Limitations of the poly(glutamic acid) reconstitution method in the reassembly of mono- and dinucleosomes.
Reconstitution of mononucleosomes and dinucleosomes at physiological ionic strength by means of poly(glutamic acid) is not efficient at physiological histone octamer:DNA ratios, unlike that with the salt dialysis method. The shorter the DNA is, the less transfer of octamers from poly(glutamic acid) to DNA occurs. By increasing the octamer:DNA ratio it is possible to involve all the DNA in the assembly, but for DNA longer than core particle length, nucleoprotein particles containing extra histones are concomitantly generated. Except for core particle and chromatosome lengths of DNA reassembled at 0.6:1 or 1:1 octamer:DNA ratio (and thus with low yield), reconstituted nucleoprotein particles proved to be different from native nucleosomes by their insolubility upon isolation. In the aggregates, DNA ends seemed to be sufficiently loose to allow exonuclease III digestion up to a certain limit. This resulted in patterns that for some cloned DNA fragments could give the impression, without knowledge of the above, of resulting from a unique octamer position. In view of the small range of length of DNA and the low yield of faithful reconstitution, the assembly method using poly(glutamic acid) is only of limited use in mono- or dinucleosome reconstitution experiments, at least in our hands.
D000818:Animals; D002645:Chickens; D004247:DNA / Q000378:metabolism*; D003852:Deoxyribonucleoproteins / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D046911:Macromolecular Substances; D009707:Nucleosomes / Q000187:drug effects / Q000648:ultrastructure*; D009994:Osmolar Concentration; D010455:Peptides / Q000494:pharmacology*; D011099:Polyglutamic Acid / Q000494:pharmacology*
D003852:Deoxyribonucleoproteins; D006657:Histones; D046911:Macromolecular Substances; D009707:Nucleosomes; D010455:Peptides; D011099:Polyglutamic Acid; D004247:DNA