Nuclear matrix isolation

Berezney R, Coffey DS (1977) Nuclear matrix, isolation and characterization of a framework structure from rat liver nuclei. J Cell Biol 73(3) 616-637... 

Whether laser flash photolysis (LFP) is used to detect RIs before they react, or matrix isolation at very low temperatures is employed to slow down or quench these reactions, spectroscopic characterization of RIs is frequently limited to infrared (IR) and/or ultraviolet-visible (UV-vis) spectroscopy. Nuclear magnetic resonance (NMR) spectroscopy, which is generally the most useful spectroscopic technique for unequivocally assigning structures to stable organic molecules, is inapplicable to many types of RI. 

The reaction of metals with energetic hydrogen or deuterium ions is important in nuclear reactors. Ion beams may be generated thermally and allowed to interact with the metal, and the reaction products then may be examined by matrix-isolation techniques. Alternatively, metal atoms are sputtered from a cathodic surface by a low-energy plasma. If or at low P is added to the discharge, then molecular species are formed by the interaction with the sputtered metal atoms. Applied to Cu this technique leads to the identification of CuH and CuD in Ar matrices by their IR spectra. The reacting species are believed to be atomic Cu and H or D formed in the hollow-cathode discharge . 

Fisher, P. A., Berrios, M., and Blobel, G. (1982). Isolation and characterization of a proteinaceous subnuclear fraction composed of nuclear matrix, peripheral lamina and nuclear pore complexes from embryos of Drosophila melanogaster J. Cell Biol 92, 674-686. 

The present experiments show that a number of ADP-ribosylated proteins in the range of 40,000-300,000 kD exist in nuclear matrices isolated from HeLa cells, and that the lamins A and B are probably modified. Furthermore, it was observed that a portion (approx. 1%) of the nuclear poly(ADP-ribose) synthetase is tightly associated with the isolated nuclear matrix. 

Figure 1 shows the proteins contained in various subnuclear fractions. Histones were not detectable in the isolated nuclear matrix by Coomassie blue staining. But these known acceptor proteins for ADP-ribose were found in the ammonium sulfate extract. Autoradiography revealed that great amounts of the self-modified poly(ADP-ribose) synthetase - in accordance with the results of Table 1 - were released by DNase, RNase digestion of the isolated nuclei (not shown). 

Since artifacts may be formed in the course of labeling experiments with permeabilized cells, the ADP-ribosylated proteins of the nuclear matrix were identified by another experimental approach. After labeling of cellular proteins by 24 h incubation with [ S]-methionine, the matrix was isolated from the intact cells. In subsequent chromatography, the ADP-ribosylated proteins were selectively isolated due to the binding of the cis-diol grouping within the sugar component of the ADP-ribose conjugates onto the boronate column. While only 2% of the total matrix proteins were bound, control... 

The question is raised as to the mechanism by which the enzyme is attached to the nuclear matrix. Even though the nuclei were digested with high DNase concentration, a residual amount of 0.06% of the original nuclear DNA was found associated with the matrix. Although the activity of the purified enzyme is known to be completely DNA dependent [18], it was observed that the synthetase reaction of the isolated matrix did not require addition of DNA. 

The bands observed at 69 and 64 kD may be due to ADP-ribosylation of lamin A and lamin B, major constituents of the nuclear matrix, as was previously suggested by Song and Adolph [26]. They reported that nuclear scaffolds isolated by mild micrococcal nuclease digestion from in vitro labeled HeLa cell nuclei contained labeled proteins at 116 kD and in the 65 to 70 kD range. 

The present results demonstrate that a portion of the nuclear poly(ADP-ribose) synthetase is found in tight association with the isolated nuclear matrix. The question is whether the enzyme, if present at this site in vivo, is required to maintain reactions proceeding in association with the nuclear matrix. These may, for example, be reactions involving DNA in the vicinity of the DNA attachment sites. In view of various indications that transcription of active genes occurs in association with the nuclear matrix [6-9], the results of Slattery et al. [27] are also of interest. The authors observed that poly(ADP-ribose) synthetase is identical with the factor TFIIC which, by inhibiting nick-induced transcription, eliminates random transcription by polymerase II. 

---