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High-Molecular-Weight DNA Associated with Intracytoplasmic A-Particle Inclusions

Laboratory of Biology, Division of Cancer Biology and Diagnosis, National Cancer Institute, NIH, Bethesda, Maryland 20014

Total nucleic acids were isolated from purified intracytoplasmic A particles and analyzed by agarose:acrylamide gel electrophoresis. A major band was observed that was found to be DNase sensitive; a minor band migrating in the 4 to 7 S region of the gel was RNase sensitive. Evidence that the DNA was not of cellular origin and a simple contaminant was obtained by several criteria. Extensive purification of the A particles, including detergent treatment followed by equilibrium sedimentation in sucrose and cesium chloride gradients, failed to effect the removal of DNA as a major nucleic acid component of purified A-particle preparations. Coisolation studies with thymidine-³H-labeled mouse embryo cells showed that cellular DNA contamination of highly purified A particles was minimal. Analytical equilibrium sedimentation analysis of A-particle-associated DNA and comparison with the buoyant density of tumor cell nuclear DNA isolated from the same tumor pool demonstrated a significantly greater density for A particle DNA (1.718 g cm^-3 and 1.701 g cm^-3, respectively). Autoradiographic grain counts obtained from electron micrographs of thymidine-³H-labeled Leydig cell tumors showed incorporation of thymidine-³H into A-particle inclusions in situ. These results correlated well with the specific activity of A-particle DNA isolated from these same tumors. The appearance of DNA in close association with putative nucleoprotein cores of mouse mammary tumor virus raises the possibility that this DNA represents the product of the viral reverse transcriptase or, conversely, that A particles are separate entities that share a time and space relationship with mouse mammary tumor virus. The answer to these questions must be resolved by molecular hybridization experiments between A-particle DNA and mouse mammary tumor virus 70 S RNA.

¹ To whom correspondence should be addressed, at National Cancer Institute, Building 37, Room 2E-16, Bethesda, Md. 20014.

² Supported in part by Damon Runyon Postdoctoral Fellowship 694.

Received 12/17/73. Accepted 6/19/74.