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)

The sensitivity and specificity of the polymerase chain reaction (PCR) in the detection of mycobacteria in paraffin-embedded tissues and in crude lysates of mycobacterial cultures were assessed. Sections of formalin-fixed, paraffin-embedded tissues were deparaffinized and then subjected to a simple proteinase K and boiling lysis procedure. These preparations were used directly for PCR amplification of the 383 bp segment of the gene encoding the 65 kDa mycobacterial surface antigen. Crude lysates of mycobacteria were used as positive controls. The specificity of the PCR products was confirmed by Southern blot using a region-specific digoxigenin-labeled oligonucleotide probe and chemiluminescent detection. The 383 bp diagnostic fragment was visualized in 11 of 12 acid-fast bacilli (AFB) stain/culture-proven-positive blocks. Crude lysates of mycobacteria were detected to a sensitivity of approximately 80 organisms. Amplified fragments from paraffin-embedded tissues and mycobacterial cultures of M. tuberculosis, M. avium-intracellulare, and saprophytic mycobacteria were distinguished by digestion with Nar 1 restriction endonuclease. These results suggest that PCR amplification followed by restriction enzyme digestion of the PCR product is a rapid, specific, and highly sensitive technique for the detection and speciation of mycobacteria in paraffin-embedded tissues.
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PMID:Rapid detection and species identification of mycobacteria in paraffin-embedded tissues by polymerase chain reaction. 134 65

During the course of a clinical study on patients with campylobacteriosis, three consecutive isolates of Campylobacter jejuni from the same patient were sent for O-serotyping. Marked differences in the specificities of the O antigens of the isolates were observed between the first and third isolates when a passive haemagglutination assay system developed for serotyping C. jejuni was used. Differences in specificity were also demonstrated by immunoblots of lipopolysaccharides (LPS) from proteinase K-digested whole cells. The three isolates could not be distinguished either by restriction endonuclease analysis of chromosomal DNA, by gel electrophoresis of whole-cell proteins or by silver-stained LPS gels, thus providing evidence that they were of the same strain and that antigenic variation had occurred in vivo.
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PMID:Variation of the O antigen of Campylobacter jejuni in vivo. 137 64

In this study, the effect of DNA single strand breaks (ssb) on the neutral (pH 9.6) filter elution of DNA from Chinese hamster ovary (CHO K1) cells containing DNA double strand breaks (dsb) was investigated. Protein associated ssb were induced by the inhibition of DNA topoisomerase I with camptothecin (cpt). Protein associated dsb were introduced by treating cells with the DNA topoisomerase II poison; etoposide (VP-16). Protein associated ssb and dsb were converted to ssb and dsb by proteinase K present in the lysis solution. In some experiments dsb were generated by the restriction endonuclease Pvu II. It was found that elution of DNA in the presence and absence of ssb was similar under neutral conditions. This finding is consistent with the view that the fast component of the bi-phasic repair kinetics observed in irradiated mammalian cells with the neutral filter elution technique is not attributable to the interference of ssb with the measurement of dsb, and thus suggests that the two components of repair observed with the neutral filter elution elution technique may represent two different types of dsb or modes of repair of dsb.
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PMID:Lack of interference of DNA single-strand breaks with the measurement of double-strand breaks in mammalian cells using the neutral filter elution assay. 164 64

DNA helicase I, the traI gene product of the Escherichia coli F factor, was shown to be associated with endonuclease activity specific for the transfer origin of the F plasmid, oriT. In the presence of Mg2+, the purified enzyme forms a complex, stable in the presence of sodium dodecylsulfate (SDS) with a negatively superhelical chimeric plasmid containing oriT. The enzyme nicks and, after this, apparently binds to the 5' nick terminus when this complex is heated in the presence of SDS and/or EDTA or treated with proteinase K. Dideoxy sequencing locates the nick site in the F DNA strand transferred during bacterial conjugation after nucleotide 138 clockwise of the mid-point of the BglII site at 66.7 kb of the F genetic map. A sequencing stop after nucleotide 137 of this strand (where oriT-nicking seems to occur in vivo) is possibly an artefact caused by helicase I protein attached to the 5' terminal nucleotide. Deletion in the amino-terminal part of the traI polypeptide abolishes the oriT-nicking activity while leaving the strand-separating activity intact. These results confirm the prediction from genetic studies that helicase I is bifunctional with site-specific endonuclease and strand-separating activities.
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PMID:Endonuclease activity of Escherichia coli DNA helicase I directed against the transfer origin of the F factor. 165 Dec 33

An ATP-dependent DNA aggregating activity was purified from rat liver by DEAE-cellulose, phosphocellulose, and novobiocin-Sepharose column chromatography. The protein aggregated superhelical, relaxed, single-, or double-stranded DNA in a divalent cation- and ATP-dependent reaction. The DNA aggregating activity was detected by retardation of a DNA-protein complex at the origin on a 1% agarose gel. The protein appeared to exist in solution as a monomer of molecular weight 66,000, and had no DNA polymerase, topoisomerase, recombinase, or ligase activity. The DNA aggregating activity was inhibited by 10 mM nalidixic acid or 1 mM novobiocin but not by 20 mM N-ethylmaleimide or camptothecin. Adenylyl(beta,gamma-methylene)-diphosphonate, adenylyl-imidodiphosphate, or adenosine-5'-O(3-thiotriphosphate) did not substitute for ATP whereas CTP, dTTP, or the ATP analog adenylyl(alpha,beta-methylene)-diphosphonate could replace ATP. The aggregated DNA was only partially dissociated by restriction endonuclease digestion but was completely dissociated by deproteinization with SDS, proteinase K, or chloroform/octanol extraction. On the basis of the molecular weight, thermostability, antigenic property, and amino acid sequence homology in the first 12 positions, we conclude that the rat liver protein is serum albumin and that the ATP-dependent DNA aggregation is a novel function of rat serum albumin.
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PMID:ATP-dependent DNA aggregation is a novel function of rat serum albumin. 189 9

The alkaline plasmid DNA extraction method of Birnboim and Doly was applied for the isolation of human papillomavirus (HPV) from warts. Tissue from common and plantar warts was digested with proteinase K, and the extrachromosomal circular covalently-closed form of HPV-DNA was rapidly extracted by alkaline sodium dodecyl sulphate and phenol-chloroform treatment. Recovery of HPV-DNA from the tissue was sufficient for determination of endonuclease restriction patterns by agarose gel electrophoresis.
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PMID:Human papillomavirus DNA from warts for typing by endonuclease restriction patterns: purification by alkaline plasmid methods. 196 33

Tissues stored as paraffin blocks are a potential source of DNA for retrospective clinicogenetic analysis. To assess the feasibility of Southern blot analysis, DNA extracted from paraffin blocks was compared with DNA obtained from fresh-frozen controls of the same tissues. Sections 50-100 microns thick cut from paraffin blocks of 11 normal tissues, 18 lymphoid lesions, and 9 gastric carcinoma samples were deparaffinized and incubated at 45 degrees C for 48 to 72 h in a sodium dodecyl sulfate (SDS)/proteinase K solution. Following organic extraction, alcohol precipitation, restriction endonuclease digestion, and gel electrophoresis, DNA was transferred to nylon membranes. 32P-labelled DNA probes for the immunoglobulin heavy-chain locus and T-cell receptor beta-chain gene were hybridized to the normal tissue and lymphoid samples; the gastric cancers were probed for the HER-2/neu protooncogene. Intact DNA was obtained from the majority of formalin-fixed samples, yielding results qualitatively similar to those from fresh tissues. Degradation is the most significant problem in analyzing DNA extracted from paraffin blocks and compromises accurate quantitation. DNA analysis using paraffin-embedded tissue has potential clinical and research applications and may be a particularly useful way to study gene abnormalities in unusual tumors infrequently available as fresh specimens.
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PMID:Extraction of DNA from paraffin blocks for Southern blot analysis. 198 65

A method is described for the rapid isolation of chromosomal deoxyribonucleic acid from species of the genus Mycoplasma. The method involves incubation of washed cells at elevated temperature in the presence of an ionic detergent, chelating agents, and proteinase K prior to the removal of residual protein and ribonucleic acid with ribonuclease and chloroform. It results in a good yield of high molecular weight material that is shown to be free of endogenous nuclease and substantially free of protein or ribonucleic acid contamination without the use of phenol. The isolated DNA is shown to be an excellent substrate for restriction endonuclease digestion and ligation with T4 DNA ligase.
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PMID:An improved method for the rapid isolation of chromosomal DNA from Mycoplasma spp. 218 71

Molecular characterization of Ehrlichia risticii, the etiological agent of Potomac horse fever, was performed. Restriction endonuclease cleavage of E. risticii DNA generated distinct patterns by different enzymes. The DNA cleavage patterns of E. risticii isolates obtained from different geographic regions were similar. Protein analysis identified thirty-five distinct proteins with molecular weights ranging from 160 to 16 kilodalton (kDa). Antigenic analysis by radioimmunoprecipitation using 125I surface labeled E. risticii and by Western blotting determined the presence of eighteen antigens (160, 110, 86, 84, 81, 70, 55, 51, 49, 44, 41, 36, 33, 31, 28, 24, 22 and 16 kDa) of which nine (110, 86, 70, 55, 51, 49, 44, 33, and 28 kDa) were major antigens. Fourteen of these antigens, which included the major antigens, were apparent surface components. There were no heat-modifiable proteins but lipopolysaccharide components of 245 and 14 kDa, resistant to proteinase K and of non-antigenic character, were detected in the organism.
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PMID:DNA restriction endonuclease cleavage pattern and protein antigen profile of Ehrlichia risticii. 224 34

Three linear DNA plasmids were found in isolate RI-64 of anastomosis group 4 (AG-4) of Rhizoctonia solani. These plasmids, designated pRS64-1, -2, and -3, possessed the same size of 2.7 kb. Restriction mapping and Southern hybridization analysis of pRS64-1, -2, and -3 revealed the presence of homologous regions at both termini. The plasmid DNAs were resistant to both 3'-exonuclease and 5'-exonuclease even after treatment with proteinase K or alkali. The length of both terminal fragments that were generated by restriction endonuclease digestion was doubled under the denaturation condition, indicating that the linear plasmid DNAs have hairpin loops at both termini. Southern blotting analysis of total DNA showed the presence of two types of dimeric forms of pRS64 DNA. One is a head-to-head dimer and the other is a tail-to-tail dimer. The role of these unique DNA structures in replication of the plasmids is discussed.
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PMID:Linear plasmid DNAs of the plant pathogenic fungus Rhizoctonia solani with unique terminal structures. 232 20


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