Fusion of adenovirus E1A to the glucocorticoid receptor by high-resolution deletion cloning creates a hormonally inducible viral transactivator.
The 289-amino-acid E1A protein of adenovirus type 2 stimulates transcription from early viral and certain cellular promoters. Its mechanism is not known, and there exist no temperature-sensitive mutants of E1A that could help to elucidate the details of E1A transcriptional activation. To create for E1A such a conditional phenotype, we fused portions of E1A to the human glucocorticoid receptor (GR) to make transactivation by E1A dependent on the presence of dexamethasone. Nested subsets of the E1A coding region, centered around the 46-amino-acid transactivating domain, were substituted for the DNA-binding domain of the GR. One of the resulting chimeric proteins (GR/E1A-99), which included the entire E1A transactivating domain, stimulated expression from a viral early promoter (E3) exclusively in the presence of hormone. GR/E1A-99 did not transactivate a GR-responsive promoter. It therefore exhibited the promoter specificity of E1A while possessing the hormone inducibility of the GR. Two smaller chimeras that contained only portions of the E1A transactivating domain failed to transactivate E3. These three chimeras were constructed by a novel strategy, high-resolution deletion cloning. In this procedure, series of unidirectional deletions were made with exonuclease III on each side of the E1A coding region at a resolution of 1 to 2 nucleotides. The large number of in-frame fragments present in the collection of deleted clones facilitated the construction of the GR/E1A chimeras and can be used to create many additional fusions.
Becker
D M
DM
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104-4268.
Hollenberg
S M
SM
Ricciardi
R P
RP
D017456:Adenovirus Early Proteins; D000595:Amino Acid Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D003907:Dexamethasone / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D015696:Gene Products, tat; D006801:Humans; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011965:Receptors, Glucocorticoid / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*
D017456:Adenovirus Early Proteins; D015696:Gene Products, tat; D009856:Oncogene Proteins, Viral; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D014157:Transcription Factors; D003907:Dexamethasone