Site-specific mutations alter in vitro factor binding and change promoter expression pattern in transgenic plants.
The 35S promoter of cauliflower mosaic virus (CaMV) is able to confer high-level gene expression in most organs of transgenic plants. A cellular factor from pea and tobacco leaf tissue, which recognizes nucleotides in a tandemly repeated TGACG motif at the -75 region of this promoter, has been detected by DNase I footprinting and gel retardation assays. This factor is named activation sequence factor 1 (ASF-1). A cellular factor binding to the two TGACG motifs can also be detected in tobacco root extracts. Mutations at these motifs inhibit binding of ASF-1 to the 35S promoter in vitro. When examined in transgenic tobacco, these mutations cause a 50% drop in leaf expression of the 35S promoter. In addition, these same mutations attenuate stem and root expression of the 35S promoter about 5- to 10-fold when compared to the level of expression in leaf. In contrast, mutations at two adjacent CCAAT-box-like sequences have no dramatic effect on promoter activity in vivo. A 21-base-pair element containing the two TGACG motifs is sufficient for binding of ASF-1 in vitro when inserted in a green-tissue-specific promoter. In vivo, the insertion of an ASF-1 binding site caused high levels of expression in root. Thus, a single factor binding site that is defined by site-specific mutations is shown to be sufficient to alter the expression pattern of promoters in vivo.
Lam
E
E
Laboratory of Plant Molecular Biology, Rockefeller University, New York, NY 10021.
Benfey
P N
PN
Gilmartin
P M
PM
Fang
R X
RX
Chua
N H
NH
D001483:Base Sequence; D003850:Deoxyribonuclease I; D007887:Fabaceae / Q000235:genetics; D015870:Gene Expression; D005786:Gene Expression Regulation; D005814:Genes, Viral; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000235:genetics; D009154:Mutation; D010944:Plants / Q000235:genetics*; D010946:Plants, Medicinal; D010957:Plasmids; D011401:Promoter Regions, Genetic
D003850:Deoxyribonuclease I