75 Integrating Membrane Transport with Male Gametophyte ... - TAIR
75 Integrating Membrane Transport with Male Gametophyte ... - TAIR
75 Integrating Membrane Transport with Male Gametophyte ... - TAIR
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91 Approaches to Study Homologous Recombination in Arabidopsis<br />
Erica Unger-Wallace, Xianyan Kuang, David Wright, Daniel Voytas, Thomas Peterson<br />
Department of Genetics, Development & Cell Biology, Iowa State University, Ames, IA, USA<br />
There are many tools available to the Arabidopsis research community for investigations of gene function. However,<br />
a critical capability currently lacking is a means to edit specific gene sequences using homologous recombination, or gene<br />
targeting. Our strategy is to utilize enzymes, such as Ac transposase, and gene-specific zinc finger nucleases to generate a<br />
double strand break (DSB) at target chromosomal loci. Such locus specific breaks are known to stimulate recombination<br />
in many systems, including plants. In addition we are testing whether expression of the yeast Rad54 protein, together<br />
<strong>with</strong> a DSB, induces a higher recombination frequency than either of these factors alone <strong>with</strong> the goal of developing<br />
an efficient approach to gene targeting in Arabidopsis. Model transgenic target loci have been generated that contain a<br />
defective GUS-NPTII gene interrupted by either a non-autonomous Ds transposable element or the recognition sequence<br />
for a zinc finger nuclease. Donor DNA, designed to repair the gene and restore function and either Ac transposase or the<br />
zinc finger nuclease, are introduced by Agrobacterium-mediated transformation. Putative recombinant plants are selected<br />
using a kanamycin resistant phenotype conferred by correction of the defective reporter gene. GUS expression and<br />
molecular analysis of these plants serves to characterize the types of recombination events recovered. Similarly, native<br />
Arabidopsis loci <strong>with</strong> Ds insertions have been selected and will be examined to determine the frequency of recombination<br />
across several locations in the Arabidopsis genome.<br />
92 Characterization of Arabidopsis mRNA Polyadenylation Machinery: Genetic and<br />
Biochemical Analysis of yPcf11p Homologous<br />
Denghui Xing, Min Mo, Q. Quinn Li<br />
Department of Botany, Miami University, Oxford, Ohio 45056<br />
The 3'-end cleavage and polyadenylation of eukaryotic pre-mRNA involves several protein complexes containing<br />
more than a dozen of subunits. Yeast (S. cerevisiae) Pcf11p is one of these subunits and is essential for appropriate 3'-end<br />
processing. yPcf11p interacts <strong>with</strong> Rna14p, Rna15p and ClpIII subunits, formimg the CFIA complex of polyadenylation<br />
machinery. In addition, through interacting <strong>with</strong> CTD domain of RNA Pol II largest subunit, yPcf11p is also a factor for<br />
transcription termination. The biochemical and biological function of yPcf11p is thought to be largely conserved between<br />
yeast and mammals. However, little is known about plant counterpart of yPcf11p. We have identified a small gene family<br />
of Arabidopsis Pcf11p homologous and other components of CFIA. To elucidate the significance of these Arabidopsis<br />
proteins in polyadenylation and transcription termination, we have tested their interaction <strong>with</strong> other polyadenylation<br />
factors using yeast two-hybrid and in vitro pull-down assays. Analysis of T-DNA knockouts of Arabidopsis genes encoding<br />
homologous of yPcf11p revealed potential interesting phenotypes. The results will be presented.
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