RECONSTITUTION OF SHORT-SPACED CHROMATIN FROM THE HISTONE OCTAMER AND EITHER HMG-14,17 OR HISTONE-H1

Two new chromatin-assembly reactions are described. The first involves the addition of phosphorylated HMG-14, 17 to the histone octamer plus DNA in high concentrations of salt and yields a repeating particle size or spacing of about 165 base-pairs. The second involves the addition of histone H1 to the acetylated histone octamer plus poly(glutamate) in low concentrations of salt, followed by the addition of DNA; and it yields a spacing of about 170 base-pairs. Plots of band size versus band number in gels, often used to determine nucleosome repeat-length, yield slopes of 138 base-pairs for the histone octamer alone, or 155 base-pairs with HMG-14, 17 or 160 base-pairs with histone H1, and intercepts of 10, 25 and 20 base-pairs, respectively, in the three cases. Attempts were made to combine the spacing activities of HMG-14, 17 and histone H1 within a single assembly reaction, to provide an even longer spacing of about 190 base-pairs (as observed in cell extracts to which H1 has been added), but our present methods did not allow this. The two assembly reactions described here will be of use for structural studies of chromatin having defined length and sequence, and potentially of practical use for the regular, ordered condensation of very long DNA. © 1993 Academic Press Limited.