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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA051792-04
Application #
3196460
Study Section
Virology Study Section (VR)
Project Start
1990-04-01
Project End
1995-03-31
Budget Start
1993-04-01
Budget End
1994-03-31
Support Year
4
Fiscal Year
1993
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
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Walker, A K; Enrietto, P J (1996) Analysis of the role of v-rel in transcriptional regulation of high mobility group 14. Oncogene 12:2515-25
Hodgson, J; Enrietto, P J (1995) Constitutive and inducible kappa B binding activities in the cytosol of v-Rel-transformed lymphoid cells. J Virol 69:1971-9
Boehmelt, G; Madruga, J; Dorfler, P et al. (1995) Dendritic cell progenitor is transformed by a conditional v-Rel estrogen receptor fusion protein v-RelER. Cell 80:341-52
Smardova, J; Walker, A; Morrison, L E et al. (1995) The role of the carboxy terminus of v-Rel in transformation and activation of endogenous gene expression. Oncogene 10:2017-26
Abbadie, C; Kabrun, N; Bouali, F et al. (1993) High levels of c-rel expression are associated with programmed cell death in the developing avian embryo and in bone marrow cells in vitro. Cell 75:899-912
Boehmelt, G; Walker, A; Kabrun, N et al. (1992) Hormone-regulated v-rel estrogen receptor fusion protein: reversible induction of cell transformation and cellular gene expression. EMBO J 11:4641-52
Morrison, L E; Boehmelt, G; Enrietto, P J (1992) Mutations in the rel-homology domain alter the biochemical properties of v-rel and render it transformation defective in chicken embryo fibroblasts. Oncogene 7:1137-47
Morrison, L E; Boehmelt, G; Beug, H et al. (1991) Expression of v-rel in a replication competent virus: transformation and biochemical characterization. Oncogene 6:1657-66
Kabrun, N; Hodgson, J W; Doemer, M et al. (1991) Interaction of the v-rel protein with an NF-kappa B DNA binding site. Proc Natl Acad Sci U S A 88:1783-7