Harold P. Rusch

Isolation and DNA content of nuclei of Physarum polycephalum

Abstract Methods have been developed for isolation of nuclei from Physarum polycephalum at various stages of the life cycle and mitotic figures and nucleoli from the plasmodial stage. Organelles of growing plasmodia and myxamoebae were isolated by Waring blending in 0.25 M sucrose, 0.1% Triton X-100, 0.01 M CaCl2 (0.001 M for nucleoli) and 0.01 M Tris buffer, pH 7.2, and centrifuging through 1 M sucrose. The same procedure was used for starving cultures, except that before homogenization starving and sporulating plasmodia were washed with 0.01 M EDTA in 0.25 M sucrose, and spherules were washed with EDTA-sucrose and broken in the French pressure cell. The DNA, RNA, protein and polysaccharide content of nuclei was determined at all stages of the life cycle. Nuclei of growing, starving and sporulating plasmodia contained 1 to 1.2 pg of DNA; viable spores, logarithmically growing amoebae and spherules contained 0.6 pg; and encysted amoebae contained 0.3 pg. It was concluded that spores and growing amoebae were in haploid, G2 phase; spherules in diploid, G1 phase; and encysted amoebae in haploid, G1 phase.

Morphological observations on growth and differentiation of Physarum polycephalum grown in pure culture

Abstract The morphological features of the vegetative and sporulative stages in the life cycle of the myxomycete Physarum polycephalum under conditions of controlled culture are described. The vegetative stage is characterized by a large multinucleate plasmodium which behaves like a single cell. When grown on a surface under optimal conditions, the plasmodium is a flat, compact, yellow, disk-shaped meshwork of tightly interwoven strands. The nuclei of such a plasmodium undergo synchronous mitoses even when the organism is 5 cm in diameter. In agitated submersed culture, the plasmodium is fragmented into microplasmodia, which in turn will coalesce when placed in intimate contact with one another on a surface. Although the microplasmodia are not in mitotic phase with one another, complete mitotic synchrony occurs in the plasmodia obtained following their coalescence. Microplasmodia in old submersed cultures form mono- and multinucleate spherules which probably are identical with the subunits of a sclerotium. While the well-nourished organism remains sedentary, a starved plasmodium migrates rapidly over the surface. During prolonged starvation many nuclei degenerate, but the remaining ones continue to undergo mitoses although at longer intervals. Sporulation is induced by exposing a partially starved plasmodium to light. During this process a rearrangement of protoplasm occurs in the absence of growth.

Nucleic acid metabolism in a slime mold with synchronous mitosis.

Abstract In the synchronously dividing slime mold, Physarum polycephalum , the synthesis of DNA as shown by incorporation of 14 C fron [6- 14 C]orotic acid into DNA-thymine, occured immediately after the nuclear division and lasted for 1–2 h. During the remainder of the interphase, which might last from 12–20 h, there was no significant labeling of the DNA. The incorporation of label into RNA at any time of the cell cycle suggested that RNA synthesis was continuous, although there was some indication that this process might be retarded at the time of division and DNA synthesis. The relative timing of DNA sythesis and nuclear division would tend to rule out the synthesis of DNA as a “trigger” for mitosis. Preliminary experiments indicated that the mold was capable of converting orotic acid to thymine nucleotides throughout the interphase; this suggests that the control of DNA synthesis must occur at some point very close to the final polymerization of the molecule.

Contractile proteins. Major components of nuclear and chromosome non-histone proteins.

Two of the major non-histone proteins from Physarum polycephalum have been isolated under nondenaturing conditions and identified as actin and myosin. A third protein has been purified from crude nuclear actomyosin and from residual nonhistone fractions and found to bind actomyosin in the presence of Mg2+. In Physarum these proteins are not components of the nuclear membrane. Based on sodium dodecylsulfate-polyacrylamide gel electrophoresis, similar proteins are also present in nuclei of HeLa cells and mouse embryo fibroblasts. Isolated metaphase chromosomes from Physarum show a several-fold enrichment in myosin and an altered ratio of actin to the Mg2+-dependent actomyosin binding protein as compared to interphase nuclei. When non-proliferative states are induced in any of these cells, the Mg2+-dependent actomyosin binding protein decreases while actin increases several fold in intranuclear concentration; concomitantly, there is a generalized condensation and inactivation of chromatin. Experiments with added purified radioactive nuclear actomyosin; comparative studies on nuclear protein during stepwise nuclear purification; and studies on isolated metaphase chromosomes indicate that these proteins exist in nuclei in vivo. These observations suggest that contractile proteins may function in the structural interconversions of chromatin and in the regulation of cell proliferation;

Localization of nucleolar and chromatin residual acidic protein changes during differentiation in Physarum polycephalum

The total nuclear proteins (30% by nuclear dry weight) of the plasmodial slime mould Physarum polycephalum have been quantitatively separated into four distinct solubility classes possessing characteristic molecular weight ranges and electrophoretic profiles. The residual acidic proteins of nuclei and nucleoli have been extracted during synchronous growth and during two forms of differentiation and separated into two fractions with buffer-saturated phenol, pH 8.2, and hot sodium dodecyl sulfate (SDS). Quantitative comparisons of electrophoretically separated proteins of both residual fractions from nucleoli and nuclei reveal numerous changes in the phenol-soluble phosphoproteins during both forms of starvation-induced differentiation, and most of the changes occur in the chromatin-associated proteins ranging in molecular weight from 32,000 to 160,000. Reversion of the changes induced by starvation is complete within 8–10 hr after refeeding and precedes the first post-feeding mitotic division by 2 hr. Only two nucleolar proteins with molecular weights of 37,000 and 41,000 are extractable with phenol and are unchanged during differentiation. Five additional residual nucleolar proteins remaining after phenol extraction can be extracted with hot SDS and one of these proteins with a molecular weight of 86,500 disappears during differentiation. Incorporation, studies with 14 C labeled amino acids demonstrate that the new proteins which appear during differentiation are newly synthesized, that proteins which disappear during differentiation incorporate no label during development of the new cell states, and that there are two periods (mid S and late G 2 ) of increased isotope incorporation during active growth. The residual nucleolar proteins as compared to the residual nuclear proteins are high in serine, glycine, and aspartic acid but low in valine and isoleucine.

The effect of actinomycin D on the timing of mitosis in Physarum polycephalum

Abstract The effect of actinomycin D on synchronous mitoses of Physarum polycephalum was investigated. Continuous exposure to 150 to 250 μg/ml of antibiotic never prevented the first, but only the second mitosis. The first mitosis was delayed only when the antibiotic was present for more than one half interphase. With 150 μg/ml of antibiotic, the second mitosis was prevented if the inhibitor was present for more than one and one half interphases, but occurred following a delay if the inhibitor was present for less than one and one half interphases. If the onset of mitosis was delayed, the duration of metaphase was also prolonged for several hours. Addition of actinomycin D for periods of 2 hr had much less effect on the plasmodium; the addition caused a slight delay in the onset of the next mitosis, whereas the subsequent interphase was of normal length. Approximately one hour following the addition of actinomycin D to the medium, the chromatin moved from the periphery of the nucleus towards the nucleolus; these changes were reversed about an hour following the removal of the antibiotic. These results indicate that RNAs required for mitosis are formed one to one and one-half cycles prior to that mitosis.

Isolation of the nuclear histones from the myxomycete, Physarum polycephalum

Abstract A method has been developed for the extraction of nuclear histones with 1 m CaCl 2 . The new procedure gave a much higher yield of histone from nuclei of Physarum polycephalum than could be obtained with the conventional mineral acid and salt extractions, and the histone preparation had a more reproducible electrophoretic pattern. Calcium chloride gave the same yields and electrophoretic patterns as mineral acids and NaCl when used with calf thymus and chick erythrocyte nuclei. Physarum nuclei contained equal amounts of histone and DNA, on a weight basis. The histone was fractionated by electrophoresis at pH 4.3 in polyacrylamide into seven characteristic bands—three large bands and one small band with higher mobility, and three smaller bands with lower mobility. The electrophoretic pattern was very similar to that of calf thymus histone and in coelectrophoresis five bands moved with thymus histones. Two of the major bands migrated with lysine-rich and arginine-rich thymus histones. The largest Physarum fraction migrated slightly slower than moderately lysine-rich histone. Physarum histone also contained about 10% of a lysine-rich band not occurring in thymus. Dye-binding capacities of the Physarum histone bands have been determined, and the arginine-rich bands were found to bind up to twice as much amido black 10B as the lysine-rich bands. The amino acid compositions of three lysine-rich components have been determined. All had compositions distinct from thymus fractions, but one, the largest band in Physarum histone, resembled moderately lysine-rich histone; and another, the band unique to Physarum, resembled very lysine-rich histone.

RNA synthesis in the mitotic cycle of physarum polycephalum

Abstract The rate of uptake of [ 3 H]uridine into RNA has been studied in synchronous cultures of Physarum polycephalum . RNA synthesis was reduced at mitosis and again in mid-interphase, and sharply increased following each of these periods. The maximum increase early in interphase coincided with the time of nucleolar reconstruction and DNA synthesis, while the second maximum occured at the time of nucleolar swelling. Such swelling is the first cytological indication of the approaching mitosis. Pretreatment with actinomycin D reduced the postmitotic synthesis one half and completely eliminated premitotic synthesis. These results suggest a different physical or chemical nature of some of the RNAs made during the two periods of synthesis.

The effect of 5-fluoro-2′-deoxyuridine on synchronous mitosis in Physarum polycephalum☆

Abstract FUDR inhibits synthesis of DNA and RNA in the plasmodia of P. polycephalum . These effects are reversible with exogenous Tdr and Ur respectively. Contact with FUDR plus Ur before the end of the S period delays the onset of the next mitosis. The length of the delay corresponds to the length of the inhibition of DNA synthesis, suggesting that in normal and inhibited cultures a minimum G 2 period of 7–8 hr precedes mitosis. It appears that DNA synthesis functions as a rate-limiting step in the preparation for mitosis.

Nuclear Acidic Protein Changes during Differentiation in Physarum polycephalum

A class of acidic nuclear phosphoproteins has been isolated throughout the mitotic cycle and at two points during differentiation in the slime mold Physarum polycephalum. The electrophoretic profiles of these proteins are reproducible and unchanging throughout the mitotic cycle, but reproducible changes occur during differentiation. The proteins are rapidly synthesized after mitosis, and their molecular weights range from 34,000 to 88,000. The proteins rapidly incorporate [32P]orthophosphate, and the content of alkali-labile phosphate increases 20 percent during the period after DNA synthesis. The proteins comprise 6.5 percent by dry weight of nuclear material while the DNA comprises about 5.5 percent. These acidic nuclear proteins may have a role in control of gene activity.