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Further Studies on the Effects of Various in Vivo and in Vitro Factors on the Priming and Template Activities of DNA Isolates in the DNA and RNA Polymerase Systems¹

State University of New York at Buffalo, School of Pharmacy and Medicine, and the Roswell Park Memorial Institute, Buffalo, New York 14214

In order to examine the possible relationship of the previously reported increased priming activity of DNA isolated from neoplastic tissues with the rate of mitosis or cancer, the priming activities of nonmalignant regenerating tissues were investigated. DNA was isolated from livers of rats at 12, 18, 24, 34, 30, 36, 40, and 72 hr following partial hepatectomy or sham operation in the controls. The priming activity with DNA polymerase was determined in the relatively crude regenerating rat liver DNA polymerase system with tritiated thymidine and deoxyribonucleotide monophosphates as substrates. The same DNA isolates were tested for template activity in the Micrococcus lysodeikticus RNA polymerase system. No differences were found in these parameters as compared to the DNA from normal rat liver. These results indicate that the priming activity of the isolated DNA is not related to the mitotic rate of the tissue of origin.

As a test of the possibility that some factors inherent in the isolation procedure could have an influence on the priming and template activities of DNA, the effects of DNase, heat, and ionic composition were investigated under the conditions of the isolation and assay procedures.

Treatment with pancreatic DNase at 0.8 unit/mg DNA significantly increased the priming activity of calf thymus DNA for DNA polymerase and markedly decreased the template activity in the RNA polymerase system. Stronger DNase treatment (8.0 units/mg DNA) decreased the priming activity and further decreased the template activity. However, when the pancreatic DNase was added to a solution of calf thymus DNA in the same buffer that was used for the isolation of DNA from tissues and this solution then was subjected to the entire DNA isolation procedure, the reisolated DNA showed the same priming activity in the DNA polymerase system as the untreated DNA standard. Addition of homogenates from normal and malignant human tissues to the above calf thymus DNA solution before the homogenization step did not affect the priming activity of the reisolated DNA. Thus the DNase present in the tissue cannot affect, under the isolation conditions used, the final assay results. In the light of available data, the effects of various in vivo and in vitro factors on the priming and template activities are discussed.

¹ This investigation was supported by USPHS Grants CA-06695 and CA-05522 from the National Cancer Institute.

Received 2/ 7/69. Accepted 9/22/69.