Multiparameter flow cytometric analysis of hepatic nuclear RNA and DNA of normal and hepatotoxin-treated mice.

Combined immunohistologic and flow cytometric methods were used to monitor the regenerative response of the mouse liver following intraperitoneal administration of the hepatonecrotic agent carbon tetrachloride (CCl4). A slight increase in the percentage of liver nuclei engaged in DNA synthesis (S-phase population) was evident 1 day after CCl4. The percentage of S-phase nuclei increased thereafter, lasting until day 6 after CCl4 with a peak at day 2 after CCl4. The method of sampling used (every 24 hours) places the period of maximal DNA synthetic activity between 48 hours and 72 hours after inoculation of the hepatonecrotic agent; at 48 hours the number of S-phase nuclei was approximately five times higher than the nonregenerating liver. Modified multiparametric flow cytofluorimetric analysis of hepatic nuclei indicated that early repopulation of the liver in response to CCl4-induced necrosis involved the selective proliferation of a specific subpopulation of liver cells which contained diploid nuclei of high RNA content. These nuclei were also observed in control animals, suggesting the existence of a subcompartment of 2C DNA content G1 hepatocytes potentially programmed for replacement proliferation.

RNA and DNA content of isolated cell nuclei measured by multiparameter flow cytometry.

Nuclei were isolated from various cell types including Chinese hamster ovary (CHO) and L1210 leukemia cell lines, primary cultures of fibroblasts, nonstimulated and stimulated human lymphocytes and mouse liver cells, by using different isolation techniques. The isolated nuclei were subsequently stained with acridine orange (AO) and their fluorescence was measured by flow cytometry. Various procedures designed to stain DNA versus RNA differentially with AO were tested, and the staining of isolated nuclei was compared with that of whole cells. Control incubations with RNase and DNase were performed to estimate in whole cells and in nuclei the contribution of DNA and RNA to the fluorescence intensity at the respective wavelength bands of maximum emission for DNA (F530) and RNA (F greater than 600). Depending on the cell type, 10-20% of total cell RNase-sensitive F greater than 600 is localized in the nuclei. The RNase-resistant portion of F greater than 600 of isolated nuclei represents the stainability of DNA. Suppression of cell proliferation in subconfluent cultures results in a decrease in both whole cell and in nuclear RNA content. Nonstimulated lymphocyte nuclei have considerably lower RNA content than nuclei from lymphocytes stimulated by pokeweed mitogen. Two subpopulations of nuclei having the same (2C) DNA content but differing in RNA content, are present in mouse liver; the cells entering S phase originate from the high RNA population.

Single-stranded fraction of deoxyribonucleic acid from Bacillus subtilis.

About 13% of the deoxyribonucleic acid (DNA) of various strains of Bacillus subtilis, independent of the stage of growth or competence for transformation, was rendered acid soluble by endonuclease S1. In a pH 11.2 CsCl gradient, 4% of the untreated DNA banded at the density typical for single-stranded molecules, whereas 9% of the remaining DNA (main band) was sensitive to endonuclease S1. Selective inhibition of DNA polymerase III, or of DNA-dependent ribonucleic acid polymerase, did not increase or abolish single-strandedness. The DNA purification procedure did affect the level of single-stranded DNA, indicating its binding to cell constituents containing ribonucleic acid, protein, and membranous material. The molecular weight of the single-stranded fraction resembled that of total denatured DNA, and its buoyant density in an alkaline CsCl gradient was centered partially at a density of 1.772 g/cm3 and partially at a density of 7.759 g/cm3. Incubation of DNA under conditions leading to renaturation of its single-stranded fraction led to an increase in transforming activity for the purA16+ marker (close to the origin of replication) relative to leu-8+ and metC3+ markers (located in the middle of the chromosome), indicating this region is the main source of the single-stranded fraction.