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)

Polymerase Chain Reaction (PCR) was used to detect and to identify Mycobacterium species. In this study, 13 out of 14 Mycobacterium species were detected by using six pairs of oligonucleotide primers. The PCR product was detected by non-isotopic southern blot hybridization even when as little as 10 fg of purified M. tuberculosis DNA was used. And 8 mycobacterial species were identified by PCR-Restriction Fragment Length Polymorphism (RFLP) using two kinds of endonuclease.
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PMID:[Detection of mycobacteria by DNA amplification]. 129 56

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

Multiple copies of an insertion sequence, IS6110, were shown to be present in the genome of members of the Mycobacterium tuberculosis complex (M. tuberculosis and M. bovis). Ten to 12 copies are present in various strains of M. tuberculosis, while strains of M. bovis contain only one to three copies. IS6110 was not detected in the DNA of other species of mycobacteria. Restriction endonuclease analysis indicated that the sequence of IS6110 is conserved across strain and species lines. Hybridization to the insertion sequence can be used to detect restriction fragment length polymorphism reflecting divergence in the sequence of regions flanking the various copies of IS6110. These differences were used to fingerprint various strains of the M. tuberculosis complex.
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PMID:IS6110: conservation of sequence in the Mycobacterium tuberculosis complex and its utilization in DNA fingerprinting. 167 28

Culture of tuberculous lesions from six of 14 captive seals yielded an organism which, on the basis of biochemical and drug sensitivity tests, was identified as Mycobacterium bovis, although the organism showed a weak cording pattern and was glycerol tolerant. It was pathogenic in rabbits and guinea pigs and after passage the organism exhibited strong cord formation and was glycerol intolerant. Restriction endonuclease analysis and sodium dodecyl-sulphate polyacrylamide gel electrophoresis indicated that the organism belonged to the Mycobacterium tuberculosis complex. Restriction patterns indicated that infection in the six seals was from a single source. Western blotting with monoclonal antibody to M bovis identified antigens at 23 and 27 kDa in M bovis which were absent from the seal isolates.
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PMID:Tuberculosis in captive seals: bacteriological studies on an isolate belonging to the Mycobacterium tuberculosis complex. 211 Mar 76

Molecular biology is beginning to revolutionise the study of tuberculosis and other mycobacterial diseases. DNA hybridization is used to classify strains at the generic and specific level and, when combined with the polymerase chain reaction, may rapidly detect very small amounts of mycobacterial DNA in clinical material. Restriction endonuclease mapping or the more refined restriction length polymorphism analysis is used to type strains at the sub-specific level for epidemiological purposes. Detection of plasmids is also an epidemiological aid and there is considerable interest in the role of plasmids as determinants of pathogenicity and virulence of some mycobacterial species. Genomic libraries provide a source of DNA probes for use in hybridization and restriction length polymorphism analysis and pure antigens in large quantities for experimental and diagnostic use. Vectors for the insertion of genes into mycobacteria provide a way of analysing genes relevant to virulence and protective immunity and a means of producing new vaccines for use against leprosy as well as tuberculosis. Finally, the ability to clone mammalian genes in bacteria enables immunological mediators to be produced in quantity for the elucidation of immune mechanisms in mycobacterial infections and possibly for therapeutic use.
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PMID:Recent developments in molecular biology of mycobacteria. 225 49

Information about specific genes specially of pathogenic mycobacteria could be used to unequivocally identify isolates of mycobacteria which are of clinical interest. Both eukaryotic and prokaryotic ribosomal RNA (rRNA) genes have been shown to comprise sequences which are conserved and others which are divergent. In the present study, rRNA genes from several cultivable mycobacteria including M. tuberculosis and armadillo derived M. leprae have been investigated. rRNA was isolated, made radioactive in vitro and then used to identify restriction fragments of DNA containing rRNA gene sequences. It was observed that restriction endonuclease patterns of rRNA genes are characteristic. By probing with homologous and heterologous rRNA probes, fragments hybridizing maximum with homologous probes could be identified and it appears that sequences flanking the rRNA genes are not identical. These fragments need to be further sequenced to identify the nucleotide sequences specific to rRNA gene cluster. It would also be necessary to analyse several isolates of each species including armadillo derived M. leprae before reaching any conclusions.
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PMID:Studies on ribosomal RNA genes of mycobacteria including M. leprae. 247 7

A genomic library of Mycobacterium tuberculosis strain Aoyama B in Escherichia coli K-12 was constructed by cloning Sau 3A I cleaved M. tuberculosis Aoyama B chromosomal DNA into pUC18, pUC181 or pUC182. Clones reacting with anti-PPDs-rabbit-serum were screened by immunoblotting among 3 x 10(4) clones. Seven clones were selected; designating pAT 01, pAT 101, pAT 102, pAT 103, pAT 104, pAT 105 and pAT 201. On Western blotting, they were shown to produce 15 kD (pAT 01, pAT 101, pAT 105), 18 kD (pAT 103) and 60 kD (pAT 102, pAT 201) peptide antigens. Restriction endonuclease map of each of the above clones was composed, and putative coding frames of anti-PPDs-rabbit-serum reactive peptides were deduced by analysing deletion derivatives. Nucleotide sequence of pAT 01, encoding for 15 kD peptide antigen was analyzed, and a Hinf I-Hinf I fragment in pAT 01 was subcloned into pUC 18 lambda CPL 1 to determine the direction of its reading frame. The origin of promoter that drives cloned mycobacterial genes in E. coli was discussed.
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PMID:[Molecular cloning and expression of Mycobacterium tuberculosis strain Aoyama B peptide antigen genes in Escherichia coli--a gene encoding 15 kD antigen (AT 01)]. 250 18

DNA preparations from 24 New Zealand isolates, two reference strains of Mycobacterium bovis, and one reference strain each of Mycobacterium microti, Mycobacterium africanum, and Mycobacterium tuberculosis were characterized by restriction endonuclease analysis. Twenty-five restriction enzymes were investigated. The clearest differences in M. bovis patterns were obtained with the enzymes BstEII and BclI. These produced four and five different patterns, respectively, for the 24 local isolates. When the results from both enzymes were considered, seven different combinations were obtained. The patterns produced for the two reference strains of M. bovis could be distinguished from each other and also from the patterns produced for the local isolates. All patterns were reproducible and are now being used for typing M. bovis isolates. With either enzyme, the patterns produced for the M. tuberculosis, M. bovis, and M. africanum strains had many features in common, but all the M. bovis patterns were clearly more similar to each other than to the M. tuberculosis patterns. The patterns produced for the M. microti strain were markedly different from those produced for the other species. Restriction endonuclease analysis is clearly a useful method for inter- and intraspecific classifications of the tuberculosis complex.
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PMID:DNA restriction endonuclease analysis of Mycobacterium bovis and other members of the tuberculosis complex. 298 47

A method for the isolation of DNA from mycobacteria propagated in vitro is described that utilizes organic solvents to extract lipoidal components from the outer membrane, and digestion with a protease (nagarse) and lysozyme to penetrate the cell wall. The mycobacterial cells were lysed by the addition of detergent and the DNA was purified by digestion with pronase, sequential phenol and chloroform extractions, and digestion with RNAase A. The isolated DNA, which was obtained in good yields, was of a relatively high Mr and could be readily digested by restriction endonucleases. By this method, the genomes of Mycobacterium avium, M. intracellulare, M. lepraemurium, 'M. lufu', M. marinum, M. phlei, M. scrofulaceum, M. smegmatis and M. tuberculosis were isolated and the restriction endonuclease digestion patterns analysed. Each species could be distinguished by the digestion patterns, indicating that this approach can be used for identifying mycobacterial species. This approach is also sufficiently sensitive to differentiate strains since we were able to distinguish two independently isolated strains of M. tuberculosis, H37 and H4. In addition, no evidence was obtained for the presence of methylcytosine residues in the sequences 5'.CCGG.3',5'.CCCGGG.3',5'.CC(A/T) GG.3' or for methyladenine at 5'.GATC.3' in the DNA of the nine mycobacterial species examined using pairs of restriction enzymes that recognize and cleave at the same nucleotide sequence but differ in their sensitivity to 5-methylcytosine or 6N-methyladenine.
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PMID:Isolation and restriction endonuclease analysis of mycobacterial DNA. 301 65

DNA restriction endonuclease analysis was used for intra-specific typing of Mycobacterium bovis isolates from 83 brush-tailed possums (Trichosurus vulpecula) obtained between 1982 and 1984 from the three major regions in New Zealand with endemic bovine tuberculosis. All the isolates were found to be genetically very similar. Differentiation of the isolates into 33 restriction types was achieved by using high-resolution electrophoresis and the combined results from separate digestions with the restriction enzymes Bst EII, Pvu II and Bcl I. The typing system was entirely reproducible. Isolates of the same type were usually found in adjacent localities and were always limited to one of the three major regions. In some cases, isolates of the same type were found in both 1982 and 1984. The phenotypic significance of the small genetic differences identified between different isolates is unknown. The typing system will be useful for monitoring the transmission of M. bovis to other species and the future spread of different M. bovis types through possum populations.
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PMID:Geographic distribution of restriction types of Mycobacterium bovis isolates from brush-tailed possums (Trichosurus vulpecula) in New Zealand. 301 75


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