Gene 1994 Sep 02;1462:227-32. doi: 10.1016/0378-1119(94)90297-6.

PCR-based construction of promoter/G-free templates for in vitro transcription analysis allows selection of plasmids with optimal activity in homologous extracts.

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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.

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Genes referenced: LOC100893523 LOC575557