Studies on the Autonomy of Pellicular DNA in Paramecium

JOAN SMITH-SONNEBORN 0 W.PLAUT 0 0 Zoology Research Building, University of Wisconsin , Madison, Wisconsin , U.S.A STUDIES SUMMARY This autoradiographic study was designed to elucidate the relationship between the macronucleus and pellicular DNA in Paramecium. The capacity of the cell to synthesize pellicular DNA in the absence of the macronucleus was established by demonstrating the incorporation of tritiated thymidine into DNase-sensitive material in the pellicles of amacronucleate cells. Moreover, using a technique which leads to selective labelling of the macronucleus in normal paramecia, we have looked for evidence of transfer of labelled DNA from the macronucleus to the pellicle with time. Finding none, we conclude that labelled pellicular DNA is not of macronuclear origin, and that labelled pellicular DNA synthesis is not directly dependent on the presence of the macronucleus. INTRODUCTION The presence of DNA in the pellicle of Paramecium has been demonstrated in our previous study (Smith-Sonneborn & Plaut, 1967). This paper addresses itself to the questions of origin and autonomy of pellicular DNA. Specifically, we are asking whether pellicular DNA can be synthesized in the absence of the macronucleus and whether there is detectable migration of DNA to the pellicle from the macronucleus in its presence. The data obtained lead us to the conclusion that little, if any, of pellicular DNA is of immediate macronuclear origin, and that the presence of the macronucleus is not essential for its synthesis. EXPERIMENTAL DESIGNS The capacity of paramecia to incorporate tritiated thymidine into the pellicle in the absence of a macronucleus was investigated using cells bearing the 'Amac' gene (Nobili, 1961). In its presence the distribution of the macronucleus during division is irregular so that a certain percentage of the fissionts will lack a macronucleus. The inability of such cells to divide was used to identify the amacronucleates. Migration of DNA from macronucleus to pellicle was studied through the use of labelled Escherichia colt cells as the source of labelled DNA precursor (Berger & Kimball, 1964). Unlike exogenously supplied tritiated thymidine, which leads to labelling of DNA in both macronucleus and pellicle, this method results in heavily labelled nuclei and unlabelled pellicles. Such selectively labelled cells were permitted to undergo several fissions in the absence of additional labelled precursors and the pellicles were examined for the appearance of labelled DNA. Labelling with exogenously supplied tritiated thymidine was carried out in normal as well as axenic medium to control the possibility of an external origin of labelled pellicular DNA with this mode of precursor administration. Cells of Paraviecium aurelia, non-kappa bearing stocks derived from stock 51, i.e. d4-i86, and d2-43 (which bears the 'Amacronucleate' gene) were kindly supplied by Professor T. M. Sonneborn, Indiana University. These cells were maintained at 27 C in Cerophyl medium inoculated 24 h before use with Aerobacter aerogenes (Sonneborn, 1950). Synchronized cells were obtained by selection of dividing cells from an exponentially growing population using a micropipette under a dissecting microscope (Kimball, Caspersson, Ivenson & Carlson, 1959). The interfission period was 4^6 h. Stock 51 paramecia, in bacteria-free medium were graciously supplied by Dr Anthony Soldo (V.A. Hospital, 1201 N.W. 16th, Miami, Florida, 33125) and were maintained in axenic medium (Soldo, Godoy & Van Wagtendonk, 1966) at 15 C. TEM-4T, an emulsifying agent which is an ingredient of the axenic medium, was received as a gift from Dr Hachmeister (Hachmeister Co., Pittsburgh, Pennsylvania, 15230). Pellicles were prepared by removing the cells from the culture medium either by centrifugation or isolation with a micropipette under a dissecting microscope. The cells were then placed in deionized distilled water. The suspension was chilled on ice for 1030 min. The water was pipetted off the settled cells and cold 0-5 % Nonidet P 40 (a non-ionic detergent (O'brien, 1964a, b)) suspended in 12 % ethanol was added. The progress of pellicle release was observed under a dissecting microscope and pellicle pieces were placed in a drop of 12% ethanol on gelatinized slides. A drop of 4 % neutral formaldehyde was added to the samples. After fixation, coverslips were applied to flatten the pellicles and were subsequently removed by freezing in liquid nitrogen. Post-fixation was carried out in a 3 :1 mixture of absolute ethanol and glacial acetic acid; after this the slides were passed through absolute and 95 % ethanol, and finally into 75 % ethanol for storage. The slides were hydrated gradually prior to further processing in aqueous solutions. Methods of labelling cells In all experiments, methyl labelled ['H]thymidine, specific activity 11 Ci/mM (Schwarz BioResearch) was used as labelled precursor. Exogenously supplied label. (A method for labelling both nuclear and pellicular DNA.) Exogenously supplied tritiated thymidine was employed for (1) normal cells, (2) cells of 'Amac' clones in bacterized medium, as well as (3) normal cells in axenic medium; 10-50 /tCi/ml tritiated thymidine-were added to the culture medium, in either bacterized Cerophyl or axenic medium. The paramecia were picked up with a micropipette under a dissecting microscope and placed in the culture fluid containing the isotope. After the cell number had doubled, the cells were removed from the radioactive medium and washed through several changes of unlabelled fluid. In the experiments using 'Amac' clones, microdrops containing 20/iCi/ml of tritiated thymidine in bacterized Cerophyl were incubated with one paramecium per drop for 24 h. Those isolates with only one cell per drop at the end of this period were selected with a micropipette under a dissecting microscope, washed, and examined as described later. Labelled Escherichia coli. (A method for selective labelling of the macronucleus.) This method was used only for cells grown in bacterized Cerophyl. E. coli 15T (thymine requiring) was kindly supplied by J. Berger, Indiana University. Exponentially growing E. coli were grown in 300-500 /tCi/ml tritiated thymidine as described by Berger & Kimball (1964). Synchronized paramecia were transferred into the washed E. coli suspended in Cerophyl medium 3 h after the previous division. After exposure to the labelled bacteria the paramecia were washed through six i-ml changes of medium. Samples were taken for (a) whole cell preparations, (6) pellicles, and (c) subsequent division in unlabelled medium. After the desired number of fissions in unlabelled medium, whole cell samples and pellicles were prepared from the latter. Enzymic treatments Enzymic digestions were carried out over 3-h periods at 37 CC. Deoxyribonuclease (Worthington, electrophoretically purified), in 4 x io~' M MgSO4 and 001 M sodium acetate, and ribonuclease (Worthington) (...truncated)