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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 different beta-glucuronidase reporter fusions. We found that the fusion protein resides primarily in the nuclei of the meristems. Also other DNA repair genes in plants are primarily expressed in meristematic tissue. This is compatible with the model that repair genes play a crucial role in assuring fertility and fitness of the progeny. Currently, we are investigating the effect of different types of stress on the AtMBD4 promotor activity. For further characterization of AtMBD4, we generated silencing and overexpression lines, which do not show a phenotype under normal conditions. To determine the impact of AtMBD4 on mutation frequency, we analyzed plants with different AtMBD4 levels, via a detection system, developed in our lab. This allows to determine the mutation frequency of cytosine to thymine via the reactivation of a deactivated GUS gene. Finally, we investigated whether AtMBD4 plays a role in the signalization of DNA damage caused by mutagenic agents, as human and mouse MBD4 not only have a function in the recognition of G:T mismatches, but also play a role in the signalization of DNA damage caused by different mutagenic agents. The results will be discussed. ---------------------------------------------------------------------------------------------------------------------------------- P - 7. Gene targeting in plants via engineered zinc finger proteins Charles Cai, Vipula Shukla, Fyodor Urnov, Jeffery Miller, Nicole Arnold, Lisa Baker, Teresa Bauer, Ryan Blue, Dave McCaskill, Jon Mitchell, Matt Simpson, Andrew Worden. Plant Genetics and Biotechnology, Dow AgroSciences, 46268 Indianapolis, IN, United States Gene targeting via homologous recombination occurs at a very low frequency in plant cells compared to random integration making targeted gene modifications impractical. Most recently, substantial increases in the frequency of homologous recombination have been observed following the induction of double stranded breaks in host cell DNA followed by the apparent stimulation of cellular repair mechanisms. Strategies to achieve targeted DNA double stranded breaks have been developed by fusing sequence-specific zinc finger DNA binding proteins with sequence-independent nuclease domains derived from Type IIS restriction endonucleases. Using this strategy, both site-specific transgene integration and targeted modification of native genes have been demonstrated in plants. Implications for novel trait development and crop improvement will be discussed. ---------------------------------------------------------------------------------------------------------------------------------- P - 8. SITE-DIRECTED HOMOLOGOUS RECOMBINATION IN TOBACCO CELL CULTURES VIA ZINC FINGER NUCLEASES Charles Cai, Michael Ainley, Trevor Collingwoo, Robbi Garrison, Lisa Schulenberg, Philip Gregory, Beth Rubin-Wilson, Joseph Petolino. Biochemistry and Molecular Biology, Dow AgroSciences, LLC., 46268 Indianapolis, United States Targeted transgene integration via homologous recombination occurs at a very low frequency in plant cells compared to random integration, even when the incoming DNA comprises large stretches of sequence homologous to host DNA. As such, gene targeting in plants via homologous recombination has not been practical. Most recently, substantial increases in the frequency of homologous recombination have been observed following the induction of DNA double stranded breaks in host cells and apparent stimulation of cellular repair mechanisms. Restriction enzymes whose recognition sites are rare in the plant genome have been shown to stimulate homologous recombination following the formation and repair of DNA double stranded breaks in the host DNA. Strategies to achieve targeted DNA double stranded breaks have been developed by fusing zinc finger DNA binding proteins with sequence-independent nuclease domains derived from Type II restriction endonucleases. In the present study, engineered zinc finger proteins fused to nuclease domains, so-called ‘zinc finger nucleases’, were used to facilitate site-specific transgene integration via homologous recombination in tobacco cell cultures. A target DNA sequence was first stably integrated into tobacco cell cultures using Agrobacterium. This target sequence contained specific zinc finger protein recognition/binding sites, Page 32 sur 56
<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 along with a non-functional test gene to be corrected. A few selected transgenic events containing a single integrated copy of the target sequence were re-transformed using Agrobacterium strains harboring different T-DNAs. One Agrobacterium strain contained a donor DNA sequence comprising the bases necessary to correct the non-functional test gene, flanked by sequences homologous to the pre-integrated target DNA. The second Agrobacterium strain contained a gene encoding a zinc finger nuclease that specifically recognized a binding site in the integrated target sequence. Gene targeting via sitedirected homologous recombination was demonstrated as evidenced by the reconstitution of a functional test gene and was confirmed via molecular and biochemical analyses. ---------------------------------------------------------------------------------------------------------------------------------- P - 9. Gene and Protein Targeting Technologies Create Novel Opportunities in Plant Biotechnology Monique Compier, Patrick Mak, Sylvia de Pater, Bert van der Zaal, Paul Hooykaas, Remko Offringa , Add2X Biosciences BV, 2333 AL Leiden, Netherlands An important platform technology that is missing in the field of plant science is gene targeting through homologous recombination. Moreover, for many economically important plant varieties, robust and efficient protocols for vegetative propagation and genetic modification are still lacking. The availability of such technologies is essential for the further development of plant biotechnology. Add2X Biosciences BV is a young biotech spin-off from Leiden University that aims to create novel opportunities in plant biotechnology, by developing innovative platform technologies and products for the directed and efficient delivery of DNA and proteins into plant cells. The Add2X portfolio includes: 1. Efficient gene targeting in plants through suppression of the non-homologous recombination pathway. Proof of concept has been obtained in different yeasts and filamentous fungi. Currently, research is in progress to confirm the applicability of this technology in plant species. 2. Agrobacterium-mediated protein translocation to produce and transiently ‘inject’ proteins of interest into plant cells in order to exert their function without permanently altering the host cells. 3. Induction of somatic embryogenesis in hitherto recalcitrant crop species. A protein family was identified that, when overexpressed, stimulates the spontaneous formation of somatic embryos from vegetative plant cells. The Add2X technologies stand well on their own, but clearly have the potential to be combined into integrated technologies or products. The technologies are developed in close collaboration with Leiden University. In addition, Add2X has established strategic alliances with other academic and industrial partners to explore novel opportunities and to achieve rapid implementation of the technologies in the biotechnology sector. ---------------------------------------------------------------------------------------------------------------------------------- P - 10. Homologous recombination between divergent sequences in Arabidopsis Eyal Emmanuel, Elizabeth Yehuda, Roy Opperman, Naomi Avivi-Ragolsky, Avraham Levy, Cathy Melamed-Bessudo. Plant Sciences Department, The Weizmann Institute of Science, 76100 Rehovot, Israel Earlier studies in plants have shown that there is a strong reduction in the rate of homologous recombination when the recombining partners are chromosomal segments that originate from related, but divergent, species. The effect of sequence divergence on homologous recombination is not well established. We found that the rates of somatic recombination between repeats that diverge only by one out of 617 otherwise identical nucleotides, is reduced by ~ 3-fold. Similarly, the rates of meiotic recombination are higher in an isogenic background than in crosses with divergent ecotypes. We analyzed the role of AtMSH2 in suppression of recombination between divergent sequences in Arabidopsis. We report that AtMSH2 has a broad range of anti-recombination effects: it Page 33 sur 56
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Page 6 and 7: 1400-1700 SESSION V: Recombination
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Page 54 and 55: Cyril Charbonnel CNRS UMR6547 Unive
Page 56 and 57: Sean Mayes University of Nottingham
Page 58: Wanda Melody Waterworth University

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