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Interaction between the tobacco DNA‐binding activity CBF and the cyt ‐1 promoter element of the Agrobacterium tumefaciens T‐DNA gene T‐ CYT correlates with cyt ‐1 directed gene expression in multiple tobacco tissue types
Author(s) -
Neuteboom Saskia T.C.,
Stoffels Arjan,
Hulleman Esther,
Memelink Johan,
Schilperoort Rob A.,
Hoge J. Harry C.
Publication year - 1993
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1993.04030525.x
Subject(s) - microbiology and biotechnology , promoter , biology , agrobacterium tumefaciens , binding site , gene , response element , core binding factor , biochemistry , gene expression , transcription factor , transgene
A novel DNA‐binding activity, designated CBF, has been identified in nuclear extracts from tobacco leaf, stem and root tissue. CBF interacts specifically with a 30 bp promoter fragment, referred to as cyt ‐1, of the Agrobacterium tumefaciens T‐DNA cytokinin (T‐ cyt ) gene. The T‐ cyt promoter, although of bacterial origin is active in planta and the 30 bp cyt ‐1 element is located within a region that is essential for T‐ cyt promoter activity in leaf, stem and root cells of tobacco plants. Gel retardation assays using different synthetic oligonucleotides and methylation interference experiments pinpointed the binding site of CBF to a GC‐rich sequence ATGCCCCACA within the cyt ‐1 element. Site‐directed mutagenesis of the CBF binding site within the T‐ cyt promoter by using PCR resulted in an almost complete loss of T‐ cyt promoter activity in transgenic tobacco plants. In a gain‐of‐function experiment a hexamer of cyt ‐1 was shown to be able to confer leaf, stem and root expression when fused upstream of a TATA box containing −55 derivative of the T‐ cyt promoter. A mutant cyt ‐1 hexamer, defective in CBF binding, did not show activity above background levels. These results indicate that binding of CBF to the cyt ‐1 element is required for cyt ‐1 directed gene expression, suggesting that CBF might act as a transcriptional activator. Apart from the ASF‐1 binding site of the CaMV 35S promoter, which is also present in the T‐DNA nopaline and octopine synthase genes, the cyt ‐1 element is the only other identified element reported until now that in combination with a TATA box is sufficient to drive gene expression in multiple tobacco tissue types.