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)

We have used the polymerase chain reaction to alter transcriptional and translational signals surrounding the hinfIM gene [encoding M.HinfI methyltransferase (MTase)] so as to achieve overexpression in Escherichia coli. The PCR-generated hinfIM gene was subcloned in a high-expression vector under control of the hybrid trp-lac promoter. In addition, the positive retroregulator stem-loop sequence derived from the crystal protein-encoding gene of Bacillus thuringiensis was inserted downstream from hinfIM. Using a similar approach, we have also constructed overproducer clones of a deletion mutant of M.HinfI MTase that has 97 amino acids from the C terminus deleted. The plasmid from the mutant clones is fully protected from HinfI restriction endonuclease digestion. It appears that the functional properties (the recognition and catalytic functions) are encoded within this mutant gene. The overproducer clones yield the wild type (wt) and the mutant enzymes to about 10% of total cellular protein upon induction with 1 mM IPTG. The wt M.HinfI and the mutant MTase were purified to near electrophoretic homogeneity by phosphocellulose, DEAE and gel chromatography. Their monomer sizes by SDS/polyacrylamide-gel electrophoresis are 43 kDa and 31 kDa, respectively, in good agreement with that predicted from the nucleotide sequence. DNA methylation experiments with purified enzymes using single-strand and double-strand M13mp18 DNA substrates indicate that while wt enzyme methylates both forms of DNA substrates, the mutant enzyme appears to preferentially methylate ss DNA substrate.
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PMID:Overproduction, purification and characterization of M.HinfI methyltransferase and its deletion mutant. 156 35

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins (WCP), immunoblot analysis and DNA restriction-endonuclease analysis (REA) were applied as potential typing methods to 31 clinically significant strains of Moraxella (Branhamella) catarrhalis, five of which came from a suspected outbreak of nosocomial infection in a respiratory-diseases ward. Twelve of 31 isolates were placed in four groups, each of which contained strains indistinguishable by the three typing techniques used. Each of a further two groups contained two strains, and they were similar by at least one technique; the remaining 15 strains were unique by all three methods. Four of five strains from the suspected outbreak were indistinguishable by SDS-PAGE of WCP, immunoblotting and REA. Results show that SDS-PAGE of WCP, immunoblotting and REA are suitable techniques for characterising M. catarrhalis and that there is a considerable degree of strain heterogeneity. Nosocomial infection with M. catarrhalis may be relatively common and further epidemiological studies with a combination of typing techniques are indicated.
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PMID:Characterisation of hospital isolates of Moraxella (Branhamella) catarrhalis by SDS-PAGE of whole-cell proteins, immunoblotting and restriction-endonuclease analysis. 162 19

Previous papers have reported that the syncytial mutant HSV-1(13)S11 carries three segregable syn mutations and exhibits its altered phenotype in four different cell lines, i.e. HEp-2, VERO, BHK and HEL both at 34 degrees C and 39 degrees C. Those studies have shown that one of three syncytial loci, designated Syn 5, is located in the Bam HI Q fragment spanning map units 0.296-0.317 of the prototype arrangement. Recombinants obtained from marker transfer experiments with donor BamHI Q fragment, have shown that locus Syn 5 is able to induce cell-to-cell fusion in VERO, BHK and HEL but not in HEp-2 cells. In this paper we have characterized the syn mutant HSV-1(13)S11 with regard to plaque morphology, synthesis of viral polypeptides and glycoproteins, thymidine kinase activity and physical map position of locus Syn 5 on the genome. Pertinent to the syn phenotype, earlier papers claimed that two different polypeptides, thymidine kinase (TK) and glycoprotein H (gH), whose genes map in BamHI Q, may be responsible for the fusion activity. Functional studies on the TK of the syn mutant HSV-1(13)S11 indicate that this polypeptide accumulates normally in infected cells and is a fully active enzyme. The other gene product, gH, has been studied with SDS-PAGE and in radioimmunoprecipitation (RIP) experiments using specific monoclonal antibodies. The results indicate that the amount of gH accumulation in the syn mutant-infected cells is greater than its parental strain. However, new marker transfer experiments described here located locus Syn 5 in 663 base pairs between SstI and EcoRI restriction endonuclease sites at the right end of the BamHI Q fragment, where TK gene overlaps in opposite orientation with UL 24 gene. Altogether these results indicate that the Syn 5 locus segregates from the gene specifying gH, to a region encompassing portions of the TK and UL 24 genes, and that the syn mutation does not affect the expression or activity of TK.
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PMID:Phenotypic and genotypic characterization of locus Syn 5 in herpes simplex virus 1. 164 2

Thirty-two isolates of Coxiella burnetii collected from various hosts ranging from arthropods to man were compared by restriction endonuclease (RE) digestion patterns of chromosomal DNA using SDS-PAGE. SDS-PAGE provided better DNA fragment separation than agarose gel electrophoresis and enabled the differentiation of these isolates into six distinct groups on the basis of DNA restriction fingerprints. Two groups of chronic disease isolates could be distinguished, each having unique RE digestion patterns of chromosomal DNA. Three similar but distinct RE digestion patterns were seen among the group of acute disease isolates. Three additional isolates included in this study exhibited a unique RE digestion pattern and also had a unique plasmid type, designated QpDG. DNA-DNA hybridization on selected isolates quantified the relatedness between several groups and supported the classification of these groups as distinct strains.
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PMID:Differentiation of Coxiella burnetii isolates by analysis of restriction-endonuclease-digested DNA separated by SDS-PAGE. 167 52

Large amounts of histones, H1, H2A, H2B, H3, and H4, were observed in total extracts of T4 lymphocytes and derived cell lines infected with the human immunodeficiency virus (HIV) type 1 or type 2. These histones were simply detectable by analysis of crude cellular extracts by polyacrylamide gel electrophoresis in SDS and staining the proteins with Coomassie blue or by immunoblot assays using specific polyclonal antibodies. The histones were found to be localized in the nucleoplasm, bound to low molecular weight (LMW) DNA in the form of nucleosomes. The mechanism responsible for the accumulation of nucleosomes during HIV infection was found to be due to fragmentation of cellular DNA, a mechanism referred to as apoptosis or programmed cell death in which a nuclear endonuclease becomes activated and cleaves DNA at internucleosomal regions. Accordingly, the LMW DNA accumulated in the course of infection was found to have a characteristic pattern of nucleosomal ladder and its accumulation was reduced in the presence of zinc, a known inhibitor of the endonuclease. Routinely in acute HIV infections, the accumulation of nucleosomes was observed at least 24 hr before lysis of infected cells. In a particular HIV-1 infection, in which the first signals of the cytopathic effect (vacuolization of cells and appearance of syncytia) was observed at Days 6-7 whereas maximal virus production occurred at Days 10-17, the accumulation of nucleosomes was at its maximal level already on Day 6 postinfection. In the nucleoplasm of chronically infected cells producing virus but not manifesting a cytopathic effect, no LMW DNA or histones were detectable. These observations indicate that the cytopathic effect of HIV infection is associated with apoptosis. The detection of histones and oligonucleosomal DNA fragments in the nucleoplasm can be used as a convenient marker for chromatin fragmentation during this process.
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PMID:The cytopathic effect of HIV is associated with apoptosis. 168 28

Variation amongst strains of Helicobacter pylori was examined by 35S-methionine-labelled protein SDS-PAGE (Radio-PAGE), immunoblot and DNA fingerprinting techniques. These methods allowed discrimination amongst isolates and showed total correlation. Pig and baboon gastric Helicobacter pylori-like organisms were found to be very similar to Helicobacter pylori by both Radio-PAGE and immunoblotting, whereas a Helicobacter mustelae isolate was markedly different. The HindIII restriction endonuclease digest patterns of Helicobacter pylori DNA illustrated the considerable genomic variation of this organism. The Radio-PAGE and immunoblot typing methods both gave precise identification of Helicobacter pylori strains, but Radio-PAGE was found to give higher resolution and represents a standardised universally applicable fingerprinting method for Helicobacter pylori.
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PMID:Evaluation of fingerprinting methods for identification of Helicobacter pylori strains. 168 29

Ionizing radiation and radiomimetic compounds, such as hydrogen peroxide and bleomycin, generate DNA strand breaks with fragmented deoxyribose 3' termini via the formation of oxygen-derived free radicals. These fragmented sugars require removal by enzymes with 3' phosphodiesterase activity before DNA synthesis can proceed. An enzyme that reactivates bleomycin-damaged DNA to a substrate for Klenow polymerase has been purified from calf thymus. The enzyme, which has a Mr of 38,000 on SDS-PAGE, also reactivates hydrogen peroxide-damaged DNA and has an associated apurinic/apyrimidinic (AP) endonuclease activity. The N-terminal amino acid sequence of the purified protein matches that reported previously for a calf thymus enzyme purified on the basis of AP endonuclease activity. Degenerate oligonucleotide primers based on this sequence were used in the polymerase chain reaction to generate from a bovine cDNA library a fragment specific for the 5' end of the coding sequence. Using this cDNA fragment as a probe, several clones containing 1.35 kb cDNA inserts were isolated and the complete nucleotide sequence of one of these determined. This revealed an 0.95 kb open reading frame which would encode a polypeptide of Mr 35,500 and with a N-terminal sequence matching that determined experimentally. The predicted amino acid sequence shows strong homology with the sequences of two bacterial enzymes that repair oxidative DNA damage, ExoA protein of S. pneumoniae and exonuclease III of E. coli.
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PMID:Isolation of cDNA clones encoding an enzyme from bovine cells that repairs oxidative DNA damage in vitro: homology with bacterial repair enzymes. 170 95

To investigate the heterogeneity of Mycoplasma ovipneumoniae, sixty isolates from three sheep on each of twenty farms were examined by restriction endonuclease analysis (REA) and SDS-PAGE. All were found to be different except for three isolates obtained from one farm. The protein and REA patterns of individual isolates were both highly reproducible and remained unchanged following long term passage (approximately 400 generations) in vitro. No plasmids were detected in the twelve strains which were examined and when two isolates were co-cultured in vitro, no genetic interchange, as judged by changes in REA patterns were detected. Since the heterogeneity of M. ovipneumoniae when examined by SDS-PAGE is too great to allow groups to be recognised, it could be advantageous for this purpose if only surface proteins were compared. As a preliminary step to this end we have identified several surface proteins of M. ovipneumoniae and found that some are common to all strains, one surface protein was shared by five of the eight strains examined and another was unique to one strain. This approach has the potential to allow the recognition of grouping of M. ovipneumoniae isolates.
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PMID:A study of the heterogeneity of isolates of Mycoplasma ovipneumoniae from sheep in New Zealand. 177 56

The heterogeneity of Mycoplasma ovipneumoniae isolates from the lungs of sheep with chronic non-progressive pneumonia (CNP) from the same flock raised the possibility that multiple isolates derived from one lung were not all identical. To test this hypothesis, thirty isolates were obtained from each of six pneumonic sheep lungs at slaughter. Four lungs had relatively severe lesions and from each of these, three or four strains of M. ovipneumonia, distinguishable by REA and in most cases by SDS-PAGE, were detected. From the lungs of each of two sheep with mild lesions, two strains of M. ovipneumoniae were detected. Four isolates from one lung were further examined by restriction endonuclease analysis (REA) using many restriction endonucleases. Those which differed with EcoRI also differed when other restriction endonucleases were used. However, partial digests occurred mainly with those restriction endonucleases which recognise cytosine-rich sequences. The presence of multiple strains of one species of microorganism in individual lesions is an unusual concept which may not be limited to one disease or to one host.
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PMID:The isolation of multiple strains of Mycoplasma ovipneumoniae from individual pneumonic sheep lungs. 177 57

An ATP-dependent DNA aggregating activity was purified from rat liver by DEAE-cellulose, phosphocellulose, and novobiocin-Sepharose column chromatography. The protein aggregated superhelical, relaxed, single-, or double-stranded DNA in a divalent cation- and ATP-dependent reaction. The DNA aggregating activity was detected by retardation of a DNA-protein complex at the origin on a 1% agarose gel. The protein appeared to exist in solution as a monomer of molecular weight 66,000, and had no DNA polymerase, topoisomerase, recombinase, or ligase activity. The DNA aggregating activity was inhibited by 10 mM nalidixic acid or 1 mM novobiocin but not by 20 mM N-ethylmaleimide or camptothecin. Adenylyl(beta,gamma-methylene)-diphosphonate, adenylyl-imidodiphosphate, or adenosine-5'-O(3-thiotriphosphate) did not substitute for ATP whereas CTP, dTTP, or the ATP analog adenylyl(alpha,beta-methylene)-diphosphonate could replace ATP. The aggregated DNA was only partially dissociated by restriction endonuclease digestion but was completely dissociated by deproteinization with SDS, proteinase K, or chloroform/octanol extraction. On the basis of the molecular weight, thermostability, antigenic property, and amino acid sequence homology in the first 12 positions, we conclude that the rat liver protein is serum albumin and that the ATP-dependent DNA aggregation is a novel function of rat serum albumin.
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PMID:ATP-dependent DNA aggregation is a novel function of rat serum albumin. 189 9


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