Regulation of beta-glucuronidase expression in transgenic tobacco plants by an A/T-rich, cis-acting sequence found upstream of a French bean beta-phaseolin gene.
A 0.8-kilobase fragment from the 5'-flanking region of a French bean beta-phaseolin gene yielded strong, temporally regulated, and embryo-specific expression of beta-glucuronidase (GUS) in transgenic tobacco plants, paralleling that found for the seed protein phaseolin [Sengupta-Gopalan, C., Reichert, N.A., Barker, R.F., Hall. T.C., and Kemp, J.D. (1985) Proc. Natl. Acad. Sci. USA 82, 3320-3324]. Gel retardation and footprinting assays using nuclear extracts from immature bean cotyledons revealed strong binding of nuclear proteins to an upstream region (-628 to -682) that contains two inverted A/T-rich motifs. Fusion of a 103-base pair fragment or a 55-base pair synthetic oligonucleotide containing these motifs to a minimal 35S promoter/GUS cassette yielded strong GUS expression in several tissues. A different pattern of GUS expression was obtained in immature embryos and germinating seedlings from the nominally constitutive, full-length, 35S promoter. Whereas GUS expression under the control of the 0.8-kilobase beta-phaseolin regulatory region is limited to immature embryos, expression from constructs containing the A/T-rich motifs is strongest in roots. These data, combined with S1 mapping, provide direct evidence that a plant upstream A/T-rich sequence that binds nuclear proteins can activate transcription in vivo. They also indicate that additional regulatory elements in the beta-phaseolin 5'-flanking region are required for embryo-specific gene expression.
Bustos
M M
MM
Department of Biology, Texas A&M University, College Station 77843-3258.
Guiltinan
M J
MJ
Jordano
J
J
Begum
D
D
Kalkan
F A
FA
Hall
T C
TC
D001482:Base Composition; D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism; D007887:Fabaceae / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009928:Organ Specificity / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified; D010946:Plants, Medicinal; D010947:Plants, Toxic; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D014026:Nicotiana / Q000235:genetics*; D014158:Transcription, Genetic
D009687:Nuclear Proteins; D010940:Plant Proteins; C048826:phaseolin protein, Phaseolus vulgaris; D004247:DNA; D005966:Glucuronidase