Structure of the octopine synthase upstream activator sequence
Abstract
Virulent strains of the soil bacterium Agrobacterium tumefaciens possess a large Ti (Tumor inducing) plasmid, a portion of which, the T-DNA (Transferred DNA), is transferred into the nuclear genome of wounded dicotyledonous (and some monocotyledonous) plants. We have been studying the regulation of a T-DNA-encoded gene, the octopine synthase (ocs) gene. Octopine synthase catalyses the reductive condensation of arginine and pyruvate to form the opine, octopine. The regulation of this gene is interesting because it is harbored on a prokaryotic plasmid and is normally silent within the bacterium. When the gene is transferred into an eukaryotic host, the gene then becomes active. Our study was initiated to determine whether the ocs gene possesses a transcriptional enhancer and to determine the cis-acting motifs that make up this putative element. Within the 5$\sp\prime$ flanking sequence of the ocs gene, we identified a transcriptional activating element that is necessary for ocs expression in tobacco calli. This element is located between 333 and 116 bp upstream from the transcription initiation site and, in both orientations, activates the ocs promoter when placed 5$\sp\prime$ of the promoter. It does not activate the ocs promoter in either orientation when placed downstream of the gene, nor can it activate the promotor when separated by a distance of 608 bp. Deletion analysis indicates that the ocs element is composed of at least four separate regions. A 16 bp palindrome is the only region that can function autonomously, but this sequence is only about one-third as active as is the entire element. Sequences flanking the palindrome, both 5$\sp\prime$ and 3$\sp\prime$ augment the activity of the palindrome. These are termed the 5$\sp\prime$ and 3$\sp\prime$ PAS (palindrome augmenting sequences). DNA sequences 5$\sp\prime$ to the 5$\sp\prime$ PAS seem to inhibit transcription, and may interact with the 3$\sp\prime$ PAS in some fashion. Although the 16 bp palindrome is sufficient for activity in calli and the leaves of small (2 cm tall) shoots, it is insufficient to activate the ocs promoter in the leaves of rooted plants. These data suggest that the palindrome is sensitive to inactivation in differentiated tissues.
Degree
Ph.D.
Advisors
Gelvin, Purdue University.
Subject Area
Molecular biology
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