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Partial Characterization of the Histones and Histone Acetylation in Cell Cultures¹

Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27706

In order to define the role of histone acetylation in cell cultures, a simple rapid method was developed to fractionate and quantitatively measure these proteins from milligram quantities of HeLa cells.

Histones were extracted from purified HeLa cell nuclei and resolved by electrophoresis in 0.1% ethylene diacrylate-15% acrylamide gels to fractionate 5 major histone components. The protein concentration in each histone band was measured quantitatively by densitometry of stained gels and radioactivity was determined by scintillation counting of sliced, base-hydrolyzed gels.

HeLa cell histones that were fractionated by differential extraction, precipitation, and Sephadex G-75 chromatography were found to be electrophoretically similar to and almost identical with calf thymus histones by amino acid analysis.

Cells grown in tritiated amino acids gave 4 radioactive peaks in acrylamide gels (the F2b and F2a2 were not resolved by this procedure). In contrast, cells pulsed with acetate-1-¹⁴C for either 30 or 45 min incorporated acetate into 3 peaks, which were identified by coelectrophoresis with amino acid-³H-labeled histone as the F3, the F2b + F2a2, and the F2a1. The fact that the acetate-¹⁴C-labeled histone fractions were consistently displaced toward the cathode in relation to amino acid-³H-labeled histone is tentatively interpreted as evidence for the loss of some of the effective positively charged -NH₃⁺ groups of the acetylated lysine residues.

¹ This work was supported in part by Grants GM 12805 from the USPHS, and ACS-P363 and IN-61H No. 10 from the American Cancer Society.

² Supported in part by a USPHS Predoctoral Traineeship GM 00233. This research was presented to the Graduate School of Duke University as partial fulfillment of the Ph.D. degree requirements. Present address: Department of Biochemistry, University of Texas M. D. Anderson Hospital at Houston, Houston, Texas 77025.

³ To whom reprint requests should be sent.

Received 4/ 1/69. Accepted 7/18/69.