutamine in the second transmembrane region (TM II) of the expressed protein, is associated with a reduction in Ca2+ permeability of the receptor channel. Thus, disturbances in GluR-2 RNA editing with alteration of intracellular Ca2+ homeostasis could lead to neuronal dysfunction and even neuronal degeneration. The present study determined the proportions of edited and unedited GluR-2 RNA in the prefrontal cortex of brains from patients with Alzheimer's disease, in the striatum of brains from patients with Huntington's disease, and in the same areas of brains from age-matched schizophrenics and controls, by using reverse transcriptase-polymerase chain reaction, restriction endonuclease digestion, gel electrophoresis and scintillation radiometry. In the prefrontal cortex of controls, < 0.1% of all GluR-2 RNA molecules were unedited and > 99.9% were edited; in the prefrontal cortex both of schizophrenics and of Alzheimer's patients approximately 1.0% of all GluR-2 RNA molecules were unedited and 99% were edited. In the striatum of controls and of schizophrenics, approximately 0.5% of GluR-2 RNA molecules were unedited and 99.5% were edited; in the striatum of Huntington's patients nearly 5.0% of GluR-2 RNA was unedited. In the prefrontal white matter of controls, approximately 7.0% of GluR-2 RNA was unedited. In the normal human prefrontal cortex and striatum, the large majority of GluR-2 RNA molecules contains a CGG codon for arginine in the TMII coding region; this implies that the corresponding AMPA receptors have a low Ca2+ permeability, as previously demonstrated for the rat brain. The process of GluR-2 RNA editing is compromised in a region-specific manner in schizophrenia, in Alzheimer's disease and Huntington's Chorea although in each of these disorders there is still a large excess of edited GluR-2 RNA molecules. Disturbances of GluR-2 RNA editing leading to excessive Ca2+ permeability, may contribute to neuronal dysfunction in schizophrenia and to neuronal death in Alzheimer's disease and Huntington's disease.
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PMID:Editing for an AMPA receptor subunit RNA in prefrontal cortex and striatum in Alzheimer's disease, Huntington's disease and schizophrenia. 861 34

The role of G protein mutations in the pathogenesis of adrenal cortex neoplasms is controversial. Two published studies disagree on the existence of a cysteine or histidine for arginine substitution at position 179 (R179C/H) of the GTP binding region of the alpha chain of an inhibitory G protein (Gi2alpha) in these tumors. Prior studies using detection by mutation-specific oligonucleotide hybridization showed either 3 of 11 or 0 of 56 tumors harbored mutations. To resolve this discrepancy and ascertain the importance of the R179C/H Gi2alpha mutation in the development of adrenal cortex tumors, we screened tumors from 29 patients (24 with adenoma, 5 with carcinoma) using a more sensitive assay employing polymerase chain reaction (PCR) and examination for restriction fragment length polymorphisms (RFLP). Detection of the potential R179C/H mutation by this technique was possible because the wild-type coding sequence includes the BSTU-1 restriction endonuclease recognition site CGCG, whereas the mutated gene does not. Results showed complete digestion of the amplified DNA samples from all 29 patients and the negative control DNA by BSTU-1, indicating that all tumor samples exhibited only the wild-type sequence. Direct sequencing of PCR product from four tumor samples confirmed the presence of only the wild-type sequence. The 0 of 29 rate of R179C/H mutations we found in Gi2alpha is different than the 3 of 11 positive rate (p < 0.05, Fishers' exact) previously reported but agrees with the report showing 0 of 56 mutations. We conclude a mutation at position 179 of Gi2alpha is not important in the pathogenesis of most adrenal cortical tumors.
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PMID:Gip-2 codon 179 oncogene mutations: absent in adrenal cortical tumors. 867 43


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