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Although several DNA response elements for HFV Bel-1 regulatory protein have been identified by mutational analysis, these show little sequence homology. It has therefore remained unclear how Bel-1 is targeted to its response elements. Using gel mobility shift analysis and DNase I footprinting, we have previously defined Bel-1 DNA binding sites in both the LTR and the internal promoter. More recently, we have identified critical residues within these two Bel-1 response elements by using modification interference analysis. THis work has defined a 22 bp minimal Bel-1 target site that is fully sufficient to give rise to efficient transcriptional activation by Bel-1, and weak activation by SFV-1 Taf, in yeast cells. A somewhat similar 22 bp sequence, derived from the SFV-1 internal promoter, allowed efficient transactivation by Taf, but not by Bel-1. At present, we are using sequence randomization, follow by selection for in vitro binding, to more fully define the precise sequence specificities displayed by Bel-1 and Taf. Data contrasting the binding site specificity of these two related proteins will be presented.
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