THE Arabidopsis trp1-100 MUTANT AS A MODEL SYSTEM FOR CHIMERIC RNA/DNA OLIGONUCLEOTIDE-DIRECTED GENE CONVERSION

¹ Kimeragen, Inc., Newtown, PA, U.S.A.,

² Boyce Thompson Institute for Plant Research, Tower Rd., Ithaca, NY, 14850.

³ Plant Biology Division, The Samuel Roberts Noble Foundation, Inc., 2510 Sam Noble Parkway Ardmore, OK 73402.

Chimeraplasty is a novel technique of in situ genome modification using chimeric oligonucleotides (or chimeraplasts) composed of DNA and modified RNA residues. The in vivo targeting of endogenous and transgenes in plants has recently been described (Zhu et al. 1999, PNAS 96:8768-8773 and Beetham et al. 1999 PNAS 96:8774-8778). Here we present the initial development of a non-selectable model system for monitoring in vivo chimeraplast-directed gene conversion in plants. Our initial results describe the in vivo modification of the Arabidopsis thaliana trp1-100 mutation site using chimeraplasty. The trp1-100 mutation has been characterized as a single nucleotide change in the phosphoribosylanthranilate transferase (PAT) gene. The PAT enzyme is involved in the second step of the tryptophan biosynthetic pathway. Our results suggest that we have successfully introduced chimeraplasts (designed to introduce the trp1-100 mutation) into A. thaliana protoplasts by electroporation and, as a result, both alleles of the PAT gene have been modified. The blue fluoresence phenotype of these protoplasts, visualized with UV light, is indicative of a build up of anthranilic acid similar to the trp1-100 mutant.