Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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A pair of oligonucleotide primers, based on the experimentally determined amino terminal sequence of Mycobacterium bovis BCG ribosomal protein S12 (MboS12) and a highly conserved sequence found in all mycobacterial ribosomal S12 proteins, was used for polymerase chain reaction (PCR) with M. bovis genomic DNA as template. The nucleotide sequence of the 338 bp fragment thus produced confirmed its origin in MboS12 gene. A 5.0 kb EcoRI fragment of M. bovis DNA hybridizing to this fragment was cloned. Its sequencing analysis revealed the presence of two open reading frames in the same strand. Their amino acid sequences deduced from DNA sequence showed high homology with Escherichia coli ribosomal proteins S12 and S7. However, the intercistronic region between S12 and S7 genes, which plays an important role for autoregulation for the str operon in E. coli, is completely absent in M. bovis.
Biochem Mol Biol Int 1995 May
PMID:Cloning and nucleotide sequence of the gene cluster encoding ribosomal proteins S12 and S7 from Mycobacterium bovis BCG. 766 16

Superoxide dismutase (SOD), purified from Mycobacterium smegmatis, was found to contain both manganese and iron. Since the Fe and Mn-reconstituted enzymes had specific activities of 190 and 2810 units/mg protein/g atom of metal/mol of subunit, respectively, the Mycobacterial SOD can be classified with SODs showing activity with either iron or manganese as the active-site metal (a cambialistic SOD). Mn-reconstituted enzyme showed an enzymatic reaction rate constant of 1.4 x 10(8) M-1 s-1 at pH 7.8. This rate only slightly increased with decreasing pH. Fe-reconstituted enzyme showed a rate constant of 2.7 x 10(7) M-1 s-1 at pH 7.8, but this rate increased with decreasing pH to become 1.7 x 10(8) M-1 s-1 at pH 5.7 with two pK values of 6.6 and 9.0. These results show that the metal specificity of the enzymatic activity of M. smegmatis superoxide dismutase shows manganese predominance at pH 7.8, but changes to be equal for either metal at acidic pH.
Biochem Mol Biol Int 1995 Jun
PMID:pH-dependent activity change of superoxide dismutase from Mycobacterium smegmatis. 766 26

The cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) are released by mononuclear phagocytes in vitro after stimulation with mycobacteria and are considered to mediate pathophysiologic events, including granuloma formation and systemic symptoms. We demonstrated that the Mycobacterium tuberculosis cell wall component lipoarabinomannan (LAM) is a very potent inducer of IL-1 beta gene expression in human monocytes and investigated the mechanism of this effect. We localized the LAM-, lipopolysaccharide (LPS)-, and TNF-alpha-inducible promoter activity to a -131/+15 (positions -131 to +15) DNA fragment of the IL-1 beta gene by deletion analysis and chloramphenicol acetyltransferase assay. Within this DNA fragment, there were two novel 9-bp motifs (-90/-82 and -40/-32) with high homology to the nuclear factor-IL6 (NF-IL6) binding site. Site-directed mutagenesis demonstrated that the two NF-IL-6 motifs could be independently activated by LAM, LPS, or TNF-alpha and that they acted in an orientation-independent manner. DNA mobility shift assay revealed specific binding of nuclear protein(s) from LAM-, LPS-, or TNF-alpha-stimulated THP-1 cells to the NF-IL6 motifs. We conclude that the two NF-IL6 sites mediate induction of IL-1 beta in response to the stimuli LAM, LPS, and TNF-alpha.
Mol Cell Biol 1993 Jun
PMID:Regulation of the interleukin-1 beta (IL-1 beta) gene by mycobacterial components and lipopolysaccharide is mediated by two nuclear factor-IL6 motifs. 768 3

A polymerase chain reaction able to amplify specifically a 205-base-pair DNA fragment of Mycobacterium avium genome was used and compared to a nonradioactive hybridization assay (AccuProbe) and to conventional biochemical identification. The PCR approach to diagnosis of M. avium infection is a valid diagnostic alternative to the more classical procedures.
Mol Cell Probes 1994 Dec
PMID:Comparison of polymerase chain reaction and non-radioactive hybridization techniques for the identification of Mycobacterium avium strains. 770 Feb 68

The toxicity of the powerful anti-tuberculosis drug isoniazid (INH) is believed to be mediated by the haem-containing enzyme catalase-peroxidase, encoded by the katG gene of Mycobacterium tuberculosis. Compelling evidence for this was obtained by studying a panel of INH-resistant clinical isolates using a novel strategy based on the polymerase chain reaction and single-strand-conformation polymorphism analysis (PCR-SSCP) to detect mutations in katG. In most cases INH resistance was associated with missense mutations while in a small number of strains the gene had been completely, or partially, deleted. The missense mutations fell into two groups, the larger of which contained several independent mutations that affected the N-terminal peroxidase domain of the protein, resulting in the production of a catalase peroxidase with strongly reduced enzyme activity and increased heat liability. The effects of these substitutions could be interpreted by means of molecular modelling using the crystal structure of the related enzyme cytochrome c peroxidase from yeast as a template. The second group comprises a frequently occurring amino acid substitution and a single mutation that are both located in the C-terminal domain but do not noticeably alter either enzyme activity or heat stability.
Mol Microbiol 1995 Jan
PMID:Missense mutations in the catalase-peroxidase gene, katG, are associated with isoniazid resistance in Mycobacterium tuberculosis. 774 45

A search for Mycobacterium smegmatis genes showing similarity to the conserved family encoding major sigma factors in diverse prokaryotes has identified two such determinants. Both genes are expressed in exponentially growing cells, as judged by Western immunoassays. A series of chromatographic steps was used to purify M. smegmatis RNA polymerase holoenzyme and it was shown that its ability to initiate in vitro transcription with a heterologous Bacillus subtilis promoter is dependent on the presence of these sigma factor(s). Reconstitution of specific in vitro transcription activity was obtained upon mixing of M. smegmatis core RNA polymerase with the major sigma factor of Bacillus subtilis. We also demonstrated in vitro transcription of the M. smegmatis rrnB promoter by the M. smegmatis RNA polymerase. Significantly, highly active B. subtilis RNA polymerase holoenzyme was unable to transcribe this gene.
Mol Microbiol 1995 Jan
PMID:Characterization of RNA polymerase and two sigma-factor genes from Mycobacterium smegmatis. 774 56

The cell wall of Mycobacterium smegmatis mc2155 was shown to be an effective permeability barrier to hydrophilic compounds. Permeability coefficients to beta-lactams ranged from 10 x 10(-7) to 0.5 x 10(-7) cms-1. Cell wall proteins were solubilized with EDTA and Genapol and were tested for channel-forming activity by reconstitution into lipid bilayers. Proteins were able to induce a voltage-gated cation-selective channel. The mycobacterial porin channel appeared to be water-filled since the single-channel conductance followed the mobility sequence of hydrated ions in the aqueous phase. On the basis of the Renkin equation and the single-channel conductance, the channel diameter was estimated to be around 3 nm. Model calculations showed that cation selectivity may be caused by four negative point-charges at the channel mouth. The permeability properties of the cell wall of intact cells were in good agreement with those of the reconstituted channel. Negatively charged cephalosporins, cefamandole and cephalothin, diffused at a 10- to 20-fold lower rate than the zwitterionic cephaloridine. The mycobacterial porin represents a major hydrophilic pathway of the cell wall of M. smegmatis.
Mol Microbiol 1994 Oct
PMID:Permeability of the cell wall of Mycobacterium smegmatis. 783 May 72

Southern blot analysis of chromosomal DNA from clinical isolates of Mycobacterium tuberculosis using cosmid DNA probes revealed extensive strain variation in the katG region of the genome. In addition to deletion of the katG gene itself in some isoniazid-resistant strains, adjacent DNA fragments were missing or altered in a range of drug-sensitive and drug-resistant isolates. A species-specific 2kb Kpnl fragment located 10kb upstream of katG in M. tuberculosis H37Rv hybridized to fragments of differing size in different clinical isolates and was characterized in detail. Sequence analysis of this fragment in detail. Sequence analysis of this fragment showed that it comprised three tandem copies of a novel 75 bp repeat element flanked by multiple copies of the previously described 10 bp major polymorphic tandem repeat of M. tuberculosis (MPTR). The copy number of the 75 bp repeat was found to vary between strains, allowing application of a polymerase chain reaction amplification strategy for strain differentiation. These results indicate that the katG region of the M. tuberculosis genome is highly variable and unstable. The presence of repetitive sequences may contribute to instability in this region of the genome.
Mol Microbiol 1994 Oct
PMID:Strain variation in the katG region of Mycobacterium tuberculosis. 783 May 74

Understanding promoter regulation and signal-transduction systems in pathogenic mycobacteria is critical for uncovering the processes that govern interactions of these bacteria with the human host. In order to develop additional genetic tools for analysis of mycobacterial promoters, the xyIE gene from Pseudomonas was tested as a transcriptional fusion reporter in fast- and slow-growing mycobacteria. Initially, its utility was demonstrated by expression behind the hsp60 promoter in Mycobacterium smegmatis and Mycobacterium bovis BCG. The presence of an active promoter in front of the promoterless xyIE cassette on a plasmid was scored by development of a bright yellow colour upon spraying of mycobacterial colonies on plates with a solution of catechol. The gene product of xyIE, catechol 2,3 dioxygenase, was measurable in sonic extracts and whole cells, permitting quantitative determination of promoter activity in both fast- and slow-growing mycobacteria. The xyIE-based mycobacterial transcriptional fusion plasmid pRCX3 was constructed and used to assess promoter activity within the sequences located upstream of the newly characterized Mycobacterium tuberculosis H37Rv response regulator mtrA, a member of the superfamily of bacterial signal-transduction systems.
Mol Microbiol 1994 Sep
PMID:Gene expression in mycobacteria: transcriptional fusions based on xylE and analysis of the promoter region of the response regulator mtrA from Mycobacterium tuberculosis. 785 20

Triplex-polymerase chain reaction technique (PCR) was developed for the detection and identification of mycobacterial DNA sequences in uncultured clinical samples. A 123 bp fragment corresponding to a specific Mycobacterium tuberculosis sequence complex, a 383 bp DNA fragment encoding for part of the 65 kD mycobacterial surface antigen, and a 268 bp fragment of the human beta-globin gene to demonstrate the presence of suitable DNA were amplified by triplex PCR. To demonstrate the applicability of this method, 206 alcohol-fixed, paraffin-embedded sputum samples from 47 patients with culture-proven tuberculosis were investigated. Of 206 samples, 157 were PCR positive, resulting in correct diagnosis of tuberculosis in 46 of 47 (97.8%) patients. Furthermore, 165 alcohol-fixed, auramin-stained sputum smears were examined in a blind trial. Triplex PCR revealed tuberculosis in 20 of 21 samples from patients with tuberculosis. In comparison, cultures were positive in 20 of 21 samples, and acid-fast organisms were found by microscopy in 18 of 21 samples. We conclude that triplex PCR is a rapid and sensitive technique for the detection of mycobacterial DNA in uncultured clinical samples and offers equivalent sensitivity (95.2%) and specificity (98.6%) as do culture methods.
Diagn Mol Pathol 1994 Dec
PMID:Rapid detection of mycobacterial DNA in clinical samples by multiplex PCR. 786 36


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