Nucleic Acids Research, 1989, Vol. 17, No. 19 7945-7963
© 1989
MOLECULAR BIOLOGY |
Detection of specific protein binding to the SV40 early promoter in vivo
Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Unité 184 de Biologie Moléculaire et de Génie Génétique de I'INSERM, Institut de Chimie Biologique, Faculté de Médecine 11 rue Humann, 67085 Strasbourg Cedex, France
*To whom correspondence should be addressed
+Present address: DNAX Research Institute, 901 California Avenue, Palo Alto, CA 94305, USA
Received March 14, 1989. Revised August 25, 1989. Accepted August 25, 1989.
The interactions in vivo between cellular proteins and the Simian Virus (SV40) early promoter region, contained in a plasnrid capable of replicating in Cos cells, have been characterized by DNasel 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 DNasel 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 DNasel 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 guaninc (G) contacts characteristic of Spl 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 S V40 early promoter.