A chromosomal phosphoprotein is preferentially released by mild micrococcal-nuclease digestion

Abstract

[32P]Pi was administered to rats (5mCi/rat) 2h before the isolation of liver nuclei. The isolated nuclei were subjected to mild micrococcal-nuclease digestion for 2.5, 5 and 10 min at 37 degrees C, and the mononucleosomal fraction was subsequently isolated by sucrose-density-gradient centrifugation. The specific radioactivity of 32P-labelled mononucleosomal fractions decreased with increased digestion times. A phosphorylated chromosomal protein, B2 (Mr 68000, pI6.5-8.2), was demonstrated immunologically in the mononucleosomal fraction by using an antibody specific to this electrophoretically purified phosphoprotein. The incorporation of 32P into this phosphoprotein, previously shown to be mainly through covalent linkage, was revealed by antibody precipitation followed by gel electrophoresis. The rate of release of acid-soluble nucleotides by micrococcal-nuclease digestion of liver nuclei from partially hepatectomized rats 16 h after operation was strikingly higher than that for sham-operated controls. After partial hepatectomy, an increase in 32P incorporation into phosphoprotein in the monomer fractions specifically precipitated by this antibody was also found. This suggests that the phosphorylated non-histone chromatin protein B2 is preferentially associated with the transcriptionally active chromatin.

Monoclonal antibodies to a phosphoprotein from chromatin of rat liver

“…In a previous study we described the production of a polyclonal antibody to a rat liver nuclear phosphoprotein (B2: Mr 68000, pI6.5-8.2) (Zhao & Liew, 1982). This non-histone chromosomal structure and subsequently demonstrated to be preferentially associated with actively transcribed nucleosomes (Liew & Chan, 1976;Chan & Liew, 1977, 1979Liew et al, 1984a). In the present report we carried this immunological approach one step further with the production of several monoclonal antibodies to this phosphoprotein.…”

“…The relationship between this protein and the phosphoprotein B2 has yet to be determined. Our previous work using regenerating liver as a model system (Liew et al, 1984a) showed that the phosphoprotein B2 showed a maximum phosphorylation at 16 h post-hepatectomy, approximately the same time that DNA synthesis was initiated (Mayfield & Bonner, 1972;Liew & Gornall, 1975). The significant change in the phosphorylation of phosphoprotein B2 following mild micrococcal nuclease digest (5-10% of DNA rendered soluble) (Liew et al, 1984a) suggest that determination of its level in the nucleus may be pertinent in determining its role in actively transcribed chromatin.…”

“…It is very likely that much of the phosphoprotein B2 is highly complexed with the nuclear structure in such a way that many of its antigenic determinants (epitopes) are in fact masked so that these monoclonal antibodies are unable to recognize and bind to them. However we have already provided evidence that the phosphoprotein B2 is preferentially released following mild micrococcal nuclease digestion (Liew et al, 1984a) and it may be that when this phosphoprotein B2 location in the more accessible regions of the genome is released by micrococcal nuclease digestion that a more accurate quantification can be achieved. In preliminary observations we found that an increase of phosphoprotein B2 is evident in the regenerating liver nuclei as compared with its sham-operated control.…”

“…In particular, our attention has been centred upon a highly phosphorylated protein (B2; Mr 68000, pI6.5-8.2) associated with these particles. A polyclonal antibody produced in rabbits to this protein has allowed us to verify further its nucleosomal location and demonstrate that B2 nucleosomes are preferentially released following limited nuclease digestion (Zhao & Liew, 1982;Liew et al, 1984a). However, this polyclonal antibody approach carried several major limitations [e.g.…”

“…It is well known that non-histone chromosomal Vol. 225 proteins are closely associated with nuclear activity (Allfrey, 1974;MacGillivray & Rickwood, 1974;Wang et al, 1976;Cartwright et al, 1982;Briggs et al, 1983;Liew et al, 1984a). For example, Hoefler & Lane (1980) recently demonstrated that a monoclonal antibody designated as DL 3C4 (IgG I) raised against the large T protein of SV40 also recognized an antigenic determinant present on a host cell nuclear protein of Mr 68000.…”