Determination of structural differences in DNA by pyrimidine isopliths ratios]

Using a modified Burton procedure, twelve pyrimidine nucleotide isopliths of DNA from five mammalian species (human, rabbit, guinea pig, rat and cattle) were determined. A method is proposed for mathematical estimation of DNA block analysis data, revealing a correlation between the specific DNA primary structure, the systemic status of the organism under investigation and the organism's radiosensitivity. In some cases DNA structural differences as determined by pyrimidine isoplith ratios help to distinguish between families of the same mammalian order. Quantitative isoplith ratios demonstrate that ionizing radiation treatment brings about certain changes in DNA primary structure. Their direction is quite the opposite to the main trend in the changes of DNA structure in the course of biological evolution.     

The estimation of deoxyribonucleic acid in the presence of sialic acid: application to analysis of human gastric washings

1. Sialic acid has been found to interfere with three colorimetric reactions used for the estimation of DNA: a modified diphenylamine reaction at 100° (Dische, 1930), the nitrophenylhydrazine method (Webb & Levy, 1955) and the diphenylamine reaction at 30° (Burton, 1956). 2. Evidence is presented that sialic acid is present in hydrolysates obtained from gastric wash-out material. 3. A mathematical method for correcting for interference from sialic acid in the diphenylamine reaction at 30° is described. 4. The diphenylamine reaction has been modified to make it suitable for the estimation of DNA in the presence of sialic acid. The modifications are to increase the concentration of diphenylamine to 2% and to perform the reaction at 6–13° for 48hr. These modifications increase the sensitivity 25% above Burton's (1956) modification of the diphenylamine reaction. 5. The precipitation, extraction and recovery of DNA from gastric wash-out material have been investigated.     

Some properties of 1 P+f bacteriophage for Bacillus brevis var. G.-B and its nucleic acid]

The 1 P+f phage, a virulent mutant of the moderate P+ phage for Bac. brevis var. G.-B., consists of a hexagonal head (90x90 nm) and a long non-contractile tail (340 nm). This phage is characterized by a relatively long latent period (90-110 min) and a low yield (40-50 particles per cell). The 1P+f phage is quite stable at pH values from 1 to 11, insensitive to osmotic shock, treatment with chloroform and acridine orange. The sensitivity of the phage to thermal treatment and UV-radiation has been studied. The nucleic acid of the P+f phage is double-stranded DNA of AT-type (GC equals 34.5 mole %) which contains 5-methylcytosine (0.18 mole %) and N6-methyladenine (0.32 mole%). The level of methylation of cytosine and adenine residues in DNA of the 1 P+f phage does not depend on the host studied (Bac. brevis, P- and S variants). The specificity of methylation of cytosine residues in the S and P- cells appears to be the same. DNA of the 1 P+f phage strongly differs from DNA of the host in nucleotide composition (GC equals 45.7 mole %). Nevertheless, phage DNA is very similar to DNA from Bac. subtilis in the character of pyrimidine distribution (the amount of different pyrimidine isopliths). This may testify to a somewhat common character of the nucleotide sequence organization in DNA of the phage and its host.     

Photomodification of nucleic acids by N-(2-hydroxyethyl)phenazine derivatives of oligonucleotides

Photomodification of a 302-membered single-stranded DNA fragment by 5'-mono- and 3',5'-di-N-(2-oxyethyl)phenazine (Phn) derivatives of oligonucleotides has been investigated. Under strong laser irradiation (lambda 532 nm; power density 2,5 GV/cm2, irradiation dose 30 J) the DNA fragment in the presence of Phn-reagents was significantly destructed (up to 70-95%). The level of complementary addressed modification (24-51%) is a direct function of the length of oligonucleotide address of the photoreagent and the amount of Phn residues, stabilizing the complementary complex. The character of the nonaddressed modification is close to the statistic one, although for a number of photoreagents a rather efficient nonspecific modification of 5'-terminal sequence of target DNA has been detected. Of interest also is an unusually broad positional direction of the DNA fragment photomodification in the area of perfect complementary coupling of 5'-Phn-reagents.     

DNA repair in cells of C57bl mice after radiation and in vivo treatment with a DNA site modulator of poly(ADP-ribose)-polymerase

Poly(ADP-ribose)-polymerase is an important cellular regulatory enzyme which can change chromatin structure and function. Action mechanisms and activation of the enzyme are described. The synthesis of poly(ADP-ribose) can be modulated by interaction of substances with the DNA binding site of the poly(ADP-ribose)-polymerase. The involvement of this enzyme in DNA repair, differentiation, carcinogenesis and DNA replication has been suggested. Unscheduled DNA synthesis in spleen cells of C57bl mice drug treated and gamma irradiated in vivo and 3 days later UV irradiated in vitro showed a slight decrease in grain numbers of treated animals. Poly(ADP-ribose)-synthesis was highest in the irradiated groups 18 hours after gamma irradiation. A higher amount of supercoils in DNA was generated by both drugs used. In one long-term experiment the gamma-irradiated group of mice had the highest incidence of lymphomas, while the combined treatment group, modulated and gamma irradiated, showed a lymphoma level like in the unirradiated control group.     

Analysis of the structure of DNA cistrons coding proteins with known sequences]

Proceeding from the amino acid sequence of a number of proteins, with the help of a special computer program we have determined the frequency of pyrimidine isopliths of different length, the degree of clustering and the degree of asymmetry of complementary chains of the corresponding DNA cistrons, as well as the range of variation of these parametres which depends on the code degeneracy. The degree of asymmetry of the chains of DNA cistrons (H/L), calculated for 255 proteins of a known composition, may vary from 0.7 to 1.8. For 90% of these proteins the mean Py/Pu ratio in the coding chain of DNA is above 1. The conclusion has been made that the majority of amino acids contained in the proteins is coded for by purine triplets. It was found that the distribution of pyrimidine isopliths between DNA cistrons coding for different proteins is other than random and has a "DNA-like" character. The degree of clustering of pyrimidines (beta) in cistrons of different proteins may vary from 6.0 to 14.3. The cistrons of some proteins were found to contain long lyrimidine fragments with about 24 residues. A positive correlation (r2 = 0.74) was found to exist between the degree of clustering of pyrimidines and the degree of asymmetry of the chains corresponding to different proteins of DNA cistrons.     

Changes in the pyrimidine blocks of thymic DNA immediately after gamma irradiation of the animal in a sublethal dosage

Pyrimidine clusters of thymus DNA were determined in normal conditions and 5-10 min after gamma-irradiation of rabbits with a dose of 2 Gy. It was shown that a relative content of isopliths I and II significantly increased and that of isopliths V and VIII decreased in the exposed DNA as compared to normal. When isopliths were divided into the components a relative content of C2T2 fragments significantly increased in the exposed DNA while that of CT3 and T5 decreased.     

Content and localisation of 5-methylcytosine in DNA of healthy and wilt-infected cotton plants.

5-Methylcytosine has been found in all pyrimidine isopliths isolated from the DNA of cotton plants, but it localizes predominantly in tri- (about 52%) and dipyrimidine (about 22%) clusters. The 5-methylcytosine distribution by pyrimidine isopliths in DNA of cotton plants is specific and quite different from that in other plant and animal DNA studied. The total 5-methylcytosine content in DNA from wilt-infected cotton plants (2.3 mol %) is less than half that found in DNA from non-infected cotton plants (4.9 mol %). No other visible differences (G+C content, Tm, deltaT, s20,w, frequencies of pyrimidine clusters and others) in these DNA have been found. This suggests that in wilt-infected plants, no essential alteration in DNA sequence or molecular population takes place. As a result of wilt infection 5-methylcytosine completely disappears from dipyrimidine oligonucleotides of cotton plant DNA; its content decreases markedly in long pyrimidine clusters (heptaoligonucleotides and longer) and in C3, C2 T, CT2 fragments. Thus, DNA in wilt-infected plant cells is specifically undermethylated (demethylated). The induced alteration in DNA methylation may be considered one of the possible mechanisms for the specific distortion of gene activity of host cells and primary fungal pathogenic action on plants.     

Preparation of fluorescent-labeled DNA and its use as a probe in molecular hybridization

A method of the fluorescent-labeled DNA preparation for visualization of the complementary nucleotide sequences has been developed. Polynucleotide probes were alkylated randomly by 4-(N-methylamino-N-2-chloroethyl)-benzylamine followed by modification with such fluorochromes as dansyl chloride or fluorescein isothiocyanate (FITC). It was found that the FITC but not dansyl-labeled polynucleotides could serve as efficient probes when about 4% of nitrogen bases were modified. The conditions minimizing the loss of the alkylated bases from DNA were determined. The procedure for hybridization with FITC-labeled DNA as a probe is described, concentration of DNA probe being about 4 ng/mm2 of the nitrocellulose filter.     

Direct determination of DNA nucleotide sequences: structure of a fragment of bacteriophage phiX172 DNA

Methods for the direct determination of nucleotide sequences in DNA have been developed and used to determine the complete primary structure of a fragment of bacteriophage φX174 DNA which is 48 residues in length. This fragment was liberated from φX DNA by digestion at low temperature and high ionic strength with the T4 phage-induced endonuclease IV. The fragment was redigested with endonuclease IV under vigorous conditions and the products fractionated two-dimensionally providing a characteristic endonuclease IV “fingerprint” of the fragment. The Burton (Burton &; Petersen, 1960) depurination reaction was used to characterize the redigestion products and identify the pyrimidine residues at their 5′ and 3′ termini. These oligonucleotide products were then fully sequenced by partial exonuclease digestion with spleen and snake venom phosphodiesterase and analysis of the fractionated digests by base composition, depurination, and 5′ end-group analysis using exonuclease I. Rules for the interpretation of two-dimensional fingerprints of partial exonuclease digests, which rapidly provide sequence information by simple inspection, were also deduced. To derive the complete structure of the fragment, the fully sequenced oligonucleotides were ordered by characterizing large, overlapping, partial endonuclease IV digestion products by means of the depurination reaction. The sequencing methods described are general and may be used for the direct determination of the primary structures of other fragments of DNA.     

The characteristics of chromosomal distribution and of the variability of the multiply repeating DNA fraction of the genome in Bos taurus L.]

The uniform distribution of satellite DNA II and IV has been revealed using in situ hybridization and differential staining in centromeric regions of autosomes. The sex chromosomes have not found such nucleotide blocks. There is only minor satellite IV block inside Y chromosome short arm. The Y chromosome has got some (TG)n enriched blocks distributed also among other parts of genome and one copy of sequences like human ZFY gene. The high repetitive fraction of bovine genomic DNA have not revealed RFLP. However, the difference has been found by blot hybridization between genomic organization of satellite IV in cattle and yak chromosomal DNA. Non-Mendelian distribution of some such nucleotide blocks has been obtained for interspecies crosses of cattle and yak.     

Diphenylamine-colorimetric method for DNA assay: a shortened procedure by incubating samples at 50 degrees C

We describe a modified procedure of the diphenylamine-colorimetric method for assay of DNA that is shorter than the procedure described by Burton. This improvement is achieved by raising the sample incubation temperature to 50 degrees C. The higher temperature hastens and stabilizes the diphenylamine reaction with DNA so that absorbances of samples can be measured as early as 3 h after the reaction is started. This shortened assay is able to detect as little as 3 micrograms of calf thymus DNA. This method is suitable for measuring DNA content of epidermal tissue.     

DNA based cladograms augment the discovery of a new Ips species from China (Coleoptera: Curculionidae: Scolytinae)

The implementation of DNA in taxonomic study is in its infancy because the association of the amount and type of nucleotide change with species boundaries has not been fully examined for most taxa. Mitochondrial cytochrome c oxidase I (COI) nucleotide data is currently the most popular molecular marker for delimiting species boundaries and a standard pair‐wise nucleotide divergence between groups of individuals has been suggested for the recognition of new species. It is unlikely that such a standard would be applicable across animal species, but the association of the amount and type of nucleotide change with species boundaries could help with the establishment of a taxon‐specific DNA taxonomy. This study utilizes DNA data from nuclear and mitochondrial genes to improve the taxonomy of an important forest beetle pest, Ips. Amount and type of nucleotide difference are associated with monophyletic species based on a cladistic analysis of these data. As a result, a new species from China is described for a clade of beetles whose nucleotide differences exceeded the amount of evolutionary change observed within currently recognized species. The COI data are analyzed independently with an expanded taxon data set, including pair‐wise nucleotide differences between recognized sister species. The wide range of average intraspecific pair‐wise nucleotide difference (0–10.0%) suggests limitations to the application of a standard percent nucleotide difference as a means to identify species boundaries. At most, average COI nucleotide intraspecific difference provides an informal guide to identify potential clades that may warrant further systematic investigation. © The Willi Hennig Society 2007.     

Contents of extracellular DNA in blood of irradiated rats]

Intact rat plasma contains high-molecular DNA which moves as a single fraction in 0.5% agarose electrophoresis. As early as 2-5 hours after gamma-irradiation in a dose of 1-100 Gy there appears low-molecular DNA (about 180 nucleotide pairs), the amount of which directly correlates with the irradiation dose 5 hours after the exposure. Blot-hybridization showed that low-molecular DNA has no common nucleotide sequences with high-molecular DNA, though has sites similar to genome repeat sequences.     

On the nature of the cytosine-methylated sequence in DNA of Bacillus brevis var. G.-B.

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of [Me-3H]methionine, practically all the radioactivity incorporated into DNA is found to exist in 5-methylcytosine and N6-methyladenine. The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of 5-methylcytosine in R-m5 C-R and R-m5 C-T-R oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring 5-methylcytosine to be identified and shows that 5-methylcytosine localizes in G-m5 C-A and G-m5 C-Tr fragments. B. brevis S DNA methylase modifying cytosine residues recognizes the GCA/TGC degenerate nucleotide sequence which is a part of the following complementary structure with a two-fold rotational axis of symmetry: (5')...N'-G-C-T-G-C-N... (3') (3')...N-C-G-A-C-G-N'... (5') (Methylated cytosine residues are askerisked). Cytosine-modifying DNA methylase activity is isolated from B. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence DNA in bacterial cells can be undermethylated. This enzyme methylates cytosine residues in native and denatured DNA in the same nucleotide sequences. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA. DNA methylases of different variants of B. brevis (R, S, P+, P-)) methylate cytosine residues in the same nucleotide sequences. It means that specificity or methylation of DNA cytosine residues in the cells of different variants of B. brevis is the same.     

Changes in DNA and surface properties of peripheral blood leukocytes in monkeys after total gamma irradiation

Changes of Macaca nemestrina and Rhesus blood DNA have been studied up to 5 days after the total uniform gamma-irradiation in doses 6.2 and 6.5 Gy. The content of nucleotide ATrich DNA has been evaluated in the fractions of leucocytes with the various surface adherence properties. The dynamics of the content nucleotide AT-DNA and blood leucocytes were similar at the both monkey species. The evaluation of structural state DNA in the blood nucleotide with the fluorescent dyes (ethidium bromide and 4; 6-diamidine-2-phenylindole) demonstrated that the important changes in the polynucleotide structure occurred from 6 to 24 h after irradiation and maintained up to 5 days. Adhesive capacity changes were reversible but they preceded the DNA structural changes. At 24 h postirradiation non-adhesive cells with relative higher AT-DNA content were found.     

Interaction of nucleotide excision repair proteins with DNA containing bulky lesion and apurinic/apyrimidinic site

The interaction of nucleotide excision repair (NER) proteins (XPC-HR23b, RPA, and XPA) with 48-mer DNA duplexes containing the bulky lesion-mimicking fluorescein-substituted derivative of dUMP (5-{3-[6-(carboxyamidofluo-resceinyl)amidocapromoyl]allyl}-2′-deoxyuridine-5′-monophosphate) in a cluster with a lesion of another type (apurinic/apyrimidinic (AP) site) has been studied. It is shown that XPC-HR23b is modified to a greater extent by the DNA duplex containing an AP site opposite nucleotide adjacent to the fluorescein residue than by DNA containing an AP site shifted to the 3′-or 5′-end of the DNA strand. The efficiency of XPA modification by DNA duplexes containing both AP site and fluorescein residue is higher than that by DNA lacking the bulky lesion; the modification pattern in this case depends on the AP site position. In accordance with its major function, RPA interacts more efficiently with single-stranded DNA than with DNA duplexes, including those bearing bulky lesions. The observed interaction between the proteins involved in nucleotide excision repair and DNA structures containing a bulky lesion processed by NER and the AP site repaired via base excision repair may be significant for both these repair pathways in cells and requires the specific sequence of repair of clustered DNA lesions.     

Construction of transferrin-coated liposomes for in vivo transport of exogenous DNA to bone marrow erythroblasts in rabbits

A method for coating liposomes with transferrin is described. Such liposomes have been used for carrying exogenous DNA to rabbit bone marrow (BM) precursor cells, in vivo. A large amount of the DNA contained in the liposomes was delivered specifically into the erythroblasts of the test animals.     

Changes in DNA secondary structure afterγ-irradiation

Native calf thymus DNA was gamma-irradiated at 500 mug/ml in 0.01 M NaCl in the presence or absence of oxygen. By irradiation, an increasing amount of DNA becomes reactive with a water-soluble carbodiimide-derivative (CMEC). In the DNA sections reactive with CMEC the nucleotide strands are separated, a phenomenon previously described as radiation-induced denaturation. The dose-effect curve for the formation of denatured DNA shows an upward-bent form; a distinct oxygen effect of about 2 is observed. By a comparative study with DNA samples, degraded partially with DNAse I, it was shown that a minor part of the radiation-induced denaturation results from the formation of the radiation-induced single strand breaks, whereas the major part is a local denaturation independent of the strand breaks. In these locally denatured regions 20 to 50 nucleotide pairs are separated.