Ds Transposon

Using the Ds element present in pDS-Lox

• pDS-Lox does not express any transposase protein

• The Ds element in pDs-Lox is completely stable in the absence of transposase

• pDS-Lox lines must be crossed to a plant expressing Ac transposase in order to mobilize the transposon

Figure 1.  pDs-Lox vector.  This vector was used to create the new Wisconsin Collection of T-DNA lines. Everything between the two Ds border sequences "hops" out of the T-DNA vector during transposition. The Ds element present in pDS-Lox will not transpose until the line carrying pDS-Lox is crossed to a plant that expresses the Ac transposase protein. T-DNA lines created using pDS-Lox can therefore be used directly for reverse-genetic analysis of single genes, and the insertions will remain stable.  Click here to view a detailed legend to Figure 1 in a new window.  pDs-Lox is a derivative of the plasmid pED204 (Medberry et al, 1995).  To view the annotated sequence of pDs-Lox  click here.

Figure 2.  Using pDS-Lox as a Ds Launch-Pad for local mutagenesis.  By crossing a T-DNA line carrying this vector to a plant that expresses the Ac transposase protein, one can cause the Ds element to transpose to linked regions of the genome. The structure of the two DNA elements that result from the excision of the Ds transposon are shown. Excision of the Ds element can be positively selected for using Hygromycin resistance. PCR can then be used to identify Ds insertions that have occured in the region of interest.

Is the Transposon functional?

We have tested the functionality of the Ds element present in pDs-Lox and found that it is indeed able to transpose.  This experiment involved taking one of the lines from our pDs-Lox population and crossing it with a plant that expresses the Ac transposase under the control of the 35S promoter.  We selected hygromycin resistant progeny from parent plants that were hemizygous for both the pDs-Lox insertion and the Ac expression construct.   TAIL PCR was then used to map the locations of several transposed Ds elements (Krysan et al, unpublished data).  These results demonstrated that the Ds element present in pDs-Lox is a functional transposon.

Because the Ac expression line that we used in these experiments is from the Landsberg ecotype, it is not the ideal line to use for crossing with pDs-Lox lines, which are in the Columbia ecotype.  We are in the process of developing Columbia lines that express the Ac transposase and will make an announcement on this web site when those lines are available.