The preparation and preliminary characterisation of chromatin from the slime mould Physarum polycephalum.

Abstract

A method is described for the preparation of nuclei from plasmodia of the slime mould, Physarum polycephalum. The nuclei can be gently lysed by suspending them in 10 mM EDTA. A method is described for the preparation of chromatin in high yield from nuclei lysed in EDTA. Between 60 and 70% of the total nuclear DNA may be recovered in the chromatin fraction which has the following chemical composition, DNA : RNA : protein = 1.0:1.0:4.0. The acid-soluble protein fraction was present in equal weight proportion to the DNA and consisted almost entirely of the five major Physarum histones resolvable by polyacrylamide-gel electrophoresis in urea. The molecular weights of these histones, determined by polyacrylamide-gel electrophoresis in sodium dodecylsulphate, were as follows: P1, 24500; P3, 14000; lysine-rich P4, 14500; arginine-rich P4, 14500; P5, 13000; P6, 10500. The lysine-rich Physarum histone, P1, differed significantly in molecular weight from the corresponding calf thymus histone, f1 (mol. wt 21000). The acid-insoluble protein fraction from the chromatin was resolved into over 30 different fractions by polyacrylamide-gel electrophoresis in dodecylsulphate. The majority of the proteins had molecular weights between 14000 and 60000. No significant differences were observed between the polyacrylamide-gel patterns of chromatin proteins isolated from chromatin prepared from the synchronised nuclei of plamodia in the middle of S and the middle of the G2 phases of the cell cycle.