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)

As an initial step in gaining a better understanding of the important clinical properties that vary between strains of mycobacteria, we attempted to find molecular markers that would define different strains of Mycobacterium tuberculosis. We used restriction fragment analysis with the endonuclease MboI and hybridization with total M. tuberculosis DNA to examine DNA differences between 15 strains of M. tuberculosis. We were able to identify different strains using this method. In order to assess the sensitivity of this method in identifying different strains, we compared it with phage typing. The 2 methods appear to be similar in sensitivity and also to be complementary. There were 2 examples where restriction fragment analysis did not separate strains with different phage types. In addition, there were 2 examples where phage typing did not separate strains with different restriction patterns. Finally, there were 2 epidemiologically unrelated strains with the same restriction pattern and the same phage type. This method of restriction fragment analysis of chromosomal DNA is potentially useful for epidemiologic studies of tuberculosis. Additionally, by analyzing the genome of M. tuberculosis, molecular markers may well be defined that will be useful in discovering the pathogenesis of the clinical properties of M. tuberculosis, which previously have been poorly understood.
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PMID:Restriction fragment analysis of chromosomal DNA defines different strains of Mycobacterium tuberculosis. 301 66

A plasmid DNA library was constructed from restriction endonuclease digested genomic deoxyribonucleic acid (DNA) of a virulent strain of Mycobacterium tuberculosis isolated from sputum of a patient. The sensitivity and specificity of two of the cloned DNA fragments in detecting M. tuberculosis and its related DNA sequences were analysed by DNA-to-DNA hybridization. The level of detection was determined to be 50 picograms of M. tuberculosis DNA, which is approximately equivalent to 10,000 mycobacterial genomes. These two M. tuberculosis DNA probes did not cross-hybridize to DNA of non-mycobacterial origin, nor with DNA from 9 out of 11 other mycobacterial species. Mycobacterial DNA sequences could be detected in 134 of 441, or 30.4%, of various types of uncultured clinical specimens from 365 patients by the DNA probes, whereas traditional culture method showed only a 19.0% positivity rate for the same specimens (p less than 0.001). The overall sensitivity and specificity of the DNA probes in detecting M. tuberculosis are 90.5% and 83.8% respectively. The DNA hybridization test may become a useful tool for the early and rapid determination of mycobacterial infection in uncultured clinical specimens.
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PMID:The detection of mycobacterial DNA sequences in uncultured clinical specimens with cloned Mycobacterium tuberculosis DNA as probes. 314 Apr 57

It is difficult to make a precise diagnosis for intestinal tuberculosis and differentiate it from Crohn's disease. For evaluating the efficacy of PCR assay in these two aspects, 36 specimens of intestinal tuberculosis and 26 of Crohn's disease from surgical resections and endoscopic biopsies were subjected to PCR assay. Oligonucleotides derived from IS 6110 sequence, which is a repeated in M. tuberculosis chromosome and highly specific for the M. tuberculosis complex, were used as a primer. The specificity of PCR products was confirmed by digestion with Sal I restrictive endonuclease and southern blot hybridization by using digoxigenin-labeled probe. The results showed that M. tuberculosis DNA was identified in 27 of the 36 specimens of intestinal tuberculosis, but none in those of 26 Crohn's disease. In conclusion, PCR is a rapid, sensitive and specific method for the diagnosis of intestinal tuberculosis, and it is also valuable in differentiating intestinal tuberculosis from Crohn's disease.
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PMID:[The value of polymerase chain reaction assay in the diagnosis of intestinal tuberculosis and differentiation from Crohn's disease]. 760 Aug 74

It is difficult to make a precise diagnosis of intestinal tuberculosis and to differentiate it from Crohn's disease. For evaluating Polymerase Chain Reaction (PCR) assay in these two aspects, 36 specimens of intestinal tuberculosis from surgical resections and endoscopic biopsies and 26 Crohn's disease samples were subjected to PCR assay. 21 specimens of normal colon tissue surrounding cancer were used as the control. Oligonucleotides derived from the IS 6110 sequence, which is repeated in M. tuberculosis chromosome and highly specific for the M. tuberculosis complex, were used as a primer. The amplified PCR products were detected by examination of ethidium-bromide-stained polyacrylamide gels. The specificity of PCR products was confirmed by digestion with Sal I restrictive endonuclease and southern blot hybridization using digoxigenin-labeled probe. The results showed that the M. tuberculosis DNA was identified in 27/36 intestinal tuberculosis, but none of 26 Crohn's disease. Acid fast bacilli were only found in 16/36 intestinal tuberculosis. In conclusion, as a rapid, sensitive, and specific pathogenic method in diagnosis of intestinal tuberculosis, PCR assay has been developed in this study, and is considered valuable in the differentiation between intestinal tuberculosis and Crohn's disease.
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PMID:Value of polymerase chain reaction assay in diagnosis of intestinal tuberculosis and differentiation from Crohn's disease. 779 30

Typing of M. bovis isolates for epidemiological purposes is possible using restriction endonuclease analysis (REA). However, the DNA fragment patterns obtained are complex and difficult to analyse due to the large number of bands produced. In an attempt to develop a less complicated typing scheme two DNA probes were used in hybridization studies to detect restriction fragment length polymorphisms (RFLP) in M. bovis. An oligonucleotide probe which matches part of the insertion sequence IS6110 produced few bands and failed to discriminate between bovine isolates of M. bovis. A probe prepared from a highly repeated DNA sequence, cloned from M. tuberculosis when used on southern blots of AluI digested M. bovis DNA, resulted in a discriminating typing scheme which was easier to perform and analyse than the REA. The RFLP typing scheme identified 27 different strains from a total of 36 isolates of M. bovis and 7 reference strains from the M. tuberculosis complex. Using REA, 24 types were identified using BclI and PvuII digests and 23 different types using BstEII digests. When results of all 3 enzyme digests were combined, the REA identified 27 types from the same strains. Ten isolates of M. bovis from 5 properties involved in an outbreak of bovine tuberculosis were all identified as the same type with both techniques.
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PMID:Use of a repetitive element isolated from Mycobacterium tuberculosis in hybridization studies with Mycobacterium bovis: a new tool for epidemiological studies of bovine tuberculosis. 790 19

Tuberculosis was diagnosed in 3 otariid seals found dead on beaches at 3 locations on the south coast of Western Australian between May 1990 and March 1991. This confirms that tuberculosis is present in the 2 native seals (Neophoca cinerea and Arctocephalus forsteri) in Western Australian waters. Mycobacterium sp isolated from the lungs of 2 of the seals were studied to determine the similarity of the strains to each other, to the strains isolated during 1986 from Australian sea lions and New Zealand fur seals kept in captivity at a marine park near Perth, Western Australia, and to a strain isolated in 1988 from a seal trainer who worked with the infected captive seals for 3 years. After restriction endonuclease analysis (REA) with the endonucleases Bst EII, Bcl I and Pvu II, one of the wild seal strains appeared to have identical DNA fragment patterns to the strains from the captive seals and the seal trainer. The other wild seal isolate had identical REA profiles using Bst EII and Bcl I, but a minor difference was detected using Pvu II. Differences in these isolates were more clearly seen in restriction fragment length polymorphisms after hybridisation with two DNA probes. The secretory protein MPB70, present in M bovis, was not detected in wild seal isolates using sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blotting techniques. Analysis of protein and DNA fragment profiles indicated that seal tuberculosis isolates form a unique cluster within the M tuberculosis complex.
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PMID:Tuberculosis in wild seals and characterisation of the seal bacillus. 809 94

Screening of a lambda gt11 genomic library has been used as an approach for molecular cloning of the Mycobacterium tuberculosis repetitive DNA shown to be present on a previously described 5.6-kb Alu I genomic fragment. The recombinant clone R18.8.2, which strongly hybridized with the radiolabeled 5.7-kb Alu fragment, carried two Eco RI inserts of 2 kb and 1.4 kb in size. Southern hybridization of each of these fragments to restriction endonuclease-cleaved M. tuberculosis DNA clearly demonstrated the 2 kb to contain the repetitive DNA sequence, while the 1.4 kb is represented in a single copy. When replica plaque lifts from the genomic library were probed, the 5.6-kb genomic fragment and the cloned 2-kb repetitive insert hybridized to an identical number of plaques, indicating the similarity and the high copy number of the repeating unit shared by the two fragments. Restriction mapping and Southern hybridization patterns indicated that the 2-kb repetitive and the 1.4-kb single-copy DNA sequences originated from a contiguous piece of genomic DNA. Both fragments were found to be unique to members of the M. tuberculosis complex, except that the 2-kb insert exhibited a weak hybridization with M. kansasii DNA. Finally, a 169-bp region from one end of the single-copy sequence has been amplified by polymerase chain reaction (PCR) in a manner specific to members of the M. tuberculosis complex. The sensitivity of the PCR was such that as few as 9-10 bacilli could be detected.
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PMID:Molecular cloning and characterization of contiguously located repetitive and single copy DNA sequences of Mycobacterium tuberculosis: development of PCR-based diagnostic assay. 837 Oct 32

Amplification of the region separating the genes coding for the two rRNA species 16S and 23S was performed to identify 56 mycobacterial strains, belonging to eleven species: Mycobacterium tuberculosis, M. avium, M. kansasii, M. gordonae, M. abscessus, M. fortuitum, M. xenopi, M. bovis, M. bovis/BCG, M. africanum and M. intracellulare. Reproducible amplification patterns were obtained with most species with the exception of M. kansasii which showed heterogeneity, confirming the existence of a genetically distinct subspecies within this species. In addition, we used the amplified products as target DNA for restriction endonuclease digestion and RAPD (randomly amplified polymorphic DNA) analysis to compare strains of M. abscessus, M. tuberculosis and M. avium. The discriminatory power of these two typing methods was higher than when whole genomic DNA is used as target. Our results demonstrate that the two-step approach to identification and typing on the basis of the hypervariability of 16S-23S spacer region is reliable, rapid and simple, and consequently could be an epidemiological tool in clinical laboratories.
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PMID:Identification and strain differentiation of Mycobacterium species on the basis of DNA 16S-23S spacer region polymorphism. 852 57

Genetic studies of Mycobacterium tuberculosis have been greatly hampered by the inability to introduce specific chromosomal mutations. Whereas the ability to perform allelic exchanges has provided a useful method of gene disruption in other organisms, in the clinically important species of mycobacteria, such as M. tuberculosis and Mycobacterium bovis, similar approaches have thus far been unsuccessful. In this communication, we report the development of a shuttle mutagenesis strategy that involves the use of long linear recombination substrates to reproducibly obtain recombinants by allelic exchange in M. tuberculosis. Long linear recombination substrates, approximately 40 to 50 kb in length, were generated by constructing libraries in the excisable cosmid vector pYUB328. The cosmid vector could be readily excised from the recombinant cosmids by digestion with PacI, a restriction endonuclease for which there exist few, if any, sites in mycobacterial genomes. A cosmid containing the mycobacterial leuD gene was isolated, and a selectable marker conferring resistance to kanamycin was inserted into the leuD gene in the recombinant cosmid by interplasmid recombination in Escherichia coli. A long linear recombination substrate containing the insertionally mutated leuD gene was generated by PacI digestion. Electroporation of this recombination substrate containing the insertionally mutated leuD allele resulted in the generation of leucine auxotrophic mutants by homologous recombination in 6% of the kanamycin-resistant transformants for both the Erdman and H37Rv strains of M. tuberculosis. The ability to perform allelic exchanges provides an important approach for investigating the biology of this pathogen as well as developing new live-cell M. tuberculosis-based vaccines.
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PMID:Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates. 855 Apr 28

Restriction endonuclease PvuII plays a central role in restriction fragment length polymorphism analysis of Mycobacterium tuberculosis complex isolates with IS6110 as a genetic marker. We have investigated the basis for an apparent dichotomy in PvuII restriction fragment pattersn observed among strains of the M. tuberculosis complex. The chromosomal regions of two modified PvuII restriction sites, located upstream of the katG gene and downstream of an IS1081 insertion sequence, were studied in more detail. An identical 10-bp DNA sequence (CAGCTGGAGC) containing a PvuII site was found in both regions, and site-directed mutagenesis analysis revealed that this sequence was a target for modification. Strain-specific modification of PvuII sites was identified in DNA from over 80% of the nearly 800 isolates examined. Furthermore, the proportion of modifying and nonmodifying strains differs significantly from country to country.
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PMID:Host-mediated modification of PvuII restriction in Mycobacterium tuberculosis. 855 Apr 46


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