Biochemical properties of the v-rel oncogene that are involved in transformatio

The research presented in this dissertation project involved designing a biological system in which the transforming potential of the v-rel oncoprotein could be correlated with the protein's biochemical characteristics. To avoid problems in analyzing transformation generated by a cytopathic helper virus, the v-rel gene was cloned into a replication competent virus. The replication competent virus (designated RCAS-rel) was demonstrated to efficiently transform avian fibroblasts and bone marrow cells; analysis of the bone marrow cells using lineage specific surface markers suggested the transformed cell may represent an early precursor hematopoietic cell. A series of deletion mutants spanning the rel-homology domain were constructed by oligonucleotide deletion mutagenesis and cloned into the RCAS viral vector. (Eleven deletion mutants were constructed, removing approximately 100 nucleotides for each mutant, except deletion one which only removes twelve amino terminal envelope residues). The deletion mutants were analyzed for nuclear localization by immunofluorescence biochemical fractionation. Mutants which removed only the amino terminal envelop residues (dl-1) and carboxy terminal residues within the rel-homology domain (dl-11) were efficiently localized to the nucleus of fibroblasts (similar to wild-type virus), whereas the other mutants (dl-2 through dl-9) did not accumulate in the nucleus. A coprecipitation assay with a v-rel specific anti-peptide antibody demonstrated that dl-1 and dl-11 still associated with several cellular proteins. In contrast, other deletions within the rel-homology domain abrogated interaction with the p36 cellular protein. Deletions within the rel-homology domain (dl-2 to dl-10) also abolished binding to a probe containing an NF-$kappa$B site; dl-1 and dl-11 retained binding similar to the wild-type v-rel. (DNA binding was analyzed using an electrophoretic mobility shift assay). Finally, deletion mutants which lost the biochemical properties described above were also found to be non-transforming in chick embryo fibroblasts. In summary, these results demonstrate the rel-homology domain may be essential for nuclear localization, complex formation, and DNA binding; maintenance of these biochemical properties also correlates with efficient transformation of fibroblasts by the v-rel oncogene.