A comprehensive study on the isolation and characterization of the HeLa S3 nuclear matrix.
Different agents have been employed to extract the histones and other soluble components from isolated HeLa S3 nuclei during nuclear matrix isolation. We report that 0.2M (NH4)2SO4 is a milder extracting agent than NaCl and LIS (lithium 3,5-diiodosalicylate), on the basis of the apparent preservation of the elaborate fibrogranular network and the residual nucleolus that resemble the in situ structures in whole cells and nuclei, minimal aggregation, and sufficient solubilization of DNA and histones. The importance of intermolecular disulfide bonds, RNA and 37 degrees C stabilization on the structural integrity of the nuclear matrix was examined in detail using sulfydryl alkylating, reducing and oxidizing agents, and RNase A. The data suggest that any disulfides formed during the isolation are not essential for maintaining the structural integrity of the in vitro matrix. However, structural integrity of the matrix is dependent upon RNA and to some degree on disulfides that presumably existed in situ. Sodium tetrathionate and 37 degrees C stabilization of isolated nuclei resulted in nuclear matrices containing an approximately twofold greater amount of protein, RNA and DNA than control preparations. The 37 degrees C incubation, unlike the sodium tetrathionate stabilization, does not appear to induce intermolecular disulfide bond formation. Neither stabilizations resulted in significant differences of the major matrix polypeptide pattern on two-dimensional (2-D) gels stained with Coomassie Blue as compared to that of unstabilized matrix. The major nuclear matrix proteins, other than the lamins, did not react to the Pruss murine monoclonal antibody (IFA) that recognizes all known intermediate filament proteins, suggesting that the internal matrix proteins are not related to the lamins in intermediate filament-like quality.
Belgrader
P
P
Department of Biological Sciences, State University of New York, Buffalo 14260.
Siegel
A J
AJ
Berezney
R
R
D015153:Blotting, Western; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D004247:DNA / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007381:Intermediate Filament Proteins / Q000737:chemistry; D007463:Iodobenzoates; D034882:Lamins; D008854:Microscopy, Electron; D015530:Nuclear Matrix / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D009687:Nuclear Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D009697:Nucleolus Organizer Region / Q000378:metabolism; D012313:RNA / Q000737:chemistry; D012459:Salicylates / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D012995:Solubility; D013696:Temperature; D013776:Tetrathionic Acid / Q000737:chemistry
D004220:Disulfides; D006657:Histones; D007381:Intermediate Filament Proteins; D007463:Iodobenzoates; D034882:Lamins; D009687:Nuclear Proteins; D012459:Salicylates; C020891:3,5-diiodosalicylic acid; D012965:Sodium Chloride; D012313:RNA; D013776:Tetrathionic Acid; D004247:DNA