STUDIES ON ISOLATED NUCLEI

Abstract

This article describes a method for the isolation of nuclei from guinea pig liver. It involves the homogenization of the tissue in 0.88 M sucrose-1.5 mM CaCl2 followed by centrifugation in a discontinuous density gradient in which the upper phase is the homogenate and the lower phase is 2.2 M sucrose-0.5 mM CaCl2. Based on DNA recovery, the isolated fraction contains 25 to 30 per cent of the nuclei of the original homogenate. Electron microscopical observations showed that ∼88 per cent of the isolated nuclei come from liver cells (the rest from von Kupffer cells and leucocytes) and that ∼90 per cent of the nuclei appear intact, with well preserved nucleoli, nucleoplasm, nuclear envelope, and pores. Cytoplasmic contamination is minimal and consists primarily of the nuclear envelope and its attached ribosomes. The nuclear fraction consists of ∼22.3 per cent DNA, ∼4.7 per cent RNA, and ∼73 per cent protein, the DNA/RNA ratio being 4.7. Data on RNA extractibility by phosphate and salt and on the base composition of total nuclear RNA are included.