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)

DNA helicase I, the traI gene product of the Escherichia coli F factor, was shown to be associated with endonuclease activity specific for the transfer origin of the F plasmid, oriT. In the presence of Mg2+, the purified enzyme forms a complex, stable in the presence of sodium dodecylsulfate (SDS) with a negatively superhelical chimeric plasmid containing oriT. The enzyme nicks and, after this, apparently binds to the 5' nick terminus when this complex is heated in the presence of SDS and/or EDTA or treated with proteinase K. Dideoxy sequencing locates the nick site in the F DNA strand transferred during bacterial conjugation after nucleotide 138 clockwise of the mid-point of the BglII site at 66.7 kb of the F genetic map. A sequencing stop after nucleotide 137 of this strand (where oriT-nicking seems to occur in vivo) is possibly an artefact caused by helicase I protein attached to the 5' terminal nucleotide. Deletion in the amino-terminal part of the traI polypeptide abolishes the oriT-nicking activity while leaving the strand-separating activity intact. These results confirm the prediction from genetic studies that helicase I is bifunctional with site-specific endonuclease and strand-separating activities.
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PMID:Endonuclease activity of Escherichia coli DNA helicase I directed against the transfer origin of the F factor. 165 Dec 33

Chinese hamster ovary (CHO) cells were treated with the restriction endonuclease AluI using various methods; namely, treatment in the presence of hypertonic concentrations of glycerol or sorbitol and electroporation. The frequencies of chromosomal aberrations induced were scored in first post-treatment metaphases. For all treatment schedules linear dose-effect relationships of polycentric chromosomes were found. The distribution of polycentric chromosomes induced by AluI was overdispersed. Inhibition of cellular energy metabolism with sodium azide and 2-deoxyglucose led to a strong reduction of the frequencies of chromosomal aberrations when the treatment with AluI was carried out in the presence of glycerol or sorbitol, but not when carried out by electroporation. This is interpreted to mean that glycerol- or sorbitol-mediated cellular uptake of AluI, but not the uptake via electroporation, is an energy-dependent mechanism.
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PMID:Induction of chromosomal aberrations with the restriction endonuclease AluI in Chinese hamster ovary cells: comparison of different treatment methods. 167 83

The intestinal anaerobic spirochetes Treponema hyodysenteriae B78T (T = type strain), B204, B169, and A-1, Treponema innocens B256T and 4/71, Treponema succinifaciens 6091T, and Treponema bryantii RUS-1T were compared by performing DNA-DNA reassociation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell proteins, restriction endonuclease analysis of DNA, and 16S rRNA sequence analysis. DNA-DNA relative reassociation experiments in which the S1 nuclease method was used showed that T. hyodysenteriae B78T and B204 had 93% sequence homology with each other and approximately 40% sequence homology with T. innocens B256T and 4/71. Both T. hyodysenteriae B78T and T. innocens B256T exhibited negligible levels of DNA homology (less than or equal to 5%) with T. succinifaciens 6091T. The results of comparisons of protein electrophoretic profiles corroborated the DNA-DNA reassociation results. We found high levels of similarity (greater than or equal to 96%) in electrophoretic profiles among T. hyodysenteriae strains, moderate levels of similarity (43 to 49%) between T. hyodysenteriae and T. innocens, and no detectable similarity between the profiles of either T. hyodysenteriae or T. innocens and those of T. succinifaciens, T. bryantii, and Escherichia coli. Restriction endonuclease analysis of DNA was not useful in assessing genetic relationships since there was heterogeneity even between strains of T. hyodysenteriae. Partial 16S rRNA sequences of the intestinal spirochetes were determined by using a modified Sanger method and were compared in order to evaluate the phylogenetic relationships among these and other spirochetes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Reclassification of Treponema hyodysenteriae and Treponema innocens in a new genus, Serpula gen. nov., as Serpula hyodysenteriae comb. nov. and Serpula innocens comb. nov. 170 92

A lambda gt11 recombinant library of Chlamydia trachomatis serovar L2 chromosomal DNA was screened with a 29-mer synthetic oligonucleotide specific to the N-terminal amino acids of a predominant 18-kDa chlamydial protein. One recombinant clone, designated lambda gt11/L2/RKA10, was selected on the basis of its strong hybridization signal. Restriction endonuclease analysis and complete nucleotide sequencing of the recombinant revealed a 2,633-bp insert containing one complete open reading frame (ORF2) and two partial ORFs (ORF1 and ORF3). The deduced amino acid sequence of ORF2 matched perfectly at its N-terminal end with the derived amino acid sequence. The 375-bp ORF is capable of encoding a protein comprising 125 amino acids with a molecular mass of 13,689. A sequence compatible with a Shine-Dalgarno ribosome-binding site was located 9 bp upstream from the initiation codon, while the sequence distal to ORF2 revealed a rho-independent terminator. The protein, designated CTH1, possesses an estimated pI of 10.71 due to its high lysine content. This highly basic protein contains no tryptophan or phenylalanine. A protein data base search identified significant homology between CTH1 and painted sea urchin histone H1. Northern (RNA) blot analysis of Chlamydia-infected host cells demonstrated transcripts at 12 h postinfection. The recombinant plasmid encoding ORF2 expressed a gene product of approximately 18 kDa, similar to the native chlamydial protein as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein appears to represent one of the few eukaryotic histonelike proteins described to date in prokaryotes.
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PMID:Identification and nucleotide sequence of a developmentally regulated gene encoding a eukaryotic histone H1-like protein from Chlamydia trachomatis. 170 78

The photochemically derived silver stain of nucleic acids in polyacrylamide gels originally described by Merril et al. (1981, Science 211, 1437-1438) was modified to reduce unspecific background staining and increase sensitivity (down to 1 pg/mm2 band cross-section). Detection limits for double-stranded DNA fragments from HaeIII endonuclease digests of phage phi X174 were maintained despite eliminating oxidation pretreatment of fixed gels and reducing silver nitrate concentration. Preexposure to formaldehyde during silver impregnation enhanced sensitivity and the inclusion of the silver-complexing agent sodium thiosulphate in the image developer decreased background staining. Higher formaldehyde concentration during image development resulted in darker bands with good contrast. The procedure almost halves the number of steps, solutions and experimental time required and can be used for the staining of DNA fragments in polyacrylamide gels bound to a polyester backing film by controlling temperature during image development. We have applied this improved staining procedure for the routine analysis of complex DNA profiles generated by DNA amplification fingerprinting (DAF).
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PMID:Fast and sensitive silver staining of DNA in polyacrylamide gels. 171 76

The production of diphtheria toxin and siderophore by the Corynebacterium diphtheriae regulatory mutant C7(beta)hm723 is resistant to the inhibitory effects of iron, and the mutant strain is defective for function of the regulatory gene dtxR. A 2.8-kb HindIII fragment carrying the C7(beta)hm723 dtxR allele was cloned and characterized in Escherichia coli. The restriction endonuclease maps of the 2.8-kb HindIII fragment from C7(beta)hm723 and the corresponding fragment from wild-type C. diphtheriae C7 were identical. RNA dot blot analysis with total RNA isolated from wild-type C. diphtheriae C7 and C7(beta)hm723 indicated that the dtxR gene was transcribed at very low but equivalent levels in both strains and was not regulated by iron. beta-Galactosidase synthesis from a tox-lacZ translational fusion construct in E. coli in high-iron medium was not repressed by the C7(beta)hm723dtxR allele, but was strongly repressed by the wild-type dtxR gene. The 28- to 29-kDa polypeptide expressed from the mutant dtxR allele in E. coli had the same electrophoretic mobility as the wild-type dtxR gene product in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The nucleotide sequence of the coding region and the 5' upstream region of the C7(beta)hm723 dtxR allele was determined and compared with the wild-type nucleotide sequence. The dtxR allele from C7(beta)hm723 contained a single-base change located 140 nucleotides from the 5' start of the gene, which resulted in replacement of arginine in the wild-type sequence by histidine in the mutant protein. These data demonstrate that C7(beta)hm723 expresses a mutant DtxR repressor protein that is severely defective in repressor activity.
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PMID:Characterization of a defective diphtheria toxin repressor (dtxR) allele and analysis of dtxR transcription in wild-type and mutant strains of Corynebacterium diphtheriae. 171 67

An endonuclease with 3'-nucleotidase activity (nuclease Le1) was purified from fruit bodies of Lentinus edodes in a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The apparent molecular weight of nuclease Le1 was about 27000. The nuclease was inactivated in the presence of ethylenediaminetetraacetic acid (EDTA) and reactivated by the addition of Zn2+. Hydrolysis of poly U by the nuclease showed many intermediate size oligomers prior to the formation of 5'-uridine monophosphate (UMP). Therefore, it was concluded that nuclease Le1 was a Zn(2+)-endonuclease similar to P1-nuclease from Penicillium citrinum. The nuclease was very sensitive to ionic strength, but pH-profiles of the hydrolysis of four 3'-nucleotides were very similar to those of P1 nuclease from P. citrinum.
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PMID:Purification and characterization of a nuclease from Lentinus edodes. 172 78

Lens is an organ composed of a layer of epithelial cells and a mass of fibers. During terminal differentiation, epithelial cells from the equatorial region elongate into fibers, nuclei change shape, the chromatin appears much condensed in the last step of differentiation and the DNA breaks down into nucleosomes. The pattern of DNAase activities has been recorded at different chick embryonic stages (11 and 18 days) using polyacrylamide gel electrophoresis with DNA substrate in the gel matrix. Two DNAases (30 and 40 kDa) have been observed in lens epithelia and fibers at both stages. However, the activities of both of the enzymes are augmented in fiber cells. The 30 kDa DNAase requires and Ca2+ and Mg2+ (5-15 mM) to hydrolyze the DNA substrate while the 40 kDa-activity is inhibited by added divalent cations (5-15 mM). The 30 kDa protein is inhibited by Na+ and is probably an endonuclease. Both nuclease activities probably are involved in the cleavage of fiber chromatin into nucleosomes during lens terminal differentiation, but variables such as chromatin configuration, unmasked DNA sequences, presence of cations, and pH gradients probably determine the extent of involvement of each DNAase.
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PMID:DNAase activities in embryonic chicken lens: in epithelial cells or in differentiating fibers where chromatin is progressively cleaved. 179 64

We used site-directed mutagenesis to introduce both a NdeI restriction endonuclease site and an initiator codon at the junction of the leader and structural gene sequences of the metallo-beta-lactamase of Bacillus cereus 5/B/6. This construct allowed us to clone just the beta-lactamase structural gene sequence into an Escherichia coli expression vector. E. coli cells were transformed with the recombinant plasmid, the B. cereus beta-lactamase was expressed, and these E. coli cells were disrupted by sonic oscillation. When the resultant suspensions were clarified by ultracentrifugation, the B. cereus beta-lactamase represented 15% of the total protein in the supernatant. Subsequent gel filtration and ion-exchange chromatography allowed the first reported purification to homogeneity of the B. cereus beta-lactamase from E. coli with an 87% recovery and an overall yield of 17 mg of enzyme per liter of cell culture. The electrophoretic mobilities of the enzyme expressed in and purified from E. coli and the enzyme purified directly from B. cereus were identical in both native and sodium dodecyl sulfate gel electrophoreses. As with the B. cereus enzyme, Km and Vmax (using cephalosporin C as substrate) for the enzyme purified from E. coli were 0.39 mM and 1333 units/mg protein, respectively. Likewise, the Co(II)-reconstituted enzyme purified from E. coli, which retained 29% of the activity of the Zn(II) enzyme, had electronic absorption spectra with maxima at 347, 551, 617, and 646 nm with extinction coefficients of 900, 250, 173, and 150 M-1 cm-1, respectively.
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PMID:Hyperexpression in Escherichia coli, purification, and characterization of the metallo-beta-lactamase of Bacillus cereus 5/B/6. 182 84

The efficacy of one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell protein patterns for fingerprinting isolates of Helicobacter pylori was assessed by means of computerized numerical analysis. Virtually all strains were found to have unique, stable, and reproducible protein profiles. The application of this technique to a collection of isolates from eight patients showed clearly that each harboured a distinct strain that was present before treatment and persisted after treatment. This suggests that relapse was due to recrudescence of the same strain rather than re-infection with a different strain. Minor differences in protein banding profiles within sets of isolates from the same patient were evident, and this was confirmed by means of both two-dimensional PAGE protein patterns and restriction endonuclease analysis of DNA on the same strains.
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PMID:Molecular techniques for studying the epidemiology of infection by Helicobacter pylori. 186 91


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