Preparation of membrane-free chromatin bodies from avian erythroid cells and analysis of chromatin acidic proteins.

Abstract

A primary objective, realized in this study, was the preparation from avian erythroid cells of chromatin free of contaminating membrane, as a prerequisiste to the study of chromatin acidic proteins from cells throughout the maturation pathway. Cells are lysed in saponin (S), washed in Nonidet-P40 (N), and plasma membrane removed by high-speed rotating knives (K). Purified SNK nuclear bodies are recovered free of membrane after centrifugation through 2.35 M sucrose. The chromatin acidic proteins from such preparations of the three major avian erythroid cell types were studied. Reticulocyte SNK chromatin was compared with reticulocyte chromatin derived from saponin lysis of cells and subsequent dispersion in EDTA solutions (Harlow et al. (1972), Cell Differ. 2, 341). The dispersed preparation has a lower acidic protein/DNA ratio, but the pattern of these proteins is more complex, presumably due to the contaminating membrane. In examining SNK acidic proteins throughout the maturation pathway it is clear that there are quantitative and qualitative differences. In the dividing erythroblast, the pattern of proteins is complex and the amount relative to DNA is 1.25:1.0. For nondividing, but transcriptionally active reticulocytes and also for transcriptionally inactive erythrocytes, the pattern is very much simpler, being dominated by three bands visible on sodium dodecyl sulfate polyacrylamide gels. The ratios of chromatin acidic proteins in these preparations relative to DNA are 0.69:1.0 and 0.36:1.0, respectively. These results, obtained with purified populations of cells from a single cell line, indicate a strong positive correlation between the transcriptional activity of the cell and both the amount and complexity of non-histone proteins associated with chromatin. The correlation does not indicate whether the proteins are the cause or result of increased transcription.