Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “NUCLEASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,243 records · Page 69Linked to original sources
PubMedDate not supplied

[Study of the secondary and tertiary structure of phage MS2 RNA using nucleases and fluorescent dyes specific for secondary structure].

The binding of ethidium bromide (EtBr) and acridine orange (AO) to RNA in native state or after hydrolysis by S1 and SV nucleases that specifically split single-stranded and double-stranded segments was studied. Nuclease S1 hydrolysis of RNA does not increase the number of EtBr strong binding sites, Tm and hyperchromic effect being also unchanged. Hydrolysis by double-stranded segments accessible to EtBr is followed by the diminishing of Tm and hyperchromism. A supposition is put forward that the main role in stabilization of the RNA tertiary structure is played by double-stranded segments arranged so that some of them are hidden and do not interact with dyes. One of the possible models may be parallel oriented intramolecular "hair-pins" forming compact "rod-like" structures.

Coliphages↗
PubMed1980

[Probing the structure of bacterial deoxyribonucleoproteins with exogenous and endogenous nucleases].

During digestion of deoxyribonucleoproteins (DNP) of gram-negative bacteria by micrococcal nuclease and Ca2+, Mg2+-dependent endonuclease in situ regular series fragments-and large nuclease-resistent fragments of DNP were revealed by electrophoresis. The DNP length of the smallest DNP-fragment was tentatively 120-140 base pairs. In investigated bacterial species DNP contained at least two basic proteins which had electrophoretic mobility similar to that of histone H4 of eucaryot. It is suggested that bacterial DNPs have common regular structure.

Bacterial Proteins↗
PubMed1980

Substrate specificity of nuclease TT1 from Thermus thermophilus HB8.

The substrate and the action mechanism of a nuclease named nuclease TT1, from the culture broth of an extreme thermophile, Thermus thermophilus HB8, were investigated. The enzyme is nonspecific for the sugar moiety and cleaves both single- and double-stranded DNAs, rRNA, tRNA and oligonucleotides irrespective of chain length to produce 5'-mononucleotides exonucleolitically. The action mechanism is processive and the enzyme shows no porality of degradation. The minimal unit as a substrate is a 5'-dinucleotide. The rate of hydrolysis is independent of a terminal phosphate group. The substrate lacking a 5'-phosphoryl group is degraded to leave the 5'-terminus and the penultimate nucleotide (NpN) as a core. The substrate possessing a 3'-phosphoryl group is degraded to leave the mononucleoside 5',3'-diphosphates (pNp). However, NpN and pNp are gradually degraded by a large dose of the enzyme to produce a 5'-mononucleotide. The enzyme is free from nonspecific phosphatase and phosphodiesterase activities. Application of this enzyme to determine the sequence of oligonucleotides is shown.

Exonucleases↗
PubMedDate not supplied

[Study of spatial organization of the RNA of phage MSZ using nucleases specific for the secondary structure].

The limited hydrolisis of bacteriophage MS2 RNA by nuclease S1 and ds-specific snake venom RNase was studied in a wide range of ionic strength, at different pH, after heating and (slow and fast) cooling and at various enzyme-substrate relations. It was shown that the RNA has exposed hydrolisis sites for both nucleases. The localizations of these sites are very specific and are not altered in all conditions studied. The hydrolisis rate was changed in some conditions, at that the fragments patterns in denaturing electrophoresis did not move. It was supposed that the RNA has strongly predetermined and predominant conformation which could not be altered by strong influences.

Coliphages↗
PubMed1983

Stereochemical course of DNA hydrolysis by nuclease S1.

Nuclease S1 hydrolyzes the Sp-diastereomer of 5'-O-(2'-deoxyadenosyl)-3'-O-thymidyl phosphorothioate in H2(18)O to [18O]deoxyadenosine 5'-O-phosphorothioate which can be phosphorylated enzymatically to the Sp-diastereomer of [alpha-18O]deoxyadenosine 5'-O-(1-thiotriphosphate). 31P nmr spectroscopy shows the oxygen-18 in this compound to be in a nonbridging position at the alpha-phosphorus, indicating that the hydrolysis reaction catalyzed by nuclease S1 proceeds with inversion of configuration at phosphorus. This result is compatible with a direct nucleophilic attack of H2O at phosphorus without the involvement of a covalent enzyme intermediate.

Aspergillus oryzae↗
PubMed1983

[Computer analysis of chromatin fragmentation with nucleases].

Computerizing of the densitograms of chromatin hydrolysate DNA is offered. It is based on searching for gaussoids depicting the peaks of individual oligomers so that the resultant curve corresponded to the experimental curve to a high enough accuracy. The method suggested was used to study the time course of mononucleosome and oligomer accumulation during chromatin fragmentation with endogenous nuclease. Comparison of the experimental data to a model calculated for break down of 500-nucleosome chromatin fragments demonstrates that in chromatin, the release of mono-, di- and trinucleosomes is shifted towards nucleosomes during break down induced by endogenous nuclease.

Animals↗
PubMed1983

Cloned mRNA sequences for two types of embryonic myosin heavy chains from chick skeletal muscle. II. Expression during development using S1 nuclease mapping.

We have examined the expression of two embryonic myosin HC mRNAs using two cDNA clones (110 and 251) which we have previously constructed from RNA isolated from 14-day-old embryonic chick skeletal muscle. Sequence divergence in the 3' nontranslated regions enabled us to analyze the differential expression of the mRNAs corresponding to the two clones using the S1 nuclease mapping procedure. Clone 251 mRNA is expressed primarily in embryonic fast muscle, where its transcripts appear to be the predominant species. This mRNA is minimally expressed in the posthatching period, but it is not detected in adult leg and breast muscle. Messenger RNA for clone 110 is also primarily expressed in embryonic fast muscle. However, in the posthatching and adult stages of development, it continues to be expressed at a low level in leg muscle but not in breast muscle. The differential expression of these mRNAs during development strongly indicates that they correspond to two different genes coding for embryonic myosin HCs. Other myosin HC mRNAs which were partially homologous to the clone 110 or 251 mRNAs were also identified by S1 nuclease mapping. Using the probes from these two clones, a minimum of four other developmentally expressed forms were detected. Two of these correspond to "neonatal" myosin HCs, while the other two code for different adult myosin HCs present in leg and in breast muscle, respectively. The results therefore suggest a much greater diversity of myosin HC mRNAs expressed during development than previously reported.

Animals↗
PubMed1983

S1 nuclease mapping analysis of ribosomal RNA processing in wild type and processing deficient Escherichia coli.

S1 nuclease mapping was used to assess rRNA processing in Escherichia coli. Single-stranded DNA probes complementary to the sequences bordering each terminus of 16 S and 23 S rRNA were end-labeled, hybridized to total E. coli RNA, and treated with S1 nuclease. The resultant DNA fragments were then displayed on denaturing polyacrylamide gels. Measurements of steady state levels of precursor rRNA species and measurements of the rates of decay of precursors after transcription arrest by rifampicin gave consistent results. 1) The rRNA precursor species identified in wild type cells corresponded to those previously identified by other means. 2) In RNase III-deficient strains, mature 16 S rRNA termini form at the same rate as in wild type cells; but the normal mature termini of 23 S rRNA are never generated. 3) RNase III cleavage at the 5' end of 23 S rRNA can occur before the 3' end of the same molecule is synthesized. 4) The cleavages that generate the mature termini of 16 S rRNA are interdependent; in the BUMMER strain, slow processing at the 5' end is accompanied by slow processing at the 3' end. Thus, the kinetically observed order of processing reactions is obligate for some cleavages but not for others, and the assumption that complete rRNA processing is required for function fails for 23 S rRNA.

Base Sequence↗
PubMedDate not supplied

[Residual chromatin of mouse spleen nuclei after intensive nuclease treatment. Isolation and properties].

Two-step treatment of mouse spleen nuclei with staphylococcal nuclease was used to isolate residual nuclear structures lacking a considerable part of chromation. Partial disruption of the nuclear envelope after the first step of digestion was shown to be essential for obtaining residual nuclear structures. Isolated residual nuclear structures contained condensed chromatin (residual chromatin) which was not solubilized upon additional staphylococcal nuclease treatment and amounted to approximately 20% of total nuclear chromatin. Residual chromatin was almost deprived of nonhistone chromosomal proteins. It contained a full complement of histones and consisted of nucleosomal chains having different lengths--from one to 50-60 nucleosomes. Some of the condensed chromatin chains were anchored to the nuclear matrix.

Animals↗
PubMedDate not supplied

[Purification and properties of intracellular nuclease of a competent strain of Bacillus subtilis].

A new nuclease was isolated from a membrane-nucleoproteid complex (MNC) of the cell lysate of the competent strain Bacillus subtilis SB25 his2trp2 and purified 674-fold. It differs from the known exonucleases of Bacillus subtilis by the place of "attacking" the phosphodiester bond, by the absence of the specificity towards the end groups of denatured DNA and by a high pH optimum. Isolation and purification were performed as follows: cell destroying by lysozyme and osmotic shock, isolating the MNC by centrifugation, treating the MNC with pancreatic RNase and dialysis with Ca+2 ions against Tris-acetate buffer, chromatography on CM-and phosphocellulose. The nuclease of the partially purified preparation was activated by Ca+2 ions with the optimal concentration 0.5-1.0 mM. It hydrolysed denatured DNA and RNA with the pH optimum at 10.0-10.5. The main products of the denatured DNA hydrolysis were mono- and dinucleotides with 5'-end phosphate. The enzyme is insensitive to the end hydroxyl and phosphate groups of denatured DNA and was completely inhibited by 1.0 mM EDTA.

Bacillus subtilis↗
PubMed1984

A sequence conserved in both the chicken and mouse alpha 2(I) collagen promoter contains sites sensitive to S1 nuclease.

We have examined the S1 nuclease sensitivity in the promoter of both the chicken and mouse alpha 2(I) collagen genes. When these DNAs are introduced into supercoiled plasmids and digested with S1 nuclease, a discrete region containing one or more cleavages is found in each promoter. These S1 cleavage sites were mapped by the distance of the S1 site from known restriction enzyme cleavage sites. In the chicken gene, the S1-sensitive segment is located 180 to 200 base pairs preceding the start site of transcription, whereas in the mouse promoter it is between -145 to -165 base pairs. This site in the chicken promoter maps to the segment that has previously been shown to be S1 and DNase I hypersensitive in chromatin. Although these S1 sites are found at different distances from the start site of transcription in the two promoters, the sequences at these sites are strongly conserved between the two species. Each sequence consists of an identical tandem repeat containing a short palindrome within each repeat. Since the DNA sequence does not exhibit the features that would favor either a left-handed Z-DNA configuration or a cruciform structure, an alternative model is discussed that could account for the S1 sensitivity of these sequences. The conservation of these sequences and their S1 sensitivity suggests they play a role in the activation or regulation of the alpha 2(I) collagen gene promoters.

Animals↗
PubMed1984

[Affinity chromatography of nucleases].

The review concerns isolation and purification of nucleases by affinity chromatography. Different stationary ligands and the methods for their immobilization on supports are described, along with diverse eluents and various procedures for a nuclease detachment from the affinity sorbents. The data on the affinity chromatography application for measuring the dissociation constants of the enzyme complexes with either immobilized or soluble ligands are compiled.

Animals↗
PubMed1981

[Low transfecting efficiency of phage lambda ring chromosomes and their fragments formed by membrane nucleases].

Transfection efficiency of a number of lambda DNA samples differing in ring to linear molecules ratio was determined. Graphic extrapolation to the zero content of linear molecules showed that efficiency of ring molecules did not exceed 5% of that of linear molecules. Probably, this difference is caused by more fast penetration of linear molecules into the cell and, therefore, by lower probability of their degradation by cell wall nucleases. Fragments of both ring and linear molecules formed by cell wall nucleases proved to be inactive in marker rescue experiments.

Bacteriophage lambda↗
PubMed1983

Isolation and comparison of two molecular species of the BAL 31 nuclease from Alteromonas espejiana with distinct kinetic properties.

The extracellular nuclease from Alteromonas espejiana sp. BAL 31 can be isolated as two distinct proteins, the "fast" (F) and "slow" (S) species, both of which have been purified to homogeneity. The F and S species of the nuclease have molecular weights, respectively, of 109 X 10(3) and 85 X 10(3), and both are single polypeptide chains with an isoelectric pH near 4.2. Both species catalyze the degradation of single-stranded and linear duplex DNAs to 5'-mononucleotides. The degradation of linear duplex DNA occurs through a terminally directed hydrolysis mechanism that results in the removal of nucleotides from both the 3' and 5' ends. Apparent Michaelis constants (Km) have been obtained for the exonuclease activities of both species and for the activity against single-stranded DNA of the S species. The Km for the hydrolysis of single-stranded DNA catalyzed by the F species has not been obtained because the reaction velocity was maximal even at the lowest substrate concentrations accessible in the photometric assay. The ratio of the turnover numbers for the exonuclease activities of the two species indicates that the F species will shorten linear duplex DNA at a rate 27 +/- 5 (S.D.) times faster than an equimolar concentration of the S species in the limit of high substrate concentration, while the corresponding ratio for the activities against single-stranded DNA (1.2 +/- 0.1) shows that the two species are similar with respect to hydrolysis of this substrate. In the limit of high substrate concentrations, the F and S species break phosphodiester bonds in single-stranded DNA at rates 1.3 +/- 0.3 and 33 +/- 2 times those for the exonucleolytic degradation of linear duplex DNA, respectively. It has not been established whether the two species are physically related.

Bacteria↗
PubMed1978

[Modification of nucleases by soluble derivatives of polysaccharides].

Preparations of pancreatic RNAase, ribonuclease Act. rimosus and nuclease Ser. marcescens covalently bound to water-soluble derivatives of polysaccharides (m-aminobenzyl-oxymethyl ether of dextran and mannan, 4beta-oxyethylsulphonylanisol, 4beta-oxyethylsulphonylaniline, 3-Cl-2-oxypropyl ethers of dextran and dialdehydedextran) have been obtained. The yields and thermal stability of immobilized nucleases depend both upon the amount and nature of the functional groups which activate the polysaccharide. Polysaccharide aminoaryl ethers capable of binding to proteins by azocoupling present special interest in view of their utilization as modifying baskings. Regulation of effectiveness of the azocoupling reaction by means of structural changes of the diazocomponent and the reaction conditions were shown. All this allows to obtain immobilized enzymes with different physico-chemical properties.

Azo Compounds↗
PubMed1995

[Serratia marcescens nucleases. II. Primary structure analysis by peptide mapping combined with plasma-desorption mass spectrometry].

The primary structures of nucleases Sm1, Sm2, and Sm3 produced by Serratia marcescens were completely characterized using plasma desorption mass spectrometry (PDMS) of proteolytic peptides isolated by reverse-phase HPLC. The isoforms were separated by anion-exchange chromatography on DEAE cellulose and subjected to hydrolysis by the lysine-specific endoproteinase Lys-C. Comparative analysis of the peptides identified by PDMS showed that all three nucleases are N-terminal variants of the same protein: Sm2 represents a "mature" protein form, whereas Sm1 and Sm3 lack three and one N-terminal amino acid residues, respectively.

Amino Acid Sequence↗
PubMed1993

Nuclease footprinting of human immunodeficiency virus reverse transcriptase/tRNA(Lys-3) complexes.

Nuclease footprinting has been used to probe features of binary complexes of type 1 human immunodeficiency virus reverse transcriptase (HIV-1 RT) with both natural and synthetic preparations of its cognate replication primer, tRNA(Lys-3). In addition to heterodimeric RT (p66/p51), ribonucleoprotein complexes containing either the p66 or p51 subunit were analyzed. Footprinting experiments employed both structure- and sequence-specific nucleases. Our results indicate a similar mode of interaction for the three RT preparations tested, suggesting contact with each loop of the tRNA primer (D, anticodon, and T psi C), as well as minor perturbation of the anticodon stem. Although there is little evidence for extensive disruption of the 3'-acceptor stem. RNase A footprinting data with natural and synthetic tRNA suggests that potential base pairing between the T psi C and D loops is disrupted in the presence of RT.

Anticodon↗
PubMed1993

Nuclease activities of Moloney murine leukemia virus reverse transcriptase. Mutants with altered substrate specificities.

RNases H are traditionally thought to degrade RNA only in RNA-DNA hybrid form. We found that the wild-type Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RT) was capable of degrading RNA in RNA-RNA duplexes as well as in RNA-DNA hybrids, as assayed by in situ gel techniques. Escherichia coli RNase H does not degrade the RNA-RNA duplex in this assay, while E. coli RNase III, a double-strand-specific ribonuclease, does. The apparent specific activity of M-MuLV RT on RNA-RNA duplexes is similar to that on RNA-DNA hybrids. Neither the DNA polymerase domain nor the RNase H domain of RT expressed individually exhibited this RNA-RNA activity. We have generated a series of mutations in the RNase H domain of M-MuLV RT, expressed the mutant enzymes in E. coli, and assayed these mutants for various activities. All RTs were as active as the wild type in the oligo(dT):poly(rA) DNA polymerase assay, and many retained both nuclease activities. Two enzymes with mutations at the carboxyl terminus of the RNase H domain retained RNA-DNA activity, but not RNA-RNA activity. Another mutant enzyme showed the opposite phenotype, retaining RNA-RNA, but not RNA-DNA, nuclease activity. Thus, we were able to genetically separate the two activities. These results may be helpful in defining enzyme-substrate interactions.

Blotting, Western↗