Deposition of newly synthesized histones: misinterpretations due to cross-linking density-labeled proteins with Lomant's reagent.
We have reinvestigated studies using Lomant's reagent to cross-link newly synthesized density-labeled histones into octameric complexes to determine the nature of histone deposition. The analysis has additionally included procedures for reversal of the cross-link in order to analyze the individual histones in these complexes. These studies indicate that density-labeled, newly synthesized histones form hybrid octameric structures composed of both new and old histones. These studies also suggest that previous interpretations by other investigators for the production of homogeneous complexes (100% dense octamer containing 100% new histones) are misinterpretations due to the presence of non-histone protein that contaminates the preparation, even under conditions where much of this non-histone protein is removed by use of ion-exchange resins. These non-histone proteins can be fractionated on density gradients as un-cross-linked proteins with molecular weights that mimic those of cross-linked histone complexes.
D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006657:Histones / Q000096:biosynthesis* / Q000302:isolation & purification; D008970:Molecular Weight; D013388:Succinimides
D002843:Chromatin; D003432:Cross-Linking Reagents; D006657:Histones; D013388:Succinimides; C011240:dithiobis(succinimidylpropionate)