Octopine synthase mRNA isolated from sunflower crown gall callus is homologous to the Ti plasmid of Agrobacterium tumefaciens

Abstract

The structural gene for octopine synthase (a crown gall-specific enzyme) is located in a central portion of the T-DNA that came from the Ti plasmid of A. tumefaciens and is expressed after it has been transferred to the plant cells. Polyadenylylated RNA was prepared from polysomes isolated from an octopine-producing crown gall callus and purified by selective hybridization to 1 of 5 recombinant plasmids. Each such plasmid contained a different fragment of T-DNA of pTi-15955 (octopine-type Ti plasmid). Purified mRNA was translated in vitro in rabbit reticulocyte lysates, and the translation products were immunoprecipitated with antibody against octopine synthase. Total and immunoprecipitated products were characterized by their molecular weights. A polypeptide of MW 40,000 (the same as authentic octopine synthase) was synthesized in vitro by crown gall mRNA selectively hybridized to 3 of the 5 fragments of T-DNA and precipitated with antibody against octopine synthase. This polypeptide was not immunoprecipitated with normal rabbit antibody nor was it synthesized when mRNA from the habituated callus was substituted. A mRNA 1500 bases long was detected when total mRNA was fractionated on an agarose gel, transferred to nitrocellulose and used for hybridization to 3 of the 5 32P-labeled T-DNA fragments. This apparent mRNA for octopine synthase hybridized to the same 3 fragments of T-DNA as the mRNA for the MW 40,000 polypeptide and was not detected in the habituated callus.