| Popis: |
The original discovery that any treatment which lead to rupture of calf thymus nuclei in crude suspension, liberating chromatin threads, also gave highly polymerized deoxyribonucleoprotein (DNP)$^{1}$ and deoxyribonucleic acid (DNA)$^{2}$, suggested that these forms were produced by reactions occurring during preparation. The overall aim of our studies, therefore, was to prepare whole, unclumped nuclei, "protected" against enzymatic activity, and isolate from them DNA, in a form approaching as closely as possible that found in vivo. A new method, involving use of the enzymatic inhibitor glycerophosphate$^{3}$ and subzero temperatures during the nuclei isolation, was thus developed$^{4}$. We found that DNA "subunits", of molecular weight lower than two million, much more homogeneous in particle size than corresponding degraded fractions described in the literature, are obtainable only from these nuclei. The smallest subunits predominantly found, had molecular weights of 500, 000 or a multiple. The subunits are obtained by special extractions, as well as by treatment of the isolated DNA with ethylenediamine tetraacetate (EDTA), a chelating agent. A still smaller subunit of about one half the smallest weight mentioned above can also be isolated under certain conditions, as described in this report. In contrast, nuclei ruptured in crude suspension, as in standard procedures for DNP and DNA${5}$, as well as our protected nuclei treated with MgATP${6}$, do not give such subunits under the same conditions of extraction. This means that two basicallydifferent forms of DNA exist: 1) a reversibly polymerized, associated state which gives subunits on EDTA treatment, and 2) an irreversibly polymerized, EDTA-stable state. Our protected nuclei give DNA of type 1, whereas ruptured or MgA TP-treated nuclei give the irreversibly polymerized type 2. lt was therefore deduced that rupture of the nuclei probably allowed reaction of the extended DNA in the released chromatin threads with the ATP in the cytoplasm. [...] |
