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)

Ampicillin-resistant strains of Shigella dysenteriae type 1 isolated in epidemics in Mexico, Central America, and Bangla Desh were examined for the presence of plasmid deoxyribonucleic acid (DNA) by gel electrophoresis. All strains contained a heterogeneous population of plasmids. Transfer experiments to Escherichia coli K-12 indicated that the ampicillin resistance determinant (Ap(r)) was located on a 5.5-megadalton (Mdal) plasmid identical in all Shiga strains examined, as judged by DNA hybridization and by its molecular properties. This 5.5-Mdal plasmid contained the ampicillin transposon (TnA) sequences. There was not a high degree of homology between the Shiga Ap(r) plasmid DNA and DNA obtained from Ap(r)Salmonella typhi strains isolated from typhoid epidemics in Mexico, previous to the dysentery outbreaks. Although low, the degree of reassociation observed indicated that probably part of the TnA sequence was present in S. typhi DNA. The DNA hybridization experiments showed, in addition, that there was a high degree of homology among Ap(r) plasmids isolated from different enterobacteria, and this identity was confirmed by restriction endonuclease activity. These results together with their similarities in molecular and replicative properties indicate that the Ap(r) plasmids, as was suggested for the Sm(r) Su(r) plasmids, possibly evolved once and then epidemiologically spread in the Enterobacteriaceae.
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PMID:Characterization of an R-plasmid associated with ampicillin resistance in Shigella dysenteriae type 1 isolated from epidemics. 32 94

Shiga-like toxin-producing Escherichia coli strains of serogroup O157 were identified in 26 of 104 patients with hemolytic-uremic syndrome and in 18 of 668 patients with diarrhea. All strains were identified by colony hybridization with DNA probes complementary to Shiga-like toxin I and Shiga-like toxin II gene sequences and characterized by biochemical tests and serotyping. Seventeen of these 44 patients had E. coli O157 strains which were unusual because they fermented sorbitol within 24 h of incubation and were positive for beta-glucuronidase activity. Culture filtrates of these sorbitol-fermenting strains were highly toxic to Vero cells in culture. Serological tests and DNA analysis performed by restriction endonuclease digestion of B-subunit toxin genes revealed that all 17 isolates produced Shiga-like toxin II. Although by using molecular probes we established a high frequency of sorbitol-fermenting E. coli O157 strains in the patients we examined, further studies on the prevalence of such isolates in other areas of endemic disease are clearly warranted.
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PMID:Molecular detection of sorbitol-fermenting Escherichia coli O157 in patients with hemolytic-uremic syndrome. 162 38

A set of synthetic oligonucleotide primers was designed for use in a polymerase chain reaction protocol to specifically detect the B subunit genes in vtx2ha and vtx2hb, which code for the production of the VT2 (Shiga-like toxin II) variant cytotoxins VT2v-a and VT2v-b, respectively. An additional set of primers amplified a fragment common to the B subunits of the VT2 and the VT2 variant genes. Subsequent restriction endonuclease digestion of this amplicon permitted prediction of specific VT2 and variant genotypes on the basis of predetermined restriction fragment length polymorphisms. Genotypes of 21 VT2-producing strains of Escherichia coli were determined using this polymerase chain reaction-restriction fragment length polymorphism procedure. Four strains contained B subunit target sequences only for VT2 genes, 9 strains contained sequences only for VT2v-a genes, and 3 strains contained sequences only for VT2v-b. For genes in combination, one strain contained B subunit genes for both VT2 and VT2v-a and two strains contained B subunit genes for VT2 and VT2v-b. Two strains of E. coli O91:H21 contained both VT2v-a and VT2v-b B subunit genes. The VT2 reference strain of E. coli, E32511, was found to contain the targeted sequences from both VT2 and VT2v-a genes, whereas the recombinant E. coli, pEB1, possessed only that of the VT2 gene. The specific activities of extracellular VT2 determined in HeLa cells ranged from 0.3 to 41.7 TCD50 per microgram of protein in strains carrying the VT2 gene target and from 0 to 50.0 TCD50 per microgram of protein in strains carrying only the VT2 variant target (TCD50 is the tissue culture dose by which 50% of the cells were affected), suggesting that phenotypic expression does not correlate with genotype.
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PMID:Identification of verotoxin type 2 variant B subunit genes in Escherichia coli by the polymerase chain reaction and restriction fragment length polymorphism analysis. 167 36

The application of nucleic acid analyses to investigations of infectious disease outbreaks has resulted in useful molecular strain markers that distinguish the epidemic clone of a particular pathogen and help identify specific vehicles of infection. We have successfully used plasmid profile analysis, restriction endonuclease digestion of plasmid and whole-cell DNAs, and nucleic acid hybridization to investigate recent outbreaks of foodborne diarrheal illness. Plasmid analysis has been important in identifying epidemic strains of Salmonella enteritidis and Escherichia coli O157:H7. In a culture survey of S. enteritidis isolates from humans and a variety of animals, including chickens and chicken eggs, we identified 16 distinct plasmid profiles and used these to differentiate strains, especially within commonly occurring phage types (Colindale 8 and 13a). HindIII digests of plasmid DNA were useful in distinguishing plasmids of similar mass but dissimilar enzyme target sequences; they clearly distinguished S. enteritidis strains causing systemic infections in children in parts of Africa from U.S. isolates. Investigations of outbreaks of hemorrhagic colitis have also been assisted by plasmid analysis. Restriction endonuclease digests of whole-cell DNA and Southern blot analysis, hybridizing with E. coli 16S and 23S rRNA (ribotyping), have been effective subtyping techniques, especially for plasmidless isolates of Campylobacter jejuni. In five outbreaks of C. jejuni infections, ribotyping of PvuII and ClaI digests distinguished individual epidemic strains within one commonly occurring C. jejuni serotype (Penner 2, Lior 4). Preliminary data show that ribotyping of NcoI digests can also distinguish individual epidemic strains of E. coli O157:H7 and may provide a more stable marker than plasmid profiles. Specific DNA probes derived from cloned virulence genes of E. coli have been invaluable in epidemic investigations and surveys. Using colony hybridization, we found in one survey of stool specimens from 174 dairy cattle that 11% of animals were asymptomatically carrying Shiga-like toxigenic E. coli other than O157:H7. We also found that newly synthesized oligonucleotide probes for the Shiga-like toxins I and II agreed 100% with cloned gene probes in a study of 613 E. coli strains. Future studies of these organisms will include the use of additional synthetic oligonucleotides as primers to amplify the toxin genes directly in patient and animal specimens by the polymerase chain reaction. There is a continuing and expanding role for molecular approaches in epidemiological investigations. The DNA methods described above are not based on the often complex expression of phenotypic characteristics, and, unlike sensitive and specific techniques such as phage typing, a single method can be used to study a variety of Gram-positive and negative bacterial pathogens.
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PMID:The use of plasmid profiles and nucleic acid probes in epidemiologic investigations of foodborne, diarrheal diseases. 201 8

The genomic fingerprints of 26 Escherichia coli O157:H7 isolates from calves on 20 farms in 16 states were determined by pulsed-field gel electrophoresis (PFGE). Digestion of genomic DNAs with the restriction enzymes SfiI and XbaI yielded 14 and 18 restriction endonuclease digestion profiles (REDP), respectively. Seventeen farms (85%) had E. coli O157:H7 with a unique REDP, and when more than one calf tested positive on a farm, the isolates displayed identical REDP. Isolates from different farms within the same state displayed distinct REDP, as did most isolates from farms in different states. The exceptions were three farms in New York, Ohio, and Washington that had calves harboring E. coli O157:H7 with the same REDP. In addition to REDP, the toxin profiles of all 26 isolates were determined using oligonucleotide probes to Shiga-like toxins (SLT) I and II. Nineteen (73%) of the E. coli O157:H7 isolates harbored the genes for both SLT and II, while the remaining seven isolates (27%) had the gene for SLT II only. Also, isolates with the same REDP had the same toxin profile. The genomic relatedness among the E. coli O157:H7 isolates was also determined by principal component analysis of Dice similarity indices of REDP. Three clusters were identified, but none of these were associated with a geographic region or toxin profile.
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PMID:Genomic analysis using pulsed-field gel electrophoresis of Escherichia coli O157: H7 isolated from dairy calves during the United State National Dairy Heifer Evaluation Project (1992-1992). 905 19

Contour-clamped homogeneous electric field pulsed-field gel electrophoresis (CHEF-PFGE) was used to compare Wisconsin isolates of Escherichia coli O157:H7, including 39 isolates from a 1994 day care center outbreak, 28 isolates from 18 individuals from the surrounding geographic area with sporadic cases occurring during the 3 months before the outbreak, and 3 isolates, collected in 1995, from patients with hemolytic-uremic syndrome (HUS) who were from eastern Wisconsin counties other than those inhabited by the day care center and sporadic-case individuals. The technique of CHEF-PFGE using XbaI identified seven highly related restriction endonuclease digestion profiles (REDPs) (93 to 98% similarity) among the 39 day care center isolates and nine XbaI REDPs (63 to 93% similarity) among the 28 isolates from sporadic-case individuals, including REDP 33, which was exhibited by both day care and sporadic-case isolates. PFGE analyses of sequential E. coli O157:H7 isolates from symptomatic day care center attendees revealed that the REDPs of 25 isolates from eight patients were indistinguishable whereas the REDPs of 2 of 6 isolates from two patients differed slightly (93 to 95% similarity). The REDPs of the three isolates from 1995 HUS patients were 78 to 83% similar, with REDP 26 being exhibited by one HUS-associated isolate and an isolate from one day care attendee who did not develop HUS. The genes for both Shiga toxins I and II (stx1 and stx2, respectively) were detected in all but one isolate (sporadic case), and Shiga toxin production by the day care center isolates was not significantly different from that of the other isolates, including the three HUS-associated isolates. Analyses of E. coli O157:H7 isolates from both the day care center outbreak and sporadic cases by CHEF-PFGE permitted us to define the REDP variability of an outbreak and geographic region and demonstrated that the day care center outbreak and a HUS case in 1995 were caused by E. coli O157:H7 strains endemic to eastern Wisconsin.
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PMID:Genomic comparisons and Shiga toxin production among Escherichia coli O157:H7 isolates from a day care center outbreak and sporadic cases in southeastern Wisconsin. 950 3

This study assessed the diversity of the enterohemorrhagic Escherichia coli (EHEC) hemolysin gene (ehxA) in a variety of Shiga toxin-producing E. coli (STEC) serotypes and the relationship between ehxA types and virulence markers on the locus for enterocyte effacement (LEE). Restriction fragment length polymorphism of the ehxA gene and flanking sequences and of the E. coli attaching and effacing (eae) gene was determined for 79 EHEC hemolysin-positive STEC isolates of 37 serotypes. Two main groups of EHEC hemolysin sequences and associated plasmids, which corresponded to the eae-positive and the eae-negative isolates, were delineated. Comparisons of the ehxA gene sequences of representative isolates of each group showed that this gene and the rest of the EHEC hemolysin operon are highly conserved. Digestion of an ehxA PCR product with the restriction endonuclease TaqI showed a unique restriction pattern for eae-negative isolates and another one for isolates of serotypes O157:H7 and O157:NM. A conserved fragment of 5.6 kb with four potential open reading frames was identified on the EHEC hemolysin plasmid of eae-positive STEC. Phylogenetic analysis of a subset of 27 STEC isolates, one enteropathogenic E. coli isolate, and a K-12 reference isolate showed that eae-positive STEC isolates all belong to a single evolutionary lineage and that the EHEC hemolysin plasmid and the ehxA gene evolved within this lineage without recent horizontal transfer. However, the eae gene and the LEE appear to have been transferred horizontally within this STEC lineage on several occasions. The reasons for the lack of transfer or maintenance of the LEE in other STEC lineages are not clear and require further study.
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PMID:Evolution of enterohemorrhagic Escherichia coli hemolysin plasmids and the locus for enterocyte effacement in shiga toxin-producing E. coli. 959 16

The specificity of 14 polymerase chain reaction (PCR) systems designed for the detection and subtyping of stx genes was tested on a set of Escherichia coli strains with known sequences of stx genes. Systems designed for the detection of genes of the stx1 type did not detect any variant genes of the stx2 type and conversely, no stx2 type-specific systems detected stx1 variant genes. Among five stx2 type-specific systems, none detected the stx2ev gene, and two detected the stx2e gene. Among systems designed for screening genes of the both stx1 and stx2 types with a single primer pair, only one system (the Lin system) was able to detect stx genes in all studied strains. Shiga-toxin-producing E. coli frequently carry more than one stx variant gene. Coamplification of stx genes present in the same strain was demonstrated by restriction of PCR products with endonucleases generating fragments of variant-specific size. The amplification product obtained by the Lin system restricted by Hincll yielded fragments of different size for stx1, stx2, stx2c, stx2e and stx2ev. Thus it was possible to identify different genes carried in a single strain with a simple two-step PCR/endonuclease restriction protocol.
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PMID:Comparison of 14 PCR systems for the detection and subtyping of stx genes in Shiga-toxin-producing Escherichia coli. 976 98

The stx-flanking regions of 49 Shiga toxin-producing Escherichia coli strains and nine Shigella dysenteriae serotype 1 strains containing either stx, stx(1), stx(2), or stx(2) variant genes, were examined. We analyzed these regions by PCR using a set of primers with one primer specific for the respective stx gene and a second primer complementary to sequences of Stx phages H-19B and 933W. We further characterized the amplification products by restriction endonuclease digestion and nucleotide sequencing. PCR products of stx(1)-containing E. coli strains of serogroups O157, O26, and 0103 showed the same lengths and similar restriction patterns. However, we failed to amplify the 3' stx-flanking region in stx(1)-harboring E. coli O111:H(-) strains. Stx2-producing E. coli strains revealed amplification products of different lengths and restriction patterns, suggesting greater heterogeneity than in stx(1)-positive strains. We also obtained specific PCR products for two Stx2c-producing and seven Stx2f-producing E. coli strains when they were subjected to PCR analysis. In nine S. dysenteriae type 1 strains, H-19B- and 933W-specific primers amplified only the 3' stx-flanking region. The results of our study demonstrate that the stx genes of all strains investigated are continuous with phage sequences. Whereas almost all strains except E. coli O111:H(-) strains were associated with a S-like gene, association with Q could not be demonstrated in nine S. dysenteriae type 1 strains and three E. coli strains. Furthermore, we showed that the organization of the stx-flanking regions is similar in all strains investigated, whereas fine-structure analysis showed subtle differences among the sequences examined. Our results support the hypothesis that stx genes in E. coli and S. dysenteriae are generally phage-borne.
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PMID:Structural analysis of phage-borne stx genes and their flanking sequences in shiga toxin-producing Escherichia coli and Shigella dysenteriae type 1 strains. 1094 97

Shiga-like toxin-producing Escherichia coli O157 isolates were characterised by random amplification of polymorphic DNA by PCR (RAPD-PCR) analysis developed to allow robust epidemiological typing of E. coli. Amplification with primer 1247 or 1290 generated a reproducible profile, but was not capable of distinguishing sufficiently between epidemiologically unrelated strains. Subsequent digestion of the amplicons with selected restriction endonucleases improved the discriminatory ability of this method for strains showing limited differentiation following RAPD-PCR analysis alone. Restriction endonuclease analysis of RAPD-PCR fragments generated from closely related strains has the potential to provide additional discriminatory information without loss of specificity.
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PMID:Restriction endonuclease analysis of RAPD-PCR amplicons derived from Shiga-like toxin-producing Escherichia coli O157 isolates. 1119 11


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