The tumor-inducing genes of the Ti plasmid harbored in Agrobacterium tumefaciens are delivered to the host plant cell where it is integrated into the chromosome by a very unique process during infection. This process involves the cleavage and release of the transferred DNA (T-DNA), which is catalyzed by enzymes encoded by virulence (vir) genes that are organized as a regulon within a 28.63-kilobase pair stretch of the Ti plasmid. The regulon contains seven vir operons (A, B, G, C, D, H & E), where the products of virA and virG make up the sensor/regulator protein pair of two-component regulatory family. VirG positively regulates the remaining vir operons when activated by VirA after the latter membrane spanning protein senses plant phenolic compounds. The expression of vir genes thus leads to T-intermediate formation. This formation is catalyzed by the products of virD and enhanced by virC, two operons that are also negatively regulated by the ros chromosomal gene. This gene encodes a repressor specific for regulating virC and virD. The product of the OS gene of pSa, which completely prevents oncogenicity, does not interfere with vir gene transcription or T-intermediate formation. Its mode of action may involve interaction with one or more products of the virB and/or virD operons and its analysis will provide insight on the T-DNA processing/transfer mechanism. The presence of two regulatory pathways raises a number of interesting questions on their regulatory mechanisms. Thus, the planned research will include generating detailed understanding of the function of: 1) the VirG protein in the positive regulation of vir genes; 2) Agrobacterium chromosomal genes with special focus on a negative regulator Ros; and 3) pSa oncogenic suppression to investigate T-DNA processing/transfer mechanisms. The research will employ a combination of genetic and biochemical techniques such as site-directed mutagenesis via polymerase chain reaction, the measurement of gene expression levels in vivo using a lux reporter system coupled with a photon imaging analytical system, in vitro and in vivo DNA-protein binding analyses, a novel in vivo affinity purification technique along with chromatographic purification of gene products, DNase and chemical footprinting, a novel cascade immunofluorescence photon imaging analytical system for analyzing perturbations of proteins; a novel transcriptional interference assay for measuring DNA binding and other techniques to facilitate generation of fundamental information on the above subjects. Much of this research will be facilitated by the availability of a number of molecular """"""""tools"""""""" and techniques developed in this laboratory during previous and present grant periods.
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