Nucleic Acids Research, 1989, Vol. 17, No. 12 4503-4516
© 1989
MOLECULAR BIOLOGY |
In vivo photocrosslinking reveals that transcription factor binding to the mammalian ATF recognitioi sequence is required for E1A-induced transactivation in injected Xenopus laevis oocytes
Worcester Foundation for Experimental Biology Shrewsbury, MA 01545, USA
Received April 3, 1989. Revised May 10, 1989. Accepted May 10, 1989.
The adenovirus El A 13S mRNA product transactivates genes injected into Xenopus laevis oocytes that are normally E1A– inducible in mammalian cells. However, ElA–stimulated transcription, but not basal (uninduced) transcription, was inhibited if oocytes were incubated in the presence of protein synthesis inhibitors. This suggests that a cellular protein(s) is required for ElA–induced transactivation, but that it is dispensable for basal transcription. In order to identify such a protein from Xenopus oocytes that interacts with the adenovirus E3 promoter, gel shift assays, a new in vivo photocrosslinking assay, and immunoselection of biotinylated oligonucleotides were employed. A protein of molecular size 75 kd, which bound to the mammalian ATF recognition sequence in vivo, was found to be essential for ElA–induced transactivation. Although cycloheximide treatment of oocytes inhibited factor binding, E1A exerted no effect on factor binding. These data suggest that E1A modulates the activity of an oocyte transcription factor, either directly or indirectly, but not its ability to bind DNA.
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