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)

High molecular weight DNA samples free of contaminating proteins and RNA obtained from one isolate (of Guangzhou origin) of Cysticercus tenuicollis and five isolates (of Tianjin, Harbin, Lanzhou, Shenyang and Zhengzhou origin) of Cysticercus cellulosae were subjected to thermal denaturation, restriction endonuclease digestion, Southern blotting and hybridization analysis. C. cellulosae DNA showed a melting temperature (Tm) of 82 degrees C corresponding to a 31% GC content whereas C. tenuicollis DNA melted at 85 degrees C suggesting 38.3% GC content. Visual inspection of ethidium bromide-stained gel showed differences not only between the DNAs of the two species of Taeniid cestodes, but also among the five isolates of C. cellulosae. Furthermore, we used two fragments (1.9kb and 5.5kb) of HindIII-derived restriction fragments of C. cellulosae DNA (Harbin origin) and pTS10 as probes to hybridize the DNAs of the Taeniid cestodes from six origins to detect inter- and intra-species genetic variation, the restriction fragment length polymorphisms (RFLPs) were identified.
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PMID:Characterization of taeniid cestodes by DNA analysis. 167 92

Total cell DNA of 14 isolates of Staphylococcus aureus from patients of an intensive care unit (ICU) and 180 unrelated strains was examined by restriction endonuclease analysis (REA). EcoRI-generated DNA fragments were either subjected to conventional REA on agarose gels and stained with ethidium bromide or separated by polyacrylamide gel electrophoresis and visualised by silver staining (SF-REA). Both methods were compared for inter-strain discriminatory ability, reproducibility and handling. All DNA-cleavage patterns of unrelated strains clearly differed from each other when subjected to SF-REA. In contrast, all S. aureus isolates from the ICU gave identical restriction fragment patterns. These findings supported the suspicion of nosocomial infection in these patients. Conventional REA proved the identity of the ICU isolates, but it failed to differentiate between some of the unrelated strains. Therefore SF-REA of total cell DNA seemed to be superior. It has proved to be a very useful technique for studying the epidemiology of S. aureus in hospitals.
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PMID:Application of small fragment restriction endonuclease analysis (SF-REA) to the epidemiological fingerprinting of Staphylococcus aureus. 170 Jan 27

RNA sequences of five flaviviruses were detected by a modified polymerase chain reaction (PCR) that incorporated a reverse transcriptase and RNase inhibitor. Oligonucleotide primer pairs were synthesized to amplify sequences from St. Louis encephalitis (SLE), Japanese encephalitis (JBE), yellow fever (YF), dengue 2 (DEN-2), and dengue 4 (DEN-4) viruses. The amplified products were visualized as bands of appropriate size on ethidium bromide-stained agarose gels. The identity of these products was confirmed by restriction endonuclease cleavage to generate fragments of predicted lengths. The reverse-transcriptase PCR (RT-PCR) successfully amplified flavivirus sequences from cell cultures, frozen brain tissue, and formalin-fixed, paraffin-embedded brain tissue. The reactions were highly specific, and the method compared favorably to two conventional assays of viral infectivity. RT-PCR followed by PCR with nesting primers (N-PCR) was 1,000-fold more sensitive in detecting virus than classical infectivity titration by intracerebral inoculation of suckling mice and nearly 1,000-fold more sensitive than amplification of virus in cell culture followed by inoculation of mice.
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PMID:Detection of flaviviruses by reverse-transcriptase polymerase chain reaction. 171 65

The availability of an epidemiologic typing system for Candida species that is sensitive, rapid, inexpensive, and easy to perform would clearly be an advantage to the mycologist, microbiologist, and epidemiologist in the ongoing struggle to understand the epidemiology and pathogenesis of candidiasis. This is particularly true given the increasing prominence of organisms such as C. albicans and C. tropicalis which are ubiquitous members of the normal flora yet are also important causes of nosocomial bloodstream infection. Unfortunately, the ideal epidemiologic typing system does not yet exist. Current data suggest that the molecular typing methods of restriction endonuclease digestion of genomic DNA with ethidium bromide staining (DEtBr typing) and electrophoretic karyotyping using pulsed-field electrophoresis offer rapid, simple, and sensitive means of discriminating strains of Candida species. These methods appear at present to be the most practical typing methods for both large- and small-scale epidemiologic studies. Other typing methods using specific DNA probes provide a powerful means of identifying strains and will undoubtedly be applied more broadly in the future. Thus far, studies employing molecular typing methods have documented that (1) most patients are colonized by one strain of Candida species, (2) isolates of Candida species recovered from blood or deep tissue sites are generally identical to those obtained from colonization sites before infection developed, and (3) nosocomial transmission of a single strain of C. albicans may occur, particularly in an intensive care unit setting. Given the limitations of the available typing methods and the complex nature of the patients at risk for candidiasis, both the epidemiologist and laboratory scientist must use these methods with clear epidemiologic objectives in mind. Whenever possible, all organisms to be typed should be typed by the same person on the same day, and typing should always include unrelated as well as epidemiologically related isolates. Additional studies, based upon sound epidemiologic principles, will be necessary to clarify the role of the various molecular typing methods as epidemiologic markers of Candida species and to further our understanding of the epidemiology and pathogenesis of candidiasis.
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PMID:The use of molecular techniques for epidemiologic typing of Candida species. 173 71

The objective of this project was to construct specific and sensitive molecular probes and amplification primers for Cryptosporidium parvum that could be used in diagnosis, retrospective tissue studies, and in epidemiologic surveys. Whole genomic DNA was extracted from oocysts of C. parvum purified from human and bovine feces. A genomic library was constructed in plasmid pUC18 and propagated in Escherichia coli DH5 alpha. Transformants were screened by colony hybridization and autoradiography. The 2.3-kilobase segment in plasmid pHC1, a clone specific for C. parvum, was sequenced by the Sanger method. Computer analysis gave a G+C content of 35%. A 400-base region (bases 470-870) was selected as an amplification target because it contained a unique restriction endonuclease site that could serve as a useful marker. Primers of 26 nucleotides each were synthesized. Sensitive and specific amplification of the target sequence was demonstrated both by ethidium bromide staining of agarose and acrylamide gels, and by hybridization with chemiluminescence-labeled synthetic oligonucleotide probes.
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PMID:DNA sequences for the specific detection of Cryptosporidium parvum by the polymerase chain reaction. 176 95

Triggering of the T cell receptor of T cell hybridomas leads to interleukin (IL) 2 secretion, inhibition of spontaneous growth, degradation of genomic DNA and cell death. We have investigated the relationship between the ability of mitochondria to convert 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DNA fragmentation and growth arrest in hybridomas stimulated with anti-CD3/T cell receptor antibodies. We describe a variant T hybridoma whose mitochondrial function remains unaffected upon stimulation with anti-CD3 antibody, although it does undergo DNA fragmentation. By contrast, treatment of another anti-CD3-stimulated T hybridoma with endonuclease inhibitor completely inhibits the DNA fragmentation response but not mitochondrial failure induced by anti-CD3 antibody. Thus, we have been able to dissociate anti-CD3-induced mitochondrial failure and DNA fragmentation, suggesting that they are separate events. Although both undoubtedly contribute to cell death induced by activation the primary cause of death may be mitochondrial failure rather than DNA fragmentation.
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PMID:Anti-CD3-induced cell death in T cell hybridomas: mitochondrial failure and DNA fragmentation are distinct events. 182 34

The plasmid profiles of six isolates of Staphylococcus epidermidis were repetitively evaluated over an 8-month period. Each isolate was subcultured and stored at three different temperatures (-70 degrees C, -20 degrees C, and room temperature) and plasmid DNA was prepared from each subculture at 0, 1, 4, and 8 months by two different methods of plasmid extraction [using mixed alkyltrimethylammonium bromide (ATAB) or Brij 58 and deoxycholate (modified Parisi)]. Plasmid DNA bands were lost from two isolates when subcultures were kept at room temperature. This plasmid loss was confirmed by repetitive extractions and electrophoresis, as well as by restriction endonuclease analysis of the ATAB preparations. Profiles were otherwise highly related to one another, with occasional exceptions being extra or missing plasmid DNA bands of high molecular size. The latter findings were not reproducible. Plasmid DNA extracted by the modified Parisi method was not reliably digested with restriction endonuclease enzymes. We conclude that the plasmid profiles of Staphylococcus epidermidis isolates are highly reproducible as long as isolates are stored at less than or equal to -20 degrees C. Minor discrepancies in the number of plasmid DNA bands of large molecular size may occur. These are resolvable by repetitive testing or restriction endonuclease analysis of ATAB-extracted plasmid DNA preparations.
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PMID:Reproducibility of Staphylococcus epidermidis plasmid profiles. 188 79

We have been investigating the structure, dynamics, and ligand-binding properties of the interface that exists between a right-handed conformation and a left-handed conformation (i.e., a B-Z junction) in synthetic DNA oligomers. Since exo- and endonuclease activity is known to be sensitive to the conformation of the template DNA, we have designed and synthesized a DNA oligonucleotide of 20 base pairs (designated as BZ-III) with an MboI recognition site (GATC) at the location of a potential B-Z junction. The activity of the MboI enzyme toward this molecule and DNA oligomers that contain multiple MboI sites located at B-Z junctions was monitored in the absence and presence of the Z-conformation-inducing reagent cobalt hexaammine. In all cases, the activity of the enzyme was enhanced in the presence of cobalt hexaammine. The activity of MboI toward BZ-III, in the presence and absence of cobalt hexaammine, was also examined when the DNA oligomer is also in the presence of the DNA binding drugs actinomycin D, ametantrone, or ethidium bromide. In all cases, the activity of the enzyme was inhibited in the presence of drug. The results suggest that B-Z junctions are structurally unique and that this uniqueness may alter nuclease activity at sites in or near the junction.
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PMID:Enhanced reactivity of a B-Z junction for cleavage by the restriction enzyme MboI. 193 82

In the present study, we examined the possibility that granulosa cell death during ovarian follicular atresia occurs by apoptosis (programmed cell death). To investigate this possibility, atresia was induced in immature female rats by injecting 15 IU PMSG. Controls received either vehicle or no treatment. PMSG-treated animals were killed on days 1-5 post-injection while controls were killed on days 1 or 5. The onset of atresia was assessed histologically by light microscopic inspection of 5 microns tissue sections and functionally by quantification of serum progesterone and estrogen levels. Apoptosis is characterized by the cleavage of genomic DNA into oligonucleosomal length fragments by a Ca2+/Mg(2+)-dependent endogenous endonuclease. Such fragments form a distinctive ladder pattern when separated electrophoretically. Accordingly, the occurrence of apoptosis in granulosa cells was assessed by examining the pattern of fragmented DNA in cell lysates after agarose gel electrophoresis. Gels were stained with ethidium bromide and DNA visualized by UV transillumination. The earliest morphological signs of atresia were detected 4 days after PMSG injection as evidenced by degeneration and detachment of granulosa cells from the basal lamina. Serum estrogen increased from basal to levels 7-fold over controls by day 3 after PMSG treatment, falling to control values by day 4 and thereafter. In contrast, progesterone remained basal for the first 3 days, rising to levels 3-fold and 8-fold above controls 4 and 5 days after PMSG treatment, respectively. Such shifts in the ratio of estrogen to progesterone production are known to be characteristic of follicular atresia. Finally, electrophoretic analysis of low mol wt DNA in granulosa cell lysates revealed a definitive ladder pattern of oligonucleosomal length DNA fragments (characteristic of apoptosis) on days 4 and 5 after PMSG injection. This pattern was not detectable on days 1 and 2 after treatment. Lysates obtained 3 days after PMSG treatment showed a faint apoptotic-like pattern of DNA fragments; a result consistent with other systems in which DNA cleavage begins before any morphological signs of death. Interestingly, a ladder pattern of DNA fragments was present in control lysates suggesting that granulosa cell death under normal (vs. induced) conditions of atresia in immature rats occurs by apoptosis. These data demonstrate an intimate association between apoptotic-like events and dying granulosa cells and thus support the possibility that apoptosis is involved in the induction of follicular atresia.
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PMID:Biochemical identification of apoptosis (programmed cell death) in granulosa cells: evidence for a potential mechanism underlying follicular atresia. 193 75

Every bulky lesion in DNA can potentially inhibit the Taq DNA polymerase and thereby decrease the amplification produced in the polymerase chain reaction. We investigated the feasibility of using this inhibition to quantify DNA lesions produced by the anticancer drug cisplatin. Products were detected by electrophoresis followed by ethidium bromide staining. Quantitation was obtained by including [32P]dCTP in the amplification reaction and subsequently assessing the incorporated radioactivity. Hamster genomic DNA was platinated in vitro to defined levels and amplified with primers that produce either a 150, 750 or 2,000 base pair fragment. The degree of inhibition of PCR agreed with the predicted level of DNA platination in each size of fragment, suggesting that the polymerase was inhibited by every cisplatin-induced lesion. This method was used to detect cisplatin-induced lesions in the adenine phosphoribosyltransferase gene of CHO cells. Cells were incubated with 0-125 microM cisplatin for 2 h, the DNA was purified and subjected to PCR. A significant decrease in amplification of the 2 kbp fragment was observed in DNA from cells incubated with cisplatin at 75 microM. The degree of inhibition agreed closely with the amount of DNA damage in the overall genome as measured by atomic absorption. No change was detected in amplification of the 150 base fragment which can therefore be used to normalize data for any variations between DNA samples. This assay has the same sensitivity as other methods currently used for the analysis of gene-specific damage. The advantage of this assay is that it obviates the need for specific endonuclease complexes to recognize and cleave DNA adducts as previously required when analyzing damage in specific genomic sequences.
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PMID:A polymerase chain reaction-based method to detect cisplatin adducts in specific genes. 195 80


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