The system employed here utilizes a transgene construct containing maize Ac/Ds transposon ends in direct orientation within an I/dSpm transposon (Ned1; Nested deletions 1). The Ned1 construct will be transformed into maize; subsequent crosses will introduce the En/Spm transposase to mobilize Ned1 to various genomic locations, and the Ac transposase to activate the deletion process. The action of Ac transposase on the Ac termini within Ned1 generates an unlimited set of nested deletions with one end anchored at the transgene locus. The Ned1 construct contains marker genes for detection of both Ned1 transpositions and Ac-induced deletions, as well as sequences for easy cloning of deletion endpoints via plasmid rescue.
If this demonstration project is successful, this approach could, in the future, be extended to the production of a set of maize lines containing Ned1 elements at dispersed sites throughout the genome. Researchers could then use these lines to isolate deletions and other rearrangements in specific regions of the genome for a variety of research applications:
1. Nested deletions will facilitate mapping of molecular and genetic markers to defined intervals. Deletions can be used in simple and robust assays for mapping molecular markers; similarly, genetic mutants are easily mapped by test crossing.
2. Deletions will be useful for assigning functions to individual gene copies within complex loci, such as disease resistance loci.
3. Deletions and duplications will be useful in gene dosage studies that seek to examine the effects of gene copy number on expression and silencing.
4. Deletion endpoints can provide landmarks that are critical for genome
sequencing projects. Such landmarks can be used for the unambiguous alignment
of YAC or BAC clones which may contain multiple repetitive sequence elements.
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