Chromatin and nucleosome organizations and DNA replication of nucleus reassembled in vitro using purified exogenous DNA and Xenopus egg extracts.

It has been demonstrated in the last ten years that the nuclear reassembly may occur in the cell-free systems from frog egg extracts added with exogenous naked DNA. However, there remains an open question: is the cell-free reassembled nucleus structurally similar to the nucleus in the intact cell? That is, does the cell-free reassembled nucleus contain nucleosomes and chromatin? For this issue, we have designed experiments for identifying the internal structures of the cell-free reassembled nucleus. These experiments show that the nucleus reassembled in vitro also contains chromatin which is composed of typical 10 nm nucleosome fibers of "beads-on-a-string", 30 nm filaments and the next higher-order structures. The digestion experiment with the enzyme micrococcal nuclease has demonstrated that the DNA in the nucleosome of the reconstituted chromatin is about 200 base pairs (bp) in length, of which 165 bp may be in the nucleosome particle, and 35 bp may be in the linker between two particles. Prolonging digestion of the 165-bp particle DNA fragment will yield a 146-bp fragment, which may be wounded in the nucleosome core. Experiments also provide evidence that the chromatin reconstitution in the cell-free reassembled nucleus is a progressive process, and that the nucleus can replicate its DNA. Based on these observations, we suppose that the chromatin of the cell-free reassembled nucleus may be structurally and functionally similar to the chromatin of the intact cells.