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.

This paper dcscribes thc snbfractionation of nuclei isolatcd from guinea pig liver by the procedure presented in thc first article of the series (8). Centrifugation in a density gradicnt system of nuclear fractions disruptcd by sonication permits the isolation of the following subfractions: (a) a nucleolar subfraction which consists mainly of nuclcoli surroundcd by a variablc amount of nuclcolus-associated chromatin and contaminated by chromatin blocks derived primarily from von Kupffcr cell nuclei; (b) and (c), two nucleoplasmic subfractions (I and If) which consist mainly of chromatin threads in a coarser (1) or finer (II) degree of fragmentation. The protein, RNA, and DNA content of these subfractions was dctermined, and their RNA's characterized in tcrms of NaCl-solubility, nucleotide composition, and in vivo nucleotidc turnover, using inorganic 32p as a markcr. The results indicate that therc are at Icast threc types of RNA in the nucleus (onc in the nucleolus and two in the nucleoplasm or chromatin), which differ from onc another in NaCl-solubility, nucleotide composition, turnover, and possibly sequcnce. Possible relations among these RNA's and thosc of the cytoplasm arc discussed.

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ZOOLOGY: MONROY ET AL. 107 recipient eggs, and none of these eggs developed normally. These results indicate a real difference in the "developmental capacity" of germ cell and somatic cell nuclei. Many of the germ cell nuclei are shown to be developmentally totipotent, while by the same test the somatic nuclei are shown to be severely restricted in capacity to promote development. The author is very grateful to Dr. Robert Briggs for his advice during the course of this investigation and for his suggestions during the preparation of this manuscript. He also wishes to express thanks to Mrs. Dorothy Barone for technical assistance.