Reparative DNA synthesis in the lymphocytes of persons coming in contact with chromium compounds in industry

The intensity of repair DNA synthesis in the lymphocytes of persons who were in contact with chromium compounds was studied depending on the contact duration. It is discovered that the intensity of repair DNA synthesis is reduced with the increase of the contact duration with chromium compounds. It is suggested that the mutagenic effect of chromium compounds is connected with the influence on the repair DNA synthesis in human cells.     

Histochemical properties of spermatozoa and somatic cells. III. Depolymerization and extraction of DNA during Feulgen acid.

The Feulgen acid hydrolysis patterns of chromatin of different biochemical composition and compactness were analyzed. It was found that the purine extraction rate during acid hydrolysis was affected by the addition of NaCl or 2-mercaptoethanol to the hydrolysis bath. The maximum DNA depolymerization rate was directly correlated to the depurination rate but the extraction rate of hydrolysed DNA was in addition dependent on the stability of the surrounding protein matrix. The results indicate that the diffusion of DNA fragments is partially obstructed in extremely stabilized chromatins (e.g. bull spermatozoa). It is assumed that the extraction pattern of DNA is mainly dependent on the size of the fragments which leave the chromatin by diffusion. It appears that basic proteins do not influence the depolymerization of DNA but there are indications that during certain experimental conditions the purine liberation is dependent upon the chromatin structure.     

Incorporation and replication of foreign metaphase chromosomes in cultured mammalian cells

It is shown that the dyes used to produce banding patterns on chromosomes, quinacrine and Giemsa, are bound to DNA, and not to non-histone protein, the other chromosomal component remaining after acetic acid fixation. Studies on fixed nuclei and on extracted DNA in gelatine films show that the amount of dye bound is not affected by whether the DNA is native or denatured, and is not directly related to the amount of DNA present. Quinacrine is bound to the DNA ionically. With Giemsa, a new magenta compound is formed in situ, consisting of two molecules of methylene blue and one of eosin; this compound is attached to the chromosome by hydrogen bonds. Both quinacrine and the magenta compound formed from Giemsa appear to be attached to DNA molecules at two separate points, and the available evidence suggests that the amount of dye bound is related to the concentration of the DNA. It is suggested that the dye molecules bridge longitudinally separated sites brought into close proximity by folding of the DNA, and that the spatial arrangement of sites in the chromosome is influenced by non-histone proteins. It is concluded that chromosome banding is thus a consequence of the reduction of dye binding in those regions where the DNA chains become sufficiently dispersed to prevent bridging by the dye molecules. Possible indirect effects of base composition and repetition on dye binding at certain chromosomal sites are discussed.     

Histochemical properties of spermatozoa and somatic cells

Summary The Feulgen acid hydrolysis patterns of chromatin of different biochemical composition and compactness were analyzed. It was found that the purine extraction rate during acid hydrolysis was affected by the addition of NaCl or 2-mercaptoethanol to the hydrolysis bath. The maximum DNA depolymerization rate was directly correlated to the depurination rate but the extraction rate of hydrolysed DNA was in addition dependent on the stability of the surrounding protein matrix. The results indicate that the diffusion of DNA fragments is partially obstructed in extremely stabilized chromatins (e.g. bull spermatozoa). It is assumed that the extraction pattern of DNA is mainly dependent on the size of the fragments which leave the chromatin by diffusion. It appears that basic proteins do not influence the depolymerization of DNA but there are indications that during certain experimental conditions the purine liberation is dependent upon the chromatin structure.     

Possible inducible nature of radioresistant DNA synthesis stimulated by 5-fluorodeoxyuridine

A study was made of the effect of cycloheximide on the radioresistant DNA synthesis stimulated by preincubation of cells with 5-fluorodeoxyuridine (FdUrd). It was shown that after the cycloheximide treatment the radioresistant DNA synthesis was absent while in FdUrd-treated cells it did occur. It is assumed that the FdUrd-stimulated radioresistant DNA synthesis is of an inducible nature.     

Intra- and interspecific variations in nuclear parameters of two closely related species of Narcissus L.

Nuclear DNA amount, nuclear area, genome volume and karyotype length were analysed in different populations of two closely related species of Narcissus. There are intra- and interspecific variations in these parameters. 4C DNA amount and karyotype length, on one hand, and 2C DNA amount and telophase nuclear area, on the other, are not correlated. It seems that DNA content and chromosome length are independent parameters. However, 4C DNA content and karyotype volume are correlated, and are also correlated to different density estimations (4C DNA to Kar.length & 2C DNA to telophase area). These facts suggest that the relative length of the chromosomes is genetically controlled and that it is independent of the DNA that they contain. It seems that the interpopulational differences in DNA content are correlated with length changes of small segments in almost all chromosomes.     

Cryptography with DNA binary strands

Biotechnological methods can be used for cryptography. Here two different cryptographic approaches based on DNA binary strands are shown. The first approach shows how DNA binary strands can be used for steganography, a technique of encryption by information hiding, to provide rapid encryption and decryption. It is shown that DNA steganography based on DNA binary strands is secure under the assumption that an interceptor has the same technological capabilities as sender and receiver of encrypted messages. The second approach shown here is based on steganography and a method of graphical subtraction of binary gel-images. It can be used to constitute a molecular checksum and can be combined with the first approach to support encryption. DNA cryptography might become of practical relevance in the context of labelling organic and inorganic materials with DNA 'barcodes'.     

Analysis of the schedule of DNA replication in heat-synchronized Tetrahymena

The temporal schedule of DNA replication in heat-synchronized Tetrahymena was studied by autoradiographic and cytofluorometric methods. It was shown that some cells, which were synchronized by selection of individual dividing cells or by temporary thymidine starvation, incorporated [³H]thymidine into macronuclei in a periodic fashion during the heat-shock treatment. It was concluded that supernumerary S periods occurred while cell division was blocked by high temperature. The proportion of cells which initiated supernumerary S periods was found to be dependent on the duration of the heat-shock treatment and on the cell cycle stage when the first heat shock was applied. Cytofluorometric measurements of Feulgen-stained macronuclei during the heat-shock treatment indicated that the DNA complement of these cells was substantially increased and probably duplicated during the course of each S period. Estimates of DNA content also suggested that the rate of DNA synthesis progressively declined during long heat-shock treatments. These results indicate that the mechanism which brings about heat-induced division synchrony is not an interruption of the process of DNA replication. Further experiments were concerned with the regulation of DNA synthesis during the first synchronized division cycle. It was shown that participation in DNA synthesis at this time increased as more cells were able to conclude the terminal S period during the preceding heat-shock treatment. It is suggested that a discrete period of time is necessary after the completion of DNA synthesis before another round of DNA synthesis can be initiated.     

Nucleotides in rat liver cells: sedimentation and fluorescence analysis; DNA, associated with residual nuclear structures

Rat hepatocyte nucleoids, obtained at different conditions, have been studied by sedimentation and fluorescence methods. Divalent metal ions have been found to play an important role in the superhelical organization of nuclear DNA. Depending on nucleoid isolation conditions, different DNA fragments become associated with the residual nuclear structure. In rapidly sedimenting nucleoids, where nuclear DNA exhibits greater compaction, two types of DNA fragments, differing is size, are associated. In slowly sedimenting nucleoids, where nuclear DNA is in a more relaxed state, one type of DNA fragments is associated. It is assumed that nuclear DNA compaction is conditioned by the formation of additional DNA binding sites with residual nuclear structures and involves divalent metal ions. It has been shown with the aid of restrictase analysis that both types of DNA fragments contain different repeated nucleotide sequences.     

Hydrodynamic and optical behavior of DNA molecule in the range of high ionic strength

The hydrodynamic and optical properties (intrinsic viscosity and optical anisotropy of DNA) have been studied at the high ionic strength mu greater than or equal to 1 M. It has been shown that the effective volume of DNA molecule doesn't depend of mu when mu greater than or equal to 1 M. In these conditions the electrostatical interactions in DNA disappear. But thermodynamic excluded volume effects do not depend on mu and play also an important role in this range of mu (mu greater than or equal to 1 M). It has been concluded that the condensation of DNA in solutions of high salt concentration is the result of local denaturation of DNA. It has been shown that the optical anisotropy of DNA increases drastically at mu congruent to 2 M but the persistence length of DNA does not change under these conditions.     

Visualization of drug-nucleic acid interactions at atomic resolution. III. Unifying structural concepts in understanding drug-DNA interactions and their broader implications in understanding protein-DNA interactions.

Structural information afforded by the X-ray crystallographic studies of ethidium-dinucleoside monophosphate crystalline complexes described in the preceding two papers has led to a detailed model for ethidium-DNA binding. Features of ethidium-DNA binding, in turn, have led to unifying structural concepts in understanding a wide range of drug-DNA interactions. It is possible that these concepts have still broader implications in understanding the nature of protein-DNA interactions.This paper begins by summarizing the stereochemical aspects of ethidium-DNA, actinomycin-DNA and irehdiamine-DNA binding, molecules that use intercalative and kinked-type geometries in binding to DNA. It then describes superhelical DNA structures formed by kinking DNA periodically varying numbers of base-pairs apart. κ-kinked B DNA, a structure formed by kinking DNA every ten base-pairs, is a left-handed superhelical structure that may be utilized in the organization of DNA within the nucleosome in chromatin. β-kinked B DNA is a right-handed superhelical structure formed by kinking DNA every two base-pairs. It is possible that premelting conformational changes occur in DNA which utilize elements of this structure. This would expose base-pairs to solvent denaturation, and could lower the activation energy necessary for strand separation during DNA denaturation. RNA polymerase and other DNA melting proteins could capitalize on this type of premelting conformational change when binding to DNA.The concept that conformational flexibility exists in DNA structure (and that drug intercalation is a phenomenon that reflects this flexibility) can, in addition, explain a wide variety of physicochemical data about DNA. In this paper we discuss the nature of these data in detail.     

Periodicity of the changes in DNA and RNA levels in Penicillium chrysogenum conidia at the stage preceding the S-period

It is ascertained, that conidia enter S-period after 11 hours of incubation on Capek-Dock medium and after 7 hours of incubation on Reistrick medium. Five peaks of DNA and RNA synthesises preceding the S-period were revealed. Moreover, DNA decay to a certain degree depending on the peak number is characteristic for every DNA synthesis peak. By the moment the cell enters into S-period the amount of DNA remains at the initial level. It is noted, that the RNA synthesis curve corresponds to the character of the DNA synthesis curve. A hypothesis is offered to explain the data obtained, which pressuposes the non-specific duplication of the greater part of the genome and its selective degradation.     

Interactions stabilizing DNA tertiary structure in the Escherichia coli chromosome investigated with ionizing radiation

The structure of the bacterial chromosome was investigated after introducing breaks in the DNA with gamma irradiation. It is demonstrated that irradiation of the chromosome in the cell prior to isolation results in partial unfolding of the isolated condensed DNA, while irradiation of the chromosome after it is released from the cell has no demonstrable effect on DNA folding. The results indicate that RNA/DNA interactions which stabilize DNA folds are unstable when breaks are introduced in the DNA prior to isolation of the chromosome. It is suggested that the supercoiled state of the DNA is required for the initial stabilization of some of the critical RNA/DNA interaction in the isolated nucleoid. However, some of these interactions are not affected by irradiation of the cells. Remnant supercoiling in partially relaxed chromosomes containing a limited number of DNA breaks has the same superhelical density as the unirradiated chromosome. This suggests that restraints on rotation of the packaged DNA are formed prior to the physical unwinding which occurs at the sites of the radiation induced DNA breaks. — Analysis of the in vitro irradiated chromosomes shows that there are 100+-30 domains of supercoiling per genome equivalent of DNA. The introduction of up to 50 double-strand breaks per nucleoid does not influence rotor speed effects of the sedimentation coefficient of the chromosome.     

Melting characteristics of highly supercoiled DNA

The effect of high supercoil densities on the melting characteristics of a supercoiled DNA has been studied. It is found that although the melting temperature increases abruptly on converting a linear DNA merely into the relaxed circular form, it falls back substantially at high supercoil densities. It is further predicted, in such cases, that the number of melted base pairs should be significantly enhanced even at the physiological temperature, which may facilitate the binding of other molecules to the highly supercoiled DNA.     

Bam HI DNA甲基化酶酶学性质的研究

以Lineweave-Burk plot双倒数作图法测得该酶对底物S-腺苷酰甲硫氨酸(SAM)的K_m=7.69×10~(-6)mol/L,在1mmol/LS-腺苷酰高半胱氨酸(SAH)存在下,Ki=7.33×10~(-4)mol/L,两条直线相交于纵轴,证明SAH是该酶的竞争性抑制剂。该酶最适pH为7.8,对热不稳定。同时还测定了该酶对不同DNA底物的专一性及盐浓度、代谢相关物’两价阳离子、某些酸根等对该酶调节性质的影响。以碘代乙酰胺修饰该酶的SH基’及用二硫苏糖醇(DTT)和巯基乙醇(MSH)保护该酶SH基所作的实验表明SH基是该酶活性中心所必需的,用高效液相色谱(HPLC)法证明该酶所甲基化的碱基为刘氏小球菌(M·L、DNA)分子中的胞嘧啶,且求得甲基化30min后所得甲基化水平为2.39％。同时也证明当用该酶将λDNA甲基化后,可使BamHI限制性核酸内切酶对甲基化后的λDNA丧失切割作用。     

Selenite: a good inhibitor of rat-liver DNA methylase

DNA methylase from rat liver was partially purified through a DEAE sephacel column and characterized in an in vitro assay with respect to time, protein, DNA and S-adenosylmethionine curves. The Km for S-adenosylmethionine was 2.5 microM. Sodium selenium inhibited the methylation of DNA in a dose dependent fashion when added to the assay. It was also demonstrated that selenite non-competitively inhibits rat-liver DNA methylase with a Ki of 6.7 microM. Dithiothreitol had no effect on selenite inhibition and increasing amounts of DNA did not alter the inhibition. However, increasing amounts of protein overcame the inhibition, suggesting that selenite is reacting with the DNA methylase protein. DNA methylase isolated from selenite treated animals had only 43% of the activity as enzyme from control rats. It appears that selenite is a good inhibitor of DNA methylase.     

Loss of CpG dinucleotides from DNA. VI. Methylation of mitochondrial and chloroplast genes

From nucleotide sequences of mitochondrial and chloroplast genes the probable frequency of the CpG----TpG + CpA substitutions was determined. These substitutions may indicate the level of prior DNA methylation. It was found that the level of this methylation is significantly lower in mitochondrial DNA (mtDNA) and chloroplast DNA (chDNA) than in nuclear DNA (nDNA) of the same species. The species (taxon) specificity of mtDNA and chDNA methylation was revealed. A correlation was found between the level of CpG methylation in nDNA, and mtDNA and chDNA in different organisms. It is shown that cytosine residues in CpG were not subjected to significant methylation in the fungi and invertebrate mtDNA and also in the algae chDNA. In contrast, the vertebrate mtDNA bears the impress of CpG-supression, which is confirmed by direct data on methylation of these DNA. Here the first data on the possible enzymatic methylation of the plant mtDNA and chDNA were obtained. It was shown that the degree of CpG-suppression in the 5S rRNA nuclear genes of lower and higher plants is significantly higher in the chloroplast genes of 4,5S and 5S rRNA. From data on pea chDNA hydrolysis with MspI and HpaII it was established that in CCGG sequences this DNA is not methylated. The role of DNA methylation in increasing the mutation rate and in accelerating the evolutionary rates of vertebrate mtDNA is discussed.     

The restoration by Escherichia coli RecA protein of the survival of gamma-irradiated HeLa cells reduced by the DNA repair inhibitors caffeine and 3-aminobenzamide

It is confirmed that inhibitors of DNA repair caffeine and 3-aminobenzamide decrease the survival of gamma-irradiated HeLa cells. It is shown that the decreased survival of irradiated cells is reversed when Escherichia coli RecA protein is introduced into cell nucleases with the aid of liposomes. This effect is more expressed in caffeine-treated (before or after irradiation) than in 3-aminobenzamide-treated (before irradiation) cells. It is suggested that E. coli 38 kD RecA protein may compensate the function of HeLa RecA-like protein, inhibited by DNA repair inhibitors, which is necessary for the repair of single-strand breaks and double-strand breaks of DNA.     

Formation of complexes between DNA and cationic amphiphile molecules by the fluorescent probe method

The formation of complexes of DNA with dodecylamine, dodecyltrimethylammonium, tetradecyltrimethylammonium, and hexadecyltrimethylammonium was studied using a fluorescent probe pyrene. The dependences of the spectral parameters of the hydrophobic pyrene probe on the concentration of the cationic amphiphile in the presence and absence of DNA were obtained and analyzed. It is shown that, in the absence of DNA, these dependences exhibit only one S-shaped region, which corresponds to the micelle formation of the amphiphile, whereas in the presence of DNA there are two S-shaped regions, which indicates the cooperative formation of two types of DNA-cationic amphiphile complexes. For each of the four cationic amphiphiles, the critical concentrations for the micelle formation in the absence of DNA (C0) and the concentrations at which the first (Cd1) and the second complex with DNA are formed were determined. It was found that the Cd1 value is 15-40 times lower than C0. The Cd1 value does not depend on DNA concentration and is determined only by the length of the hydrocarbon chain and the structure of the amphiphile ionic fragment. The Cd1 value increases as the length of the aliphatic chain decreases and upon replacement of mobile hydrogen atoms in the ammonium fragment by methyl groups. It was shown that hydrophobic clusters of amphiphile arising upon complex formation with DNA play the role of cross-links promoting DNA aggregation, or DNA compactization in the case of dilute solution of high-molecular weight DNA. The structures of the first and second DNA-cationic amphiphile complexes are proposed, and the mechanism and nature of interactions that determine their formation are discussed.