Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells and attempts to isolate the anthranilate synthase gene from plants

Analysis of the structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension cultured cell lines selected for glyphosate resistance was carried out to determine the mechanism of gene amplification in this plant system. Cloning of the EPSPS gene and 5;In attempts to isolate the anthranilate synthase (AS) gene from plants, antibodies were prepared from the purified carrot protein and purified bacterial AS to be used to screen a gene expression library. Carrot AS activity in native PAGE was localized and the region of enzyme activity was used as an antigen to raise antibodies in a rabbit. That antibody, denoted anti-Gel II, and an antibody against AS from Serratia marcescens did not inhibit AS activity of carrot. Anti-Gel II reacted to show two major bands of 65 kd and 67.5 kd in western blots of carrot, tobacco and potato, which could be good candidates to be the large subunit of AS as known by subunit size studies with microorganisms.;Bacterial and fungal AS genes were used to probe Southern blots of genomic DNA of many plant species to see if the heterologous probe can be used to screen the genomic DNA library. The approach using heterologous probes was not adequate for the isolation of the AS gene of plants due to the low homology of amino acids and nucleic acids sequences of the AS gene.;Attempts were made to isolate the Blue fluorescent gene (Bf1) which could be the AS gene by transposon tagging.The cosegregation of the restriction fragment with the mutant phenotype was followed with the Orf1 fragment of the En element as probe. We could not observe any bands cosegregating with the mutable allele with one parental line and two mutable lines using many methylation sensitive enzymes. Thus we could not clone the gene so this project has been discontinued.