Sequence organization in nuclear deoxyribonucleic acid from Physarum polycephalum. Physical properties of foldback sequences.

An investigation was performed with the use of physical techniques, to determine the nature and organization of inverted repeat sequences in nuclear DNA fragments from Physarum polycephalum. From the average size of foldback duplexes (550 nucleotide pairs), and the foldback duplex yield as determined by treatment of DNA with S1 deoxyribonuclease followed by hydroxyapatite chromatography, it is estimated that there are at least 25000 foldback sequences in the Physarum genome. Foldback DNA molecules exhibit properties intermediate between single-stranded DNA and native duplexes on elution from hydroxyapatite with a salt gradient. In addition, thermal-elution chromatography of foldback DNA from hydroxyapatite crystals shows that foldback duplexes are less stable than native DNA. These properties can be explained on the basis that inverted repeat sequences are mismatched when in the foldback configuration. The results of experiments in which the binding of foldback DNA molecules to hydroxyapatite was determined, by using fragments of different single-chain size, agree with previous studies indicating that inverted repeat sequences are located, on average, every 7000 residues throughout the Physarum genome. The inverted repeats are derived from both the repetitive and single-copy components in Physarum nuclear DNA.     

Natural Occurrence of Cross-Linked Vaccinia Virus Deoxyribonucleic Acid

The molecular weight of native vaccinia deoxyribonucleic acid (DNA) is 1 to 1.17 times that of native T4 DNA. Sedimentation of denatured vaccinia DNA through alkaline sucrose gradients yields an apparent molecular weight greater than twice that of denatured T4 DNA, implying that the complementary strands of vaccinia DNA do not separate upon denaturation. When alkali-denatured vaccinia DNA is neutralized, it has the physical chemical properties of native DNA when tested by sedimentation through neutral sucrose gradients, banding in CsCl, and by hydroxylapatite chromatography. We conclude that almost all mature vaccinia DNA molecules contain a small number of naturally occurring cross-links.     

The Isolation of DNA from Plant Materials

Total DNA was isolated from seedling and tissues of many plants. The nuclei and chloroplasts were prepared from plant tissues, and then the nuclear DNA and chloroplast DNA were isolated from them. According to the chemical analysis and the physical properties determined by ultraviolet absorbance, hyperchromicities, ultracentrifugation, gel electrophoresis and the electro-microscopical observations it is suggested that DNA obtained possessed a considerable purity and to a certain extent retained the natural status of the large molecule.     

Identification of a new DNA polymerase activity in human KB cells.

We have isolated and partially characterized a new DNA polymerase activity from the purified nuclear fraction of cultured human KB cells. The physical and chemical properties of this enzyme which we have designated DNA polymerase N3 define it as a unique entity that can be distinguished from the several classes of eukaryotic D-DNA and R-DNA polymerase activity that have been recognized to date.     

In vitro incorporation of tritium into native DNA

An exchange method is described for producing tritium-labeled native DNA in vitro with minimal physical damage to the DNA. Tritium-labeled calf thymus DNA prepared in this way has a specific activity of about 100 μCi/mmole of nucleotide (i.e., about 2 × 10⁸ dpm/mmole). Sedimentation velocity at neutral and alkaline pH indicate that the product has an average of two single strand breaks per duplex molecule of molecular weight 6 × 10⁶ daltons. The optical and thermal denaturation properties of the product are those of native DNA. The method should be particularly useful for labeling DNA from organisms that cannot be labeled conveniently in vivo.     

The relationship between two murine DNA-dependent DNA polymerases from the cytosol and the low molecular weight DNA polymerase.

After aqueous subcellular fractionation and partial purification by phosphocellulose chromatography, murine cells are found to contain a low molecular weight DNA-dependent DNA polymerase (beta) in the nuclear fraction and two distinguishable DNA-dependent DNA polymerases (C-I and C-II) in the cytosol. Both C-I and C-II are found in testis, liver, and regenerating liver; the amount of C-I being several fold increased in the regenerating liver and in immature testis. C-I and C-II are distinguishable by the criteria of salt sensitivity, inhibition by single-stranded DNA, elution from phosphocellulose, inhibition by 0.3 mM N-ethylmaleimide, template preference, and sedimentation coefficient. C-II is dissociated by 0.25 M KC1 to an active form of DNA polymerase of sedimentation coefficient 3.5 S while C-I is not dissociated, maintaining its sedimentation coefficient of 7.2 S. Many similar chemical and physical properties of C-II and the low molecular weight nuclear DNA polymerase (beta) suggest that C-II may represent an aggregate state of beta monomers, The size, reaction properties and the increase in enzyme activity under conditions of rapid cellular proliferation suggest C-I is analogous to the alpha DNA polymerase.     

Isolation of neuronal parvalbumin by high-performance liquid chromatography. Characterization and comparison with muscle parvalbumin

Neuronal parvalbumin has been isolated from rat brain and purified to homogeneity by high-performance liquid chromatography (HPLC) on reverse-phase supports. This procedure includes four consecutive chromatographic steps with an overall protein recovery of 74% and a 26 400-fold purification. The concentration of parvalbumin was found to be approximately 10 mg/kg wet weight in brain tissue, which is about 100 times lower than that in rat muscle. The physical properties of brain parvalbumin are described and compared with those of the muscle counterpart. These proteins were identical in their molecular weights (12 000), isoelectric points (4.9), retention times on C-18 reverse-phase HPLC columns, Ca2+ content (two per molecule), amino acid compositions, and immunological properties. A comparison of the tryptic peptide maps of brain and muscle parvalbumin by analytical HPLC also revealed identity and showed that the isolation method described here did not alter the chemical structure of the protein.     

Formation, detection and repair of AP sites

The paper is an outline review of the main aspects concerning the formation and repair of AP (apurinic/apyrimidinic) sites in DNA as well as some of the chemical properties allowing their quantitative determination. A new method for the measurement of AP sites based on their reaction with [14C]methoxyamine is described. It has been applied to the measurement of AP sites produced in DNA either by physical (gamma-rays) or chemical (methyl methanesulphonate, osmium tetroxide) agents. The method has also been used to quantify the excision of abnormal bases from DNA under the action of specific DNA glycosylases and to prevent the chemical or enzymatic degradation of DNA containing AP sites. The paper contains data about the purification and characterization of uracil-DNA glycosylase and AP endodeoxyribonuclease from carrot cells, two enzymes involved in the first steps of base excision repair through AP site intermediates. The biological effects of unrepaired AP sites are also discussed.     

Screening of isolated DNA for sequences released from anchorage sites in nuclear matrix

Isolated chromosomal DNA is associated with polypeptides that are not released from DNA by several methods designed to purify DNA, e.g. treatment with sodium dodecyl sulphate. DNA fragments associated with these very tight DNA/protein complexes show high affinity to nitrocellulose filters in the presence of salt concentrations of 500 mM or greater. Consequently, a fraction of AluI-fragmented native DNA comprising the complexes and 0.2 to 0.3 micron of vicinal DNA can be isolated by one filtration step. This fraction of DNA shows characteristics of residual DNA sequences retained in nuclei after extraction with nucleases and high salt (nuclear matrix). The DNA fragments retained on filters are highly enriched in replicative DNA; and their degree of hybridization with poly(A)+ RNA points to enrichment in actively transcribed sequences. The results support previous work indicating that the very tight DNA/polypeptide complexes co-isolating with DNA under conditions that release other peptide materials from DNA may be anchorage sites of DNA in the nuclear matrix. Moreover, the method described here allows isolation of replicating and actively transcribed DNA sequences directly from isolated total genomic DNA by skipping artefact-prone isolations of the nuclear matrix.     

外来入侵植物和本地植物核DNA C-值的比较及其与入侵性的关系

用流式细胞仪测定了8科10属13种外来入侵植物、6种本地植物和1种外来非入侵植物的核DNAC-值。结果表明:作为整体,外来入侵植物的平均核DNAC-值显著低于本地种和外来非入侵种,但对同属不同类型植物进行比较,未发现一致的规律;在4个既包含外来入侵种又包含本地种的属中,泽兰属(Eupatorium)和鬼针草属(Bidens)外来入侵种的核DNAC-值显著低于同属本地种,莲子草属(Alternanthera)的2种外来入侵植物中仅有1个种的核DNAC-值显著低于同属本地种,而草胡椒属(Peperomia)外来入侵种的核DNAC-值显著高于同属本地种;表明核DNAC-值与外来植物入侵性无必然联系。     

Comparative study of powdered and microcrystalline cellulose samples of a various natural origins: Physical and chemical characteristics

This project studies the dependence of the physical and chemical properties of powdered cellulose on their natural origin and conditions of preparation. We studied the effect of chemical composition and properties of the original native cellulose on the general characteristics of the powdered and microcrystalline cellulose obtained from arboreal and herbaceous cellulose, which were prepared under identical conditions of hydrolysis.     

Transformation in pneumococcus: existence and properties of a complex involving donor deoxyribonucleate single strands in eclipse.

Donor deoxyribonucleic acid (DNA) single strands exist in a complex during the eclipse phase in pneumococcal transformation. This eclipse complex exhibited specific physical properties distinct from those of both pure DNA single strands and native DNA. These included a lower affinity for diethylaminoethyl-cellulose and hydroxylapatite than that of single-strand DNA, faster sedimentation than the DNA chains that it contains, and a buoyant density in Cs2SO4 lower than that of native DNA. The complex was dissociated by treatments with sodium dodecyl sulfate, NaOH, guanidine-hydrochloride, chloroform, and proteinase K but was insensitive to ribonuclease.     

Properties of Nucleoprotein Complexes Containing Replicating Polyoma DNA

Short-lived nucleoprotein complexes (r-py complex) containing replicating polyoma DNA were isolated from infected cells after lysis with Triton X-100. The Triton lysing procedure of Green, Miller, and Hendler (1971) releases most complexes containing supercoiled viral DNA (py complex) from nuclei, but liberates only a portion of r-py complexes. r-py Complexes are associated more strongly with nuclear sites but can be extracted by prolonged incubation of nuclei in lysing solution. Complexes containing replicating polyoma DNA appear to be precursors to stable complexes containing supercoiled DNA. Sedimentation and buoyant density studies indicate that protein is bound to both r-py complexes and py complexes at a ratio of protein to DNA of about 1 to 2/1. Both types of complexes sediment as if the viral DNA is more compact than free DNA and both undergo major reversible configurational changes with increased salt concentration. Changes resulting from enzymatic and chemical treatment indicate that there may be two or more protein components in both r-py complex and py complex. One component is digested by Pronase and trypsin while another is resistant to the enzymes but released by deoxycholate. The abundance and similarity in chemical and physical properties of protein bound to all forms of polyoma DNA suggest that part of the protein molecules may serve in a structural capacity.     

Conversion of DNA polymerase extracted from rat ascites hepatoma cells

DNA polymerase extracted fresh from rat ascites hepatoma cells possesses high molecular weight, maximal activity at neutral pH, and high sensitivity to N-ethylmaleimide (NEM). After physical and chemical treatment of the enzyme fraction, the appearance of low molecular weight DNA polymerase was detected by means of Sephadex gel filtration or sucrose density gradient centrifugation. This low molecular weight DNA polymerase possesses alkaline pH optimum, preference of native DNA as template/primer, and relative resistance to NEM.     

PROPERTIES OF CELL NUCLEI ISOLATED FROM VARIOUS REGIONS OF RAT BRAIN: DIVERGENT CHARACTERISTICS OF CEREBELLAR CELL NUCLEI

Abstract— Cell nuclei were isolated in yields ranging from 38 to 61 per cent from six anatomically defined brain regions of the albino rat. To provide basic information for further studies of altered genomic activity in brain cell nuclei, various properties of these isolated nuclei were measured, including counts of their number, estimates of the distribution of sizes, amounts of RNA, DNA and protein, and endogenous RNA polymerase activity. DNA content per nucleus approximated the accepted value of 6 pg per diploid set of chromosomes. Distributions of nuclear size showed a sensitivity to the concentration of divalent cation, with a shift toward larger nuclear diameters as the Mg concentration was reduced. Cell nuclei from hippocampus, hypothalamus-preoptic region, cerebral cortex, amygdala and midbrain plus brainstem were generally similar in yield, distribution of size, and RNA, DNA and protein content. Cell nuclei from cerebellum differed from those of other brain regions, in all of these parameters. The cerebellum contained a high content of DNA and had an enormous number (8 × 10⁸ per g wet wt.) of cell nuclei of predominantly very small size and characterized by lower ratios of RNA, histones and non-histone protein to DNA and lower endogenous activity of RNA polymerase than nuclei from other brain structures. These properties correlated well with properties of cerebellar tissue, namely, high content of small granule neurons and low ratio of RNA to DNA, and suggest that the small cerebellar nuclei may have relatively inactive genomes. The relationship of 'large' and 'small' cell nuclei to cell types in the brain is discussed.     

Adenovirus gene function required for induction of nuclear acidic protein synthesis: binding of these proteins to adenovirus DNA.

Two different viral DNA-defective temperature-sensitive mutants of adenovirus 12 (H 12) were defective in their ability to induce the synthesis of various molecular weight classes of nuclear acidic proteins, both virion and nonvirion components, after lytic infection of human embryo kidney (HEK) cells at the restrictive temperature. This finding indicates that the induction of nuclear acidic protein synthesis is an adenovirus gene function(s). Treatment of infected cells with actinomycin D at an early stage of virus maturation suppressed the synthesis of an acidic virion protein (hexon), but allowed the synthesis of other classes of nuclear nonvirion acidic proteins during the subsequent late maturation period, suggesting that different mechanisms control virion and nonvirion polypeptide synthesis. The interaction of the nuclear acidic proteins isolated from H 12-infected cells with native-labeled H 12 DNA was studied using the membrane filter technique. Measurements of the ability of different DNA preparations to inhibit the H 12 DNA-acidic protein complex formation suggest that the nuclear acidic proteins bound to native H 12 or HEK cell DNA with much higher affinity than to native calf thymus DNA. Moreover, native H 12 DNA was able to bind the acidic proteins more efficiently than did denatured H 12 DNA. The acidic proteins isolated from the cytoplasm of H 12-infected cells bound approximately 100-fold less to native H 12 DNA than did the nuclear proteins. Furthermore, the H 12 DNA binding affinity of the nuclear acidic proteins from uninfected cells, or from H 12-infected and 1-beta-D-arabinofuranosylcytosine-treated cells, was somewhat lower than that of the nuclear proteins from infected (untreated) cells.     

Studies on Spinach Chloroplast and Nuclear DNA Using Large-scale Tissue Preparations

A method for the large-scale preparation of spinach chloroplasts using the Spinco Model L-4 zonal ultracentrifuge and for the extraction of DNA from the chloroplasts is described. Thirty-five percent of the chloroplast DNA (rho = 1.706 g/cc) differs from nuclear DNA (rho = 1.695 g/cc) in buoyant density. T(m), base composition, and renaturation properties. Sixty-five percent of the chloroplast DNA (rho = 1.696 g/cc) has the same buoyant density and T(m) as nuclear DNA, but it differs in base composition and renaturation properties.     

Purification and properties of type 1 topoisomerase from chicken erythrocytes: mechanism of eukaryotic topoisomerase action

A simple method for the purification of the major topoisomerase (topoisomerase 1) from chicken erythrocytes is described. Because of the generally repressed state of the chromatin from these nuclei, the heterogeneity of the non-histone proteins is reduced, and it is possible to purify this enzyme from a nuclear extract by a single chromatographic step. The chicken erythrocyte topoisomerase appears to be similar to previously described eukaryotic type I topoisomerases with respect to its physical and enzymological properties. The pattern of intermediate products generated during the action of chicken erythrocyte topoisomerase on a supercoiled closed circular DNA substrate has been examined quantitatively and has been shown to be consistent with a mechanism in which the enzyme closes its substrate DNA molecular after the removal of each superhelical turn and in which dissociation of the enzyme substrate complex may, but does not necessarily, occur after each cycle of the reaction.     

Multiple forms of mammalian deoxyribonucleic acid polymerase. An attempt to relate their interactions with nuclei and free deoxyribonucleic acid in vitro with their possible functions in vivo

THE DNA POLYMERASES OF THE FOLLOWING EUKARYOTIC TISSUES WERE STUDIED: regenerating rat liver, normal rat liver, rat thymus, normal mouse liver and Ehrlich ascites-tumour cells. In all cases two main polymerase forms are observed, one of mol.wt. 200000, preferring denatured DNA to native calf thymus DNA primer, designated type I, and the other, designated type II, of mol.wt. 100000, showing a variable and slight preference for native calf thymus DNA primer. Some catalytic properties of these polymerases are described. Nuclei have been isolated from some of these tissues by using two different buffer systems. The ionic composition of the isolation medium is found to affect greatly the amounts and types of polymerase that bind to the nuclei, and also affects the kinetic properties of the polymerases. The way the polymerases and nuclei change properties as the ionic composition of the buffers is changed suggests that ionic effects may be a significant factor in the control of DNA synthesis in vivo. These ionic effects also explain much of the previous confusion over the localization of specific DNA polymerases.     

How does a cell repair damaged DNA?

DNA in living cells is constantly subjected to different chemical and physical factors of the environment and to cell metabolites. Some changes altering DNA structure occur spontaneously. This raises the potential danger of harmful mutations that could be transmitted to offspring. To avoid the danger of mutations and changing genetic information, a cell is capable to switch on multiple mechanisms of DNA repair that remove damage and restore native structure. In many cases, removal of the same damage may involve several alternative pathways; this is very important for DNA repair under the most unfavorable conditions. This review summarizes data about all known mechanisms of eukaryotic DNA repair including excision repair (base excision repair and nucleotide excision repair), mismatch repair, repair of double-strand breaks, and cross-link repair. Special attention is given to the regulation of excision repair by different proteins—proliferating cell nuclear antigen (PCNA), p53, and proteasome. The review also highlights problem of bypassing irremovable lesions in DNA.Translated from Biokhimiya, Vol. 70, No. 3, 2005, pp. 341–359.Original Russian Text Copyright © 2005 by Sharova.