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.
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 promotor 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.
Neuteboom
S T
ST
Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, The Netherlands.
Stoffels
A
A
Hulleman
E
E
Memelink
J
J
Schilperoort
R A
RA
Hoge
J H
JH
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D050660:Core Binding Factors; D004269:DNA, Bacterial / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D017343:Genes, Plant; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins; D009687:Nuclear Proteins / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism* / Q000382:microbiology; D014157:Transcription Factors / Q000378:metabolism*
D050660:Core Binding Factors; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D010940:Plant Proteins; C036483:T-DNA; D014157:Transcription Factors