High-resolution fractionation of nucleosomes: minor particles, "whiskers," and separation of mononucleosomes containing and lacking A24 semihistone.

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Abstract
Staphylococcal nuclease digests of [human cervical carcinoma] HeLa chromatin, fractionated on low ionic strength nucleoprotein gels, were further analyzed by 2nd-dimension DNA and protein gel electrophoresis. In vivo radioactive labeling of chromatin components and the use of longer gels allowed a higher sensitivity and resolution than was previously reported for this approach. A number of nonhistone protein spots and about 20 DNA spots can be detected in the mononucleosomal region of the 2nd-dimension gel. In particular, there are 3 DNA spots identical in DNA size that correspond to 3 discrete kinds of core mononucleosomes resolved on the 1st-dimension nucleoprotein gel. Analysis of protein composition shows that the most rapidly migrating particle contains all 4 core histones but no A24 semihistone (A24 is a covalent conjugate of histone H2A and a specific nonhistone protein, ubiquitin), whereas the other 2 core mononucleosomes contain A24 semihistone. The A24-lacking core mononucleosomes can now be quantitatively separated from those containing A24; this makes it possible to directly address the question of whether A24 is associated with nucleosomes containing a specific subset of DNA sequences. Additional features of 2-dimensional nucleoprotein-DNA patterns are whiskers, which run slower than core mononucleosomes in the nucleoprotein dimension and both faster and slower than core-length DNA in the DNA dimension. In more extensive digests, secondary whiskers are observed, which run faster than core mononucleosomes in both dimensions and appear to coincide with previously described subnucleosomal particles SN7 and SN8. Possible mechanisms of whisker formation are discussed.