Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:BIOD00001 (DNase I)
 8,324 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasmid pAL5000 from Mycobacterium fortuitum encodes two proteins necessary for replication: RepA (307 amino acid residues) and RepB (119 residues). A single RNA species encoding these proteins was characterized, and its 5' end was defined. The proteins were expressed as maltose-binding protein fusions in Escherichia coli. The RepB protein was shown in vitro to bind specifically to a previously defined 435-bp region of pAL5000 containing the origin of replication (ori). The precise RepB binding sites were defined by DNase I footprinting experiments. RepB binds to two motifs in the ori region: a high-affinity site within its own promoter region, implying autoregulation of its expression, and a low-affinity site further upstream, presumably the origin of replication itself. The binding to the latter motif seems to occur on one DNA strand only. The high-affinity binding site contains several palindromic sequences. Gel retardation assays were performed with the different binding sites as templates, and the binding constant to each site was estimated from protein titrations. This is the first molecular dissection of mycobacterial DNA-binding proteins and their interactions with their targets.
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PMID:Protein-DNA interactions in the ori region of the Mycobacterium fortuitum plasmid pAL5000. 895 84

Lysogenization of mycobacteriophage L5 involves integration of the phage genome into the Mycobacterium smegmatis chromosome. Integration occurs by a site-specific recombination event between a phage attachment site, attP, and a bacterial attachment site, attB, which is catalyzed by the phage-encoded integrase protein. DNase I footprinting reveals that L5 integrase binds to two types of sites within attP which span an unexpectedly large region of 413 bp: seven arm-type sites (P1 to P7) each of which correspond to a consensus sequence 5'-TGCaaCtcYy, and core-type sites at the points of strand exchange. Mutational analyses indicate that not all of the arm-type sites are required for integration, and that the P3 site and the rightmost pair of sites (P6 and P7) are dispensable for integration. We show that a 252 bp segment of attP DNA is sufficient for efficient integrative recombination and that int can be provided in trans for simple and efficient transformation of the mycobacteria.
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PMID:Characterization of the mycobacteriophage L5 attachment site, attP. 905 72

Exochelin is the primary extracellular siderophore of Mycobacterium smegmatis, and the iron-regulated fxbA gene encodes a putative formyltransferase, an essential enzyme in the exochelin biosynthetic pathway (E. H. Fiss, Y. Yu, and W. R. Jacobs, Jr., Mol. Microbiol. 14:557-569, 1994). We investigated the regulation of fxbA by the mycobacterial IdeR, a homolog of the Corynebacterium diphtheriae iron regulator DtxR (M. P. Schmitt, M. Predich, L. Doukhan, I. Smith, and R. K. Holmes, Infect. Immun. 63:4284-4289, 1995). Gel mobility shift experiments showed that IdeR binds to the fxbA regulatory region in the presence of divalent metals. DNase I footprinting assays indicated that IdeR binding protects a 28-bp region containing a palindromic sequence of the fxbA promoter that was identified in primer extension assays. fxbA regulation was measured in M. smegmatis wild-type and ideR mutant strains containing fxbA promoter-lacZ fusions. These experiments confirmed that fxbA expression is negatively regulated by iron and showed that inactivation of ideR results in iron-independent expression of fxbA. However, the levels of its expression in the ideR mutant were approximately 50% lower than those in the wild-type strain under iron limitation, indicating an undefined positive role of IdeR in the regulation of fxbA.
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PMID:Transcriptional control of the iron-responsive fxbA gene by the mycobacterial regulator IdeR. 1034 51

A mycobacterial cell wall complex prepared from the non-pathogenic microorganism Mycobacterium phlei, where mycobacterial DNA is preserved and complexed to cell wall fragments, possesses anticancer and immunomodulatory activity. DNA from a number of prokaryotes has been found to modulate the immune system and to induce cytokine synthesis. We have therefore determined whether the DNA associated with this complex has the ability to induce the synthesis of interleukin-12 (IL-12), a potent anticancer cytokine. Mycobacterial DNA complexed with cell wall fragments or DNA purified from M. phlei induced IL-12 synthesis by murine and human monocytes and macrophages in vitro, and was capable of inducing IL-12 synthesis in vivo in mice following i.p. administration. Neutralization of DNA with cationic liposomes or digestion with DNase I significantly decreased the ability of the cell wall complex to induce IL-12. CpG methylation of DNA extracted from these cell walls or from M. phlei did not affect the induction of IL-12 synthesis by monocytes and macrophages. In contrast, CpG methylation of DNA from Escherichia coli abolished its ability to induce IL-12 synthesis. These results demonstrate that unmethylated CpG motifs present in M. phlei DNA are not a prerequisite for the induction of IL-12 synthesis. The size of the mycobacterial DNA, in the range of 5 bp to genomic DNA, did not influence its capacity to induce IL-12. Our results emphasize that M. phlei DNA associated with the cell wall complex makes a significant contribution to the overall immunomodulatory and anticancer activity of this mycobacterial cell wall preparation and that these activities are not correlated with the presence of CpG motifs.
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PMID:Modulation of interleukin-12 synthesis by DNA lacking the CpG motif and present in a mycobacterial cell wall complex. 1094 15

We have investigated interaction of Mycobacterium smegmatis topoisomerase I at its specific recognition sequence. DNase I footprinting demonstrates a large region of protection on both the scissile and non-scissile strands of DNA. Methylation protection and interference analyses reveal base-specific contacts within the recognition sequence. Missing contact analyses reveal additional interactions with the residues in both single and double-stranded DNA, and hence underline the role for the functional groups associated with those bases. These interactions are supplemented by phosphate contacts in the scissile strand. Conformation specific probes reveal protein-induced structural distortion of the DNA helix at the T-A-T-A sequence 11 bp upstream to the recognition sequence. Based on these footprinting analyses that define parameters of topoisomerase I-DNA interactions, a model of topoisomerase I binding to its substrate is presented. Within the large protected region of 30 bp, the enzyme makes direct contact at two locations in the scissile strand, one around the cleavage site and the other 8-12 bases upstream. Thus the enzyme makes asymmetric recognition of DNA and could carry out DNA relaxation by either of the two proposed mechanisms: enzyme bridged and restricted rotation.
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PMID:A novel bipartite mode of binding of M. smegmatis topoisomerase I to its recognition sequence. 1155 91

Hybrids of the Streptomyces coelicolor conjugative plasmid SCP2* and the Mycobacterium plasmid pAL5000 were transferred from Streptomyces coelicolor or Streptomyces lividans to Mycobacterium smegmatis mc2155 in plate crosses. Inactivation of the SCP2* transfer function did not prevent or reduce plasmid transfer. This transfer was DNase I sensitive and thus involved release of DNA from Streptomyces, followed by transformation of M. smegmatis. M. smegmatis growing on specific solid media was also transformed by pure CCC and linear plasmid DNA. Small plasmids were taken up intact but large plasmids suffered deletions. Competence developed within 24 h of incubation at 30 degrees C or 37 degrees C, and up to 400 transformants were obtained per microg of CCC plasmid DNA. Transformation frequencies were higher when M. smegmatis was co-cultivated with plasmid-free Streptomyces, but unaffected by resident homologous sequences or inactivation of recA in M. smegmatis. Spontaneous transformation was also observed with a circular Streptomyces transposable element which inserted into chromosomal sites. Transformative plasmid transfer was also shown to occur between M. smegmatis strains. This is the first report of non-artificially induced, spontaneous plasmid transformation in Mycobacterium.
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PMID:Plasmid transfer from Streptomyces to Mycobacterium smegmatis by spontaneous transformation. 1184 42

The TrcRS two-component system of Mycobacterium tuberculosis is comprised of the TrcS histidine kinase and the TrcR response regulator, which is homologous to the OmpR class of DNA binding response regulators. Reverse transcription-PCRs with total RNA showed that the trcR and trcS two-component system genes are transcribed in broth-grown M. tuberculosis. Analysis of the trcR and trcS genes using various SCOTS (selective capture of transcribed sequences) probes also confirmed that these genes are expressed in broth-grown cultures and after 18 h of M. tuberculosis growth in cultured human primary macrophages. To determine if the TrcR response regulator is autoregulated, a trcR-lacZ fusion plasmid and a TrcR expression plasmid were cotransformed into Escherichia coli. Upon induction of the TrcR protein, there was a >500-fold increase in beta-galactosidase activity from the trcR-lacZ fusion, indicating that TrcR is involved in transcriptional autoactivation. Gel mobility shift assays with the trcR promoter and TrcR established that the response regulator was autoregulating via direct binding. By use of a delimiting series of overlapping trcR PCR fragments in gel mobility shift assays with TrcR, an AT-rich region of the trcR promoter was shown to be essential for TrcR binding. Additionally, this AT-rich sequence was protected by TrcR in DNase I protection assays. To further analyze the role of the AT-rich region in TrcR autoregulation, the trcR promoter was mutated and analyzed in lacZ transcriptional fusions in the presence of TrcR. Alteration of the AT-rich sequence in the trcR promoter resulted in the loss of trcR transcriptional activation in the presence of TrcR. This report indicates that the M. tuberculosis TrcR response regulator activates its own expression by interacting with the AT-rich sequence of the trcR promoter.
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PMID:Expression, autoregulation, and DNA binding properties of the Mycobacterium tuberculosis TrcR response regulator. 1191 51

We have previously reported that NF-kappa B and stimulating factor 1 elements within the proximal mouse Toll-like receptor 2 (TLR2) promoter region are required for the transcriptional activation of TLR2 expression following infection with Mycobacterium avium. In the present study, we found that a rapid increase in both DNase I sensitivity and restriction enzyme accessibility at the TLR2 promoter region occurred following infection with M. avium. Increase in restriction enzyme accessibility at the TLR2 promoter region covering the NF-kappa B and stimulating factor 1 elements was associated with the induction of TLR2 expression at the mRNA level. Furthermore, the increase in restriction enzyme accessibility at the TLR2 promoter region did not appear to result from binding of NF-kappa B, but rather depended on a TLR2-myeloid differentiation factor 88 signaling pathway. Together our results indicate that chromatin remodeling occurs at TLR2 promoter region following infection with M. avium, allowing the access of transcription factors to initiate the transcription of TLR2.
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PMID:Rapid chromatin remodeling of Toll-like receptor 2 promoter during infection of macrophages with Mycobacterium avium. 1209 82

In a systematic approach to understand the transcriptional machinery of mycobacteria, we had previously isolated and characterized mycobacterial promoter regions. In this study, we have investigated molecular interactions between mycobacterial RNA polymerase holoenzyme, reconstituted with different sigma subunits and the promoter element of the Mycobacterium tuberculosis gene pknH (Rv1266c), a representative of promoters belonging to the 'extended -10' class. In vitro transcription assays using the pknH promoter and reconstituted RNA polymerase holoenzyme demonstrated that transcription from the pknH promoter is specifically initiated by sigmaA, the principal sigma factor of mycobacteria. DNase I protection assay and deletion studies with the pknH promoter revealed that the minimal region required for optimal transcription carries the sequence from position -37 to position +6. Moreover, mutation in the TGN motif of the pknH promoter resulted in the loss of >75% of its activity. Binding of RNA polymerase with wild-type promoter as well as its TG- mutant revealed that the TGN motif is required for the transition from a close complex into an open complex. Further, it was observed that the presence of the TGN motif reduces the thermal energy required for the conversion of a close complex into an open complex, necessary for initiation of transcription.
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PMID:Role of 5'-TGN-3' motif in the interaction of mycobacterial RNA polymerase with a promoter of 'extended -10' class. 1290 24

The Mycobacterium tuberculosis oriC (the origin of chromosomal replication) region contains 13 non-perfect DnaA boxes. The M. tuberculosis initiator protein, DnaA, was overexpressed in Escherichia coli as a soluble His-tagged fusion protein. The purified protein His6MtDnaA was investigated for its binding properties to DnaA boxes from the oriC region. Gel retardation demonstrated that the DnaA from M. tuberculosis requires two DnaA boxes for efficient binding. Electron microscopy as well as DNase I footprinting showed that the His6MtDnaA protein binds to four specific regions, which correspond to the location of 11 out of 13 previously identified DnaA boxes within the M. tuberculosis oriC. Probably, in M. tuberculosis, DnaA molecules by co-operative binding of numerous 'non-perfect' DnaA boxes assemble along the oriC region and subsequently form a massive nucleoprotein complex.
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PMID:Mycobacterium tuberculosis DnaA initiator protein: purification and DNA-binding requirements. 1513 7


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