The transposable elements Activator/Dissociation ( Ac/Ds ) were first discovered in maize, yet they have not been used extensively in their native host for gene-tagging experiments. This can be attributed largely to the low forward mutation rate and the propensity for closely linked transpositions associated with Ac and its nonautonomous deletion derivative Ds . To overcome these limitations, we are developing a series of nearly isogenic maize lines, each with a single active Ac element positioned at a well-defined location. These Ac elements are distributed at 10-to 20-centimorgan intervals throughout the genome for use in regional mutagenesis. Here, we demonstrate the utility of this Ac-based gene-tagging approach through the targeted mutagenesis of the pink scutellum1/viviparous7 ( ps1/vp7 ) locus. Using a novel PCR-based technique, the Ps1 gene was cloned and Ac elements positioned precisely in each of the seven alleles recovered. The Ps1 gene is predicted to encode lycopene ␤ -cyclase and is necessary for the accumulation of both abscisic acid and the carotenoid zeaxanthin in mature maize embryos. This study demonstrates the utility of an Ac mutagenesis program to efficiently generate allelic diversity at closely linked loci in maize.

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A collection of Activator (Ac)-containing, near-isogenic W22 inbred lines has been generated for use in regional mutagenesis experiments. Each line is homozygous for a single, precisely positioned Ac element and the Ds reporter, r1-sc:m3. Through classical and molecular genetic techniques, 158 transposed Ac elements (tr-Acs) were distributed throughout the maize genome and 41 were precisely placed on the linkage map utilizing multiple recombinant inbred populations. Several PCR techniques were utilized to amplify DNA fragments flanking tr-Ac insertions up to 8 kb in length. Sequencing and database searches of flanking DNA revealed that the majority of insertions are in hypomethylated, low-or single-copy sequences, indicating an insertion site preference for genic sequences in the genome. However, a number of Ac transposition events were to highly repetitive sequences in the genome. We present evidence that suggests Ac expression is regulated by genomic context resulting in subtle variations in Ac-mediated excision patterns. These tr-Ac lines can be utilized to isolate genes with unknown function, to conduct fine-scale genetic mapping experiments, and to generate novel allelic diversity in applied breeding programs.

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The Mutator system of maize consists of more than eight different classes of transposable elements each of which can be found in multiple copies. All Mu elements share the approximately 220-bp terminal inverted repeats, whereas each distinct element class is defined by its unique internal sequences. The regulation of instability of this system has been difficult to elucidate due to its multigenic inheritance. Here we present genetic experiments which demonstrate that there is a single locus, MuR1, which can regulate the transposition of Mu1 elements. We describe the cloning of members of a novel class of Mu elements, MuR, and demonstrate that a member of the class is the regulator of Mutator activity, MuR1. This conclusion is based on several criteria: MuR1 activity and a MuR-homologous restriction fragment cosegregate; when MuR1 undergoes a duplicative transposition, an additional MuR restriction fragment is observed, and MuR1 activity and the cosegregating MuR fragment are simultaneously lost within clonal somatic sectors. In addition, the MuR element hybridizes to transcripts in plants with Mutator activity. Our genetic experiments demonstrate that the MuR1 transposon is necessary to specify Mutator activity in our lines.