Site-specific mutations alter in vitro factor binding and change promoter expression pattern in transgenic plants.

E. Lam, P. N. Benfey, P. M. Gilmartin, R. X. Fang, N. H. Chua

Research output: Contribution to journalArticlepeer-review

323 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)7890-7894
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume86
Issue number20
DOIs
StatePublished - Oct 1989
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • General

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