Nuclear proteins. II. Similarity of nonhistone proteins in nuclear sap and chromatin, and essential absence of contractile proteins from mouse liver nuclei.

Abstract

High resolution SDS slab gel electrophoresis has been used to examine the distribution of nonhistone proteins (NHP) in the saline-EDTA, Tris, and 0.35 M NaCI washes of isolated mouse liver nuclei. These studies led to the following conclusions: (a) all the prominent NHP which remain bound to DNA are also present in somewhat similar proportions in the saline-EDTA, Tris, and 0.35 M NaCI washes of nuclei; (b) a protein comigrating with actin is prominent in the first saline-EDTA wash of nuclei, but present as only a minor band in the subsequent washes and on washed chromatin; (e) the presence of nuclear matrix proteins in all the nuclear washes and cytosol indicates that these proteins are distributed throughout the cell; (d) a histone-binding protein (J2) analogous to the HMG1 protein of K. V. Shooter, G. H. Goodwin, and E. W. Johns (Eur J. Biochem. 4"/:263-270) is a prominent nucleoplasmic protein; (e) quantitation of the major NHP indicates that they are present in a range of 2.2 • 105-5.2 • l06 copies per diploid nucleus. Most of the electrophoretically visible NHP are probably structural rather than regulatory proteins; 09 actin, myosin, tubulin, and tropomyosin, if present at all, constitute a very minor fraction of the nuclear NHP. Contractile proteins constitute a major portion of the NHP only when the chromatin is prepared from crude cell lysates instead of from purified nuclei. These studies support the conclusion that there are no clear differences between many nucleoplasmic and chromatin-bound nonhistone proteins. Except for the histones, many of the intranuclear proteins appear to be in equilibrium between DNA, HnRNA, and the nucleoplasm.Most studies of nonhistone proteins begin with chromatin purified by repeated washing in dilute buffers. The washes are usually discarded. Since the development of high-resolution SDS gel electrophoresis there have been few studies to examine whether the proteins removed by the nuclear washes represent a unique set of proteins or whether they are essentially identical to the nonhistone proteins that remain bound to DNA. Using a relatively low resolution SDS gel electropho-440

Reversible translocation of cytoplasmic actin into the nucleus caused by dimethyl sulfoxide.

“…The recent experiments of Rungger et al (24), who microinjected actin antibody into Xenopus oocyte nuclei and prevented chromosome condensation, suggest an important role for nuclear actin in the precise distribution of genetic material during proliferation. Despite biochemical work that indicates that actin is a genuine constituent of the nuclei of several cell types (1-7), it has not been found in purified nuclear preparations of other cells, for example, mouse hepatocytes (8). Experiments with microinjected proteins have shown that actin readily diffuses from the cytoplasm into the nucleus in amoebae (3,25) and Xenopus oocytes (1).…”

Microinjection of actin-binding proteins and actin antibodies demonstrates involvement of nuclear actin in transcription of lampbrush chromosomes

“…Actin has been identified in chromatin and nuclear fractions isolated from organisms as diverse as slime molds (Jockusch et aI., 1974;LeStourgeon et aI., 1975;Pederson , 1977) and mammals (Ohnishi et aI., 1964;Oouvas et aI., 1975;Paulin et aI., 1976;Peterson and McConkey, 1976; for review see LeStourgeon, 1978). However, such studies do not prove unequivocally that the actin is localized in the nuclei since cytoplasmic contamination could not be rigorously excluded (for detailed discussion of this problem see Comings and Harris, 1976;LeStourgeon, 1978). More convincing are the demonstrations that manually isolated and cleaned nuclei of ameba and amphibian oocytes contain relatively large amounts of actin (Clark and Merriam , 1977;Goldstein et al,1977aGoldstein et al, , 1977bOeRobertis et aI.…”

Differential staining of actin in metaphase spindles with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and fluorescent DNase: is actin involved in chromosomal movement?

“…Strong evidence exists that tubulin is a spindle component, although there remains some uncertainty regarding the amount present (5)(6)(7). Electron micrographs show microtubules inserted into chromosomal kinetochores, structures thought to be tubulin-nucleation centers (5).…”

“…Some experiments have indicated the presence of actin in nuclear fractions (9,10), but others have indicated that actin in these fractions may be a cytoplasmic contaminant. By using isolated nuclei, Comings and Harris found that actin and myosin constitute at most a very small fraction of nonhistone proteins (6).…”