Subnuclear associations of the v-myb oncogene product and actin are dependent on ionic strength during nuclear isolation.

Abstract

The method used to isolate nuclei has a direct effect on the subnuclear association of the v-myb product, p48v-myb and nuclear actin. Analysis of nuclei subjected to various isolation procedures showed that disruption of native nuclear structure during hypotonic treatment resulted in dissociation of p48v-.Yb from the nuclear matrix.The product of the oncogene v-myb of avian myeloblastosis virus (AMV) in AMV-transformed myeloblasts is a 48,000-Mr nuclear protein, p48V--myb (4,13). To help define the molecular role of p48v-myb in leukemogenesis, several investigators have sought to identify and define its nuclear interactions (1,2,8,12,14,15). The association between p48v-myb and nuclear elements has been analyzed in fractionated nuclei isolated from AMV-transformed cells (BM2) by two different strategies (1,8). One isolation procedure has been previously described (1, 3), in which nuclei are obtained from cells lysed under isotonic conditions in buffer containing 5 mM NaH2PO4 (pH 7.4), 50 mM NaCl, 150 mM sucrose, 5 mM KCl, 2 mM dithiothreitol (DTT), 1 mM MgCl2, 0.5 mM CaCl2, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1% Kyro EOB detergent (isotonic nuclei). After extraction of these isotonic nuclei with isotonic Nonidet P-40 (NP-40) buffer (0.5% NP-40, 10 mM NaH2PO4, pH 7.4, 120 mM NaCl, and 0.1 mM PMSF) (1), brief digestion with micrococcal nuclease at 37°C, and then extraction is 2.0 M NaCl (high salt), p48v-myb was removed from the nucleus in varying amounts in three distinct fractions: approximately 29% was contained in nucleoplasm, 7% was in nuclease-sensitive chromatin, and 64% was associated with the nuclear matrix/lamina complex (1). The other procedure we used to isolate nuclei relies on osmotic and shear force to separate the plasma membrane by disrupting cells which had been preincubated in hypotonic buffer (RSB: 10 mM NaCl, 10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 7.4, 1.5 mM MgCl2, 0.1% sodium deoxycholate [DOC], and 0.1 mM PMSF) with a Dounce homogenizer as described by Evan and Hancock (8).After fractionation of these nuclei (hypotonic nuclei) by digestion with DNase I at low temperature (4°C) followed by extraction in high salt, most or all of p48v-myb was eluted from nuclei along with soluble nucleoplasm and chromatin (Fig. 1A) as previously described (8). However, when these hypotonic nuclei were transiently exposed to temperatures near 37°C during nuclease digestion, most or all of p48v-myb remained associated with the resulting insoluble nuclear complex (Fig. 1A) 92138-9216. isolated by this hypotonic procedure is an important factor in nuclear subfractionation. These results could suggest that localization of p48V-myb within the nuclear matrix fraction is an artifact induced by a temperature-dependent insolubilization of nuclear proteins.Alternatively, the isolation procedure alone, not the subsequent method of subnuclear fractionation, may affect the associations of some nuclear proteins, including p48vmYb. In nuclei isolated under hypotonic conditio...

Absence of high levels of DNA polymerase α activity in the nuclear matrix prepared from mouse erythroleukemia cells

“…For example, 0.2 M CNH4)Z S04 has been claimed to be a milder extracting agent in comparison with 2 M NaCi and it should better preserve the interactions between enzymes and the nucleoskeleton but in our hands this was not the case (He et aI., 1990;Belgrader et aI., 1991).Also the sequence in which the extraction is performed has been demonstrated to produce changes in the properties of the nuclear matrix (Djondjurov et al, 1986) but we were unable to find differences between matrices which have been prepared by extracting first with high salt followed by DNase I digestion (the so called "reverse" procedure) and those obtained by the "direct" procedure. Moreover, also the technique used for nuclear isolation could have deep consequences on the association of particular proteins with the nuclear matrix , as has been demonstrated for the v-myb oncogene product and nuclear actin (Boyle and Baluda, 1987). Nevertheless, in our system, similar results were obtained both with nuclei isolated by a hypotonic method or by a iso-osmotic lysis.…”

Changes in the subnuclear distribution of two RNA metabolism-related proteins can be detected in nuclear scaffold or matrix prepared by different techniques

“…In the past, much emphasis has been placed on the detrimental effects exerted by Mg ++ ions and hypotonic buffers during preparation of nuclei (Boyle and Baluda 1987;Gasser and Laemmli 1987;Cook 1988) and polyamines have been proposed as substitutes (Gasser and Laemmli 1987). However, following in vitro stabilization by heat, nuclei kept in buffer with polyamines also exhibited changes very similar to those described for nuclei heat-stabilized in a Mg ++ -containing buffer, i.e., chromatin condensation and clumping, as well as enlargement of the interchromatin spaces (Martelli et al 1991).…”

Different Concentrations of Mg++Ions Affect Nuclear Matrix Protein Distribution During Thermal Stabilization of Isolated Nuclei

“…Surprisingly, however, the effect of different concentrations of Mg ++ ions during nuclear preparation to obtain the matrix has never been investigated. Only the effect of the ionic strength in general has been examined (Boyle and Baluda 1987). Moreover, all of the aforementioned investigations dealt with the ultrastructural morphology of the nuclear matrix prepared under different conditions and its overall protein composition.…”

“…Changes have been observed in the distribution of three nuclear scaffold proteins when heat treatment was employed to stabilize nuclei and nuclear scaffolds (Neri et al 1997b). During the thermal stabilization of isolated nuclei, different concentrations of Mg ++ , ranging from 1 to 5 mM, have been employed (Boyle and Baluda 1987;Humphrey and Pigiet 1987;McConnell et al 1987;Martelli et al 1990;Belgrader et al 1991;Kaufmann and Shaper 1991;Martelli et al 1991;Neri et al 1994). It is established that different concentrations of Mg ++ have a marked effect on nuclear structure (e.g., Leake et al 1972;Laval and Bouteille 1973;Dixon and Burkholder 1984;Papa et al 1988).…”