Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A promoter.
A deletion analysis of the Arabidopsis thaliana rbcS-1A promoter defined a 196 bp region (-320 to -125) sufficient to confer light-regulated expression on a heterologous Arabidopsis alcohol dehydrogenase (Adh) reporter gene in transgenic Nicotiana tabacum (tobacco) leaves. This region, which contains DNA sequences I, G and GT boxes, with homology to other ribulose-1,5-bisphosphate carboxylase small subunit (RBCS) gene promoter sequences, directed expression independent of orientation and relative position in the Adh promoter. Site-specific mutagenesis of these conserved sequences and subsequent expression analysis in transgenic tobacco showed that both G box and I box mutations in the context of the full (-1700 to +21) rbcS-1A promoter substantially reduced the expression of Adh and beta-glucuronidase (GUS) reporter genes. The G box has previously been shown to specifically bind in vitro a factor isolated from nuclear extracts of tomato and Arabidopsis. This factor (GBF) is distinct from the factor GT-1 which binds to adjacent GT boxes in the pea rbcS-3A promoter. Multiple mutations in putative Arabidopsis rbcS-1A promoter GT boxes had no pronounced affect on expression, possibly due to a redundancy of these sites. Experiments in which rbcS-1A promoter fragments were fused to truncated 35S CaMV (cauliflower mosaic virus) promoter--GUS reporter constructs showed that cis-acting CaMV promoter elements could partially restore expression to G-box-mutated rbcS-1A sequences.
Donald
R G
RG
Department of Biology, University of Pennsylvania, Philadelphia 19104.
Cashmore
A R
AR
D000426:Alcohol Dehydrogenase / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D004357:Drug Synergism; D005966:Glucuronidase / Q000235:genetics; D008027:Light; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000235:genetics; D009154:Mutation; D010944:Plants / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics*
D004247:DNA; D000426:Alcohol Dehydrogenase; D005966:Glucuronidase; D012273:Ribulose-Bisphosphate Carboxylase