The long-term objective of this project is to examine the interaction of the product of the rel oncogene with other cellular proteins. Characterizing these interactions may provide insight into the function and regulation of the rel gene in the transformation of avian lymphoid cells. By examining the interactions that oncogenes have with other cellular proteins, it is also possible to gain insight into the regulatory events that govern normal cell growth and differentiation.
The specific aims of this project are the following: 1) To map the binding sites on p59v-rel for the associated cellular proteins p36, p115, and p124. This will be done using site-directed deletion mutagenesis of the rel gene. 2) To correlate the binding of p36, p115, and p124 with biological properties of the virus. Mutant viruses will be tested for their ability to complex with p36, p115, and p124 in vitro and in vivo. They will be tested for transformation of avian lymphoid cells in vitro. In ovo assays will be performed with mutant viruses to test their ability to induce tumors. Mutant rel proteins will be examined to determine their localization in the infected cell, modification by phosphorylation, and for associated kinase activity. 3) The cellular proteins found in complex with rel, p36, p115, and p124 will be purified using immunoaffinity column chromatography. They will then be injected into mice and rabbits in order to develop monoclonal and polyclonal antibodies. Resulting antibodies will be used to characterize p36, p115, and p124 in detail. 4) The genes encoding p36, p115, and p124 will be molecularly cloned using one of two approaches. Gamma gt 11 libraries will be screened using antibodies directed against p36, p115, and p124. Alternatively, p36, p115, and p124 will be purified by immunoaffinity column chromatography. The proteins will be sequenced and, from the sequence, oligonucleotides will be designed. These oligonucleotides will then be used to screen a cDNA library made from the avian REV-transformed lymphoid cell line NPB4.