Site-directed mutagenesis of the enhancer region of the 780 gene promoter of T-DNA

Abstract

Potential regulatory sequences within the enhancer-like region of the 780 gene promoter (Agrobacterium tumefaciens T-DNA) were identified by site-directed mutagenesis. Transcriptional activity of the mutated promoter was analyzed by S1 nuclease mapping of RNA from crown gall tumors of sunflower incited using a T-DNA-based vector. Variability in expression levels were minimized by the use of an internal reference gene and the pooling of at least 200 tumors per construct tested. This approach identified numerous sequences that influence transcriptional activity in either a positive or negative manner. Eight regions of positive influence and three of negative were identified from analysis of those mutations that exhibited low variability in expression (P < 0.005) and affected activity by at least 20%.