Free ribonucleotides in plasmodia of Physarum polycephalum a t different stages of the synchronous mitotic cycle were determined by anion exchange chromatography. The nucleotide pattern of this myxomycete resembled that obtained from rat liver, The contents of total adenine nucleotides based on protein content were almost identical in both tissues, whereas higher contents of guanine-, uracil-, and cytosine-nucleotides were found in the myxomycete. During the mitotic cycle, the levels of nucleoside triphosphates increased just prior to mitosis and decreased in the following reconstruction period. It is suggested that the accumulation of triphosphates results from a diminished demand for RNA precursors due to decreasing RNA synthesis during late interphase.Physarum polycephulum, a myxomycete, forms giant multinuclear plasmodia, which exhibit naturally synchronous mitosis every 8 to I0 hours [l-31. The following communication reports the pattern of free ribonucleotides in acid soluble extracts of this slime mold, measured by anion exchange chromatography with an automated analyzer [4,5]. Significant changes in the ribonucleoside triphosphate levels were observed in relation to the mitotic cycle.
METHODSDisc-shaped macroplasmodia of Physarunz polycephalum with a diameter of4 to 6 cm were cultivated in Petri dishes on sterile millipore filter as described previously [3]. At various stages of the synchronous mitotic cycle, small pieces (1 to 2 cm2, 20 to 40 mg wet weight) were cut from a plasmodium with a scalpel, carefully removed from the supporting filter with a stainless steel spatula and quickly frozen by pressing the spatula against an aluminium block precooled in liquid nitrogen. The frozen samples were transferred to a closed coolbox (-20"), weighed, mixed with 10 volumes 7.5'3/, perchloric acid in 40°/, ethanol, and homogenized by sonification. After centrifugation, the supernatant wag adjusted to pH3-4 with KOH. Aliquots, equivalent to 10 to 20 mg of the original sample, were chromatographed according to Schnitger et al. [4-61. This automated method permits the quantitative analysis of nanomolar amounts of nucleotides. The apparatus consists of a short "storage part" and the main "separation part", both filled with a strong basic anion exchange resin (Dowex 1 x 8, Fluke). The acid soluble extract is adsorbed onto the storage part of the column and subsequently eluted through the separation part with increasing concentrations (concave gradient) of a formic acid-ammonium formatc mixture. The eluent passes a quartz cuvette in anEppendorf spectrophotometer, which alternately monitors the absorbancies at 265mp and 280mp. I n the chromatogram, individual nucleotides were identified by the relative peak position and by the ratio of the absorbance a t 265 to that at 280 mp. The quantities of the different nucleotides were calculated on the basis of their known extinction coefficients [6,7].Protein was measured by the Biuret Method [8] using bovine albumin as a standard. Mitotic stages of nuclei were observed in eth...
