CHARACTERIZATION OF A GENE ENCODING A DNA-BINDING PROTEIN THAT INTERACTS IN-VITRO WITH VASCULAR SPECIFIC CIS-ELEMENTS OF THE PHENYLALANINE AMMONIA-LYASE PROMOTER

A study of the expression of a bean phenylalanine ammonia-lyase (PAL) promoter/beta-glucuronidase gene fusion in transgenic tobacco has show n that the PAL2 promoter has a modular organization. Expression of the PAL;! promoter in the vascular system involves positive and negative regulatory cis elements. Among these elements is an AC-rich motif impl icated in xylem expression and a suppressing cis element for phloem ex pression. Using radiolabelled complementary oligonucleotides bearing t he AC-rich motif, a cDNA clone encoding a DNA-binding protein has been isolated from a tobacco lambda gt11 expression library. This factor, named AC-rich binding factor (ACBF), showed binding specificity to the AC-rich region. The specificity of ACBF for the AC-rich region was al so shown using a gel retardation assay with an ACBF recombinant protei n extract. The deduced amino acid sequence from ACBF contains a long r epeat of glutamine residues as found in well characterized transcripti on factors. Interestingly, ACBF shared sequence similarity to conserve d amino acid motifs found in RNA-binding proteins. Genomic gel blot an alysis indicated the presence of a small gene family of sequences rela ted to ACBF within the tobacco nuclear genome. Analysis of tobacco mRN A using the ACBF cDNA as probe showed that while ACBF mRNA was present in all tissues examined, the highest transcript accumulation occurred in stem tissues. The functional characteristics of the AC-rich sequen ce were examined in transgenic tobacco. A heptamer of the AC-rich sequ ence, in front of a minimal 35S promoter from cauliflower mosaic Virus (-46 to +4), conferred specific expression in xylem.