A molecular study of chromatin containing integrated murine leukemia virus DNA will be performed. These experiments will provide model systems to investigate 1) chromatin configuration around gene regulatory regions and 2) chromosomal position effect. Moloney murine leukemia viruses (M-MuLV's) containing altered LTR's will be generated by recombinant DNA manipulations. DNA sequences conferring hormone or metal responsiveness will be inserted, as will heterologous transcriptional enhancersers. M-MuLV's carrying the modified LTR's will be recovered by transfection, and tested for hormone or metal responsiveness or change in host range. The chromatin structure of the altered proviruses will be investigated, and the appearance of new DNAse I hypersensitive sites or bound proteins will be correlated with the biological activity. Promoter activity of the altered LTR's will also be assessed by transient expression assays. Chromatin structure of transcribed and non-transcribed M-MuLV proviruses from a cell line chronically infected with M-MuLV will be investigated. Recombinant clones of individual M-MuLV proviruses will be used to identify these proviruses. Nuclear run-off transcription will be employed. The chromatin state of the unoccupied host site as well as the proviral integration will be investigated by DNase I digestion. Cellular proteins which associate with the M-MuLV LTR will be identified by procedures which reveal specific DNA-binding proteins. The location of protein binding in the LTR will be mapped, and LTR's which contain deletions and insertions will be studied. Extracts from different cell types will be tested. Correlation with the in vivo chromatin studies may reveal specific proteins responsible for particular features of chromatin structure.
