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Journal of Biochemistry May 1981Phasing of nucleosomes on SV40 DNA was studied by the reconstitution of chromatin from SV40 DNA form I and core histones. The reconstituted chromatin sedimented with...
Phasing of nucleosomes on SV40 DNA was studied by the reconstitution of chromatin from SV40 DNA form I and core histones. The reconstituted chromatin sedimented with increasing S values as the histone : DNA ratios increased, and the buoyant densities in CsCl decreased concomitantly. The average repeat lengths of nucleosomes in the chromatin reconstituted at ratios of 1.0 and 1.5 were estimated to be 168 and 143 base pairs, respectively, by electrophoretic analysis of DNA fragments generated by micrococcal nuclease digestion. The chromatin generated a series of DNA bands that differed in size by about 10 nucleotides upon DNase I digestion followed by heat-denaturation. Phasing of nucleosomes was probed by the use of single-site restriction endonucleases, EcoRI, BamH1, BglI, and HpaII: the latter two cleave DNA at and near the origin of DNA replication and transcription. The form I DNA in the chromatin reconstituted at ratios of 0.5, 1.0, and 1.5 was cleaved up to 60 to 80%, 20 to 60%, and 0 to 10%, respectively. Although the frequency of cleavage by these enzymes was not very different at the ratio 0.5, the BglI site became relatively more susceptible than the other sites at the ratio 1.0. At the ratio 1.5, the DNA was almost resistant to these enzymes, though a significant amount (10%) was cleaved by BglI. These results suggest that the origin is the site unfavorable for nucleosome phasing although the region can be almost completely covered with nucleosomes at higher histone : DNA ratios. The fraction of chromatin immunoprecipitated with anti-T serum after in vitro T antigen binding also decreased with increase in the histone : DNA ratios. The results suggest that T antigen binds preferentially to the internucleosomal region. T antigen preferentially bound to the chromatin reconstituted with the DNA fragment containing the origin. Inefficient phasing of nucleosomes at the origin of DNA replication may facilitate the binding of T antigen to the origin.
Topics: Antigens, Neoplasm; Antigens, Viral; Antigens, Viral, Tumor; Base Sequence; Chemical Precipitation; DNA Restriction Enzymes; DNA, Viral; Deoxyribonuclease BamHI; Deoxyribonuclease HpaII; Deoxyribonucleases, Type II Site-Specific; Histones; Immunologic Techniques; Nucleosomes; Simian virus 40
PubMed: 6168635
DOI: 10.1093/oxfordjournals.jbchem.a133329 -
The Biochemical Journal May 1979The organization of chromatin in three rat liver nuclear populations, namely diploid stromal, diploid parenchymal, and tetraploid parenchymal nuclei, which were...
The organization of chromatin in three rat liver nuclear populations, namely diploid stromal, diploid parenchymal, and tetraploid parenchymal nuclei, which were separated by zonal centrifugation, was studied by digestion with micrococcal nuclease and pancreatic deoxyribonuclease in 3-week-old rats in which the parenchymal cells contain diploid nuclei and in 2-and 4-month-old rats with a high proportion of tetraploid nuclei. Digestion by micrococcal nuclease allowed the estimation of DNA-repeat length in chromatin. Parenchymal nuclei have shorter repeat length than stromal nuclei and DNA-repeat length increases with the age in all three nuclei populations. The kinetics of digestion by micrococcal nuclease showed that nuclei with shorter repeat length are more sensitive to micrococcal nuclease and that the sensitivity of chromatin decreases with age for all the types of nuclei in this study. The kinetics of digestion by pancreatic deoxyribonuclease showed that sensitivity of chromatin is related to the repeat length and that the sensitivity decreases with the ages.
Topics: Age Factors; Animals; Cell Nucleus; Chromatin; DNA; Deoxyribonucleases; Electrophoresis, Polyacrylamide Gel; In Vitro Techniques; Kinetics; Liver; Male; Micrococcal Nuclease; Ploidies; Rats
PubMed: 486082
DOI: 10.1042/bj1790291 -
The Journal of Biological Chemistry Apr 1981An endodeoxyribonuclease from HeLa cells acting on apurinic/apyrimidinic (AP) sites has been purified to apparent homogeneity as judged by sodium dodecyl sulfate...
An endodeoxyribonuclease from HeLa cells acting on apurinic/apyrimidinic (AP) sites has been purified to apparent homogeneity as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The presence of Triton X-100 was necessary throughout the purification for stabilization and stimulation of activity. The endonuclease has an apparent native molecular weight of 32,000 determined by molecular sieving and an apparent subunit molecular weight of 41,000 as judged by its electrophoretic mobility in SDS-polyacrylamide gels. The activity has an absolute requirement for Mg2+ or Mn2+ and a broad pH optimum between 6.7 and 9.0 with maximal activity near pH 7.5. The enzyme has no detectable exonuclease activity, nor any endonuclease activity on untreated duplex or single-stranded DNA. It is inhibited by adenine, hypoxanthine, adenosine, AMP, ADP-ribose, and NAD+, but it is unaffected by caffeine, the pyrimidine bases, ADP, ATP, or NADH. The use of a variety of damaged DNA substrates provided no indication that the enzyme acts on other than AP sites. The enzyme appears to cleave AP DNA so as to leave deoxyribose-5-phosphate at the 5' terminus and a 3'-OH at the 3' terminus; it also removes deoxyribose-5-phosphate from AP DNA which has deoxyribose at the 3' terminus. Specific antibody has been produced in rabbits which interacts only with a 41,000-dalton protein present in the purified enzyme (presumably the enzyme itself), as well as with partially purified AP endonuclease fractions from human placenta and fibroblasts.
Topics: Animals; Cattle; DNA-(Apurinic or Apyrimidinic Site) Lyase; Deoxyribonuclease IV (Phage T4-Induced); Deoxyribonucleases; Endonucleases; Female; HeLa Cells; Humans; Kinetics; Liver; Macromolecular Substances; Magnesium; Manganese; Molecular Weight; Organ Specificity; Placenta; Pregnancy; Species Specificity; Substrate Specificity; Thymus Gland
PubMed: 6259165
DOI: No ID Found -
BioTechniques Sep 2000The stability of restriction enzymes as supplied by manufacturers without any modification has been examined. No reduction in activity was observed for three enzymes...
The stability of restriction enzymes as supplied by manufacturers without any modification has been examined. No reduction in activity was observed for three enzymes (HindIII, EcoRI and Tsp509I) held at ambient temperature or 4 degrees C for the period of study (12 months), while activity was observed for up to 12 weeks after storage at 37 degrees C, which was considerably better than following desiccation with trehalose, a recognized preservation technique. A larger trial of 23 different restriction enzymes held at room temperature for one week showed that all enzymes retained significant activity. As a practical demonstration of the usefulness of this finding, enzymes were posted to Africa by conventional mail (cost $1 US) and shown to retain activity upon arrival after three weeks in transit (compared to a cost of $1000 US by cold-chain transportation). Supplying enzymes to third-world markets should now be possible by removing the necessity for cold-chain transport. After arrival, enzymes can simply be stored in a standard domestic refrigerator.
Topics: Cold Temperature; DNA Restriction Enzymes; Deoxyribonuclease EcoRI; Deoxyribonuclease HindIII; Deoxyribonucleases, Type II Site-Specific; Developing Countries; Enzyme Stability; Temperature; Time Factors; Transportation; Trehalose
PubMed: 10997268
DOI: 10.2144/00293st06 -
The Journal of Biological Chemistry May 1978The deoxyribonuclease induced in KB cells by herpes simplex virus (HSV) type 1 and type 2 has been purified. Both enzymes are able to completely degrade single- and...
The deoxyribonuclease induced in KB cells by herpes simplex virus (HSV) type 1 and type 2 has been purified. Both enzymes are able to completely degrade single- and double-stranded DNA yielding 5'-monophosphonucleotides as the sole products. A divalent cation, either Mg2+ or Mn2+, is an absolute requirement for catalysis and a reducing agent is necessary for enzyme stability. The maximum rate of reaction is achieved with 5 mM MgCl2 for both HSV-1 and HSV-2 DNase. The optimum concentration for Mn2+ is 0.1 to 0.2 mM and no exonuclease activity is observed when the concentration of Mn2+ is greater than 1 mM. The rate of reaction at the optimal Mg2+ concentration is 3- to 5-fold greater than that at the optimal Mn2+ concentration. In the presence of Mg2+, the enzymes are inhibited upon the addition of Mn2+, Ca2+, and Zn2+. The enzymatic reaction is also inhibited by spermine and spermidine, but not by putrescine. Crude and purified HSV-1 and HSV-2 DNase can degrade both HSV-1 and HSV-2 DNA, but native HSV-1 DNA is hydrolyzed at only 22% of the rate and HSV-2 DNA at only 32% of the rate of Escherichia coli DNA. Although HSV-1 and HSV-2 DNase were similar, minor differences were observed in most other properties such as pH optimum, inhibition by high ionic strength, activation energy, and sedimentation coefficient. However, the enzymes differ immunologically.
Topics: Cell Line; Deoxyribonucleases; Enzyme Induction; Immunoassay; Kinetics; Polyamines; Simplexvirus
PubMed: 206546
DOI: No ID Found -
Journal of Virology Jul 1984Epstein-Barr virus (EBV), isolated from P3HR-1 cells, induces early antigen and viral capsid antigen upon infection of human B-lymphoblasts. The strong early antigen-...
Epstein-Barr virus (EBV), isolated from P3HR-1 cells, induces early antigen and viral capsid antigen upon infection of human B-lymphoblasts. The strong early antigen- and viral capsid antigen-inducing activity is only observed in P3HR-1 virus preparations harboring particles with defective genomes, suggesting that this biological activity is directly associated with the defective DNA population. After infection of EBV genome-carrying Raji or EBV genome-negative BJAB cells, defective genomes of P3HR-1 EBV DNA are replicated in excess, depending on the multiplicity of infecting EBV particles. Hybridization of the DNA from such infected cells with 32P-labeled EBV DNA after HindIII cleavage reveals six hypermolar fragments. Mapping of these fragments shows that they form one defective genome unit containing four nonadjacent regions (alpha, beta, gamma, and delta) of the nondefective P3HR-1 EBV DNA. Two of the segments (alpha and beta) contain ca. 17 and 13 megadaltons, respectively, from the terminal regions of the P3HR-1 genome, whereas the two smaller segments (gamma and delta) contain ca. 3.7 and 3.0 megadaltons, respectively, originating from the central portion of the genome. In the defective molecule, the regions gamma and delta are present in the opposite orientation compared with nondefective P3HR-1 EBV DNA. Tandem concatemers are formed by fusion of the alpha and beta regions. Our model suggests that tandem concatemers of three defective genome units can be packaged into virions in P3HR-1 cells.
Topics: Base Sequence; Cell Line; DNA Restriction Enzymes; DNA, Viral; Deoxyribonuclease BamHI; Deoxyribonucleases, Type II Site-Specific; Herpesvirus 4, Human; Nucleic Acid Conformation; Nucleic Acid Hybridization
PubMed: 6328039
DOI: 10.1128/JVI.51.1.199-207.1984 -
Nucleic Acids Research Mar 1982The ability of thirty Type II restriction endonucleases to cleave five different types of highly modified DNA has been examined. The DNA substrates were derived from... (Comparative Study)
Comparative Study
The ability of thirty Type II restriction endonucleases to cleave five different types of highly modified DNA has been examined. The DNA substrates were derived from relatively large bacteriophage genomes which contain all or most of the cytosine or thymine residues substituted at the 5-position. These substituents were a proton (PBS1 DNA), a hydroxymethyl group (SP01 DNA), a methyl group (XP12 DNA), a glucosylated hydroxymethyl group (T4 DNA), or a phosphoglucuronated, glucosylated 4,5-dihydroxypentyl group (SP15 DNA). Although PBS1 DNA and SP01 DNA were digested by most of the enzymes, they were cleaved much more slowly than was normal DNA by many of them. 5-Methylcytosine-rich XP12 DNA and the multiply modified T4 and SP15 DNAs were resistant to most of these endonucleases. The only enzyme that cleaved all five of these DNAs was TaqI, which fragmented them extensively.
Topics: Base Sequence; DNA Restriction Enzymes; DNA, Viral; Deoxyribonuclease I; Deoxyribonucleases; Endonucleases; Kinetics; Phosphoric Diester Hydrolases; Substrate Specificity
PubMed: 6280151
DOI: 10.1093/nar/10.5.1579 -
The Journal of Biological Chemistry Dec 1987K562 cells express embryonic (epsilon) and fetal (gamma) globins and hemoglobins but not adult (beta) globin. To define the cis acting regulatory elements involved in...
K562 cells express embryonic (epsilon) and fetal (gamma) globins and hemoglobins but not adult (beta) globin. To define the cis acting regulatory elements involved in the discrimination between gamma and beta genes, we have constructed chimeric genes composed of portions of gamma and beta and evaluated their expression in stable K562 transfectants. A gamma beta fusion gene containing gamma 5' sequences to the EcoRI site in exon 3 and beta sequences 3' is expressed at 10-40% that of the endogenous gamma level. In 50% of the lines, this fusion gene appropriately increases its expression in response to hemin, an inducer of endogenous globin gene expression in K562 cells. In contrast, a beta gamma fusion gene, containing beta sequences 5' to the EcoRI site in exon 3 and gamma sequences 3', is neither expressed nor correctly initiated. A beta gene containing gamma-intervening sequence (IVS) 2 accumulates an mRNA transcript when analyzed with a 3' beta probe. However, no correctly initiated beta mRNA is observed. A gamma gene with beta-IVS 2 is only inducible in one of six expressing clones. All the results are consistent with the presence of stage-specific trans acting factors in K562 cells that stimulate expression of gamma genes and suggest a significant role for gamma-IVS 2 in gamma gene expression.
Topics: Cell Line; DNA Restriction Enzymes; Deoxyribonuclease EcoRI; Deoxyribonuclease HpaII; Deoxyribonucleases, Type II Site-Specific; Endonucleases; Gene Expression Regulation; Globins; Humans; Leukemia, Erythroblastic, Acute; Plasmids; Single-Strand Specific DNA and RNA Endonucleases; Transfection
PubMed: 2445753
DOI: No ID Found -
Nucleic Acids Research Sep 1981The precise number of base pairs per turn of the DNA double helix in the nucleosome core particle has been the subject of controversy. In this paper the positions of...
The precise number of base pairs per turn of the DNA double helix in the nucleosome core particle has been the subject of controversy. In this paper the positions of nuclease cutting sites are analysed in three dimensions. Using this midpoint of the DNA on the nucleosome dyad as origin, the cutting site locations measured along a strand of DNA are mapped onto models of the nucleosome core containing DNA of different helical periodicities. It is found that a helical periodicity of 10.5 base pairs per turn leads to cutting site positions which are sterically inaccessible. In contrast, a periodicity of 10.0 base pairs per turn leads to cutting site positions which are not only sterically sound, but which fall into a pattern such as would be expected when the access of the nuclease to the DNA is restricted by the presence of the histone core on one side and of the adjacent superhelical turn of DNA on the other. As proposed earlier by us (1), a value for the helical periodicity close to 10 base pairs per turn on the nucleosome, taken together with a periodicity close to 10.5 for DNA in solution - a value now established - resolves the so-called linkage number paradox.
Topics: Chemical Phenomena; Chemistry, Physical; DNA; Deoxyribonuclease I; Deoxyribonucleases; Endonucleases; Genetic Linkage; Nucleic Acid Conformation; Nucleosomes; Periodicity
PubMed: 6272202
DOI: 10.1093/nar/9.17.4267 -
The Journal of Biological Chemistry Dec 2003Current biochemical characterizations of cystic fibrosis (CF) sputum do not address the high degree of microheterogeneity in the rheological properties of the mucosal...
Current biochemical characterizations of cystic fibrosis (CF) sputum do not address the high degree of microheterogeneity in the rheological properties of the mucosal matrix and only provide bulk-average particle diffusion coefficients. The viscoelasticity of CF sputum greatly reduces the diffusion rates of colloidal particles, limiting the effectiveness of gene delivery to underlying lung cells. We determine diffusion coefficients of hundreds of individual amine-modified and carboxylated polystyrene particles (diameter 100-500 nm) embedded in human CF sputum with 5 nm and 33 ms of spatiotemporal resolution. High resolution multiple particle tracking is used to calculate the effective viscoelastic properties of CF sputum at the micron scale, which we relate to its macroscopic viscoelasticity. CF sputum microviscosity, as probed by 100- and 200-nm particles, is an order of magnitude lower than its macroviscosity, suggesting that nanoparticles dispersed in CF sputum are transported primarily through lower viscosity pores within a highly elastic matrix. Multiple particle tracking provides a non-destructive, highly sensitive method to quantify the high heterogeneity of the mucus pore network. The mean diffusion coefficient becomes dominated by relatively few but fast-moving particles as particle size is reduced from 500 to 100 nm. Neutrally charged particles with a diameter <200 nm undergo more rapid transport in CF sputum than charged particles. Treatment with recombinant human DNase (Pulmozyme) reduces macroviscoelastic properties of CF sputum by up to 50% and dramatically narrows the distribution of individual particle diffusion rates but surprisingly does not significantly alter the ensemble-average particle diffusion rate.
Topics: Adolescent; Adult; Biological Transport; Carbon; Cystic Fibrosis; Deoxyribonuclease I; Deoxyribonucleases; Diffusion; Female; Humans; Hydrogen-Ion Concentration; Lung; Male; Microscopy, Confocal; Microscopy, Electron; Mucus; Polystyrenes; Rheology; Sputum; Time Factors
PubMed: 13679362
DOI: 10.1074/jbc.M309026200