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PLANT<br />
DNA Repair<br />
& Recombination<br />
Invited Speakers<br />
Anne Britt USA<br />
Zac Cande USA<br />
Greg Copenhaver USA<br />
Chris Franklin UK<br />
Jim Haber USA<br />
John Hays USA<br />
Barbara Hohn Switzerland<br />
Paul Hooykaas Holland<br />
Shigeru Lida Japan<br />
Avraham Levy Israel<br />
Rob Martienssen USA<br />
Jerzy Paszkowski Switzerland<br />
Holger Puchta Germany<br />
Karel Riha Austria<br />
Dorothy Shippen USA<br />
Dan Voytas USA<br />
Cliord Weil USA<br />
Charles White France<br />
Co-Sponsors<br />
BIOGEMMA<br />
www.biogemma.com<br />
Centre National de la Recherche Scientique<br />
www.cnrs.fr<br />
CSREES - USDA<br />
http://www.usda.gov/wps/portal/usdahome<br />
DOW Agrosciences<br />
http://www.dowagro.com/homepage/index.htm<br />
National Science Foundation<br />
http://www.nsf.gov<br />
Pioneer-DuPont<br />
http://www.pioneer.com<br />
Enquiries<br />
cw@univ-bpclermont.fr<br />
http://cwp.embo.org/w30-07<br />
<strong>EMBO</strong> WORKSHOP<br />
Session Titles<br />
31 May–3 June | 2007<br />
Presqu’île de Giens | France<br />
Somatic DNA repair and recombination<br />
Chromatin and Epigenetics<br />
Chromosome maintenance and architecture<br />
Recombination and Meiosis<br />
Recombination and related technologies<br />
Organisers<br />
Barbara Hohn<br />
Friedrich Miescher Institute for Biomedical Research, Switzerland<br />
Avraham Levy<br />
Weizmann Institute of Science, Israel<br />
Holger Puchta<br />
Karlsruhe University, Germany<br />
Dorothy Shippen<br />
Texas A&M University, USA<br />
Cliord Weil<br />
Purdue University, USA<br />
Charles White<br />
Centre National de la Recherche Scientique, France
<strong>EMBO</strong> Plant DNA Repair & Recombination<br />
Workshop<br />
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Presqu'île de Giens, France<br />
31 May - 3 June 2007<br />
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15.00-18.00 Check in and registration<br />
19.00-20.15 Dinner<br />
<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
31 May 2007<br />
20.15-20.30 Welcome and Announcements (Charles White)<br />
20.30-21.30 Jim Haber<br />
Multiple pathways to repair double-strand breaks and thoughts about gene<br />
targeting.<br />
21.30-22.30 Barbara Hohn<br />
Homologous recombination as a sensor to environmental<br />
challenges.<br />
22.30 Poster set-up and wine<br />
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1 June 2007<br />
8.00-11.20<br />
SESSION I: Somatic DNA repair and recombination I<br />
(Chairs: Anne Britt and Charles White)<br />
8-8.30 Anne Britt<br />
Regulation of IR-induced checkpoint responses<br />
8.30-8.50 Toon Cools<br />
Arabidopsis WEE1 Kinase Controls Cell Cycle Arrest in Response to Activation of the<br />
DNA Integrity Checkpoint.<br />
8.50-9.10 Pascal Genschik<br />
The CUL4-DDB1-DDB2 complex and genome integrity.<br />
9.10-9.30 Wei Xiao<br />
Arabidopsis thaliana Ubc13-Uev complexes and DNA damage tolerance<br />
9.30-9.50 Charles White<br />
Roles of the Rad51 paralogs in mitosis and meiosis<br />
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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
9.50-10.10 Coffee break<br />
10.10-10.30 Marie-Edith Chabouté<br />
New partner in DSB-induced gammaH2AX foci in Arabidopsis<br />
10.20-10.40 Seiichi Toki<br />
Homologous recombinational repair and cell cycle regulation in Arabidopsis.<br />
10.40-11 Ayako Sakamoto<br />
Functional analysis of DNA polymerase zeta and REV1 protein in Arabidopsis<br />
11-11.20 John Hays<br />
Requirements of DNA translesion polymerases Zeta and/or Eta for coordinated<br />
tissue growth in Arabidopsis developing ovaries and UVB-irradiated root meristems<br />
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16.00-18.00<br />
SESSION II: Somatic DNA repair and recombination II<br />
(Chairs: Holger Puchta and Barbara Hohn)<br />
16-16.30 Holger Puchta<br />
Role of Human Disease Genes for Genome Stability in Arabidopsis thaliana<br />
16.30-16.50 Peter Schlögelhofer<br />
The Arabidopsis thaliana AtGR1/COM1 gene is essential for DNA repair and meiosis<br />
and relates yeast's COM1/SAE2 to the human CtIP gene<br />
16.50-17.10 Thomas Peterson<br />
Transposon-Induced Genome Rearrangements in Maize and Arabidopsis<br />
17.10-17.30 Didier G. Schaefer<br />
Genetic analysis of transformation in the moss Physcomitrella patens<br />
17.30-17.50 Bernd Reiss<br />
RAD51 genes have different roles in Physcomitrella patens and Arabidopsis<br />
thaliana.<br />
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20.00-22.00<br />
SESSION III: Chromatin and Epigenetics<br />
(Chair: Jurek Paszkowski)<br />
20-20.30 Jurek Paszkowski<br />
DNA methylation and other epigenetic marks in Arabidopsis.<br />
20.30-21 Rob Martienssen<br />
Silent running: RNA interference and heterochromatic modifications in plants and<br />
fission yeast.<br />
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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
21-21.20 Marcelina Garcia-Aguilar<br />
A functional and comparative analysis of chromatin changes associated with the<br />
control of reproductive development in sexual and apomictic plants.<br />
21.20-21.40 Teresa Roldan-Arjona<br />
DNA Demethylation by 5-methylcitosine excision, a new mechanism of epigenetic<br />
reprograming in plants.<br />
21.40-22.00 Corina Belle Villar<br />
Mechanisms of Polycomb group-mediated gene silencing in Arabidopsis<br />
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2 June 2007<br />
0800-1120<br />
SESSION IV: Chromosome maintenance and architecture<br />
(Chairs: Dorothy Shippen and Karel Riha)<br />
8-8.30 Dorothy Shippen<br />
Chromosome end protection in Arabidopsis<br />
8.30-9 Karel Riha<br />
Role of the Ku70/Ku80 heterodimer in telomere metabolism<br />
9-9.20 Sue Armstrong<br />
Dynamics of telomere behaviour in Arabidopsis thaliana meiosis<br />
9.20-9.40 Ingo Schubert<br />
HR versus NHEJ at the chromosomal level<br />
9.40-10 Coffee break<br />
10-10.20 Maria Gallego<br />
AtRad1/AtErcc1 endonuclease and telomere stability in Arabidopsis<br />
10.20-10.40 Laurent Vespa<br />
A role of ATM in telomere length regulation in Arabidopsis<br />
10.40-11 Jiri Fajkus<br />
Mapping of interaction domains of putative plant telomere proteins<br />
11-11.20 Koichi Watanabe<br />
Positional sister chromatid alignment in a mutant of the Arabidopsis homolog of<br />
Structural Maintenance of Chromosome 6 (SMC6)<br />
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1400-1700<br />
SESSION V: Recombination and meiosis<br />
(Chairs: Clifford Weil and Zac Cande)<br />
14-14.30 Cliff Weil<br />
Forward Foreword: mutations that alter recombination rate and crossover<br />
interference in maize<br />
14.30-14.50 Chris West<br />
Arabidopsis NBS1: roles in meiosis and DNA repair<br />
14.50-15.10 Zac Cande<br />
Meiotic prophase chromosome architecture revealed by ultrahigh resolution<br />
structured illumination (SI) microscopy<br />
15.10-15.30 Graham Moore<br />
It's not size but coordination that matters<br />
15.30-15.50 Greg Copenhaver<br />
Using Arabidopsis tetrads to understand interference<br />
15.50-16.10 break<br />
<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
16.10-16.30 Chris. Franklin<br />
Co-ordination of meiotic recombination by ASY1 in Arabidopsis thaliana<br />
16.30-16.50 Franck L'Huissier<br />
MLH1 marks a subset of strongly interefering crossovers in tomato<br />
16.50-17.10 Mathilde Grelon<br />
Identification of meiotic DSB forming proteins in Arabidopsis thaliana<br />
17.10-17.30 Tom Gerats<br />
Recombination and genetic maps in Petunia<br />
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19.00 - Drinks and Congress Dinner<br />
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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
3 June 2007<br />
08.00-11.00<br />
SESSION VI: Recombination and related Technologies<br />
(Chairs: Avi Levy and Dan Voytas)<br />
8-8.30 Paul Hooykaas<br />
Modulation of the recombination machinery for gene targeting<br />
8.30-9.00 Shigeru Iida<br />
Gene Targeting by Homologous Recombination in Rice<br />
9:00-9:30 Daniel Voytas<br />
Gene targeting in plants using zinc finger nucleases<br />
9.30-9.50 coffee break<br />
9:50-10:05 Avraham Levy<br />
Stimulating Gene Targeting into the Arabidopsis genome<br />
10:05-10:20 Paul Bundock<br />
Mutation scanning by massive parallel sequencing<br />
10:20-10:35 Vipula Shukla<br />
Site-directed homologous recombination in tobacco cell cultures via zinc-finger<br />
nucleases.<br />
10:35-10:50 Frédéric Van Ex<br />
Development of a gene targeting system based on in planta presentation of<br />
homologous donor DNA during meiosis.<br />
10:50-11:05 Frédéric Pâques<br />
Meganucleases with tailored cleavage specificity can induce efficient homologous<br />
gene targeting<br />
11:05-11:20 Tzvi Tzfira<br />
Toward zinc finger nucleases-mediated gene targeting in plants<br />
11:20-11:35 Sylvie DeBuck<br />
Generation of Single-Copy DNA Transformants by the Cre/LoxP Recombination<br />
Mediated Resolution System<br />
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11.35-12.00 Closing remarks<br />
12.00-13.30 Lunch<br />
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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
Abstracts of Talks<br />
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T - 1. Arabidopsis WEE1 Kinase Controls Cell Cycle Arrest in Response to<br />
Activation of the DNA Integrity Checkpoint.<br />
Toon Cools, Anelia Iantcheva, Kristof De Schutter, Jerome Joubes, Francois-Yves Bouget,<br />
Dirk Inze, Lieven De Veylder Plant Systems Biology, Ghent University / VIB, 9052<br />
Zwijnaarde, Belgium<br />
Upon the incidence of DNA stress, the ataxia telangiectasia-mutated (ATM) and Rad3related<br />
(ATR) signaling kinases activate a transient cell cycle arrest that allows cells to<br />
repair DNA before proceeding into mitosis. Although the ATM-ATR pathway is highly<br />
conserved over species, the mechanisms by which plant cells stop their cell cycle in<br />
response to the loss of genome integrity are unclear. Previously, we have demonstrated<br />
that the cell cycle regulatory WEE1 kinase gene of Arabidopsis thaliana is<br />
transcriptionally activated upon the cessation of DNA replication or DNA damage in an<br />
ATR- or ATM-dependent manner, respectively. In accordance with a role for WEE1 in DNA<br />
stress signaling, WEE1-deficient plants showed no obvious cell division or<br />
endoreduplication phenotype when grown under nonstress conditions but were<br />
hypersensitive to agents that impair DNA replication (De Schutter et al., 2007). To study<br />
the effects of a deficient DNA replication checkpoint in more detail, a microarray analysis<br />
was performed. These data illustrated that in the WEE1-deficient plants upregulation of<br />
DNA-repair genes is not accompanied with the downregulation of cell cycle genes,<br />
contrary to what is observed in wild type plants. These data illustrate that WEE1 solely is<br />
responsible for the cell cycle arrest seen in wild-type plants upon activation of the DNA<br />
replication checkpoint and that a correct cell cycle arrest is essential for survival. To<br />
uncover the proteins that build up the cascade that results into WEE1 induction upon<br />
genotoxic stress, a mutagenesis screen was preformed. Additionally, a promoter deletion<br />
analysis was initiated to identify the possible cis-acting regulatory elements that are<br />
responsible for the induction of WEE1 upon DNA stress. De Schutter et al. (2007)<br />
Arabidopsis WEE1 Kinase Controls Cell Cycle Arrest in Response to Activation of the DNA<br />
Integrity Checkpoint. Plant Cell, In press.<br />
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T - 2. The CUL4-DDB1-DDB2 complex and genome integrity.<br />
Jean Molinier, Esther Lechner, Pascal Genschik , Institut de Biologie Moleculaire des Plantes<br />
CNRS, 67084 Strasbourg , France<br />
Plants due to their sessile life style are constantly exposed to UV irradiation which trigger<br />
cellular damage especially on DNA. As response, repair pathways are activated in order to<br />
guarantee genome integrity. The predominant DNA lesions, induced by UV radiation, are<br />
the cyclobutane pyrimidine dimers (CPDs) but also pyrimidine [6-4]pyrimidinone dimers.<br />
The main repair pathways of these lesions is the direct repair performed by photolyases.<br />
In addition to this photoreactivation process, most organisms have also a sophisticated<br />
general repair mechanism, termed nucleotide excision repair (NER). In this mechanism,<br />
the heterodimeric DDB protein complex, formed by DDB1 and DDB2, binds tightly to UVdamaged<br />
DNA and might act as a sensor to detect a subset of conformational changes on<br />
DNA. DDB1-DDB2 are part of a E3 ubiquitin ligase complex together with Cullin4. We will<br />
present recent results demonstarting the role of the CUL4-DDB1-DDB2 complex in DNA<br />
excision repair and maintenance of genome intergrity<br />
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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
T - 3. Arabidopsis thaliana Ubc13-Uev complexes and DNA damage tolerance<br />
Rui Wen, Lindsay Newton, Antonio Torres-Acosta, Hong Wang, Wei Xiao Department of<br />
Microbiology and Immunology, University of Saskatchewan, S7N 5E5 Saskatoon, Canada<br />
DNA damage tolerance (DDT) is a newly defined pathway in eukaryotes. In budding yeast,<br />
this is achieved by covalent modifications of the proliferating cell nuclear antigen (PCNA).<br />
A ubiquitin conjugating enzyme Ubc13 and a Ubc enzyme variant (Uev) are required for a<br />
unique Lys63-linked polyubiquitination of PCNA. We isolated two UBC13 and four UEV<br />
genes from Arabidopsis thaliana. All four AtUev1 proteins are able to form a stable<br />
complex with AtUbc13 as well as Ubc13 from yeast and human. These genes are able to<br />
functionally replace the corresponding yeast UBC13 or MMS2 genes for the DDT<br />
functions in vivo. Although all two AtUBC13 and four AtUEV1 genes are ubiquitously<br />
expressed in most tissues, AtUEV1D appears to be expressed at a much higher level in<br />
germinating seeds and in pollen. We obtained a T-DNA insertion line and created Atuev1d<br />
null plants. Compared with wild type and heterozygous mutant plants, seeds from the<br />
Atuev1d null plant germinated poorly when treated with a DNA damaging agent, while<br />
those germinated grew slower and majority ceased growth within two weeks. To our<br />
knowledge, this is the first report of Ubc13-Uev functions in tolerating DNA damage in a<br />
multicellular organism.<br />
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T - 4. Roles of the Rad51 paralogs in mitosis and meiosis<br />
Le Goff, S., Abe, K., Gallego, M.E. and Charles I. White. CNRS UMR6547, Université Blaise<br />
Pascal, Clermont Ferrand, France.<br />
By permitting the exchange of genetic information between DNA molecules, genetic<br />
recombination processes contribute both to genetic diversity and to the maintenance of<br />
genome integrity. In addition, recombination plays key roles in the transmssion of the<br />
genome via sexual reproduction, assuring the proper segregation of chromosomes at the<br />
first, reductional meiotic division. We are studying the nature of DNA<br />
recombination/repair pathways and the interrelationships between them in Arabidopsis<br />
thaliana and will present our data on the Rad51 paralogues. In addition to the meiosisspecific<br />
recombinase Dmc1, five Rad51 paralog proteins are known and all five are<br />
implicated in recombination, chromosomal stability and the DNA repair. The inviability of<br />
mutants of these genes in animals has complicated study of their roles, particularly in<br />
meiosis, and makes Arabidopsis a model of particular interest. We have reported the<br />
identification and characterisation of four of the proteins: Rad51B, Rad51C, Xrcc2, and<br />
Xrcc3, and have shown that the Arabidopsis xrcc3 and rad51C mutants are sterile due to<br />
fragmentation of the genome at meiotic prophase I. We have now identified a mutant for<br />
the fifth paralog, Rad51D, and will present data on the roles of this protein and the other<br />
paralogs in meiotic and mitotic recombination in Arabidopsis.<br />
T - 5. New partner in DSB-induced gammaH2AX foci in Arabidopsis<br />
julien lang, lenin sanchez-calderon, ondrej smetana, guy houlne, marie-edith<br />
chaboute department of cellular biology, IBMP/CNRS, 67000 strasbourg, France<br />
Occurrence of DNA double-strand breaks (DSBs) constitutes a major threat for the<br />
survival of the cell. Over the last years several studies have outlined the signalling<br />
pathway triggered by such damage and shown that it was conserved through evolution.<br />
Thus the ATM-dependent phosphorylated form of the histone variant H2AX (called<br />
gamaH2AX), participating both in the correct recruitment of repair proteins (Celeste A. et<br />
al., Nat Cell Biol 2003; 5: 675-679) as well as in the maintenance of the cell cycle<br />
checkpoints until complete recovery (Chowdhury et al., Mol Cell 2005; 20: 801-809), has<br />
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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
been pointed out at as a reliable marker of DSBs. Using the plant model Arabidopsis<br />
thaliana (At) we further analysed the repair function of the two putative AtH2AX<br />
orthologs, especially the nature and relevance of their accumulation, under<br />
phosphorylated state, with other partners at sites of DSBs. Thanks to an artificial miRNA<br />
line we highlighted the contribution of AtgamaH2AX in plant growth, repair and genomic<br />
stability. More interestingly our first results also hinted at an involvement, upon DSBs<br />
induction, of some cell cycle regulators E2Fs factors whose role in DNA repair is still<br />
poorly understood. As Nicotiana tabaccum E2Fs relocalize in Arabidopsis root cells, after<br />
bleomycin treatment, and form some clear and discrete foci together with AtgamaH2AX,<br />
we emitted the hypothesis that some AtE2F factors may be instrumental in responses to<br />
DNA damage and started to confirm it by comparing different genotoxic effects in an<br />
Ate2fa mutant<br />
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T - 6. Homologous recombinational repair and cell cycle regulation in<br />
Arabidopsis.<br />
Masaki Endo, Keishi Osakabe, Kiyomi Abe, Masaaki Umeda, Seiichi Toki Plant Engineering<br />
Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences, 305-<br />
8602 Tsukuba, Ibaraki, Japan<br />
Homology-based replacement of an endogenous gene by an introduced gene, termed gene<br />
targeting is a prime goal in the precise engineering of higher plants. In the previous<br />
study, we indicated that disfunction of chromatin assembly factor 1 (CAF-1) increased<br />
frequency of somatic homologous recombination (HR) and T-DNA integration in<br />
Arabidopsis. We thought that a decreased rate of histone loading onto newly replicated<br />
chromosomal DNA may leave this DNA more accessible to DNA repair and recombination<br />
enzymes, and to integrating T-DNA. Furthermore, we observed increased levels of DNA<br />
double-strand breaks, elevated levels of mRNAs coding for proteins involved in<br />
homologous recombination and G2 phase retardation in CAF-1 mutants. Interestingly,<br />
some factors, which operate cell cycle and DNA repair were reported in yeast and animal<br />
(CycA1, CDK2 etc.). Since the relationship between cell cycle regulation and DNA repair<br />
machinery remains elucidated in higher plants, we isolated Arabidopsis mutants<br />
depleting cell cycle regulating factors and analyzed the intrachromosomal homologous<br />
recombination frequency and DNA damage sensitivity in these mutants. We hope we<br />
could discuss the potential use of the cell cycle regulators and other related factors on<br />
the improvement of gene targeting system in higher plants.<br />
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T - 7. Functional analysis of DNA polymerase zeta and REV1 protein in<br />
Arabidopsis<br />
Ayako Sakamoto, Mayu Nakagawa, Shinya Takahashi, Atsushi Tanaka Radiation-Applied<br />
Biology , Japan Atomic Energy Agency, 370-1292 Takasaki, Japan<br />
Translesion synthesis (TLS) is one of the cellular processes to overcome the lethal effect<br />
of unrepaired DNA damage. During screening for genes accounting for the UV-resistance<br />
in Arabidopsis, we first identified AtREV3 that encodes a catalytic subunit of DNA<br />
polymerase zeta (Pol z). Pol z is present in almost all eukaryotes and thought to bypass<br />
damaged DNA in an error-prone manner. We subsequently identified AtREV7, a<br />
regulatory subunit of Pol z, and AtREV1 that is thought to cooperate with Pol z in<br />
bypassing of apurine/apyrimidine (AP) sites. Disruption of any of AtREV3, AtREV7, or<br />
AtREV1 made the plants more sensitive to UVB than the wild type, suggesting that these<br />
REV proteins are required for plants to tolerate to the UV-induced damage. We further<br />
analyzed the UV-induced mutation frequency in rev3 and rev1. The point-mutated nonfunctional<br />
uidA genes were introduced into the Arabidopsis plants and reversion events<br />
were detected by blue sectors on the somatic tissues. We found that the disruption of<br />
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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
AtREV3 or AtREV1 reduced the mutation frequency to 1/4 of the level of the wild type.<br />
These results are consistent with an idea of that the Pol z and AtREV1 are involved in the<br />
UV-induced mutation in Arabidopsis. However, by using the bacterially expressed protein,<br />
although we detected AtREV1 inserted a dCMP at the opposite of the AP site in vitro,<br />
AtREV1 failed to bypass UV-induced damage as reported in other organisms. Thus, the<br />
mechanism by which the REV1 functions remains to be cleared.<br />
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T - 8. Requirements of DNA translesion polymerases Zeta and/or Eta for<br />
coordinated tissue growth in Arabidopsis developing ovaries and UVBirradiated<br />
root meristems<br />
Marc Curtis, Peter Hoffman, John Hays Dept. of Environmental and Molecular Toxicology,<br />
Oregon State University, 97331-7301 Corvallis. Oregon, United States<br />
Specialized DNA translesion polymerases (TLPs) help rescue replication forks arrested<br />
when environmentally induced or endogenous DNA lesions, or perhaps difficult contexts<br />
in undamaged DNA, stall replicative polymerases. We analyzed, by various light<br />
microscopy techniques, UVB-irradiated Arabidopsis root meristems deficient in one or<br />
both of two key TLPs, AtPolEta and AtPolZeta. A threshold UV-B dose that induced<br />
roughly 0.03 cyclobutane pyrimidine dimers (CPDs) per kbp had little effect on wild-type<br />
(wt) roots. However, this dose inhibited root growth, suppressed division of stem cells<br />
and transiently-amplifying (TA) cells and specifically killed stem cells, in the increasing<br />
severity order Eta? < Zeta? < Eta? Zeta?. In UVB-irradiated Eta? Zeta? roots, alterations in<br />
tissue development persisted several days after CPDs had disappeared. Even unirradiated<br />
Eta? Zeta? roots showed substantial stem-cell death. Seed set in (unirradiated) selfed-<br />
Arabidopsis plants heterozygous (-/ ) for Pol? was roughly 50% of wt ( / ), although<br />
progeny showed no selection against mutant (-) or wt ( ) alleles. Correspondingly, about ½<br />
of ovules in Pol?-/ ovaries arrested after meiosis and failed to progress through gamete<br />
mitosis. Remarkably, Pol?-/- ovaries showed no post-meiotic arrest or reduced seed set.<br />
Recombinant PolEta proteins from Arabidopsis (AtPolEta), humans (HsPolEta), and yeast<br />
(ScPolEta) were overexpressed in E. coli and purified to homogeneity. Comprehensive<br />
analyses of their abilities to synthesize DNA past template CPDs reveals high biochemical<br />
similarity between AtPolEta and HsPolEta and interesting differences between these two<br />
and ScPolEta. (Supported by National Science Foundation grant MCB 03455061 to J.B.H.)<br />
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T - 9. Role of Human Disease Genes for Genome Stability in Arabidopsis thaliana<br />
Holger Puchta, Stefanie Suer, Daniela Kobbe, Sandra Blanck, Wim Reidt, Rebecca Wurz,<br />
Manfred Focke, Frank Hartung Botany II, University Karlsruhe, 76128 Karlsruhe,<br />
Germany<br />
During the elucidation of the genomic sequences of plants it became clear that multiple<br />
homologues of genes, which in their mutated form cause specific human diseases, are<br />
present in the plant genome. The main interest of our group focuses around breast<br />
cancer and the Blooms and Werner syndrome. In case of the two latter, mutations in two<br />
different members of the human RecQ DNA helicase genes family are responsible for the<br />
diseases. Arabidopsis harbours 7 RecQ homologues in its genome. In a long term project<br />
we are trying to define the biological role of individual family members. On one side we<br />
express AtRecQ ORFs in E. coli and characterized the biochemical properties in vitro. First<br />
result indicate that particular helicases indeed differ in their biochemical properties e.g.<br />
differences in the ability to process specific DNA structures could be defined. On the<br />
other side we try to analyse the role of the genes in vivo with sensitivity assays against<br />
DNA damaging agents and determination of homologous recombination frequencies.<br />
Additionally we started to test the effect of these mutations in combination with mutants<br />
of other genes also involved in the preservation of genome stability. Our results indicate<br />
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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
that at least several members of the RecQ family have non-redundant partly even<br />
antagonistic functions in Arabidopsis.<br />
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T - 10. The Arabidopsis thaliana AtGR1/COM1 gene is essential for DNA repair and<br />
meiosis and relates yeastâ€s COM1/SAE2 to the human CtIP gene<br />
Clemens Uanschou, Tanja Siwiec, Andrea Pedrosa Harand, Claudia Kerzendorfer, Eugenio<br />
Sanchez-Moran, Maria Novatchkova, Svetlana Akimcheva, Franz Klein, Peter<br />
Schlogelhofer Dept. of Chromosome Biology, Max F. Perutz Laboratories/University of<br />
Vienna, A-1030 Vienna, Austria<br />
Meiotic recombination is initiated by DNA double strand breaks (DSBs), generated by a<br />
homolog of the archaebacterial topoisomerase subunit Top6A Spo11. As an intermediate<br />
of the DNA cleavage reaction, Spo11 is covalently linked to 5†termini of DNA. Release<br />
of Spo11 is mediated by cleaving ssDNA next to the DSB site, thereby liberating the Spo11<br />
protein attached to a few nucleotides. In budding yeast this release requires Rad50,<br />
Mre11 and Com1/Sae2. This study describes the identification and characterisation of the<br />
first COM1/SAE2 homologue of a higher eukaryote, the Arabidopsis thaliana<br />
AtGR1/COM1 gene. We provide evidence that AtGR1/COM1 is essential for male and<br />
female meiosis and for conferring resistance against mitomycin C (MMC) in plants.<br />
Mutations in the corresponding gene lead to defects in chromosome pairing, to DNA<br />
fragmentation and subsequently to sterile plants. We demonstrate that AtCOM1 acts<br />
downstream of AtSPO11-1 and upstream of AtDMC1 during meiosis. Furthermore, our<br />
data show that regular turn-over of AtSPO11-1 at DSB sites and DSB processing depends<br />
on AtGR1/COM1. Importantly, this study creates a so far unsuspected link between the<br />
yeast Com1/Sae2 and the mammalian CtIP protein, a DNA repair related protein, well<br />
conserved in higher eukaryotes.<br />
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T - 11. Transposon-Induced Genome Rearrangements in Maize and Arabidopsis<br />
Thomas Peterson, Jianbo Zhang, Chuanhe Yu, Lakshminarasimhan Krishnaswamy, David<br />
Weber Genetics, Development and Cell Biology, Iowa State University, 50011 Ames Iowa,<br />
United States<br />
Since their discovery by McClintock, transposable elements have been associated with the<br />
generation of a variety of genome rearrangements, including deletions, direct and<br />
inverted duplications, and translocations. In addition to providing dispersed sequence<br />
homologies for ectopic recombination, transposons can induce genome rearrangements<br />
through alternative transposition reactions that utilize the termini of different elements.<br />
Transposition reactions involving transposon termini in direct orientation can generate<br />
deletions and inverted duplications. In addition, pairs of Ac termini in reversed<br />
orientation can undergo transposition reactions resulting in inversions, deletions, and<br />
translocations. In each of these cases, the rearrangement breakpoints are bounded by the<br />
characteristic footprint or target site duplications typical of Ac transposition reactions.<br />
These results show how alternative transposition reactions could contribute significantly<br />
to genome evolution by generating chromosome rearrangements, and by creating new<br />
genes through shuffling of coding and regulatory sequences (Zhang, Zhang and Peterson,<br />
2006). These alternative transposition reactions were first observed in natural maize<br />
variants, and have now been reproduced in transgenic maize and Arabidopsis plants. The<br />
system utilizes transgene constructs containing maize Ac termini in direct or reversed<br />
orientation. The action of Ac transposase on the Ac termini generates a variety of<br />
rearrangements, including deletions, inversions and translocations. In these<br />
rearrangements, one endpoint is at the Ac termini, and the other endpoint is at another<br />
genomic site. This transposition-based system provides an alternative to the cre-lox<br />
system for genome modifications. The current state of the project will be presented. To<br />
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view an animation of the alternative transposition model, see<br />
http://jzhang.public.iastate.edu/Transposition.html. This research is supported by NSF<br />
awards 0450243 to T. Peterson and J. Zhang, and 0450215 to D. Weber.<br />
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T - 12. Genetic analysis of transformation in the moss Physcomitrella patens<br />
Didier G. Schaefer, Sandrine Choinard, Florence Charlot, Fabien Nogué SGAP, Station de<br />
génétique et amélioration des plantes, INRA, Institut National de Recherche Agronomique,<br />
78026 Versailles, France<br />
With an efficiency of gene targeting following direct gene transfer comparable to that<br />
observed in S.cerevisiae, the moss Physcomitrella patens is unique among multicellular<br />
eukaryotes. Yet the integration pattern of replacement cassette differs from that<br />
observed in yeast: (a) gene conversions mediated by double HR and targeted insertions<br />
mediated by a combination of HR and NHEJ are observed at similar frequencies and (b)<br />
tandem direct repeats of the transforming DNA likely generated by DNA replication are<br />
frequently detected at the targeted locus. Furthermore, we recently demonstrated that GT<br />
efficiencies in this moss is reduced ca 2 orders of magnitude following Agrobacterium<br />
mediated transformation, an observation that is in contrast with data from previous<br />
analysis of gene targeting efficiencies following direct versus Agrobacterium mediated<br />
gene transfer in plants and yeast. In Eukaryotes, several DNA repair pathways have been<br />
shown to be involved in gene targeting. To gain insight into the genetic determinants that<br />
control gene targeting in P.patens, we have generated loss of function mutants for the<br />
following genes: Rad 50, Rad 51a1, Rad 51a2, Rad 51c, Rad 51d, Rad 54, Mre11, LigIV,<br />
Rad1, Rad10 and Msh2. We shall present our initial analysis of the role of these genes in<br />
P.patens during Agrobacterium versus PEG-mediated transformation and in response to<br />
DNA damaging agents.<br />
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T - 13. RAD51 genes have different roles in Physcomitrella patens and Arabidopsis<br />
thaliana.<br />
Ulrich Markmann-Mulisch, Edelgard Wendeler, Bozena Chrost, Hans-Henning Steinbiss,<br />
Bernd Reiss Department Coupland, MPI für Züchtungsforschung, 50829 Köln, Germany<br />
The eukaryotic RecA homologue RAD51 plays an essential role in homologous<br />
recombination and DNA damage repair in yeast. The lethality of rad51 mutants in<br />
vertebrates suggests that this gene has acquired additional functions at the interface of<br />
recombination and cell-cycle control in some animals. However, viability and unaltered<br />
vegetative development in a RAD51 knockout mutant in Arabidopsis thaliana suggests<br />
that this additional function is absent in plants. The moss Physcomitrella patens is<br />
exceptional in the plant kingdom in its gene targeting efficiency, a process based on<br />
homologous recombination. To analyse the recombination apparatus of Physcomitrella<br />
and to compare a gene targeting efficient and inefficient organism, we have isolated<br />
RAD51 homologues of Physcomitrella. The Physcomitrella genome contains two<br />
duplicated and highly homologous RAD51 genes, PpRAD51A and PpRAD51B. Both genes<br />
were inactivated individually by gene targeting and double mutants produced by crossing<br />
and re-transformation. Development of the mutants and DNA damage repair was<br />
analysed. The PpRAD51A and PpRAD51B single mutants had a mild developmental<br />
phenotype. However, loss of both genes caused sterility and seberely affected vegetative<br />
development. This result suggests that RAD51 is important for both, vegetative growth<br />
and meiosis. In addition, inactivation of both genes caused hypersensitivity to DNA<br />
damage induced by UV and Bleomycin. This result suggests that RAD51 is important for<br />
somatic DNA damage repair. Supporting this conclusion, the transcription of both RAD51<br />
genes was induced significantly by these agents. To compare this result to Arabidopsis,<br />
the Atrad51 mutant was treated with the damaging agents Mitomycin C and Bleomycin.<br />
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Among other DNA damage, both agents induce double-strand breaks. However,<br />
Mitomycin C induces double-strand breaks preferentially in the replicative phase while<br />
Bleomycin acts independently of the phase in the cell cycle. In contrast to Physcomitrella,<br />
the Arabidopsis rad51 mutant barely showed hypersensitivity to bleomycin, but was<br />
highly sensitive to Mitomycin C. This result suggests that RAD51 in Arabidopsis has no<br />
essential role in the repair of somatic double-strand breaks, but is important for the<br />
repair of such lesions during replication. Therefore, RAD51 in Arabidopsis is likely to be<br />
important for post-replicative, but not for somatic DNA damage repair. These data<br />
suggest that Physcomitrella and Arabidopsis differ in the use of homologous<br />
recombination for DNA damage repair, and possibly in the role of RAD51 in development.<br />
Thus gene targeting efficient plants differ from inefficient plants in respect to<br />
recombination. This difference could be in the recombination genes themselves, their<br />
regulation or in the mechanisms of recombination.<br />
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T - 14. A functional and comparative analysis of chromatin changes associated<br />
with the control of reproductive development in sexual and apomictic<br />
plants.<br />
Marcelina Garcia-Aguilar, Daniel Grimanelli Plant Genome and Development Laboratory,<br />
Institute for research and Development (IRD), 34394 Montpellier, France<br />
The unique plants life strategy, with alterning sporophytic and gametophytic generations,<br />
requires the correct activation or repression of transcriptional programs specific to each<br />
developmental transition. In certain plant species, apomixis replaces sexual reproduction.<br />
By avoiding meiosis and fertilization, it results in the formation of clonal seeds. Apomixis<br />
is associated with the heterochronic expression of sexual reproductive programs, and<br />
therefore eventually on modifications of the control of transcriptional transitions.<br />
Chromatin structure and remodeling plays pivotal functions among epigenetic<br />
mechanisms allowing plant cells to control gene expression in a flexible way during<br />
development. In Arabidopsis and maize, chromatin-remodeling factors have been shown<br />
to be involved controlling the orderly progression of reproductive development; however,<br />
their potential role during female meiosis, gametogenesis and fertilization remains<br />
poorly understood. The overall objective of this project is to determine the function of<br />
chromatin dynamics in the control of reproductive differentiation between the sexual and<br />
apomictic pathways. This study is focuses on histone and DNA modifications to compare<br />
reproductive cell nuclei in the ovule of maize and apomictic maize lines. Based on<br />
cytological and molecular expression studies, functional analysis of candidate regulators<br />
of these processes are being carried out through reverse genetic, combined to RNAi<br />
approach using stage-specific promotors to mis-regulate expression pro<strong>file</strong>s. Results<br />
from this project will improve our understanding of the epigenetic mechanisms<br />
controlling developmental transitions in flowering plants, and will also shed light on the<br />
molecular and cellular events leading to asexual reproduction in apomixis, an uniquely<br />
high-value trait for crops biotechnology.<br />
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T - 15. DNA Demethylation by 5-methylcitosine excision, a new mechanism of<br />
epigenetic reprograming in plants.<br />
Teresa Morales-Ruiz, Ana-Pilar Ortega-Galisteo, M. Isabel Ponferrada-Marin, M. Isabel<br />
Martinez-Macias, Rafael R. Ariza, Teresa Roldan-Arjona Departamento de Genetica,<br />
Universidad de Cordoba, 14071 Cordoba, Spain<br />
Methylation of cytosine at carbon 5 of the pyrimidine ring (5-meC) is a stable epigenetic<br />
mark for transcriptional gene silencing and plays important roles in development and<br />
genome defence against parasitic mobile elements. Although the enzymes responsible for<br />
the establishment and maintenance of DNA methylation have been well characterized, the<br />
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mechanisms of DNA demethylation are not well understood. We and others have found<br />
genetic and biochemical evidence suggesting that the Arabidopsis DNA glycosylase<br />
domain-containing proteins ROS1 (REPRESSOR OF SILENCING 1) and DME (DEMETER) are<br />
DNA demethylases. ROS1 was identified in a screen for mutants with deregulated<br />
expression of the repetitive RD29A-LUC transgene. Whereas in wild plants the transgene<br />
and the homologous endogenous gene are expressed, ros1 mutants display<br />
transcriptional silencing and hypermethylation of both loci. The protein encoded by ROS1<br />
shares a high degree of sequence similarity to the product of DME, that was<br />
independently identified in a search for mutations causing parent-of-origin effects on<br />
seed viability and is required for the expression of the maternal alleles of the imprinted<br />
genes MEA and FWA. ROS1 and DME catalyze the release of 5-meC from DNA by a<br />
glycosylase/lyase mechanism. Both enzymes also remove thymine, but not uracil,<br />
mismatched to guanine. DME and ROS1 show a preference for 5-meC over thymine in the<br />
symmetric dinucleotide CpG context, where most plant DNA methylation occurs.<br />
Nevertheless, they also have significant activity on both substrates at CpApG and<br />
asymmetric sequences, which are additional methylation targets in plant genomes. These<br />
findings suggest that a function of ROS1 and DME is to initiate erasure of 5-meC through<br />
a base excision repair process, and provide strong evidence for the existence of an active<br />
DNA demethylation pathway in plants.<br />
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T - 16. Mechanisms of Polycomb group-mediated gene silencing in Arabidopsis<br />
Corina Belle Villar, Grigory Makarevich, Claudia Köhler Plant Developmental Biology, ETH<br />
Zurich, Institute of Plant Sciences, 8092 Zurich, Switzerland<br />
The type I MADS-box gene PHERES1 is expressed dependent on the parent-of-origin, a<br />
phenomenon known as genomic imprinting. The paternal allele of PHERES1 is expressed,<br />
whereas the maternal allele is silenced. PHERES1 is repressed by the FIS Polycomb-group<br />
complex that includes MEDEA (MEA), FERTILIZATION INDEPENDENT ENDOSPERM (FIE),<br />
FERTILIZATION INDEPENDENT SEED 2 (FIS2),and MULTICOPY SUPPRESSOR of ira1 (MSI1).<br />
PHERES1 repression by the FIS complex is mediated through chromatin modification, i.e.,<br />
histone methylation. However, our research revealed that FIS Polycomb group mediated<br />
repression of PHERES1 is not sufficient to establish PHERES1 imprinting. We are<br />
investigating the nature of the imprint and how this imprint is established and<br />
maintained. In particular we are looking at the role of tandem repeats as possible<br />
sequence elements necessary for imprinting establishment. Our progress in elucidating<br />
the imprinting mechanism of PHERES1 is going to be presented.<br />
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T - 17. Chromosome end protection in Arabidopsis<br />
Dorothy Shippen. Department of Biochemistry and Biophysics, College Station, Texas<br />
77843, USA.<br />
Telomeres are specialized nucleoprotein structures that facilitate the complete<br />
replication of chromosome ends and allow the cell to distinguish the natural<br />
chromosome terminus from a double-strand break. We are employing genetic and<br />
biochemical approaches in Arabidopsis to investigate fundamental aspects of telomere<br />
function and maintenance. We previously showed that telomere tracts in plants lacking<br />
telomerase undergo progressive shortening and after six generations suffer end-to-end<br />
chromosome fusions that ultimately lead to developmental and growth arrest. Currently,<br />
our efforts focus on the identification of proteins that provide protection for the<br />
chromosome terminus. Here we describe a novel Arabidopsis mutant, dubbed cit1<br />
(Critical for the Integrity of Telomeres), which displays severe developmental defects in<br />
the first generation. Telomere length in cit1 is deregulated, the single strand G-overhang<br />
is grossly elongated, and chromosomes are highly vulnerable to telomere fusion. Thus,<br />
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CIT1 appears to encode a factor crucial for distinguishing the chromosome terminus<br />
from a double-strand break. We also report the characterization of a family of singlestrand<br />
telomere binding proteins, termed POT (Protection of Telomeres). Arabidopsis is<br />
unusual as it encodes three highly divergent POT-like genes, which appear to represent<br />
separation of function alleles. Interestingly, AtPOT1 has evolved to function as a<br />
component of the telomerase RNP necessary for telomere maintenance in vivo. To<br />
further investigate the evolution of POT proteins, we obtained POT coding regions from<br />
over twenty organisms within the plant kingdom, ranging from green algae to the most<br />
advanced flowering plants. Unlike Arabidopsis, most plant genomes encode only a single<br />
POT protein. We are currently analyzing the wealth of information obtained from plant<br />
POT1 sequences with the goal of identifying residues under positive selection and their<br />
relationship to protein structure and function.<br />
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T - 18. Role of the Ku70/Ku80 heterodimer in telomere metabolism<br />
Barbara Zellinger, Svetlana Akimcheva, Jasna Puizina, Martina Schirato, Karel Riha ,<br />
Austrian Academy of Sceinces, Gregor Mendel Institute, 1030 Vienna, Austria<br />
Telomeres distinguish natural chromosome ends from being processed (repaired) as<br />
deleterious DNA double strand breaks (DSBs). However, many factors required for DSB<br />
repair, such as Ku70/80 heterodimer, are also involved in telomere metabolism. Ku70/80<br />
complex is crucial component of nonhomologous end-joining (NHEJ) DNA repair pathway,<br />
but it is also localized to telomeres and its deficiency impairs telomere function in most<br />
organisms where it has been studied. We use Arabidopsis as a model system to study<br />
molecular mechanisms underlying Ku function at telomeres. Our initial characterization<br />
of the Arabidopsis KU70 gene revealed that mutants carrying a T-DNA insertion in the<br />
gene have much longer telomeres than wild-type plants. Subsequently, we showed that<br />
Ku70 acts as a negative regulator of telomerase at telomeres and at the same time,<br />
prevents excessive nucleolytic resection of the 5’ chromosome end [1, 2]. Although Kudeficient<br />
telomeres are fully functional, additional inactivation of telomerase causes<br />
accelerated telomere shortening and an early onset of chromosome end-to-end fusions<br />
and developmental defects [1]. The accelerated loss of telomeres in the absence of Ku<br />
could be, to a large extent, attributable to the exonucleolytic resection of the telomeric Cstrand.<br />
However, it could also be caused by an increased rate of aberrant homologous<br />
recombination resulting in telomere rapid deletion (TRD). To investigate this possibility,<br />
we developed a novel highly sensitive assay for assessing the level of extrachromosomal<br />
telomeric circles (t-circles), which are products of TRD. We found that the Ku heterodimer<br />
suppresses t-circle formation and intrachromatid recombination at Arabidopsis<br />
telomeres. Furthermore, we show that Ku prevents engagement of the telomeraseindependent<br />
ALT pathway for telomere maintenance. These data demonstrate that Ku<br />
acts as a key regulator of homologous recombination at Arabidopsis telomeres. [1] K.<br />
Riha and D.E. Shippen, Proc. Natl. Acad. Sci. USA 100 (2003) 611-5. [2] K. Riha, J.M.<br />
Watson, J. Parkey and D.E. Shippen, <strong>EMBO</strong> J. 21 (2002) 2819-26.<br />
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T - 19. Dynamics of telomere behaviour in Arabidopsis thaliana meioisis<br />
Susan Armstrong, Nicola Roberts School of Biosciences, University of Birmingham, B152TT<br />
Birmingham, United Kingdom<br />
There is growing evidence that telomeres play a key role in the initiation of chromosome<br />
pairing at meiosis but little is known about how this is achieved. The primary role of the<br />
telomere is to stabilise the ends of the eukaryotic chromosome, but it is apparent that the<br />
telomere also has a role in the highly conserved meiotic pathway. It has been suggested<br />
that the telomeres play a role in this process by aligning (pairing) the homologous<br />
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chromosomes preceding recombination and synapsis. Recent attention has been paid to<br />
the bouquet, a nearly universal event, during which the telomeres cluster in early<br />
prophase. It has been suggested that because the telomeres are in close proximity this<br />
would enhance the pairing of the homologues. We have shown in Arabidopsis that we do<br />
not observe a classical bouquet, rather the telomeres are organised already around the<br />
prophase nucleolus. The homologous telomeres are paired at the transition from G2 to<br />
leptotene during the assembly of the axial elements. As the chromosomes synapse during<br />
zygotene, the telomeres reveal only a loose clustering, which may represent a relic<br />
bouquet. To investigate the nature of chromosome pairing we are carrying out a<br />
functional analysis of pairing in recombination mutants. We have already shown in a<br />
range of Arabidopsis asynaptic meiotic mutants (spo11, asy1, mre11, dmc1, rad 51c) that<br />
the telomeres appear to be paired in early leptotene but this is not permanent and<br />
appears to be lost as meiotic prophase progresses. We are using FISH with unique<br />
chromosomal paints to find out if our observations are due purely to random<br />
associations or are we observing homologous telomeric pairing Our analysis will uncover<br />
whether initial homologous pairing requires DSB, recombination proteins MRE11, DMC1,<br />
RAD51C or structural meiotic components.<br />
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T - 20. HR versus NHEJ at the chromosomal level<br />
Ingo Schubert, Koichi Watanabe, Ales Pecinka Cytogenetics and Genome Analysis, Leibniz-<br />
Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Germany<br />
HR versus NHEJ at the chromosomal level Ingo Schubert, Koichi Watanabe, Ales<br />
Pecinka,IPK, D-06466 Gatersleben, Germany According to molecular studies, HR is the<br />
major mechanism for double-strand break (DSB) repair in yeast, while in somatic cells of<br />
vertebrates and of seed plants NHEJ is considered to be predominant. A low HR<br />
frequency is coincident with a low frequency of homologous gene targeting in seed<br />
plants. However, at least at the level of genotoxin-mediated structural rearrangements<br />
between freshly replicated chromatids, HR is remarkably frequent; apparently 100% of<br />
SCEs and up to 8-fold more chromatid-type aberrations than expected at random are the<br />
outcome of HR. Both phenomena can be quantified reliably only during the first mitotic<br />
division after the S-phase of their origin. Later such events are either not (unambiguously)<br />
detectable or the carrier cells are eliminated. Thus it could be possible that quite a<br />
proportion of HR is overlooked, the more so as it remains to be elucidated which<br />
proportion of the preclastogenic DSBs are processed via HR into SCE or chromatid-type<br />
aberrations. Therefore it is needed to determine i) the probability of DSB processing into<br />
the different endpoints, including restoration of the pre-break situation, and ii) how are<br />
the spatial requirements for HR achieved. As a prerequisite to address the first aim, a<br />
screening system to monitor the induction of DSBs per nucleus at a specific locus is being<br />
established. In chromatin mutants with a hyper-recombination phenotype and an up to<br />
100-fold increase of intrachromosomal somatic HR (in cis), chromosome territory<br />
arrangement and homologous pairing frequencies are not significantly altered. Contrarily,<br />
clastogenic doses of X-rays cause a significant increase of the positional homologous<br />
pairing frequencies in A.thaliana that decays during 1h after irradiation. These data<br />
indicate that for homologous intrachromatid recombination chromatin relaxation might<br />
be sufficient, while interchromatid recombination likely requires an active search for<br />
homology.<br />
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T - 21. AtRad1/AtErcc1 endonuclease and telomere stability in Arabidopsis.<br />
Jean-Baptiste Vannier, Annie Depeiges, Charles White, Maria Eugenia Gallego CNRS UMR<br />
6547, Universite Blaise Pascal, 63177 Aubiere, France<br />
Telomeres are the specific chromatin structures present at the ends of chromosomes<br />
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serving to avoid chromosomal shortening at replication and recombination. Proteins<br />
known to be involved in DNA repair and recombination have been found associated to<br />
telomeres, however, their specific roles in the maintenance of functional chromosome<br />
ends are poorly understood. The XPF/Ercc1 complex, a structure-specific endonuclease<br />
that acts in nucleotide excision repair and recombination, has been shown to be<br />
associated to telomeres. Arabidopsis plants mutated for either Xpf (AtRad1) or Ercc1<br />
(AtErcc1) orthologs develop normally and show wild-type telomere length. However in the<br />
absence of telomerase, mutation of either of these two genes induces an earlier onset of<br />
chromosomal instability. End-to-end chromosome fusions are detected at generation 1 as<br />
compared to generation 5 in telomerase mutant plants. Furthermore a significantly<br />
higher number of anaphase bridges per cell are observed in telomerase mutant plants<br />
lacking either AtRad1 or AtErcc1 as compared to single telomerase mutants. Plants<br />
showing fertility defects are observed at generation 2 both in AtRad1 and AtErcc1<br />
telomerase double mutants. This early appeareance of uncapped telomeres is not related<br />
to a general acceleration of telomeric repeat loss. Fluorescent in situ hybridization show<br />
the presence of extrachromosomal DNA fragments, containing both subtelomeric and<br />
telomeric repeat signals, which are not observed in telomerase single mutant plants<br />
presenting a similar numbers of anaphase bridges. Anaphase bridges present in nuclei<br />
with extrachromosomal fragments never show both subtelomeric and telomeric signals.<br />
These data and the possible roles of AtRad1/AtErcc1 in Arabidopsis telomere stability<br />
will be discussed.<br />
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T - 22. A role of ATM in telomere length regulation in Arabidopsis<br />
Laurent Vespa, Ross Warrington, Jiri Siroky, Petr Mokros, Dorothy Shippen Biochemistry<br />
and Biophysics, Texas A&M University, TX 77840 College Station, United States<br />
Telomeres are the indispensable nucleoprotein complexes that protect linear<br />
chromosomes from replicative attrition and from the DNA damage response machinery.<br />
Without them, chromosome ends would be recognized as double-strand breaks and fused<br />
to each other, leading to deleterious chromosomal rearrangements. This function has led<br />
to investigations of the relationship between telomeres and the DNA damage response<br />
machinery. Paradoxically, several of these factors have an essential role in the physical<br />
integrity of telomeres. We have taken advantage of the outstanding resistance of<br />
Arabidopsis to genomic instability to study the role of several DNA damage response<br />
factors at telomeres, including the central sensor/activator protein kinase ATM. We have<br />
shown that a combined deficiency in ATM and TERT, the reverse transcriptase subunit of<br />
telomerase, leads to a premature onset of telomere uncapping. However the nature of the<br />
fusion events in atm/tert are different from the single tert mutants. Our telomere-fusion<br />
amplification assay mostly failed to detect products in the double atm tert mutant, while<br />
cytogenetic experiments showed very high number of anaphase bridges, a hallmark for<br />
chromosome fusions. Therefore, we have characterized telomere fusions arising in atm<br />
tert mutants by FISH analysis using telomeric probes and an extensive series of<br />
subtelomeric BAC probes. Our results indicate that one particular chromosome end is<br />
involved in most of the fusions. We also revealed that about 40% of the fusions in atm<br />
tert (versus ~5% in tert) do not involve chromosome ends, which suggests that ATM may<br />
prevent multiple breakage-fusion-breakage events as a cell cycle checkpoint controller. A<br />
precise telomere length analysis of individual chromosome ends was then performed by<br />
PETRA (Primer Extension Telomere Repeat Amplification). These data showed that the<br />
chromosome end involved in the fusions was much shorter than its counterparts, and<br />
that this shortening event happened in early generations (G2-G3). This finding suggests<br />
that a particularly large Telomere Rapid Deletion (TRD) event occurred in the double<br />
mutant. We then addressed the putative role of ATM in controlling TRD by performing<br />
parent/progeny PETRA analysis on a large number of tert and atm tert plants. We found<br />
that large size telomeres are more prone to TRD in the double mutant, which can lead to<br />
the stochastic generation of critically shortened telomeres. We conclude that ATM has a<br />
role in preventing TRD or in preventing cells that have undergone large deletions from<br />
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T - 23. Mapping of interaction domains of putative plant telomere proteins<br />
Milan Kuchar, Petra Schrumpfova, Iva Santruckova, Jan Palecek, Ctirad Hofr, Jiri<br />
Fajkus Functional Genomics and Proteomics, Masaryk University and Institute of Biophysics<br />
ASCR, 625 00 Brno, Czech Republic<br />
Although a number of candidate proteins have been identified by homology searches in<br />
plant genome databases and tested for their affinity to telomeric DNA sequences in vitro,<br />
data relevant to their telomeric function are mostly missing. Recently we reported on<br />
interactions among three myb-like proteins of the single myb histone (Smh) family<br />
AtTRB2, AtTRB3 binding telomeric dsDNA in vitro and AtTRB1, and on interaction of<br />
AtTRB1 with AtPo1-2 that binds G-rich strand of telomeric DNA strand by<br />
oligonucleotide-binding (OB)-fold and is involved in telomere length regulation. The Smh<br />
family has been described to bear a unique triple motif structure containing N-terminal<br />
myb-like domain, central GH1/GH5 histone globular domain and C-terminal coiled-coil<br />
domain. We report here on mapping of protein domains responsible for these<br />
interactions using the yeast two-hybrid system and independent biophysical techniques<br />
like surface plasmon resonance. Surprisingly, the domain responsible for the interaction<br />
of AtPot1-2 with AtTRB1 has been mapped to the region of N-terminal OB-fold domain.<br />
Protein AtTRB1 uses the central GH1/GH5 histone globular domain in interactions with<br />
AtTRB2, AtTRB3, AtPot1-2, AtTRP1 and with itself. Its C-terminal coiled-coil domain is<br />
involved in the interactions with AtTRB2, AtTRB3 and itself and increases interaction<br />
stability. Our research is supported by Grant Agency of the Czech Republic (521/050055),<br />
Grant Agency of the Czech Acad. Sci (IAA600040505), Ministry of Education (LC LC06004)<br />
and institutional funding (MSM0021622415, AVOZ50040507)<br />
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T - 24. Positional sister chromatid alignment in a mutant of the Arabidopsis<br />
homolog of STRUCTURAL MAINTENANCE of CHROMOSOME 6 (SMC6).<br />
Koichi Watanabe, Andrea Weißleder, Michael Florian Mette, Veit Schubert, Ingo Schubert ,<br />
Leibniz Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben,<br />
Germany<br />
DNA double-strand breaks (DSBs) can be repaired either by non-homologous end-joining<br />
(NHEJ) or by homologous recombination (HR). Most cells of a plant are not proliferating<br />
or they perform endocycles omitting G2 and mitosis. It is not yet clear how DSBs are<br />
repaired in plant cells during endocycle. To better understand the DSBs repair mechanism<br />
in endopolyploid plant cells, we examined sister chromatid alignment in Arabidopsis<br />
thaliana nuclei after DSBs formation. Since the physical proximity between the donor and<br />
acceptor strands is critical for the DNA strand exchange process during HR, it is possible<br />
that alignment of homologous or sister chromatids is a prerequisite for DSB repair via<br />
HR. FISH experiments indicated an increased frequency of positional sister chromatid<br />
alignment in wild type nuclei of 4C or 8C DNA content immediately after X ray irradiation<br />
compared to unirradiated cells. Sister chromatid alignment is considered to be mediated<br />
by the cohesin complex. One of the SMC proteins, SMC6, is involved in DNA repair and<br />
required to recruit the cohesin complex to DSB sites. To examine whether the fluctuation<br />
in the frequency of positional sister chromatid alignment observed in irradiated plants<br />
depends on recruitment of cohesin, we compare sister chromatid alignment frequencies<br />
between wild-type and mutants of the Arabidopsis homolog of SMC6.<br />
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T - 25. Mutations Affecting Meiotic Recombination Rate in Maize<br />
Sanzhen Liu, Stephen Baluch, Mitzi Wilkening, Alice Eggleston, An-Ping Hsia, Hugo Dooner,<br />
William Eggleston, Patrick Schnable, Clifford Weil Dept of Agronomy, Purdue University,<br />
47907-2054 West Lafayette, United States<br />
1 Iowa State University, Ames, IA, 2 Purdue University, West Lafayette, IN 3 Virginia<br />
Commonwealth University, Richmond, VA 4 Waksman Institute, Piscataway, NJ The<br />
mechanisms and genetic regulation of meiotic recombination are still not wellunderstood.<br />
Although great progress has been made in other model systems, the<br />
underlying genes of recombination and the control of these steps remain elusive in<br />
plants, including maize. We have used EMS mutagenesis on two differentially marked<br />
maize populations to identify mutations that significantly alter recombination (p < .05).<br />
Using two different crossing schemes and testcross assays that eliminate meiotic defects<br />
resulting in sterility, we monitored crossovers in the C1-Wx1 interval of chromosome 9S<br />
and the A1-Et1 interval of chromosome 3L. Thus far, the two screens have produced 56<br />
candidate mutations segregating in M2 and M3 families (30 from the 9S screen and 26<br />
from the 3L screen), including 39 putative mutations that significantly increase<br />
recombination, seven that significantly decrease recombination, and four families in<br />
which both a significant increase and a significant decrease in recombination frequency<br />
over the same interval segregate in the same family. In addition, nine families segregate<br />
for an increased frequency of double crossovers (rarely observed in maize, where<br />
crossover interference is extremely high); four of these correspond to mutants with a<br />
general increase in recombination, suggesting the other mutants in this class may be<br />
defective for genes involved in enforcing crossover interference.<br />
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T - 26. A key role for AtNBS1 in plant cell responses to DNA double strand breaks<br />
Christopher E. West, Wanda M Waterworth, Cagla Altun, Susan J Armstrong, Nicola<br />
Roberts, Philip J Dean, Kim Young, Clifford F Weill, Clifford M Bray Centre for Plant<br />
Sciences, University of Leeds, LS2 9JT Leeds, United Kingdom<br />
Centre for Plant Sciences, University of Leeds, Leeds, LS2 9JT, UK; †Faculty of Life<br />
Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, UK; ‡Plant<br />
Biology Program and §Dept. of Agronomy, Purdue University, West Lafayette IN 47907<br />
USA; School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK DNA<br />
double strand breaks (DSBs) in genomic DNA are caused by a combination of<br />
environmental and endogenous factors including irradiation and reactive metabolites.<br />
Inability to correctly repair DSBs can result in the loss of large amounts of genetic<br />
information and mutagenesis. However, programmed DSBs play an important role during<br />
meiosis in initiating recombination between homologous chromosomes. In most<br />
Arabidopsis cells the major pathway for DSB repair is non-homologous end joining<br />
(NHEJ), a process involving the protein complexes AtKU80/AtKU70 and AtLIG4/XRCC4. A<br />
multifunctional protein complex of MRE11, RAD50 and NBS1 is involved in both<br />
homologous recombination (HR) and NHEJ pathways. Arabidopsis atmre11 and atrad50<br />
mutant plants are sterile, reflecting the essential role that these proteins play in meiotic<br />
HR. In addition, these mutants displayed hypersensitivity to DNA damaging reagents<br />
including methyl-methanesulfonate indicating a probable role for this complex in NHEJ in<br />
vegetative tissues. Here we report a functional characterisation of AtNBS1 and find that it<br />
forms part of the Arabidopsis MRN complex and has a role in the DNA damage response<br />
in plant vegetative tissues. In addition, phenotypic analysis shows that atnbs1 deficient<br />
plants are fully fertile suggesting that AtNBS1 may be dispensable for meiosis, in contrast<br />
to AtMRE11 and AtRAD50. However, AtNBS1 is essential for the residual fertility in plants<br />
lacking AtATM, consistent with roles of AtNBS1 independent of AtATM signalling.<br />
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T - 27. Meiotic prophase chromosome architecture revealed by ultrahigh resolution<br />
structured illumination (SI) microscopy<br />
W.Z. Cande, Chung-Ju Rachel Wang, Pete Carlton, John Sadat Dept of Molecular and Cell<br />
biology, Univeersity of California, Berkeley, 94720-3200 Berkeley, CA, United States<br />
1 Department of Molecular and Cell Biology, University of California at Berkeley 2<br />
Department of Biochemistry and Biophysics, University of California, San Francisco An<br />
integrative view of meiotic prophase chromosome structure and function has been<br />
difficult to come by. Meiotic chromosomes are large, complex structures that can be tens<br />
of microns in length, yet the size of many structural elements of interest such as axial<br />
element organization during synapsis is just beyond the resolution of the conventional<br />
wide field microscopy. To overcome these limitations we have used structured<br />
illumination (SI) microscopy with a resolution less than 100 nm in the XY and Z axis<br />
(compared to 250 nm in the Xy and 500 nm in the Z axis by conventional light<br />
microscopy) to analyze the architecture of the pachytene chromosome. During leptotene,<br />
each chromosome develops a linear proteinaceous structure called an axial element (AE).<br />
At that time, recombination and the homology search are initiated by the formation of<br />
double strand breaks. In zygotene, homologous chromosomes synapse via the<br />
polymerization of a central element between the two homologous AEs, forming the<br />
synaptonemal complex (SC). During pachytene, synapsis and recombination are<br />
completed. We have analyzed chromomere and chromatin organization by using<br />
antibodies to monitor the distribution of histone modifications associated with<br />
heterochromatin (H3K9 dimethyl) and euchromatin (H3K9 acetylation, H3K4 dimethyl)<br />
and analyzed axial element organization by monitoring the distribution of AFD1 (a rec8<br />
homolog) and HOP1. Chromomeres of paired chromosomes are bilaterally symetrical and<br />
have a variable left handed helical pitch. Chromosome and axial element organization<br />
differ at centromeres. AFD1 and HOP1 are distributed as alternating islands of variable<br />
morphology along the axial element and display a bilaterial symmetry on the paired axes.<br />
Unsynapsed regions at late zygotene are always associated with interlocks suggesting<br />
that resolution of interlocks between chromosomes may be a rate limiting step to<br />
complete synapsis. We also found evidence for a process of pre-synaptic adjustment in<br />
unsynapsed regions of zygotene chromosomes, as the homologs in the unsynapsed<br />
regions differ in length.<br />
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T - 28. Ph1 in wheat-its not size but coordination that matters<br />
Graham Moore Crop Genetics, John Innes Centre, NR4 7UH Norwich, United Kingdom<br />
Hexaploid wheat (Triticum aestivum 2n=6x=42) possesses three ancestral genomes or 7<br />
sets of six related chromosomes. For hexpaloid wheat to be fertile, true homologues must<br />
pair amongst the 6 related chromosomes during meiosis. The Ph1 locus controls this<br />
pairing mechanism. We have recently defined the Ph1 locus to a cdk-like gene complex<br />
containing a segment of heterochromatin. This gene complex is related to the IME2 and<br />
cdk2 meiotic checkpoint genes of yeast and humans involved in initiating meiosis. Cell<br />
biological studies reveal that the Ph1 locus triggers the initiation and coordination of<br />
chromatin remodelling required to enable chromosomes to become competent to pair<br />
and recombine during meiosis. However unlike many species, it seems that the presence<br />
of Ph1 in wheat links this chromatin remodelling to homology<br />
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T - 29. A new pollen-based meiotic recombination assay.<br />
Kirk Francis, Sandy Lam, Alexandra Bey, Luke Berchowitz, Gregory Copenhaver BASF<br />
Plant Sciences, BASF, 27709 Research Triangle Park, United States<br />
Meiotic recombination, in the form of crossovers (CO) and gene conversions (GC), is a<br />
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highly conserved process. Recombination helps ensure chromosome segregation and<br />
promotes allelic diversity. Defects in the recombination machinery are often catastrophic<br />
for meiosis and may result in sterility. To facilitate investigation of the critical processes<br />
relating to recombination we have developed an Arabidopsis-based visual assay capable<br />
of detecting crossovers, crossover interference, and gene conversion events. The assay<br />
utilizes transgene constructs encoding three distinct pollen-expressed fluorescent<br />
proteins in the tetrad-producing qrt mutant background. By observing the segregation of<br />
the fluorescent alleles in pollen tetrads we demonstrate (i) a correlation between<br />
developmental position and CO frequency, (ii) a temperature dependence for CO<br />
frequency, (iii) the ability to detect meiotic GC events and (iv) the ability to rapidly asses<br />
CO interference. Additional applications of this system to identify and evaluate<br />
developmental, environmental, and genetic factors that influence meiotic recombination<br />
will be discussed.<br />
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T - 30. Co-ordination of meiotic recombination by ASY1 in Arabidopsis thalian<br />
F. Chris. H. Franklin Eugenio Sanchez-Moran, Gareth H. Jones . School of Biosciences,<br />
University of Birmingham, Birmingham B15 2TT UK<br />
We are investigating the formation of meiotic crossovers (COs) in Arabidopsis and how<br />
components of the chromosome axes and synaptonemal complex (SC) interface with the<br />
recombination machinery to influence this process. Here we focus on a recent study<br />
aimed at understanding the role of ASY1 during the early stages of the meiotic<br />
recombination pathway. This analysis has provided new insight into the role of ASY1, the<br />
formation of DNA double-strand breaks (DSBs) and the co-ordination of strand-invasion.<br />
Meiotic recombination is initiated by the formation of AtSPO11-1 catalysed DSBs. DSB<br />
formation is coincident with the formation of the chromosome axes which occurs ~2h<br />
after initial AtSPO11-1 localization. ASY1 is a HORMA domain protein that is initially<br />
detected in G2 as chromatin-localized foci that associate with the nascent chromosome<br />
axes at late G2/early leptotene to form a linear signal. Loss of ASY1 results in asynapsis<br />
and a dramatic reduction in chiasma/CO formation leading to the presence of univalent<br />
chromosomes at metaphase I. Studies indicate that DSB formation is normal in an asy1<br />
mutant. In wild-type, localization of the strand exchange proteins AtRAD51 and AtDMC1<br />
to the chromatin occurs asynchronously, shortly after DSB formation with AtDMC1<br />
localizing in advance of AtRAD51. Both recombinases form numerous foci that persist for<br />
~12h before gradually decreasing in number. In asy1, initial localization of AtDMC1 is<br />
normal, but declines abruptly such that inter-homolog recombination is severely<br />
compromised. As a result virtually all DSB repair proceeds via AtRAD51 mediated intersister<br />
recombination. Limited ASY1-independent, AtDMC1-dependent inter-homolog<br />
recombination remains, but is restricted to sub-telomeric sequences where the homologs<br />
are fortuitously in register due to nucleolus-associated telomere clustering. We propose<br />
that the meiotic bias in favour of inter-homolog recombination is established by an<br />
asynchrony in the expression of the recombinases together with ASY1-mediated<br />
repression of AtRAD51 during the early stages of strand invasion. In the absence of ASY1<br />
AtRAD51 is able to displace AtDMC1 such that inter-sister chromatid repair<br />
predominates with a subsequent loss of CO formation and synapsis.<br />
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T - 31. MLH1 marks a subset of strongly interefering crossovers in tomato<br />
Franck Lhuissier, Hildo Offenberg, Peter Wittich, Norbert Vischer, Christa<br />
Heyting Upstream research, Keygene (company), 6700AE Wageningen, Netherlands<br />
In most eukaryotes, the prospective chromosomal positions of meiotic crossovers are<br />
marked during meiotic prophase by protein complexes called late recombination nodules<br />
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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
(LNs). In tomato (Solanum lycopersicum), a cytological recombination map has been<br />
constructed based on LN positions. We demonstrate that the mismatch repair protein<br />
MLH1 occurs in LNs. We determined the positions of MLH1 foci along the 12 tomato<br />
bivalents during meiotic prophase and compared the map of MLH1 focus positions with<br />
that of LN positions. On all 12 bivalents, the number of MLH1 foci was app. 70% of the<br />
number of LNs. Bivalents with zero MLH1 foci were rare, which argues against random<br />
failure of detecting MLH1 in the LNs. We inferred that there are two types of LNs, MLH1positive<br />
and MLH1-negative LNs, and that each bivalent gets an obligate MLH1-positive<br />
LN. The two LN types are differently distributed along the bivalents. Furthermore,<br />
cytological interference among MLH1 foci was much stronger than interference among<br />
LNs, implying that MLH1 marks the positions of a subset of strongly interfering<br />
crossovers. Based on the distances between MLH1 foci or LNs, we propose that MLH1positive<br />
and MLH1-negative LNs stem from the same population of weakly interfering<br />
precursors.<br />
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T - 32. Identification of meiotic DSB forming proteins in Arabidopsis thaliana<br />
Arnaud De Muyt, Lucie Pereira, Daniel Vezon, Ghislaine Gendrot, Mathilde Grelon Genetic<br />
and Plant Breeding, INRA, 78026 Cedex Versailles, France<br />
In budding yeast, meiotic recombination events are initiated by double strand breaks<br />
(DSBs) catalysed by the Spo11 protein. In this species, DSB formation also requires nine<br />
other proteins whose molecular function is poorly understood. Nevertheless, recent<br />
studies tend to group them into subcomplexes, indicating that they can be intimately<br />
linked and can form a large recombination initiation complex in which DSBs are made.<br />
Many investigations have been carried out to identify in other organisms, homologs of<br />
these DSB forming proteins. Unfortunately, unlike Spo11, most of these are not conserved<br />
across kingdoms. Furthermore, even when DSB proteins are conserved, their role in<br />
meiotic DSB formation is missing. A large-scale forward genetic approach to isolate plant<br />
meiotic genes has revealed the existence of new meiotic functions that could be<br />
necessary for meiotic DSB formation. We will present data concerning the isolation of one<br />
of these genes : AtPRD1 for Arabidopsis thaliana Putative Recombination initiation Defect<br />
1. In Atprd1 mutants, meiotic recombination rates fall dramatically, early recombination<br />
markers (e.g. DMC1 foci) are absent, but meiosis progresses until achiasmatic univalents<br />
are formed. Besides, Atprd1 mutants suppress DSB repair defects of a large range of<br />
meiotic mutants, showing that AtPRD1 is involved in meiotic recombination and is<br />
required for meiotic DSB formation. Furthermore, we showed that AtPRD1 and AtSPO11-1<br />
interact in a yeast two hybrid assay, suggesting that AtPRD1 is a partner of AtSPO11-1.<br />
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T - 33. Recombination and genetic maps in Petunia<br />
tom gerats IWWR/Plant Genetics, Radboud University, 6525ED Nijmegen, Netherlands<br />
Most Petunia species have 2n=14 chromosomes; hybrids between these species are easy<br />
to obtain. Despite this genetic maps are rather enigmatic and highly variable in length,<br />
with varying clusters of no recombination, depending on the parental lines used in the<br />
specific cross. A gene has been defined that modulates meiotic recombination<br />
frequencies. Moreover, it is shown that chromosome structure in itself can modulate<br />
recombination frequencies: increasing length of deletions in the short arm of<br />
chromosome six induce increased recombination between two markers on the tip of the<br />
long arm. Deletion of the complete arm leads to the markers behaving independently,<br />
while restoration to a complete chromosome leads to the original situation: the markers<br />
behave as nearly completely linked.<br />
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T - 34. Modulation of the recombination machinary for gene targeting<br />
Paul Hooykaas Molecular and Developmental Genetics, Leiden University /Institute of<br />
Biology Leiden (IBL), 2333AL Leiden, Netherlands<br />
Transgenes ,introduced into eukaryotic cells by a variety of methods, are best maintained<br />
by integration into one of the host chromosomes. The molecular mechanisms underlying<br />
DNA integration and the enzymes involved are only partially known. It seems that<br />
depending on the host either the enzymes involved in homologous recombiation(HR) are<br />
preferably used or the enzymes mediating non-homologous end-joining(NHEJ). In yeasts<br />
and fungi genes are almost exclusively integrated by homologous recombination after<br />
disruption of the NHEJ-genes, and vice versa after deletion of the HR-genes DNAintegration<br />
occurs by NHEJ. In plants (and animal cells) the enzymatic machinery is more<br />
complex as after inactivation of the NHEJ-genes DNA-integration still occurs<br />
predominantly by non-homologous recombination. Alternative methods to promote gene<br />
targeting in these organisms will be discussed.<br />
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T - 35. Gene Targeting by Homologous Recombination in Rice<br />
Rie Terada, Yasuyo Johzuka-Hisatomi, Takaki Yamauchi, Shigeru Iida Division of<br />
Molecular Genetics, National Institute for Basic Biology, 444-8585 Okazaki, Japan<br />
Gene Targeting by Homologous Recombination in Rice Rie Terada1, Yasuyo Johzuka-<br />
Hisatomi1, Takaki Yamauchi1,2 and Shigeru Iida1 1National Institute for Basic Biology,<br />
Okazaki 444-8585, Japan 2Faculty of Horticulture, Chiba University, Chiba 271-8510,<br />
Japan. The modification of an endogenous gene into a designed sequence by homologous<br />
recombination, termed gene targeting, has broad implications for basic and applied<br />
research. Rice (Oryza sativa L.), with a sequenced genome of 389 Mb, is one of the most<br />
important crops and a model plant for cereals, and the single-copy gene Waxy on<br />
chromosome 6 has been disrupted with a frequency of 1% per surviving callus by gene<br />
targeting using a strong positive-negative selection. Since the strategy is independent of<br />
gene-specific selection or screening, it is in principle applicable to any gene. However, a<br />
gene in the multigene family or a gene carrying repetitive sequences may preclude<br />
efficient homologous recombination-promoted gene targeting due to the occurrence of<br />
ectopic recombination. Here, we describe an improved gene targeting procedure whereby<br />
we obtained nine independent transformed calli having the Adh2 gene for alcohol<br />
dehydrogenase 2 disrupted with a frequency of approximately 2% per surviving callus<br />
and subsequently isolated eight fertile transgenic plants without the concomitant<br />
occurrence of undesirable ectopic events, even though the rice genome carries four Adh<br />
genes, including a newly characterized Adh3 gene, and a copy of highly repetitive<br />
retroelements is present adjacent to the Adh2 gene. By analyzing the surviving calli<br />
containing randomly integrated and truncated T-DNA segments that carry a positive<br />
selection marker without intact negative markers, we can speculate certain aspects of T-<br />
DNA integration processes leading to homologous and nonhomologous recombination<br />
events in gene targeting with positive-negative selection. In addition to generating gene<br />
knockout rice plants, we are attempting to generate transgenic rice plants carrying<br />
knockin modification at target genes.<br />
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T - 36. Gene targeting in plants using zinc finger nucleases<br />
David Wright, Jeff Townsend, Ronnie Winfrey, Fengli Fu, Jeffry Sander, Drena Dobbs, Keith<br />
Joung, Dan Voytas Dept. of Genetics, Development & Cell Biology, Iowa State University,<br />
50011 Ames, United States<br />
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Engineered zinc-finger nucleases can stimulate high frequency gene targeting at specific<br />
genomic loci in plant cells (Plant Journal, 44:693). ZFNs consist of a Cys2His2 zinc finger<br />
domain engineered to bind a particular gene sequence and the non-specific nuclease<br />
domain of the FokI restriction enzyme. These artificial proteins introduce doublestranded<br />
breaks at specific DNA sequences and thereby vastly increase the rate of<br />
homologous recombination at the cleaved locus. ZFN-mediated gene targeting has great<br />
potential for applications in both basic and applied plant biology; however, the general<br />
application of this promising technology depends critically on the ability to design zinc<br />
finger domains targeted to any desired DNA sequence. To address this need, the Zinc<br />
Finger Consortium was established to promote continued research and development of<br />
engineered zinc finger technology (www.zincfingers.org). In initial work, the Consortium<br />
developed a unified, robust, and user-friendly zinc finger engineering platform. A<br />
comprehensive archive of plasmids was created that encode more than 140 wellcharacterized<br />
zinc-finger modules together with complementary web-based software<br />
(termed ZiFiT) for identifying potential zinc-finger target sites in a gene of interest. The<br />
Consortium also developed protocols for rapidly testing the DNA-binding activities of<br />
assembled multi-finger arrays in bacterial and yeast cell-based reporter assays as well as<br />
vectors for the expression of zinc finger nucleases in plants.<br />
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T - 37. Stimulating Gene Targeting into the Arabidopsis genome<br />
Hezi Shaked, Cathy Melamed-Bessudo, Michal Lieberman-Lazarovich, Aviva Samach,<br />
Naomi Avivi-Ragolsky, Yonat Eshchar, Avraham Levy Plant Sciences, Weizmann Institute of<br />
Sciences, 76100 Rehovot, Israel<br />
Gene targeting, the homologous recombination-mediated integration of an extrachromosomal<br />
DNA segment into a chromosomal target sequence, enables the precise<br />
disruption or replacement of any gene. Despite its importance, gene targeting remains<br />
inefficient in higher plants and in several eukaryotic species. In species such as chicken,<br />
mouse and yeast, earlier reports have shown that the chromatin remodeling activity of<br />
Rad54-like proteins is important for efficient gene targeting. We have developed a new<br />
high-throughput assay, based on the use of a green fluorescent seed marker, to study the<br />
effect of chromatin remodeling on gene targeting in plants. We report that expression of<br />
the yeast RAD54 gene enhances gene targeting in Arabidopsis by one to two orders of<br />
magnitude, from 10-4 to 10-3 in the wildtype plants to 10-2 to 10-1. These findings<br />
suggest that chromatin remodeling is rate-limiting for gene targeting in plants and<br />
improve the prospects for using gene targeting for the precise modification of plant<br />
genomes. We describe the structure of both precise gene replacement events and of<br />
ectopic targeting events. In addition we have developed a new gene targeting assay, based<br />
on the use of a red fluorescent seed marker. This assay, combined with the precise<br />
expression of additional recombination-enhancing proteins in the target cells will be used<br />
for further optimization of the targeting process.<br />
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T - 38. Mutation scanning by massive parallel sequencing<br />
Paul Bundock, Rene Hogers, Antoine Janssen, Diana Rigola, Harrie Schneiders, Nathalie<br />
van Orsouw, Hein van der Poel, Michiel van Eijk, Michiel de Both Upstream Research,<br />
Keygene N.V., 6700 AE Wageningen, Netherlands<br />
In order to rapidly screen mutant libraries for the detection of mutations in specific<br />
genes we utilize massive parallel sequencing. We apply a 3D pooling strategy on DNA of<br />
EMS mutagenized plants followed by amplification of the target gene with PCR primers<br />
carrying a pool identifier tag specific to each of the 3D pools. PCR products undergo ultra<br />
deep sequencing using the automated pyrosequencer GS20. Mutations in the gene of<br />
interest are observed at a much higher frequency than random sequencing errors.<br />
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Bioinformatics analysis allows the idenification of the mutant pool and the corresponding<br />
mutant is then retrieved. In addition, the sequence around the mutation is directly<br />
available for further analysis and assay development, which is not the case with common<br />
CEL-1 based TILLING methods. Using this approach a complete mutant library can be<br />
screened efficiently for mutations in a specific locus in one straightforward experiment.<br />
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T - 39. Application of Designed Zinc-Finger Protein Technology in Plants<br />
Vipula Shukla, Teresa Bauer, Nicole Arnold, Jon Mitchell, Matt Simpson, Sarah Worden,<br />
Fyodor Urnov, Jeffrey Miller, Jeremy Rock, Erica Moehle, Yannick Doyon, Lei<br />
Zhang Discovery R&D, Dow AgroSciences, LLC, 46268 Indianapolis, IN, United States<br />
The primary sequence of the genome at endogenous loci can be altered with high<br />
efficiency in mammalian cells using designed zinc finger nucleases (ZFNs; Urnov et al.<br />
Nature 435: 646). Ongoing studies indicate that a precisely-placed double-strand break<br />
(DSB) induced by engineered ZFNs can stimulate integration of long DNA stretches into a<br />
predetermined genomic location in human cells, resulting in site-specific gene addition.<br />
Zinc finger protein technology represents a significant breakthrough relative to the ability<br />
to edit and engineer genomes in a precise manner. In this presentation, results from a<br />
collaboration between Dow AgroSciences LLC and Sangamo Biosciences that is focused on<br />
applications of designed zinc-finger protein technology in plant species will be described.<br />
Multiple zinc-finger proteins, including zinc-finger nucleases and zinc-finger<br />
transcription factors, have been designed to target specific genes in model and<br />
agriculturally important plant species. Validation of this technology and examples of its<br />
utility for plant biotechnology will be discussed.<br />
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T - 40. Development of a gene targeting system based on in planta presentation of<br />
homologous donor DNA during meiosis.<br />
Frederic Van Ex, Dimitri Verweire, Kristof Verleyen, Koen Peeters, Eric Dewale, Geert<br />
Angenon Laboratory of Plant Genetics, Vrije Universiteit Brussel (V.U.B.), 1050 Brussels,<br />
Belgium<br />
Keywords: Gene Targeting/Homologous recombination/Cre-Lox/meiosis Currently, we<br />
are developing a system that allows efficient gene targeting in Arabidopsis thaliana. The<br />
outline of our approach is based on two main features: 1. the intrinsic characteristics – in<br />
regard to homologous recombination – of cells during meiosis 2. the in planta<br />
presentation of homologous donor DNA in the aforementioned cells Our current assay is<br />
based on the restoration of a GUS:nptII gene, defective for kanamycin resistance. Target<br />
lines were created by introducing such a defective GUS:nptII reporter gene (containing a<br />
3’ nptII deletion) under control of a 35S promoter. Two separate target lines – both<br />
homozygous for a single copy of the defective GUS:nptII reporter gene – were selected. A<br />
targeting vector – carrying a lox cassette containing a promoterless gus:NPTII reporter<br />
gene (having a 5’ gus deletion) and a downstream 5000 bp stretch of sequence<br />
homologous to the target sequence – was introduced in the two target lines. In the final<br />
step of our approach a Cre gene under control of a promoter active in the prophase I of<br />
the meiosis (promoter of the A. thaliana Solo Dancers (SDS) gene, AT1G14750, Azumi et<br />
al., 2002) was introduced into the target targeting lines. Placing the expression of the Cre<br />
gene under control of the SDS promoter allows the induction of the Cre/lox<br />
recombination during the meiotic prophase I. The Cre/lox recombination will result in the<br />
formation of a circular donor DNA that can be presented for homologous recombination<br />
with the target sequence. Homologous recombination between both gus:NPTII reporter<br />
genes will lead to repair of the target GUS:nptII reporter gene (controlled by a 35S<br />
promoter) resulting in kanamycin resistance. First results show that in planta<br />
presentation of homologous donor DNA can lead to homologous recombination.<br />
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Extensive analysis will identify the nature of the observed recombination events in order<br />
to discriminate between ‘true’ and ectopic gene targeting events. We recently extended<br />
the strategy to different promoters allowing activation of our gene targeting system in<br />
other specific cell types where homologous recombination is suspected to be the<br />
predominant recombination mechanism.<br />
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T - 41. Meganucleases with tailored cleavage specificity can induce efficient<br />
homologous gene targeting<br />
Sylvain Arnould, Jean-Pierre Cabaniols, Philippe Duchateau, Aymeric Duclert, Jean-Charles<br />
Epinat, Agnès Gouble, Sylvestre Grizot, Christophe Perez, Julianne Smith, Frédéric<br />
Pâques Scientific Department, Cellectis S.A., 93 235 Romainville, France<br />
Homologous gene targeting is the best way to modify a genome in a precise and rational<br />
way, but has proved to be very inefficient in plants. This limit can be alleviated by<br />
meganucleases, sequence-specific endonucleases recognizing large (>12 bp) cleavage<br />
sites. These proteins can stimulate homologous gene targeting by a 1000-fold factor or<br />
induce mutagenesis in the vicinity of their target site, and these findings have opened<br />
novel perspectives for genome engineering in plants. However, the use of this technology<br />
has long been limited by the repertoire of natural meganucleases: the probability of<br />
finding a sequence cleaved by a natural meganuclease in a chosen gene is extremely low.<br />
Therefore, the design of artificial endonucleases with chosen specificities is under intense<br />
investigation. Given their exceptional specificity, natural meganucleases should provide<br />
ideal scaffolds to derive genome engineering tools.We have developed a combinatorial<br />
approach to redesign the DNA-binding interface of I-CreI, a Chlamydomonas reinhardti<br />
protein belonging to the LAGLIDADG family of meganucleases. First, we collect large<br />
numbers of locally engineered I-CreI derivatives with altered specificity. Second, we<br />
assemble these mutants into entirely redesigned endonucleases binding a priori chosen<br />
targets. The engineered proteins keep the essential properties of natural meganucleases<br />
in terms of folding, activity and specificity, and can be used to induce recombination and<br />
site directed mutagenesis in chromosomal sequences. Thus, our strategy provides the<br />
means to engineer a very large number of sequences in a very specific way. We will<br />
illustrate both the protein engineering and genome engineering steps.<br />
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T - 42. Toward zinc finger nucleases-mediated gene targeting in plants<br />
Andriy Tovkach, Vardit Zeevi, Tzvi Tzfira Department of Molecular, Cellular, &<br />
Developmental Biology, University of Michigan, 48109-1048 Ann Arbor, MI, United States<br />
Double-strand breaks (DSBs) in plant genomes are typically repaired by the plant nonhomologous<br />
end-joining (NHEJ) machinery, which usually leads to local mutagenesis due<br />
to small deletions at the repair site. We have shown that double stranded T-DNA<br />
molecules preferentially integrate into DSBs in transgenic plants carrying an I-SceI<br />
endonuclease recognition site which, upon cleavage with I-SceI, generates a DSB. In<br />
addition, using AscI, an 8 base cutter recognizes ca. 80 sites in the Arabidopsis genome<br />
we discovered that DSBs acts as attractants of T-DNA molecules at different genomic<br />
locations. This phenomenon could potentially be used for targeting foreign DNA<br />
molecules into specific sites in the plant genome using zinc finger nucleases (ZFNs). ZFNs<br />
are a new type of artificial restriction enzymes which are custom-designed to recognize<br />
and cleave specific DNA sequences, producing DSBs. However, technical difficulties in the<br />
design, assembly and analysis of ZFNs have hindered the use of this new technology for<br />
plant gene targeting. We have recently designed a set of constructs and cloning,<br />
biochemical and in-planta analysis procedures for the newly designed ZFNs. Cloning<br />
begins with de-novo assembly of the DNA-binding regions of new ZFNs from overlapping<br />
oligos containing modified helices responsible for DNA triplet recognition, and their<br />
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insertion between a nuclear localization signal and the FokI endonuclease domain.<br />
Following the transfer of fully assembled ZFNs into E. coli expression vectors, bacterial<br />
lysates were found to be most suitable for in-vitro digestion analysis of palindromic<br />
target sequences. An in-planta activity test was also developed to confirm the nucleic<br />
activity of ZFNs in plant cells. The assay is based on reconstruction of GUS expression<br />
following bombardment of a reporter and ZFN-expressing plasmids into mesophyll cells.<br />
Our new procedures, plasmids and assays bring us one step closer to efficient<br />
implementation of ZFN-based technology for gene targeting in plant species.<br />
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T - 43. Generation of Single-Copy T-DNA Transformants by the Cre/LoxP<br />
Recombination Mediated Resolution System<br />
Sylvie De Buck, Annelies De Paepe, Ingrid Peck, Gordana Marjanac, Ann Depicker Plant<br />
Systems Biology, VIB, UGent, 9052 Gent, Belgium<br />
Transgene loci obtained after Agrobacterium tumefaciens transformation are considered<br />
to be less complex than those obtained after direct gene transfer, but integration of<br />
multiple T-DNA copies into direct or inverted repeats is fairly common. Recently, it was<br />
convincingly shown that transgenic plants with high and stable heterologous protein<br />
accumulation levels can be enriched for by screening for single-copy T-DNA<br />
transformants (De Buck et al., 2004). The aim was to evaluate a transformation system to<br />
generate efficiently single T-DNA copy transformants. Therefore, a strategy was designed<br />
to reduce the number of transgene copies by making use of the site-specific Cre/loxP<br />
recombination system. A lox-T-DNA vector containing two invertedly oriented loxP<br />
sequences located both inside and immediately adjacent to the T-DNA border ends was<br />
constructed. In the presence of the CRE recombinase, recombination between the<br />
outermost loxP sequences in direct orientation should resolve multiple copies into a<br />
single T-DNA copy, regardless of the orientation and number of T-DNAs integrated at one<br />
locus. Two different strategies were followed to test the approach. Firstly, several<br />
parental plants containing multiple lox-T-DNA copies at one locus were crossed with creexpressing<br />
plants. Secondly, cre-expressing plants were retransformed with the lox-T-<br />
DNA construct. For both methods, we could demonstrate the resolvement of multimeric<br />
T-DNAs to one T-DNA copy with a satisfactory frequency. Furthermore, this reduction in<br />
T-DNA copies led in most cases to an increased transgene expression level. We therefore<br />
propose that the described lox-T-DNA vector is a tool to obtain transformants with high<br />
and stable transgene expression. Reference: De Buck, S., Windels, P., De Loose, M., and<br />
Depicker, A. (2004). Single-copy beta-glucuronidase transgenes integrated at different<br />
positions in the Arabidopsis genome show uniform and comparable expression. Cell. Mol.<br />
Life Sci., 61: 2632-2645.<br />
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Abstracts of Posters<br />
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P - 1. Analysis of Meiotic Recombination in Rad51 Paralog Mutants in<br />
Arabidopsis.<br />
Kiyomi Abe*, Jean-Yves Bleuyard, Charles White.. CNRS UMR6547, Université Blaise Pascal,<br />
63177 Aubière, France. *Plant Genetic Engineering Research Unit, National Institute of<br />
Agrobiological Sciences, 305-8602 Tsukuba, Japan<br />
Recent studies indicate that two Rad51 paralogs (Xrcc3 and Rad51C) are involved in both<br />
DNA repair and meiotic recombination. To determine further details about the role of<br />
these proteins in meiotic recombination, we analyzed meiosis in atrad51C and xrcc3<br />
mutants. Cytological observation showed apparently normal figures up to<br />
zygotene/pachytene and dramatic abnormalities afterwards through telophase I in both<br />
atrad51C and xrcc3 mutants, indicating that pairing does occur at zygotene but that full<br />
chromosomal synapsis and synaptonemal complex formation depends upon the presence<br />
of the Xrcc3 and Rad51C proteins. Using fluorescence in situ hybridization (FISH) analysis<br />
we observe pairing of centromeres but not of the tested euchromatic region at pachytene<br />
in both mutants. These results suggest that Xrcc3 and Rad51C proteins play important<br />
roles in homologous pairing of the euchromatic regions of chromosomes.<br />
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P - 2. Genome-wide analysis of rye specific chromosome regions<br />
Olena Alkhimova, Marie Kubalakova, Olena Martynenko, Jaroslav Dolezel . National<br />
Academy of Sciences of Ukraine, Institute of Molecular Biology and Genetics, 03143 Kyiv,<br />
Ukraine<br />
Telomeres are specialized chromosome structure, which play important role in<br />
chromosome stability and behaviour. Together with subtelomeres, they form terminal<br />
regions of chromosomes. These are dynamic regions and might help organisms to adapt<br />
to new environmental conditions and promote genomic rearrangements. Large<br />
heterochromatic blocks, which are present near the telomeres of rye chromosomes, are<br />
notoriously difficult for physical mapping because of enrichment by different types of<br />
repetitive DNA sequences. We used wheat-rye addition lines and performed fluorescence<br />
in situ hybridization (FISH) on metaphase stretched flow-sorted chromosomes and<br />
extended DNA fibers for comparison of the terminal regions of the individual rye<br />
chromosomes. Each wheat-rye addition line produced a chromosome-specific large-scale<br />
organization of the tandem repeat arrays. The FISH signals on DNA fibers showed the<br />
multiple patterns of co-linear monomers arrangement of repetitive families as well as of<br />
authentic telomeric probe from Arabidopsis. The primary structure of some spacer<br />
sequences revealed the scrambled regions of similarity to various known repetitive<br />
elements. Using PCR analysis, we determined the DNA sequences adjacent to the tandem<br />
repeats array (spacers). They are enriched of short direct, complementary, inverted and<br />
symmetrical repeats which appear to be associated with recombination events. These<br />
spacers may be a powerful source of tandem array rearrangements. We mapped some of<br />
them on metaphase stretched chromosomes, and the distribution of the signals was in<br />
good agreement with the localization of the breakpoints for deletions and for<br />
translocations of 1R short arm with different wheat chromosomes. This work was<br />
supported by the INTAS grant (03-51-5908).<br />
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P - 3. Immunolocalization of Mre11 and Rad50 proteins during prophase I in<br />
tomato<br />
Lorinda Anderson, Leslie Lohmiller, Hildo Offenberg, Christa Heyting . Biology, Colorado<br />
State University, 80523-1878 Fort Collins, Colorado, United States<br />
Mre11 and Rad50 proteins act together in a protein complex that is involved in DNA<br />
double-strand break (DSB) repair, and the proteins also have roles in meiotic synapsis and<br />
recombination. mre11 and rad50 mutants in Arabidopsis thaliana lack homologous<br />
synapsis, suffer extensive chromosome fragmentation during prophase I, and are sterile.<br />
Current models predict the presence of both Mre11 and Rad50 in early recombination<br />
nodules (ENs) that are thought to represent sites of DSBs. Here, we used<br />
immunofluorescence to examine the meiotic behavior of these two proteins in Solanum<br />
lycopersicum (tomato) microsporocytes during early prophase I. Mre11 was present as<br />
numerous immunofluorescent foci that associated preferentially with the axes of tomato<br />
chromosomes before, during and immediately after synapsis. As many as 800 Mre11 foci<br />
were observed in leptotene nuclei (more than twice the maximum number of ENs<br />
observed at zygotene), and Mre11 foci were most common in distal euchromatic regions<br />
of chromosomes (as are ENs). Electron microscopic immunogold localization<br />
demonstrated that Mre11 is a component of only some ENs at mid-late zygotene while<br />
accumulations of Mre11 along the axial/lateral elements are common. In addition, Mre11<br />
foci are far more numerous than ENs on asynapsed euchromatic segments of leptotene<br />
and early zygotene chromosomes. Like Mre11, Rad50 foci preferentially associate with<br />
chromosomal axes during early prophase I. Unexpectedly, the number of Rad50 foci was<br />
less than that of Mre11 foci (although more similar to EN numbers), and Rad50 and<br />
Mre11 foci did not often colocalize. The presence of Rad50 in ENs is still under<br />
investigation, but our results indicate that most Mre11 foci do not correspond to ENs.<br />
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P - 4. Human Rad51 binding and the subsequent unwinding of duplex DNA<br />
requires different nucleotide cofactors<br />
kamakshi Balakrishnan, Rekha Govekar, Basuthkar J. Rao . Department of Biological<br />
sciences, Tata Institute of Fundamental Research (TIFR), 400006 Mumbai, India<br />
Homologous recombination is one of the central mechanisms for removing DNA double<br />
strand breaks in humans and other eukaryotes. Human Rad51 (hRad51) is a recombinase,<br />
which brings about this process in concert with other recombination mediator proteins<br />
such as Rad52, Replication Protein A (RPA), Rad54 and other accessory proteins by<br />
performing DNA homology search and pairing, followed by DNA strand exchange. With<br />
the aim of understanding the roles of different nucleotide cofactors such as ATP, ADP,<br />
ATP gammaS (a slow hydrolysable ATP analog), GTP and GDP on: (a) hRad51 binding to<br />
duplex DNA and (b) hRad51 mediated unwinding of duplex DNA , we carried out binding<br />
and topological assays. Our results suggest that binding of hRad51 to circular duplex<br />
DNA is efficient in the presence of nucleotide cofactors such as ATP, ATP gammaS or<br />
GTP, and results in the formation of higher order protein-DNA complexes. However, in<br />
absence of such nucleotide cofactors or in presence of ADP, lower order complexes<br />
result. We observe rapid unwinding only in the presence of ATP, whereas the unwinding<br />
kinetics slow down in presence of ATP gammaS. This indicates that hRad51 mediated<br />
duplex DNA unwinding depends on ATP hydrolysis, unlike binding. Although providing<br />
GTP as a cofactor enhances hRad51 binding to duplex DNA as much as ATP does, the<br />
unwinding kinetics mediated by GTP are poorer compared to ATP. ADP and GDP on the<br />
other hand, neither facilitate binding nor unwinding. Upon inclusion of hRad52 however,<br />
in the reaction system hRad51 unwinds dsDNA utilizing ADP as cofactor. Same is not<br />
true for GDP. Our results thus indicate that (a) hRad51 mediated unwinding of duplex<br />
DNA is kinetically favored when ATP can be hydrolyzed as against the slower unwinding<br />
observed with ATP gammaS, (b) A new role for hRad52 in the unwinding reaction carried<br />
out by hRad51 when ADP is provided as cofactor, (c) GTP can also serve as a cofactor for<br />
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unwinding reaction although not as efficient as ATP.<br />
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P - 5. Towards plant gene targeting – Enhancing homologous recombination<br />
frequency in tobacco.<br />
Abdellah Barakate, Claire Halpin. based at Scottish Crop Research Institute, University of<br />
Dundee , DD2 5DA Dundee, United Kingdom<br />
Gene targeting (GT) is a powerful tool that allows a homology-based replacement of an<br />
endogenous gene by an in vitro manipulated copy. Developing this technology has proved<br />
particularly difficult in plants where foreign DNA is integrated predominantly by nonhomologous<br />
recombination. The main competing pathways of recombination repair(1)<br />
could be manipulated to increase homologous recombination (HR) efficiency. One<br />
strategy to stimulate HR is to express recombinases involved in this pathway. We have<br />
expressed multiple bacterial (RecA, RecG, RuvC) and human (hRad51, hDMC1 and<br />
hRad52) recombinases in a tobacco line (N1DC4) that already contains a transgene as an<br />
artificial substrate for intrachromosomal recombination(2) (ICR). The transgene for ICR<br />
consists of a direct repeat of two defective b-glucoronidase (GUS) genes that can<br />
recombine to generate a functional GUS, which can be detected by histochemical staining<br />
of plant material. In order to co-ordinate the expression of multiple recombinases in a<br />
single plant, we have used an artificial self-dissociating polyprotein system. This unique<br />
and novel function was taken from the 2A region of foot-and-mouth disease virus(3).<br />
Several tobacco transgenic lines expressing multiple recombinases were selected and<br />
their ICR assessed both in pollen and six-week-old seedlings. While ICR stimulation in<br />
seedlings remained modest, a huge increase (up to 1000 fold) was detected in pollen of<br />
some transgenic lines. In addition, most transgenic lines showed various degrees of<br />
sterility and their meiosis will be analysed. This plant material will also be used to<br />
determine whether ICR stimulation will translate into efficient gene targeting using our<br />
newly designed vectors. References: 1) Trends Genet. (2005) 21, 172-181. 2) <strong>EMBO</strong> J.<br />
(1994) 13, 484-489. 3) Plant Physiol. (2004) 135, 16-24. Acknowledgements: Barbara Hohn<br />
(Basel) for N1DC4 line, Stephen West (London) for human recombinases. Funding: BBSRC,<br />
Scottish Enterprise Tayside, The Leverhulme Trust.<br />
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P - 6. Repair of G:T mismatches in plants<br />
Joke Baute, Gert Van der Auwera, Ann Depicker. Plant Systems Biology, VIB - Ghent<br />
University, 9052 Ghent, Belgium<br />
G:T mismatches are the result of replication errors or of hydrolytic deamination of 5methylcytosine<br />
that occurs spontaneously in the cell. In mammals, G:T mismatches<br />
caused by deamination of methylated cytosines, are repaired by base excision repair. Two<br />
DNA glycosylases recognize this mismatch: thymine DNA glycosylase (TDG) and methylbinding<br />
domain 4 protein (MBD4). Both enzymes specifically convert G:T mismatches to<br />
G:C. In plants, the methylation level is higher than in animals, suggesting that plants also<br />
developed systems to repair G:T mismatches caused by deamination of 5-methylcytosine.<br />
To analyze this in plants, we are performing a functional analysis of MBD4 homologs in<br />
Arabidopsis thaliana. We identified only one annotated coding sequence in Arabidopsis<br />
thaliana that shows sequence homology to the glycosylase domain of MBD4. The human<br />
MBD4 consists of two DNA binding domains: a methyl-CpG-binding domain and a<br />
mismatch specific DNA glycosylase domain, while the Arabidopsis protein (AtMBD4) has<br />
the glycosylase domain but lacks the methyl-CpG-binding domain. Using Y2H, we are<br />
currently investigating with which proteins AtMBD4 interacts. We have found different<br />
splicing variants of AtMBD4 in different tissues, what is also the case for human MBD4<br />
and for many other DNA glycosylases, and these will be presented. To learn more on the<br />
function of the AtMBD4 gene, we analyzed the AtMBD4 promotor activity by using<br />
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different beta-glucuronidase reporter fusions. We found that the fusion protein resides<br />
primarily in the nuclei of the meristems. Also other DNA repair genes in plants are<br />
primarily expressed in meristematic tissue. This is compatible with the model that repair<br />
genes play a crucial role in assuring fertility and fitness of the progeny. Currently, we are<br />
investigating the effect of different types of stress on the AtMBD4 promotor activity. For<br />
further characterization of AtMBD4, we generated silencing and overexpression lines,<br />
which do not show a phenotype under normal conditions. To determine the impact of<br />
AtMBD4 on mutation frequency, we analyzed plants with different AtMBD4 levels, via a<br />
detection system, developed in our lab. This allows to determine the mutation frequency<br />
of cytosine to thymine via the reactivation of a deactivated GUS gene. Finally, we<br />
investigated whether AtMBD4 plays a role in the signalization of DNA damage caused by<br />
mutagenic agents, as human and mouse MBD4 not only have a function in the recognition<br />
of G:T mismatches, but also play a role in the signalization of DNA damage caused by<br />
different mutagenic agents. The results will be discussed.<br />
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P - 7. Gene targeting in plants via engineered zinc finger proteins<br />
Charles Cai, Vipula Shukla, Fyodor Urnov, Jeffery Miller, Nicole Arnold, Lisa Baker, Teresa<br />
Bauer, Ryan Blue, Dave McCaskill, Jon Mitchell, Matt Simpson, Andrew Worden. Plant<br />
Genetics and Biotechnology, Dow AgroSciences, 46268 Indianapolis, IN, United States<br />
Gene targeting via homologous recombination occurs at a very low frequency in plant<br />
cells compared to random integration making targeted gene modifications impractical.<br />
Most recently, substantial increases in the frequency of homologous recombination have<br />
been observed following the induction of double stranded breaks in host cell DNA<br />
followed by the apparent stimulation of cellular repair mechanisms. Strategies to achieve<br />
targeted DNA double stranded breaks have been developed by fusing sequence-specific<br />
zinc finger DNA binding proteins with sequence-independent nuclease domains derived<br />
from Type IIS restriction endonucleases. Using this strategy, both site-specific transgene<br />
integration and targeted modification of native genes have been demonstrated in plants.<br />
Implications for novel trait development and crop improvement will be discussed.<br />
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P - 8. SITE-DIRECTED HOMOLOGOUS RECOMBINATION IN TOBACCO CELL<br />
CULTURES VIA ZINC FINGER NUCLEASES<br />
Charles Cai, Michael Ainley, Trevor Collingwoo, Robbi Garrison, Lisa Schulenberg, Philip<br />
Gregory, Beth Rubin-Wilson, Joseph Petolino. Biochemistry and Molecular Biology, Dow<br />
AgroSciences, LLC., 46268 Indianapolis, United States<br />
Targeted transgene integration via homologous recombination occurs at a very low<br />
frequency in plant cells compared to random integration, even when the incoming DNA<br />
comprises large stretches of sequence homologous to host DNA. As such, gene targeting<br />
in plants via homologous recombination has not been practical. Most recently, substantial<br />
increases in the frequency of homologous recombination have been observed following<br />
the induction of DNA double stranded breaks in host cells and apparent stimulation of<br />
cellular repair mechanisms. Restriction enzymes whose recognition sites are rare in the<br />
plant genome have been shown to stimulate homologous recombination following the<br />
formation and repair of DNA double stranded breaks in the host DNA. Strategies to<br />
achieve targeted DNA double stranded breaks have been developed by fusing zinc finger<br />
DNA binding proteins with sequence-independent nuclease domains derived from Type II<br />
restriction endonucleases. In the present study, engineered zinc finger proteins fused to<br />
nuclease domains, so-called ‘zinc finger nucleases’, were used to facilitate site-specific<br />
transgene integration via homologous recombination in tobacco cell cultures. A target<br />
DNA sequence was first stably integrated into tobacco cell cultures using Agrobacterium.<br />
This target sequence contained specific zinc finger protein recognition/binding sites,<br />
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along with a non-functional test gene to be corrected. A few selected transgenic events<br />
containing a single integrated copy of the target sequence were re-transformed using<br />
Agrobacterium strains harboring different T-DNAs. One Agrobacterium strain contained a<br />
donor DNA sequence comprising the bases necessary to correct the non-functional test<br />
gene, flanked by sequences homologous to the pre-integrated target DNA. The second<br />
Agrobacterium strain contained a gene encoding a zinc finger nuclease that specifically<br />
recognized a binding site in the integrated target sequence. Gene targeting via sitedirected<br />
homologous recombination was demonstrated as evidenced by the reconstitution<br />
of a functional test gene and was confirmed via molecular and biochemical<br />
analyses.<br />
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P - 9. Gene and Protein Targeting Technologies Create Novel Opportunities in<br />
Plant Biotechnology<br />
Monique Compier, Patrick Mak, Sylvia de Pater, Bert van der Zaal, Paul Hooykaas, Remko<br />
Offringa , Add2X Biosciences BV, 2333 AL Leiden, Netherlands<br />
An important platform technology that is missing in the field of plant science is gene<br />
targeting through homologous recombination. Moreover, for many economically<br />
important plant varieties, robust and efficient protocols for vegetative propagation and<br />
genetic modification are still lacking. The availability of such technologies is essential for<br />
the further development of plant biotechnology. Add2X Biosciences BV is a young biotech<br />
spin-off from Leiden University that aims to create novel opportunities in plant<br />
biotechnology, by developing innovative platform technologies and products for the<br />
directed and efficient delivery of DNA and proteins into plant cells. The Add2X portfolio<br />
includes: 1. Efficient gene targeting in plants through suppression of the non-homologous<br />
recombination pathway. Proof of concept has been obtained in different yeasts and<br />
filamentous fungi. Currently, research is in progress to confirm the applicability of this<br />
technology in plant species. 2. Agrobacterium-mediated protein translocation to produce<br />
and transiently ‘inject’ proteins of interest into plant cells in order to exert their function<br />
without permanently altering the host cells. 3. Induction of somatic embryogenesis in<br />
hitherto recalcitrant crop species. A protein family was identified that, when overexpressed,<br />
stimulates the spontaneous formation of somatic embryos from vegetative<br />
plant cells. The Add2X technologies stand well on their own, but clearly have the<br />
potential to be combined into integrated technologies or products. The technologies are<br />
developed in close collaboration with Leiden University. In addition, Add2X has<br />
established strategic alliances with other academic and industrial partners to explore<br />
novel opportunities and to achieve rapid implementation of the technologies in the<br />
biotechnology sector.<br />
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P - 10. Homologous recombination between divergent sequences in Arabidopsis<br />
Eyal Emmanuel, Elizabeth Yehuda, Roy Opperman, Naomi Avivi-Ragolsky, Avraham Levy,<br />
Cathy Melamed-Bessudo. Plant Sciences Department, The Weizmann Institute of Science,<br />
76100 Rehovot, Israel<br />
Earlier studies in plants have shown that there is a strong reduction in the rate of<br />
homologous recombination when the recombining partners are chromosomal segments<br />
that originate from related, but divergent, species. The effect of sequence divergence on<br />
homologous recombination is not well established. We found that the rates of somatic<br />
recombination between repeats that diverge only by one out of 617 otherwise identical<br />
nucleotides, is reduced by ~ 3-fold. Similarly, the rates of meiotic recombination are<br />
higher in an isogenic background than in crosses with divergent ecotypes. We analyzed<br />
the role of AtMSH2 in suppression of recombination between divergent sequences in<br />
Arabidopsis. We report that AtMSH2 has a broad range of anti-recombination effects: it<br />
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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
suppresses recombination between divergent direct repeats in somatic cells or between<br />
homologs from different ecotypes during meiosis. We describe the involvement of other<br />
genes from the mismatch repair family on homologous recombination and discuss the<br />
implications of these results for plant improvement via gene transfer across species.<br />
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P - 11. Transient inactivation of NHEJ proteins<br />
Sylvia de Pater, Vanessa Costa, Paul Hooykaas. Institute Biology Leiden, Leiden University,<br />
2333 AL Leiden, Netherlands<br />
Agrobacterium tumefaciens T-DNA normally integrates into random sites in the plant<br />
genome and frequencies of gene targeting (GT) in plants are very low. It would be an<br />
advantage for analysis of gene function and for exploitation of genetically modified<br />
organisms if the frequency of targeted integration could be increased. Experiments with<br />
yeast mutants have shown that GT can be enhanced by mutations in the DNA repair<br />
pathway of non-homologous end joining (NHEJ)(1). Subsequently, Arabidopsis NHEJ T-<br />
DNA insertion mutants have been tested in GT experiments, and preliminary results show<br />
that GT frequency is improved somewhat in these mutants. Since constitutive inhibition<br />
of the NHEJ DNA repair pathway results in telomere lengthening (2,3) and maybe other<br />
DNA rearrangements, we applied the peptide aptamer technology (4) combined with an<br />
inducible promoter system for the temporary inactivation of NHEJ proteins. A peptide<br />
aptamer has been isolated that binds to AtKu80 and yKu80 in yeast (Y2H) and in vitro<br />
and expression in yeast and Arabidopsis results in more sensitivity for DNA damage<br />
inducing agents. 1. van Attikum H and Hooykaas PJJ (2003) Genetic requirements for<br />
targeted integration of Agrobacterium T-DNA in Saccharomyces cereviciae. Nucl Acid Res<br />
31, 826-832. 2. Bundock P, van Atticum H, Hooykaas P (2002) Increased telomere length<br />
and hypersensitivity to DNA damaging agents in an Arabidopsis KU70 mutants. Nucleic<br />
Acids Res 30, 3395-3400. 3. Bundock P and Hooykaas P (2002) Severe developmental<br />
defects, hypersensitivity to DNA-damaging agents, and lengthened telomeres in<br />
Arabidopsis MRE11 mutants. Plants Cell 14, 2451-2462. 4. Colas P, Cohen B, Jessen T,<br />
Grishina, McCoy J, Brent R (1996) Genetic selection of peptide aptamers that recognize<br />
and inhibit cyclin-dependent kinase 2. Nature 380, 548-550.<br />
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P - 12. ADI IS A NOVEL COMPONENT OF THE ARABIDOPSIS SOMATIC DNA<br />
DOUBLE STRAND BREAK RESPONSE AND IS ESSENTIAL FOR MEIOSIS<br />
Philip Dean, Susan Armstrong, Christopher West. CENTRE FOR PLANT SCIENCES,<br />
UNIVERSITY OF LEEDS, UK, LS2 9JT Leeds, United Kingdom<br />
Here we describe ATM DEPENDENT INDUCTION (ADI), a plant specific gene which shows a<br />
large induction specifically in response to genotoxic stress. Quantitative PCR<br />
demonstrates that the induction of ADI is dependent on the protein kinase ATM, placing<br />
ADI in an ATM pathway. ADI is also essential for Arabidopsis meiosis as adi knockout<br />
mutant plants are both male and female sterile. Detailed cytological analysis of these<br />
mutants reveals extensive chromosome fragmentation during meiosis. Studies in an adi<br />
heterozygous background correlate with ADI also being required for pollen mitosis as<br />
there is a failure to complete mitotic cell divisions in approximately half the pollen.<br />
Preliminary yeast 2-hybrid interaction studies indicate ADI may interact with components<br />
required for both DNA double strand break repair and cell cycle progression.<br />
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P - 13. A Cre::FLP fusion protein recombines FRT or loxP sites in transgenic maize<br />
plants<br />
Vesna Djukanovic, Brian Lenderts, Alex Lyznik. Crop Genetics Research, Pioneer Hi-Bred<br />
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International, Inc, IA 50131 Johnston, United States<br />
The coding sequences of Cre (site-specific recombinase from the bacteriophage P1) and<br />
FLP (the yeast 2mm plasmid site-specific recombinase) were fused in frame to produce a<br />
novel, dual-function site-specific recombinase gene. Transgenic maize plants containing<br />
the Cre::FLP-fusion expression vector were crossed to transgenic plants containing either<br />
loxP or FRT excision substrates. Complete and precise excisions of chromosomal<br />
fragments flanked by the respective target sites were observed in the F1 and F2 progeny<br />
plants. The episomal DNA recombination products were frequently lost. Some<br />
chromosomal recombination substrates survived in the F1 plants producing the chimeric<br />
F1 plants for the excision products. They became stabilized in the F2 generation after the<br />
cre::FLP gene segregated out. These observations may indicate that the efficiency of sitespecific<br />
recombination is affected by plant developmental stages with site-specific<br />
recombination being more prevalent in the developing embryos. Both FLP and Cre<br />
activities were comparable, producing clean and precise chromosomal excision products<br />
in maize. The crossing strategy proved to be suitable for genetic engineering of maize<br />
using the FLP or Cre site-specific recombination systems.<br />
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P - 14. In search of a role for Dss1 in the model plant Arabidopsis thaliana<br />
Emeline Dubois, Fabien Delacote, Marie-Pascale Doutriaux. Institut de Biotechnologie des<br />
Plantes-UMR8618, Université Paris XI-CNRS, 91405 Orsay, France<br />
In human, mutations of the BRCA2 gene are responsible for a hereditary form of breast<br />
cancer predisposition (1,2). One important role of Brca2 is to act on homologous<br />
recombination by loading Rad51 onto ssDNA at the site of the double strand breaks.<br />
Dss1 is a partner of Brca2 in human and Ustilago maydis (3,4). In Ustilago, Dss1 is<br />
essential to the Brca2 function in somatic cells (5). In addition, Dss1 was shown to belong<br />
to the 26S proteasome in human and yeast (6,7), whereas there is no Brca2 in yeast. Two<br />
isoforms of Brca2 and two isoforms of Dss1 are encoded for in the Arabidopsis thaliana<br />
genome. A potential role for AtDss1 in the proteasome has been revealed by the<br />
functional complementation of sem1 (the Dss1 ortholog) mutants in yeast. We are now<br />
examining the role of Dss1 in relation to Brca2 in Arabidopsis. One of the two Brca2<br />
isoforms interacts with both AtDss1 isoforms, whereas the other one interacts with only<br />
one (8). RNAi constructs have been established to inactivate Brca2 or Dss1 constitutively<br />
(p35S) or at meiosis (pDMC1). Plants transformed with the RNAi/BRCA2 constructs were<br />
sterile, due to aberrant meiosis (9). Plants transformed with the RNAi/DSS1 constructs<br />
are fertile. Thus, a role of AtDss1 at meiosis seems to be excluded. To further investigate<br />
the role of Brca2 and Dss1 in somatic DNA repair, we have transformed plants containing<br />
recombination substrates (10), with both RNAi constructs (under the control of the<br />
constitutive p35S promoter). Those plants are now being evaluated in terms of their<br />
sensitivity to genotoxic stresses and for their recombination rate. 1. Wooster et al (1995)<br />
Identification of the breast cancer susceptibility gene BRCA2. Nature 378: 789-792 2.<br />
Scully , Livingston (2000) In search of the tumour-suppressor functions of BRCA1 and<br />
BRCA2. Nature 408: 429-432 3. Marston, et al (1999) Interaction between the product of<br />
the breast cancer susceptibility gene BRCA2 and DSS1, a protein functionally conserved<br />
from yeast to mammals. Mol. Cell. Biol. 19 4633-42. 4. Kojic et al (2003) The BRCA2interacting<br />
protein DSS1 is vital for DNA repair, recombination, and genome stability in<br />
Ustilago maydis. Mol. Cell. 12:1043-9. 5. Kojic et al (2005) Brh2-Dss1 interplay enables<br />
properly controlled recombination in Ustilago maydis.Mol Cell Biol. 25:2547-57. 6.<br />
Funakoshi et al (2004) Sem1, the yeast ortholog of a human BRCA2-binding protein, is a<br />
component of the proteasome regulatory particle that enhances proteasome stability. J<br />
Cell Sci. 117:6447-54. 7. Krogan, et al (2004) Proteasome involvement in the repair of<br />
DNA double-strand breaks. Mol. Cell. 16 : 1027-34. 8. Dray et al (2006) Interaction<br />
between Arabidopsis Brca2 and Its Partners Rad51, Dmc1, and Dss1. Plant Physiol.<br />
140:1059-69.. 9. Siaud et al (2004) Brca2 is involved in meiosis in Arabidopsis thaliana as<br />
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suggested by its interaction with Dmc1. <strong>EMBO</strong> J 23:1392-1401 10. Molinier et al (2004)<br />
Interchromatid and Interhomolog Recombination in Arabidopsis thaliana. The Plant Cell,<br />
16 : 342-35<br />
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P - 15. AtMUS81 and its Genetic Interactions with RecQ-Helicases in Arabidopsis<br />
thaliana<br />
Frank Hartung, Stefanie Suer, Andreas Braun, Tina Bergmann, Holger Puchta. Botanical<br />
Institute II, University of Karlsruhe, 76128 Karlsruhe, Germany<br />
The endonuclease MUS81 has been shown in a variety of organisms to be involved in DNA<br />
repair in mitotic and meiotic cells. Homologues of the MUS81 gene exist in the genomes<br />
of all eukaryotes, pointing to a conserved role of the protein. However, the biological role<br />
and meiotic significance of MUS81 varies between different eukaryotes. For example,<br />
while loss of the gene results in strongly impaired fertility in S. cerevisiae and nearly<br />
complete sterility in S. pombe, it is not essential for meiosis in mammals. We identified a<br />
structural and functional homologue (AtMUS81/At4g30870) in the genome of<br />
Arabidopsis thaliana and determined the full-length cDNA of this gene, using RACE<br />
technology. Analysing two independent T-DNA insertion mutant lines of AtMUS81, we<br />
found that they are sensitive to the mutagens MMS and MMC but not to bleomycin. In<br />
contrast to yeast, no meiotic defect of Atmus81-1 and 2 was detectable resulting in a<br />
normal fertility. Crosses of Atmus81-1 with a hyperrecombinogenic mutant of the<br />
AtRECQ4A helicase resulted in synthetic lethality in the double mutant, whereas other<br />
AtRECQ genes tested did not show this effect. Thus, the nuclease AtMUS81 and the<br />
helicase AtRECQ4A seem to be involved in two alternative pathways of resolution of<br />
replicative DNA structures in somatic cells and the knock out of both pathways leads to<br />
plant lethality.<br />
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P - 16. Meiotic recombination frequency analysis in Arabidopsis thaliana mutants<br />
Veena Hedatale, Tony Connors, Tom Gerats, Janny Peters. Plant Genetics, Radboud<br />
University, 6525 ED Nijmegen, India<br />
Using sequence data from cDNA AFLP-based transcript pro<strong>file</strong>s in Petunia undergoing<br />
male meiosis (Cnudde et al., 2006), we are trying to understand the roles of the<br />
corresponding Arabidopsis genes in homologous recombination. In order to estimate<br />
recombination frequency in meiotic mutants, we are employing a previously described<br />
fluorescent seed-based assay (Melamed-Bessudo et al, 2005). In addition to the available<br />
tester line from Melamed-Bessudo and colleagues, four new tester lines were developed,<br />
consisting of GFP and RFP markers linked in cis on three different chromosomes. In the<br />
future we plan to generate meiotic testers for all five Arabidopsis chromosomes. The<br />
available tester lines were crossed to several mutants to monitor the effect of meiotic<br />
genes on meiotic recombination. From the F2 population plants heterozygous for the<br />
fluorescent markers and homozygous for the mutant or wild-type locus were selected. By<br />
backcrossing the selected plants to a male sterile line, the recombination rate in the GFP-<br />
RFP marker interval of these plants can be compared to determine the effect of the<br />
mutation on recombination frequency. References: 1. Cnudde F, Hedatale V, de Jong H,<br />
Pierson ES, Rainey DY, Zabeau M, Weterings K, Gerats T, Peters JL (2006), Changes in gene<br />
expression during male meiosis in Petunia hybrida, Chromosome Res. 14:919-932. 2.<br />
Melamed-Bessudo C, Yehuda E, Stuitje AR, Levy AA. (2005), A new seed-based assay for<br />
meiotic recombination in Arabidopsis thaliana, Plant J 43:458-466.<br />
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P - 17. Functional analysis of the OsEZ polycomb group gene in rice<br />
Doolyi Kim, Myungsuk Jung, Insun Yoon, Yeonhee Lee, Seokcheol Suh. Cell and Genetics,<br />
National Institute of Agricultural Biotechnology, 441-707 Suwaon, South Korea<br />
The epigenetic control of gene expression is equally important in plants. Genetic and<br />
molecular analyses in recent years have started to illuminate how products of these<br />
multiple genes interact to initiate seed development. PcG proteins are known to form<br />
multimeric complexes that modulate gene expression by changing higher order<br />
chromatin structure. In this study, EZ gene was isolated and characterized from rice. A<br />
full-length EZ cDNA was obtained by reverse transcriptase (RT)-PCR. EZ gene is producted<br />
to encode a protein of 895 amino acid with an estimated molecular 99.8 kDa. The EZ<br />
protein contains a characteristic SET domain. To identified interaction of EZ gene with<br />
polycomb group protein, we also present evidence, from yeast two-hybrid experiment, of<br />
physical interaction of EZ and FIE proteins, as would be expected in a polycomb type<br />
system. Also, the EZ cDNA was inserted into binary vector pMJ-Cytc. The vectors<br />
described above were introduced into Agrobacterium tumefaciens strain LBA4404 to<br />
obtain a transgenic plants. We have obtained transgenic lines from rice. The phenotype of<br />
transgenic plants were investigated for functional characterization of EZ gene.<br />
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P - 18. A Novel Model for Chloroplast Replication Mechanism in Green Plants<br />
Neeraja Krishnan, Basuthkar Rao. Department of Biological Sciences, Tata Institute of<br />
Fundamental Research, 400005 Mumbai, India<br />
A Novel Model for Chloroplast Replication Mechanism in Green Plants Neeraja M.<br />
Krishnan*, Basuthkar J. Rao Correspondence Address: B-202, Department of iological<br />
Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai –<br />
400 005 INDIA Email Address: neeraja@tifr.res.in, bjrao@tifr.res.in Abstract Chloroplast<br />
replication mechanism, despite 30 years of work, is still sketchy and not well understood.<br />
The initial proposition of rolling circle, bi-directional replication mechanism in tobacco<br />
and pea chloroplast genomes is now being questioned by the new hypothesis of<br />
homologous recombination-mediated replication based on complex, branched multimeric<br />
forms of these molecules. We address this issue of uni- vs. bi-directional replication by<br />
analyzing nucleotide composition in the regions between known replication origins, with<br />
an aim of revealing any cytosine to thymine deamination gradients. These gradients<br />
typically result from accumulation of deaminations over the extent of singlestrandedness<br />
experienced by the genome and can, therefore, be used as a strong<br />
signature of single-strandedness. Such analyses can thus, throw more light on the singlestrandedness<br />
levels experienced by these regions during replication. Fine-mapping of<br />
replication origins on tobacco chloroplast genome revealed two pairs of replication<br />
origins (A which folds into a simple linear hair-pin form, and B with a complex D-Loop<br />
like form), one pair on each inverted repeat. We found homologues of these replication<br />
origins on other Viridiplantae chloroplast genomes, using NCBI pair-wise BLAST tool. Our<br />
linear regression analyses on the nucleotide compositions of the non-coding regions and<br />
the synonymous third codon position of the coding regions, between any two replication<br />
origins, reveal existence of C to T deamination-type mutation gradients. We find<br />
increasing gradients for C to T deaminations in the regions interspersed between the<br />
complex origins B on either inverted repeat, only on one strand, suggesting unidirectional<br />
replication. These C to T gradients were however, found for both the strands<br />
in the region between origins A and B, on each inverted repeat. This indicates singlestrandedness<br />
on both strands, suggesting bi-directional replication. The larger region<br />
between the pair of origins, A on each inverted repeat again reveals increasing C to T<br />
deaminations only on one strand, suggesting uni-directional replication. We summarize<br />
and present these results in the form of a model for replication mechanism in chloroplast<br />
genomes.<br />
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P - 19. Majority of DNA double strand breaks are in plant genome rapidly<br />
removed independently of NHEJ repair pathways.<br />
Jaroslav Kozak, Chris West, Charles White, Karel J Angelis . Molec. Farming and DNA<br />
Repair, Inst. Experimental Botany, 160 00 Praha 6, Czech Republic<br />
Plants developed during evolution repair capacity to protect genome integrity against<br />
environmental genotoxines like UV or ionizing radiation. Among DNA lesions induced by<br />
radiation, the most lethal are double strand breaks (DSB) when only few, if unrepaired,<br />
can cause cell death. Repair of DSB induced in plant DNA by ionizing radiation or<br />
radiomimetic mutagens has been shown earlier. Several repair mechanisms were<br />
proposed for DSB elimination and nonhomologous joining of DNA ends (NHEJ) suggested<br />
as the major repair pathway in mammals and higher plants. However, the role of<br />
individual genes on the kinetic and extent of DSB repair in genomic DNA have not been<br />
established yet. Here we show that genes of NHEJ pathway do not have key role in overall<br />
repair of DSB and other repair pathway that quickly removes majority of DSB is active in<br />
higher plants. DSB detected by electrophoretic single cell (comet) assay under neutral<br />
conditions are in NHEJ pathway mutants AtLig4 and AtKu80 repaired faster (t½ = 3.5 and<br />
3.4 minutes respectively) than in w.t. Arabidopsis (t½ = 5 min) and AtLig1a (t½ = 9 min),<br />
which was tested as possible alternative ligase to complete DSB sealing. Also<br />
chromosome maintenance proteins AtMim and AtBru1 (t½ = 50 and 15 min. respectively)<br />
were found to have dramatic effect on kinetics of overall DSB repair. Our results<br />
demonstrate that role of individual genes, if mutated, could be substituted by other<br />
genes, e.g. Lig4 in ligation step by Lig1. We also show dominant role of genes involved in<br />
structural maintenance of DNA to repair DSB.<br />
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P - 20. Zinc finger transcription factors to discover Arabidopsis mutants with<br />
enhanced homologous recombination<br />
Beatrice Lindhout, Johan Pinas, Paul Hooykaas, Bert van der Zaal. Molecular and<br />
developmental genetics, Institute of Biology, Leiden University, 2333 AL Leiden,<br />
Netherlands<br />
A library of genes for zinc finger artificial transcription factors (ZF-ATF) was generated by<br />
fusion of DNA sequences encoding three-fingered Cys2His2 ZF domains to the VP16<br />
activation domain and put under control of the promoter of the ribosomal protein gene<br />
RPS5A of Arabidopsis thaliana. After introduction of this library into an Arabidopsis<br />
homologous recombination (HR) indicator line, we selected primary transformants<br />
exhibiting multiple somatic recombination events. After PCR-mediated rescue of ZF<br />
sequences, reconstituted ZF-ATFs were reintroduced in the target line. In this manner, a<br />
ZF-ATF was identified that led to a 200 to 1000-fold increase in somatic HR, also in an<br />
independent second target line. A mutant plant line, expressing the HR-inducing ZF-ATF<br />
exhibited increased resistance to the DNA-damaging agent bleomycin and was more<br />
sensitive to methyl methanesulfonate (MMS), a combination of traits not described<br />
before. Our results demonstrate that the use of ZF-ATF pools is highly rewarding when<br />
screening for novel dominant phenotypes in Arabidopsis.<br />
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P - 21. Addressing the role of chromatin structure on gene targeting rates<br />
Marie Mirouze, Jon Reinders, Jerzy Paszkowski. Laboratory of Plant Genetics, Université de<br />
Genève Sciences III, 1211 Genève, Switzerland<br />
Addressing the role of chromatin structure on gene targeting rates Marie Mirouze, Jon<br />
Reinders and Jerzy Paszkowski Laboratory of Plant Genetics, University of Geneva,<br />
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Sciences III, Quai Ernest-Ansermet 30, CH-1211 Genève, Switzerland We describe an<br />
experimental system which is currently under development in our laboratory to address<br />
to which extent epigenetic modifications affect gene targeting rates in Arabidopsis<br />
thaliana. For this we use PPOX gene encoding protoporphyrinogen oxidase, an enzyme<br />
involved in chlorophyll biosynthesis. Inhibition of PPOX by the herbicide Butafenacil<br />
results in rapid plant death due to oxidative stress. However, the combination of two<br />
mutations in PPOX gene confers resistance towards Butafenacil. It was shown that the<br />
introduction of these mutations to the chromosomal copy of PPOX gene by gene targeting<br />
is possible and can be used to assess gene targeting frequencies. We first examined<br />
epigenetic marks at the PPOX locus. DNA methylation analyses indicate that in the wild<br />
type plants PPOX locus is associated with substantial levels of DNA methylation,<br />
therefore, using mutants impaired in maintenance of DNA methylation may alter the<br />
epigenetic status of this locus. We developed an EpiRILs population comprising F8 lines<br />
from a cross between Col WT and met1-3, a mutant affected in a methyltransferase<br />
responsible for CpG methylation maintenance. These EpiRILs have the same genetic Col<br />
background, but differ in their cytosine methylation pro<strong>file</strong>s. We will describe the interest<br />
of using this epigenetic material to assess the role of chromatin structure in homologous<br />
recombination rates. This research is supported by the European Community in the frame<br />
of the TAGIP project (No. 018785).<br />
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P - 22. Genetic modification of rice plants by using Zinc Finger technology.<br />
Keishi Osakabe, Kiyomi Abe, Masaki Endo, Hiroaki Saika, Seiichi Toki. Division of Plant<br />
Sciences, National Institute of Agrobiological Sciences, 305-8602 Tsukuba, Ibaraki, Japan<br />
Zinc finger nucleases (ZFNs) are synthetic nucleases consisting of a zinc finger DNAbinding<br />
domain fused to the cleavage domain of the restriction endonuclease, Fok I. ZFNs<br />
can be used to introduce double-strand DNA breaks (DSBs) in specific DNA sequences,<br />
and promote site-specific homologous recombination (gene targeting) and targeted<br />
genome modification at the desired genomic loci including deletion- and insertion-type<br />
mutations. To apply this technology to the genetic modification of rice, we made a<br />
system to identify potential zinc-finger target sites in a gene of interest with the FASTA<br />
program based on the finding by Barbas's group (1). We already established the efficient<br />
transformation system of rice (2) and the gene targeting system with the rice acetolactate<br />
synthase (OsALS) gene locus (3). Thus, we chose the OsALS gene for the initial gene<br />
targeting experiment with ZFNs. We identified a ZFN recognition sequence at the Cterminal<br />
region of OsALS using our searching system, and designed ZFNs (ZFNosals_L<br />
and ZFNosals_R). We also confirmed that the combination of ZFNosals_L and ZFNosals_R<br />
could digest its recognition site on the OsALS gene in in vitro. We are further confirming<br />
digestion in in vivo by the ZFNs, and testing whether the introduction of the ZFNs can<br />
increase the efficiency of gene targeting at the OsALS gene locus. References 1. Mandell,<br />
J.G. and Barbas III, C.F. (2006) Nucleic Acids Res., 34, W516-W523. 2. Toki, S., et al. (2006)<br />
Plant J., 46, 969-976. 3. Endo, M., et al. (2006) 8th International Congress of Plant<br />
Molecular Biology, Book of Abstracts, p153.<br />
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P - 23. Two unlinked double-strand breaks can induce reciprocal exchanges in<br />
plant genomes via homologous recombination and non-homologous endjoining<br />
Michael Pacher, Waltraud Schmidt-Puchta, Holger Puchta. Molecular Biology &<br />
Biochmeistry of plants / Prof. Puchta, University of Karlsruhe / Institute of Botany II, 76128<br />
Karlsruhe, Germany<br />
Using the rare-cutting endonuclease I-SceI we were able to demonstrate before that the<br />
repair of a single DSB in a plant genome can be mutagenic due to insertions and<br />
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deletions. However, during replication or due to irradiation several breaks might be<br />
induced simultaneously. To analyze the mutagenic potential of such a situation we<br />
established an experimental system in tobacco harboring two unlinked transgenes each<br />
carrying an I-SceI site. After transient expression of I-SceI a kanamycin resistance marker<br />
could be restored by joining two previously unlinked broken ends, either by homologous<br />
recombination (HR) or by non-homologous end-joining (NHEJ). Indeed, we were able to<br />
recover HR and NHEJ events with similar frequencies. Despite of the fact that no selection<br />
was applied for joining the two other ends, the respective linkage could be detected in<br />
most cases tested, demonstrating that the respective exchanges were reciprocal. The<br />
frequencies obtained indicate that DSB-induced translocation is up to two orders of<br />
magnitude more frequent in somatic cells than ectopic gene conversion. Thus, DSB<br />
induced reciprocal exchanges might play a significant role in plant genome evolution. The<br />
technique applied in this study may also be useful for the controlled exchange of<br />
unlinked sequences in plant genomes.<br />
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P - 24. ATM-mediated transcriptional and developmental responses to ?-rays in<br />
Arabidopsis<br />
Lilian Ricaud, Caroline Proux, Jean-Pierre Renou, Olivier Pichon, Sylvain Fochesato, Phiippe<br />
Ortet, Marie-Hélène Montané. Direction des Sciences du vivant, CEA, 13108 St Paul-lez-<br />
Durance cedex, France<br />
(1) CEA, DSV, Institut de Biologie Environnementale et de Biotechnologie (iBEB), Service de<br />
biologie végétale et de microbiologie environnementales (SBVME), F-13108 Saint Paul-lez-<br />
Durance, France. (2) Unité de Recherche en Génomique Végétale, INRA, 2 rue Gaston<br />
Crémieux, F-91057 Evry, France (3) Present address: Plate-forme puces à ADN, Genopole,<br />
Institut Pasteur, 28 rue du Docteur Roux, F-75724 Paris cedex 15, France *E-mail: mariehelene.montane@cea.fr<br />
ATM (Ataxia Telangiectasia Mutated) is an essential checkpoint<br />
kinase that signals DNA double-strand breaks in eukaryotes. Its depletion causes meiotic<br />
and somatic defects in Arabidopsis and progressive motor impairment accompanied by<br />
several cell deficiencies in patients with ataxia telangiectasia (AT). To obtain a<br />
comprehensive view of the ATM pathway in plants, we performed a time-course analysis<br />
of seedling responses by combining confocal laser scanning microscopy studies of root<br />
development and genome-wide expression profiling of wild-type (WT) and homozygous<br />
ATM-deficient mutants challenged with a dose of ?-rays (IR) that is sublethal for WT<br />
plants. Early morphologic defects in meristematic stem cells indicated that AtATM, an<br />
Arabidopsis homolog of the human ATM gene, is essential for maintaining the quiescent<br />
center and controlling the differentiation of initial cells after exposure to IR. Results of<br />
several microarray experiments performed with whole seedlings and roots up to 5 h post-<br />
IR, which were compiled in a single table; sequence and function homology searches;<br />
import of cell cycling, and mutant-constitutive expression characteristics; and a<br />
simplified functional classification system were used to identify novel genes. The<br />
transcription burst was almost exclusively AtATM-dependent or weakly AtATRdependent,<br />
and followed two major trends of expression in atm: (i)-loss or severe<br />
attenuation and delay, and (ii)-inverse and/or stochastic, as well as specific, enabling one<br />
to distinguish IR/ATM pathway constituents. The hundreds of radiomodulated genes<br />
identified were not a random collection, but belonged to functional pathways such as<br />
those of the cell cycle; cell death and repair; DNA 3R; and transcription; translation; and<br />
signaling, indicating the strong cell reprogramming and double-strand break abrogation<br />
functions of ATM checkpoints. Accordingly, genes in all functional classes were either<br />
down or up-regulated concomitantly with downregulation of chromatin deacetylases or<br />
upregulation of acetylases and methylases, respectively. Determining the early<br />
transcriptional indicators of prolonged S-G2 phases that coincided with cell proliferation<br />
delay, or an anticipated subsequent auxin increase, accelerated cell differentiation or<br />
death, was used to link IR-regulated hallmark functions and tissue phenotypes after IR.<br />
Our data provide a large resource for studies on the interaction between plant<br />
checkpoints of the cell cycle, development, hormone response, and DNA repair functions,<br />
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because IR-induced transcriptional changes partially overlap with the response to<br />
environmental stress. Putative connections of ATM to stem cell maintenance pathways,<br />
and to non-DSB/DSBs repair mechanisms in plants after IR are discussed.<br />
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P - 25. Cellular and molecular understanding of plant DNA damage response<br />
through ATM, ATR, H2AX, E2F and RNR proteins.<br />
Hélène Roa, Julien Lang, Lenin Sanchez-Calderon, Ondrej Smetana, Frédéric Lincker,<br />
Chamseddine Mediouni, Guy Houlné. plant cellular biology, IBMP/CNRS UPR2357<br />
Strasbourg, 67084 Strasbourg, France<br />
Plants are continuously exposed to high levels of genotoxic stresses which can induce<br />
DNA damage, and according to its could lead to cellular death. So through evolution,<br />
plants have fixed efficient DNA repair mechanisms. Among key factors involved in DNA<br />
repair, Ribonucleotide reductase has to be highly regulated for providing the cell with<br />
dNTPs. In Arabidopsis the 3 AtRNR2 encoding the small R2 subunit present specific<br />
pro<strong>file</strong> of gene induction, R2-3b is significantly induced in response to IR or the<br />
radiomimetic bleomycin (ATM-dependency) but not upon HU contrarily to R2-3 and R2-5<br />
genes. These genes presented E2F-elements in their promoters and as described in<br />
tobacco, we demonstrate that E2F factor plays an important role to drive their gene<br />
induction upon DNA damage in Arabidopsis (replicative stress or DSB) under the control<br />
of ATM, ATR or alternative pathways. In addition GFP-NtE2F protein fusion presents a<br />
nuclear localization in both tobacco and Arabidopsis cells and forms some clear and<br />
discrete nuclear foci when these cells are submitted to bleomycin. Most of these NtE2F<br />
nuclear foci colocalized with Atgamma H2AX, a marker of DSBs, and their number was<br />
increasing in a time-dependent manner upon BLM treatment. Such NtE2F foci formation is<br />
ATM-dependent and occurs with a higher density in the root meristem. Interestingly,<br />
increasing time of BLM treatment leads also to increased cell death concomitant with<br />
strong gene induction of particular PCD marker genes. Works is in progress to<br />
understand the role of E2F in DNA repair and PCD cellular processes.<br />
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P - 26. CHARACTERIZATION OF ARABIDOPSIS EXONUCLEASE 1<br />
Max Roessler, Karel Riha , Austrian Academy of Sceinces, Gregor Mendel Institute, 1030<br />
Vienna, Austria<br />
Exonuclease 1 (EXO1) is a multitasking nuclease, with 5' - 3' exonuclease activity, which is<br />
present in virtually all eukaryotic organism from yeast to mammals. Studies performed<br />
mainly in yeast have demonstrated that EXO1 is involved in mismatch DNA repair (MMR).<br />
It is also implicated in the resection of DNA double strand breaks, homologous<br />
recombination, telomere maintenance, meiosis, and DNA replication. However, these<br />
functions of EXO1 are less well understood. We found that the Arabidopsis genome<br />
encodes two paralogous proteins, which we refer to as AtEXO1A and AtEXO1B.<br />
Arabidopsis is the only known organism containing two putative EXO1 genes. The<br />
homology between the AtEXO1A and AtEXO1B proteins is limited to the N-terminal<br />
nuclease domain suggesting that the proteins have evolved different functions.<br />
Supporting this idea we found that expression levels of EXO1A and EXO1B transcripts<br />
differes in various tissues. We characterized mutant lines harbouring T-DNA insertions in<br />
the AtEXO1A and AtEXO1B genes. These lines showed no sensitivity to DNA damage<br />
inducing agents (Mitomycin C, Menadion, Hydroxy Urea, Methyl-Methane Sulfonate,<br />
Bleocin). Further experiments are underway to examine the function of the EXO1<br />
paralogues at telomeres, in MMR and in homologous recombination (HR).<br />
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P - 27. Relationships between genetic and physical maps in bread wheat:<br />
Recombination studies on chromosome 3B.<br />
cyril saintenac, pierre sourdille. UMR 1095 Amelioration et Sante des plantes, INRA, 63000<br />
Clermont-Ferrand, France<br />
Recombination and population history seems to play an important role at several levels<br />
of the gene evolution dynamics (duplications, deletions, mutations. It is admitted that<br />
recombination increases the sequence divergence rate (polymorphism) as well as the<br />
deletion-duplication rate. However, the balance between these two phenomena remains<br />
poorly understood. In wheat, we recently showed that recombination varies at the<br />
chromosome scale where it increases gradually with the distance from the centromere<br />
with some local variations. However, we still have the idea neither of the distribution of<br />
the recombination along the chromosome nor of the relationships between its intensity<br />
and gene evolution as well as their polymorphism. Recent progresses in wheat genomics<br />
have made possible the elaboration of the first chromosome-specific BAC library (Safar et<br />
al, 2004, Plant J., 39: 960-968) from the wheat chromosome 3B. We recently started to<br />
build the physical map of this chromosome by fingerprinting and contiguing the 68,000<br />
BAC clones from this library. The physical map is now made of about 2,000 contigs<br />
covering more than 85% of the chromosome. The aims of the PhD will be to use this<br />
resource to study the recombination at different levels: 1- At the level of the whole<br />
chromosome, the recombination gradient will be studied by refining the position of the<br />
break-points observed using deletion lines. The relationships between genetic and<br />
physical distances will be precised by: (1) increasing the number of deletion lines<br />
evaluated; (2) using the genetically anchored contigs within a deletion bin. We will also<br />
evaluate the influence of the length of the chromosome arm on the recombination by<br />
studying segregating populations issued from the cross between the deletion lines and a<br />
normal cultivar (Renan). 2- At the level of a deletion bin known to be gene-rich (bin 3BL7,<br />
~200 Mb) and where the recombination is frequent, we will increase the density of<br />
markers (1/5Mb). We will then be able to compare the recombination in gene-rich (contigs<br />
with at least one EST assigned) and gene-poor (contigs without any EST assigned) regions.<br />
Moreover we want to know if the distribution of recombination is conserved between two<br />
different segregating populations. 3- At the level of a sequenced region. In the course of<br />
the IWGSC, it was planned to establish a physical contig spanning the region covering<br />
several resistance loci (FHB, Rph7, Sr2, Sn2) and to define a Minimal Tiling Path (MTP) that<br />
will be completely sequenced and analysed (estimated to be between 14 and 23 Mb). This<br />
region covers between 6 and 12 cM according the segregating populations studied and is<br />
thus highly recombinogenic. At the present time, 60 markers have been identified in the<br />
region and it is planned to map those that are polymorphic and to increase the density of<br />
markers by using additional ISBPs in order to identify putative recombination hot-spots.<br />
All the results will bring a new insight in the recombination in wheat which will be<br />
helpful for further positional cloning of genes of agronomical interest on chromosome 3B<br />
as well as on other regions of the genome.<br />
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P - 28. Characterisation of AtTRB1 binding properties<br />
Iva Santruckova, Ctirad Hofr, Petra Schrumpfova, Jiri Fajkus. Functional Genomics and<br />
Proteomics, Masaryk University, 625 00 Brno, Czech Republic<br />
Telomeres play an essential role in maintenance of chromosome integrity. Their<br />
protective function is dependent on dynamic association of telomeric DNA with histones<br />
and specific telomeric proteins. Here we report on characterization of AtTRB1<br />
(At1g49950, 33kDa), a member of plant-specific group of putative telomere-binding<br />
proteins – the Smh (Single myb histone) family. This family is defined by the presence Nterminal<br />
Myb-like domain, centrally localized histone H1/H5 domain and a C-terminal<br />
coiled-coil. In Arabidopsis, the family includes 5 members – AtTRB1-5. Two of them -<br />
AtTRB2 and AtTRB3 – have been characterized previously in respect of their DNA-protein<br />
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and protein-protein interactions. The functional importance of AtTRB1 at telomeres<br />
comes from the results of our recent protein-protein interaction studies which showed<br />
(using yeast two-hybrid assay and IP) an interaction between AtTRB1 and AtPot1-2, a<br />
member of AtPot family in Arabidopsis. The focus of this study has been directed<br />
towards the description of the DNA binding capability of AtTRB1 and its truncated<br />
variants. AtTRB1 and its fragments were cloned and expressed in bacterial system and<br />
the purified proteins were used in Electrophoretic Mobility Shift Assay (EMSA) and<br />
Surface Plasmon Resonance (SPR). It has been shown that myb-like domain of AtTRB1<br />
preferentially binds telomeric over non-telomeric dsDNA. However, AtTRB1 lacking the<br />
Myb-like domain also shows preference to telomeric dsDNA. Histone H1/H5 domain<br />
alone is not able to show any binding preference. These data suggest a positive role of Cterminal<br />
coiled-coil domain in sequence-specific binding of AtTRB1 to telomeric DNA<br />
sequence. Our research is supported by Grant Agency of the Czech Republic<br />
(521/050055), Grant Agency of the Czech Acad. Sci (IAA600040505), Ministry of<br />
Education (LC LC06004) and institutional funding (MSM0021622415)<br />
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P - 29. A HYPOMORPHIC ALLELE OF TELOMERASE DELAYS ONSET OF GROWTH<br />
DEFECTS IN ARABIDOPSIS WITHOUT PREVENTING TELOMERE<br />
SHORTENING<br />
Martina Schirato, Barbara Zellinger, Karel Riha , Austrian Academy of sciences - Gregor<br />
Mendel Institute, 1030 Vienna, Austria<br />
Disruption of Arabidopsis telomerase reverse transcriptase (TERT) gene leads to a<br />
gradual telomere shortening and an onset of developmental defects after six generations<br />
of telomerase deficiency. To investigate whether telomere shortening and occurrence of<br />
growth abnormalities can be reverted by reintroduction of telomerase, we transformed<br />
fourth generation (G4) of Arabidopsis tert mutants with a construct expressing TERT<br />
cDNA from a strong constitutive 35S promoter (35S::TERT). Overexpression of the TERT<br />
re-established telomerase activity in tert mutants, but failed to rescue telomere<br />
shortening. In fact, telomeres in tert plants ectopically expressing TERT cDNA shortened<br />
at a similar rate as telomeres in control tert mutants or in tert lines overexpressing<br />
catalytically inactive TERT(D860N) construct. However, while the tert and tert<br />
35S::TERT(D860N) lines started exhibiting developmental defects at G6 and could not be<br />
propagated beyond G8, the onset of growth defects was significantly delayed in the lines<br />
complemented with the 35S::TERT construct and they could be propagated for at least 13<br />
generations. Interestingly, no telomeric DNA could be detected in these plants from G10<br />
onwards by standard techniques suggesting that chromosome termini contain no or only<br />
very short arrays of telomeric repeats. These data suggest that the hypomorphic<br />
35S::TERT allele significantly extends proliferation capacity of cells with critically short<br />
telomeres.<br />
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P - 30. Molecular analysis of Phaseolus coccineus PCNA<br />
Wojciech Strzalka, Anna Kaczmarek, Barbara Naganowska, Alicja Ziemienowicz. Faculty<br />
of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow,<br />
Poland<br />
Molecular analysis of Phaseolus coccineus PCNA Strzalka W1, Kaczmarek A2,<br />
Naganowska B2 and Ziemienowicz A1 1Department of Molecular Genetics, Faculty of<br />
Biochemistry, Biophysics and Biotechnology Jagiellonian University, Gronostajowa 7, 30-<br />
387 Kraków, Poland. 2Institute of Plant Genetics, Polish Academy of Science,<br />
Strzeszynska 34, 60-479 Poznan, Poland Proliferating cell nuclear antigen (PCNA) is one<br />
of the most conserved proteins present both in animal and plant organisms. PCNA<br />
functions as a auxiliary factor of DNA polymerase delta and naturally forms a trimeric<br />
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ring encircling DNA that serves as a platform for binding various proteins involved in<br />
DNA metabolism. Moreover, it also plays an important role in DNA repair and in<br />
regulation of the cell cycle. Although the role of PCNA in animal and yeast cells has been<br />
studied extensively, only few information concerning the role of PCNA in plant cells,<br />
especially in DNA repair, is currently available. The aim of our study was molecular<br />
analysis of P. coccineus PCNA. First, by using 5’ and 3’ RACE technique we identified<br />
cDNA encoding PcPCNA1 ad PcPCNA2. The identity between these two proteins on the<br />
amino acid level was 54%. Second, we purified the recombinant PcPCNA1 and PcPCNA2<br />
proteins and compared their biochemical properties. Our results showed that PcPCNA1<br />
was able to stimulate the activity of human DNA polymerase delta, whereas no<br />
stimulatory effect was observed for PcPCNA2. Moreover, we observed that PcPCNA1 but<br />
not PcPCNA2 was (i) present as a trimer in solution, (ii) was able interact with p21<br />
peptide, and (iii) was recognized by anti-human PCNA antibody PC10. Furthermore, we<br />
analysed the patterns of PcPCNA1 and PcPCNA2 gene expression during seed germination<br />
and in the runner bean organs. Real-time RT-PCR analysis showed changes in the<br />
expression of both genes during germination with maximum levels reached 48-72 h after<br />
imbibing. The transcripts could be also detected in seed micropylar region, and they were<br />
present at very low level in roots, stem and leaves of 10 day old plants. Finally, the copy<br />
number of PcPCNA genes in the genome of P. coccineus and their chromosomal<br />
localisation was analysed using Southern blotting and PRINS techniques. The<br />
hybridization pattern suggested the presence of at least two PCNA genes in the runner<br />
bean genome. On the other hand, the presence of single dot signals observed on<br />
metaphase chromosomes (PRINS analysis) indicated that both PcPCNA1 and PcPCNA2<br />
genes were localised most probably in a single locus in a centromeric region of a<br />
submetacentric chromosome. These results implicate a number of the intriguing<br />
questions concerning the role of PCNA in plants that we are going to answer in the<br />
coming future. Moreover, our studies may help us to understand the evolution route from<br />
bacterial sliding clamp to eukaryotic PCNA.<br />
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P - 31. DNA damage repair/toleration protein Drt112 may be involved in<br />
integration of T-DNA into the plant genome<br />
Wojciech Strzalka, Tomasz Herjan, Monika Kapusniak, Karolina Peplowska, Alicja<br />
Ziemienowicz. Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian<br />
University, 30-387 Krakow, Poland<br />
Agrobacterium tumefaciens, a plant natural pathogen, has an ability of interkingdom<br />
DNA transfer. In favourable conditions this bacterium is able to transfer a large fragment<br />
of the resident Ti-plasmid (T-DNA: transferred DNA) to the plant cell, where T-DNA gets<br />
integrated into the host genome. T-DNA integration into the plant chromosomes occurs<br />
via illegitimate recombination, involving various pathways of DNA repair. This process<br />
requires plant factors, such as DNA ligase and other enzymes involved in DNA<br />
repair/recombination, although the role of bacterial factors, such as VirD2 protein,<br />
cannot be excluded. VirD2 was shown previously not to act as a ligase for T-DNA<br />
integration, however it may play a crucial role in recruiting plant factors to the site of T-<br />
DNA integration. By employing yeast two-hybrid system, we have identified as a VirD2interacting<br />
factor a precursor of Drt112 protein, that may be involved in T-DNA<br />
integration. Two cDNA, DRT111 and DRT112, have been previously selected from E. coli<br />
mutants lacking RuvC endonuclease (recombination resolvase) transformed with<br />
plasmids expressing Arabidopsis thaliana cDNA library (Pang Q. et al., Nucleic Acid<br />
Research, 1993, 21(7): 1647-53). Expression of these plant cDNAs increased the resistance<br />
of the bacterial mutant to DNA damaging factors, indicating involvement of the Drt<br />
proteins in DNA repair and recombination (Drt: DNA-damage-repair/toleration). By using<br />
far-Western blotting we have shown that only Drt112 but not Drt111 protein interacted<br />
with VirD2, suggesting the potential involvement of the Drt112 protein in the T-DNA<br />
integration process, most likely by resolving T-DNA integration intermediates. Further<br />
studies aiming to discover the role of Drt proteins in plants will help us to reveal the<br />
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mechanism of Drt-mediated DNA repair/recombination pathway employed for T-DNA<br />
integration.<br />
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P - 32. Positional cloning of the hus2 locus in Arabidopsis thaliana: A new player<br />
in the cell-cycle arrest response to replication blocks<br />
Paul Sweeney, Daniel Lynch, Anne Britt, Kevin Culligan. Biochemistry and Molecular<br />
Biology, University of New Hampshire, 03824 Durham, United States<br />
Cell-cycle arrest is an integral component of the cellular response to DNA damage in<br />
eukaryotic cells. Persisting DNA damage can block replication during S-phase, resulting in<br />
G2-phase arrest. This arrest prevents chromosome loss by blocking cell-cycle progress<br />
into M-phase with an incompletely replicated genome, and also allows time for repair and<br />
additional DNA synthesis. ATR, a phosphoinositide (PI)-3-like protein kinase, controls this<br />
G2/M transition in response to blocked replication. We have shown previously that<br />
Arabidopsis plants defective in ATR are hypersensitive to the replication-blocking agent<br />
HU (hydroxyurea), likely due to a G2-arrest defect, and display a short, “hairy” root<br />
phenotype when grown on HU-containing plates. To further define molecular steps in the<br />
ATR-dependent cell-cycle response, we developed a genetic screen to isolate Arabidopsis<br />
mutants that display a similar (atr-like) phenotype when grown on HU plates. One<br />
resultant mutant, termed hus2 (for HydroxyUrea Sensitive), is hypersensitive to<br />
replication blocking agents and is defective in G2 arrest, similar to the atr mutant.<br />
However, complementation studies suggest hus2 is an independent locus from ATR. To<br />
identify the mutation responsible for the hus2 phenotype, we genetically mapped the<br />
hus2 locus to the distal end of chromosome 5 between the genetic (CAPS) markers RPS4<br />
and PDC2. To refine the location of the hus2 mutation between these markers, we<br />
generated additional (dCAPS) markers and found the hus2 mutation is located within an<br />
80 Kb region (18,470 Kb to 18,550 Kb), which encodes ~18 putative genes. Of these 18<br />
genes, none have a characterized or predicted role in the DNA damage response in plants.<br />
To identify the mutated locus in hus2, we are currently employing a combination of<br />
complementation (TAC clones), T-DNA knockouts, and sequencing. The results of these<br />
experiments will be presented.<br />
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P - 33. Isolation of Rice FOX Lines Tolerance to Ultraviolet-B Stress<br />
Shinya Takahashi, Tomoko Kuriyama, Takanari Ichikawa, Youichi Kondou, Yukako<br />
Hasegawa, Mika Kawashima, Shu Muto, Hirohiko Hirochika, Minami Matsui . Plant<br />
Functional Genomics Research Team, RIKEN Plant Science Center, 230-0045 Yokohama,<br />
Japan<br />
To identify large-scale useful rice gene, we are established rice FOX lines using 13,000<br />
non-redundant rice full length cDNAs to introduce them into Arabidopsis plants.<br />
Ultraviolet-B (UV-B) radiation can damage plants due to destruct several cell components,<br />
such as DNA, and reduce gene expressions. In higher plants, there are some protective<br />
mechanisms such as DNA repair, ROS scavenging system and accumulation of UV-B<br />
absorbing pigments. To isolate useful rice genes that confer UV-B stress tolerance, we try<br />
to screen UV-B tolerant mutants from rice FOX lines. We already isolated 49 UV-B tolerant<br />
mutant candidates in T2 generation from ca. 4,500 lines by root bending assay screening.<br />
Each isolated mutant contained one or two copies of rice cDNA which may encode cellcycle<br />
related genes, stress responsive genes, transcription factors and some unknown<br />
function genes. Now we differentiate the isolated mutants based on phenotypes of root<br />
growth with or without UV-B irradiation. This research is supported by the Special<br />
coordination funds for Promoting Science and Technology entitled Rapid identification of<br />
useful traits using rice full-length cDNAs.<br />
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P - 34. Towards efficient gene targeting in plants<br />
Seiichi Toki, Masaki Endo, Ryuya Ohishi, Masaaki Umeda, Kiyomi Abe, Hiroaki Saika, Keishi<br />
Osakabe. Plant Genetic Engineering Research Unit, National Institute of Agrobiological<br />
Scienced, Ibaraki 305-8602 Tsukuba, Japan<br />
Modification of genes by gene targeting (GT) should be the most precise gene<br />
manipulation technique and direct method for future molecular breeding in plants. In<br />
order to improve the frequency of GT, we are currently applying several approaches<br />
including the use of the zinc-finger nucleases, chromatin remodeling factors and cell<br />
cycle regulators. We are also trying to establish the model gene targeting system in<br />
Arabidopsis and rice. In this paper we would like to present current status of our study<br />
and discuss potential use of our study for the practical molecular breeding in plants.<br />
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P - 35. High-frequency site directed modification of the tobacco SuRB locus<br />
mediated by zinc-finger nucleases<br />
Jeffrey A. Townsend, David A. Wright, Ronnie J. Winfrey, Stacey Thibodeau-Beganny,<br />
Magdalena Eichtinger, J. Keith Joung, Daniel F. Voytas. Genetics, Cell and Developmental<br />
Biology, Iowa State University, 50011-3650 Ames, Iowa, United States<br />
Zinc-finger (ZF) proteins can be engineered to recognize unique chromosomal sites. When<br />
fused to a nuclease domain they make targeted double-strand breaks that stimulate<br />
homologous recombination between the chromosome and an extrachromosomal DNA<br />
donor. Here we demonstrate high frequency, site directed modification of the tobacco<br />
SuRB locus using engineered zinc-finger nucleases. The SuRB gene codes for<br />
acetohydroxyacid synthase (AHAS) the target enzyme for sulfonylurea and imidazolinone<br />
herbicides. Single amino acid changes in the enzyme confer herbicide resistance.<br />
Substitutions at amino acid P196A and S647T confer sulfonylurea and imidazolinone<br />
resistance respectively, while a W573L substitution confers resistance to both herbicide<br />
classes. Promotorless, nonfunctional fragments of the SuRB gene that encode these<br />
substitutions plus a novel restriction site have been cloned and introduced into tobacco<br />
protoplasts with pairs of zinc-finger nucleases designed to recognize specific SuRB<br />
sequence. The ZF proteins were generated by selection using a bacterial cell-based twohybrid<br />
(B2H) scheme. Herbicide resistant colonies occurred at a rate of more than 3% of<br />
that given by the control, functional SuRB mutants (3% of illegitimate recombination). The<br />
herbicide resistance pro<strong>file</strong> of the recombinants in all cases matches that expected given<br />
the donor DNA used.<br />
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P - 36. Designed zinc finger nucleases as genome editing tools<br />
Fyodor Urnov, Jeffrey Miller, Russell DeKelver, Erica Moehle, Jeremy Rock, Kenneth Kim,<br />
Edward Rebar, Philip Gregory, Michael Holmes. Genomics, Sangamo BioSciences, 94804<br />
Richmond, United States<br />
Designed zinc finger nucleases (ZFNs) provide a generally applicable solution to a<br />
fundamental problem in biology: the efficient alteration of genotype in plant and animal<br />
cells to an investigator-specified allelic form. Work by Jasin et al on the homing<br />
endonuclease I-SceI (1) and the invention of ZFNs by Chandrasegaran et al (2) provided<br />
the basis for work by Carroll et al on ZFN-driven homologous recombination in Xenopus<br />
(3), followed by studies of Porteus and Baltimore on reporter gene targeting in human<br />
cells (4), which, importantly, have been recapitulated in Arabidopsis by Drews et al (5)<br />
and in tobacco cells by Voytas et al (6). The transition to editing native loci of one’s<br />
choosing was enabled by the development of designed zinc finger proteins (7) that allow<br />
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one to target a DSB to essentially any position in the genome of any cell, and yield rapid,<br />
selection-free, high efficiency gene correction (8) and gene addition (9) at endogenous<br />
loci. We will describe recent progress in designed ZFN technology: our development of<br />
massively parallel tools for studying genome editing, the structure-based redesign of<br />
ZFNs aimed at ensuring single-locus specificity of the editing process, and the remarkable<br />
efficiency of single-step endogenous gene disruption we have observed in a broad range<br />
of cell types. 1. Rouet, P., et al. 1994. Proc Natl Acad Sci U S A 91:6064-8. 2. Kim, Y. G., et<br />
al. 1996. Proc Natl Acad Sci U S A 93:1156-60. 3. Bibikova, M., et al. 2001. Mol Cell Biol<br />
21:289-97. 4. Porteus, M. H., and D. Baltimore. 2003. Science 300:763. 5. Lloyd, A., et al.<br />
2005. Proc Natl Acad Sci U S A 102:2232-7. 6. Wright, D. A., et al. 2005. Plant J 44:693-<br />
705. 7. Pabo, C. O., et al. 2001. Annu Rev Biochem 70:313-340. 8. Urnov, F. D., et al. 2005.<br />
Nature 435:646-51. 9. Moehle, E. A., et al. 2007. Proc Natl Acad Sci U S A 104:3055-3060.<br />
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P - 37. Strategies to modify and monitor gene targeting and meiotic<br />
recombination in crops.<br />
Philippe Vain, Vera Thole, Barbara Worland, Katie Mayes, Upendra Devisetty, Chanate<br />
Malumpong, Sean Mayes. Agricultural and Environmental Sci (Biosciences), University of<br />
Nottingham, LE12 5RD Loughborough, United Kingdom<br />
Significant progress has been made recently in perturbing genetic recombination<br />
processes in model plants, particularly Arabidopsis thaliana and tobacco. While these<br />
approaches have not yet coalesced into practical and efficient methods to achieve Gene<br />
Targeting (GT) and to modify meiotic recombination, a firm foundation is being laid. We<br />
have been looking at opportunities to test promising results in model di- and<br />
monocotyledonous species, with an ultimate aim to manipulate these processes in crop<br />
species, particularly wheat. We have developed a deficient GUS-based marker system,<br />
which could be repaired by GT. As this system does not require plant regeneration, we<br />
can rapidly examine very large numbers of events. A number of genes involved in<br />
recombination, particularly in rice have been cloned into expression vectors and we are<br />
beginning to characterise the effect of these genes in A. thaliana as a first step. The<br />
system we have developed should allow the same recombination genes to be tested in a<br />
number of species (A. thaliana, tobacco and rice), allowing rapid follow up of positive<br />
results from models to crops. We will use the same transgenic lines that have been tested<br />
for GT effects to assess effects in meiotic recombination, through crossing and<br />
microsatellite analysis. In addition to this, we are currently screening neutron deletion<br />
material of wheat to identify deletion of specific homeologues of RAD51 and DMC1<br />
(which may not cause full sterility in this hexaploid) and intend to compare these with<br />
characterisation of rice Tos17 insertional inactivation lines for the rice equivalents (where<br />
two versions of each gene exist). Results to date and our future plans will be presented.<br />
----------------------------------------------------------------------------------------------------------------------------------<br />
P - 38. Reverse Progeny Mapping<br />
Aat Vogelaart, Johan Schut, Rob Dirks, Cilia Lelivelt. Celbiology, Rijk Zwaan Breeding BV,<br />
4793 RS Fijnaart, Netherlands<br />
A method is provided for mapping traits in organisms, in particular in plants. The<br />
method comprises a) providing a population of SDR-0 organisms, that each arise from<br />
one member of a population of unreduced cells resulting from second division<br />
restitution, in particular a population of unreduced spores; b) producing SDR-I progeny<br />
populations of each of these SDR-0 organisms; c) phenotyping the SDR-I progeny<br />
populations to identify segregating traits within each SDR-I progeny population; d) if<br />
segregating progeny is present in a SDR-I progeny population genotyping the<br />
corresponding SDR-0 organism and comparing the genotype thereof with the genotype of<br />
the other SDR-0 organism to identify heterozygous chromosomal regions associated with<br />
Page 47 sur 56
<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
the occurrence of the segregating trait identified in the said SDR-I progeny population.<br />
This method provides a reliable method for the mapping of complex traits and is a very<br />
efficient alternative to the development and use of heterogeneous inbred families. WO<br />
2006/094774.<br />
----------------------------------------------------------------------------------------------------------------------------------<br />
P - 39. A functional analysis of DNA ligases from Arabidopsis thaliana<br />
Wanda M. Waterworth, Ghzaleh Masnavi, Claire Provost, Paul Sunderland, Christopher E.<br />
West, Clifford M. Bray. Molecules to Cells, University of Manchester, UK, M13 9PT<br />
Manchester, United Kingdom Faculty of Life Sciences, University of Manchester, Manchester<br />
M13 9PT, U.K.; Centre for Plant Sciences, University of Leeds, Leeds, LS2 9JT, UK<br />
Eukaryotic organisms possess multiple DNA ligases with distinct roles in DNA<br />
metabolism. In plants, DNA ligases are crucial for the maintenance of nuclear,<br />
mitochondrial and plastid genome stability and both the levels and their intracellular<br />
distribution are tightly regulated. The three DNA ligase genes expressed in Arabidopsis<br />
(DNA ligases I, IV and VI; AtLIG1, AtLIG4 and AtLIG6) have been functionally<br />
characterized in our laboratory. AtLIG1 has roles in both DNA repair and replication, and<br />
possibly recombination, and is indispensable for cell viability. AtLIG1 expresses one<br />
major and two minor mRNA transcripts differing only in the length of the 5’ untranslated<br />
leader sequences preceding a common ORF. Intracellular targeting of DNA ligase 1 is<br />
controlled via an evolutionarily conserved posttranscriptional mechanism that involves<br />
the use of alternative start codons to translate multiple ligase activities from a single<br />
transcript. A further level of control involves hierarchical dominance of the different<br />
targeting sequences when more than one targeting sequence is present in the same ligase<br />
isoform. The maintenance of genomic integrity involves the function of all three DNA<br />
ligases in Arabidopsis. As with many DNA repair factors, the ligases are likely to form<br />
dynamic multi protein complexes to repair DNA damage. Ongoing studies are concerned<br />
with the identification and characterisation of interacting protein partners for AtLig1 and<br />
the physiological functions and importance of the novel plant specific ligase 6.<br />
----------------------------------------------------------------------------------------------------------------------------------<br />
P - 40. I-SceI-stimulated intrachromosomal homologous recombination in maize<br />
Sophie Wehrkamp-Richter, Wyatt Paul, Julien Levy, Samuel LeGoff, Jean-Baptiste Laffaire,<br />
Fabienne Degroote, Pascual Perez, Georges Picard. Gene Function and Maize Traits Group,<br />
Biogemma, 63000 Clermont-Ferrand, France<br />
Gene targeting (GT) by homologous recombination (HR) allows the precise modification of<br />
any gene. GT has become an indispensable tool in yeast and mouse, however, this tool is<br />
still missing in higher plants since the frequency of homologous recombination in plants<br />
is too low (10-4 to 10-5) compared to the frequency of illegitimate recombination (NHEJ).<br />
However, in model plants, induction of a double strand break at the target locus by the<br />
expression of the meganuclease I-SceI results in a dramatic stimulation of HR. In<br />
collaboration with Cellectis (who develop meganuclease technologies) we are applying<br />
these results to maize in order to develop an efficient gene targeting system for a major<br />
crop plant. We have generated maize plants which contain a reporter of<br />
intrachromosomal HR and plants with constitutive I-SceI expression. In this assay the<br />
frequency of HR is estimated by the reconstitution by HR, of GUS in pollen. Preliminary<br />
data suggests that I-SceI -induced DSBs can stimulate HR in maize. We are now<br />
developing an inducible meganuclease in order to control HR and develop vectors that<br />
both measure RH and NHEJ in maize.<br />
----------------------------------------------------------------------------------------------------------------------------------<br />
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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
P - 41. Programmed response to IR-induced genomic stress<br />
Kaoru Yoshiyama, Clare Robertson, Kevin Culligan, Anne Britt. Plant Biology, University fo<br />
California, Davis, 95616 Davis, United States<br />
Cellular responses to DNA damage are many and varied. Many of these responses are<br />
programmed, and the complete response requires the presence of multiple signal<br />
transduction pathways. The genetic requirements for these responses can be difficult to<br />
sort out in mammals, as many defects in damage repair or response lead to the induction<br />
of apoptosis. Plants, fortunately, are not as exquisitely sensitive to mismanaged damage,<br />
and so make excellent model systems for the study of the roles of these proteins both in<br />
response to acute damage, and in the general maintenance of unperturbed mitotic and<br />
meiotic cells. Whole-genome analysis indicates that plants, unlike yeasts and mammals,<br />
exhibit a robust transcriptional response to DNA damage that includes many genes<br />
relevant to DNA repair and cell cycle regulation. However, this response is governed, at<br />
least in part, by the same signal transduction components that are required for other<br />
types of damage response in yeasts and mammals. Thus plants may make an excellent<br />
model system for the genetic analysis of DNA damage response. In this presentation we<br />
will focus on our efforts, suing both classical genetic and reverse genetic techniques, to<br />
identify components of the plant DNA damage response pathway.<br />
----------------------------------------------------------------------------------------------------------------------------------<br />
P - 42. Identification of genes required for DNA-damage-induced cell cycle arrest<br />
in Arabidopsis<br />
Kaoru Yoshiyama, Anne Britt. Plant Biology, University of California Davis, 95616 Davis,<br />
United States<br />
The genome of a living organism is subjected to a wide variety of genotoxic stresses of<br />
both endogenous and environmental origin. DNA-damage-induced cell cycle checkpoints<br />
are a crucial component of genome maintenance. These checkpoints sense DNA damage,<br />
pause the cell division cycle to provide time for repair, and finally release the cell cycle<br />
from arrest. The mammalian ATM and ATR protein kinases are key proteins involved in<br />
the cell cycle arrest, and Arabidopsis has ATM and ATR homologs. However, in contrast<br />
to yeast or mammalian cells, little is known about DNA damage checkpoint in plants.<br />
AtXPF is a homolog of human repair endonuclease XPF. The xpf mutant of Arabidopsis<br />
can’t form true leaves after irradiation as seeds, showing sensitivity to gamma-radiation.<br />
We screened for suppressor mutants in the xpf background using EMS and isolated sog<br />
(suppressor of gamma radiation) mutants that can form true leaves following radiation,<br />
suggesting that this cell cycle arrest is due to a signaling mechanism rather than to<br />
inhibition of cell cycle by physically blocking the cellular machinery. We have observed<br />
that xpf sog1-1 seeds are hypersensitive to HU (hydroxyurea), an inhibitor of<br />
ribonucleotide reductase. They could germinate on the HU plates, but after that, they<br />
were dead. Surprisingly, when the seeds are grown on “no HU” plates for 5 days, and the<br />
seedlings are transferred to HU plates, the plants can grow as well as wild type. These<br />
results suggest that SOG might be important during or immediately after germination. We<br />
reported that in wild-type Arabidopsis, hundreds of genes are induced in response to<br />
gamma-radiation, and the induction is dependent on ATM. BRCA1, RAD51 and CYCB1;1<br />
transcriptions increased rapidly and strongly. Interestingly, the transcription inductions<br />
of these genes were compromised severely in the xpf sog1-1 mutant, suggesting that the<br />
SOG gene plays an important role in upstream of signal transduction like ATM. (The xpf<br />
mutation does not affect the induction of the genes.) To identify the SOG gene, we’re<br />
currently mapping the mutation, and we know that the SOG gene lies in the vicinity of<br />
CIW12 marker on chromosome I. We are hoping that identifying the function of SOG gene<br />
is a good way to clarify the mechanism of checkpoint mechanism in plants and, perhaps,<br />
in animals.<br />
----------------------------------------------------------------------------------------------------------------------------------<br />
Page 49 sur 56
<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
Page 50 sur 56
Participants<br />
----------------------------------------------------------------------------------------------------------------------------------<br />
Kiyomi Abe<br />
National Institute of Agrobiological Sciences<br />
Plant Genetic Engineering Research Unit<br />
Japan<br />
kiyomi@affrc.go.jp<br />
Olena Alkhimova<br />
Institute of Molecular Biology and Genetics<br />
National Academy of Sciences of Ukraine<br />
Ukraine<br />
o.g.alkhimova@imbg.org.ua<br />
Lorinda Anderson<br />
Colorado State University<br />
Biology<br />
United States<br />
lorinda.anderson@colostate.edu<br />
Karel J. Angelis<br />
Inst. Experimental Botany<br />
Molec. Farming and DNA Repair<br />
Czech Republic<br />
angelis@ueb.cas.cz<br />
Geert Angenon<br />
Vrije Universiteit Brussel<br />
Plant Genetics<br />
Belgium<br />
Geert.Angenon@vub.ac.be<br />
Susan Armstrong<br />
University of Birmingham<br />
School of Biosciences<br />
United Kingdom<br />
s.j.armstrong@bham.ac.uk<br />
<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
Naomi Avivi-Ragolsky<br />
Weizmann Institute of Science<br />
Plant Sciences<br />
Israel<br />
lpavivi@wicc.weizmann.ac.il<br />
Kamakshi Balakrishnan<br />
Tata Institute of Fundamental Research (TIFR)<br />
Department of Biological sciences<br />
India<br />
kamakshihema@tifr.res.in<br />
Abdellah Barakate<br />
University of Dundee<br />
based at Scottish Crop Research Institute<br />
United Kingdom<br />
abdellah.barakate@scri.ac.uk<br />
Joke Baute<br />
VIB - Ghent University<br />
Plant Systems Biology<br />
Belgium<br />
joke.baute@psb.ugent.be<br />
Dagmar Berenova<br />
Institute of Experimental botany ASCR<br />
Laboratory of DNA repair<br />
Czech Republic<br />
berenova@ueb.cas.cz<br />
Christian Biesgen<br />
SunGene GmbH<br />
Molecular Enabling Technologies<br />
Germany<br />
Christian.Biesgen@sungene.de<br />
Dita Bohdanecka<br />
Institute of experimental botany,<br />
Academy of sciences<br />
Molecular farming and DNA Repair Laboratory<br />
Czech Republic<br />
bohdanecka@ueb.cas.cz<br />
Anne Britt<br />
University fo California, Davis<br />
Plant Biology<br />
United States<br />
abbritt@ucdavis.edu<br />
Jitka Brouzdova<br />
Institute of Experimental Botany<br />
Laboratory of DNA repair and molecule farm<br />
Czech Republic<br />
brouzdova@ueb.cas.cz<br />
Paul Bundock<br />
Keygene N.V.<br />
Upstream Research<br />
Netherlands<br />
paul.bundock@keygene.com<br />
Tamara Byk-Tennenbaum<br />
Evogene Ltd<br />
Israel<br />
tamara.byk@evogene.com<br />
Charles Cai<br />
Dow AgroSciences, LLC.<br />
Biochemistry and Molecular Biology<br />
United States<br />
ccai2@dow.com<br />
W. Zacheus Cande<br />
Univeersity of California, Berkeley<br />
Dept of Molecular and Cell biology<br />
United States<br />
zcande@berkeley.edu<br />
Marie-Edith Chabouté<br />
IBMP/CNRS UPR2357 Strasbourg<br />
plant cellular biology<br />
France<br />
marie-edith.chaboute@ibmp-ulp.u-strasbg.fr<br />
Page 51 sur 56
Cyril Charbonnel<br />
CNRS UMR6547<br />
Université Blaise Pascal<br />
France<br />
cycharbo@univ-bpclermont.fr<br />
Monique Compier<br />
Add2X Biosciences BV<br />
Netherlands<br />
compier@add2x.com<br />
Toon Cools<br />
Ghent University / VIB<br />
Plant Systems Biology<br />
Belgium<br />
tocoo@psb.ugent.be<br />
Gregory Copenhaver<br />
The University of North Carolina at<br />
Chapel Hill<br />
Dept. of Biology and The Carolina Center<br />
for Genome Sciences<br />
United States<br />
gcopenhaver@bio.unc.edu<br />
Kevin Culligan<br />
University of New Hampshire<br />
Biochemistry and Molecular Biology<br />
United States<br />
k.culligan@unh.edu<br />
Kathleen D'Halluin<br />
Bayer BioScience N.V.<br />
Research<br />
Belgium<br />
Kathleen.D'Halluin@bayercropscience.com<br />
Sylvie De Buck<br />
VIB, UGent<br />
Plant Systems Biology<br />
Belgium<br />
sybuc@psb.ugent.be<br />
<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
Sylvia de Pater<br />
Leiden University<br />
Institute Biology Leiden<br />
Netherlands<br />
b.s.de.pater@biology.leidenuniv.nl<br />
PHILIP DEAN<br />
UNIVERSITY OF LEEDS, UK<br />
CENTRE FOR PLANT SCIENCES<br />
United Kingdom<br />
bmbpjd@leeds.ac.uk<br />
Fabien Delacote<br />
Université paris sud / CNRS<br />
Institut de biotechnologie des plantes<br />
UMR8618<br />
France<br />
fabien.delacote@u-psud.fr<br />
Annie Depeiges<br />
CNRS UMR6547<br />
Université Blaise Pascal<br />
France<br />
annie.depeiges@univ-bpclermont.fr<br />
Rob Dirks<br />
Rijk Zwaan Breeding BV<br />
Biotechnologial laboratory<br />
Netherlands<br />
d.van.noort@rijkzwaan.nl<br />
Marie-Pascale Doutriaux<br />
CNRS Université Paris XI<br />
Institut de Biotechnologie des Plantes<br />
France<br />
marie-pascale.doutriaux@u-psud.fr<br />
manuel dubald<br />
Bayer CropScience<br />
BioScience<br />
France<br />
manuel.dubald1@bayercropscience.com<br />
Emeline Dubois<br />
Université Paris XI-CNRS<br />
Institut de Biotechnologie des Plantes-<br />
UMR8618<br />
France<br />
emeline.dubois@u-psud.fr<br />
Masaki Endo<br />
National Institute of Agrobiological Sciences<br />
Plant Engineering Research Unit,<br />
Division of Plant Sciences<br />
Japan<br />
mendo@affrc.go.jp<br />
Yonat Eshchar<br />
Weizmann Institute of Science<br />
Plant Sciences<br />
Israel<br />
yonat.eshchar@wicc.weizmann.ac.il<br />
Jiri Fajkus<br />
Masaryk University and Institute of<br />
Biophysics ASCR<br />
Functional Genomics and Proteomics<br />
Czech Republic<br />
fajkus@sci.muni.cz<br />
Chris Franklin<br />
University of Birmingham<br />
School of Biosciences<br />
United Kingdom<br />
F.C.H.Franklin@bham.ac.uk<br />
Maria Gallego<br />
Universite Blaise Pascal<br />
CNRS UMR 6547<br />
France<br />
megalleg@univ-bpclermont.fr<br />
Marcelina Garcia Aguilar<br />
Institute for research and Development (IRD)<br />
Plant Genome and Development Laboratory<br />
France<br />
garciam@mpl.ird.fr<br />
Pascal Genschik<br />
Institut de Biologie Moleculaire<br />
des Plantes CNRS<br />
France<br />
Pascal.Genschik@ibmp-ulp.u-strasbg.fr<br />
Page 52 sur 56
tom gerats<br />
Radboud University<br />
IWWR/Plant Genetics<br />
Netherlands<br />
t.gerats@science.ru.nl<br />
Mathilde Grelon<br />
INRA<br />
Genetic and Plant Breeding<br />
France<br />
grelon@versailles.inra.fr<br />
Manju Gupta<br />
Dow AgroSciences<br />
Plant Genetics and Biotechnology<br />
United States<br />
mgupta@dow.com<br />
James E. Haber<br />
Brandeis University<br />
Rosenstiel Basic Medical Sciences Research<br />
Center<br />
United States<br />
haber@brandeis.edu<br />
Claire Halpin<br />
University of Dundee<br />
Plant Research Unit, College of Life Sciences<br />
United Kingdom<br />
c.halpin@dundee.ac.uk<br />
Frank Hartung<br />
University of Karlsruhe<br />
Botanical Institute II<br />
Germany<br />
frank.hartung@bio.uka.de<br />
John Hays<br />
Oregon State University<br />
Dept. of Environmental and<br />
Molecular Toxicology<br />
United States<br />
haysj@science.oregonstate.edu<br />
Veena Hedatale<br />
Radboud University<br />
Plant Genetics<br />
India<br />
v.hedatale@science.ru.nl<br />
Barbara Hohn<br />
Freidrich Meischer Institut<br />
Switzerland<br />
barbara.hohn@fmi.ch<br />
<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
Paul Hooykaas<br />
Leiden University /Institute of Biology<br />
Leiden (IBL)<br />
Molecular and Developmental Genetics<br />
Netherlands<br />
p.j.j.hooykaas@biology.leidenuniv.nl<br />
Shigeru Iida<br />
National Institute for Basic Biology<br />
Division of Molecular Genetics<br />
Japan<br />
shigiida@nibb.ac.jp<br />
Doolyi Kim<br />
National Institute of<br />
Agricultural Biotechnology<br />
Cell and Genetics<br />
South Korea<br />
dykim@rda.go.kr<br />
Jaroslav Kozak<br />
Academy of Sciences of the Czech Republic<br />
Institute of Experimental Botany,<br />
Laboratory of DNA repair<br />
Czech Republic<br />
kozak@ueb.cas.cz<br />
Neeraja Krishnan<br />
Tata Institute of Fundamental Research<br />
Department of Biological Sciences<br />
India<br />
neeraja@tifr.res.in<br />
julien lang<br />
IBMP/CNRS<br />
department of cellular biology<br />
France<br />
Julien.Lang@ibmp-ulp.u-strasbg.fr<br />
samuel le goff<br />
CNRS UMR6547<br />
Université Blaise Pascal<br />
France<br />
samuel.le_goff@univ-bpclermont.fr<br />
Cilia Lelivelt<br />
Rijk Zwaan Breeding BV<br />
Celbiology<br />
Netherlands<br />
d.van.noort@rijkzwaan.nl<br />
Avraham Levy<br />
Weizmann Institute of Sciences<br />
Plant Sciences<br />
Israel<br />
avi.levy@weizmann.ac.il<br />
Franck Lhuissier<br />
Keygene<br />
Upstream research<br />
Netherlands<br />
franck.lhuissier@keygene.com<br />
Michal Lieberman - Lazarovich<br />
Weizmann Institute of Science<br />
Plant Sciences department<br />
Israel<br />
michal.lieberman@weizmann.ac.il<br />
Beatrice Lindhout<br />
Institute of Biology, Leiden University<br />
Molecular and developmental genetics<br />
Netherlands<br />
b.i.lindhout@biology.leidenuniv.nl<br />
L. Alexander Lyznik<br />
Pioneer Hi-Bred International, Inc<br />
Crop Genetics Research<br />
United States<br />
alex.lyznik@pioneer.com<br />
Page 53 sur 56
Sean Mayes<br />
University of Nottingham<br />
Agricultural and Environmental Sci<br />
(Biosciences)<br />
United Kingdom<br />
sean.mayes@nottingham.ac.uk<br />
Cathy Melamed Bessudo<br />
The Weizmann Institute of Science<br />
Plant Sciences Department<br />
Israel<br />
cathy.bessudo@weizmann.ac.il<br />
Raphaël Mercier<br />
INRA<br />
genetics<br />
France<br />
rmercier@versailles.inra.fr<br />
Marie Mirouze<br />
Université de Genève Sciences III<br />
Laboratory of Plant Genetics<br />
Switzerland<br />
marie.mirouze@bioveg.unige.ch<br />
Jon Mitchell<br />
Dow AgroSciences LLC<br />
R&D/Dept. of Biochemistry<br />
and Molecular Biology<br />
United States<br />
jcmitchell@dow.com<br />
Marie-Hélène Montané<br />
CEA<br />
Direction des Sciences du vivant<br />
France<br />
marie-helene.montane@cea.fr<br />
Graham Moore<br />
John Innes Centre<br />
Crop Genetics<br />
United Kingdom<br />
graham.moore@bbsrc.ac.uk<br />
Keishi Osakabe<br />
National Institute of Agrobiological Sciences<br />
Division of Plant Sciences<br />
Japan<br />
kosakabe@affrc.go.jp<br />
Michael Pacher<br />
University of Karlsruhe / Institute of Botany II<br />
Molecular Biology & Biochmeistry of plants<br />
Germany<br />
Michael.Pacher@botanik2.uni-karlsruhe.de<br />
Frédéric Pâques<br />
Cellectis S.A.<br />
France<br />
paques@cellectis.com<br />
<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
Jerzy Paszkowski<br />
Université de Genève<br />
Switzerland<br />
Jerzy.Paszkowski@bioveg.unige.ch<br />
Wyatt PAUL<br />
Biogemma<br />
Cereal Functional Analysis Group<br />
United Kingdom<br />
wyatt.paul@biogemma.com<br />
Janny Peters<br />
Radboud University Nijmegen<br />
Plant Genetics<br />
Netherlands<br />
jl.peters@science.ru.nl<br />
Thomas Peterson<br />
Iowa State University<br />
Genetics, Development and Cell Biology<br />
United States<br />
thomasp@iastate.edu<br />
Holger Puchta<br />
University Karlsruhe<br />
Botany II<br />
Germany<br />
holger.puchta@bio.uka.de<br />
Bernd Reiss<br />
MPI für Züchtungsforschung<br />
Department Coupland<br />
Germany<br />
reiss@mpiz-koeln.mpg.de<br />
Karel Riha<br />
Austrian Academy of Sceinces,<br />
Gregor Mendel Institute<br />
Austria<br />
karel.riha@gmi.oeaw.ac.at<br />
Eddy Risseeuw<br />
National Research Council<br />
Plant Biotechnology Institute<br />
Canada<br />
risseeuwe@nrc.ca<br />
Max Roessler<br />
Austrian Academy of Sceinces,<br />
Gregor Mendel Institute<br />
Austria<br />
max.roessler@gmi.oeaw.ac.at<br />
Teresa Roldan-Arjona<br />
Universidad de Cordoba<br />
Departamento de Genetica<br />
Spain<br />
ge2roarm@uco.es<br />
Cyril Saintenac<br />
INRA<br />
UMR 1095 Amelioration et Sante des plantes<br />
France<br />
saintena@clermont.inra.fr<br />
Ayako Sakamoto<br />
Japan Atomic Energy Agency<br />
Radiation-Applied Biology<br />
Japan<br />
sakamoto.ayako@jaea.go.jp<br />
Aviva Samach<br />
Weizmann Institute of sciences<br />
Dept. of plant sciences<br />
Israel<br />
aviva.samach@weizmann.ac.il<br />
Page 54 sur 56
Iva Santruckova<br />
Masaryk University<br />
Functional Genomics and Proteomics<br />
Czech Republic<br />
iva.sant@mail.muni.cz<br />
Didier Georges Schaefer<br />
INRA,<br />
Station de génétique et<br />
amélioration des plantes<br />
France<br />
dschaefer@versailles.inra.fr<br />
Martina Schirato<br />
Austrian Academy of sciences<br />
Gregor Mendel Institute<br />
Austria<br />
martina.schirato@gmi.oeaw.ac.at<br />
Peter Schlögelhofer<br />
Max F. Perutz Laboratories/<br />
University of Vienna<br />
Dept. of Chromosome Biology<br />
Austria<br />
peter.schloegelhofer@univie.ac.at<br />
Ingo Schubert<br />
Leibniz-Institute of Plant Genetics<br />
and Crop Plant Research<br />
Cytogenetics and Genome Analysis<br />
Germany<br />
schubert@ipk-gatersleben.de<br />
Dorothy Shippen<br />
Texas A&M University<br />
Biochemistry and Biophysics<br />
United States<br />
dshippen@tamu.edu<br />
Vipula Shukla<br />
Dow AgroSciences, LLC<br />
Discovery R&D<br />
United States<br />
vkshukla@dow.com<br />
<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
Marketa Smidkova<br />
IEB ASCR<br />
Laboratory of molecular farming<br />
and DNA repair<br />
Czech Republic<br />
m.smidkova@seznam.cz<br />
Pierre SOURDILLE<br />
INRA UMR1095<br />
Amélioration et Santé des Plantes<br />
Structure, Function and Evolution<br />
of Wheat Genomes<br />
France<br />
pierre.sourdille@clermont.inra.fr<br />
Wojciech Strzalka<br />
Jagiellonian University<br />
Faculty of Biochemistry, Biophysics<br />
and Biotechnology<br />
Poland<br />
strzalka@if.uj.edu.pl<br />
Shinya Takahashi<br />
RIKEN Plant Science Center<br />
Plant Functional Genomics Research Team<br />
Japan<br />
shinya@psc.riken.jp<br />
Seiichi Toki<br />
National Institute of Agrobiological Scienced<br />
Plant Genetic Engineering Research Unit<br />
Japan<br />
stoki@affrc.go.jp<br />
Jeffrey Townsend<br />
Iowa State University<br />
Genetics, Cell and Developmental Biology<br />
United States<br />
townend@iastate.edu<br />
Tzvi Tzfira<br />
University of Michigan<br />
Department of Molecular, Cellular, &<br />
Developmental Biology<br />
United States<br />
ttzfira@umich.edu<br />
Fyodor Urnov<br />
Sangamo BioSciences<br />
Genomics<br />
United States<br />
furnov@sangamo.com<br />
Frédéric Van Ex<br />
Vrije Universiteit Brussel (V.U.B.)<br />
Laboratory of Plant Genetics<br />
Belgium<br />
fvex@vub.ac.be<br />
Jean-Baptiste VANNIER<br />
CNRS UMR6547<br />
Université Blaise Pascal<br />
France<br />
jbvannie@univ-bpclermont.fr<br />
Laurent Vespa<br />
Texas A&M University<br />
Biochemistry and Biophysics<br />
United States<br />
vespa@tamu.edu<br />
Corina Belle Villar<br />
ETH Zurich, Institute of Plant Sciences<br />
Plant Developmental Biology<br />
Switzerland<br />
cvillar@ethz.ch<br />
Dan Voytas<br />
Iowa State University<br />
Dept. of Genetics, Development & Cell Biology<br />
United States<br />
voytas@iastate.edu<br />
Koichi Watanabe<br />
Leibniz Institute of Plant Genetics<br />
and Crop Plant Research<br />
Germany<br />
watanabe@ipk-gatersleben.de<br />
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Wanda Melody Waterworth<br />
University of Manchester, UK<br />
Molecules to Cells<br />
United Kingdom<br />
mqbsswmw@manchester.ac.uk<br />
Sophie Wehrkamp-Richter<br />
Biogemma<br />
Gene Function and Maize Traits Group<br />
France<br />
sophie.wehrkamp@biogemma.com<br />
Clifford Weil<br />
Purdue University<br />
Dept of Agronomy<br />
United States<br />
cweil@purdue.edu<br />
Chris West<br />
University of Leeds<br />
Centre for Plant Sciences<br />
United Kingdom<br />
c.e.west@leeds.ac.uk<br />
<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007<br />
Charles White<br />
CNRS UMR6547<br />
Université Blaise Pascal<br />
France<br />
chwhite@univ-bpclermont.fr<br />
Wei Xiao<br />
University of Saskatchewan<br />
Department of Microbiology and Immunology<br />
Canada<br />
wei.xiao@usask.ca<br />
Kaoru Yoshiyama<br />
University of California Davis<br />
Plant Biology<br />
United States<br />
kyoshiyama@ucdavis.edu<br />
Alicja Ziemienowicz<br />
Jagiellonian University<br />
Faculty of Biochemistry, Biophysics<br />
and Biotechnology<br />
Poland<br />
alicja@mol.uj.edu.pl<br />
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