Changes in the template activity of chromatin isolated from sarcoma-180 ascites cells treated with mitomycin C and gamma irradiation in vivo.

The murine ascites sarcoma 180 cells were used to test the in vivo effectiveness of mitomycin C (MMC) and gamma-radiation applied in combination. The action of intraperitoneal administration of MMC and/or whole-body gamma irradiation on sarcoma 180 tumor bearing Swiss albino mice was investigated by studying the template activity of isolated tumor chromatin. The Km value for transcription of 10 Gy-irradiated chromatin was found to decrease with time implying an increase in the template efficiency with respect to that of the unirradiated control. Maximum decrease in Km was observed after 24 h of irradiation. MMC treatment (7 mg/kg body weight of mouse) for 18 h resulted in an inhibition of the transcription rate. Severe inhibition in the template activity was found when cells were subjected to MMC treatment 18 h prior to irradiation with 10 Gy. Susceptibility of tumor chromatin to DNase II followed the same pattern as observed in the case of transcription indicating structural alteration of the treated chromatin. The data showed that DNA damage and its consequences produced in the ascites cells by prior treatment of MMC were not repaired during the 18 h period after which the application of radiation enhanced cytotoxicity.

Effect of carcinogenic adducts on transcription by T7 RNA polymerase.

The effect of modifying the T7 promoter-containing plasmid pDR100 with aminofluorene (AF), acetylaminofluorene (AAF), and benzo[a]pyrene (B[a]P) adducts on RNA synthesis by the T7 RNA polymerase was determined. We found that increasing levels of each of the three adducts caused a progressive decline in RNA synthesis, but that the inhibition produced by benzo[a]pyrene adducts was substantially greater than that caused by either AF or AAF adducts. Polyacrylamide gel electrophoresis analysis confirmed that the B[a]P adducts more strongly inhibited chain elongation since their presence resulted in the production of RNA fragments having average chain lengths very close to that predicted if each adduct permanently blocked the polymerase during transcription. We have also determined the effect of each type of adduct on the initiation of transcription and show that the T7 RNA polymerase initiates as efficiently on both AF or AAF-containing templates as it does on unmodified DNA, while B[a]P adducts were found to increase in the number of chain starts.

Thymine glycols and urea residues in M13 DNA constitute replicative blocks in vitro.

Thymine glycols were produced in M13 DNA in a concentration dependent manner by treating the DNA with osmium tetroxide (OsO4). For the formation of urea-containing M13 DNA, OsO4-oxidized DNA was hydrolyzed in alkali (pH 12) to convert the thymine glycols to urea residues. With both thymine glycol- and urea-containing M13 DNA, DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment was decreased in proportion to the number of damages present in the template DNA. Sequencing gel analysis of the products synthesized by E. coli DNA polymerase I and T4 DNA polymerase showed that DNA synthesis terminated opposite the putative thymine glycol site and at one nucleotide before the putative urea site. Substitution of manganese for magnesium in the reaction mix resulted in increased processivity of DNA synthesis so that a base was incorporated opposite urea. With thymine glycol-containing DNA, processivity in the presence of manganese was strongly dependent on the presence of a pyrimidine 5' to the thymine glycol in the template.

Inactivation of the DNA template in HeLa cells treated with chlorambucil.

The effect of chlorambucil on the colony-forming ability of HeLa cells following either treatment for 1 h or continuous treatment has been measured. Concentrations of chlorambucil which had only minimal effects on cell survival inhibited the rate of DNA but not RNA and protein synthesis within 1 h of treatment. Nevertheless, cells continued to synthesize DNA for many hours after this treatment in the absence of cell division. Synchronous populations of HeLa cells treated prior to DNA synthesis, in the G1 phase of the cell cycle, were not delayed in their progression into the S phase where they exhibited a marked dose-dependent inhibition of the rate of DNA synthesis. Cells in which DNA synthesis had been depressed showed a prolongation of the S phase and this was accompanied by a corresponding dose-dependent mitotic delay. Treatment during the G2 phase of the cell cycle did not induce any delay or block in the next mitosis, but did inhibit the rate of DNA synthesis in the following cell cycle in a dose-dependent manner; this depression of DNA synthesis was followed by a delay in the next mitosis. Cross-linking of either isolated DNA or DNA present in treated HeLa cells was demonstrated, and in the latter case calculated to be of the same order as that which occurred with other difunctional agents at comparable toxic concentrations.

Mode of action of phleomycin on Bacillus subtilis.

Phleomycin appears to act on the cell wall and membrane to induce the release of membrane-associated deoxyribonucleic acid (DNA) and the degradation of the DNA. Degradation occurs in a series of energy-requiring endonuclease and exonuclease reactions. These produce, first, single-strand breaks, then double-strand breaks, and finally almost complete solubilization of the cellular DNA. The in vivo inhibition of DNA synthesis by phleomycin is probably a secondary effect caused by the destruction of template DNA.

Structural alteration in isolated rat liver nuclei after removal of template restriction by polyanions.

Specific polyanions release DNA template restrictions for DNA synthesis in isolated rat liver nuclei. The degree to which DNA synthesis is enhanced can be correlated with a spectrum of changes in nuclear structure Each polyanion which is effective in the release of template restriction produces a characteristic alteration in nuclear ultrastructure. Polyanions which have no effect on DNA synthesis do not appear to cause any change in nuclear organization or ultrastructure. Parallel measurements of nuclear DNA release and nuclear volume changes also indicate that template-activating polyanions cause remarkable changes in the structural organization of the treated nuclei. These results indicate that DNA template activation involves direct interactions between polyanions and nuclear constituents and suggest the possibility that naturally occurring polyanions might have a role in the control of gene activity

Mechanism of antiviral action of acetone on rabbitpox virus replication.

Acetone added to the maintenance medium in a 1% concentration reduces the yield of infectious rabbitpox virus in L-cell monolayer cultures by 90 to 97%. This concentration of the inhibitor is not toxic for cells. It was established that there is an inhibitor-sensitive stage late in the infectious cycle. Acetone exerted no significant influence on production of early viral messenger ribonucleic acid, formation of polyribosomes, deoxyribonucleic acid (DNA) replication, or protein synthesis. In the presence of acetone, the assembly of so-called "acetone particles" occurred. These particles are similar to normal virions by morphological and sedimentation properties but are slightly different from them in buoyant density. The amount of virus-specific DNA and the optical density of the "acetone particles" are the same as those of normal virions despite a 10- to 25-fold difference in the infectivity of the preparations.