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)

A major endodeoxyribonulcease was isolated from a mutant of the transformable Bacillus subtilis 168. The magnesium-dependent endonuclease was purified approximately 750-fold to electrophoretic homogeneity. The enzyme had a molecular weight of about 31 000, as determined by gel filtration and polyacrylamide gel electrophoresis. The protein appears to be composed of two subunits. The nuclease was dependent on magnesium or maganese ions for hydrolytic activity. The purified nuclease degraded DNA from several species of Bacillus, as well as Escherichia coli DNA, alkylated, depurinated, and thymine-dimer containing B. subtilis DNA, and hydroxymethyluracil-containing phage DNA. The enzyme also hydrolyzed single-stranded DNA, although native DNA was the preferred substrate. However, the nuclease was unable to degrade ribosomal RNA. The cleavage products of the DNA hydrolysis have 5'-phosphate and 3'-hydroxyl ends. The enzyme could be activated in crude extracts by heat treatment or treatment with guanidine hydrochloride. The nuclease activity was inhibited by phosphate and by high concentrations of NaCl. A possible function for this endonuclease in bacterial transformation is discussed.
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PMID:Isolation, characterization, and activation of the magnesium dependent endodeoxyribonuclease from Bacillus subtilis. 1 15

Complementary oligonucleotide primers which flank a 675-bp DNA fragment encompassing part of the putative gene for the capsid protein of chicken anemia virus (CAV) were used for the enzymatic amplification of CAV DNA by the polymerase chain reaction (PCR). Application of a dot blot hybridization assay by using a 32P-labeled cloned CAV DNA probe allowed PCR product amplified from as little as 0.1 fg of the target DNA sequence to be detected. When it was used for PCR amplification, DNA extracted from thymus tissue by a guanidine isothiocyanate-based method proved to be more efficient than that extracted by methods involving phenol or boiling. DNAs specified by 14 CAV isolates originating in the United Kingdom, Ireland, Germany, Sweden, the United States, Japan, and Australia were amplified. Restriction endonuclease analysis of the PCR-amplified DNAs with the enzymes HaeIII, HinfI, and HpaII indicated that the 14 CAV isolates can be assigned to seven groups, with isolates from different countries usually exhibiting the greatest number of restriction site differences.
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PMID:Detection and differentiation of chicken anemia virus isolates by using the polymerase chain reaction. 132 Nov 65

Recombinant Mu gam gene protein (Mu GAM) synthesized in Escherichia coli accumulates in the form of insoluble inclusion bodies which, after cell lysis and low-speed centrifugation, can be recovered in the pellet fraction. This property was utilized in a purification procedure for Mu GAM based on guanidine hydrochloride denaturation-renaturation followed by a single DEAE-cellulose chromatographic step. The purified Mu GAM was shown by nitrocellulose-filter-binding experiments to bind with high affinity to linear double-stranded DNA and more weakly to supercoiled and single-stranded forms. Mu GAM protects linear DNA from degradation by a variety of exonucleases, but only weakly inhibits endonuclease activity. These results are in accord with a model of Mu GAM conferring protection from exonuclease activity by binding to the ends of DNA.
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PMID:Purification of overexpressed gam gene protein from bacteriophage Mu by denaturation-renaturation techniques and a study of its DNA-binding properties. 216 62

Hepatitis B virus (HBV) DNA labelled with [3H] or [alpha-32P]dTTP in vitro was isolated from Dane particles by CsCl-guanidine hydrochloride density gradient centrifugation. Virtually all HBV DNA extracted as above contained a knob on the double-stranded region visible by electron microscopy. Proteinase K removes the know from HBV DNA. The HBV DNA-protein complex was efficiently bound to nitrocellulose membrane filters. When the radioactively labelled HBV DNA-protein complex was digested with restriction endonuclease Hae III or Hind II and subjected to polyacrylamide gel electrophoresis, some radioactivity did not enter the gel. Restriction endonuclease fragments did not remain on the top of the gel after proteinase K treatment and no new band was detected. The possible role of the knob protein in the replication of HBV DNA is discussed.
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PMID:DNA-protein complex from hepatitis B virus. 633 Apr 5

EcoRII was the first restriction endonuclease (ENase) reported needing the cooperative interaction with at least two DNA sites for activity. We constructed an EcoRII-overproducing strain of Escherichia coli by placing the coding sequence under control of the T7 gene 10 regulatory elements. The yield of EcoRII expression could be increased to about 10% of total soluble cellular protein. Inclusion bodies are formed that mainly consist of insoluble EcoRII molecules. After solubilization by 6 M guanidine hydrochloride refolding of the enzyme was achieved by dilution into appropriate buffer. The endonuclease was purified to homogeneity from both the soluble protein fraction and the protein renatured from inclusion bodies. Their identity was proven by circular dichroism and analysis of enzyme activity with respect to the special substrate requirements of EcoRII. It is shown that EcoRII cleavage of oligodeoxyribonucleotide duplexes (oligo duplexes) with only one recognition site follows a sigmoidal concentration dependence, i.e., they cannot be cleaved below a distinct low DNA concentration where simultaneous interaction with two substrate molecules is no longer possible. We demonstrate that the restriction of oligo duplexes containing two recognition sites does not show this concentration dependence, confirming an intramolecular site cooperativity.
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PMID:Hyperexpressed EcoRII renatured from inclusion bodies and native enzyme both exhibit essential cooperativity with two DNA sites. 775 33

A rapid direct-extraction method was used to obtain DNA from environmental soil samples. Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonuclease PalI. The extraction method was used to obtain DNA from a variety of plants, bacteria, and fungi including Gossypium hirsucum (cotton), Pseudomonas, Bacillus, Streptomyces, and Colletotrichum. Up to 100 micrograms DNA/g (wet weight) of soil and 400 micrograms DNA/g of plant material were recovered. Restriction endonuclease analysis patterns of amplified rDNA from pure microbial cultures and plant species contained three to five different DNA fragments. Amplified rDNA of mixed population DNA extracts from soil samples, digested with the restriction endonuclease PalI, contained 12-20 DNA fragments, appearing as sample "fingerprints." Results from eight environmental soil samples that were analyzed suggest that the amplified rDNA fingerprints can be used to help characterize the genetic and biological diversity of the microbial populations in these samples.
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PMID:An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis. 776 20

A HindIII restriction endonuclease analysis (REA) typing system for total genomic Clostridium difficile DNA including a rapid and efficient method of DNA extraction and a scheme for organizing unique electrophoretic DNA band patterns was developed. REA typing was performed by two extraction methods for 1,965 C. difficile isolates obtained from patients with symptomatic C. difficile disease, asymptomatic patients who were C. difficile culture positive, and environmental surfaces. This isolate collection yielded 206 unique REA types, which were organized into 75 groups. A reference strain representing each unique REA type was chosen for DNA band pattern comparisons, cytotoxin testing, and plasmid analysis. The DNA band patterns utilizing a guanidine thiocyanate-EDTA-Sarkosyl DNA extraction method were 94% reproducible, while the original and sporadically problematic diethyl pyrocarbonate-sodium dodecyl sulfate DNA extraction method was 98% reproducible when readable patterns were obtained. Reference strains from 43 of the 75 groups were cytotoxin positive, 28 groups were cytotoxin negative, and 4 groups included both toxigenic and nontoxigenic strains. Cytotoxicity of isolates with a particular REA type was always consistent with the toxicity of the reference strain for that type. REA typing was able to discriminate strain differences within types identified by the immunoblot (89 isolates), bacteriophage-bacteriocin (44 isolates), and ribotyping (23 isolates) methods. REA typing is a sensitive, discriminating, reproducible, and rapid method for differentiating C. difficile strains and is suitable for large-scale epidemiologic studies.
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PMID:Development of a rapid and efficient restriction endonuclease analysis typing system for Clostridium difficile and correlation with other typing systems. 839 78

Plasma membrane Na+/H+ exchanger (NHE) isoforms NHE1 and NHE3 exhibit very different sensitivities to amiloride and its 5-amino-substituted analogues, benzoyl guanidinium derivatives (e.g. (3-methylsulfonyl-4-piperidinobenzoyl)guanidine methanesulfonate (HOE694)), and cimetidine. To define structural domains that confer differential sensitivity to these antagonists, unique restriction endonuclease sites were engineered into cDNAs for each isoform near the regions that encode the putative membrane-spanning domains. These new sites did not modify their pharmacological properties and allowed several chimeric Na+/H+ exchangers to be constructed by exchanging homologous segments. The modified parental (E1' and E3') and chimeric molecules were stably expressed in exchanger-deficient Chinese hamster ovary AP-1 cells and assayed for their sensitivities to amiloride, ethylisopropylamiloride, HOE694, and cimetidine. Most chimeras showed drug sensitivities corresponding to the dominant parental segment. However, interchanging a 66-amino acid segment containing the putative ninth transmembrane (M9) domain and its adjacent loops caused reciprocal alterations in the sensitivities of E1' and E3' to all antagonists. In addition, substituting the first five putative membrane-spanning domains of E3' with the corresponding region of E1' modestly reduced the transporter's sensitivity to cimetidine but not the other compounds. These data indicate that the protein segment between M8 and M10 may be a major site of interaction with these antagonists, although other regions modestly influence sensitivity to certain drugs.
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PMID:Delineation of transmembrane domains of the Na+/H+ exchanger that confer sensitivity to pharmacological antagonists. 870 6

We have previously described an allelic variant of the human H2R, nominated the H2R649G allele. This allele contains an adenine-->guanidine substitution at base 649, which introduces an additional TaqI restriction endonuclease site into the gene. With this in mind, we have investigated allelic polymorphism of this receptor and its association with schizophrenia. H2R DNA from 47 schizophrenic patients and 46 control subjects was amplified by the polymerase chain reaction (PCR). These PCR products were analyzed by observing TaqI cleavage patterns and single-stranded conformational polymorphisms. It was found that the H2R649G allele was 1.8 times more frequent in the schizophrenic population (chi 2 test P < 0.01). In addition, schizophrenic individuals were 2.8 times more likely to be homozygous for the H2R649G allele than the control population, (chi 2 test P < 0.05). These data place the attributable fraction for possession of the H2R649G allele at 28.4%.
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PMID:Individuals with schizophrenia have an increased incidence of the H2R649G allele for the histamine H2 receptor gene. 915 48

A new method for isolation of prawn baculovirus and subsequent extraction of viral DNA was developed. No density gradient centrifugation, ultracentrifugation or phenol-chloroform extraction steps were involved. Phenylmethylsulfonyl fluoride (PMSF) was used to prevent proteinase degradation, DNase and RNase were used to degrade prawn DNA and RNA respectively. The nucleocapsid was a bacilliform virion, about 58 62 nm in width and 300-350 nm in length as observed by transmission electron microscopy. Intact viral DNA was obtained by lysing nucleocapsids with guanidine hydrochloride and degrading protein with proteinase K. As the viral DNA was digested with restriction endonuclease and separated by electrophoresis, restriction fragments were clearly shown on the agarose gel. The size of the DNA was estimated approximately to be 290 kb. The virus which appeared to be a prawn baculovirus was named prawn white spot baculovirus (PWSBV) due to the white spots which appeared on the inside surface of the crust of infected prawns.
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PMID:A simple and efficient method for purification of prawn baculovirus DNA. 927 11


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