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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A series of Escherichia coli-mycobacteria shuttle plasmids for the isolation and study of gene regulatory sequences was constructed. These pJEM vectors contain an efficient transcription terminator and multiple cloning sites and allow either operon or gene fusions to lacZ. By constructing operon fusions with pJEM15, we assessed various previously characterized mycobacterial promoters in the fast-growing species Mycobacterium smegmatis and the slow-growing species M. bovis BCG. Our results suggest that M. smegmatis and M. bovis BCG RNA polymerases do not share the same specificity. To isolate new mycobacterial promoters, an M. tuberculosis DNA library was generated, using pJEM13, and screened in M. smegmatis. Several Lac+ clones were isolated, and the beta-galactosidase activity was measured.
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PMID:Escherichia coli-mycobacteria shuttle vectors for operon and gene fusions to lacZ: the pJEM series. 796 29

Sera from lepromatous leprosy patients were used to screen a Mycobacterium leprae lambda gt11 library. Three positive plaques were picked, and lysogens were constructed. Immunoblot analysis showed that all of the lysogens expressed an apparently identical beta-galactosidase fusion protein which reacted strongly with the sera. The 1.7-kbp insert from one clone was subcloned into the lacZ gene in pUR290; sequence analysis of the end fused to lacZ revealed an open reading frame with no significant homology to previously published sequences. The insert was used to screen an M. leprae cosmid library, and five clones were isolated. The insert was also found to hybridize to clones expressing the M. leprae antigen which had previously been designated class III and 25L. A 1.8-kbp HindIII fragment was subcloned from one of the cosmids and sequenced. The sequence revealed a 1,227-bp open reading frame, encoding a 408-amino-acid protein with a predicted molecular mass of 42,466 Da. The protein contains amino- and carboxy-terminal hydrophobic domains and a hydrophilic central domain; the amino-terminal domain shows some homology to a 51-kDa hypothetical antigen of Mycobacterium tuberculosis, while the hydrophilic region contains a high proportion of serine residues, and we have therefore designated the protein serine-rich antigen (Sra). Some repeated motifs are present in the protein, but their significance is unknown. Seventy-eight percent of serum samples from multibacillary leprosy patients and 68% of serum samples from paucibacillary leprosy patients recognized the fusion protein, showing that this is a major M. leprae antigen. In contrast, all serum samples from endemic controls were negative, while 26% of serum samples from tuberculosis patients were weakly positive.
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PMID:Sequence and immunological characterization of a serine-rich antigen from Mycobacterium leprae. 847 4

A gene conferring low-level isoniazid (INH) resistance on Mycobacterium smegmatis was isolated from a cosmid library of the genome of an INH-resistant Mycobacterium bovis strain. The gene had good homology with ahpC, the product of which is a subunit of alkyl hydroperoxide reductase, and also with a family of thiol-specific antioxidant enzymes. A mutation was found in the promoter upon comparison with the equivalent DNA sequence from the INH-sensitive parent strain. Promoter sequences from other INH-sensitive and INH-resistant M. bovis and Mycobacterium tuberculosis strains were sequenced and the mutation was found only in the INH-resistant strains. An INH-resistant M. tuberculosis strain also had an additional mutation in the promoter region. The wild-type promoter and promoters with one and two mutations were ligated into a reporter plasmid containing the lacZ gene. The presence of the first mutation resulted in a sixfold induction of beta-galactosidase activity, and the presence of both mutations caused a 10-fold induction. Increased expression of AhpC may account for some of the INH resistance of strains of the M. tuberculosis complex.
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PMID:ahpC, a gene involved in isoniazid resistance of the Mycobacterium tuberculosis complex. 883 Feb 60

D-Cycloserine is an effective second-line drug against Mycobacterium avium and Mycobacterium tuberculosis. To analyze the genetic determinants of D-cycloserine resistance in mycobacteria, a library of a resistant Mycobacterium smegmatis mutant was constructed. A resistant clone harboring a recombinant plasmid with a 3.1-kb insert that contained the glutamate decarboxylase (gadA) and D-alanine racemase (alrA) genes was identified. Subcloning experiments demonstrated that alrA was necessary and sufficient to confer a D-cycloserine resistance phenotype. The D-alanine racemase activities of wild-type and recombinant M. smegmatis strains were inhibited by D-cycloserine in a concentration-dependent manner. The D-cycloserine resistance phenotype in the recombinant clone was due to the overexpression of the wild-type alrA gene in a multicopy vector. Analysis of a spontaneous resistant mutant also demonstrated overproduction of wild-type AlrA enzyme. Nucleotide sequence analysis of the overproducing mutant revealed a single transversion (G-->T) at the alrA promoter, which resulted in elevated beta-galactosidase reporter gene expression. Furthermore, transformants of Mycobacterium intracellulare and Mycobacterium bovis BCG carrying the M. smegmatis wild-type alrA gene in a multicopy vector were resistant to D-cycloserine, suggesting that AlrA overproduction is a potential mechanism of D-cycloserine resistance in clinical isolates of M. tuberculosis and other pathogenic mycobacteria. In conclusion, these results show that one of the mechanisms of D-cycloserine resistance in M. smegmatis involves the overexpression of the alrA gene due to a promoter-up mutation.
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PMID:Overexpression of the D-alanine racemase gene confers resistance to D-cycloserine in Mycobacterium smegmatis. 926 Sep 45

Mycobacterium aurum is considered a surrogate of M. tuberculosis and recently has been proposed as test organism in high throughput screening of antituberculosis drugs (3). In this investigation, we suggest use of a recombinant M. aurum expressing E. coli lacZ gene, in which beta-galactosidase production is the reporter system as recently reported by us (6). The assay is based on production of beta-galactosidase in presence of drugs during growth. Enzyme production was inhibited within 4 h by frontline antimycobacterial drugs like streptomycin, rifampicin, isoniazid, ethambutol, ofloxacin, and sparfloxacin at their MICs. The assay could be performed conveniently in 96-well microtiter plate format.
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PMID:Recombinant Mycobacterium aurum expressing Escherichia coli beta-galactosidase in high throughput screening of antituberculosis drugs. 939 99

To gain a better understanding of the pathological role of interferon-gamma (IFN-gamma) in specific granuloma formation, IFN-gamma gene-deficient mice (BALB/c and C57BL/6) were produced. The IFN-gamma gene in embryonic stem (ES) cells was disrupted by inserting the beta-galactosidase gene (lacZ) and the neomycin resistance gene (neo) at the translation initiation site in exon 1 by homologous recombination. Six-week-old IFN-gamma-deficient and wild-type mice were inoculated with 10(3)-10(7) bacilli of various strains of Mycobacterium tuberculosis (Kurono, H37Rv, H37Ra and BCG Pasteur) through their tail veins. The mice were examined 7 weeks later for granuloma formation. The avirulent BCG Pasteur and H37Ra strains (10(3)-10(4) bacilli/ml) induced granulomas in the spleen, liver and lungs of IFN-gamma-deficient mice. The granulomas consisted of epithelioid macrophages and Langhans multinucleate giant cells, but lacked caseous necrosis. The virulent Kurono and H37Rv strains induced disseminated abscesses but not granulomas in various organs of IFN-gamma-deficient mice and Mac-3-positive macrophages were not detected in the abscess lesions. These results suggest that IFN-gamma may be primarily responsible for macrophage activation and that other factor(s) may be involved in the granuloma formation mechanism.
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PMID:Induction of granulomas in interferon-gamma gene-disrupted mice by avirulent but not by virulent strains of Mycobacterium tuberculosis. 978 10

Mycobacterium tuberculosis is a slow-growing pathogen and is characterized by a low content of RNA per unit of DNA. rRNAs represent a major proportion of the total RNA pool, and the entire requirement for rRNA is met by transcription from a single rrn operon that is driven by two promoters, P1 and P3. This study attempted to analyze the specific role of the rrn promoter in determining the characteristically low levels of RNA in M. tuberculosis. For this purpose, the activity of the M. tuberculosis rrn promoter as a function of the growth rate was studied by rrn-lacZ promoter fusion, hybridization, and primer extension analysis in M. smegmatis. rrn promoter signals were faithfully recognized in M. smegmatis cultures harboring the rrn-lacZ promoter construct. In M. smegmatis cultures that displayed doubling times varying between 3.06 and 6.5 h, beta-galactosidase activity increased approximately sixfold in proportion to the growth rate (mu). There was a corresponding increase in the amount of lacZ-specific mRNA, while the plasmid copy number remained essentially unchanged. For any given mu, the P3 promoter was approximately twofold more efficiently utilized than the P1 promoter. Since both promoters of the M. tuberculosis rrn operon are regulatable as a function of growth rate in M. smegmatis cultures, it is implied that the inherent structure or sequence of the rrn promoter per se is not primarily responsible for the observed lack of modulation of RNA synthesis in M. tuberculosis.
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PMID:Mycobacterium tuberculosis rrn promoters: differential usage and growth rate-dependent control. 1040 May 91

A polyclonal serum sample from a lepromatous leprosy (LL) patient, which presented a specific recognition pattern for leprosin, was used to screen a Mycobacterium leprae genomic library constructed with DNA isolated from human lepromas. One clone, designated ML4-1, which expressed a specific antigenic determinant of M. leprae as part of a beta-galactosidase fusion protein, was isolated. The 1.932 bp M. leprae-derived genomic fragment was sequenced, and it had an incomplete open-reading frame shown to code for a 644 amino-acid polypeptide (72.3 kDa). Some partial nucleotide homology to the M. tuberculosis MTCY9C4 cosmid and the M. leprae B1913 cosmid were found. Southern blot assays using the 584 bp Eco RI-Bam HI fragment excised from the ML4-1 clone revealed that this sequence is present only in the M. leprae genome and not in the 24 different mycobacterial DNA tested. Two oligonucleotides based on the genomic sequence were also synthesized and used as amplifiers for a polymerase chain reaction (PCR) test, giving a positive signal exclusively in M. leprae DNA. Furthermore, 32 sequential synthetic peptides, 20 amino-acids long, spanning the entire protein corresponding to the hypothetical ML4-1 clone sequence, were synthesized and evaluated by ELISA. A peptide included in the 221-240 region was significantly recognized by either lepromatous leprosy or healthy tuberculosis contact patient sera. Thus, PCR amplification of this fragment, along with the recognition of its protein sequence by leprosy patient sera, could be a useful tool for a potential diagnostic method in the detection of M. leprae infection in the future.
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PMID:Isolation, characterization, molecular cloning and amplification of a species-specific M. leprae antigen. 1070 Sep 13

By subtractive hybridization, we isolated genes, differentially expressed in virulent strain (dev), that are expressed at higher levels in the virulent Mycobacterium tuberculosis H37Rv strain in comparison to its avirulent counterpart, H37Ra, and consequently may be associated with the virulence phenotype of M. tuberculosis. A two-component system, devR-devS, was identified by DNA sequencing of a dev clone. DevR, the predicted gene product of devR, is a response regulator (RR) in the NarL/ UhpA subfamily of two-component systems. The devS gene product displayed homology with histidine protein kinases (HPKs) including UhpB, NarX and NarQ. The devR-devS locus is preceded by gene Rv3134c that encodes a putative alanine-aline- rich protein. This locus was conserved in M. tuberculosis and M. bovis BCG but not in other mycobacteria. A devR -lacZ transcription fusion demonstrated beta-galactosidase activity in M. smegmatis and in M. tuberculosis. The devR and devS genes were cotranscribed and the levels of their transcripts were lower in two isolates of the avirulent H37Ra strain in comparison to the virulent H37Rv strain of M. tuberculosis. The level of DevR protein was also lower in one of the H37Ra strains in comparison to the H37Rv strain. However, in a third isolate of H37Ra, RNA and protein expression was equivalent to that in the H37Rv strain. Electron microscopic immunogold analysis of M. tuberculosis grown in laboratory medium and within human monocytes revealed specific labelling for DevR protein within the bacteria and the phagosomal lumen of infected monocytes. These findings collectively suggest a potential role for devR-devS in the regulation of genetic programmes unique to the tubercle bacillus.
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PMID:Characterization of a two-component system, devR-devS, of Mycobacterium tuberculosis. 1097 Jul 62

The uptake and assimilation of nitrogen sources is effectively regulated in bacteria. In the Gram-negative enterobacterium Escherichia coli, the NtrB/C two-component system is responsible for the activation of transcription of different enzymes and transporters, depending on the nitrogen status of the cell. In this study, we investigated regulation of ammonium uptake in Corynebacterium glutamicum, a Gram-positive soil bacterium closely related to Mycobacterium tuberculosis. As shown by Northern blot hybridizations, regulation occurs on the level of transcription upon nitrogen starvation. In contrast to enterobacteria, a repressor protein is involved in regulation, as revealed by measurements of methylammonium uptake and beta-galactosidase activity in reporter strains. The repressor-encoding gene, designated amtR, was isolated and sequenced. Deletion of amtR led to deregulation of transcription of amt coding for the C. glutamicum (methyl)ammonium uptake system. E. coli extracts from amtR-expressing cells were applied in gel retardation experiments, and binding of AmtR to the amt upstream region was observed. By deletion analyses, a target motif for AmtR binding was identified, and binding of purified AmtR protein to this motif, ATCTATAGN1-4ATAG, was shown. Furthermore, the binding of AmtR to this sequence was proven in vivo using a yeast one-hybrid system. Subsequent studies showed that AmtR not only regulates transcription of the amt gene but also of the amtB-glnK-glnD operon encoding an amt paralogue, the signal transduction protein PII and the uridylyltransferase/uridylyl-removing enzyme, key components of the nitrogen regulatory cascade. In summary, regulation of ammonium uptake and assimilation in the high G+C content Gram-positive bacterium C. glutamicum differs significantly from the mechanism found in the low G+C content Gram-positive model organism Bacillus subtilis and from the paradigm of nitrogen control in the Gram-negative enterobacteria.
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PMID:AmtR, a global repressor in the nitrogen regulation system of Corynebacterium glutamicum. 1097 15


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