Binding behaviour on DNA-agar of a naturally occurring cross-linked fraction in rodent DNA

Summary A naturally occurring, cross-linked fraction, constituting about 1% of the total DNA, has been isolated from the DNA of mice and rats. In sharp contrast to the rest of the rapidly renaturing DNA, this material shows poor binding ability during incubation in agar with DNA of the same species.     

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.     

Circular dichroism studies of ethidium bromide binding to the isolated nucleolus.

Circular dichroism in the 300-360 nm region and fluorescence induced by intercaltating binding of ethidum bromide to both DNA and RNA components were studied in isolated HeLa nucleoli. Both DNA and RNA compoents contribute to the induced dichroic elliticity. Digestion of nucleoli by RNase or DNase shows that most of the induced ellipticity comes from the DNA component. In nucleoli with an RNA/DNA = 0.8/1.0 the RNA component gives only 20% of the total ellipticity when measured at an ethidium bromide/DNA = 0.25. Spectro-fluorometric titration shows that ethidium bromide intercalates mostly into DNA in nucleoli. Both circular dichroism and fluorescence studies indicate that both DNA and RNA components in isolated nucleoli are less accessible to intercalating binding by ethidium bromide when compared to purified nucleolar DNA, DNA in chromatin or purified ribosomal RNA. Circular dichroic measurements of intercalating binding of ethidium bromide to to nucleoli may be used to study changes in nucleoli under different physiological or pathological conditions.     

The methionine initiator tRNA genes of yeast

We have isolated three distinct tRNAimet genes from a yeast DNA clone bank. The complete sequence of two shows that these genes are colinear with the mature tRNAimet and supports the RNA sequence of tRNAimet. Southern analysis of yeast genomic DNA indicates the presence of four copies of tRNAimet gene per haploid genome.     

Rate of DNA replication fork movement within a single mammalian cell

Autoradiography of replicating DNA molecules isolated from individual human cells shows that the rate of DNA replication fork movement within a single cell varies from 0.2 to 1.2 μm/min with an average value of 0.5 to 0.6 μm/min. These data suggest that replication forks move at substantially different rates in different parts of the genome within a single cell.     

Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid.

Purified nuclei, isolated from appropriately infected HeLa cells, are shown to synthesize large amounts of either herpes simplex virus (HSV) or vaccinia virus DNA in vitro. The rate of synthesis of DNA by nuclei from infected cells is up to 30 times higher than the synthesis of host DNA in vitro by nuclei isolated from uninfected HeLa cells. Thus HSV nuclei obtained from HSV-infected cells make DNA in vitro at a rate comparable to that seen in the intact, infected cell. Molecular hybridization studies showed that 80% of the DNA sequences synthesized in vitro by nuclei from herpesvirus-infected cells are herpesvirus specific. Vaccinia virus nuclei from vaccinia virus-infected cells, also produce comparable percentages of vaccinia virus-specific DNA sequences. Adenovirus nuclei from adenovirus 2-infected HeLa cells, which also synthesize viral DNA in vitro, have been included in this study. Synthesis of DNA by HSV or vaccinia virus nuclei is markedly inhibited by the corresponding viral-specific antisera. These antisera inhibit in a similar fashion the purified herpesvirus-induced or vaccinia virus-induced DNA polymerase isolated from infected cells. Phosphonoacetic acid, reported to be a specific inhibitor of herpesvirus formation and the herpesvirus-induced DNA polymerase, is equally effective as an inhibitor of HSV DNA synthesis in isolated nuclei in vitro. However, we also find phosphonoacetic acid to be an effective inhibitor of vaccinia virus nuclear DNA synthesis and the purified vaccinia virus-induced DNA polymerase. In addition, this compound shows significant inhibition of DNA synthesis in isolated nuclei obtained from adenovirus-infected or uninfected cells and is a potent inhibitor of HeLa cell DNA polymerase alpha.     

Isolation of mitochondrial DNA from cytoplasmic male sterile and maintainer lines of pearl millet,Pennisetum americanum (L.) Leeke

Summary Mitochondrial DNA has been isolated from paired lines of pearl millet maintainer and cytoplasmic male sterile plants. Evaluation of the DNA by agarose gel electrophoresis shows that good quality DNA of high molecular weight can be obtained from mitochondria of both maintainer and male sterile pearl millet.     

A four-stranded DNA from Bacillus subtilis which may be an intermediate in genetic recombination

Summary DNA of Bacillus subtilis strain UVSS 19-8 M, of high ultraviolet sensitivity, was isolated after cultivating in medium containing bromouracil. Isopycnic banding in CsCl shows an unusual pattern with four bands, including an extra one halfway between those for hybrid and for DNA fully substituted with bromouracil. DNA of this band, amounting to 15–25% of the total DNA mass in one preparation, was isolated and investigated. The characteristics found for this DNA are in agreement with a fourstranded DNA unit similar to one of the structures postulated by Holliday as intermediates during genetic recombination.UVSS 19-8 M from which this DNA has been isolated is shown to be defective for transformation and transfection, and can be regarded as rec ^-.     

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.     

Specificity of methylation of cytosine risidues in DNA of Bacillus brevis var. G-B]

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of (methyl-3H)-methionine practically the total radioactivity included into DNA is found to exist in 5-methylcytosine (MC) and 6N-methyladenine (MA). The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of MC in Pur-MC-Pur and Pur-MC-T-Pur oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring with MC to be revealed and shows that MC localizes in G-MC-A and G-MC-T-Pu fragments. Bac. brevis S DNA-methylase modifying cytosine residues recognizes the GCAT GC degenerative nucleotide sequence which is a part of the following complementary structure with rotational symmetry: (5') ... N'--G--MC--T--G--C--N ... (3') (3') ... N--C--G--A--MC--G--N' ... (5') Cytosine modifying DNA-methylase activity is isolated from Bac. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence, DNA in bacterial cells can be partially undermethylated. This enzyme methylates cytosine residues in native and deneaturated DNA in the same nucleotide sequences. As compared to the native DNA, the denaturated DNA is indicative of a decrease in the level of methylation of adenine, rather than cytosine residues. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA (calf thymus, Pseudomonas aeruginosa etc.). DNA-methylases of different variants of Bac. brevis (R, S, P+, P-) methylate cytosine residues in the same nucleotide sequences. It means that specificity of methylation of DNA cytosine residues in the cells of different variants of Bac. brevis is the same.     

The characterization of arabms1: an Arabidopsis thaliana sequence homologous to bacteriophage M13 repeat element

A genomic DNA fragment was isolated from the genome of Arabidopsisthaliana via hybridization with bacteriophage M13 protein IIIrepeat element. A 45 bp region of the A. thaliana DNA fragmenthas a repeating 12 bp structure that shows sequence homologyto both the M13 repeat and to a rice minisatellite-like element.Hybridization of digests of A. thaliana genomic DNA with theminisatellite DNA generates a multilocus DNA fingerprint withlow polymorphism. Key words: Minisatellite, M13 repeat element, Arabidopsis thaliana     

Isolation of a DNA fraction highly enriched in transfer RNA genes from Xenopus laevis.

A DNA fraction highly enriched in tRNA genes can be isolated from the Xenopus laevis genome by the use of Ag+/Cs2SO4 density gradients. Ag+ shows a low affinity for some tRNA cistrons, allowing their separation from bulk DNA upon equilibrium centrifugation in a Cs2SO4 density gradient. Contaminating DNA in the resulting tDNA fraction is further removed by two additional CsCl density gradient centrifugations. The final DNA fraction is 60-fold enriched in tRNA genes, compared to the starting DNA material.     

A DNA study of rat liver oligonucleosomes enriched by transcriptionally active genes during induction due to the administration of an amino acid mixture

A highly active fraction of rat liver oligonucleosome DNA has been isolated and studied by means of thermal denaturation after induction by amino acid mixture or hydrocortisone. A considerable redistribution of DNA content has been shown in sucrose gradient fractions during these forms of induction. The changes are revealed in melting temperature, differential melting profile of DNA, isolated from actively transcribed chromatine fractions. Analysis of melting profiles shows changes of GC content of oligonucleosome DNA, suggesting that there are differences in activation during two studied forms of induction.     

Small peptides controlling transcription in vitro are bound to chromatin DNA

Low-molecular-weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction at pH 9.5. The level of the active peptide fraction ranges between 10 and 35 micrograms/mg DNA. The removal of peptides from DNA causes a relevant amplification of DNA template capacity for prokaryotic and eukaryotic RNA polymerases. Gel filtration on Sephadex G-25 or BioGel P4 shows that the chromatin peptide fraction from purified DNA migrates as a sharp peak with an elution volume corresponding to a molecular weight of about 1000. The chromatin peptides are further purified by Sephadex G-10 and high-performance liquid chromatography. Four active fractions are isolated, one of which shows very high inhibition activity on the RNA synthesis in vitro. The amino acid analysis and the inhibition mechanism of the purified peptides are reported.     

Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells

Mature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15–22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA. © 1993 Wiley-Liss, Inc.     

Isolation and characterization of the DNA fraction of rat liver chromatin which binds polylysine.

The structure of eukaryotic chromatin has been investigated by isolating and analyzing the "accessible" DNA fraction of rat liver chromatin. This DNA fraction has been isolated by titrating the chromatin with the protese-resistant D isomer of polylysine to bind the "accessible" DNA sites. After removal of chromosomal proteins by digestion with pronase, all DNA not protected from attack by bound polylysine was removed by digestion with DNase. Even after exhaustive treatment with pronase and DNase approximately 30% of the chromatin DNA remains resistant to nuclease attack. Analysis of the isolated DNA shows it to be mainly double-stranded with an average size of 200-250 base pairs. The DNA is slightly A-T rich and contains both repetitive and "single-copy" nuleotide sequences. The results suggest that there are extensive regions in chromatin where the DNA is not tightly complexed with protein. Furthermore, the DNA of these regions is similar in gross properties to the DNA of the total genome.     

Cloning and Sequencing of Coat Protein Gene of Papaya Ringspot Virus and Sequence Comparison of Strains of PRV-P and PRV-YS

Papaya rinspot virus (PRV) was isolated and purified from infected papaya (Carica papaya L. )leaves in Hainan Island. The first strand of cDNA was synthesized with Olig(dT)as a primer. The coat protein gene of PRV was obtained by PCR techniques with primers synthesized accoding to the DNA sequence of PRV-P. Full length of cDNA clone encoding coat protein of PRV was sequenced and analysed. The result shows that the strain of PRV we isolated contains 881 nucleotides with 287 amino acids. Sequences among several strains of PRV were compared and it indicates an over 90% homology in DNA sequence of PRV-G the strain we isolated with PRV-P and PRV-YS. Highly convered sequence was located in carboxyl end and interestingly highly variable region was in N-terminal.