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

K W Adolph,J P Phelps

doi:10.1016/s0021-9258(18)34246-7

Abstract

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.

Highlights

As partof a studyof the role of non-histoneproteins particles which maintain a high degree of DNA organization in chromosome structure, thseynthesis of non-histones [14,15,16]
Mosomes were characterized by two-dimensional understood [26], but the nature of the involvement of nongel electrophoresis andcompared to theproteins of histones is less clear
The pattern of non-histones is The experimentsdescribed inthis paper arceoncerned with notidentical with that ofinterphasechromatin,alinvestigating the cell cycle-dependent changeisn the synthesis though a number of major species may be shared by of chromatin-associated non-histones

Summary

CELL CYCLE VARIATIONS IN PROTEIN SYNTHESIS*

Chromatin Preparation-To measure the synthesis of chromatinassociated non-histonesduringthe cell cycle, portions of cultures synchronized with double blocks of thymidine or hydroxyurea were withdrawn at intervalsof 4 h following release from the CI/S boundary. The synthesis during the cell cycle of non-histoneproteins associatedwith interphasechromatin wasinvestigated by pulse-labelingsynchronizedsuspension cultures of HeLa cells with['"'Slmethionine for 1.0 h, isolating nuclei, and preparing chromatinby digestion withmicrococcal pH 7.4,5 mM MgCI?, 0.1 mM phenylmethylsulfonyl fluoride at 0 "C, and resuspended in the buffer at a cell concentration of ahout 10"/mI. The peak of chromatin was removed from such gradients and the associated non-histones characterized by SDS-polyacrylamide gel electrophoresis. Cell Cycle Variations in Non-histone Synthesis i ; 1 L i Ub. the patterns with fractionated chromatin are similar. Non-histone species are seen tboe associated with chromatin purified on sucrose density gradients. Cells at 16-17 h after GI/Shave progressed into G I of the nextcell cycle, and protein synthesins G I is seen to resemble thaitn early S phase

Densitometer scansof fluorograms of chromatin labeled a t

Findings

The synthesis of nuclear scaffold proteins during the cell