Liza J. Conrad

Transposon tagging is an especially important tool for gene identification and characterization in many cereal crops. In maize the Activator/Dissociation (Ac/Ds) tranpsoson family has been used extensively to clone and characterize genes. Specifically, a collection of Ac-containing, near-isogenic lines has been generated for use in regional mutagenesis experiments. Through molecular genetic techniques, 158 transposed Ac elements (tr-Acs) were distributed throughout the genome and 41 were placed on the linkage map. These tr-Ac lines can be utilized to isolate and characterize genes with unknown function. I have identified and characterized an immobilized Ac derivative (Ac-im) that is incapable of excision yet contributes to the negative dosage effect of Ac. Ac-im is identical to Ac with the exception of a 10 bp deletion of sequences at the left end of the element. A model for the generation of Ac-im was proposed in which Ac-im was generated as an aberrant transposition event that resulted in the deletion of Ac end sequences. To understand the mechanism of Ds formation, a high-throughput genetic and molecular screen was developed to identify rare Ds derivatives generated from Ac insertions in the genome. Using these methods 15 new Ds elements derived from Ac insertions at 8 different loci were discovered. We propose that all Type II Ds elements in maize are generated through a process of slip mispairing during error-prone DNA repair. The endogenous Activator (Ac)/Dissociation (Ds) transposons of maize are tightly regulated in the genome. In an attempt to identify regions of Ac involved in Ac regulation in maize a chemical mutagenesis experiment was performed using ethyl methanesulfonate (EMS). Several derivatives of Ac-im were discovered that has altered expression patterns. Although most alleles were associated with increased methylation, a single EMS-induced allele was recovered. The Ac-im'-0534 derivative displays a positive dosage effect instead of the canonical negative dosage of a wild-type Ac and accumulates nearly 4-fold lower transcript levels compared to wild-type. The EMS mutation introduces a translation start codon in the 5' UTR of the Ac-im'0534 derivative. Understanding Ac regulation in maize could lead to the manipulation Ds excision rates in mutagenesis programs.