Detection of specific protein binding to the SV40 early promoter in vivo.

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RESUMO

The interactions in vivo between cellular proteins and the Simian Virus (SV40) early promoter region, contained in a plasmid capable of replicating in Cos cells, have been characterized by DNaseI and dimethyl sulfate (DMS) footprinting. The relative contribution of each GC-motif within the 21 bp repeat upstream element to transcription was first determined after transfection of Cos cells with either the wild type 21 bp repeats or recombinants where the GC-motifs were mutated either individually or in neighboring pairs. Mutation of GC-motifs III and VI was the most detrimental, mutation of GC-I, -IV and -V also decreased promoter activity but to a lesser extent, while mutation of GC-II had little effect on transcription. All six GC-motifs of the wild type 21 bp repeats were found protected from DNaseI nuclease attack in vivo though to varying degrees. Motifs GC-III, -V and -VI were more strongly protected than GC-I, -II and -IV. In vivo DNaseI footprinting of recombinants bearing mutations in the GC-motifs demonstrated the specificity of factor interaction and further indicated that, in agreement with the previously published in vitro results, the binding of factor(s) to each of the GC-motifs was independent. DMS protection experiments identified specific guanine (G) contacts characteristic of Sp1 binding to the GC-motifs and this in vivo pattern was compared to that obtained in vitro using a crude nuclear extract. These results indicate that the transcription factor Sp1 interacts in vivo with the GC-motifs of the SV40 early promoter.

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