Plant Genome I Conference
Town & Country Conference Center, San Diego, CA, November, 1992.
PG-I: A METHOD FOR MUTATION, GENOMIC REARRANGEMENT AND MOLECULAR
CLONING BASED ON THE DS TRANSPOSON AND THE CRE-LOX RECOMBINATION
SYSTEM
A METHOD FOR MUTATION, GENOMIC REARRANGEMENT AND MOLECULAR
CLONING BASED ON THE DS TRANSPOSON AND THE CRE-LOX RECOMBINATION
SYSTEM.
Brian I. Osborne, Uwe Wirtz, and Barbara Baker, Plant Gene
Expression Center, U.C. Berkeley/U.S.D.A., 800 Buchanan St.,
Albany, CA, USA 94710.
We have developed a series of T-DNA vectors for insertion
and deletion mutagenesis, genomic rearrangement, and molecular
cloning for use in Arabidopsis and other species. The basic
design uses genetically marked Ds transposons and transcriptional
fusions which place the Ac transposase ORF under the control of
strong promoters. The Ds will transpose from its T-DNA in hybrid
plants bearing both Ds and a promoter::Ac transposase gene -
subsequent segregation of Ds away from the transposase gene will
stabilize Ds insertions. We have included lox sites in both Ds
and its T-DNA, derived from the Cre-lox site-specific
recombination system of bacteriophage PI. Plants bearing
transposed, mapped, stable Ds-lox and mapped T-DNA-lox will be
crossed to plants bearing a gene expressing Cre recombinase.
Recombination between the lox sites in the two elements in vivo
will generate chromosomal rearrangements. Since Ds transposes
preferentially to linked sites, we expect precise deletions to be
generated. In addition, we expect to generate inversions and
translocations with molecularly defined endpoints. The T-DNA-lox
and Ds-lox both contain plant selectable markers, as well as
yeast selectable markers, E. coli selectable markers, and
"rare-cutter" sites. These elements should allow cloning of the
DNA within or adjacent to the rearranged interval.
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