Analysis of chromatin assembled in vivo using exogenous histones in Physarum polycephalum.

ABSTRACT

Histones are involved in the regulation of almost all events within the eukaryotic cell nucleus that utilize DNA as a substrate. We have developed a novel approach for examining the function of histone proteins and specific domains of these proteins in these various nuclear processes, and in particular assembly of chromatin throughout the cell cycle. This approach exploits several unique characteristics of the slime mold Physarum polycephalum, including the natural synchrony of all (approximately 10(8)) nuclei throughout the cell cycle and the ability of this organism to take up exogenous proteins. Here, culture techniques and biochemical procedures for the incorporation of exogenous core histones into Physarum chromatin in vivo are described. The procedures for subsequent verification of the assembly of exogenous proteins into bona fide nucleosomes are also described.