Characterization of Deoxyribonucleic Acid of Entamoeba histolytica by Cesium Chloride Density Centrifugation

Abstract

Buoyant density of deoxyribonucleic acid (DNA) from Entamoeba histolytica was determined in cesium chloride gradients in the analytical ultracentrifuge. The DNA was extracted from fresh or lyophilized cells in a solution containing 8 M urea. It was purified by absorption on and elution from hydroxylapatite, and on a cesium chloride gradient in the preparative ultracentrifuge. DNA from axenically grown amebae contained a single component and it banded in the region of 1.69 g/ml. DNA prepared from 13 strains of amebae grown with penicillin-inhibited bacterial cells also banded in this region. Some of the latter preparations contained a second component of buoyant density 1.708 g/ml which is attributed to the food bacteria, Bacteroides symbiosus. Buoyant density of amebal DNA ranged from 1.6881 to 1.6913 g/ml corresponding to guanine plus cytosine contents of 27.5 to 30.8 mole per cent. Buoyant density is not a sensitive parameter for making interstrain distinctions among strains of E. histolytica, but it appears to offer promise in an area where little else has succeeded. A number of statistically significant interstrain differences were encountered. Thus low density DNA from NRS or HK9 strains was distinguished from DNA of intermediate density from DKB or 200 strains and from the high density DNA of the F22 strain at probability levels to 0.001. Guanine plus cytosine contents calculated from thermal hyperchromicity (Tin) were lower than those calculated from buoyant density data. The DNA from Bacteroides symbiosus was calculated to contain 48.2 mole per cent guanine plus cytosine, using buoyant density data. Knowledge concerning the base composition of DNA has been useful in taxonomic studies on bacteria. The less extensive work with protozoa was reviewed by Mandel (1967). Recently Adam, Blewett, and Flamm (1969) encountered differences in base composition among DNAs from various Acanthamoeba. The purpose of this investigation was to probe for evidence of different base composition among DNAs from various strains of Entamoeba histolytica. We were particularly interested in comparing the group of high-temperature strains which grow in vitro between 33 and 39 C with the group of low-temperature strains which grow at temperatures of 25, or less, to about 37 C. The chemical method for determining DNA base composition by separation and quantitation of the hydrolyzed bases requires more purified DNA than we could afford for this work. Most of our data were obtained by the buoyant density method which requires only about 1 I/g of DNA for a single centrifuge run. This method has the added capability of resolving DNAs of different densities which may be present in a single preparation. Thus we were able to survey DNA not only from four Received for publication 13 April 1971. axenically grown strains, but also from a larger number of strains grown with penicillin-inhibited cells of the associate organism, Bacteroides symbiosus. Many of the latter strains have not yet been placed in axenic culture. MATERIALS AND METHODS Amebae were grown in axenic culture by the method of Diamond (1968), or with penicillininhibited cells of Bacteroides symbiosus by the method of Reeves and Ward (1965). The hightemperature strains of E. histolytica were grown at 36 to 37 C, the others at 25 to 31 C. Cultures were harvested soon after population peaks had been reached. Cells were collected by centrifugation and washed 3 times with a solution containing 0.11 M NaCl, 0.02 M KC1, 1 mM potassium phosphate (pH 7.4), 0.5 mM MgCl2, and 0.1 mM CaCI2. Centrifugally packed cell volumes were noted and cell counts were made in a Fuchs-Rosenthal counting chamber. The final wash fluid was withdrawn as completely as possible from the packed cells and the cell pellet was quickly frozen in an acetone-dry ice bath. The frozen cells were lyophilized, sealed in glass under vacuum, and stored at -20 C until used. Source of the axenic cultures has been reported by Diamond (1968), and of the monoxenic cultures by Reeves et al. (1967), except for the N strain which is from South Africa and was obtained from Dr. Shirley Madison.