IT has been previously shown that during the feeding to rats of thioacetamide and 4-dimethylaminoazobenzene (DAB), parenchymal liver cell nuclei show an increase in their chemical constituents (Rees and Rowland, 1961). These measurements were carried out at regular time intervals throughout the tumour induction period of approximately 6 months. In the case of thioacetamide feeding it was observed that mar-ked changes in nuclear composition were already established by 2 weeks. This would reflect the histological finding (Gupta, 1956) of enlargement of the nucleus and nucleolus after a few days of such feeding. With the development of a technique of sub-nuclear fractionation (Rees, Rowland and Varcoe, 1963) it is now possible to examine changes in composition of the nucleolus and other Farts of the nucleus during the very early stages of chemical carcinogenesis.In the present investigation subnuclear proteins have been studied at various time intervals from the livers of rats fed thioacetamide for up to 3 weeks. As a comparison, similar studies have been made on the livers of rats fed with DAB for up to 5 weeks, no histological changes being apparent before 3 weeks with this carcinogen (Rees and Rowland, 1961). In addition to the determination of chemical composition of these fractions the incorporation in vivo has been followed of 32P into ribonucleic acid (RNA) and phosphotlipid.
It was shown by Rees & Rowland (1961) that rat-liver nuclei isolated in 025 M-sucrose incorporated amino acids into protein, and nucleotides into RNA. Although the incorporation was inhibited to varying degrees by anoxia and by several inhibitors of oxidative phosphorylation, it was not affected by detergents, by freezing and thawing the nuclei, or by disruption of the nuclei by ultrasonic vibration (Rees, Ross & Rowland, 1961). We have now studied isolated nucleoli and other subnuclear fractions in order to investigate the active site of synthesis of proteins and nucleic acids within the nucleus. In addition the enzymic and chemical composition of the various fractions has been examined. Part of this work has been published in a preliminary form (Rowland, Rees & Varcoe, 1962). MATERIALS AND METHODS Animals. Male albinorats,weighing200-250g., were used. Reagent8. These were as described by Rees & Rowland (1961). In addition [1-34C]valine and [1-14C]leucine were obtained from The Radiochemical Centre, Amersham, Bucks. Nuclear and8ubnuclearpreparation&. Nuclei were isolated from rat liver in 0*25M-sucrose as described by Rees & Rowland (1961). Scheme 1 shows the method used for isolating nucleoli and other subnuclear fractions. Batches of isolated nuclei, suspended in 15-20 ml. of 0 25m-sucrose, were subjected to ultrasonic vibration (20 keyc./sec.) at 20 in a MSE ultrasonic disintegrator (60w) with a titanium probe of 1 cm. diam. until all nuclei were disrupted (usually 7 min.). The resulting suspension was centrifuged for 30 sec. at 2500g to remove probe debris and coagulated protein. The nucleoli I Sediment Recentrifuged twice in water (see text) Nucleoli (fraction A) Disrupted nuclei Centrifuged at 412 x 104g,^min.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.