GEL-ELECTROPHORETIC ANALYSIS OF NUCLEAR MATRIX FRACTIONS-ISOLATED FROM DIFFERENT HUMAN CELL-LINES

The nuclear matrix is operationally defined as the structure that rema ins after nuclei are extracted with nonionic detergent and with high s alt and are digested with nucleases. Thus the nuclear matrix protein c omposition is critically dependent on the isolation conditions. We hav e compared nuclear matrices isolated from human cell lines by two diff erent methods. First, isolated nuclei were extracted as above to obtai n a matrix fraction. This method showed a substantial contamination by cytoplasmic intermediate filaments but immunization of mice resulted in antibodies recognizing nuclei and the mitotic spindle apparatus. Se cond, a nuclear matrix fraction was made by extracting whole cells as above and dissolving the residue in urea and dialysing against an asse mbly buffer to precipitate intermediate filament proteins (Fey, E. G. and Penman, S., Proc, Natl. Acad. Sci. USA 1988, 85, 121-125). Such fr actions showed complex protein patterns in silver-stained two-dimensio nal gels for four cell lines: HeLa, MCF-7, SW13 and the U333CG/343MG g lioma line. While some proteins in the nuclear matrix fraction were co mmon to all cell lines, others appeared cell-line specific. Two-dimens ional gels and the immunoresponse in mice again showed contamination o f these preparations with cytoplasmic proteins. These results clearly show the difficulties associated with protein chemical analysis of nuc lear matrices: the preparations have substantial cytoplasmic contamina tion, the polypeptide composition is extremely complex and the yield o f individual polypeptides is low. Thus, without further experiments on e cannot say which proteins are true nuclear matrix components. In add ition, our results underline the value of dissecting the nuclear matri x by analyzing its individual components with monoclonal antibodies, a nd we show examples of the nuclear staining patterns of three differen t classes of monoclonal antibodies isolated by this approach.