-
Microbiology and Immunology 1981The DNAs from virulent strain BC-1 and avirulent strains C2(A) of Marek's disease virus (MDV) were compared by electrophoresis on 0.5% agarose gels of the products... (Comparative Study)
Comparative Study
The DNAs from virulent strain BC-1 and avirulent strains C2(A) of Marek's disease virus (MDV) were compared by electrophoresis on 0.5% agarose gels of the products obtained with the restriction endonucleases Bam H1, Sal 1, and Sma 1. The patterns of the fragments of the DNAs from these two strains were very similar, but showed some significant differences in the number and mobility of the DNA bands. The DNA fragments obtained with the restriction endonucleases, and especially Sma 1, were mostly present in equal molar ratios, but a few of those obtained with Bam H1 and Sal 1 were present in submolar amounts. In addition, several fragments obtained with Bam H1 and Sal 1 were present in greater than molar quantities, suggesting the presence of reiterated sequences in MDV DNA. The terminal fragments of MDV DNA were identified by their sensitivity to lambda 5'-exonuclease. The terminal fragments obtained with Bam H1 A and Sal 1B showed heterogeneous electrophoretic mobility and contained sequences with high content of guanosine and cytosine, suggesting the presence of reiterated sequences at the end of the MDV DNA molecule.
Topics: Centrifugation, Isopycnic; DNA Restriction Enzymes; DNA, Viral; Deoxyribonuclease BamHI; Deoxyribonucleases, Type II Site-Specific; Electrophoresis, Agar Gel; Genes, Viral; Herpesvirus 2, Gallid; Repetitive Sequences, Nucleic Acid
PubMed: 6268948
DOI: 10.1111/j.1348-0421.1981.tb00070.x -
The Journal of Biological Chemistry Jan 1986The chromatin structure of the 3'-nontranscribed spacer of the linear rRNA gene molecules of Tetrahymena thermophila was examined. This region includes the transcription...
The chromatin structure of the 3'-nontranscribed spacer of the linear rRNA gene molecules of Tetrahymena thermophila was examined. This region includes the transcription termination site, two sets of recently identified conserved spacer repeats (Type IV and V repeats (Challoner, P. B., Amin, A. A., Pearlman, R. E., and Blackburn, E. H. (1985) Nucleic Acids Res. 13, 2661-2680], and the terminus of the molecule. Using sensitivity to nucleases as a probe, a unique chromatin structure was found in this rDNA region. Proceeding from the end of the rDNA molecule, the telomeric repeated sequence, (CCCCAA)n, was packaged in a non-nucleosomal complex adjacent to three phased nucleosomes. This nucleosomal structure was disrupted at the Type V repeat region, which, compared with the neighboring nucleosomal region, was more accessible to nucleases and, from both micrococcal nuclease and DNase I digestion results, was packaged in chromatin differently from the sequences flanking it on both sides. The region between the Type V repeats and adjacent to the transcription termination site was in yet another distinguishable chromatin structure as judged by its sensitivity to nucleases. It includes sites protected in chromatin and sites which were cleaved in chromatin but not detectably digested in DNA controls, suggesting that specific proteins are also associated with this region.
Topics: Animals; Base Sequence; Chromatin; DNA Restriction Enzymes; DNA, Ribosomal; Deoxyribonuclease I; Deoxyribonucleases, Type II Site-Specific; Micrococcal Nuclease; RNA, Ribosomal; Repetitive Sequences, Nucleic Acid; Tetrahymena
PubMed: 3001053
DOI: No ID Found -
The Journal of Biological Chemistry Jun 1975A deoxyribonuclease specific for methylated DNA was isolated from Diplococcus pneumoniae. The enzyme, an endonuclease, degrades DNA for Escherichia coli to fragments of...
A deoxyribonuclease specific for methylated DNA was isolated from Diplococcus pneumoniae. The enzyme, an endonuclease, degrades DNA for Escherichia coli to fragments of average molecular weight about half a million; it forms discrete fragments from phage lambda DNA. Methyl-deficient E. coli DNA is not attacked, neither is DNA from Micrococcus radiodurans, which contains no methylated adenine or cytosine. Nor is DNA from D. pneumoniae or phage T7 attacked. However, DNA from M. radiodurans, D. pneumoniae, and T7 is attacked after methylation with and E. coli extract. Methylated T7 DNA is degraded to discrete fragments. Although the genetic transforming activity of normal DNA from D. pneumoniae is not affected by the enzyme, transforming activity of methylated DNA is destroyed. The enzyme is designated endonuclease R Dpn I. Under certain conditions another enzyme of complementary specificity can be isolated. This enzyme, designated endonuclease R Dpn II, produces a similar pattern of fragments from the DNA of T7 without prior methylation of the DNA. It also degrades normal DNA for D. pneumoniae. It is suggested that this pair of enzymes plays a role in some unknown control process, which would involve a large fraction of the specific base sequences that are methylated in E. coli DNA and are present but not methylated in DNA from other sources.
Topics: Chromatography, Gel; Coliphages; DNA Viruses; DNA, Bacterial; DNA, Viral; Deoxyribonucleases; Electrophoresis; Electrophoresis, Polyacrylamide Gel; Endonucleases; Escherichia coli; Methylation; Sepharose; Species Specificity; Streptococcus pneumoniae; Transformation, Genetic; Viscosity
PubMed: 236309
DOI: No ID Found -
Scientific Reports Jul 2017The stratum corneum of the epidermis constitutes the mammalian skin barrier to the environment. It is formed by cornification of keratinocytes, a process which involves...
The stratum corneum of the epidermis constitutes the mammalian skin barrier to the environment. It is formed by cornification of keratinocytes, a process which involves the removal of nuclear DNA. Here, we investigated the mechanism of cornification-associated DNA degradation by generating mouse models deficient of candidate DNA-degrading enzymes and characterizing their epidermal phenotypes. In contrast to Dnase1l2 mice and keratinocyte-specific DNase2 knockout mice (Dnase2 ), Dnase1l2 Dnase2 mice aberrantly retained nuclear DNA in the stratum corneum, a phenomenon commonly referred to as parakeratosis. The DNA within DNase1L2/DNase2-deficient corneocytes was partially degraded in a DNase1-independent manner. Isolation of corneocytes, i.e. the cornified cell components of the stratum corneum, and labelling of DNA demonstrated that corneocytes of Dnase1l2 Dnase2 mice contained DNA in a nucleus-shaped compartment that also contained nucleosomal histones but lacked the nuclear intermediate filament protein lamin A/C. Parakeratosis was not associated with altered corneocyte resistance to mechanical stress, changes in transepidermal water loss, or inflammatory infiltrates in Dnase1l2 Dnase2 mice. The results of this study suggest that cornification of epidermal keratinocytes depends on the cooperation of DNase1L2 and DNase2 and indicate that parakeratosis per se does not suffice to cause skin pathologies.
Topics: Animals; DNA; Deoxyribonucleases; Endodeoxyribonucleases; Epidermis; Keratinocytes; Mice, Knockout; Mice, Transgenic; Parakeratosis
PubMed: 28743926
DOI: 10.1038/s41598-017-06652-8 -
Genetics Aug 1990Mutagen-sensitive strains that identify 16 different Drosophila genes have been screened for alterations in DNA metabolic enzymes. A characteristic defect in an...
Mutagen-sensitive strains that identify 16 different Drosophila genes have been screened for alterations in DNA metabolic enzymes. A characteristic defect in an acid-active deoxyribonuclease was observed in strains carrying the six available mutant alleles of the mus308 gene. Since that enzyme is detected at normal levels in a mutant strain that is deficient in the previously identified enzymes DNase 1 and DNase 2, it represents a new Drosophila nuclease that is designated Nuclease 3. The mus308 mutants were originally distinguished from all other mutagen-sensitive mutants of Drosophila because they exhibit hypersensitivity to the DNA cross-linking agent nitrogen mustard without expressing a concurrent sensitivity to the monofunctional agent methyl methanesulfonate. Further observations of hypersensitivity to the mutagens trimethylpsoralen, diepoxybutane and cis-platinum now establish a more general sensitivity of these mutants to agents capable of generating DNA cross-links. In spite of the hypersensitivity of the mus308 mutants to DNA cross-linking agents, the initial incision step of DNA cross-link repair is normal in mus308 cells as assayed by the alkaline elution procedure. The Drosophila mus308 mutants show promise of providing a useful model for analogous defects in other organisms including man.
Topics: Animals; Cross-Linking Reagents; DNA; DNA Repair; Deoxyribonucleases; Drosophila; Ficusin; Genes; Mechlorethamine; Mutagens; Mutation
PubMed: 2397884
DOI: 10.1093/genetics/125.4.813 -
The Biochemical Journal Sep 1998The cytotoxicity of the bacterial toxin colicin E9 is due to a non-specific DNase that penetrates the cytoplasm of the infected organism and causes cell death. We report... (Comparative Study)
Comparative Study
The cytotoxicity of the bacterial toxin colicin E9 is due to a non-specific DNase that penetrates the cytoplasm of the infected organism and causes cell death. We report the first enzymological characterization of the overexpressed and purified 15 kDa DNase domain (E9 DNase) from this class of toxin. CD spectroscopy shows the E9 DNase to be structured in solution, and analytical ultracentrifugation data indicate that the enzyme is a monomer. The nuclease activity of the E9 DNase was compared with the well-studied, non-specific DNase I by using a spectrophotometric assay with calf thymus DNA as the substrate. Both enzymes require divalent metal ions for activity but, unlike DNase I, the E9 DNase is not activated by Ca2+ ions. Somewhat surprisingly, the E9 DNase shows optimal activity and linear kinetics in the presence of transition metals such as Ni2+ and Co2+ but displays non-linear kinetics with metals such as Mg2+ and Ca2+. Conversely, Ni2+ and other transition metals showed poor activity in a plasmid-based nicking assay, yielding significant amounts of linearized plasmid, whereas Mg2+ was very active, with the main intermediate being open-circle DNA. The results suggest that, on entry into bacterial cells, the E9 DNase is likely to exhibit primarily Mg2+-dependent nicking activity against chromosomal DNA, although other metals could also be utilized to introduce both single- and double-strand cleavages.
Topics: Bacterial Proteins; Circular Dichroism; Colicins; DNA; Deoxyribonuclease I; Deoxyribonucleases; Escherichia coli; Escherichia coli Proteins; Kinetics; Magnesium; Plasmids; Protein Conformation
PubMed: 9716496
DOI: 10.1042/bj3340387 -
Journal of Biochemistry Feb 19781. Four DNases were found in the dried liver extract of a top shell, Turbo cornutus. The major one was purified 120-fold by phosphocellulose column chromatography,...
1. Four DNases were found in the dried liver extract of a top shell, Turbo cornutus. The major one was purified 120-fold by phosphocellulose column chromatography, sulfoethylcellulose column chromatography and gel-filtration on Sephadex G-150. The yield was 2.7%. 2. The enzyme activity was not affected by Mg2+ (10(-3)--10(-2)M), EDTA (10(-3)--10(-2)M), or NaCl (10(-1)M). It showed a pH optimum of 4.7--4.8. Ionic strength was found to be critical for the maximal activity. The isoelectric point was 8.5--9.0. On heating at 50 degrees C C for 5 min the enzymic activity fell to half the initial value. 3. The enzyme preparation degraded native as well as heat-denatured DNA, but not RNA. It degraded heat-denatured DNA endonucleolytically to give oligonucleotides with 3'-phosphates. 4. The 3'-phosphate and 5'-hydroxy termini of oligonucleotides were investigated. At both the 3'- and 5'-terminal positions, purine nucleotides were predominant.
Topics: Deoxyribonucleases; Edetic Acid; Hot Temperature; Hydrogen-Ion Concentration; Liver; Magnesium; Mollusca; Nucleotides; Oligonucleotides
PubMed: 24627
DOI: 10.1093/oxfordjournals.jbchem.a131948 -
Cellular Physiology and Biochemistry :... 2008Inhaled rhDNase may improve sputum viscosity and mucociliary clearance by cleavage of extracellular DNA derived for instance from dead leukocytes in purulent, highly...
BACKGROUND
Inhaled rhDNase may improve sputum viscosity and mucociliary clearance by cleavage of extracellular DNA derived for instance from dead leukocytes in purulent, highly viscous patient sputum.
METHODS
Here we established a method to quantify rhDNase-mediated DNA fragmentation in sputum using gel electrophoresis. Sputum of Pseudomonas aeruginosa colonized cystic fibrosis (CF) patients with (CF+) or without (CF-) rhDNase treatment or mechanically ventilated non-CF patients receiving rhDNase (non-CF+) or not (non-CF-) was analyzed. DNA measurements from T-lymphocytes served as controls. Absolute DNA content and the relative quantity within eight molecular mass ranges (12000 to 200 bp) was determined by gel electrophoresis and densitometric analysis.
RESULTS
Geometric mean sputum DNA concentrations were 0.41 mg/dl for CF- (n=54), 0.78 mg/dl for CF+ (n=60), 0.053 mg/dl for non-CF- (n=41) and 0.049 mg/dl for non-CF+ (n=28). Treatment with rhDNase resulted in fragmentation of DNA that was quantified by separation and densitometric analysis of the DNA on agarose gels. The new analysis method permits analysis of DNA cleavage with high accuracy.
CONCLUSION
This new monitoring method facilitates DNA quantification and in vitro monitoring of rhDNase in sputum.
Topics: Administration, Inhalation; DNA; DNA Fragmentation; Deoxyribonucleases; Electrophoresis; Humans; Jurkat Cells; ROC Curve; Recombinant Proteins; Sensitivity and Specificity; Sputum
PubMed: 18769062
DOI: 10.1159/000149813 -
Nucleic Acids Research Jul 1981A method for DNA sequencing has been developed that utilises libraries of cloned randomly-fragmented DNA. The DNA to be sequenced is first subjected to limit attach by a...
A method for DNA sequencing has been developed that utilises libraries of cloned randomly-fragmented DNA. The DNA to be sequenced is first subjected to limit attach by a non-specific endonuclease (DNase I in the presence of Mn++), fractionated by size and cloned in a single-stranded phage vector. Clones are then picked at random and used to provide a template for sequencing by the dideoxynucleotide chain termination method. This technique was used to sequence completely a 4257 bp EcoRI fragment of bovine mitochondrial DNA. The cloned fragments were evenly distributed with respect to the EcoRI fragment, and completion of the entire sequence required the construction of only a single library. In general, once a clone library has been prepared, the speed of this approach (greater than 1000 nucleotides of randomly selected sequence per day) is limited mainly by the rate at which the data can be processed. Because the clones are selected randomly, however, the average amount of new sequence information per clone is substantially diminished as the sequence near completion.
Topics: Base Sequence; Cloning, Molecular; Computers; DNA; DNA Restriction Enzymes; DNA, Recombinant; Deoxyribonuclease I; Deoxyribonucleases; Endonucleases; Substrate Specificity
PubMed: 6269069
DOI: 10.1093/nar/9.13.3015 -
Journal of Virology Sep 1983Murine cytomegalovirus (MCMV) Smith strain DNA is cleaved by restriction endonuclease HindIII into 16 fragments, ranging in size from 0.64 to 22.25 megadaltons. Of the...
Murine cytomegalovirus (MCMV) Smith strain DNA is cleaved by restriction endonuclease HindIII into 16 fragments, ranging in size from 0.64 to 22.25 megadaltons. Of the 16 HindIII fragments, 15 were cloned in plasmid pACYC177 in Escherichia coli HB101 (recA). The recombinant plasmid clones were characterized by cleavage with the enzymes XbaI and EcoRI. In addition, fragments generated by double digestion of cloned fragments with HindIII and XbaI were inserted into the plasmid vector pACYC184. The results obtained after hybridization of 32P-labeled cloned fragments to Southern blots of MCMV DNA cleaved with HindIII, XbaI, EcoRI, BamHI, ApaI, ClaI, EcoRV, or KpnI allowed us to construct complete physical maps of the viral DNA for the restriction endonucleases HindIII, XbaI, and EcoRI. On the basis of the cloning and mapping experiments, it was calculated that the MCMV genome spans about 235 kilobase pairs, corresponding to a molecular weight of 155,000,000. All fragments were found to be present in equimolar concentrations, and no cross-hybridization between any of the fragments was seen. We conclude that the MCMV DNA molecule consists of a long unique sequence without large terminal or internal repeat regions. Thus, the structural organization of the MCMV genome is fundamentally different from that of the human cytomegalovirus or herpes simplex virus genome.
Topics: Animals; Cloning, Molecular; Cytomegalovirus; DNA Restriction Enzymes; DNA, Viral; Deoxyribonuclease EcoRI; Deoxyribonuclease HindIII; Deoxyribonucleases, Type II Site-Specific; Genes, Viral; Mice
PubMed: 6312075
DOI: 10.1128/JVI.47.3.421-433.1983