NUCLEOSOMES RECONSTITUTED IN-VITRO ON MOUSE MAMMARY-TUMOR VIRUS-B REGION DNA OCCUPY MULTIPLE TRANSLATIONAL AND ROTATIONAL FRAMES

The mouse mammary tumor virus acquires a highly reproducible chromatin structure when integrated into cellular DNA. Previous studies have su ggested that the LTR is arranged as a series of six phased nucleosomes , that occupy specific positions on the LTR. On the basis of nucleosom e reconstitution studies using DNA from the B region of the LTR, it ha s been argued that this sequence directs a uniquely positioned nucleos ome. Here we demonstrate in vitro that reconstituted B region nucleoso mes adopt at least five distinct translational positions in two rotati onal frames on a 206 bp fragment of DNA. We have resolved an initial r econstitute into its component species using nondenaturing gel electro phoresis, and precisely mapped the positions of each species using a h ydroxyl radical footprinting assay. To confirm the nucleosome position s determined with the hydroxyl radical assay, nucleosome boundaries we re mapped using exonuclease III. Comparison of the results from the hy droxyl radical footprinting and exonuclease III assays revealed a symm etrical pattern of overdigestion by exonuclease III which made unequiv ocal determination of nucleosome boundaries dubious. We conclude that the general use of exonuclease III to map the positions of nucleosomes may lead to incorrect assignment of position, and that assignment of position through the determination of the nucleosome pseudo-dyad from hydroxyl radical footprinting data represents a superior method of ana lysis.