In vivo photocrosslinking reveals that transcription factor binding to the mammalian ATF recognition sequence is required for E1A-induced transactivation in injected Xenopus laevis oocytes.
The adenovirus E1A 13S mRNA product transactivates genes injected into Xenopus laevis oocytes that are normally E1A-inducible in mammalian cells. However, E1A-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 E1A-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 E1A-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.
Richter
J D
JD
Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
D017456:Adenovirus Early Proteins; D000818:Animals; D001483:Base Sequence; D001710:Biotin; D003432:Cross-Linking Reagents; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D015345:Oligonucleotide Probes / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000502:physiology*; D009865:Oocytes / Q000378:metabolism*; D010777:Photochemistry; D014157:Transcription Factors / Q000378:metabolism* / Q000502:physiology; D014982:Xenopus laevis
D017456:Adenovirus Early Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D009856:Oncogene Proteins, Viral; D014157:Transcription Factors; D001710:Biotin; D003513:Cycloheximide