The primary goal of this project is the detailed characterization of cis- and transacting promoter elements involved in the transcriptional regulation of three T-DNA genes- the """"""""780"""""""" gene, and the genes encoding agropine synthase (AGS) and transcript 5 of T-left. The promoters for the 780 gene and AGS show little specificity and will serve as models for constitutive promoter design. The gene 5 promoter seems to be regulated in response to auxin/cytokinin levels in the plant and may yield information regarding developmentally regulated plant promoters. The specific objectives are: 1) to identify by linker scan mutagenesis cis-acting promoter elements; 2) to assess the function of cis- elements with regard to spacing, position, and polarity; 3) to characterize the hormone and tissue specificity of gene 5 expression by in situ hybridization using regenerated plants; and 4) to assess binding of trans-acting factors by in vivo and in vitro footprinting studies. An in depth characterization of cis-elements is required not only for understanding promoter structure, but to better evaluate the significance and possible function of trans- acting factor interactions. Analysis of transcription in tumors and regenerated plants using a reference gene as an internal standard will provide quantitative data regarding the fine structure of constitutively expressed and hormonally regulated T- DNA promoters active in calli and differentiated tissue. Nuclear factors will be characterized with regard to specificity of binding to DNA, factor abundance in different plant organs and tissues, and conservation of factors between plant species. DNA binding cooperatively experiments with purified nuclear factors may give insight regarding mechanisms of transcriptional initiation and induction. Just as mammalian viral genes have provided much basic information regarding eukaryotic promoter structure, the T-DNA genes are well suited to serve as models for promoter design in higher plants. The benefits of wide host range derived by Agrobacterium have driven selection in T-DNA promoters for utilization of eukaryotic transcriptional control mechanisms that are highly conserved among a wide range of higher plant species. Although T-DNA genes may be bacterial in evolutionary origin, and certainly by lineage, they have achieved a close mimicry of plant genes from a functional standpoint and in their interactions with the trans-acting factors.
| O'Grady, K; Gurley, W B (1995) Site-directed mutagenesis of the enhancer region of the 780 gene promoter of T-DNA. Plant Mol Biol 29:99-108 |
| Adams, E C; Gurley, W B (1994) Nuclear protein 780BP from cauliflower binds an element in the 780 gene promoter of T-DNA. Plant Mol Biol 26:377-92 |
