DNA Moiety Research Articles

Protein damages in irradiated deoxyribonucleoprotein.

When the solid-state deoxyribonucleoprotein (DNP) was irradiated by gamma rays, Lucke-Huhle et al.1) found that single strand breaks of DNA were produced more frequently in the DNP than in pure DNA. Ormerod2) and Kuwabara and Yoshii3) reported that the ESR spectra of DNP after irradiation showed radicals to be located preferentially on the DNA moiety, and the results were explained by the electron transfer from the protein moiety to the DNA moiety. From studies on the luminescence decay curve of DNP when irradiated with electron pulses, Lillicrap and Fielden4) also found that the excitation energy formed in the protein moiety was transferred to the DNA moiety. When the DNP solution was irradiated by gamma rays, Weiss and Wheelers5) and Hayashi et al.6) found that the template activity of DNP for RNA synthesis was considerably enhanced by irradiation (10-20 krad), whereas Hagen et al.7) found that the template activity of purified DNA was markedly reduced. Using an irradiated DNP template, Mee et al.8) also reported that changes in the base composition of the synthesized RNA were found at doses of 10-20 krad.

The specificity of S1nuclease toward RNA-DNA hybrids as studied using isotopes of phosphorus-32 and phosphorus-33

Hybrids were formed from Bacillus cereus DNA and ribosomal RNA. They were treated with various combination of S1 nuclease and ribonuclease, and the molar ratios of the RNA and DNA moieties remaining in the treated hybrids were determined using a 32P-33P dual-label technique. It was found that both S1 nuclease and ribonuclease are required to give hybrid with RNA and DNA in a perfect 1:1 molar ratio. It was noted that the dual-label technique which employs orthophosphate as the sole phosphorus source for both labels gives unambiguous molar ratios and obviates the need to calculate specific activities, make quench corrections, or correct for base content.

Immunofluorescence for the detection of photochemical lesions in intracellular DNA

The ability of methylene blue to photooxidize the guanine moiety of intracellular DNA of living human cells was demonstrated by immunofluorescence using an antibody with a specificity for the unpaired cytosine residue of DNA. These findings which indicate that sufficient amounts of the dye penetrate the mammalian cell to cause biological effects may present the investigator with the unique opportunity of selectively altering the guanine residue in DNA and thereby determining the genetic consequences of this reaction.

Multiple forms of nuclear ribonuclease H from Tetrahymena pyriformis.

Three types of ribonuclease H were detected in the isolated macronuclei of Tetrahymena pyriformis GL, partially purified and characterized. They were eluted at around 0.15 M, 0.3 M and 0.4 M of ammonium chloride in phosphocellulose chromatography, and termed H-1, H-2 and H-3, respectively. The partially purified enzymes have following properties. a) These enzymes specifically hydrolyze the RNA moiety of RNA . DNA hybrid. Neither DNA moiety of RNA . DNA hybrid nor the RNA molecule dissociated by heat from RNA . DNA hybrid is hydrolyzed by these enzymes. b) The enzymes are most active at pH 8.5-9.0. c) They require divalent cations such as Mg2+ or Mn2+ for their activities. The optimal concentrations of these cations are different among these enzymes. d) The mode of cleavage by these enzymes is endonucleoytic, producing mostly oligonucleotides and a small amount of mononucleotides which possess 3'-hydroxyl and 5'-phosphate termini.

Cesium chloride gradients of chromatin after treatment with micrococcal nuclease

Cesium chloride equilibrium density centrifugation shows that treatment of rat liver nuclei with low concetrations of micrococcal nuclease for extremely short periods of time results in the appearance of chromatin fractions of low protein/DNA ratio and even free DNA. The DNA of these chromatin fractions is shorter than the DNA moiety of one chromatin subunit. The amount of high buoyant density material is decreased with increasing digestion time. We conclude that this material belongs to the minor chromatin fraction which is not organized according to the subunit model.

Electron transfer between protein and DNA in gamma-irradiated deoxyribonucleoprotein

When deoxyribonucleoprotein-proflavine complexes were studied by electron spin-resonance spectroscopy following gamma-irradiation, it was found that stable free radicals were not formed at random on the complex but were preferentially located on proflavine. Since proflavine intercalated to DNA bases serves as a final acceptor of electrons liberated by ionization, the result of our experiment was regarded as suggesting that the electron transfer from the protein moiety to the DNA moiety occurred in the irradiated deoxyribonucleoprotein.

Decapsidation of polyoma virus: Identification of subviral species

Mouse embryo cells that had been exposed to polyoma virus (Py) labeled in its protein and/or DNA moiety were fractionated at various times after infection. Sedimentation analysis of the cytoplasm in 1 M NaCl solution revealed the presence of a labeled viral DNA-protein complex, termed NP, which is absent from virus preparations used for infection. This complex has a sedimentation coefficient of about 135 S and a DNA/protein ratio higher than that of virus, reflecting primarily loss of histone-like polypeptides. Another newly recognized species, a DNA-free complex (DFC) sedimenting at about 185 S, was identified in the cytoplasm. It contains reduced amounts of histone-like polypeptides and little or no DNA, as compared with virus. Unlike the material sedimenting like “full” virus particles which was also found in the cytoplasm, neither NP or DFC was stable in 2.7 M CsCl. Histone-like polypeptides lost from the above-mentioned species were detected in the cytoplasm as slowly sedimenting material. Radioactive material sedimenting as free DNA in 1 M NaCl solution was recovered from the nucleus and, occasionally, from the cytoplasm. It never represented more than 1.5% of the labeled DNA molecules present in virus at the time of infection. We propose a scheme for the decapsidation of Py in the cell. As the viron meanders in the cytoplasm, histone-like polypeptides are lost and NP is generated. The ensuing step is transfer of DNA to the nucleus, with a simultaneous reorganization of the capsid proteins into DFC. The viral DNA in the nucleus presumably is instrumental in directing the infectious process.

Microhemagglutination test for the detection of nucleoprotein antibody in systemic lupus erythematosus.

A simple and practical microhemagglutination test that detects two antibodies, one showing reactivity to the DNA moiety of soluble nucleoprotein (sNP) and the other to the DNA-histone complex of sNP, is described. The method incorporates human erythrocytes formalinized at 30 C., tanned, and coated with sNP at 37 C. Antibody specificity was determined by inhibition experiments performed on sera with added DNA or sNP antigen. With the indirect LE cell technic, evidence that the anti-sNP antibody detected in this work is related to the serum LE factor commonly associated with the LE cell phenomenon was obtained. The hemagglutination test is helpful in establishing the specific diagnosis of systemic lupus erythematosus (SLE) and may be used to follow the course of the disease and response to therapy in SLE patients, as this is a semiquantitative technic and rise or fall in titer or antibody can be determined.

The nature of the deoxyribonucleosides involved in the binding of carcinogenic hydrocarbons to the DNA of mouse embryo cells.

The DNA of mouse embryo cells was specifically labelled in the purine moieties with (G(3)H)-deoxyadenosine or in the cytosine moieties with (5-(3)H)-deoxycytidine. These cells were then treated with 7-methylbenz (a) anthracene (7MBA) or benzo (a)-pyrene (B(a)P) and the DNA isolated, degraded and fractionated by LH20 Sephadex column chromatography. When the purines of the DNA were tritium-labelled, radioactive hydroccarbondeoxyribonucleoside products were obtained. No such products were found with deoxycytidine pre-labelled DNA. Contrary to an earlier suggestion, these results indicate that it is the purine moieties of DNA which react with the metabolically activated hydrocarbon derivative in vivo.

Demonstration of infectious DNA in transformed cells. III. Correlation of detection of infectious DNA-protein complexes with persistence of virus in simian adenovirus SA7-induced tumor cells.

Primary tumors induced in newborn hamsters by simian adenovirus SA7 were investigated in transfection experiments. Infectious DNA-protein complexes were readily detected in both the supernatant and pellet fractions obtained by a modified Hirt extraction procedure; DEAE-dextran was required for infectivity to become manifest. Infectivity could be abolished by exposure to DNase, but it was unaffected by SA7-specific antiserum or RNase, and only partially inactivated by trypsin treatment. SA7 tumor cells serially passaged either in tissue culture or in hamsters yielded infectious DNA complexes much less frequently and appeared to evolve into nonyielder cell lines. When large numbers of cells were lysed, intact virus could be recovered from all the primary tumors and from some of the subcultured cell lines. There was a correlation between the persistence of complete virus and the presence of infectious DNA-protein complexes. When the tumor cells carried very small amounts of intact virus, infectious DNA complexes could still be detected; when virus could no longer be detected in the tumor cells, infectious DNA complexes could no longer be found. The results suggest that a portion of the infectious DNA moieties exists as viral DNA-protein complexes in the tumor cells.

Induction of free radicals in DNA by proflavine and visible light: influence of oxygen and ionic strength.

Abstract—The photosensitization of native DNA is observed as an induction of free radicals in the DNA moiety of proflavine‐DNA complexes. The intensity of the electron paramagnetic resonance spectra (at 77 K) is a measure of the number of free radicals present in frozen solutions of DNA‐proflavine complexes after irradiation with visible light (Λ > 320 nm). In the absence of O2, the photosensitization is significant but very low; it increases slightly with increasing NaCl ionic strength; it appears to be due to intercalated dye molecules and the qualitative analysis of the spectra obtained shows that mainly thymidine is involved. The reaction measured after saturation with O2 is the same as the reaction in air but is quantitatively higher; the free radicals observed are peroxides. This induction of free radicals appears to be due to the intercalated dye molecules, each molecule acting independently. The important observation is a very sharp and large (around a hundred‐fold) increase in the photosensitizing efficiency of the bound dye molecules occurring in NaCl between μ, # 0–25 and μ= 0–5 and in MgCl2 between μ# 0–01 and μ=0–1.

Enzymatic repair of X-ray-damaged DNA: I. Labeling of the precursor of a malonaldehyde-like material in X-irradiated DNA and the enzymatic excision of labeled lesions

As a result of X-irradiation of DNA the precursor of a malonaldehyde-like material is formed, which remains covalently bound to DNA. This precursor derives from the damaged deoxyribose moiety of DNA, as indicated by lack of its appearance when free bases or ribonucleotides are irradiated. The precursor can be labeled with tritium by reduction with NaB 3H 4. The 3H-labeled lesions are excised from the DNA as parts of small oligonucleotides by an enzyme which was partially purified from rat liver extract. The enzyme preparation has high affinity for X-ray-damaged DNA. Its activity has a pH optimum between 8 and 9, requires Mg 2+ for activity and is inhibited by Ca 2+.

Characterization of chromatin from Triticum aestivum

Chromatin was isolated from wheat seedlings using chromatin aggregation in the presence of cations and dispersion in the absence of these cations. The preparation contained some contaminating RNA. The protein, DNA and RNA moieties were separated from each other by ion exchange chromatography on hydroxyapatite in the presence of 5 M urea and 2 M NaCl. The histone and non-histone proteins were subsequently separated on CM-cellulose and the histones partially separated from one another. The various protein fractions were characterized by urea-acetic acid and sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Purification and characteristics of hybridase (ribonuclease H) from rat-liver cytosol.

Ribonuclease H or hybridase, the enzyme cleaving the RNA moiety of DNA · RNA hybrids has been extensively purified from rat‐liver cytosol by ammonium sulphate precipitation, DEAE‐cellulose chromatography, Sephadex G‐200 and G‐150 superfine gel filtration, isoelectric focusing, phosphocellulose and hydroxyapatite chromatography. Neither native or denatured DNA, nor single or double‐stranded natural or synthetic ribopolynucleotides are degraded by the enzyme. The pH optimum of the reaction is 7.9. Enzyme activity is totally dependent on the divalent cations Mg2+ and Mn2+, Mg2+ being optimal for the reaction. The enzyme acts on the hybrid as an endonuclease resulting in end products with a chain length less than 15, having a 5′‐phosphate and a free 3′‐hydroxyl end group. Synthetic homopolymer hybrids are digested only if they are composed of purine bases. The enzyme has a molecular weight of 120000–125000 and seems to be composed of two subunits with molecular weights of 85000 and 43000.

Effects of Ca++ Ion on the Formation of Free Radicals in Irradiated Deoxyribonucleoprotein

ESR studies of dry deoxyribonucleoprotein in the presence or absence of Ca++ ion revealed that the addition of Ca++ ions to deoxyribonucleoprotein resulted in a decrease of electrons transferred from protein moiety to DNA moiety.

Enzymatic and Physical Studies on the Triplex dTn∙dAn∙rUn

The triplex dTn∙dAn∙rUn was studied as a model system both enzymatically and physically with a view that a rational approach for attempting to isolate triplexes from in vivo situations might emerge. The triplex was characterized by mixing curves and by its equilibrium buoyant density. In 5 mM Na phosphate, pH 7.3, for KCl concentrations below 0.4 M the triplex dissociated into dTn∙dAn + rUn, dissociation being complete at about 0.3 M KCl. If MgCl2 replaced KCl a strongly cooperative dissociation occurred at 1 mM MgCl2. Whereas with alkaline titration of dTn∙dAn∙rUn, rUn was dissociated first followed by dTn∙dAn, acidic titration resulted in the whole triplex dissociating together. Both transcription and replication of dTn∙dAn were strongly inhibited by formation of a triplex. The DNA and RNA moieties in the triplex are somewhat protected against DNase I and pancreatic RNase degradation, when compared with duplex dTn∙dAn or rUn. Spermine binds equally well to dTn∙dAn and dTn∙dAn∙rUn which is consistent with spermine binding in the minor groove and RNA in the major groove of DNA.

Localization of free radicals on irradiated deoxyribonucleohistone.

Deoxyribonucleohistone (DNH) from calf thymus was studied by ESR spectroscopy following gamma irradiation. Free radicals formed in gamma irradiated DNH were mainly localized in DNA moiety. The G-value for radical production in DNH was 1.8, while the G-values in DNA and histone were 1.1 and 2.1, respectively. Thus the spectrum obtained in DNH could be explained as a consequence of an electron transfer from histone to DNA. This observation was also confirmed from ESR spectrum of the reconstituted DNH. (auth)

Letter: Free radicals in the sugar moiety of x-irradiated DNA.

A brief description is presented of an esr study of x-irradiated solutions of D-ribose, deoxy-D-ribose, their corresponding nucleosides and nucleotides, and DNA. Solutions were respectively in water, 6MH/sub 2/SO/sub 4/, and 0.5N NaOH. It is concluded that, next to hydrogen abstraction radicals, either in C/sub 5/ or C/sub 4/, one must consider the existence of radicals corresponding to the abstraction of an OH group in isolated sugars, and to the liberation of a phosphate group in the nucleotide, or in DNA itself. (UK)

Labelling of RNA and DNA moieties of mammalian embryos in-vivo by 14 C-U-glucose and 32 P-orthophosphate.

1. Uniformly labelled 14C-glucose and 32P-orthophosphate were applied intravenously to pregnant rats on day 12 of gestation. DNA and RNA were found to be labelled by both of these precursors. 2. Highly purified RNA and DNA were isolated from the embryos and hydrolyzed to mononucleotides and bases, the four nucleotides or bases were separated and the specific activities determined. According to the alkaline hydrolysis, a nearest-neighbour analysis (2′–3′-nucleotides) is achieved in the case of the RNA nucleotides whereas the enzymic hydrolysis of the DNA allows the preparation of 5′-nucleotides. 3. The ribosomal RNA bases were found to be labelled rather equally (≃1.4dpm/nmol) except for Uracil where the specific activity showed a 3 times higher value (≃4 dpm/nmol). 4. The base moieties of the DNA show an almost equal rate of incorporation of 14C-glucose fragments. The 32P-radioactivity found in DNA nucleotides is in the range of the corresponding 14C-data except for that of TMP. In this case a higher rate of 32P was found compared to the 14C-radioactivity.

Isolation of a high-molecular-weight hybrid between ribosomal RNA and DNA.

The genes for rRNA of Bacillus subtilis have been purified as high molecular weight DNA: RNA hybrids with a DNA/RNA ratio of 1. Two different methods were used. (1) High molecular weight denatured DNA (Mr= 2.2 × 107) was hybridized with rRNA and the excess of single‐stranded DNA was digested with Neurospora crassa endonuclease and Escherichia coli exonuclease I. The hybrid was then purified on Sepharose 4B and two subsequent CsCl gradients. This is a marked improvement on the previously published procedure of Sgaramella, Spadari and Falaschi (1968) insofar as it gives high molecular weight products (8 × 105 for the DNA moiety of the hybrid). (2) A simplified non‐enzymic procedure that used relatively short single‐stranded DNA molecules (Mr= 4 × 106) and several subsequent CsCl gradients led to a hybrid with a DNA: RNA ratio of 1.1 and a molecular weight higher than the previous procedure (1.7 × 106 for the DNA moiety). The DNA of the isolated hybrid, after removal of the RNA moiety, rehybridizes completely with rRNA.After hybridization of rRNA with DNA having a single‐stranded molecular weight of 2.2 × 107, the hybrid bands mainly at a density corresponding to that of denatured DNA; hybrid with a density corresponding to that of 1:1 DNA/RNA ratio can be obtained only if the DNA has been reduced by enzymic digestion or shearing to a single‐stranded molecular weight close to 2 × 106; this indicates that between each set of 16‐S + 23‐S rRNA genes, there must be regions of DNA non‐complementary to rRNA; these regions have a single‐stranded molecular weight that can be estimated as greater than 1.8 × 107.When the isolated DNA is treated with N. crassa endonuclease, in conditions that cause 95% hydrolysis of denatured DNA and 5% digestion of native DNA, it is digested only up to 50%. This suggests the presence of self‐annealing sequences in the rRNA genes, in agreement with the secondary structure of rRNA.