Most class B (II) promoter regions from higher eukaryotes contain the TATA box and upstream and enhancer elements. Both the upstream and enhancer elements and their cognate factors have regulatory functions, whereas the TATA sequence interacts with the TATA box factor BTF1 to position RNA polymerase B and its ancillary initiation factors (STF, BTF2 and BTF3) to direct the initiation of transcription approximately 30 base pairs downstream. In many respects, class B promoter regions from the unicellular eukaryote Saccharomyces cerevisiae are similarly organized, containing upstream activating sequences that bear many similarities to enhancers. Although they are essential for initiation, the yeast TATA sequences are located at variable distances and further from the start sites (40-120 base pairs), whose locations are primarily determined by an initiator element. The basic molecular mechanisms that control initiation of transcription are known to be conserved from yeast to man: the yeast transcriptional transactivator GAL4 can activate a minimal TATA box-containing promoter in human HeLa cells, and a human inducible enhancer factor, the oestrogen receptor, can activate a similar minimal promoter in yeast. This striking evolutionary conservation prompted us to look for the presence in yeast of an activity that could possibly substitute for the human TATA box factor. We report here the existence of such an activity in yeast extracts.

An activity (designated BTF1Y) in extracts of Saccharomyces cerevisiae can substitute for the human TATA box-binding factor BTF1 in a reconstituted transcription system containing the adenovirus 2 major late promoter, RNA polymerase B (II), and the basic transcription factors BTF2, BTF3, and STF. We have purified BTF1Y to homogeneity, using as assays reconstitution of in vitro transcription and DNase I footprinting on the TATA element. Both activities copurified with a 27-kDa polypeptide as determined by SDS/PAGE. Gel Accurate and specific initiation of transcription in vitro from minimal promoter elements of protein-coding genes requires at least four transcription factors in addition to RNA polymerase B (II) (1-5). The first step in transcription initiation corresponds to the binding of the factor BTF1 (the TATA box-binding factor, also known as TFIID and DB) to the TATA box element (6-8). This binding step is crucial in the initiation pathway, since it results in the formation of a stable "committed" complex onto which the other factors and RNA polymerase B assemble to form a multiprotein complex at the promoter proximal site (3, 4, 9-11).Although numerous attempts have been made to purify mammalian BTF1, notably from human HeLa cell extracts, purification was only partial (e.g., ref. MATERIALS AND METHODSPurification of BTF1Y Protein. S100 extract (34 mg of protein per ml) was prepared from frozen S. cerevisiae cells as described (19). After overnight dialysis against buffer DB [10 mM Tris'HCl, pH 7.9/5 mM MgCl2/0.5 mM dithiothreitol/17.4% (vol/vol) glycerol/0.2 mM phenylmethanesulfonyl fluoride (PMSF)] containing 0.05 M KCI, 125 ml of 2-folddiluted S100 was loaded onto a 450-tnl heparin-Ultrogel (IBF) column equilibrated in buffer DB containing 0.1 M KCI. After washing, proteins were eluted stepwise with buffer DB containing 0.15 M KCI, 0.37 M KCI (HEPO.37 fraction), and 0.6 M KCl. BTF1Y activity was found in the HEPO.37 fraction. After dialysis in buffer B (50 mM Tris-HCl, pH 7.9/8.7% glycerol/0.1 mM EDTA/0.5 mM dithiothreitol/0.1 mM PMSF) containing 0.08 M KCI, 2 liters of HEPO.37 fraction was loaded onto a 500-mI DEAE-Sephacel column. The flow-through fraction (DEFT80), which contained the BTF1Y activity, was made 50 mM potassium phosphate (pH 6.3) and loaded onto a sulfopropyl-5PW (Toyo Soda, Tokyo) HPLC column equilibrated in the same buffer. Proteins were eluted with a linear 0.08-0.60 M KCl gradient. Active BTF1Y fractions, which eluted at 0.4-0.5 M KCl (SPO.4 fraction), were dialyzed against buffer B (without PMSF) containing 0.05 M KCl, and 25 ml was loaded onto a heparin-5PW (Toyo Soda) HPLC column. Proteins were eluted with a linear 0.05-0.60 M KCI gradient, with BTF1Y activity recovered at -0.5 M KCl (HEPO.5 fraction). Ten milliliters of the HEPO.5 fraction was dialyzed against buffer C (50 mM Tris-HCl, pH 7.9/17.4% glycerol/0.1 mM EDTA/0.5 mM dithiothreitol/5 mM MgCI2) containing 0..1 M KCl and loaded onto a 400 /ul/heparin-Ultrogel column, equilibrated in the same buffer. Proteins w...

We present evidence that transcription factor TFIID, known for its central role in transcription by RNA polymerase II, is also involved in RNA polymerase III transcription of the human U6 snRNA gene. Recombinant human TFIID, expressed either via a vaccinia virus vector in HeLa cells or in Escherichia coli, affects U6 transcription in three different in vitro assays. First, TFIID‐containing fractions stimulate U6 transcription in reactions containing rate‐limiting amounts of HeLa nuclear extract. Second, TFIID addition relieves transcriptional exclusion between two competing U6 templates. Third, TFIID can replace one of two heat labile fractions essential for U6 transcription. Thus, at least one basal transcription factor is involved in transcription by two different RNA polymerases.

Intramuscular administration of plasmid expressing fulllength human dystrophin in dystrophin-deficient adult mdx mice resulted in humoral and weak specific T cell responses against the human dystrophin protein. Following plasmid injection, human dystrophin was detected in the injected muscles at 7 days, but decreased thereafter. Anti-dystrophin antibodies were found 21 days following plasmid injection, which coincided with transient myositis. This immune rejec-