Joyce Mohberg

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.

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.

Nuclear DNA content and chromosome numbers in the myxomycete Physarum polycephalum.

Abstract An improved method is described for making chromosome spreads of the plasmodium of the myxomycete, Physarum polycephalum . It consists of isolating metaphase nuclei, spreading the chromosomes with hot lactic acid, and staining with acetic-orcein. Most sublines derived from the Backus Wis 1 sclerotium had about 1 pg of DNA per nucleus, and had nuclei with 50 and 75 chromosomes in both the growing and sporulating plasmodium. Mature spores contained 0.6 pg of DNA, and hatching amoebae had 20–25 chromosomes and 0.6 pg of DNA. Plasmodia of the homothallic Colonia strain had a nuclear DNA content of about 1 pg, and had 35–40 chromosomes during growth and sporulation. Polyploid plasmodial sublines were found which had 1.5 and 3 times the normal DNA content and chromosome number. The polyploid sublines had the same plasmodial protein:DNA and RNA:DNA ratios as normal cultures. DNA content of nuclei varied directly with nuclear surface area. Ploidy was determined by the parent amoebae and therefore can serve as a genetic marker. A simple technique is given for completing the life cycle of P. polycephalum axenically. Germinating spores are plated without bacteria on one-tenth strength semidefined plasmodial growth medium, containing 2% agar. Plasmodia are visible in 2–4 days.

Nuclear histones in Physarum polycephalum during growth and differentiation.

Abstract The electrophoretic composition of the nuclear histones of Physarum polycephalum has been determined at various times during the mitotic cycle of the plasmodium and at all stages of the life cycle. During the mitotic cycle there were no marked changes in the relative amounts of the major histone components, but there were changes in a triplet of minor bands, with one band decreasing and another increasing during the period from early prophase to telophase. Histone:DNA ratios remained at about 1, on a weight basis, throughout the mitotic cycle but were slightly higher at the time of mitosis. Histone synthesis could occur when DNA synthesis was blocked by 5-fluorodeoxyuridine, and histone:DNA ratios could be increased to 3 by prolonged treatment with the drug. The nuclei of starving and sporulating plasmodia, and of spores, amoebae, and spherules all contained the seven histone bands characteristic of the growing plasmodia, but the relative amounts of the bands varied. The histone:DNA ratio was about 1 in amoebae and 0.8 in starving and sporulating plasmodia.