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)

Amphizoic small amoebic protozoa are capable of existing both in 'free-living' and in 'parasitic' form depending on the actual conditions. Two genera (Naegleria and Acanthamoeba) have become recognised as opportunist human parasites. Since the first description in 1965 of a lethal case of primary amoebic meningoencephalitis (PAM) caused by Naegleria, many more (mostly lethal) cases have been reported, while granulomatous amoebic encephalitis (GAE), as well as eye (keratinitis, conjunctivitis, etc.), ear, nose, skin and internal organ infections caused by Acanthamoeba have also occurred in rapidly increasing numbers. Both pathogenic and non-pathogenic species of Naegleria and Acanthamoeba are found worldwide in water, soil and dust, where they provide a potential source of infection. Successful differential diagnosis and appropriate (specific) therapy depends on precise laboratory identification of the 'free-living' amoebae. In most cases, isolation from the environment can be achieved, but identification and differentiation of the pathogenic and non-pathogenic strains is not easy. The methods presently available do not fulfil completely the requirements for specificity, sensitivity and reliability. Morphological criteria are inadequate, while thermophilic character, pH dependency and even virulence in infected mice, are not unambiguous features of pathogenicity of the different strains. More promising are molecular methods, such as restriction endonuclease digestion of whole-cell DNA or mitochondrial DNA, as well as iso-enzyme profile analysis after iso-electric focusing and staining for acid phosphatase and propionyl esterase activity. Use of appropriate monoclonal antibodies has also yielded promising results in the differentiation of human pathogenic and non-pathogenic strains. However, quicker, simpler, more specific and reliable methods are still highly desirable. The significance of endosymbiosis (especially with Legionella strains) is not well understood. The results of a systematic survey in Hungary for the isolation and identification of 'free-living' amoebae, including an investigation of the Hungarian amoebic fauna, the isolation of possibly pathogenic Naegleria strains and of some Acanthamoeba strains from eye diseases, as well as the finding of a case of endosymbiosis, are also reported here.
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PMID:Isolation, identification and increasing importance of 'free-living' amoebae causing human disease. 978 20

Bursts of free radicals produced by ionization of water in close vicinity to DNA can produce clusters of opposed DNA lesions and these are termed multiply damaged sites (MDS). How MDS are processed by the Escherichia coli DNA glycosylases, endonuclease (endo) III and endo VIII, which recognize oxidized pyrimidines, is the subject of this study. Oligonucleotide substrates were constructed containing a site of pyrimidine damage or an abasic (AP) site in close proximity to a single nucleotide gap, which simulates a free radical-induced single-strand break. The gap was placed in the opposite strand 1, 3 or 6 nt 5' or 3' of the AP site or base lesion. Endos III and VIII were able to cleave an AP site in the MDS, no matter what the position of the opposed strand break, although cleavage at position one 5' or 3' was reduced compared with cleavage at positions three or six 5' or 3'. Neither endo III nor endo VIII was able to remove the base lesion when the gap was positioned 1 nt 5' or 3' in the opposite strand. Cleavage of the modified pyrimidine by endo III increased as the distance increased between the base lesion and the opposed strand break. With endo VIII, however, DNA breakage at the site of the base lesion was equivalent to or less when the gap was positioned 6 nt 3' of the lesion than when the gap was 3 nt 3' of the lesion. Gel mobility shift analysis of the binding of endo VIII to an oligonucleotide containing a reduced AP (rAP) site in close opposition to a single nucleotide gap correlated with cleavage of MDS substrates by endo VIII. If the strand break in the MDS was replaced by an oxidized purine, 7,8-dihydro-8-oxoguanine (8-oxoG), neither endo VIII cleavage nor binding were perturbed. These data show that processing of oxidized pyrimidines by endos III and VIII was strongly influenced by the position and type of lesion in the opposite strand, which could have a significant effect on the biological outcome of the MDS lesion.
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PMID:Multiply damaged sites in DNA: interactions with Escherichia coli endonucleases III and VIII. 946 50

A 14-month longitudinal study was conducted on four dairy farms (C, H, R, and X) in Wisconsin to ascertain the source(s) and dissemination of Escherichia coli O157:H7. A cohort of 15 heifer calves from each farm were sampled weekly by digital rectal retrieval from birth to a minimum of 7 months of age (range, 7 to 13 months). Over the 14 months of the study, the cohort heifers and other randomly selected cattle from farms C and H tested negative. Farm R had two separate periods of E. coli O157:H7 shedding lasting 4 months (November 1995 to February 1996) and 1 month (July to August 1996), while farm X had at least one positive cohort animal for a 5-month period (May to October 1996). Heifers shed O157:H7 strains in feces for 1 to 16 weeks at levels ranging from 2.0 x 10(2) to 8.7 x 10(4) CFU per g. E. coli O157:H7 was also isolated from other noncohort cattle, feed, flies, a pigeon, and water associated with the cohort heifers on farms R and/or X. When present in animal drinking water, E. coli O157:H7 disseminated through the cohort cattle and other cattle that used the water source. E. coli O157:H7 was found in water at < 1 to 23 CFU/ml. Genomic subtyping by pulsed-field gel electrophoresis demonstrated that a single O157:H7 strain comprised a majority of the isolates from cohort and noncohort cattle, water, and other positive samples (i.e., from feed, flies, and a pigeon, etc.) on a farm. The isolates from farm R displayed two predominant XbaI restriction endonuclease digestion profiles (REDP), REDP 3 and REDP 7, during the first and second periods of shedding, respectively. Six additional REDP that were > or = 89% similar to REDP 3 or REDP 7 were identified among the farm R isolates. Additionally, the REDP of an O157:H7 isolate from a heifer on farm R in 1994 was indistinguishable from REDP 3. Farm X had one O157:H7 strain that predominated (96% of positive samples had strains with REDP 9), and the REDP of an isolate from a heifer in 1994 was indistinguishable from REDP 9. These results suggest that E. coli O157:H7 is disseminated from a common source on farms and that strains can persist in a herd for a 2-year period.
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PMID:Longitudinal study of Escherichia coli O157:H7 dissemination on four dairy farms in Wisconsin. 954 76

Phage typing (PT) combined with pulsed-field gel electrophoresis (PFGE) and a random amplified polymorphic DNA (RAPD) fingerprinting method was used to characterize Salmonella enteritidis strains. Twenty-four epidemiologically unrelated isolates, sampled from diverse ecological niches and fifteen isolates from four well-defined outbreaks of foodborne gastroenteritis, were studied. Seven phage types, with a predominance of PT 4 (63% of isolates), were observed when analysing the epidemiologically unrelated group. PT 4 was detected in all of the ecological niches studied, including food and fecally polluted river and beach water. The discriminatory power for phage typing, the average probability that the typing system will assign a different type to two unrelated strains randomly sampled in the microbial population, was 0.62. Ten PFGE pattern types were obtained with Xba I restriction endonuclease enzyme among the unrelated isolates; thirteen isolates belonged to PFGE pattern type 1 and the rest of the PFGE types were assigned to one or two isolates. The Dice coefficient clustered the similarities of the PFGE patterns between 80-100%. PFGE showed a discriminatory power of 0.72. Five clearly distinct RAPD patterns were observed with the OPS-19 oligonucleotide, but the discrimination obtained was low (0.46). The combination of the three typing methods increased the number of types to seventeen, giving high discrimination (0.92). Seven of the isolates recovered from various ecological niches belonged to the combination PT 4/PFGE 1/RAPD A and other combinations were unique or included only two strains. The four epidemiologically well-defined foodborne outbreaks were associated with the PT 4 phage type. In two of the outbreaks, other phage types (PT 7a and RDNC) were also observed in two isolates. Most of the isolates belonging to the foodborne outbreaks had an identical PFGE pattern (PFGE pattern type 1), but a difference in a restriction band was observed in an isolate belonging to an outbreak. Two RAPD patterns were observed in the outbreaks; RAPD pattern type A was detected in three of the four outbreaks. When the combined typing method was applied to the study, high concordance was observed and most of the outbreak strains belonged to the combination PT 4/PFGE 1/RAPD A. It is concluded that the combination of phage type with PFGE and RAPD provides a powerful discriminatory tool for the epidemiological analysis of unrelated and related strains of S. enteritidis.
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PMID:Phage typing combined with pulsed-field gel electrophoresis and random amplified polymorphic DNA increases discrimination in the epidemiological analysis of Salmonella enteritidis strains. 960 Jun 7

In restriction-modification systems, cleavage of substrate sites in cellular DNA by the restriction endonuclease is prevented by the action of a cognate methyltransferase that acts on the same substrate sites. The PvuII restriction endonuclease (R.PvuII) has been structurally characterized in a complex with substrate DNA (Cheng et al., 1994) and as an apoenzyme (Athanasiadis et al., 1994). We report here a structure, determined to 1.9 A resolution by crystallography, of a complex between R.PvuII and iodinated DNA. The presence of an iodine at the 5-carbon of the methylatable cytosine results in the following changes in the protein: His84 moved away from the modified base; this movement was amplified in His85 and disrupts an intersubunit hydrogen bond; and the base modification disturbs the distribution of water molecules that associate with these histidine residues and the area of the scissile bond. Considering these observations, hypotheses are given as to why a similar oligonucleotide, where a methyl group resides on the 5-carbon of the methylatable cytosine, is slowly cleaved by R.PvuII (Rice et al., 1995).
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PMID:How is modification of the DNA substrate recognized by the PvuII restriction endonuclease? 962 37

The phospholipase D (PLD) superfamily includes enzymes of phospholipid metabolism, nucleases, as well as ORFs of unknown function in viruses and pathogenic bacteria. These enzymes are characterized by the invariant sequence motif, H(X)K(X)4D. The endonuclease member Nuc of the PLD family was over-expressed in bacteria and purified to homogeneity. Mutation of the conserved histidine to an asparagine in the endonuclease reduced the kcat for hydrolysis by a factor of 10(5), suggesting that the histidine residue plays a key role in catalysis. In addition to catalyzing hydrolysis, a number of phosphohydrolases will catalyze a phosphate (oxygen)-water exchange reaction. We have taken advantage of this observation and demonstrate that a 32P-labeled protein could be trapped when the enzyme was incubated with 32P-labeled inorganic phosphate. The phosphoenzyme intermediate was stable in 1 M NaOH and labile in 1 M HCl and 1 M hydroxylamine, suggesting that the enzyme forms a phosphohistidine intermediate. The pH-stability profile of the phosphoenzyme intermediate was consistent with phosphohistidine and the only radioactive amino acid found after alkaline hydrolysis was phosphohistidine. These results suggest that the enzymes in the PLD superfamily use the conserved histidine for nucleophilic attack on the substrate phosphorus atom and most likely proceed via a common two-step catalytic mechanism.
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PMID:Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate. 968 58

The 2.1-A cocrystal structure of EcoRV endonuclease bound to 5'-CGGGATATCCC, in a crystal lattice isomorphous with the cocrystallized undecamer 5'-AAAGATATCTT previously determined, shows novel base recognition in the major groove of the DNA flanking the GATATC target site. Lys104 of the enzyme interacts through water molecules with the exocyclic N-4 amino groups of flanking cytosines. Steric exclusion of water molecule-binding sites by the 5-methyl group of thymine drives the adoption of alternative water-mediated contacts with AT versus GC flanks. This structure provides a rare example of structural adaptability in the recognition of different DNA sequences by a protein and suggests preferred strategies for the expansion of target site specificity by EcoRV.
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PMID:Recognition of flanking DNA sequences by EcoRV endonuclease involves alternative patterns of water-mediated contacts. 970 8

The epidemiology of Yersinia pseudotuberculosis infections in a limited area of Shimane Prefecture, Japan, was examined by serotyping and restriction endonuclease analysis of virulence plasmid DNA of Y. pseudotuberculosis strains isolated from humans, wildlife animals and river water. Almost all isolates from three sources belonged to serotype 1b REAP pattern D and serotype 4b REAP patterns B, G and L. The identity of the distribution of serotype and REAP patterns among isolates from humans, wildlife animals and river water shows that Y. pseudotuberculosis is transmitted to humans through environmental substances contaminated by wildlife animals infected with this species.
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PMID:Restriction endonuclease analysis of plasmid DNA of Yersinia pseudotuberculosis infections in Shimane Prefecture, Japan. 981 Jun 74

The 2.15-A resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and Ca2+ ions reveals two divalent metals bound in one of the active sites. One of these metals is ligated through an inner-sphere water molecule to the phosphate group located 3' to the scissile phosphate. A second inner-sphere water on this metal is positioned approximately in-line for attack on the scissile phosphate. This structure corroborates the observation that the pro-SP phosphoryl oxygen on the adjacent 3' phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV, EcoRI, PvuII, and BamHI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construction of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step by a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.
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PMID:Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases. 981 27

We generated variants of the restriction endonuclease EcoRV that discriminate between recognition sites with different flanking sequences. This was achieved by designing new contacts to the bases in the major groove of the DNA preceding and following the EcoRV recognition site. We selected Ala181 as the starting point for the extension of the site specificity of EcoRV because, according to the structure of the specific EcoRV x DNA complex, this residue is involved in a water mediated contact with the bases flanking the recognition sequence on the 5' side. A substitution of this alanine residue by other amino acid residues changes the protein-DNA interface in this region and potentially creates new contacts, such that EcoRV variants could have an extended specificity, i.e. a greater selectivity for EcoRV sites within a particular sequence context. EcoRV variants with naturally occurring amino acid residues at position 181 were produced and their selectivity analyzed with oligodeoxynucleotide and plasmid substrates that differ only in the base pairs immediately flanking the EcoRV site. Some variants, having amino acid residues with long or bulky side chains at position 181 showed altered preferences for the base pairs flanking the recognition sequence with oligodeoxynucleotide substrates without loosing their catalytic efficiency. One variant, A181K, is able to discriminate between purine and pyrimidine bases on the 5' side of the recognition sequence, probably by means of a new hydrogen bond to the N7 of the purine base. Another variant, A181E, strongly prefers a thymine base on the 5' side of the recognition sequence, presumably due to a hydrogen bond formed between the protonated glutamic acid residue and the O4 of thymine.
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PMID:Protein engineering of the restriction endonuclease EcoRV--structure-guided design of enzyme variants that recognize the base pairs flanking the recognition site. 985 8


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