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PubMedDate not supplied

[Application of the duplex-specific nuclease preference method to the analysis of point mutations in human genes].

A new modification of the single nucleotide polymorphism (SNP) analysis (DSNP, duplex-specific nuclease preference) method using the duplex-specific nuclease from the king crab was proposed. The method was used to study SNPs in the following human genes: kRAS, nRAS, hRAS, and p53, the genes of blood coagulation factor V, methyltetrahydrofolate reductase, prothrombin, and apolipoprotein E and a deletion in the BRCA1 gene. DSNP was shown to be useful for the estimation of the mutant allele content in DNA samples. A system for the simultaneous identification of several adjacent single-nucleotide polymorphisms in the kRAS gene was proposed. The approaches could be used to develop test systems for the detection of SNPs in human genes. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.

Alleles↗
PubMed1990

[Fecal contamination with nuclease-positive bacteria as one of the indices of intestinal dysbacteriosis].

A comparative study of the levels of fecal contamination with opportunistic anaerobic nuclease-positive microorganisms was carried out in 114 normal subjects with normal intestinal microflora and in 82 somatic patients with disordered microflora in order to find new integral indicators of disturbed microflora of the intestine; along with routine methods, new techniques were employed. Integral assessment of the intestinal microflora was carried out by scintillation of the total count of facultative anaerobic microorganisms and the share of nuclease-positive bacteria, that yielded nucleic acid depolymerization areas 2 mm or higher from the border of the colonies.

Bacterial Infections↗
PubMed1991

[A comparative study of the functions of recBCD-nuclease from Escherichia coli and "recombinase", encoded by plasmid R1drd-19].

The RecBCD nuclease of Escherichia coli and "recombinase" determined by R1drd-19 plasmid (the latter is able to replace at least partially the indicated cellular enzyme) were shown to differ from each other in some essential features. The product encoded by the plasmid as distinct from RecBCD nuclease practically is not sensitive to inhibition by GamS protein of the lambda phage. Earlier, it was found that the presence of R1drd-19 plasmid in the recBC cells restores the level of the total ATP-dependent exonuclease activity because of appearance in such cells of a new exonuclease activity also ATP-dependent. The exonuclease activity determined by R1drd-19 plasmid was found to differ from the corresponding activity of the RecBCD enzyme. The plasmid enzyme was able to prevent reproduction of T4g2- mutant on recBC cells. The ability of the plasmid "recombinase" to some stimulation of intrachromosomal recombination in recA mutant witness to incomplete RecA-dependence of its function. No significant homology was registered between Escherichia coli DNA fragment containing the recB, recC, recD genes and the EcoRI-C-fragment of R1drd-19 carrying the sequences responsible for recombination and repair functions of the plasmid.

Bacteriophage lambda↗
PubMed1986

On the recognition of helical RNA by cobra venom V1 nuclease.

The V1 nuclease from cobra venom preferentially hydrolyzes double helical RNA and has been used extensively for detecting RNA secondary structure. To increase the utility of this enzyme as an RNA structure probe, we have investigated its properties and substrate specificity, using assays for polynucleotide hydrolysis based on fluorescent polynucleotide derivatives. Enzymatic activity requires both Na+ and Mg2+, with optima at 100 and 0.3 mM, respectively. From the sharp decrease in enzyme activity above 100 mM Na+ we estimate that 3-4 ionic interactions between the protein and polynucleotide phosphates take place. Analysis of products remaining after extensive V1 digestion also shows that the minimum size substrate is 4-6 nucleotides long. Helical RNAs and DNAs have Michaelis constants a factor of 3-10 times lower than most single-stranded RNAs. However, poly(epsilon A) has a Michaelis constant equal to the best synthetic double helices tested and is hydrolyzed at a rate comparable to helical RNA. The major V1 cutting sites in yeast tRNAPhe have Michaelis constants lower than any synthetic polymers. These data suggest that V1 nuclease recognizes any 4-6-nucleotide segment of polynucleotide backbone with an approximately helical conformation, but does not require that the bases be paired in a helix. A few single-stranded V1 cleavage sites are known in tRNA and rRNA, and their structures are consistent with the suggested V1 recognition site.

Base Sequence↗
PubMed1989

A sensitive assay for Staphylococcus aureus nucleases.

A sensitive assay for staphylococcal nuclease involving incubation of the enzyme sample with heat-denatured [3H]thymidine labelled DNA from E. coli, precipitation with trichloroacetic acid and measurement of the radioactivity of acid-soluble nucleotides released has been developed. The assay is sensitive enough to be used for comparing the levels of nucleases elaborated by different strains of S. aureus as well as for determining the extent of contamination of S. aureus in food and water samples even at levels at which the conventional spectrophotometric and toluidine blue-DNA methods are totally inadequate.

Deoxyribonucleases↗
PubMed1989

Quantification of aminofluorene adduct formation and repair in defined DNA sequences in mammalian cells using the UVRABC nuclease.

Using the UVRABC nuclease as a reagent coupled with DNA restriction and hybridization analysis we have developed a method to quantify N-acetoxy-2-acetylaminofluorene (NAAAF)-induced DNA damage in the coding and noncoding sequences of the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells. High performance liquid chromatography analysis shows that the only DNA adduct formed in NAAAF-treated CHO cells is N-(deoxyguanosine-C8-yl)-2-aminofluorene (dG-C8-AF). DNA sequencing analysis demonstrates that the UVRABC nuclease incises at all potential sites in which dG-C8-AF adduct may form in linear DNA fragments. We have found that the formation and removal of dG-C8-AF adducts in the coding and 3' downstream noncoding sequences of the DHFR domain are similar in cells treated with 10 microM NAAAF (3.1 adducts/14 kilobases); DNA adduct removal attains 70% for both sequences within 24 h. This result contrasts with that obtained for the repair of cyclobutane dipyrimidines in the DHFR gene, in which the repair efficiency is much higher in the coding region than in the 3' downstream noncoding region. Our results suggest that in CHO cells the repair pathway for aminofluorene DNA adducts is not the same as that for cyclobutane dipyrimidines. This new technique has the potential to detect a variety of chemical carcinogen induced DNA adducts at the gene level in cultured cells and in DNA isolated from animal tissues.

2-Acetylaminofluorene↗
PubMed1987

Flow cytometric evaluation of DNA digestion with S1 nuclease.

The digestion of DNA with the S1 nuclease has been investigated by flow cytometry on two cultured cell lines and on splenic lymphocytes. The nucleolytic activity of the enzyme has been determined by measuring the decrease in fluorescence intensity of DNA stained with Propidium Iodide (PI). The reaction kinetics could be estimated through the shift of the G1 peak, since the shape of the DNA histograms obtained from cell submitted to S1 digestion did not show any particular variation as compared with the histograms of undigested control cells. DNA digestion was found to be more pronounced when cells were previously damaged with agents known to induce strand breaks, such as gamma-rays or Adriamycin. The assessment of DNA sensitivity to S1 nuclease with flow cytometry may thus provide a fast and reliable procedure for evaluating chromatin structural changes, both in physiological and pathological conditions.

Animals↗
PubMed1988

[Changes in the interaction of DNA with chromatin and nuclear matrix proteins during digestion of nuclei with restrictases and nuclease Bal 31].

Earlier experiments with the use of nucleoprotein-celite chromatography revealed that DNA is bound to a replicative complex localized in the nuclear matrix by a topologically tight bond. Induction of site-specific DNA breaks by restriction nucleases in isolated nuclei of proliferating cells causes a gradual concentration-dependent liberation of DNA from the tight binding to the nuclear matrix. The DNA involved in the tight interaction with matrix proteins is especially sensitive to digestion by Sau 3A1, EcoRI, PstI, BCNI and Bam HI restrictases. One-strand DNA-specific nuclease Bal 31 also destroys the tight DNA-matrix bond. The tightness of DNA-protein bonds in chromatin particles formed after the digestion of nuclei with restrictases is dependent on the particle size. The data are summarized in a model of a topological DNA-matrix bond.

Chromatin↗
PubMed1986

[Dinucleosome as a product of initial chromatin cleavage by endogenous nucleases].

The ability of nucleic endonucleases to recognize dinucleosomal level of chromatin structure was studied. Rat liver chromatin endonucleases were shown to be capable of DNA cleavage at dinucleosome linkers. The cleavage was observed mainly at initial stages of chromatin autohydrolysis, i.e. up to 10-20th min of incubation in the medium containing 5 mmol of magnesium chloride and 2 mmol of calcium chloride. Thus, mononucleosomal DNA in dinucleosomes and other even chromatin subunits was larger and more homogeneous than in odd subunits. The cleavage of autodigested chromatin by bovine spleen and snake venom exonucleases and by nuclease S1 has shown that dinucleosomal structure is independent of differences in the length of internal and external dinucleosome linkers. The initial endonucleolysis appears to be characterized by different accessibility of the linkers for chromatin nucleases.

Animals↗
PubMed1986

Human acid beta-glucosidase: Northern blot and S1 nuclease analysis of mRNA from HeLa cells and normal and Gaucher disease fibroblasts.

Human acid beta-glucosidase (beta-Glc) mRNA was evaluated by dot blot, Northern blot, and S1 nuclease analyses of extracts of HeLa cells and cultured fibroblasts from normal individuals and Gaucher disease patients. Dot blot quantitation indicated an equal concentration of specific mRNA in all sources. Northern blot analyses demonstrated the presence of three poly (A)+ mRNAs of about 5,600, 2,500, and 2,000 nucleotides in length from all cell extracts. All three mRNAs were present in normal amounts in fibroblast extracts from several subtypes and variants of Gaucher disease. The largest poly (A)+ mRNA species was thought to represent an unspliced nuclear precursor for the two smaller beta-Glc mRNAs. S1 nuclease analyses, using SP6 transcripts of beta-Glc cDNA, indicated that the 2,000 nucleotide mRNA differs from the 2,500 nucleotide form at both the 5' and 3' ends. These results are consistent with the use of an alternate 5' splice site and 3' polyadenylation signal. These results also suggest that the subtype and variants of Gaucher disease result from single base alterations that lead to the synthesis of defective beta-Glc.

Cells, Cultured↗
PubMed1987

Treatment with dilute alkali-nuclease S1 permits the analysis of DNA damage: cells treated with platinum analogues.

We describe here an approach to characterize various lesions induced in DNA by drug treatments, using three parameters: (a) release of single-stranded DNA fragments by cell lysis in dilute alkali, which result from enzymatic strand scission during DNA repair or chemical alterations of DNA; (b) the presence of high molecular weight DNA in cells after lysis in dilute alkali followed by nuclease S1 treatment which, due to drug-induced DNA cross-links and its level is a measure of the amount of DNA-containing cross-links; and (c) the appearance of small double-stranded DNA fragments, when the cell lysis is followed by digestion with nuclease S1 to remove single-stranded DNA. This DNA shows the same characteristics as DNA of untreated cells, but it may contain monoadducts. By following the flow of label through the three parameters, one can characterize both the lesions induced in DNA and how the lesions are repaired. We report here results of three platinum analogues: cis-Pt(II), trans-Pt(II), and cis-FLAP(II). A large proportion of DNA in treated cells appears as fragments (parameter c). The cis- compounds and trans- compounds differ with regard to appearance of high molecular weight DNA (parameter b) and the initial release of fragments (parameter a).

Cell Line↗
PubMed1977

[Antitumor action of Ser. marcescens nuclease covalently bound to soluble dextrans].

The antitumor effect of Ser. marcescens nuclease, modified by covalent binding using the method of diazocoupling with m-aminobenzyloxymethyl dextran of molecular weight 20 000, 40 000, 60 000, exceeds 3--4 times the antitumor effect of the native enzyme in relation to Ehrlich ascites carcinoma in contact tumor cells exposure. In intramuscular injection of the enzyme the nuclease preparation modified by dextran of molecular weight 40 000 showed the greatest antitumor activity against Ehrlich carcinoma and sarcoma 37.

Animals↗
PubMed1979

[Rat brain nuclease activity during learning with emotionally different reinforcement].

Acid and alkaline activity of nucleases of the rats trained with emotional positive or negative reinforcement was estimated in the neocortex, hippocampus, midbrain, and in caudal portions of the brain-stem, using native and denaturated DNA as a substrate. The results showed the total increase in nuclease activity during learning. Nevertheless the dynamics of enzyme activation was different depending on the emotional state of rats during learning. The most active enzyme was found in the caudal portion of the brain-stem.

Animals↗
PubMed1979

[Nuclease-antibodies in infections with Serratia marcescens (author's transl)].

A total of 467 presumable healthy men of age eighteen yielded titres of anti-serratia-nuclease that ranged from 1:4 to 1:6. One third of 56 patients, who had been infected with Serratia marcescens (urinary-, wound-, respiratory-infections, and bacteremia) yielded raised anti-nuclease titres, which amounted maximaly 1:16. The diagnostic utility of this serological test was discussed.

Adolescent↗
PubMed1985

[Identification of Staphylococcus aureus by the sero-inhibition of nuclease in blood culture broths].

Identification of Staphylococcus aureus in blood cultures by sero-inhibition nuclease test is described in this paper. No positive results were found in 392 Staphylococcus other than Staphylococcus aureus bacteremia. Only 0,8% Staphylococcus aureus bacteremia could not be detected. This sero-inhibition nuclease test provides a rapid, less than 3 hours, easy to perform, reliable method to identify Staphylococcus aureus in blood cultures. Results are obtained 24 hours earlier than the conventional methods.

Culture Media↗
PubMedDate not supplied

[Nucleases of cell fractions of rat brain tissues].

Nucleases are found in different fractions of nerve cells in the rat brain. The nuclease denaturating preferably the one-chain DNA at pH 8.0 is located chiefly in the neuron nuclei. Fractions of cell nuclei containg mainly neuron nuclei or glial nuclei were obtained by the method of ultracentrifugation within the sucrose gradient.

Animals↗