Nuclear matrix of the lower eukaryote Physarum polycephalum and the mammalian epithelial LLC‐PK₁ cell line

Abstract

Agarose-encapsulated nuclear matrix preparations of the lower eukaryote Physarum polycephalum and the mammalian renal epithelial LLC-PKJ cell line were analyzed after various experimental protocols with respect to the protein composition. The effect of the mode of deproteinization (2 M NaC1,0.25 M ammonium sulfate or 25 mh4 lithium diiodosalicylate), presence of 2-mercaptoethanol, Ca", Cu2+, chelating agents, the sequence of protein extraction and nuclease digestion, the use of RNase, the temperature at which the experimental manipulations were performed and the use of hypotonic or isotonic conditions was investigated. No significant differences in the final nuclear matrix composition could be observed, regardless of the experimental procedure applied. In Physarum, the major nuclear matrix proteins range over 12-70 kDa with prominent bands at 24, 31, 37 and 45 kDa; the proteins of the matrix in U C -P K J cells extend predominantly over 40-80 kDa.Furthermore, no essential differences in the protein composition could be observed when type I and type I1 nuclear matrices from the highly differentiated LLC-PK, cell line were compared. The same was found for analogous matrix preparations of Physarum. Therefore, in both systems a distinction between type I/II matrix is questionable. Immunoblotting of the matrix preparations with a variety of antibodies against intermediate filament proteins and with antinuclear autoantibodies revealed the presence of intermediate filament proteins as components of the nuclear matrix. We conclude that the nuclear matrix represents a much more stable and reproducible structure than has been proposed so far, largely independent of changes in the preparation protocol.

Cloning and Characterization of Physarum polycephalumTectonins

“…The many nuclei within a single plasmodium progress through the cell cycle synchronously, and at the end of the G 2 phase undergo closed mitosis. Because of these characteristics, several investigators have examined the P. polycephalum nuclear matrix (1)(2)(3)(4) and reported that, as with mammalian nuclear matrix, the P. polycephalum matrix contained a number of proteins ranging from approximately 40 kDa to more than 100 kDa but that it differed from mammalian nuclear matrix by having two dominant proteins with reported molecular masses of 23-28 and 35-38 kDa as determined by SDS-PAGE. 1 The same proteins have also been found associated with purified rDNA chromatin (5).…”

Tropomyosin is localized in the nuclear matrix and chromosome scaffold of physarum polycephalum

“…Grabber et al [14] and Eberharter et al [15] demonstrated that intermediate fibre protein was the component of the nuclear matrix. Some previous experiments indicated that a 37 kD polypeptide which is equivalent to tropomyosin in molecular weight existed in the nucler matrix and chromosome scaffold [15,[17][18][19][20], and this polypeptide might represent tropomyosin [19]. Our experiments of protein analyses showed that a 37 kD polypeptide was present in the nuclear matrix and chromosome scaffold of P. polycephalum, which is consistent wf previous reports.…”

“…The main component of nuclear matrix and chromosome scaffold is nonhistone proteins [1][2][3][4], [10]. Although many biochemical and immunocytochemical analyses have been conducted concerning protein composition of the nuclear matrix and chromosome scaffold, only a few proteins (such as topoisomerase II) have been identified [11][12][13][14][15][16]. Some previous biochemical analyses indicated that a polypeptide of about 37 kD, which is equivalent to tropomyosin in molecular weight, existed in the nuclear matrix and chromosome scaffold [15], [17][18][19][20] and this polypeptide might represent tropomyosin [19].…”

“…Although many biochemical and immunocytochemical analyses have been conducted concerning protein composition of the nuclear matrix and chromosome scaffold, only a few proteins (such as topoisomerase II) have been identified [11][12][13][14][15][16]. Some previous biochemical analyses indicated that a polypeptide of about 37 kD, which is equivalent to tropomyosin in molecular weight, existed in the nuclear matrix and chromosome scaffold [15], [17][18][19][20] and this polypeptide might represent tropomyosin [19]. In order to gain a clear idea whether tropomyosin exists in the nuclear matrix and chromosome scaffold, we analysed the protein composition of the nuclear matrix and chromosome scaffold of P. polycephalum with SDS-PAGE and localized tropomyosin in the nuclear matrix and chromosome scaffold with immunodotting, immunofluorescence and immunoelectron microscopy.…”

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

“…In these reports the investigators have taken into consideration the effect on nuclear matrix structure of RNAse A (Kaufmann et al 1981;Belgrader et al 1991), of reducing or oxidative chemicals (Dijkwel and Wenink 1984;Kaufmann and Shaper 1984;Stuurman et al 1992a), and of different extracting agents (Belgrader et al 1991;Eberharter et al 1993). Surprisingly, however, the effect of different concentrations of Mg ++ ions during nuclear preparation to obtain the matrix has never been investigated.…”