Extraction, separation and labeling with 14 C-glucose of hela cell polyglucose, RNA and DNA and comparison of the molecular weights and buoyant densities of polyglucose from poliovirus-infected and noninfected cultures

Abstract

The aqueous phase of phenol extracts of HeLa cells contains polyglucose (CHO) n , RNA, and DNA. These macromolecules were precipitated together and removed from 50% (v/v) ethanol solutions with a stirring rod. The viscous precipitate had the classical white appearance of DNA, but contained an average of 439, 670, and 220 Μg (from 3 × 10 7 cells) of (CHO) n , RNA, and DNA, respectively. The (CHO) n was separated from the RNA, either by CsCl density gradient centrifugation or by precipitating the RNA with trichloroacetic acid (TCA). Both methods of separation resulted in preparations of (CHO) n with similar specific activities (radioactive counts/Μg min). However, electron micrographs showed that the (CHO) n separated by using TCA had a greater variation in particle size when compared with (CHO) n separated by CsCl centrifugation. With the CsCl methods, the number-average molecular weights, as determined by electron microscope particle-counting, and the buoyant densities of (CHO) n whose synthesis was stimulated by poliovirus infection and (CHO) n from noninfected cultures, were found to be similar. When the (CHO) n was extracted from HeLa cells with TCA, rather than phenol, the yield was 1.68-fold greater and its specific activity was an average of twice that of the (CHO) n extracted with phenol. The time at which cells were pulse-labeled with 14 C-glucose, after reducing the glucose in the culture medium to 0.01 of normal, was found to be important, in that the specific activity of the (CHO) n increased 23.4-fold over a 4-hr period and the amount extracted decreased 8.2-fold. The increase in the specific activities of RNA and DNA was not as large as that of the (CHO) n and the amounts extracted were not significantly changed. The sedimentation coefficients of RNA and (CHO) n which were separated from each other with TCA were 6.4 and 116 S, respectively, whereas, without separation, two peaks were seen, with values of 25.4 and 31.4 S. Chloride ions reduce the sensitivity of the Burton test for DNA. However, the Burton reagent will detect (CHO) n even in the presence of DNA if the assay mixture is heated. Chloride ions increase the sensitivity of the Burton reagent to detect melizitose and, at concentrations above l.5 M , synthetic- polyglucose by increasing the absorption of the colored (CHO) n reaction product(s).