title: PCR-based construction of promoter/G-free templates for in vitro transcription analysis allows selection of plasmids with optimal activity in homologous extracts.
abstract: In vitro transcription has been used for dissecting transcriptional controls in many eukaryotic systems. One modification which greatly reduces background non-specific transcription is the placement of a guanosine-free (G-free) region of DNA immediately downstream from a promoter [Sawadogo and Roeder, Proc. Natl. Acad. Sci. USA 82 (1985) 4394-4398]; transcription in the presence of RNase T1 and 3' O-Me-GTP eliminates non-specific transcripts, and produces the G-free transcripts initiated at the promoter. Restriction site-based fusion of a G-free cassette downstream from promoters is complicated by the requirement for G nucleotides to be excluded from the coding strand downstream from the transcription start points. We present an approach to add a G-free template onto a eukaryotic promoter by combining PCR-based termini construction and terminal deoxynucleotidyl transferase extension. The pisatin demethylase promoter (PDA1p) of the filamentous fungus Nectria haematococca was used as the test promoter. Three PDA1p/G- free constructs were tested in heterologous Drosophila melanogaster and HeLa and homologous N. haematococca transcription extracts. Each extract produced a PDA1p-specific transcript from each construct, but the relative level of transcription between constructs varied, particularly in the homologous extract. Since the choice of G-free sequence influences transcription differently among systems, this method for producing multiple G-free constructs should be useful for constructing and selecting optimal promoter/G-free templates for in vitro transcription in other homologous systems.
authors.affiliation: Department of Botany, University of Maryland, College Park, 20742.
authors.forename: Y
authors.initials: Y
authors.lastname: Ruan
authors.forename: D C
authors.initials: DC
authors.lastname: Straney
mesh_terms: D000818:Animals; D001483:Base Sequence; D004331:Drosophila melanogaster / Q000235:genetics; D005670:Fusarium / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics* / Q000502:physiology; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics*; D006151:Guanosine; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
chemical_list: D006151:Guanosine