Asexual conversion of amoebae to plasmodia was studied in the Colonia isolate of the myxomycete, Physarum polycephalum. When a culture of Colonia amoebae is grown on a bacterial lawn, a period of amoebic growth precedes the appearance of cells committed to the plasmodial state. The onset of plasmodium production appears to be related to amoebic nutrition since cultures supplied with fewer bacteria display earlier differentiation. For a period of time after differentiation is initiated, conversion of amoebae to plasmodia is rapid and proceeds as an exponential function of time. A filter-transmissible substance, apparently released by differentiating cells, is implicated in the control of this rapid conversion. The myxomycete Physarum polycephalum displays two strikingly different vegetative forms: microscopic, uninucleate, colorless amoebae and macroscopic, multinucleate, yellow plasmodia (1, 2). Amoebae of the Colonia isolate, which carry the allele mth at the mating type locus, readily undergo an asexual conversion to the plasmodial state. Because the change occurs without genetic alteration (3) and results in major, stable phenotypic alterations, the material provides a model system for studies on the control of cell differentiation. Mutants affecting the differentiation can be isolated (refs. 4 and 5; L. Davidow and C. E. Holt, manuscript in preparation; P. N. Adler, manuscript in preparation), and the present work provides a beginning for physiological and biochemical studies on the process of commitment to the plasmodial state.We report here the development of a technique which permits a quantitative analysis of the time course and extent of differentiation in a Colonia culture. With this technique, we have demonstrated that a differentiating culture of Colonia cells can induce early differentiation in a neighboring culture separated by filters which prevent direct cell contact between the two populations. The results favor the conclusion that differentiating cells elaborate a diffusible inducer of differentiation. MATERIALS AND METHODSMedia. Dilute plasmodial rich medium (dPRM) agar and liver infusion agar were made as described previously (4, 6). Agar containing dPRM adjusted to pH7 (dPRM7) rather than pH 4.6 was also used. Buffer-streptomycin agar was made by adding 0.25 g streptomycin sulfate (Sigma Chemical Co.) and 10 ml of 1 M citrate buffer (pH 5) to 1 liter of 1.5% agar. Final concentrations were 250,ug/ml and 0.01 M, respectively.Preparation of Amoebae. Plasmodia-free amoebae for use in starting kinetics experiments were prepared by growing the amoebae on agar plates at 300 and harvesting the amoebae prior to the onset of plasmodium formation. For experiments with live bacteria, the amoebae were grown on lawns of live Escherichia coli on liver infusion agar. For experiments with formalin-killed bacteria (7), the amoebae were subcultured serially on formalin-killed E. coli covered buffer-streptomycin plates to ensure elimination of live bacteria. Kinetic Experiments. At time zero, replica...

DNA metabolism in the slime mold Physarum polycephalum was studied by centrifugation in CsCl of lysates of cultures labeled with radioactive thymidine at various times in the cell cycle. During the G 2 (premitotic) phase of the cell cycle, two components of the DNA are labeled. One component is lighter (buoyant density 1.686 g/cc) than the mean of the principal DNA (1.700 g/cc), and one is heavier (approximately 1.706 g/cc). The labeled light DNA was identified chemically by its denaturability, its susceptibility to DNase, and the recovery of its radioactivity in thymine. Cell fractionation studies showed that the heavy and the principal DNA components are located in the nucleus and that the light DNA is in the cytoplasm. The light DNA comprises approximately 10 % of the DNA. About 1/-1 of the light DNA is synthesized during the S period, and the remainder is synthesized throughout G 2 (there is no G 1 in Physarum). The light DNA is metabolically stable. A low, variable level of incorporation of radioactive thymidine into the principal, nuclear DNA component was observed during G 2 .The rates of DNA, RNA, and protein synthesis vary throughout the mitotic cycle in eucaryotic cells. Nuclear DNA synthesis is limited to a portion of the cycle, the "S phase" (Howard and Pelc, 1953), and both RNA and protein synthesis show minimal rates at the time of mitosis (Robbins and Scharff, 1966). A second minimum later in the cell cycle has been reported for Physarum (Mittermayer et al., 1964(Mittermayer et al., , 1966. Synthesis of certain enzymes has been shown to occur at particular times in the cycle (Gorman et al., 1964), and recent evidence confirms that the ratio of the rate of histone synthesis to the rate of DNA synthesis is remarkably constant throughout the cell cycle (Borun et al., 1967).The discovery of cytoplasmic DNA has raised the question as to the extent to which the synthesis and function of this DNA are coupled to the events of the cell cycle. While information on this question can be provided by radioautographic studies, experiments with synchronously dividing systems have several advantages, one of them being the opportunity for a more complete characterization of macromolecules labeled by pulses of radioactivity.We report here on the properties and metabolism of a cytoplasmic DNA from Physarum polycephalum. This acellular slime mold displays a natural precise synchrony in the nuclear division of each plasmodium (Howard, 1932). Mitosis is followed immediately by anS period of about 3 hr, and the succeeding G 2 period lasts 4-16 hr Nygaard et al., 1960). Methods have been developed by Rusch and collaborators

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Mating inPhysarum polycephalum involves the fusion of two haploid amoebae and the differentiation of the resulting diploid zygote into a multinucleate plasmodium. Mating proceeds optimally with amoebae growing on an agar medium at pH 5.0. At pH 6.2, the amoebae still grow normally, but mating is completely blocked. The barrier at pH 6.2 is not in the differentiation step, since preformed diploids readily convert to plasmodia at this pH. The barrier can be overcome by raising the ionic strength of the agar medium; the effect, moreover, is not ion-specific. We have discovered a genetic locus,imz (ionicmodulation of zygote formation), that affects the upper pH limit for mating; the respective limits associated with the two known alleles,imz-1 andimz-2, are pH 5.6 and pH 6.0 at low ionic strength. Animz-1×imz-2 mating displays the pH 6.0 limit;imz-2 is therefore "dominant". We suggest that this new gene affects a cell component that is exposed to the exterior of the amoeba and is involved in the fusion step of mating.