extrachromosomal ribosomal chromatin in rapidly growing Dictyostelium discoideum and Physarum polycephalum cells

When chromatin is photoreacted with psoralen, crosslinks occur preferentially in the linker DNA between nucleosomes. The pattern of these crosslinks can be analysed by exonuclease digestion of random DNA fragments, since the exonucleases tested stop at sites of psoralen-crosslinks. Further digestion of these fragments with Sl-nuclease leads to DNA fragments of nucleosomal and polynucleosomal size, which presumably carry psoralencrosslinks at both ends. This method of analysis of chromatin structure complements the classical micrococcal nuclease digestion analysis, since it can be performed in vitro as well as in vivo, and since it is independent of pH and ionic conditions. INTRODUCTION It has been shown that treatment of cells, isolated nuclei or soluble chromatin with psoralen derivatives and long wave length ultraviolet (UV) irradiation leads to crosslinks in the DNA which are preferentially located in the linker between nucleosomes (1). Even under saturating conditions of psoralencrosslinking the structure of soluble rat liver chromatin appears to remain undisturbed to a large extent as judged by micrococcal nuclease digestion and electron microscopy (2). These conditions of crosslinking appeared to be powerful in mapping, at the level of the purified DNA, the active extrachromosomal ribosomal chromatin in rapidly growing Dictyostelium discoideum and Physarum polycephalum cells (3,4). The transcribed regions appear to be mostly devoid of nucleosomes (3,5) in contrast to the adjacent non-transcribed spacer. Moreover, this technique also allowed the analysis of in vivo transcribing SV40 minichromosomes (5). The results suggested that in this system transcription through nucleosome© IRL Press Limited, Oxford, England. 7 0 1 3 Nucleic Acids Research like particles is possible. On the other hand, the same technique used for analysis of in vivo replicating SV40 minichromosomes indicated that the nucleosome in front of the replication fork dissociates and reassociates randomly on one of the newly synthezised daughter strands (6). In the work reported above the analysis of the crosslinking pattern in the DNA was mainly performed by electron microscopy and therefore was restricted to gene systems which can be purified, like nucleoli or SV40 minichromosomes. However, psoralen crosslinks can be reversed by irradiation with short wave length UV light and psoralen-crosslinked DNA fragments, after reversal of the crosslinks, can be detected with radioactive hybridization probes on Southern blots (3,4). We have therefore developed a procedure to analyze the psoralencrosslinking pattern by gel electrophoresis. Exonucleases appear to stop at sites of crosslinks, so after extensive digestion of random fragments of psoralen-crosslinked chromatin DNA with exonuclease and Si nuclease, fragments are produced which are of nucleosomal and of polynucleosomal size. MATERIALS AND METHODS Standard buffers 5 mM triethanolamine chloride at pH 7 or pH 8, 0.2 #1 EDTA is called "0 mM", pH 7 or pH 8, respectively. 5 mM diethanolamine chloride at pH 9 or pH 10, 0.2 mM EDTA is called "0 mM", pH 9 or pH 10, respectively. "0 mM" buffers at pH 7 or pH 8 or pH 9 or pH 10 containing 10 mM NaCl, or 40 mM NaCl or 100 mM NaCl are called "10 mM", "40 mM", "100 mM", pH 7 or pH 8 or pH 9 or pH 10, respectively. Isolation of nuclei and preparation of chromatin Nuclei of rat liver were prepared as described by Hewish & Burgoyne (7). Preparation of soluble chromatin and of Hl-depleted chromatin were performed according to Thoma et al. (8). Psoralen crosslinking Chromatin samples (1.5 3 0D_6(./ml) were first dialyzed for 4 h against "10 mM", pH 7 buffer. Aliquots were then dialyzed against "0 mM" or 10 mM" or 40 mM" or "100 mM", pH 7, or pH 8,