ABSTRACT
A relatively rapid chalone assay using inhibition of purified calf thymus DNA polymerase alpha by Ehrlich Ascites Cell (EAC) chalone has been performed. The DNA polymerase alpha was inhibited in a concentration-related fashion by partially purified EAC chalone ranging from 10 to 200 micrograms/ml. Spermidine was also tested since there has been some suggestion that chalone may be spermidine; we found no effect of spermidine at 170 and 230 microM, but marked inhibition at 33 mM. This assay should facilitate chalone purification, since chalone appears to non-specifically inhibit DNA polymerase alpha.
Subject(s)
Carcinoma, Ehrlich Tumor/analysis , Growth Inhibitors/analysis , Animals , Cattle , DNA Polymerase II/antagonists & inhibitors , Growth Inhibitors/pharmacology , Thymus Gland/enzymologyABSTRACT
Ehrlich Ascites Tumor (EAT) chalone has been shown to inhibit nascent DNA synthesis by inhibiting DNA polymerase alpha and beta (Nakai, 1976), but one of the problems in studying eurkaryotic DNA replication has been the relative impermeability of the cell membrane to precursors and macromolecules; hence, to circumvent this restriction without sacrificing the integrity of the replication process, a broken cell system utilizing nuclei in aqueous media was investigated. Isolated nuclei appear to continue the process of DNA replication that was proceeding in vivo before their isolation and under optimal concitions are able to initiate new synthesis (Fraser & Huberman, 1977). The effects of partially purified EAT chalone on nascent DNA could be studied directly in this nuclear system, which excluded effects of the cell membrane, nucleotide pools and other cytosol elements. A concentration-related inhibition of [3H]thymidine triphosphate ([3H]dTTP) incorporation was noted over a chalone range of 50-200 micrograms/ml. It appears that chalone can inhibit DNA polymerase alpha directly within the nucleus without an intermediate step such as a cell membrane receptor.
Subject(s)
Cell Nucleus/metabolism , DNA/biosynthesis , Growth Inhibitors/pharmacology , Animals , Carcinoma, Ehrlich Tumor , Cell Fractionation , DNA/metabolism , Dose-Response Relationship, Drug , Kinetics , Thymine Nucleotides/metabolismABSTRACT
EAT chalone effects on nascent DNA synthesis and DNA polymerase were examined. Concentration related inhibition of 3H-thymidine (3H-TdR) incorporation into EAT cell DNA was noted over a chalone range of 50-200 mug/ml. RNA synthesis was not affected, but protein synthesis decreased an average of 82% during 3 hr. Nascent DNA pulse-labeled for 2 min was normally incorporated into bulk DNA in the presence of chalone, but crude alpha- and beta-polymerase activities were inhibited. Crude DNA polymerase for C3H mouse kidney and spleen was also partially inhibited by EAT chalone, suggesting non-specific inhibition of DNA polymerase. Preincubation studies of chalone with crude EAT DNA polymerase or 'gapped' DNA primer had no effect on chalone activity. Chalone may control mitotic activity by inhibiting alpha- and beta-polymerase activity, thereby decreasing nascent DNA synthesis. Nascent DNA is incorporated normally into bulk DNA in the presence of chalone, indicating the DNA ligase is not inhibited.
Subject(s)
Carcinoma, Ehrlich Tumor , DNA Polymerase II/metabolism , DNA Polymerase I/metabolism , DNA, Neoplasm/biosynthesis , DNA-Directed DNA Polymerase/metabolism , Growth Inhibitors/pharmacology , Animals , Cell Line , Dose-Response Relationship, Drug , RNA, Neoplasm/biosynthesisABSTRACT
Ehrlich ascites tumor cell putative nuclear pre-messenger RNA (pre-mRNA) was isolated under conditions minimizing RNA degradation by ribonucleases, aggregation, and non-specific protein-RNA interaction. Isolated under these conditions, it sedimented 10 to 12 S; proteinase K, a powerful proteolytic enzyme with a broad action spectrum, gave similar sedimentation values and polyacrylamide gel electrophoresis revealed the major component migrating ahead of 16S E. Coli rRNA marker. Cesium chloride buoyant density analysis of pre-mRNA revealed 2 components (1.51 and 1.68 g/cm3). Therefore, pre-mRNA appeared to be smaller than some previous reports.
Subject(s)
Carcinoma, Ehrlich Tumor/analysis , RNA, Messenger , Animals , Cell Nucleus , Molecular Weight , Peptide Hydrolases , RNA, Messenger/analysisABSTRACT
Ehrlich ascites tumor cell messenger ribonucleoprotein (mRNP) and putative messenger RNA (mRNA) were isolated under conditions minimizing RNA degradation by ribonucleases and non-specific protein-RNA interaction, mRNP dissociated from polysomes by puromycin and EDTA sedimented to 185S. Putative messenger RNA released by proteinase K sedimented from 50 to 97S, with a maximum molecular weight approximating 6-25 X 10(6) daltons.
