FIG. 1.

Tax transactivation in vitro is dependent upon CREB and the viral CRE. (A) Schematic representation of the linearized promoter templates used in the in vitro transcription reactions. The HTLV-1 promoter carries the full transcriptional control region of the virus, including the three 21-bp repeats (viral CREs). The heterologous promoters each contained three tandem copies of either the viral CRE (from the promoter-proximal 21-bp repeat) or the cellular CRE (from the human chorionic gonadotropin gene), cloned immediately upstream of the herpesvirus thymidine kinase (TK) minimal promoter (at position −32). (B) In vitro transcription assay showing that Tax activation of transcription is dependent upon the viral CREs. Transcription reaction mixtures contained the HTLV-1 template fragment (50 ng) (lanes 2 to 4), the viral CRE promoter template fragment (100 ng) (lanes 5 to 7), or the cellular CRE promoter template fragment (25 ng) (lanes 8 to 10) in the presence of 32 μg of nuclear extract from the HTLV-1-negative human T-lymphocyte cell line CEM. A 50-ng amount of purified, recombinant CREB and/or 125 ng of purified recombinant Tax was added to the indicated reaction mixtures. A labeled DNA fragment was added to each reaction prior to RNA isolation to measure the recovery of the labeled RNA transcript. The positions of the recovery standard, the RNA transcripts, and the size of the radiolabeled DNA markers are indicated.