Alterations in nuclear anatomy by chemical modification of proteins in isolated rat liver nuclei.
Whole rat liver nuclei were treated with citraconic anhydride, a reagent specific for primary amines. Dramatic changes were observed in nuclear morphology and light scattering properties. An analysis for DNA and RNA content suggested that DNA was released from the nuclei with a short half-time, approximately 2-4s demonstrating a biphasic release profile. RNA was similarly released but with a monophasic profile. Analysis of SDS-PAGE gels of modified nuclei demonstrated a progressive enrichment of nuclear matrix (lamins) polypeptides with extent of modification. H1 histone was quantitatively lost as a function of modification reagent concentration, while approx. 50% of the nucleosomal histones cosedimented with DNA- and RNA-free nuclei. Modification in the presence of 2 mM EGTA released all the DNA and RNA [less than or equal to 1% remaining) while retaining structures characteristic of nuclear matrix, nucleoli, and ribonucleoprotein (predominantly hnRNA group A and B). These nucleic acid-deficient structures have been termed nuclear fossils to differentiate them from high salt detergent-prepared empty nuclear sacks, nuclear remnants, or nuclear scaffolds. Modification in the presence of 2% Triton X-100 results in structures similar to the nuclear fossils (EGTA treatment), but missing the double bilayer and a 51K polypeptide that is a major component of the other structures. The use of chemical modification on the nucleus provides an experimental approach for examining the role of ionic interactions in controlling nuclear structure. Citraconylation may thus serve two functions: (a) as a protein-specific perturbant of nuclei capable of simply and rapidly preparing a range of structural variants for the analysis of nuclear interactions; (b) offer a paradigm for control of nucleic acid-polypeptide interactions based on post-translational alterations in protein charge.
D000818:Animals; D002467:Cell Nucleus / Q000032:analysis / Q000187:drug effects / Q000648:ultrastructure*; D002949:Citraconic Anhydrides / Q000494:pharmacology*; D004247:DNA / Q000032:analysis*; D004533:Egtazic Acid / Q000494:pharmacology; D005663:Furans / Q000494:pharmacology*; D006657:Histones / Q000032:analysis; D008099:Liver / Q000648:ultrastructure; D008297:Male; D008854:Microscopy, Electron; D009698:Nucleoproteins / Q000032:analysis*; D017830:Octoxynol; D010658:Phenylglyoxal / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D012313:RNA / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D012261:Ribonucleoproteins / Q000032:analysis; D013387:Succinic Anhydrides / Q000494:pharmacology
D002949:Citraconic Anhydrides; D005663:Furans; D006657:Histones; D009698:Nucleoproteins; D012261:Ribonucleoproteins; D013387:Succinic Anhydrides; D011092:Polyethylene Glycols; D004533:Egtazic Acid; D012313:RNA; C031801:succinic anhydride; D017830:Octoxynol; D004247:DNA; D010658:Phenylglyoxal