Role of non-histones in chromosome structure. Cell cycle variations in protein synthesis.

As part of a study of the role of non-histone proteins in chromosome structure, the synthesis of non-histones associated with interphase chromatin was investigated. Synchronized suspension cultures of HeLa cells were pulse-labeled with [35S]methionine, and chromatin was prepared by mild micrococcal nuclease digestion. Two-dimensional polyacrylamide gel electrophoresis, in addition to one-dimensional electrophoresis, was used to resolve the patterns of incorporation of radioactive label. Significant variations in non-histone synthesis were seen during the cell cycle. A strong correlation was not found between DNA synthesis in mid-S phase and variations in non-histone synthesis. The non-histone proteins of purified metaphase chromosomes were also characterized by two-dimensional gel electrophoresis and compared to the proteins of interphase chromatin. The pattern of non-histones is not identical with that of interphase chromatin, although a number of major species may be shared by interphase chromatin and metaphase chromosomes. The HeLa nuclear scaffold, the framework that maintains the overall morphology of the interphase nucleus, shows relatively few proteins on two-dimensional gels. The synthesis of nuclear scaffold proteins was quantitated by excising each of 19 proteins from two-dimensional gels and determining the incorporated radioactivity by scintillation counting. Substantial variations in protein synthesis were found, with several species showing changes of about 2-fold in the percentage of incorporation.