Identification of a cellular protein that binds to a T cell-specific element in the SL3-3 enhancer
Boral, Anthony Lloyd
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The murine leukemia virus, SL3-3, induces T-cell lymphomas 60 to 120 days after injection into newborn mice, while the closely related virus, Akv, induces no disease. Recombination studies between these viruses have shown that the enhancer is the most important determinant of SL3-3 leukemogenicity, and transient expression assays have shown that it is about ten fold more active than the Akv enhancer in a variety of T-lymphoma cell lines. In the experiments described in this thesis, transient expression assays were used to demonstrate that a single base-pair difference between the SL3-3 and Akv enhancers is important for the T-cell specificity of the SL3-3 enhancer. This base-pair lies within the enhancer core, a nine base-pair element known to be important for the activity of a number of viral and cellular enhancers. DNA-protein complexes comprising the SL3-3 or Akv cores and one or more nuclear proteins were identified. One of these complexes, S, formed preferentially with a SL3-3 probe; a second, S/A, formed equally well with both SL3-3 and Akv probes. The protein component of the S complex (S-CBF) was purified to near homogeneity by DNA-affinity chromatography. Three tryptic peptide sequences were obtained for the most abundant protein in the preparation of S-CBF, and a mouse cDNA clone was isolated and sequenced. S-CBF showed high homology to the nucleic acid binding domain common to a group of RNP proteins and single-stranded DNA binding proteins. Theoretical translation of the clone revealed no canonical double-stranded DNA binding motifs. Two synthetic peptides deduced from the sequence of the clone were used to raise antibodies in chickens. Serum from immune chickens reacted with all observable bands in a western blot of purified S-CBF, but failed either to block the formation or alter the electrophoretic mobility of the S-CBF/DNA complex. S-CBF DNA-binding activity and antibody reactivity continued to co-purify, following additional chromatographic techniques. Attempts to recover sequence-specific DNA binding activity following expression in E. coli suggested that the cloned gene encoded S-CBF. ftn*All degree requirements completed in 1991, but degree will be granted in 1993.