Dose-dependent preferential binding of polycyclic aromatic hydrocarbons to reiterated DNA of murine skin cells in culture.
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The distribution of active metabolites of polycyclic aromatic hydrocarbons bound to reiterated or unique regions of murine DNA has been studied by a DNA-DNA renaturation technique. Murine skin cells were exposed to different doses of radioactive polycyclic aromatic hydrocarbons for 24 hr; then the hydrocarbon-labeled DNA was isolated, fragmented, and denatured. Renaturation kinetics and thermal stabilities of DNA-DNA duplexes were studied. At high carcinogen doses, polycyclic aromatic hydrocarbon adducts seem to be distributed equally among the DNA of all reiteration frequencies. At low carcinogen doses, however, a dose-dependent preferential binding to reiterated DNA sequences occurs. An inverse linear relationship appears to exist between the enrichment of hydrocarbon adducts in reiterated DNA sequences and the logarithm of the amount of total hydrocarbon bound to DNA. (+info)
Estimation of SELEX pool size by measurement of DNA renaturation rates.
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We report a general method of measuring the complexity of SELEX pools. In analogy to measurements of genome size by C0t analysis, the complexity of a SELEX pool is measured by determining the reannealing rate of its double-stranded PCR product. We applied this technique to study the selection dynamics of a recently reported SELEX to neutrophil elastase. We found that the number of sequences decreased from 10(7) in round 6 to approximately 60 by round 15, the final round. The intermediate rounds are a mixture of a high abundance/low complexity pool with a low abundance/high complexity pool. As the SELEX progresses, the former pool expands at the expense of the latter. This technique should be useful for studying and optimizing SELEX dynamics, as well as for monitoring the progress of SELEX experiments. (+info)
Distribution and virogenic effects of 5-bromodeoxyuridine in synchronized rat embryo cells.
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Rat embryo cell cultures were synchronized by a double thymidine block. The DNA replication phase (S) was divided into an early, middle, and late period. Cell cultures in the early, middle, or late S phase were pulsed with 0.1 muM 5-bromo[(3)H]deoxyuridine (BrdU) or equimolar [(3)H]dT. DNA-DNA reassociation experiments of each sample revealed that [(3)H]BrdU was more concentrated in the intermediate repetitive than the repetitive or unique DNA sequences of the early and middle S phase. In contrast, [(3)H]dT was nearly uniformly jistributed throughout all nucleotide sequences during the entire S phase. synchronized rat cells were pulsed during various portions of the S phase with unlabeled 0.1 mM or 0.1 muM BrdU and examined for sytoplasmic immumofluorescence against the 30,000 molecular weight group-specific antigen (p30) of Friend mouse leukemia virus. Equally strong fluorescence was detected 12 hr later in cells treated with each concentration of BrdU. Furthermore, incorporation of BrdU during late S phase was suffieient to elicit maximal antigen expression. (+info)
Metastable structures and refolding kinetics in hok mRNA of plasmid R1.
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Programmed cell death by hok/sok of plasmid R1 and pnd/pndB of R483 mediates plasmid maintenance by killing of plasmid-free cells. It has been previously suggested that premature translation of the plasmid-mediated toxin is prevented during transcription of the hok and pnd mRNAs by the formation of metastable hairpins in the mRNA at the 5' end. Here, experimental evidence is presented for the existence of metastable structures in the 5' leader of the hok and pnd mRNAs in vitro. The kinetics of refolding from the metastable to the stable structure in the isolated fragments of the 5' ends of both the hok and pnd mRNAs could be estimated, in agreement with the structural rearrangement in this region, as predicted to occur during transcription and mRNA activation. The refolding rates of hok and pnd structures are slow enough to allow for the formation of downstream hairpin structures during elongation of the mRNAs, which thereby helps to stabilize the metastable structures. Thus, the kinetic refolding parameters of the hok and pnd mRNAs are consistent with the proposal that the metastable structures prevent premature translation and/or antisense RNA binding during transcription. (+info)
Site-specific cleavage of single-stranded DNA by a Hemophilus restriction endonuclease.
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Single-stranded viral DNA of bacteriophage f1 is cleaved into specific fragments by endo R-HaeIII, a restriction endonuclease isolated from Hemophilus aegyptius. The sites of the single strand cleavage correspond to those of the double strand cleavage. A single-stranded DNA fragment containing only one HaeIII site is also cleaved by this enzyme. This observation suggests that the reaction of single-stranded DNA cleavage does not require the formation of a symmetrical double-stranded structure that would result from the intramolecular base-pairing between two different HaeIII sites. Other restriction endonucleases may also cleave single-stranded DNA. (+info)
Analysis of DNA of isolated chromatin subunits.
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Partial digestion of rat liver nuclei with staphylococcal nuclease results in the liberation of nucleo-protein complexes consisting of one or more upsilon bodies. By velocity centrifugation we have isolated the monomeric subunit in relatively pure form. We find that this subunit contains 185 base pairs of DNA and 240,000 daltons of protein, resulting in a protein to DNA ratio identical to that of unperturbed chromatin. The isolated monomeric particle is further susceptible to internal nuclease attack resulting in the solubilization of 46% of the monomeric DNA. Analysis of the resistant DNA reveals a complex but highly reproducible pattern of DNA fragments ranging from 160 to 60 base pairs in length. Analysis of the reassociation kinetics of the isolated subunit DNA reveals that most, if not all genomic sequences, are involved in this basic subunit structure. No special frequency class of DNA is absent from upsilon bodies. Furthermore, virtually all liver sequences transcribed into mRNA are present in upsilon body DNA. These results indicate that upsilon body formation may be random with respect to DNA sequence and suggest that the mere presence of upsilon bodies over a specific region of DNA is not sufficient to restrict its transcription. (+info)
Distribution of 5-bromodeoxyuridine and thymidine in the DNA of developing chick cartilage.
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In order to study the mechanism of the irreversible effects of BrdUrd on the differentiation of limb bud mesenchyme to cartilage, the reannealing behavior of DNA obtained from such cells was examined. Cells incubated with [3H]thymidine ([3H]dThd) during days 1 and 2 of culture incorporated label into repetitive, moderately repetitive, and unique classes of DNA. In contrast, when 5-bromo-2'-[3H]deoxyuridine ([3H]Brd Urd) was added during the first 48 hr (in the presence of 32 muM BrdUrd), the label was preferentially incorporated into a late moderately repetitive region. Simultaneous incubation of unlabeled BrdUrd and [3H]dThd revealed a selective inhibition of [3H]dThd incorporation into moderately repetitive regions. Cultures incubated during days 3 and 4 with [3H]dThd incorporated label into all three classes of DNA; however, when [3H]dThd was present during days 3 and 4 in cultures previously incubated with BrdUrd during days 1 and 2, the [3H]dThd was incorporated preferentially in the late moderately repetitive region. The melting behavior of this reannealed DNA was identical with that of the reannealed 1-2 day [3H]BrdUrd-labeled, late moderately repetitive DNA. Turnover experiments revealed that whereas there was no loss of [3H]deoxycytidine or [3H]dThd, 37% of [3H]BrdUrd activity was lost from the DNA in 2 days after removal of the isotopes. (+info)
Intracellular forms of adenovirus DNA: integrated form of adenovirus DNA appears early in productive infection.
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In KB cells productively infected with adenovirus type 2, alkali-stable greater than 100S and 40-100S viral DNAs are synthesized starting 2-4 hr postinfection, i.e., before unit length (34 S) viral DNA is made. The amount of greater than 100S and 40-100S viral DNA increases when 34S viral DNA synthesis begins, and at 16-18 hr postinfection, the 40-100S viral DNA represents 5-20% of the total intracellular viral DNA. The 40-100S viral DNA is synthesized throughout infection. Part of the 40-100S DNA synthesized 5-8 hr postinfection has a density in alkaline CsCl gradients intermediate between those of viral and cellular DNAs. This finding indicates that newly synthesized viral DNA is covalently linked to cellular DNA. Viral sequences can be excised from the cellular DNA of infected cells with the EcoRI restriction endonuclease. Fragments of viral DNA are detected in polyacrylamide-agarose gels by DNA-DNA hybridization, and these fragments correspond in size to most of the known EcoRI fragments of adenovirus 2 DNA. Viral DNA sequences in size-classes between the EcoRI-A and -C fragments are also found and probably represent viral DNA linked to cellular sequences. (+info)