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)

Protein splicing is a self-catalyzed process involving the excision of an intervening polypeptide sequence, the intein, and joining of the flanking polypeptide sequences, the extein, by a peptide bond. We have studied the in vitro splicing of erythropoietin (EPO) using a truncated form of the Mycobacterium tuberculosis RecA mini-intein in which the homing endonuclease domain was replaced with a hexahistidine sequence (His-tag). The intein was inserted adjacent to cysteine residues to assure that the spliced product had the natural amino acid sequence. When expressed in Escherichia coli, intein-containing EPO was found entirely as inclusion bodies but could be refolded in soluble form in the presence of 0.5 M arginine. Protein splicing of the refolded protein could be induced with a reducing agent such as DTT or tris(2-carboxyethyl)phosphine and led to the formation of EPO and mini-intein along with some cleavage products. Protein splicing mediated by the RecA intein requires the presence of a cysteine residue adjacent to the intein insertion site. We compared the efficiencies of protein splicing adjacent to three of the four cysteine residues of EPO (Cys29, Cys33 and Cys161) and found that insertion of intein adjacent to Cys29 allowed far more efficient protein splicing than insertion adjacent to Cys33 or Cys161. For ease of purification, our experiments involved a His-tagged EPO fusion protein and a His-tagged intein and the spliced products (25 kDa EPO and 24 kDa mini-intein) were identified by Western blotting using anti-EPO and anti-His-tag antibodies and by mass spectroscopy. The optimal splicing yield at Cys29 (40%) occurred at pH 7.0 after refolding at 4 degrees C and splicing for 18 h at 25 degrees C in the presence of 1 mM DTT.
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PMID:In vitro splicing of erythropoietin by the Mycobacterium tuberculosis RecA intein without substituting amino acids at the splice junctions. 1252 16

Mycobacterium tuberculosis RecA intein (PI-MtuI), a LAGLIDADG homing endonuclease, displays dual target specificity in response to alternative cofactors. While both ATP and Mn(2+) were required for optimal cleavage of an inteinless recA allele (hereafter referred to as cognate DNA), Mg(2+) alone was sufficient for cleavage of ectopic DNA sites. In this study, we have explored the ability of PI-MtuI to catalyze ATP hydrolysis in the presence of alternative metal ion cofactors and DNA substrates. Our results indicate that PI-MtuI displays maximum ATPase activity in the presence of cognate but not ectopic DNA. Kinetic analysis revealed that Mn(2+) was able to stimulate PI-MtuI catalyzed ATP hydrolysis, whereas Mg(2+) failed to do so. Using UV crosslinking, limited proteolysis and amino acid sequence analysis, we show that (32)P-labeled ATP was bound to a 14 kDa peptide containing the putative Walker A motif. Furthermore, the limited proteolysis approach disclosed that cognate DNA was able to induce structural changes in PI-MtuI. Mutation of the presumptive metal ion-binding ligands (Asp122 and Asp222) in the LAGLIDADG motifs of PI-MtuI impaired its affinity for ATP, thus resulting in a reduction in or loss of its endonuclease activity. Together, these results suggest that PI-MtuI is a (cognate) DNA- and Mn(2+)-dependent ATPase, unique from the LAGLIDADG family of homing endonucleases, and implies a possible role for ATP hydrolysis in the recognition and/or cleavage of homing site DNA sequence.
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PMID:Mycobacterium tuberculosis RecA intein, a LAGLIDADG homing endonuclease, displays Mn(2+) and DNA-dependent ATPase activity. 1285 36

Genome sequencing projects have focused attention on the problem of discovering the functions of protein domains that are widely distributed throughout living species but which are, as yet, largely uncharacterized. One such example is the PIN domain, found in eukaryotes, bacteria, and Archaea, and with suggested roles in signaling, RNase editing, and/or nucleotide binding. The first reported crystal structure of a PIN domain (open reading frame PAE2754, derived from the crenarchaeon, Pyrobaculum aerophilum) has been determined to 2.5 A resolution and is presented here. Mapping conserved residues from a multiple sequence alignment onto the structure identifies a putative active site. The discovery of distant structural homology with several exonucleases, including T4 phage RNase H and flap endonuclease (FEN1), further suggests a likely function for PIN domains as Mg2+-dependent exonucleases, a hypothesis that we have confirmed in vitro. The tetrameric structure of PAE2754, with the active sites inside a tunnel, suggests a mechanism for selective cleavage of single-stranded overhangs or flap structures. These results indicate likely DNA or RNA editing roles for prokaryotic PIN domains, which are strikingly numerous in thermophiles, and in organisms such as Mycobacterium tuberculosis. They also support previous hypotheses that eukaryotic PIN domains participate in RNAi and nonsense-mediated RNA degradation.
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PMID:Distant structural homology leads to the functional characterization of an archaeal PIN domain as an exonuclease. 1473 48

Mycobacterial isolates were obtained by radiometric culture from 33 different species of captive or free-ranging animals (n = 106) and environmental sources (n = 3) from six geographic zones within the United States. The identities of all 109 isolates were confirmed by using mycobactin J dependence and characterization of five well-defined molecular markers, including two integration loci of IS900 (loci L1 and L9), one Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis)-specific sequence (locus 251), and one M. avium subsp. avium-specific marker (IS1245), as well as hsp65 and IS1311 restriction endonuclease analyses. Seventy-six acid-fast isolates were identified as M. paratuberculosis, 15 were identified as belonging to the M. avium-M. intracellulare complex (but not M. paratuberculosis), and the remaining 18 were identified as mycobacteria outside the M. avium-M. intracellulare complex. Fingerprinting by multiplex PCR for IS900 integration loci clustered 67 of the 76 M. paratuberculosis strains into a single clade (designated clade A18) and had a Simpson's diversity index (D) of 0.53. In contrast, sequence-based characterization of a recently identified M. paratuberculosis short sequence repeat (SSR) region enabled the differentiation of the M. paratuberculosis isolates in clade A18 into seven distinct alleles (D = 0.75). The analysis revealed eight subtypes among the 33 species of animals, suggesting the interspecies transmission of specific strains. Taken together, the results of our analyses demonstrate that SSR analysis enables the genetic characterization of M. paratuberculosis isolates from different host species and provide evidence for the host specificity of some M. paratuberculosis strains as well as sharing of strains between wild and domesticated animal species.
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PMID:Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species. 1507 Oct 28

PCR-restriction endonuclease analysis (PRA) was used for direct identification of Mycobacterium haemophilum in clinical specimens from immunocompromised patients. PRA correctly identified M. haemophilum in four smear-positive specimens. Direct identification by PRA takes 2 to 3 working days compared to the 3 to 5 weeks required for culture isolation and identification by conventional methods.
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PMID:Direct identification of Mycobacterium haemophilum in skin lesions of immunocompromised patients by PCR-restriction endonuclease analysis. 1524 10

Mutations at embB gene codons 306 and 497 and iniA gene codon 501 occur frequently in ethambutol (EMB)-resistant Mycobacterium tuberculosis strains worldwide. The identification of these mutations in resistant strains has been achieved by labor-intensive DNA sequencing or by tedious amplification protocols followed by restriction endonuclease digestion. In this report, we describe PCR-restriction fragment length polymorphism (RFLP)-based methods for determining substitutions at embB codons 306 and 497 and iniA codon 501 directly in BACTEC cultures of M. tuberculosis isolates. The wild-type and mutant alleles are revealed by easily interpretable and different RFLP patterns. The methods optimized initially on reference strains were tested directly on BACTEC cultures of 25 randomly selected clinical M. tuberculosis isolates, seven of which were determined to contain EMB-resistant strains by phenotypic drug susceptibility testing. The PCR-RFLP methods identified mutations in four of seven EMB-resistant strains with three isolates containing mutated embB codon 306 and one isolate containing mutated embB codon 497. The results of PCR-RFLP were confirmed by DNA sequencing. The worldwide prevalence figures for mutations at embB codons 306 and 497 and iniA codon 501 suggest that nearly half of EMB-resistant M. tuberculosis strains could be identified within one working day even in developing countries equipped with simple PCR technology instead of weeks required for phenotypic drug susceptibility testing. Further, since EMB resistance is also associated with multiple-drug resistance from some geographical locations, detection of EMB resistance may also lead to rapid identification of multidrug-resistant strains of M. tuberculosis.
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PMID:Rapid detection of ethambutol-resistant Mycobacterium tuberculosis strains by PCR-RFLP targeting embB codons 306 and 497 and iniA codon 501 mutations. 1529 17

Heat shock protein 65 (HSP65) is one of the most important protective immunogens against the tuberculosis infection. The signal sequence of antigen 85B and the whole HSP65 DNA sequence of human Mycobacterium tuberculosis (M. tuberculosis) were amplified from BCG genome and plasmid pCMV-MTHSP65 respectively by polymerase chain reactions (PCR). These two sequences were cloned into the plasmid pBCG-2100 under the control of the promoter of heat shock protein 70 (HSP70) from human M. tuberculosis, yielding the prokaryotic shuttle expression plasmid pBCG-SP-HSP65. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis showed that the two cloned DNA sequences were consistent with those previously reported, and the direction of their inserting into the recombinant was correct and the reading frame had been maintained. The recombinants were electroporated into BCG to construct the recombinant BCG vaccine and induced by heating. The induced expression detected by SDS-PAGE showed that the content of 65 kD protein expressed in recombinant BCG was 35.69% in total bacterial protein and 74.09% in the cell lysate supernatants, suggesting that the recombinant HSP65 gene could express in BCG with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive recombinant proteins could specifically combine with antibody against M. tuberculosis HSP65, indicating that the recombinant proteins possess the biological activity of HSP65.
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PMID:Construction, expression and identification of a recombinant BCG vaccine encoding human Mycobacterium tuberculosis heat shock protein 65. 1531 55

Resistance to antituberculous agents is an important cause of ineffectiveness of antimicrobial therapy. The resistance of M. tuberculosis to antituberculous agents is a result of mutations in genes participating in those agent's action. The antituberculous drug--isoniazid can be activated by Mycobacterium tuberculosis either through a hydroperoxidase I/II or a superoxide-dependent oxyferrous pathway. The present study analyzed the frequency of the mutations occurring in codons 315 and 463 in katG gene of Mycobacterium tuberculosis strains, isolated from patients with pulmonary tuberculosis from Silesia, Poland. In this study 23 isoniazid-resistant Mycobacterium tuberculosis strains were analyzed. For RFLP analysis, a 620 bp amplified fragment of katG gene was digested with restriction endonuclease MspI. Among 24 isoniazid-resistant strains, isolated from patients between 2000-2001, point mutations were found in 30% of analyzed isoniazid-resistant strains in codons 315 or 463 (7 strains). In contrast, no mutations in codons 315 and/or 463 katG gene were found in 16 strains (70%). Obtained results suggests that point mutations S315T (AGC-->ACC) and R463L in katG gene are infrequent in the analyzed population.
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PMID:PCR-RFLP analysis of a point mutation in codons 315 and 463 of the katG gene of Mycobacterium tuberculosis isolated from patients in Silesia, Poland. 1547 53

Cerebrospinal fluid (CSF) obtained from patients with partially treated and culture-negative meningitis was subjected to PCR using 16S rDNA universal primers followed by restriction endonuclease digestion. In all, 43 patients and 7 controls were enrolled in this study. Twenty-one meningitic samples were positive by PCR. Mycobacterium tuberculosis was the causative agent in seven cases followed by Haemophilus influenzae (four), Streptococcus pneumoniae (two), Listeria monocytogenes (one), Escherichia coli (one), Pseudomonas aeruginosa (one) and Staphylococcus aureus (one). Only two meningitic CSF samples were culture-positive. In this study, PCR using bacterial 16S rDNA specific universal primers was found to be superior to conventional methods in the diagnosis of partially treated meningitis.
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PMID:Diagnosis of partially treated culture-negative bacterial meningitis using 16S rRNA universal primers and restriction endonuclease digestion. 1588 61

Rifampin resistance in Mycobacterium tuberculosis is mainly due to a small number of mutations in the rpoB gene coding for the beta subunit of RNA polymerase. Heteroresistance, which is defined as a mixture of wildtype and resistant subpopulations in the same culture, can influence the sensitivity of molecular tests for determining drug resistance by leading to false negative or indeterminable results. In our laboratory, we identified one heteroresistant strain during sequencing of the rpoB gene mutations, which are responsible for Rifampin resistance in M. tuberculosis. The sequence analysis of this isolate demonstrated the mixture of different strains, and the exact nature of the mutation could not be determined. In order to solve this problem, the possible mutation site was digested with Tsp509I restriction endonuclease, which made us separate the different strains. Sequencing of the separated mutated product revealed the mutation responsible for Rifampin resistance. From this result, we propose that, when the suggested mutation site creates and/or deletes a restriction endonuclease recognition site in heteroresistant populations, restriction endonuclease analysis can be used to ease the determination of resistance mutations by sequencing.
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PMID:Restriction endonuclease analysis as a solution for determining rifampin resistance mutations by automated DNA sequencing in heteroresistant Mycobacterium tuberculosis strains. 1591 Feb 27


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