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)

Size separation after UV-endonuclease digestion of DNA from UV-irradiated human cells using denaturing conditions fractionates the genome based on cyclobutane pyrimidine dimer content. We have examined the largest molecules available (50-80 kb; about 5% of the DNA) after fractionation and those of average size (5-15 kb) for content of some specific genes. We find that the largest molecules are not a representative sampling of the genome. Three contiguous genes located in a G+C-rich isochore (tyrosine hydroxylase, insulin, insulin-like growth factor II) have concentrations two to three times greater in the largest molecules. This shows that this genomic region has fewer pyrimidine dimers than most other genomic regions. In contrast, the beta-actin genomic region, which has a similar G+C content, has an equal concentration in both fractions as do the p53 and beta-globin genomic regions, which are A+T-rich. These data show that DNA damage in the form of cyclobutane pyrimidine dimers occurs with different probabilities in specific isochores. Part of the reason may be the relative G+C content, but other factors must play a significant role. We also report that the transcriptionally inactive insulin region is repaired at the genome-overall rate in normal cells and is not repaired in xeroderma pigmentosum complementation group C cells.
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PMID:Identification of a large genomic region in UV-irradiated human cells which has fewer cyclobutane pyrimidine dimers than most genomic regions. 748 Jan 36

Ribonuclease H (RNase H) is an endonuclease that cleaves only the RNA strand of an RNA-DNA hybrid to produce 5'-phosphate and 3'-hydroxy termini and lacks useful sequence specific recognition properties. A mutant form of the E. coli enzyme has been prepared that is suited for selective chemical modification at a site proximal to the substrate binding region. The chemical derivatization involves the formation of a disulfide linkage to a modified octadeoxyribonucleotide. The conjugate retains only 0.3% of the normal sequence independent RNase H activity demonstrating that substrate recognition can be modulated by a covalent appendage. A beta-globin RNA transcript containing a sequence complementary to that of the octadeoxyribonucleotide was cleaved in a catalytic fashion to two products upon treatment with the conjugate. The selectivity in the phosphodiester bond cleavage mediated by the conjugate was found to be different than that displayed by the nonderivatized enzyme. These results demonstrate the potential of semi-synthetic RNase H conjugates for mechanistic studies and their application as RNA targeted diagnostic or therapeutic agents.
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PMID:Sequence specific cleavage of messenger RNA by a modified ribonuclease H. 752 7

Coelomic fluid, placental tissue and maternal blood were collected at 7-10 weeks gestation from each of 58 women undergoing elective termination of pregnancy for psychological indications. In all samples, a 364 bp fragment of the human beta-globin gene spanning positions -23 to 341 was amplified. The restriction endonuclease Ddel was used to detect the sickle mutation which abolishes its restriction site. beta-Globin DNA was successfully amplified from all samples. In 53 cases a normal maternal beta-globin genotype was detected. In three out of five cases, where the maternal haemoglobin phenotype was HbAS, heterozygosity for the sickle mutation was demonstrated on analysis of coelomic fluid. In the remaining two cases a normal beta-globin genotype was observed. Three further coelomic fluid samples were found to be heterozygous for the sickle mutation. In these instances the maternal haemoglobin phenotype was normal, indicating paternal transmission of the sickle gene. The results of the present study have established that the diagnosis of sickle cell anaemia, and potentially other human single gene disorders, is feasible by coelocentesis.
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PMID:Detection of sickle gene by coelocentesis in early pregnancy: a new approach to prenatal diagnosis of single gene disorders. 765 81

Hemoglobin (Hb) M-Saskatoon, a beta variant of methemoglobin, is characterized by mild hemolysis. It is caused by the substitution of a histidine by a tyrosine at the 63rd amino acid residue of the beta-globin chain. Amplification and sequence analysis of genomic beta-globin DNA from an Indonesian boy diagnosed as having the more severe disease thalassemia demonstrated the presence of a C to T transition at nucleotide 473 in one of the two beta-globin genes resulting in a histidine to tyrosine substitution at 63rd residue. This amino acid change matched with that reported in Hb M-Saskatoon. This nucleotide change abolished a recognition site for the restriction endonuclease NlaIII. NlaIII digestion of the corresponding beta-globin DNA amplified from the patient's parents indicated that the mutation was inherited through from his mother. This result shows that the world-wide distribution of Hb M-Saskatoon extends to Indonesia, where it was not previously identified. Possible causes of the unusually severe symptoms observed in the case are discussed.
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PMID:C to T transition at the first nucleotide of codon 63 of the beta-globin gene corresponding to hemoglobin M-Saskatoon in an Indonesian boy. 766

Using a combination of oligonucleotide probes and restriction endonuclease enzymes, we characterize beta-thalassemic mutations in 91 homozygous patients and 86 unrelated carriers. Overall, 268 beta-thalassemic genes were obtained. Eleven beta-globin mutations were identified, confirming the wide molecular heterogeneity of beta-thalassemia in Calabria. Information from the present study represents the mainstay for the development of a program of early prenatal diagnosis by direct detection of mutations in Calabria.
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PMID:Spectrum of beta-thalassemia mutations in Calabria: implications for prenatal diagnosis. 784 28

Characterization of beta-thalassemia mutations was attempted for 13 unrelated Japanese patients heterozygous for beta-thalassemia. We have systematically analyzed beta-thalassemia genes using polymerase-chain-reaction-related techniques; dot blot hybridization with oligonucleotide probes complementary to known mutations, restriction endonuclease assay and direct sequencing of amplified genomic DNA. Seven different mutations were detected. Six of them are an amber mutation in codon 90 (GAG to TAG), a four-base-pair deletion in codons 41 and 42 causing premature termination due to frameshift, a C-T substitution at position 654 of IVS-2, a G-A substitution at position 1 of IVS-2 and a C-G substitution at position 848 of IVS-2, leading to splicing defects, and an ocher mutation (GAA-TAA) in codon 121 causing a thalassemia intermedia phenotype with inclusion body formation in erythrocytes. A silent mutation (CTG-TTG) was also detected in codon 91 of the allele with the IVS-2 position 1 mutation. These mutations have been reported previously in the Japanese population. The other mutation is a novel one in the Japanese, an amber mutation (TGG-TAG) in codon 15, causing a beta zero-thalassemia phenotype by premature termination of the beta-globin chain synthesis. We analyzed haplotypes of chromosomes bearing each beta-thalassemia mutation. Origins and a spectrum of mutations in comparison with those detected in malaria-endemic regions are discussed.
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PMID:Molecular basis of beta-thalassemia in Japan: heterogeneity and origins of mutations. 809 35

DNA of yeast artificial chromosomes (YACs) was prepared for microinjection by separation from most of the natural yeast chromosomes on a pulsed-field gel, treatment with agarase, and centrifugation. A salt concentration of 100 mM NaCl was necessary to protect the DNA from shear during these procedures. Injection of a 590-kb YAC, yGART2, into Chinese hamster ovary cells gave rise to cells expressing the 40-kb human GART gene carried on the YAC. Nine of 12 cell lines analyzed contained an intact stretch of at least 110 kb of YAC DNA surrounding the GART gene, and one cell line contained at least 480 kb, but not the entire 590 kb, intact. Mouse L A-9 cells were similarly injected with DNA of a 230-kb YAC containing the human beta-globin gene cluster and a mammalian selectable marker. Seven of 10 of the resulting cell lines contained both YAC vector arms plus the intact 140-kb SfiI fragment spanning the beta-globin gene. Three cell lines were analyzed by RecA-assisted restriction endonuclease (RARE) cleavage and found to contain the entire intact 210-kb YAC insert. Introduction of similarly prepared DNA into mammalian cells by lipofection gave rise to cell lines with multiple YAC fragments that were generally shorter than the YAC fragments found in microinjected cell lines. The results show that microinjection of gel-purified YAC DNA into mammalian cells is an efficient method of transferring DNA fragments several hundred kilobase pairs in size into mammalian cells.
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PMID:Microinjection of intact 200- to 500-kb fragments of YAC DNA into mammalian cells. 838 48

A membrane-independent morphogenetic viral signal peptide is identified within bacteriophage T4 internal protein III (IPIII). Utilizing a phagederived expression-packaging-processing system, which packages foreign proteins fused with IPIII into the phage capsid, a synthetic cleavage site introduced at the C terminus of IPIII, is demonstrated to be functional and permits processing of fusion proteins. IPIII, which possesses a native P21 cleavage site at its N terminus, is altered to possess a second P21 cleavage site at its C terminus where cleavage occurs by means of the scaffold proteinase P21 within the capsid. The altered IPIII was inserted into an expression vector to permit the creation of fusion proteins with staphylococcal nuclease, EcoRI endonuclease, beta-globin, and luciferase. Western immunoblot analysis of packaged T4eG326 indicates that the IPIII:fusion-proteins are packaged into phage and proteolytically processed, thus the synthetic P21 cleavage site positioned at the C terminus of IPIII is demonstrated to be functional, and 20 to 200 protein molecules are packaged per capsid. Truncation experiments identified the minimal portion of IPIII required to achieve targeting into the phage capsid as a ten amino acid residue from the N terminus, which includes the N-terminal methionine residue and the proteinase P21 cleavage site, designated the CTS (capsid targeting sequence). The addition of the CTS to a fragment of luciferase permits the protein to be packaged and processed, which demonstrates that the CTS is by itself sufficient to target foreign protein to the capsid. The imputed dual function of the CTS is supported by site-directed PCR mutagenesis, which reveals two functionally separate domains of the CTS for targeting and processing. The CTS appears to function in a core-related targeting mechanism that directs a polymorphic set of proteins into the T-even capsid or scaffold. Although structure formation is often assumed to involve extended protein interfaces, the analysis shows that a limited but specific sequence, the CTS, drives the interaction required to achieve targeting.
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PMID:Capsid targeting sequence targets foreign proteins into bacteriophage T4 and permits proteolytic processing. 878 Jul 80

We have investigated the endonuclease activity of the influenza A virus RNA polymerase in an in vitro assay with an artificial influenza-like mRNA containing a cap structure at its 5' terminus, followed by a 10 nt beta-globin mRNA sequence, and the 5' and 3' conserved termini of a truncated nucleoprotein (NP) cRNA influenza sequence. Results showed that partially purified virion ribonucleoprotein complexes (RNPs) and micrococcal nuclease treated RNPs cleaved the artificial influenza-like mRNA substrate specifically at positions near the 5' terminus to generate capped 14 and 15 nucleotide long RNA fragments which subsequently served as primers to initiate transcription. The endonuclease activity was completely blocked by addition of cap analog and competitively inhibited by added globin mRNA. Furthermore, an in vitro reconstituted influenza RNA transcription reaction containing a truncated NP vRNA as template, micrococcal nuclease treated RNPs and globin mRNA as primer, synthesized capped and uncapped full length (+) sense products. Enzyme kinetics showed that capped RNA was made earlier in the reaction; it reached a peak at 120 min and then declined. However, uncapped cRNA synthesis appeared later and remained as the dominant product later in the reaction. The nature of these products was confirmed by ribonuclease protection assays and by primer extension.
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PMID:Influenza A virus RNA-dependent RNA polymerase cleaves influenza mRNA in vitro. 880 82

Prenatal diagnostic strategies applied today are based mainly on polymerase chain reaction (PCR) analytical protocols. In Greece a wide range of mutations underlie the thalassaemic haemoglobinopathies, and consequently a variety of PCR-based methods are required to facilitate diagnosis of all potential abnormal genotypes. PCR protocols include those which are relatively simple and others that are technically challenging, but very few have been designed for high through-put clinical diagnostics. Over a period of 18 months we carried out prenatal diagnosis of 147 pregnancies (150 fetal samples) at risk for a wide range of haemoglobinopathies. This involved the precise characterization of parental genotypes and the subsequent analysis of fetal DNA samples. In this series, 18 different mutations in the alpha- or beta-globin clusters were identified. For the characterization of these mutations, five PCR-based protocols were selected: denaturing gradient gel electrophoresis (DGGE), amplification refractory mutation system (ARMS) PCR, restriction endonuclease analysis of PCR fragments, oligonucleotide hybridization and 'gap' PCR for detection of deletions. To avoid spurious diagnosis due to contamination of fetal samples, two additional methods were used to genotype polymorphic variable nucleotide tandem repeat (VNTR) regions of the genome in parental and fetal samples. Through analysis of the results we assess the advantages and drawbacks of the selected PCR-based protocols for providing routine clinical diagnostics.
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PMID:Prenatal diagnosis of the thalassaemia syndromes by rapid DNA analytical methods. 923 42


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