Probing with DNase I of nucleosomal core particles assembled in vitro in the presence of polyglutamic acid

Limitations of the poly(glutamic acid) reconstitution method in the reassembly of mono- and dinucleosomes

“…We thus confirm the results of Retief et al (1984) obtained from reconstitutions on long DNA, who showed that the poly(glutamic acid) aiding the assembly is only associated with the octamer in a transient fashion.…”

“…Poly(glutamic acid) also permits very fast reconstitution. After 30 min, the association maximum is attained (Retief et al, 1984). Yet, in spite of its advantages, our studies show that this method may not be well suited for reconstituting mono-and dinucleosomal length of DNA.…”

A novel nucleosome assembly procedure (with a little help from pectin)

“…We have chosen to use this last method for the preparation of hybrid core particles since, in cases where the histone of choice cannot be directly reconstituted into histone octamers, the polyglutamic acid overcomes the barriers of histone association and allows the assembly of polycores on the DNA [1,3]. Two problems exist with the current methodology -the cost of the polyglutamic acid and the fact that the polyglutamic acid cannot be completely removed from the assembled core particle preparation even by sucrose gradient centrifugation [7]. We have therefore explored the possibility that the acidic polysaccharide pectin (a-1 ,Cpolygalacturonic acid) could substitute for polyglutamic acid.…”

Nucleosome cores assembled in vitro occupy two preferred frames flanking the histone H1 gene from Psammechinus miliaris

“…Histone octamers were prepared and stored at -20 °C in 50% (w/v) glycerol, 1 M NaCl, and 10 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 8.0 (Greyling et al, 1983). Nucleosome core assembly reactions were carried out by mixing 10 fig of poly(glutamic acid), from a stock solution of 10 mg/mL in 10 mM phosphate buffer, pH 6.9, to 5.4 fig of histone octamers (Retief et al, 1984). The NaCl concentration of the mixture was then adjusted to 125 mM NaCl from a 0.5 M NaCl stock solution.…”

“…These plasmids had been developed to facilitate chemical DNA sequencing, but we have found them to be particularly suitable for studying DNA-protein interactions at high resolution. We have used this technique in conjunction with a poly(glutamic acid)-assisted assembly reaction (Stein et al, 1979;Retief et al, 1984) to faithfully assemble nucleosome cores on such strand specifically end labeled fragments. The analysis of the protection pattern of the core-associated DNA, by DNase I digestions followed by denaturing DNA gel electrophoresis, can then be used to accurately map the positions of the assembled cores on unique DNA sequences.…”