pdf file - Events - EMBO

PLANT 

DNA Repair 

& Recombination 

Invited Speakers 

Anne Britt USA 

Zac Cande USA 

Greg Copenhaver USA 

Chris Franklin UK 

Jim Haber USA 

John Hays USA 

Barbara Hohn Switzerland 

Paul Hooykaas Holland 

Shigeru Lida Japan 

Avraham Levy Israel 

Rob Martienssen USA 

Jerzy Paszkowski Switzerland 

Holger Puchta Germany 

Karel Riha Austria 

Dorothy Shippen USA 

Dan Voytas USA 

Cliord Weil USA 

Charles White France 

Co-Sponsors 

BIOGEMMA 

www.biogemma.com 

Centre National de la Recherche Scientique 

www.cnrs.fr 

CSREES - USDA 

http://www.usda.gov/wps/portal/usdahome 

DOW Agrosciences 

http://www.dowagro.com/homepage/index.htm 

National Science Foundation 

http://www.nsf.gov 

Pioneer-DuPont 

http://www.pioneer.com 

Enquiries 

cw@univ-bpclermont.fr 

http://cwp.embo.org/w30-07 

EMBO WORKSHOP 

Session Titles 

31 May–3 June | 2007 

Presqu’île de Giens | France 

Somatic DNA repair and recombination 

Chromatin and Epigenetics 

Chromosome maintenance and architecture 

Recombination and Meiosis 

Recombination and related technologies 

Organisers 

Barbara Hohn 

Friedrich Miescher Institute for Biomedical Research, Switzerland 

Avraham Levy 

Weizmann Institute of Science, Israel 

Holger Puchta 

Karlsruhe University, Germany 

Dorothy Shippen 

Texas A&M University, USA 

Cliord Weil 

Purdue University, USA 

Charles White 

Centre National de la Recherche Scientique, France

EMBO Plant DNA Repair & Recombination 

Workshop 

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Presqu'île de Giens, France 

31 May - 3 June 2007 

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15.00-18.00 Check in and registration 

19.00-20.15 Dinner 

EMBO Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 

31 May 2007 

20.15-20.30 Welcome and Announcements (Charles White) 

20.30-21.30 Jim Haber 

Multiple pathways to repair double-strand breaks and thoughts about gene 

targeting. 

21.30-22.30 Barbara Hohn 

Homologous recombination as a sensor to environmental 

challenges. 

22.30 Poster set-up and wine 

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1 June 2007 

8.00-11.20 

SESSION I: Somatic DNA repair and recombination I 

(Chairs: Anne Britt and Charles White) 

8-8.30 Anne Britt 

Regulation of IR-induced checkpoint responses 

8.30-8.50 Toon Cools 

Arabidopsis WEE1 Kinase Controls Cell Cycle Arrest in Response to Activation of the 

DNA Integrity Checkpoint. 

8.50-9.10 Pascal Genschik 

The CUL4-DDB1-DDB2 complex and genome integrity. 

9.10-9.30 Wei Xiao 

Arabidopsis thaliana Ubc13-Uev complexes and DNA damage tolerance 

9.30-9.50 Charles White 

Roles of the Rad51 paralogs in mitosis and meiosis 

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9.50-10.10 Coffee break 

10.10-10.30 Marie-Edith Chabouté 

New partner in DSB-induced gammaH2AX foci in Arabidopsis 

10.20-10.40 Seiichi Toki 

Homologous recombinational repair and cell cycle regulation in Arabidopsis. 

10.40-11 Ayako Sakamoto 

Functional analysis of DNA polymerase zeta and REV1 protein in Arabidopsis 

11-11.20 John Hays 

Requirements of DNA translesion polymerases Zeta and/or Eta for coordinated 

tissue growth in Arabidopsis developing ovaries and UVB-irradiated root meristems 

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16.00-18.00 

SESSION II: Somatic DNA repair and recombination II 

(Chairs: Holger Puchta and Barbara Hohn) 

16-16.30 Holger Puchta 

Role of Human Disease Genes for Genome Stability in Arabidopsis thaliana 

16.30-16.50 Peter Schlögelhofer 

The Arabidopsis thaliana AtGR1/COM1 gene is essential for DNA repair and meiosis 

and relates yeast's COM1/SAE2 to the human CtIP gene 

16.50-17.10 Thomas Peterson 

Transposon-Induced Genome Rearrangements in Maize and Arabidopsis 

17.10-17.30 Didier G. Schaefer 

Genetic analysis of transformation in the moss Physcomitrella patens 

17.30-17.50 Bernd Reiss 

RAD51 genes have different roles in Physcomitrella patens and Arabidopsis 

thaliana. 

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20.00-22.00 

SESSION III: Chromatin and Epigenetics 

(Chair: Jurek Paszkowski) 

20-20.30 Jurek Paszkowski 

DNA methylation and other epigenetic marks in Arabidopsis. 

20.30-21 Rob Martienssen 

Silent running: RNA interference and heterochromatic modifications in plants and 

fission yeast. 

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21-21.20 Marcelina Garcia-Aguilar 

A functional and comparative analysis of chromatin changes associated with the 

control of reproductive development in sexual and apomictic plants. 

21.20-21.40 Teresa Roldan-Arjona 

DNA Demethylation by 5-methylcitosine excision, a new mechanism of epigenetic 

reprograming in plants. 

21.40-22.00 Corina Belle Villar 

Mechanisms of Polycomb group-mediated gene silencing in Arabidopsis 

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2 June 2007 

0800-1120 

SESSION IV: Chromosome maintenance and architecture 

(Chairs: Dorothy Shippen and Karel Riha) 

8-8.30 Dorothy Shippen 

Chromosome end protection in Arabidopsis 

8.30-9 Karel Riha 

Role of the Ku70/Ku80 heterodimer in telomere metabolism 

9-9.20 Sue Armstrong 

Dynamics of telomere behaviour in Arabidopsis thaliana meiosis 

9.20-9.40 Ingo Schubert 

HR versus NHEJ at the chromosomal level 

9.40-10 Coffee break 

10-10.20 Maria Gallego 

AtRad1/AtErcc1 endonuclease and telomere stability in Arabidopsis 

10.20-10.40 Laurent Vespa 

A role of ATM in telomere length regulation in Arabidopsis 

10.40-11 Jiri Fajkus 

Mapping of interaction domains of putative plant telomere proteins 

11-11.20 Koichi Watanabe 

Positional sister chromatid alignment in a mutant of the Arabidopsis homolog of 

Structural Maintenance of Chromosome 6 (SMC6) 

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1400-1700 

SESSION V: Recombination and meiosis 

(Chairs: Clifford Weil and Zac Cande) 

14-14.30 Cliff Weil 

Forward Foreword: mutations that alter recombination rate and crossover 

interference in maize 

14.30-14.50 Chris West 

Arabidopsis NBS1: roles in meiosis and DNA repair 

14.50-15.10 Zac Cande 

Meiotic prophase chromosome architecture revealed by ultrahigh resolution 

structured illumination (SI) microscopy 

15.10-15.30 Graham Moore 

It's not size but coordination that matters 

15.30-15.50 Greg Copenhaver 

Using Arabidopsis tetrads to understand interference 

15.50-16.10 break 


16.10-16.30 Chris. Franklin 

Co-ordination of meiotic recombination by ASY1 in Arabidopsis thaliana 

16.30-16.50 Franck L'Huissier 

MLH1 marks a subset of strongly interefering crossovers in tomato 

16.50-17.10 Mathilde Grelon 

Identification of meiotic DSB forming proteins in Arabidopsis thaliana 

17.10-17.30 Tom Gerats 

Recombination and genetic maps in Petunia 

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19.00 - Drinks and Congress Dinner 

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3 June 2007 

08.00-11.00 

SESSION VI: Recombination and related Technologies 

(Chairs: Avi Levy and Dan Voytas) 

8-8.30 Paul Hooykaas 

Modulation of the recombination machinery for gene targeting 

8.30-9.00 Shigeru Iida 

Gene Targeting by Homologous Recombination in Rice 

9:00-9:30 Daniel Voytas 

Gene targeting in plants using zinc finger nucleases 

9.30-9.50 coffee break 

9:50-10:05 Avraham Levy 

Stimulating Gene Targeting into the Arabidopsis genome 

10:05-10:20 Paul Bundock 

Mutation scanning by massive parallel sequencing 

10:20-10:35 Vipula Shukla 

Site-directed homologous recombination in tobacco cell cultures via zinc-finger 

nucleases. 

10:35-10:50 Frédéric Van Ex 

Development of a gene targeting system based on in planta presentation of 

homologous donor DNA during meiosis. 

10:50-11:05 Frédéric Pâques 

Meganucleases with tailored cleavage specificity can induce efficient homologous 

gene targeting 

11:05-11:20 Tzvi Tzfira 

Toward zinc finger nucleases-mediated gene targeting in plants 

11:20-11:35 Sylvie DeBuck 

Generation of Single-Copy DNA Transformants by the Cre/LoxP Recombination 

Mediated Resolution System 

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11.35-12.00 Closing remarks 

12.00-13.30 Lunch 

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Abstracts of Talks 

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T - 1. Arabidopsis WEE1 Kinase Controls Cell Cycle Arrest in Response to 

Activation of the DNA Integrity Checkpoint. 

Toon Cools, Anelia Iantcheva, Kristof De Schutter, Jerome Joubes, Francois-Yves Bouget, 

Dirk Inze, Lieven De Veylder Plant Systems Biology, Ghent University / VIB, 9052 

Zwijnaarde, Belgium 

Upon the incidence of DNA stress, the ataxia telangiectasia-mutated (ATM) and Rad3related 

(ATR) signaling kinases activate a transient cell cycle arrest that allows cells to 

repair DNA before proceeding into mitosis. Although the ATM-ATR pathway is highly 

conserved over species, the mechanisms by which plant cells stop their cell cycle in 

response to the loss of genome integrity are unclear. Previously, we have demonstrated 

that the cell cycle regulatory WEE1 kinase gene of Arabidopsis thaliana is 

transcriptionally activated upon the cessation of DNA replication or DNA damage in an 

ATR- or ATM-dependent manner, respectively. In accordance with a role for WEE1 in DNA 

stress signaling, WEE1-deficient plants showed no obvious cell division or 

endoreduplication phenotype when grown under nonstress conditions but were 

hypersensitive to agents that impair DNA replication (De Schutter et al., 2007). To study 

the effects of a deficient DNA replication checkpoint in more detail, a microarray analysis 

was performed. These data illustrated that in the WEE1-deficient plants upregulation of 

DNA-repair genes is not accompanied with the downregulation of cell cycle genes, 

contrary to what is observed in wild type plants. These data illustrate that WEE1 solely is 

responsible for the cell cycle arrest seen in wild-type plants upon activation of the DNA 

replication checkpoint and that a correct cell cycle arrest is essential for survival. To 

uncover the proteins that build up the cascade that results into WEE1 induction upon 

genotoxic stress, a mutagenesis screen was preformed. Additionally, a promoter deletion 

analysis was initiated to identify the possible cis-acting regulatory elements that are 

responsible for the induction of WEE1 upon DNA stress. De Schutter et al. (2007) 

Arabidopsis WEE1 Kinase Controls Cell Cycle Arrest in Response to Activation of the DNA 

Integrity Checkpoint. Plant Cell, In press. 

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T - 2. The CUL4-DDB1-DDB2 complex and genome integrity. 

Jean Molinier, Esther Lechner, Pascal Genschik , Institut de Biologie Moleculaire des Plantes 

CNRS, 67084 Strasbourg , France 

Plants due to their sessile life style are constantly exposed to UV irradiation which trigger 

cellular damage especially on DNA. As response, repair pathways are activated in order to 

guarantee genome integrity. The predominant DNA lesions, induced by UV radiation, are 

the cyclobutane pyrimidine dimers (CPDs) but also pyrimidine [6-4]pyrimidinone dimers. 

The main repair pathways of these lesions is the direct repair performed by photolyases. 

In addition to this photoreactivation process, most organisms have also a sophisticated 

general repair mechanism, termed nucleotide excision repair (NER). In this mechanism, 

the heterodimeric DDB protein complex, formed by DDB1 and DDB2, binds tightly to UVdamaged 

DNA and might act as a sensor to detect a subset of conformational changes on 

DNA. DDB1-DDB2 are part of a E3 ubiquitin ligase complex together with Cullin4. We will 

present recent results demonstarting the role of the CUL4-DDB1-DDB2 complex in DNA 

excision repair and maintenance of genome intergrity 

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T - 3. Arabidopsis thaliana Ubc13-Uev complexes and DNA damage tolerance 

Rui Wen, Lindsay Newton, Antonio Torres-Acosta, Hong Wang, Wei Xiao Department of 

Microbiology and Immunology, University of Saskatchewan, S7N 5E5 Saskatoon, Canada 

DNA damage tolerance (DDT) is a newly defined pathway in eukaryotes. In budding yeast, 

this is achieved by covalent modifications of the proliferating cell nuclear antigen (PCNA). 

A ubiquitin conjugating enzyme Ubc13 and a Ubc enzyme variant (Uev) are required for a 

unique Lys63-linked polyubiquitination of PCNA. We isolated two UBC13 and four UEV 

genes from Arabidopsis thaliana. All four AtUev1 proteins are able to form a stable 

complex with AtUbc13 as well as Ubc13 from yeast and human. These genes are able to 

functionally replace the corresponding yeast UBC13 or MMS2 genes for the DDT 

functions in vivo. Although all two AtUBC13 and four AtUEV1 genes are ubiquitously 

expressed in most tissues, AtUEV1D appears to be expressed at a much higher level in 

germinating seeds and in pollen. We obtained a T-DNA insertion line and created Atuev1d 

null plants. Compared with wild type and heterozygous mutant plants, seeds from the 

Atuev1d null plant germinated poorly when treated with a DNA damaging agent, while 

those germinated grew slower and majority ceased growth within two weeks. To our 

knowledge, this is the first report of Ubc13-Uev functions in tolerating DNA damage in a 

multicellular organism. 

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T - 4. Roles of the Rad51 paralogs in mitosis and meiosis 

Le Goff, S., Abe, K., Gallego, M.E. and Charles I. White. CNRS UMR6547, Université Blaise 

Pascal, Clermont Ferrand, France. 

By permitting the exchange of genetic information between DNA molecules, genetic 

recombination processes contribute both to genetic diversity and to the maintenance of 

genome integrity. In addition, recombination plays key roles in the transmssion of the 

genome via sexual reproduction, assuring the proper segregation of chromosomes at the 

first, reductional meiotic division. We are studying the nature of DNA 

recombination/repair pathways and the interrelationships between them in Arabidopsis 

thaliana and will present our data on the Rad51 paralogues. In addition to the meiosisspecific 

recombinase Dmc1, five Rad51 paralog proteins are known and all five are 

implicated in recombination, chromosomal stability and the DNA repair. The inviability of 

mutants of these genes in animals has complicated study of their roles, particularly in 

meiosis, and makes Arabidopsis a model of particular interest. We have reported the 

identification and characterisation of four of the proteins: Rad51B, Rad51C, Xrcc2, and 

Xrcc3, and have shown that the Arabidopsis xrcc3 and rad51C mutants are sterile due to 

fragmentation of the genome at meiotic prophase I. We have now identified a mutant for 

the fifth paralog, Rad51D, and will present data on the roles of this protein and the other 

paralogs in meiotic and mitotic recombination in Arabidopsis. 

T - 5. New partner in DSB-induced gammaH2AX foci in Arabidopsis 

julien lang, lenin sanchez-calderon, ondrej smetana, guy houlne, marie-edith 

chaboute department of cellular biology, IBMP/CNRS, 67000 strasbourg, France 

Occurrence of DNA double-strand breaks (DSBs) constitutes a major threat for the 

survival of the cell. Over the last years several studies have outlined the signalling 

pathway triggered by such damage and shown that it was conserved through evolution. 

Thus the ATM-dependent phosphorylated form of the histone variant H2AX (called 

gamaH2AX), participating both in the correct recruitment of repair proteins (Celeste A. et 

al., Nat Cell Biol 2003; 5: 675-679) as well as in the maintenance of the cell cycle 

checkpoints until complete recovery (Chowdhury et al., Mol Cell 2005; 20: 801-809), has 

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been pointed out at as a reliable marker of DSBs. Using the plant model Arabidopsis 

thaliana (At) we further analysed the repair function of the two putative AtH2AX 

orthologs, especially the nature and relevance of their accumulation, under 

phosphorylated state, with other partners at sites of DSBs. Thanks to an artificial miRNA 

line we highlighted the contribution of AtgamaH2AX in plant growth, repair and genomic 

stability. More interestingly our first results also hinted at an involvement, upon DSBs 

induction, of some cell cycle regulators E2Fs factors whose role in DNA repair is still 

poorly understood. As Nicotiana tabaccum E2Fs relocalize in Arabidopsis root cells, after 

bleomycin treatment, and form some clear and discrete foci together with AtgamaH2AX, 

we emitted the hypothesis that some AtE2F factors may be instrumental in responses to 

DNA damage and started to confirm it by comparing different genotoxic effects in an 

Ate2fa mutant 

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T - 6. Homologous recombinational repair and cell cycle regulation in 

Arabidopsis. 

Masaki Endo, Keishi Osakabe, Kiyomi Abe, Masaaki Umeda, Seiichi Toki Plant Engineering 

Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences, 305- 

8602 Tsukuba, Ibaraki, Japan 

Homology-based replacement of an endogenous gene by an introduced gene, termed gene 

targeting is a prime goal in the precise engineering of higher plants. In the previous 

study, we indicated that disfunction of chromatin assembly factor 1 (CAF-1) increased 

frequency of somatic homologous recombination (HR) and T-DNA integration in 

Arabidopsis. We thought that a decreased rate of histone loading onto newly replicated 

chromosomal DNA may leave this DNA more accessible to DNA repair and recombination 

enzymes, and to integrating T-DNA. Furthermore, we observed increased levels of DNA 

double-strand breaks, elevated levels of mRNAs coding for proteins involved in 

homologous recombination and G2 phase retardation in CAF-1 mutants. Interestingly, 

some factors, which operate cell cycle and DNA repair were reported in yeast and animal 

(CycA1, CDK2 etc.). Since the relationship between cell cycle regulation and DNA repair 

machinery remains elucidated in higher plants, we isolated Arabidopsis mutants 

depleting cell cycle regulating factors and analyzed the intrachromosomal homologous 

recombination frequency and DNA damage sensitivity in these mutants. We hope we 

could discuss the potential use of the cell cycle regulators and other related factors on 

the improvement of gene targeting system in higher plants. 

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T - 7. Functional analysis of DNA polymerase zeta and REV1 protein in 

Arabidopsis 

Ayako Sakamoto, Mayu Nakagawa, Shinya Takahashi, Atsushi Tanaka Radiation-Applied 

Biology , Japan Atomic Energy Agency, 370-1292 Takasaki, Japan 

Translesion synthesis (TLS) is one of the cellular processes to overcome the lethal effect 

of unrepaired DNA damage. During screening for genes accounting for the UV-resistance 

in Arabidopsis, we first identified AtREV3 that encodes a catalytic subunit of DNA 

polymerase zeta (Pol z). Pol z is present in almost all eukaryotes and thought to bypass 

damaged DNA in an error-prone manner. We subsequently identified AtREV7, a 

regulatory subunit of Pol z, and AtREV1 that is thought to cooperate with Pol z in 

bypassing of apurine/apyrimidine (AP) sites. Disruption of any of AtREV3, AtREV7, or 

AtREV1 made the plants more sensitive to UVB than the wild type, suggesting that these 

REV proteins are required for plants to tolerate to the UV-induced damage. We further 

analyzed the UV-induced mutation frequency in rev3 and rev1. The point-mutated nonfunctional 

uidA genes were introduced into the Arabidopsis plants and reversion events 

were detected by blue sectors on the somatic tissues. We found that the disruption of 

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AtREV3 or AtREV1 reduced the mutation frequency to 1/4 of the level of the wild type. 

These results are consistent with an idea of that the Pol z and AtREV1 are involved in the 

UV-induced mutation in Arabidopsis. However, by using the bacterially expressed protein, 

although we detected AtREV1 inserted a dCMP at the opposite of the AP site in vitro, 

AtREV1 failed to bypass UV-induced damage as reported in other organisms. Thus, the 

mechanism by which the REV1 functions remains to be cleared. 

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T - 8. Requirements of DNA translesion polymerases Zeta and/or Eta for 

coordinated tissue growth in Arabidopsis developing ovaries and UVBirradiated 

root meristems 

Marc Curtis, Peter Hoffman, John Hays Dept. of Environmental and Molecular Toxicology, 

Oregon State University, 97331-7301 Corvallis. Oregon, United States 

Specialized DNA translesion polymerases (TLPs) help rescue replication forks arrested 

when environmentally induced or endogenous DNA lesions, or perhaps difficult contexts 

in undamaged DNA, stall replicative polymerases. We analyzed, by various light 

microscopy techniques, UVB-irradiated Arabidopsis root meristems deficient in one or 

both of two key TLPs, AtPolEta and AtPolZeta. A threshold UV-B dose that induced 

roughly 0.03 cyclobutane pyrimidine dimers (CPDs) per kbp had little effect on wild-type 

(wt) roots. However, this dose inhibited root growth, suppressed division of stem cells 

and transiently-amplifying (TA) cells and specifically killed stem cells, in the increasing 

severity order Eta? < Zeta? < Eta? Zeta?. In UVB-irradiated Eta? Zeta? roots, alterations in 

tissue development persisted several days after CPDs had disappeared. Even unirradiated 

Eta? Zeta? roots showed substantial stem-cell death. Seed set in (unirradiated) selfed- 

Arabidopsis plants heterozygous (-/ ) for Pol? was roughly 50% of wt ( / ), although 

progeny showed no selection against mutant (-) or wt ( ) alleles. Correspondingly, about ½ 

of ovules in Pol?-/ ovaries arrested after meiosis and failed to progress through gamete 

mitosis. Remarkably, Pol?-/- ovaries showed no post-meiotic arrest or reduced seed set. 

Recombinant PolEta proteins from Arabidopsis (AtPolEta), humans (HsPolEta), and yeast 

(ScPolEta) were overexpressed in E. coli and purified to homogeneity. Comprehensive 

analyses of their abilities to synthesize DNA past template CPDs reveals high biochemical 

similarity between AtPolEta and HsPolEta and interesting differences between these two 

and ScPolEta. (Supported by National Science Foundation grant MCB 03455061 to J.B.H.) 

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T - 9. Role of Human Disease Genes for Genome Stability in Arabidopsis thaliana 

Holger Puchta, Stefanie Suer, Daniela Kobbe, Sandra Blanck, Wim Reidt, Rebecca Wurz, 

Manfred Focke, Frank Hartung Botany II, University Karlsruhe, 76128 Karlsruhe, 

Germany 

During the elucidation of the genomic sequences of plants it became clear that multiple 

homologues of genes, which in their mutated form cause specific human diseases, are 

present in the plant genome. The main interest of our group focuses around breast 

cancer and the Blooms and Werner syndrome. In case of the two latter, mutations in two 

different members of the human RecQ DNA helicase genes family are responsible for the 

diseases. Arabidopsis harbours 7 RecQ homologues in its genome. In a long term project 

we are trying to define the biological role of individual family members. On one side we 

express AtRecQ ORFs in E. coli and characterized the biochemical properties in vitro. First 

result indicate that particular helicases indeed differ in their biochemical properties e.g. 

differences in the ability to process specific DNA structures could be defined. On the 

other side we try to analyse the role of the genes in vivo with sensitivity assays against 

DNA damaging agents and determination of homologous recombination frequencies. 

Additionally we started to test the effect of these mutations in combination with mutants 

of other genes also involved in the preservation of genome stability. Our results indicate 

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that at least several members of the RecQ family have non-redundant partly even 

antagonistic functions in Arabidopsis. 

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T - 10. The Arabidopsis thaliana AtGR1/COM1 gene is essential for DNA repair and 

meiosis and relates yeastâ€s COM1/SAE2 to the human CtIP gene 

Clemens Uanschou, Tanja Siwiec, Andrea Pedrosa Harand, Claudia Kerzendorfer, Eugenio 

Sanchez-Moran, Maria Novatchkova, Svetlana Akimcheva, Franz Klein, Peter 

Schlogelhofer Dept. of Chromosome Biology, Max F. Perutz Laboratories/University of 

Vienna, A-1030 Vienna, Austria 

Meiotic recombination is initiated by DNA double strand breaks (DSBs), generated by a 

homolog of the archaebacterial topoisomerase subunit Top6A Spo11. As an intermediate 

of the DNA cleavage reaction, Spo11 is covalently linked to 5â€ termini of DNA. Release 

of Spo11 is mediated by cleaving ssDNA next to the DSB site, thereby liberating the Spo11 

protein attached to a few nucleotides. In budding yeast this release requires Rad50, 

Mre11 and Com1/Sae2. This study describes the identification and characterisation of the 

first COM1/SAE2 homologue of a higher eukaryote, the Arabidopsis thaliana 

AtGR1/COM1 gene. We provide evidence that AtGR1/COM1 is essential for male and 

female meiosis and for conferring resistance against mitomycin C (MMC) in plants. 

Mutations in the corresponding gene lead to defects in chromosome pairing, to DNA 

fragmentation and subsequently to sterile plants. We demonstrate that AtCOM1 acts 

downstream of AtSPO11-1 and upstream of AtDMC1 during meiosis. Furthermore, our 

data show that regular turn-over of AtSPO11-1 at DSB sites and DSB processing depends 

on AtGR1/COM1. Importantly, this study creates a so far unsuspected link between the 

yeast Com1/Sae2 and the mammalian CtIP protein, a DNA repair related protein, well 

conserved in higher eukaryotes. 

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T - 11. Transposon-Induced Genome Rearrangements in Maize and Arabidopsis 

Thomas Peterson, Jianbo Zhang, Chuanhe Yu, Lakshminarasimhan Krishnaswamy, David 

Weber Genetics, Development and Cell Biology, Iowa State University, 50011 Ames Iowa, 

United States 

Since their discovery by McClintock, transposable elements have been associated with the 

generation of a variety of genome rearrangements, including deletions, direct and 

inverted duplications, and translocations. In addition to providing dispersed sequence 

homologies for ectopic recombination, transposons can induce genome rearrangements 

through alternative transposition reactions that utilize the termini of different elements. 

Transposition reactions involving transposon termini in direct orientation can generate 

deletions and inverted duplications. In addition, pairs of Ac termini in reversed 

orientation can undergo transposition reactions resulting in inversions, deletions, and 

translocations. In each of these cases, the rearrangement breakpoints are bounded by the 

characteristic footprint or target site duplications typical of Ac transposition reactions. 

These results show how alternative transposition reactions could contribute significantly 

to genome evolution by generating chromosome rearrangements, and by creating new 

genes through shuffling of coding and regulatory sequences (Zhang, Zhang and Peterson, 

2006). These alternative transposition reactions were first observed in natural maize 

variants, and have now been reproduced in transgenic maize and Arabidopsis plants. The 

system utilizes transgene constructs containing maize Ac termini in direct or reversed 

orientation. The action of Ac transposase on the Ac termini generates a variety of 

rearrangements, including deletions, inversions and translocations. In these 

rearrangements, one endpoint is at the Ac termini, and the other endpoint is at another 

genomic site. This transposition-based system provides an alternative to the cre-lox 

system for genome modifications. The current state of the project will be presented. To 

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view an animation of the alternative transposition model, see 

http://jzhang.public.iastate.edu/Transposition.html. This research is supported by NSF 

awards 0450243 to T. Peterson and J. Zhang, and 0450215 to D. Weber. 

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T - 12. Genetic analysis of transformation in the moss Physcomitrella patens 

Didier G. Schaefer, Sandrine Choinard, Florence Charlot, Fabien Nogué SGAP, Station de 

génétique et amélioration des plantes, INRA, Institut National de Recherche Agronomique, 

78026 Versailles, France 

With an efficiency of gene targeting following direct gene transfer comparable to that 

observed in S.cerevisiae, the moss Physcomitrella patens is unique among multicellular 

eukaryotes. Yet the integration pattern of replacement cassette differs from that 

observed in yeast: (a) gene conversions mediated by double HR and targeted insertions 

mediated by a combination of HR and NHEJ are observed at similar frequencies and (b) 

tandem direct repeats of the transforming DNA likely generated by DNA replication are 

frequently detected at the targeted locus. Furthermore, we recently demonstrated that GT 

efficiencies in this moss is reduced ca 2 orders of magnitude following Agrobacterium 

mediated transformation, an observation that is in contrast with data from previous 

analysis of gene targeting efficiencies following direct versus Agrobacterium mediated 

gene transfer in plants and yeast. In Eukaryotes, several DNA repair pathways have been 

shown to be involved in gene targeting. To gain insight into the genetic determinants that 

control gene targeting in P.patens, we have generated loss of function mutants for the 

following genes: Rad 50, Rad 51a1, Rad 51a2, Rad 51c, Rad 51d, Rad 54, Mre11, LigIV, 

Rad1, Rad10 and Msh2. We shall present our initial analysis of the role of these genes in 

P.patens during Agrobacterium versus PEG-mediated transformation and in response to 

DNA damaging agents. 

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T - 13. RAD51 genes have different roles in Physcomitrella patens and Arabidopsis 

thaliana. 

Ulrich Markmann-Mulisch, Edelgard Wendeler, Bozena Chrost, Hans-Henning Steinbiss, 

Bernd Reiss Department Coupland, MPI für Züchtungsforschung, 50829 Köln, Germany 

The eukaryotic RecA homologue RAD51 plays an essential role in homologous 

recombination and DNA damage repair in yeast. The lethality of rad51 mutants in 

vertebrates suggests that this gene has acquired additional functions at the interface of 

recombination and cell-cycle control in some animals. However, viability and unaltered 

vegetative development in a RAD51 knockout mutant in Arabidopsis thaliana suggests 

that this additional function is absent in plants. The moss Physcomitrella patens is 

exceptional in the plant kingdom in its gene targeting efficiency, a process based on 

homologous recombination. To analyse the recombination apparatus of Physcomitrella 

and to compare a gene targeting efficient and inefficient organism, we have isolated 

RAD51 homologues of Physcomitrella. The Physcomitrella genome contains two 

duplicated and highly homologous RAD51 genes, PpRAD51A and PpRAD51B. Both genes 

were inactivated individually by gene targeting and double mutants produced by crossing 

and re-transformation. Development of the mutants and DNA damage repair was 

analysed. The PpRAD51A and PpRAD51B single mutants had a mild developmental 

phenotype. However, loss of both genes caused sterility and seberely affected vegetative 

development. This result suggests that RAD51 is important for both, vegetative growth 

and meiosis. In addition, inactivation of both genes caused hypersensitivity to DNA 

damage induced by UV and Bleomycin. This result suggests that RAD51 is important for 

somatic DNA damage repair. Supporting this conclusion, the transcription of both RAD51 

genes was induced significantly by these agents. To compare this result to Arabidopsis, 

the Atrad51 mutant was treated with the damaging agents Mitomycin C and Bleomycin. 

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Among other DNA damage, both agents induce double-strand breaks. However, 

Mitomycin C induces double-strand breaks preferentially in the replicative phase while 

Bleomycin acts independently of the phase in the cell cycle. In contrast to Physcomitrella, 

the Arabidopsis rad51 mutant barely showed hypersensitivity to bleomycin, but was 

highly sensitive to Mitomycin C. This result suggests that RAD51 in Arabidopsis has no 

essential role in the repair of somatic double-strand breaks, but is important for the 

repair of such lesions during replication. Therefore, RAD51 in Arabidopsis is likely to be 

important for post-replicative, but not for somatic DNA damage repair. These data 

suggest that Physcomitrella and Arabidopsis differ in the use of homologous 

recombination for DNA damage repair, and possibly in the role of RAD51 in development. 

Thus gene targeting efficient plants differ from inefficient plants in respect to 

recombination. This difference could be in the recombination genes themselves, their 

regulation or in the mechanisms of recombination. 

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T - 14. A functional and comparative analysis of chromatin changes associated 

with the control of reproductive development in sexual and apomictic 

plants. 

Marcelina Garcia-Aguilar, Daniel Grimanelli Plant Genome and Development Laboratory, 

Institute for research and Development (IRD), 34394 Montpellier, France 

The unique plants life strategy, with alterning sporophytic and gametophytic generations, 

requires the correct activation or repression of transcriptional programs specific to each 

developmental transition. In certain plant species, apomixis replaces sexual reproduction. 

By avoiding meiosis and fertilization, it results in the formation of clonal seeds. Apomixis 

is associated with the heterochronic expression of sexual reproductive programs, and 

therefore eventually on modifications of the control of transcriptional transitions. 

Chromatin structure and remodeling plays pivotal functions among epigenetic 

mechanisms allowing plant cells to control gene expression in a flexible way during 

development. In Arabidopsis and maize, chromatin-remodeling factors have been shown 

to be involved controlling the orderly progression of reproductive development; however, 

their potential role during female meiosis, gametogenesis and fertilization remains 

poorly understood. The overall objective of this project is to determine the function of 

chromatin dynamics in the control of reproductive differentiation between the sexual and 

apomictic pathways. This study is focuses on histone and DNA modifications to compare 

reproductive cell nuclei in the ovule of maize and apomictic maize lines. Based on 

cytological and molecular expression studies, functional analysis of candidate regulators 

of these processes are being carried out through reverse genetic, combined to RNAi 

approach using stage-specific promotors to mis-regulate expression profiles. Results 

from this project will improve our understanding of the epigenetic mechanisms 

controlling developmental transitions in flowering plants, and will also shed light on the 

molecular and cellular events leading to asexual reproduction in apomixis, an uniquely 

high-value trait for crops biotechnology. 

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T - 15. DNA Demethylation by 5-methylcitosine excision, a new mechanism of 

epigenetic reprograming in plants. 

Teresa Morales-Ruiz, Ana-Pilar Ortega-Galisteo, M. Isabel Ponferrada-Marin, M. Isabel 

Martinez-Macias, Rafael R. Ariza, Teresa Roldan-Arjona Departamento de Genetica, 

Universidad de Cordoba, 14071 Cordoba, Spain 

Methylation of cytosine at carbon 5 of the pyrimidine ring (5-meC) is a stable epigenetic 

mark for transcriptional gene silencing and plays important roles in development and 

genome defence against parasitic mobile elements. Although the enzymes responsible for 

the establishment and maintenance of DNA methylation have been well characterized, the 

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mechanisms of DNA demethylation are not well understood. We and others have found 

genetic and biochemical evidence suggesting that the Arabidopsis DNA glycosylase 

domain-containing proteins ROS1 (REPRESSOR OF SILENCING 1) and DME (DEMETER) are 

DNA demethylases. ROS1 was identified in a screen for mutants with deregulated 

expression of the repetitive RD29A-LUC transgene. Whereas in wild plants the transgene 

and the homologous endogenous gene are expressed, ros1 mutants display 

transcriptional silencing and hypermethylation of both loci. The protein encoded by ROS1 

shares a high degree of sequence similarity to the product of DME, that was 

independently identified in a search for mutations causing parent-of-origin effects on 

seed viability and is required for the expression of the maternal alleles of the imprinted 

genes MEA and FWA. ROS1 and DME catalyze the release of 5-meC from DNA by a 

glycosylase/lyase mechanism. Both enzymes also remove thymine, but not uracil, 

mismatched to guanine. DME and ROS1 show a preference for 5-meC over thymine in the 

symmetric dinucleotide CpG context, where most plant DNA methylation occurs. 

Nevertheless, they also have significant activity on both substrates at CpApG and 

asymmetric sequences, which are additional methylation targets in plant genomes. These 

findings suggest that a function of ROS1 and DME is to initiate erasure of 5-meC through 

a base excision repair process, and provide strong evidence for the existence of an active 

DNA demethylation pathway in plants. 

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T - 16. Mechanisms of Polycomb group-mediated gene silencing in Arabidopsis 

Corina Belle Villar, Grigory Makarevich, Claudia Köhler Plant Developmental Biology, ETH 

Zurich, Institute of Plant Sciences, 8092 Zurich, Switzerland 

The type I MADS-box gene PHERES1 is expressed dependent on the parent-of-origin, a 

phenomenon known as genomic imprinting. The paternal allele of PHERES1 is expressed, 

whereas the maternal allele is silenced. PHERES1 is repressed by the FIS Polycomb-group 

complex that includes MEDEA (MEA), FERTILIZATION INDEPENDENT ENDOSPERM (FIE), 

FERTILIZATION INDEPENDENT SEED 2 (FIS2),and MULTICOPY SUPPRESSOR of ira1 (MSI1). 

PHERES1 repression by the FIS complex is mediated through chromatin modification, i.e., 

histone methylation. However, our research revealed that FIS Polycomb group mediated 

repression of PHERES1 is not sufficient to establish PHERES1 imprinting. We are 

investigating the nature of the imprint and how this imprint is established and 

maintained. In particular we are looking at the role of tandem repeats as possible 

sequence elements necessary for imprinting establishment. Our progress in elucidating 

the imprinting mechanism of PHERES1 is going to be presented. 

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T - 17. Chromosome end protection in Arabidopsis 

Dorothy Shippen. Department of Biochemistry and Biophysics, College Station, Texas 

77843, USA. 

Telomeres are specialized nucleoprotein structures that facilitate the complete 

replication of chromosome ends and allow the cell to distinguish the natural 

chromosome terminus from a double-strand break. We are employing genetic and 

biochemical approaches in Arabidopsis to investigate fundamental aspects of telomere 

function and maintenance. We previously showed that telomere tracts in plants lacking 

telomerase undergo progressive shortening and after six generations suffer end-to-end 

chromosome fusions that ultimately lead to developmental and growth arrest. Currently, 

our efforts focus on the identification of proteins that provide protection for the 

chromosome terminus. Here we describe a novel Arabidopsis mutant, dubbed cit1 

(Critical for the Integrity of Telomeres), which displays severe developmental defects in 

the first generation. Telomere length in cit1 is deregulated, the single strand G-overhang 

is grossly elongated, and chromosomes are highly vulnerable to telomere fusion. Thus, 

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CIT1 appears to encode a factor crucial for distinguishing the chromosome terminus 

from a double-strand break. We also report the characterization of a family of singlestrand 

telomere binding proteins, termed POT (Protection of Telomeres). Arabidopsis is 

unusual as it encodes three highly divergent POT-like genes, which appear to represent 

separation of function alleles. Interestingly, AtPOT1 has evolved to function as a 

component of the telomerase RNP necessary for telomere maintenance in vivo. To 

further investigate the evolution of POT proteins, we obtained POT coding regions from 

over twenty organisms within the plant kingdom, ranging from green algae to the most 

advanced flowering plants. Unlike Arabidopsis, most plant genomes encode only a single 

POT protein. We are currently analyzing the wealth of information obtained from plant 

POT1 sequences with the goal of identifying residues under positive selection and their 

relationship to protein structure and function. 

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T - 18. Role of the Ku70/Ku80 heterodimer in telomere metabolism 

Barbara Zellinger, Svetlana Akimcheva, Jasna Puizina, Martina Schirato, Karel Riha , 

Austrian Academy of Sceinces, Gregor Mendel Institute, 1030 Vienna, Austria 

Telomeres distinguish natural chromosome ends from being processed (repaired) as 

deleterious DNA double strand breaks (DSBs). However, many factors required for DSB 

repair, such as Ku70/80 heterodimer, are also involved in telomere metabolism. Ku70/80 

complex is crucial component of nonhomologous end-joining (NHEJ) DNA repair pathway, 

but it is also localized to telomeres and its deficiency impairs telomere function in most 

organisms where it has been studied. We use Arabidopsis as a model system to study 

molecular mechanisms underlying Ku function at telomeres. Our initial characterization 

of the Arabidopsis KU70 gene revealed that mutants carrying a T-DNA insertion in the 

gene have much longer telomeres than wild-type plants. Subsequently, we showed that 

Ku70 acts as a negative regulator of telomerase at telomeres and at the same time, 

prevents excessive nucleolytic resection of the 5’ chromosome end [1, 2]. Although Kudeficient 

telomeres are fully functional, additional inactivation of telomerase causes 

accelerated telomere shortening and an early onset of chromosome end-to-end fusions 

and developmental defects [1]. The accelerated loss of telomeres in the absence of Ku 

could be, to a large extent, attributable to the exonucleolytic resection of the telomeric Cstrand. 

However, it could also be caused by an increased rate of aberrant homologous 

recombination resulting in telomere rapid deletion (TRD). To investigate this possibility, 

we developed a novel highly sensitive assay for assessing the level of extrachromosomal 

telomeric circles (t-circles), which are products of TRD. We found that the Ku heterodimer 

suppresses t-circle formation and intrachromatid recombination at Arabidopsis 

telomeres. Furthermore, we show that Ku prevents engagement of the telomeraseindependent 

ALT pathway for telomere maintenance. These data demonstrate that Ku 

acts as a key regulator of homologous recombination at Arabidopsis telomeres. [1] K. 

Riha and D.E. Shippen, Proc. Natl. Acad. Sci. USA 100 (2003) 611-5. [2] K. Riha, J.M. 

Watson, J. Parkey and D.E. Shippen, EMBO J. 21 (2002) 2819-26. 

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T - 19. Dynamics of telomere behaviour in Arabidopsis thaliana meioisis 

Susan Armstrong, Nicola Roberts School of Biosciences, University of Birmingham, B152TT 

Birmingham, United Kingdom 

There is growing evidence that telomeres play a key role in the initiation of chromosome 

pairing at meiosis but little is known about how this is achieved. The primary role of the 

telomere is to stabilise the ends of the eukaryotic chromosome, but it is apparent that the 

telomere also has a role in the highly conserved meiotic pathway. It has been suggested 

that the telomeres play a role in this process by aligning (pairing) the homologous 

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chromosomes preceding recombination and synapsis. Recent attention has been paid to 

the bouquet, a nearly universal event, during which the telomeres cluster in early 

prophase. It has been suggested that because the telomeres are in close proximity this 

would enhance the pairing of the homologues. We have shown in Arabidopsis that we do 

not observe a classical bouquet, rather the telomeres are organised already around the 

prophase nucleolus. The homologous telomeres are paired at the transition from G2 to 

leptotene during the assembly of the axial elements. As the chromosomes synapse during 

zygotene, the telomeres reveal only a loose clustering, which may represent a relic 

bouquet. To investigate the nature of chromosome pairing we are carrying out a 

functional analysis of pairing in recombination mutants. We have already shown in a 

range of Arabidopsis asynaptic meiotic mutants (spo11, asy1, mre11, dmc1, rad 51c) that 

the telomeres appear to be paired in early leptotene but this is not permanent and 

appears to be lost as meiotic prophase progresses. We are using FISH with unique 

chromosomal paints to find out if our observations are due purely to random 

associations or are we observing homologous telomeric pairing Our analysis will uncover 

whether initial homologous pairing requires DSB, recombination proteins MRE11, DMC1, 

RAD51C or structural meiotic components. 

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T - 20. HR versus NHEJ at the chromosomal level 

Ingo Schubert, Koichi Watanabe, Ales Pecinka Cytogenetics and Genome Analysis, Leibniz- 

Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Germany 

HR versus NHEJ at the chromosomal level Ingo Schubert, Koichi Watanabe, Ales 

Pecinka,IPK, D-06466 Gatersleben, Germany According to molecular studies, HR is the 

major mechanism for double-strand break (DSB) repair in yeast, while in somatic cells of 

vertebrates and of seed plants NHEJ is considered to be predominant. A low HR 

frequency is coincident with a low frequency of homologous gene targeting in seed 

plants. However, at least at the level of genotoxin-mediated structural rearrangements 

between freshly replicated chromatids, HR is remarkably frequent; apparently 100% of 

SCEs and up to 8-fold more chromatid-type aberrations than expected at random are the 

outcome of HR. Both phenomena can be quantified reliably only during the first mitotic 

division after the S-phase of their origin. Later such events are either not (unambiguously) 

detectable or the carrier cells are eliminated. Thus it could be possible that quite a 

proportion of HR is overlooked, the more so as it remains to be elucidated which 

proportion of the preclastogenic DSBs are processed via HR into SCE or chromatid-type 

aberrations. Therefore it is needed to determine i) the probability of DSB processing into 

the different endpoints, including restoration of the pre-break situation, and ii) how are 

the spatial requirements for HR achieved. As a prerequisite to address the first aim, a 

screening system to monitor the induction of DSBs per nucleus at a specific locus is being 

established. In chromatin mutants with a hyper-recombination phenotype and an up to 

100-fold increase of intrachromosomal somatic HR (in cis), chromosome territory 

arrangement and homologous pairing frequencies are not significantly altered. Contrarily, 

clastogenic doses of X-rays cause a significant increase of the positional homologous 

pairing frequencies in A.thaliana that decays during 1h after irradiation. These data 

indicate that for homologous intrachromatid recombination chromatin relaxation might 

be sufficient, while interchromatid recombination likely requires an active search for 

homology. 

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T - 21. AtRad1/AtErcc1 endonuclease and telomere stability in Arabidopsis. 

Jean-Baptiste Vannier, Annie Depeiges, Charles White, Maria Eugenia Gallego CNRS UMR 

6547, Universite Blaise Pascal, 63177 Aubiere, France 

Telomeres are the specific chromatin structures present at the ends of chromosomes 

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serving to avoid chromosomal shortening at replication and recombination. Proteins 

known to be involved in DNA repair and recombination have been found associated to 

telomeres, however, their specific roles in the maintenance of functional chromosome 

ends are poorly understood. The XPF/Ercc1 complex, a structure-specific endonuclease 

that acts in nucleotide excision repair and recombination, has been shown to be 

associated to telomeres. Arabidopsis plants mutated for either Xpf (AtRad1) or Ercc1 

(AtErcc1) orthologs develop normally and show wild-type telomere length. However in the 

absence of telomerase, mutation of either of these two genes induces an earlier onset of 

chromosomal instability. End-to-end chromosome fusions are detected at generation 1 as 

compared to generation 5 in telomerase mutant plants. Furthermore a significantly 

higher number of anaphase bridges per cell are observed in telomerase mutant plants 

lacking either AtRad1 or AtErcc1 as compared to single telomerase mutants. Plants 

showing fertility defects are observed at generation 2 both in AtRad1 and AtErcc1 

telomerase double mutants. This early appeareance of uncapped telomeres is not related 

to a general acceleration of telomeric repeat loss. Fluorescent in situ hybridization show 

the presence of extrachromosomal DNA fragments, containing both subtelomeric and 

telomeric repeat signals, which are not observed in telomerase single mutant plants 

presenting a similar numbers of anaphase bridges. Anaphase bridges present in nuclei 

with extrachromosomal fragments never show both subtelomeric and telomeric signals. 

These data and the possible roles of AtRad1/AtErcc1 in Arabidopsis telomere stability 

will be discussed. 

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T - 22. A role of ATM in telomere length regulation in Arabidopsis 

Laurent Vespa, Ross Warrington, Jiri Siroky, Petr Mokros, Dorothy Shippen Biochemistry 

and Biophysics, Texas A&M University, TX 77840 College Station, United States 

Telomeres are the indispensable nucleoprotein complexes that protect linear 

chromosomes from replicative attrition and from the DNA damage response machinery. 

Without them, chromosome ends would be recognized as double-strand breaks and fused 

to each other, leading to deleterious chromosomal rearrangements. This function has led 

to investigations of the relationship between telomeres and the DNA damage response 

machinery. Paradoxically, several of these factors have an essential role in the physical 

integrity of telomeres. We have taken advantage of the outstanding resistance of 

Arabidopsis to genomic instability to study the role of several DNA damage response 

factors at telomeres, including the central sensor/activator protein kinase ATM. We have 

shown that a combined deficiency in ATM and TERT, the reverse transcriptase subunit of 

telomerase, leads to a premature onset of telomere uncapping. However the nature of the 

fusion events in atm/tert are different from the single tert mutants. Our telomere-fusion 

amplification assay mostly failed to detect products in the double atm tert mutant, while 

cytogenetic experiments showed very high number of anaphase bridges, a hallmark for 

chromosome fusions. Therefore, we have characterized telomere fusions arising in atm 

tert mutants by FISH analysis using telomeric probes and an extensive series of 

subtelomeric BAC probes. Our results indicate that one particular chromosome end is 

involved in most of the fusions. We also revealed that about 40% of the fusions in atm 

tert (versus ~5% in tert) do not involve chromosome ends, which suggests that ATM may 

prevent multiple breakage-fusion-breakage events as a cell cycle checkpoint controller. A 

precise telomere length analysis of individual chromosome ends was then performed by 

PETRA (Primer Extension Telomere Repeat Amplification). These data showed that the 

chromosome end involved in the fusions was much shorter than its counterparts, and 

that this shortening event happened in early generations (G2-G3). This finding suggests 

that a particularly large Telomere Rapid Deletion (TRD) event occurred in the double 

mutant. We then addressed the putative role of ATM in controlling TRD by performing 

parent/progeny PETRA analysis on a large number of tert and atm tert plants. We found 

that large size telomeres are more prone to TRD in the double mutant, which can lead to 

the stochastic generation of critically shortened telomeres. We conclude that ATM has a 

role in preventing TRD or in preventing cells that have undergone large deletions from 

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eing propagated. 


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T - 23. Mapping of interaction domains of putative plant telomere proteins 

Milan Kuchar, Petra Schrumpfova, Iva Santruckova, Jan Palecek, Ctirad Hofr, Jiri 

Fajkus Functional Genomics and Proteomics, Masaryk University and Institute of Biophysics 

ASCR, 625 00 Brno, Czech Republic 

Although a number of candidate proteins have been identified by homology searches in 

plant genome databases and tested for their affinity to telomeric DNA sequences in vitro, 

data relevant to their telomeric function are mostly missing. Recently we reported on 

interactions among three myb-like proteins of the single myb histone (Smh) family 

AtTRB2, AtTRB3 binding telomeric dsDNA in vitro and AtTRB1, and on interaction of 

AtTRB1 with AtPo1-2 that binds G-rich strand of telomeric DNA strand by 

oligonucleotide-binding (OB)-fold and is involved in telomere length regulation. The Smh 

family has been described to bear a unique triple motif structure containing N-terminal 

myb-like domain, central GH1/GH5 histone globular domain and C-terminal coiled-coil 

domain. We report here on mapping of protein domains responsible for these 

interactions using the yeast two-hybrid system and independent biophysical techniques 

like surface plasmon resonance. Surprisingly, the domain responsible for the interaction 

of AtPot1-2 with AtTRB1 has been mapped to the region of N-terminal OB-fold domain. 

Protein AtTRB1 uses the central GH1/GH5 histone globular domain in interactions with 

AtTRB2, AtTRB3, AtPot1-2, AtTRP1 and with itself. Its C-terminal coiled-coil domain is 

involved in the interactions with AtTRB2, AtTRB3 and itself and increases interaction 

stability. Our research is supported by Grant Agency of the Czech Republic (521/050055), 

Grant Agency of the Czech Acad. Sci (IAA600040505), Ministry of Education (LC LC06004) 

and institutional funding (MSM0021622415, AVOZ50040507) 

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T - 24. Positional sister chromatid alignment in a mutant of the Arabidopsis 

homolog of STRUCTURAL MAINTENANCE of CHROMOSOME 6 (SMC6). 

Koichi Watanabe, Andrea WeiÃŸleder, Michael Florian Mette, Veit Schubert, Ingo Schubert , 

Leibniz Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, 

Germany 

DNA double-strand breaks (DSBs) can be repaired either by non-homologous end-joining 

(NHEJ) or by homologous recombination (HR). Most cells of a plant are not proliferating 

or they perform endocycles omitting G2 and mitosis. It is not yet clear how DSBs are 

repaired in plant cells during endocycle. To better understand the DSBs repair mechanism 

in endopolyploid plant cells, we examined sister chromatid alignment in Arabidopsis 

thaliana nuclei after DSBs formation. Since the physical proximity between the donor and 

acceptor strands is critical for the DNA strand exchange process during HR, it is possible 

that alignment of homologous or sister chromatids is a prerequisite for DSB repair via 

HR. FISH experiments indicated an increased frequency of positional sister chromatid 

alignment in wild type nuclei of 4C or 8C DNA content immediately after X ray irradiation 

compared to unirradiated cells. Sister chromatid alignment is considered to be mediated 

by the cohesin complex. One of the SMC proteins, SMC6, is involved in DNA repair and 

required to recruit the cohesin complex to DSB sites. To examine whether the fluctuation 

in the frequency of positional sister chromatid alignment observed in irradiated plants 

depends on recruitment of cohesin, we compare sister chromatid alignment frequencies 

between wild-type and mutants of the Arabidopsis homolog of SMC6. 

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T - 25. Mutations Affecting Meiotic Recombination Rate in Maize 

Sanzhen Liu, Stephen Baluch, Mitzi Wilkening, Alice Eggleston, An-Ping Hsia, Hugo Dooner, 

William Eggleston, Patrick Schnable, Clifford Weil Dept of Agronomy, Purdue University, 

47907-2054 West Lafayette, United States 

1 Iowa State University, Ames, IA, 2 Purdue University, West Lafayette, IN 3 Virginia 

Commonwealth University, Richmond, VA 4 Waksman Institute, Piscataway, NJ The 

mechanisms and genetic regulation of meiotic recombination are still not wellunderstood. 

Although great progress has been made in other model systems, the 

underlying genes of recombination and the control of these steps remain elusive in 

plants, including maize. We have used EMS mutagenesis on two differentially marked 

maize populations to identify mutations that significantly alter recombination (p < .05). 

Using two different crossing schemes and testcross assays that eliminate meiotic defects 

resulting in sterility, we monitored crossovers in the C1-Wx1 interval of chromosome 9S 

and the A1-Et1 interval of chromosome 3L. Thus far, the two screens have produced 56 

candidate mutations segregating in M2 and M3 families (30 from the 9S screen and 26 

from the 3L screen), including 39 putative mutations that significantly increase 

recombination, seven that significantly decrease recombination, and four families in 

which both a significant increase and a significant decrease in recombination frequency 

over the same interval segregate in the same family. In addition, nine families segregate 

for an increased frequency of double crossovers (rarely observed in maize, where 

crossover interference is extremely high); four of these correspond to mutants with a 

general increase in recombination, suggesting the other mutants in this class may be 

defective for genes involved in enforcing crossover interference. 

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T - 26. A key role for AtNBS1 in plant cell responses to DNA double strand breaks 

Christopher E. West, Wanda M Waterworth, Cagla Altun, Susan J Armstrong, Nicola 

Roberts, Philip J Dean, Kim Young, Clifford F Weill, Clifford M Bray Centre for Plant 

Sciences, University of Leeds, LS2 9JT Leeds, United Kingdom 

Centre for Plant Sciences, University of Leeds, Leeds, LS2 9JT, UK; †Faculty of Life 

Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, UK; ‡Plant 

Biology Program and §Dept. of Agronomy, Purdue University, West Lafayette IN 47907 

USA; School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK DNA 

double strand breaks (DSBs) in genomic DNA are caused by a combination of 

environmental and endogenous factors including irradiation and reactive metabolites. 

Inability to correctly repair DSBs can result in the loss of large amounts of genetic 

information and mutagenesis. However, programmed DSBs play an important role during 

meiosis in initiating recombination between homologous chromosomes. In most 

Arabidopsis cells the major pathway for DSB repair is non-homologous end joining 

(NHEJ), a process involving the protein complexes AtKU80/AtKU70 and AtLIG4/XRCC4. A 

multifunctional protein complex of MRE11, RAD50 and NBS1 is involved in both 

homologous recombination (HR) and NHEJ pathways. Arabidopsis atmre11 and atrad50 

mutant plants are sterile, reflecting the essential role that these proteins play in meiotic 

HR. In addition, these mutants displayed hypersensitivity to DNA damaging reagents 

including methyl-methanesulfonate indicating a probable role for this complex in NHEJ in 

vegetative tissues. Here we report a functional characterisation of AtNBS1 and find that it 

forms part of the Arabidopsis MRN complex and has a role in the DNA damage response 

in plant vegetative tissues. In addition, phenotypic analysis shows that atnbs1 deficient 

plants are fully fertile suggesting that AtNBS1 may be dispensable for meiosis, in contrast 

to AtMRE11 and AtRAD50. However, AtNBS1 is essential for the residual fertility in plants 

lacking AtATM, consistent with roles of AtNBS1 independent of AtATM signalling. 

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T - 27. Meiotic prophase chromosome architecture revealed by ultrahigh resolution 

structured illumination (SI) microscopy 

W.Z. Cande, Chung-Ju Rachel Wang, Pete Carlton, John Sadat Dept of Molecular and Cell 

biology, Univeersity of California, Berkeley, 94720-3200 Berkeley, CA, United States 

1 Department of Molecular and Cell Biology, University of California at Berkeley 2 

Department of Biochemistry and Biophysics, University of California, San Francisco An 

integrative view of meiotic prophase chromosome structure and function has been 

difficult to come by. Meiotic chromosomes are large, complex structures that can be tens 

of microns in length, yet the size of many structural elements of interest such as axial 

element organization during synapsis is just beyond the resolution of the conventional 

wide field microscopy. To overcome these limitations we have used structured 

illumination (SI) microscopy with a resolution less than 100 nm in the XY and Z axis 

(compared to 250 nm in the Xy and 500 nm in the Z axis by conventional light 

microscopy) to analyze the architecture of the pachytene chromosome. During leptotene, 

each chromosome develops a linear proteinaceous structure called an axial element (AE). 

At that time, recombination and the homology search are initiated by the formation of 

double strand breaks. In zygotene, homologous chromosomes synapse via the 

polymerization of a central element between the two homologous AEs, forming the 

synaptonemal complex (SC). During pachytene, synapsis and recombination are 

completed. We have analyzed chromomere and chromatin organization by using 

antibodies to monitor the distribution of histone modifications associated with 

heterochromatin (H3K9 dimethyl) and euchromatin (H3K9 acetylation, H3K4 dimethyl) 

and analyzed axial element organization by monitoring the distribution of AFD1 (a rec8 

homolog) and HOP1. Chromomeres of paired chromosomes are bilaterally symetrical and 

have a variable left handed helical pitch. Chromosome and axial element organization 

differ at centromeres. AFD1 and HOP1 are distributed as alternating islands of variable 

morphology along the axial element and display a bilaterial symmetry on the paired axes. 

Unsynapsed regions at late zygotene are always associated with interlocks suggesting 

that resolution of interlocks between chromosomes may be a rate limiting step to 

complete synapsis. We also found evidence for a process of pre-synaptic adjustment in 

unsynapsed regions of zygotene chromosomes, as the homologs in the unsynapsed 

regions differ in length. 

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T - 28. Ph1 in wheat-its not size but coordination that matters 

Graham Moore Crop Genetics, John Innes Centre, NR4 7UH Norwich, United Kingdom 

Hexaploid wheat (Triticum aestivum 2n=6x=42) possesses three ancestral genomes or 7 

sets of six related chromosomes. For hexpaloid wheat to be fertile, true homologues must 

pair amongst the 6 related chromosomes during meiosis. The Ph1 locus controls this 

pairing mechanism. We have recently defined the Ph1 locus to a cdk-like gene complex 

containing a segment of heterochromatin. This gene complex is related to the IME2 and 

cdk2 meiotic checkpoint genes of yeast and humans involved in initiating meiosis. Cell 

biological studies reveal that the Ph1 locus triggers the initiation and coordination of 

chromatin remodelling required to enable chromosomes to become competent to pair 

and recombine during meiosis. However unlike many species, it seems that the presence 

of Ph1 in wheat links this chromatin remodelling to homology 

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T - 29. A new pollen-based meiotic recombination assay. 

Kirk Francis, Sandy Lam, Alexandra Bey, Luke Berchowitz, Gregory Copenhaver BASF 

Plant Sciences, BASF, 27709 Research Triangle Park, United States 

Meiotic recombination, in the form of crossovers (CO) and gene conversions (GC), is a 

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highly conserved process. Recombination helps ensure chromosome segregation and 

promotes allelic diversity. Defects in the recombination machinery are often catastrophic 

for meiosis and may result in sterility. To facilitate investigation of the critical processes 

relating to recombination we have developed an Arabidopsis-based visual assay capable 

of detecting crossovers, crossover interference, and gene conversion events. The assay 

utilizes transgene constructs encoding three distinct pollen-expressed fluorescent 

proteins in the tetrad-producing qrt mutant background. By observing the segregation of 

the fluorescent alleles in pollen tetrads we demonstrate (i) a correlation between 

developmental position and CO frequency, (ii) a temperature dependence for CO 

frequency, (iii) the ability to detect meiotic GC events and (iv) the ability to rapidly asses 

CO interference. Additional applications of this system to identify and evaluate 

developmental, environmental, and genetic factors that influence meiotic recombination 

will be discussed. 

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T - 30. Co-ordination of meiotic recombination by ASY1 in Arabidopsis thalian 

F. Chris. H. Franklin Eugenio Sanchez-Moran, Gareth H. Jones . School of Biosciences, 

University of Birmingham, Birmingham B15 2TT UK 

We are investigating the formation of meiotic crossovers (COs) in Arabidopsis and how 

components of the chromosome axes and synaptonemal complex (SC) interface with the 

recombination machinery to influence this process. Here we focus on a recent study 

aimed at understanding the role of ASY1 during the early stages of the meiotic 

recombination pathway. This analysis has provided new insight into the role of ASY1, the 

formation of DNA double-strand breaks (DSBs) and the co-ordination of strand-invasion. 

Meiotic recombination is initiated by the formation of AtSPO11-1 catalysed DSBs. DSB 

formation is coincident with the formation of the chromosome axes which occurs ~2h 

after initial AtSPO11-1 localization. ASY1 is a HORMA domain protein that is initially 

detected in G2 as chromatin-localized foci that associate with the nascent chromosome 

axes at late G2/early leptotene to form a linear signal. Loss of ASY1 results in asynapsis 

and a dramatic reduction in chiasma/CO formation leading to the presence of univalent 

chromosomes at metaphase I. Studies indicate that DSB formation is normal in an asy1 

mutant. In wild-type, localization of the strand exchange proteins AtRAD51 and AtDMC1 

to the chromatin occurs asynchronously, shortly after DSB formation with AtDMC1 

localizing in advance of AtRAD51. Both recombinases form numerous foci that persist for 

~12h before gradually decreasing in number. In asy1, initial localization of AtDMC1 is 

normal, but declines abruptly such that inter-homolog recombination is severely 

compromised. As a result virtually all DSB repair proceeds via AtRAD51 mediated intersister 

recombination. Limited ASY1-independent, AtDMC1-dependent inter-homolog 

recombination remains, but is restricted to sub-telomeric sequences where the homologs 

are fortuitously in register due to nucleolus-associated telomere clustering. We propose 

that the meiotic bias in favour of inter-homolog recombination is established by an 

asynchrony in the expression of the recombinases together with ASY1-mediated 

repression of AtRAD51 during the early stages of strand invasion. In the absence of ASY1 

AtRAD51 is able to displace AtDMC1 such that inter-sister chromatid repair 

predominates with a subsequent loss of CO formation and synapsis. 

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T - 31. MLH1 marks a subset of strongly interefering crossovers in tomato 

Franck Lhuissier, Hildo Offenberg, Peter Wittich, Norbert Vischer, Christa 

Heyting Upstream research, Keygene (company), 6700AE Wageningen, Netherlands 

In most eukaryotes, the prospective chromosomal positions of meiotic crossovers are 

marked during meiotic prophase by protein complexes called late recombination nodules 

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(LNs). In tomato (Solanum lycopersicum), a cytological recombination map has been 

constructed based on LN positions. We demonstrate that the mismatch repair protein 

MLH1 occurs in LNs. We determined the positions of MLH1 foci along the 12 tomato 

bivalents during meiotic prophase and compared the map of MLH1 focus positions with 

that of LN positions. On all 12 bivalents, the number of MLH1 foci was app. 70% of the 

number of LNs. Bivalents with zero MLH1 foci were rare, which argues against random 

failure of detecting MLH1 in the LNs. We inferred that there are two types of LNs, MLH1positive 

and MLH1-negative LNs, and that each bivalent gets an obligate MLH1-positive 

LN. The two LN types are differently distributed along the bivalents. Furthermore, 

cytological interference among MLH1 foci was much stronger than interference among 

LNs, implying that MLH1 marks the positions of a subset of strongly interfering 

crossovers. Based on the distances between MLH1 foci or LNs, we propose that MLH1positive 

and MLH1-negative LNs stem from the same population of weakly interfering 

precursors. 

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T - 32. Identification of meiotic DSB forming proteins in Arabidopsis thaliana 

Arnaud De Muyt, Lucie Pereira, Daniel Vezon, Ghislaine Gendrot, Mathilde Grelon Genetic 

and Plant Breeding, INRA, 78026 Cedex Versailles, France 

In budding yeast, meiotic recombination events are initiated by double strand breaks 

(DSBs) catalysed by the Spo11 protein. In this species, DSB formation also requires nine 

other proteins whose molecular function is poorly understood. Nevertheless, recent 

studies tend to group them into subcomplexes, indicating that they can be intimately 

linked and can form a large recombination initiation complex in which DSBs are made. 

Many investigations have been carried out to identify in other organisms, homologs of 

these DSB forming proteins. Unfortunately, unlike Spo11, most of these are not conserved 

across kingdoms. Furthermore, even when DSB proteins are conserved, their role in 

meiotic DSB formation is missing. A large-scale forward genetic approach to isolate plant 

meiotic genes has revealed the existence of new meiotic functions that could be 

necessary for meiotic DSB formation. We will present data concerning the isolation of one 

of these genes : AtPRD1 for Arabidopsis thaliana Putative Recombination initiation Defect 

1. In Atprd1 mutants, meiotic recombination rates fall dramatically, early recombination 

markers (e.g. DMC1 foci) are absent, but meiosis progresses until achiasmatic univalents 

are formed. Besides, Atprd1 mutants suppress DSB repair defects of a large range of 

meiotic mutants, showing that AtPRD1 is involved in meiotic recombination and is 

required for meiotic DSB formation. Furthermore, we showed that AtPRD1 and AtSPO11-1 

interact in a yeast two hybrid assay, suggesting that AtPRD1 is a partner of AtSPO11-1. 

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T - 33. Recombination and genetic maps in Petunia 

tom gerats IWWR/Plant Genetics, Radboud University, 6525ED Nijmegen, Netherlands 

Most Petunia species have 2n=14 chromosomes; hybrids between these species are easy 

to obtain. Despite this genetic maps are rather enigmatic and highly variable in length, 

with varying clusters of no recombination, depending on the parental lines used in the 

specific cross. A gene has been defined that modulates meiotic recombination 

frequencies. Moreover, it is shown that chromosome structure in itself can modulate 

recombination frequencies: increasing length of deletions in the short arm of 

chromosome six induce increased recombination between two markers on the tip of the 

long arm. Deletion of the complete arm leads to the markers behaving independently, 

while restoration to a complete chromosome leads to the original situation: the markers 

behave as nearly completely linked. 

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T - 34. Modulation of the recombination machinary for gene targeting 

Paul Hooykaas Molecular and Developmental Genetics, Leiden University /Institute of 

Biology Leiden (IBL), 2333AL Leiden, Netherlands 

Transgenes ,introduced into eukaryotic cells by a variety of methods, are best maintained 

by integration into one of the host chromosomes. The molecular mechanisms underlying 

DNA integration and the enzymes involved are only partially known. It seems that 

depending on the host either the enzymes involved in homologous recombiation(HR) are 

preferably used or the enzymes mediating non-homologous end-joining(NHEJ). In yeasts 

and fungi genes are almost exclusively integrated by homologous recombination after 

disruption of the NHEJ-genes, and vice versa after deletion of the HR-genes DNAintegration 

occurs by NHEJ. In plants (and animal cells) the enzymatic machinery is more 

complex as after inactivation of the NHEJ-genes DNA-integration still occurs 

predominantly by non-homologous recombination. Alternative methods to promote gene 

targeting in these organisms will be discussed. 

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T - 35. Gene Targeting by Homologous Recombination in Rice 

Rie Terada, Yasuyo Johzuka-Hisatomi, Takaki Yamauchi, Shigeru Iida Division of 

Molecular Genetics, National Institute for Basic Biology, 444-8585 Okazaki, Japan 

Gene Targeting by Homologous Recombination in Rice Rie Terada1, Yasuyo Johzuka- 

Hisatomi1, Takaki Yamauchi1,2 and Shigeru Iida1 1National Institute for Basic Biology, 

Okazaki 444-8585, Japan 2Faculty of Horticulture, Chiba University, Chiba 271-8510, 

Japan. The modification of an endogenous gene into a designed sequence by homologous 

recombination, termed gene targeting, has broad implications for basic and applied 

research. Rice (Oryza sativa L.), with a sequenced genome of 389 Mb, is one of the most 

important crops and a model plant for cereals, and the single-copy gene Waxy on 

chromosome 6 has been disrupted with a frequency of 1% per surviving callus by gene 

targeting using a strong positive-negative selection. Since the strategy is independent of 

gene-specific selection or screening, it is in principle applicable to any gene. However, a 

gene in the multigene family or a gene carrying repetitive sequences may preclude 

efficient homologous recombination-promoted gene targeting due to the occurrence of 

ectopic recombination. Here, we describe an improved gene targeting procedure whereby 

we obtained nine independent transformed calli having the Adh2 gene for alcohol 

dehydrogenase 2 disrupted with a frequency of approximately 2% per surviving callus 

and subsequently isolated eight fertile transgenic plants without the concomitant 

occurrence of undesirable ectopic events, even though the rice genome carries four Adh 

genes, including a newly characterized Adh3 gene, and a copy of highly repetitive 

retroelements is present adjacent to the Adh2 gene. By analyzing the surviving calli 

containing randomly integrated and truncated T-DNA segments that carry a positive 

selection marker without intact negative markers, we can speculate certain aspects of T- 

DNA integration processes leading to homologous and nonhomologous recombination 

events in gene targeting with positive-negative selection. In addition to generating gene 

knockout rice plants, we are attempting to generate transgenic rice plants carrying 

knockin modification at target genes. 

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T - 36. Gene targeting in plants using zinc finger nucleases 

David Wright, Jeff Townsend, Ronnie Winfrey, Fengli Fu, Jeffry Sander, Drena Dobbs, Keith 

Joung, Dan Voytas Dept. of Genetics, Development & Cell Biology, Iowa State University, 

50011 Ames, United States 

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Engineered zinc-finger nucleases can stimulate high frequency gene targeting at specific 

genomic loci in plant cells (Plant Journal, 44:693). ZFNs consist of a Cys2His2 zinc finger 

domain engineered to bind a particular gene sequence and the non-specific nuclease 

domain of the FokI restriction enzyme. These artificial proteins introduce doublestranded 

breaks at specific DNA sequences and thereby vastly increase the rate of 

homologous recombination at the cleaved locus. ZFN-mediated gene targeting has great 

potential for applications in both basic and applied plant biology; however, the general 

application of this promising technology depends critically on the ability to design zinc 

finger domains targeted to any desired DNA sequence. To address this need, the Zinc 

Finger Consortium was established to promote continued research and development of 

engineered zinc finger technology (www.zincfingers.org). In initial work, the Consortium 

developed a unified, robust, and user-friendly zinc finger engineering platform. A 

comprehensive archive of plasmids was created that encode more than 140 wellcharacterized 

zinc-finger modules together with complementary web-based software 

(termed ZiFiT) for identifying potential zinc-finger target sites in a gene of interest. The 

Consortium also developed protocols for rapidly testing the DNA-binding activities of 

assembled multi-finger arrays in bacterial and yeast cell-based reporter assays as well as 

vectors for the expression of zinc finger nucleases in plants. 

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T - 37. Stimulating Gene Targeting into the Arabidopsis genome 

Hezi Shaked, Cathy Melamed-Bessudo, Michal Lieberman-Lazarovich, Aviva Samach, 

Naomi Avivi-Ragolsky, Yonat Eshchar, Avraham Levy Plant Sciences, Weizmann Institute of 

Sciences, 76100 Rehovot, Israel 

Gene targeting, the homologous recombination-mediated integration of an extrachromosomal 

DNA segment into a chromosomal target sequence, enables the precise 

disruption or replacement of any gene. Despite its importance, gene targeting remains 

inefficient in higher plants and in several eukaryotic species. In species such as chicken, 

mouse and yeast, earlier reports have shown that the chromatin remodeling activity of 

Rad54-like proteins is important for efficient gene targeting. We have developed a new 

high-throughput assay, based on the use of a green fluorescent seed marker, to study the 

effect of chromatin remodeling on gene targeting in plants. We report that expression of 

the yeast RAD54 gene enhances gene targeting in Arabidopsis by one to two orders of 

magnitude, from 10-4 to 10-3 in the wildtype plants to 10-2 to 10-1. These findings 

suggest that chromatin remodeling is rate-limiting for gene targeting in plants and 

improve the prospects for using gene targeting for the precise modification of plant 

genomes. We describe the structure of both precise gene replacement events and of 

ectopic targeting events. In addition we have developed a new gene targeting assay, based 

on the use of a red fluorescent seed marker. This assay, combined with the precise 

expression of additional recombination-enhancing proteins in the target cells will be used 

for further optimization of the targeting process. 

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T - 38. Mutation scanning by massive parallel sequencing 

Paul Bundock, Rene Hogers, Antoine Janssen, Diana Rigola, Harrie Schneiders, Nathalie 

van Orsouw, Hein van der Poel, Michiel van Eijk, Michiel de Both Upstream Research, 

Keygene N.V., 6700 AE Wageningen, Netherlands 

In order to rapidly screen mutant libraries for the detection of mutations in specific 

genes we utilize massive parallel sequencing. We apply a 3D pooling strategy on DNA of 

EMS mutagenized plants followed by amplification of the target gene with PCR primers 

carrying a pool identifier tag specific to each of the 3D pools. PCR products undergo ultra 

deep sequencing using the automated pyrosequencer GS20. Mutations in the gene of 

interest are observed at a much higher frequency than random sequencing errors. 

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Bioinformatics analysis allows the idenification of the mutant pool and the corresponding 

mutant is then retrieved. In addition, the sequence around the mutation is directly 

available for further analysis and assay development, which is not the case with common 

CEL-1 based TILLING methods. Using this approach a complete mutant library can be 

screened efficiently for mutations in a specific locus in one straightforward experiment. 

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T - 39. Application of Designed Zinc-Finger Protein Technology in Plants 

Vipula Shukla, Teresa Bauer, Nicole Arnold, Jon Mitchell, Matt Simpson, Sarah Worden, 

Fyodor Urnov, Jeffrey Miller, Jeremy Rock, Erica Moehle, Yannick Doyon, Lei 

Zhang Discovery R&D, Dow AgroSciences, LLC, 46268 Indianapolis, IN, United States 

The primary sequence of the genome at endogenous loci can be altered with high 

efficiency in mammalian cells using designed zinc finger nucleases (ZFNs; Urnov et al. 

Nature 435: 646). Ongoing studies indicate that a precisely-placed double-strand break 

(DSB) induced by engineered ZFNs can stimulate integration of long DNA stretches into a 

predetermined genomic location in human cells, resulting in site-specific gene addition. 

Zinc finger protein technology represents a significant breakthrough relative to the ability 

to edit and engineer genomes in a precise manner. In this presentation, results from a 

collaboration between Dow AgroSciences LLC and Sangamo Biosciences that is focused on 

applications of designed zinc-finger protein technology in plant species will be described. 

Multiple zinc-finger proteins, including zinc-finger nucleases and zinc-finger 

transcription factors, have been designed to target specific genes in model and 

agriculturally important plant species. Validation of this technology and examples of its 

utility for plant biotechnology will be discussed. 

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T - 40. Development of a gene targeting system based on in planta presentation of 

homologous donor DNA during meiosis. 

Frederic Van Ex, Dimitri Verweire, Kristof Verleyen, Koen Peeters, Eric Dewale, Geert 

Angenon Laboratory of Plant Genetics, Vrije Universiteit Brussel (V.U.B.), 1050 Brussels, 

Belgium 

Keywords: Gene Targeting/Homologous recombination/Cre-Lox/meiosis Currently, we 

are developing a system that allows efficient gene targeting in Arabidopsis thaliana. The 

outline of our approach is based on two main features: 1. the intrinsic characteristics – in 

regard to homologous recombination – of cells during meiosis 2. the in planta 

presentation of homologous donor DNA in the aforementioned cells Our current assay is 

based on the restoration of a GUS:nptII gene, defective for kanamycin resistance. Target 

lines were created by introducing such a defective GUS:nptII reporter gene (containing a 

3’ nptII deletion) under control of a 35S promoter. Two separate target lines – both 

homozygous for a single copy of the defective GUS:nptII reporter gene – were selected. A 

targeting vector – carrying a lox cassette containing a promoterless gus:NPTII reporter 

gene (having a 5’ gus deletion) and a downstream 5000 bp stretch of sequence 

homologous to the target sequence – was introduced in the two target lines. In the final 

step of our approach a Cre gene under control of a promoter active in the prophase I of 

the meiosis (promoter of the A. thaliana Solo Dancers (SDS) gene, AT1G14750, Azumi et 

al., 2002) was introduced into the target targeting lines. Placing the expression of the Cre 

gene under control of the SDS promoter allows the induction of the Cre/lox 

recombination during the meiotic prophase I. The Cre/lox recombination will result in the 

formation of a circular donor DNA that can be presented for homologous recombination 

with the target sequence. Homologous recombination between both gus:NPTII reporter 

genes will lead to repair of the target GUS:nptII reporter gene (controlled by a 35S 

promoter) resulting in kanamycin resistance. First results show that in planta 

presentation of homologous donor DNA can lead to homologous recombination. 

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Extensive analysis will identify the nature of the observed recombination events in order 

to discriminate between ‘true’ and ectopic gene targeting events. We recently extended 

the strategy to different promoters allowing activation of our gene targeting system in 

other specific cell types where homologous recombination is suspected to be the 

predominant recombination mechanism. 

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T - 41. Meganucleases with tailored cleavage specificity can induce efficient 

homologous gene targeting 

Sylvain Arnould, Jean-Pierre Cabaniols, Philippe Duchateau, Aymeric Duclert, Jean-Charles 

Epinat, Agnès Gouble, Sylvestre Grizot, Christophe Perez, Julianne Smith, Frédéric 

Pâques Scientific Department, Cellectis S.A., 93 235 Romainville, France 

Homologous gene targeting is the best way to modify a genome in a precise and rational 

way, but has proved to be very inefficient in plants. This limit can be alleviated by 

meganucleases, sequence-specific endonucleases recognizing large (>12 bp) cleavage 

sites. These proteins can stimulate homologous gene targeting by a 1000-fold factor or 

induce mutagenesis in the vicinity of their target site, and these findings have opened 

novel perspectives for genome engineering in plants. However, the use of this technology 

has long been limited by the repertoire of natural meganucleases: the probability of 

finding a sequence cleaved by a natural meganuclease in a chosen gene is extremely low. 

Therefore, the design of artificial endonucleases with chosen specificities is under intense 

investigation. Given their exceptional specificity, natural meganucleases should provide 

ideal scaffolds to derive genome engineering tools.We have developed a combinatorial 

approach to redesign the DNA-binding interface of I-CreI, a Chlamydomonas reinhardti 

protein belonging to the LAGLIDADG family of meganucleases. First, we collect large 

numbers of locally engineered I-CreI derivatives with altered specificity. Second, we 

assemble these mutants into entirely redesigned endonucleases binding a priori chosen 

targets. The engineered proteins keep the essential properties of natural meganucleases 

in terms of folding, activity and specificity, and can be used to induce recombination and 

site directed mutagenesis in chromosomal sequences. Thus, our strategy provides the 

means to engineer a very large number of sequences in a very specific way. We will 

illustrate both the protein engineering and genome engineering steps. 

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T - 42. Toward zinc finger nucleases-mediated gene targeting in plants 

Andriy Tovkach, Vardit Zeevi, Tzvi Tzfira Department of Molecular, Cellular, & 

Developmental Biology, University of Michigan, 48109-1048 Ann Arbor, MI, United States 

Double-strand breaks (DSBs) in plant genomes are typically repaired by the plant nonhomologous 

end-joining (NHEJ) machinery, which usually leads to local mutagenesis due 

to small deletions at the repair site. We have shown that double stranded T-DNA 

molecules preferentially integrate into DSBs in transgenic plants carrying an I-SceI 

endonuclease recognition site which, upon cleavage with I-SceI, generates a DSB. In 

addition, using AscI, an 8 base cutter recognizes ca. 80 sites in the Arabidopsis genome 

we discovered that DSBs acts as attractants of T-DNA molecules at different genomic 

locations. This phenomenon could potentially be used for targeting foreign DNA 

molecules into specific sites in the plant genome using zinc finger nucleases (ZFNs). ZFNs 

are a new type of artificial restriction enzymes which are custom-designed to recognize 

and cleave specific DNA sequences, producing DSBs. However, technical difficulties in the 

design, assembly and analysis of ZFNs have hindered the use of this new technology for 

plant gene targeting. We have recently designed a set of constructs and cloning, 

biochemical and in-planta analysis procedures for the newly designed ZFNs. Cloning 

begins with de-novo assembly of the DNA-binding regions of new ZFNs from overlapping 

oligos containing modified helices responsible for DNA triplet recognition, and their 

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insertion between a nuclear localization signal and the FokI endonuclease domain. 

Following the transfer of fully assembled ZFNs into E. coli expression vectors, bacterial 

lysates were found to be most suitable for in-vitro digestion analysis of palindromic 

target sequences. An in-planta activity test was also developed to confirm the nucleic 

activity of ZFNs in plant cells. The assay is based on reconstruction of GUS expression 

following bombardment of a reporter and ZFN-expressing plasmids into mesophyll cells. 

Our new procedures, plasmids and assays bring us one step closer to efficient 

implementation of ZFN-based technology for gene targeting in plant species. 

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T - 43. Generation of Single-Copy T-DNA Transformants by the Cre/LoxP 

Recombination Mediated Resolution System 

Sylvie De Buck, Annelies De Paepe, Ingrid Peck, Gordana Marjanac, Ann Depicker Plant 

Systems Biology, VIB, UGent, 9052 Gent, Belgium 

Transgene loci obtained after Agrobacterium tumefaciens transformation are considered 

to be less complex than those obtained after direct gene transfer, but integration of 

multiple T-DNA copies into direct or inverted repeats is fairly common. Recently, it was 

convincingly shown that transgenic plants with high and stable heterologous protein 

accumulation levels can be enriched for by screening for single-copy T-DNA 

transformants (De Buck et al., 2004). The aim was to evaluate a transformation system to 

generate efficiently single T-DNA copy transformants. Therefore, a strategy was designed 

to reduce the number of transgene copies by making use of the site-specific Cre/loxP 

recombination system. A lox-T-DNA vector containing two invertedly oriented loxP 

sequences located both inside and immediately adjacent to the T-DNA border ends was 

constructed. In the presence of the CRE recombinase, recombination between the 

outermost loxP sequences in direct orientation should resolve multiple copies into a 

single T-DNA copy, regardless of the orientation and number of T-DNAs integrated at one 

locus. Two different strategies were followed to test the approach. Firstly, several 

parental plants containing multiple lox-T-DNA copies at one locus were crossed with creexpressing 

plants. Secondly, cre-expressing plants were retransformed with the lox-T- 

DNA construct. For both methods, we could demonstrate the resolvement of multimeric 

T-DNAs to one T-DNA copy with a satisfactory frequency. Furthermore, this reduction in 

T-DNA copies led in most cases to an increased transgene expression level. We therefore 

propose that the described lox-T-DNA vector is a tool to obtain transformants with high 

and stable transgene expression. Reference: De Buck, S., Windels, P., De Loose, M., and 

Depicker, A. (2004). Single-copy beta-glucuronidase transgenes integrated at different 

positions in the Arabidopsis genome show uniform and comparable expression. Cell. Mol. 

Life Sci., 61: 2632-2645. 

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Abstracts of Posters 

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P - 1. Analysis of Meiotic Recombination in Rad51 Paralog Mutants in 

Arabidopsis. 

Kiyomi Abe*, Jean-Yves Bleuyard, Charles White.. CNRS UMR6547, Université Blaise Pascal, 

63177 Aubière, France. *Plant Genetic Engineering Research Unit, National Institute of 

Agrobiological Sciences, 305-8602 Tsukuba, Japan 

Recent studies indicate that two Rad51 paralogs (Xrcc3 and Rad51C) are involved in both 

DNA repair and meiotic recombination. To determine further details about the role of 

these proteins in meiotic recombination, we analyzed meiosis in atrad51C and xrcc3 

mutants. Cytological observation showed apparently normal figures up to 

zygotene/pachytene and dramatic abnormalities afterwards through telophase I in both 

atrad51C and xrcc3 mutants, indicating that pairing does occur at zygotene but that full 

chromosomal synapsis and synaptonemal complex formation depends upon the presence 

of the Xrcc3 and Rad51C proteins. Using fluorescence in situ hybridization (FISH) analysis 

we observe pairing of centromeres but not of the tested euchromatic region at pachytene 

in both mutants. These results suggest that Xrcc3 and Rad51C proteins play important 

roles in homologous pairing of the euchromatic regions of chromosomes. 

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P - 2. Genome-wide analysis of rye specific chromosome regions 

Olena Alkhimova, Marie Kubalakova, Olena Martynenko, Jaroslav Dolezel . National 

Academy of Sciences of Ukraine, Institute of Molecular Biology and Genetics, 03143 Kyiv, 

Ukraine 

Telomeres are specialized chromosome structure, which play important role in 

chromosome stability and behaviour. Together with subtelomeres, they form terminal 

regions of chromosomes. These are dynamic regions and might help organisms to adapt 

to new environmental conditions and promote genomic rearrangements. Large 

heterochromatic blocks, which are present near the telomeres of rye chromosomes, are 

notoriously difficult for physical mapping because of enrichment by different types of 

repetitive DNA sequences. We used wheat-rye addition lines and performed fluorescence 

in situ hybridization (FISH) on metaphase stretched flow-sorted chromosomes and 

extended DNA fibers for comparison of the terminal regions of the individual rye 

chromosomes. Each wheat-rye addition line produced a chromosome-specific large-scale 

organization of the tandem repeat arrays. The FISH signals on DNA fibers showed the 

multiple patterns of co-linear monomers arrangement of repetitive families as well as of 

authentic telomeric probe from Arabidopsis. The primary structure of some spacer 

sequences revealed the scrambled regions of similarity to various known repetitive 

elements. Using PCR analysis, we determined the DNA sequences adjacent to the tandem 

repeats array (spacers). They are enriched of short direct, complementary, inverted and 

symmetrical repeats which appear to be associated with recombination events. These 

spacers may be a powerful source of tandem array rearrangements. We mapped some of 

them on metaphase stretched chromosomes, and the distribution of the signals was in 

good agreement with the localization of the breakpoints for deletions and for 

translocations of 1R short arm with different wheat chromosomes. This work was 

supported by the INTAS grant (03-51-5908). 

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P - 3. Immunolocalization of Mre11 and Rad50 proteins during prophase I in 

tomato 

Lorinda Anderson, Leslie Lohmiller, Hildo Offenberg, Christa Heyting . Biology, Colorado 

State University, 80523-1878 Fort Collins, Colorado, United States 

Mre11 and Rad50 proteins act together in a protein complex that is involved in DNA 

double-strand break (DSB) repair, and the proteins also have roles in meiotic synapsis and 

recombination. mre11 and rad50 mutants in Arabidopsis thaliana lack homologous 

synapsis, suffer extensive chromosome fragmentation during prophase I, and are sterile. 

Current models predict the presence of both Mre11 and Rad50 in early recombination 

nodules (ENs) that are thought to represent sites of DSBs. Here, we used 

immunofluorescence to examine the meiotic behavior of these two proteins in Solanum 

lycopersicum (tomato) microsporocytes during early prophase I. Mre11 was present as 

numerous immunofluorescent foci that associated preferentially with the axes of tomato 

chromosomes before, during and immediately after synapsis. As many as 800 Mre11 foci 

were observed in leptotene nuclei (more than twice the maximum number of ENs 

observed at zygotene), and Mre11 foci were most common in distal euchromatic regions 

of chromosomes (as are ENs). Electron microscopic immunogold localization 

demonstrated that Mre11 is a component of only some ENs at mid-late zygotene while 

accumulations of Mre11 along the axial/lateral elements are common. In addition, Mre11 

foci are far more numerous than ENs on asynapsed euchromatic segments of leptotene 

and early zygotene chromosomes. Like Mre11, Rad50 foci preferentially associate with 

chromosomal axes during early prophase I. Unexpectedly, the number of Rad50 foci was 

less than that of Mre11 foci (although more similar to EN numbers), and Rad50 and 

Mre11 foci did not often colocalize. The presence of Rad50 in ENs is still under 

investigation, but our results indicate that most Mre11 foci do not correspond to ENs. 

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P - 4. Human Rad51 binding and the subsequent unwinding of duplex DNA 

requires different nucleotide cofactors 

kamakshi Balakrishnan, Rekha Govekar, Basuthkar J. Rao . Department of Biological 

sciences, Tata Institute of Fundamental Research (TIFR), 400006 Mumbai, India 

Homologous recombination is one of the central mechanisms for removing DNA double 

strand breaks in humans and other eukaryotes. Human Rad51 (hRad51) is a recombinase, 

which brings about this process in concert with other recombination mediator proteins 

such as Rad52, Replication Protein A (RPA), Rad54 and other accessory proteins by 

performing DNA homology search and pairing, followed by DNA strand exchange. With 

the aim of understanding the roles of different nucleotide cofactors such as ATP, ADP, 

ATP gammaS (a slow hydrolysable ATP analog), GTP and GDP on: (a) hRad51 binding to 

duplex DNA and (b) hRad51 mediated unwinding of duplex DNA , we carried out binding 

and topological assays. Our results suggest that binding of hRad51 to circular duplex 

DNA is efficient in the presence of nucleotide cofactors such as ATP, ATP gammaS or 

GTP, and results in the formation of higher order protein-DNA complexes. However, in 

absence of such nucleotide cofactors or in presence of ADP, lower order complexes 

result. We observe rapid unwinding only in the presence of ATP, whereas the unwinding 

kinetics slow down in presence of ATP gammaS. This indicates that hRad51 mediated 

duplex DNA unwinding depends on ATP hydrolysis, unlike binding. Although providing 

GTP as a cofactor enhances hRad51 binding to duplex DNA as much as ATP does, the 

unwinding kinetics mediated by GTP are poorer compared to ATP. ADP and GDP on the 

other hand, neither facilitate binding nor unwinding. Upon inclusion of hRad52 however, 

in the reaction system hRad51 unwinds dsDNA utilizing ADP as cofactor. Same is not 

true for GDP. Our results thus indicate that (a) hRad51 mediated unwinding of duplex 

DNA is kinetically favored when ATP can be hydrolyzed as against the slower unwinding 

observed with ATP gammaS, (b) A new role for hRad52 in the unwinding reaction carried 

out by hRad51 when ADP is provided as cofactor, (c) GTP can also serve as a cofactor for 

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unwinding reaction although not as efficient as ATP. 

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P - 5. Towards plant gene targeting – Enhancing homologous recombination 

frequency in tobacco. 

Abdellah Barakate, Claire Halpin. based at Scottish Crop Research Institute, University of 

Dundee , DD2 5DA Dundee, United Kingdom 

Gene targeting (GT) is a powerful tool that allows a homology-based replacement of an 

endogenous gene by an in vitro manipulated copy. Developing this technology has proved 

particularly difficult in plants where foreign DNA is integrated predominantly by nonhomologous 

recombination. The main competing pathways of recombination repair(1) 

could be manipulated to increase homologous recombination (HR) efficiency. One 

strategy to stimulate HR is to express recombinases involved in this pathway. We have 

expressed multiple bacterial (RecA, RecG, RuvC) and human (hRad51, hDMC1 and 

hRad52) recombinases in a tobacco line (N1DC4) that already contains a transgene as an 

artificial substrate for intrachromosomal recombination(2) (ICR). The transgene for ICR 

consists of a direct repeat of two defective b-glucoronidase (GUS) genes that can 

recombine to generate a functional GUS, which can be detected by histochemical staining 

of plant material. In order to co-ordinate the expression of multiple recombinases in a 

single plant, we have used an artificial self-dissociating polyprotein system. This unique 

and novel function was taken from the 2A region of foot-and-mouth disease virus(3). 

Several tobacco transgenic lines expressing multiple recombinases were selected and 

their ICR assessed both in pollen and six-week-old seedlings. While ICR stimulation in 

seedlings remained modest, a huge increase (up to 1000 fold) was detected in pollen of 

some transgenic lines. In addition, most transgenic lines showed various degrees of 

sterility and their meiosis will be analysed. This plant material will also be used to 

determine whether ICR stimulation will translate into efficient gene targeting using our 

newly designed vectors. References: 1) Trends Genet. (2005) 21, 172-181. 2) EMBO J. 

(1994) 13, 484-489. 3) Plant Physiol. (2004) 135, 16-24. Acknowledgements: Barbara Hohn 

(Basel) for N1DC4 line, Stephen West (London) for human recombinases. Funding: BBSRC, 

Scottish Enterprise Tayside, The Leverhulme Trust. 

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P - 6. Repair of G:T mismatches in plants 

Joke Baute, Gert Van der Auwera, Ann Depicker. Plant Systems Biology, VIB - Ghent 

University, 9052 Ghent, Belgium 

G:T mismatches are the result of replication errors or of hydrolytic deamination of 5methylcytosine 

that occurs spontaneously in the cell. In mammals, G:T mismatches 

caused by deamination of methylated cytosines, are repaired by base excision repair. Two 

DNA glycosylases recognize this mismatch: thymine DNA glycosylase (TDG) and methylbinding 

domain 4 protein (MBD4). Both enzymes specifically convert G:T mismatches to 

G:C. In plants, the methylation level is higher than in animals, suggesting that plants also 

developed systems to repair G:T mismatches caused by deamination of 5-methylcytosine. 

To analyze this in plants, we are performing a functional analysis of MBD4 homologs in 

Arabidopsis thaliana. We identified only one annotated coding sequence in Arabidopsis 

thaliana that shows sequence homology to the glycosylase domain of MBD4. The human 

MBD4 consists of two DNA binding domains: a methyl-CpG-binding domain and a 

mismatch specific DNA glycosylase domain, while the Arabidopsis protein (AtMBD4) has 

the glycosylase domain but lacks the methyl-CpG-binding domain. Using Y2H, we are 

currently investigating with which proteins AtMBD4 interacts. We have found different 

splicing variants of AtMBD4 in different tissues, what is also the case for human MBD4 

and for many other DNA glycosylases, and these will be presented. To learn more on the 

function of the AtMBD4 gene, we analyzed the AtMBD4 promotor activity by using 

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different beta-glucuronidase reporter fusions. We found that the fusion protein resides 

primarily in the nuclei of the meristems. Also other DNA repair genes in plants are 

primarily expressed in meristematic tissue. This is compatible with the model that repair 

genes play a crucial role in assuring fertility and fitness of the progeny. Currently, we are 

investigating the effect of different types of stress on the AtMBD4 promotor activity. For 

further characterization of AtMBD4, we generated silencing and overexpression lines, 

which do not show a phenotype under normal conditions. To determine the impact of 

AtMBD4 on mutation frequency, we analyzed plants with different AtMBD4 levels, via a 

detection system, developed in our lab. This allows to determine the mutation frequency 

of cytosine to thymine via the reactivation of a deactivated GUS gene. Finally, we 

investigated whether AtMBD4 plays a role in the signalization of DNA damage caused by 

mutagenic agents, as human and mouse MBD4 not only have a function in the recognition 

of G:T mismatches, but also play a role in the signalization of DNA damage caused by 

different mutagenic agents. The results will be discussed. 

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P - 7. Gene targeting in plants via engineered zinc finger proteins 

Charles Cai, Vipula Shukla, Fyodor Urnov, Jeffery Miller, Nicole Arnold, Lisa Baker, Teresa 

Bauer, Ryan Blue, Dave McCaskill, Jon Mitchell, Matt Simpson, Andrew Worden. Plant 

Genetics and Biotechnology, Dow AgroSciences, 46268 Indianapolis, IN, United States 

Gene targeting via homologous recombination occurs at a very low frequency in plant 

cells compared to random integration making targeted gene modifications impractical. 

Most recently, substantial increases in the frequency of homologous recombination have 

been observed following the induction of double stranded breaks in host cell DNA 

followed by the apparent stimulation of cellular repair mechanisms. Strategies to achieve 

targeted DNA double stranded breaks have been developed by fusing sequence-specific 

zinc finger DNA binding proteins with sequence-independent nuclease domains derived 

from Type IIS restriction endonucleases. Using this strategy, both site-specific transgene 

integration and targeted modification of native genes have been demonstrated in plants. 

Implications for novel trait development and crop improvement will be discussed. 

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P - 8. SITE-DIRECTED HOMOLOGOUS RECOMBINATION IN TOBACCO CELL 

CULTURES VIA ZINC FINGER NUCLEASES 

Charles Cai, Michael Ainley, Trevor Collingwoo, Robbi Garrison, Lisa Schulenberg, Philip 

Gregory, Beth Rubin-Wilson, Joseph Petolino. Biochemistry and Molecular Biology, Dow 

AgroSciences, LLC., 46268 Indianapolis, United States 

Targeted transgene integration via homologous recombination occurs at a very low 

frequency in plant cells compared to random integration, even when the incoming DNA 

comprises large stretches of sequence homologous to host DNA. As such, gene targeting 

in plants via homologous recombination has not been practical. Most recently, substantial 

increases in the frequency of homologous recombination have been observed following 

the induction of DNA double stranded breaks in host cells and apparent stimulation of 

cellular repair mechanisms. Restriction enzymes whose recognition sites are rare in the 

plant genome have been shown to stimulate homologous recombination following the 

formation and repair of DNA double stranded breaks in the host DNA. Strategies to 

achieve targeted DNA double stranded breaks have been developed by fusing zinc finger 

DNA binding proteins with sequence-independent nuclease domains derived from Type II 

restriction endonucleases. In the present study, engineered zinc finger proteins fused to 

nuclease domains, so-called ‘zinc finger nucleases’, were used to facilitate site-specific 

transgene integration via homologous recombination in tobacco cell cultures. A target 

DNA sequence was first stably integrated into tobacco cell cultures using Agrobacterium. 

This target sequence contained specific zinc finger protein recognition/binding sites, 

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along with a non-functional test gene to be corrected. A few selected transgenic events 

containing a single integrated copy of the target sequence were re-transformed using 

Agrobacterium strains harboring different T-DNAs. One Agrobacterium strain contained a 

donor DNA sequence comprising the bases necessary to correct the non-functional test 

gene, flanked by sequences homologous to the pre-integrated target DNA. The second 

Agrobacterium strain contained a gene encoding a zinc finger nuclease that specifically 

recognized a binding site in the integrated target sequence. Gene targeting via sitedirected 

homologous recombination was demonstrated as evidenced by the reconstitution 

of a functional test gene and was confirmed via molecular and biochemical 

analyses. 

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P - 9. Gene and Protein Targeting Technologies Create Novel Opportunities in 

Plant Biotechnology 

Monique Compier, Patrick Mak, Sylvia de Pater, Bert van der Zaal, Paul Hooykaas, Remko 

Offringa , Add2X Biosciences BV, 2333 AL Leiden, Netherlands 

An important platform technology that is missing in the field of plant science is gene 

targeting through homologous recombination. Moreover, for many economically 

important plant varieties, robust and efficient protocols for vegetative propagation and 

genetic modification are still lacking. The availability of such technologies is essential for 

the further development of plant biotechnology. Add2X Biosciences BV is a young biotech 

spin-off from Leiden University that aims to create novel opportunities in plant 

biotechnology, by developing innovative platform technologies and products for the 

directed and efficient delivery of DNA and proteins into plant cells. The Add2X portfolio 

includes: 1. Efficient gene targeting in plants through suppression of the non-homologous 

recombination pathway. Proof of concept has been obtained in different yeasts and 

filamentous fungi. Currently, research is in progress to confirm the applicability of this 

technology in plant species. 2. Agrobacterium-mediated protein translocation to produce 

and transiently ‘inject’ proteins of interest into plant cells in order to exert their function 

without permanently altering the host cells. 3. Induction of somatic embryogenesis in 

hitherto recalcitrant crop species. A protein family was identified that, when overexpressed, 

stimulates the spontaneous formation of somatic embryos from vegetative 

plant cells. The Add2X technologies stand well on their own, but clearly have the 

potential to be combined into integrated technologies or products. The technologies are 

developed in close collaboration with Leiden University. In addition, Add2X has 

established strategic alliances with other academic and industrial partners to explore 

novel opportunities and to achieve rapid implementation of the technologies in the 

biotechnology sector. 

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P - 10. Homologous recombination between divergent sequences in Arabidopsis 

Eyal Emmanuel, Elizabeth Yehuda, Roy Opperman, Naomi Avivi-Ragolsky, Avraham Levy, 

Cathy Melamed-Bessudo. Plant Sciences Department, The Weizmann Institute of Science, 

76100 Rehovot, Israel 

Earlier studies in plants have shown that there is a strong reduction in the rate of 

homologous recombination when the recombining partners are chromosomal segments 

that originate from related, but divergent, species. The effect of sequence divergence on 

homologous recombination is not well established. We found that the rates of somatic 

recombination between repeats that diverge only by one out of 617 otherwise identical 

nucleotides, is reduced by ~ 3-fold. Similarly, the rates of meiotic recombination are 

higher in an isogenic background than in crosses with divergent ecotypes. We analyzed 

the role of AtMSH2 in suppression of recombination between divergent sequences in 

Arabidopsis. We report that AtMSH2 has a broad range of anti-recombination effects: it 

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suppresses recombination between divergent direct repeats in somatic cells or between 

homologs from different ecotypes during meiosis. We describe the involvement of other 

genes from the mismatch repair family on homologous recombination and discuss the 

implications of these results for plant improvement via gene transfer across species. 

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P - 11. Transient inactivation of NHEJ proteins 

Sylvia de Pater, Vanessa Costa, Paul Hooykaas. Institute Biology Leiden, Leiden University, 

2333 AL Leiden, Netherlands 

Agrobacterium tumefaciens T-DNA normally integrates into random sites in the plant 

genome and frequencies of gene targeting (GT) in plants are very low. It would be an 

advantage for analysis of gene function and for exploitation of genetically modified 

organisms if the frequency of targeted integration could be increased. Experiments with 

yeast mutants have shown that GT can be enhanced by mutations in the DNA repair 

pathway of non-homologous end joining (NHEJ)(1). Subsequently, Arabidopsis NHEJ T- 

DNA insertion mutants have been tested in GT experiments, and preliminary results show 

that GT frequency is improved somewhat in these mutants. Since constitutive inhibition 

of the NHEJ DNA repair pathway results in telomere lengthening (2,3) and maybe other 

DNA rearrangements, we applied the peptide aptamer technology (4) combined with an 

inducible promoter system for the temporary inactivation of NHEJ proteins. A peptide 

aptamer has been isolated that binds to AtKu80 and yKu80 in yeast (Y2H) and in vitro 

and expression in yeast and Arabidopsis results in more sensitivity for DNA damage 

inducing agents. 1. van Attikum H and Hooykaas PJJ (2003) Genetic requirements for 

targeted integration of Agrobacterium T-DNA in Saccharomyces cereviciae. Nucl Acid Res 

31, 826-832. 2. Bundock P, van Atticum H, Hooykaas P (2002) Increased telomere length 

and hypersensitivity to DNA damaging agents in an Arabidopsis KU70 mutants. Nucleic 

Acids Res 30, 3395-3400. 3. Bundock P and Hooykaas P (2002) Severe developmental 

defects, hypersensitivity to DNA-damaging agents, and lengthened telomeres in 

Arabidopsis MRE11 mutants. Plants Cell 14, 2451-2462. 4. Colas P, Cohen B, Jessen T, 

Grishina, McCoy J, Brent R (1996) Genetic selection of peptide aptamers that recognize 

and inhibit cyclin-dependent kinase 2. Nature 380, 548-550. 

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P - 12. ADI IS A NOVEL COMPONENT OF THE ARABIDOPSIS SOMATIC DNA 

DOUBLE STRAND BREAK RESPONSE AND IS ESSENTIAL FOR MEIOSIS 

Philip Dean, Susan Armstrong, Christopher West. CENTRE FOR PLANT SCIENCES, 

UNIVERSITY OF LEEDS, UK, LS2 9JT Leeds, United Kingdom 

Here we describe ATM DEPENDENT INDUCTION (ADI), a plant specific gene which shows a 

large induction specifically in response to genotoxic stress. Quantitative PCR 

demonstrates that the induction of ADI is dependent on the protein kinase ATM, placing 

ADI in an ATM pathway. ADI is also essential for Arabidopsis meiosis as adi knockout 

mutant plants are both male and female sterile. Detailed cytological analysis of these 

mutants reveals extensive chromosome fragmentation during meiosis. Studies in an adi 

heterozygous background correlate with ADI also being required for pollen mitosis as 

there is a failure to complete mitotic cell divisions in approximately half the pollen. 

Preliminary yeast 2-hybrid interaction studies indicate ADI may interact with components 

required for both DNA double strand break repair and cell cycle progression. 

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P - 13. A Cre::FLP fusion protein recombines FRT or loxP sites in transgenic maize 

plants 

Vesna Djukanovic, Brian Lenderts, Alex Lyznik. Crop Genetics Research, Pioneer Hi-Bred 

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International, Inc, IA 50131 Johnston, United States 

The coding sequences of Cre (site-specific recombinase from the bacteriophage P1) and 

FLP (the yeast 2mm plasmid site-specific recombinase) were fused in frame to produce a 

novel, dual-function site-specific recombinase gene. Transgenic maize plants containing 

the Cre::FLP-fusion expression vector were crossed to transgenic plants containing either 

loxP or FRT excision substrates. Complete and precise excisions of chromosomal 

fragments flanked by the respective target sites were observed in the F1 and F2 progeny 

plants. The episomal DNA recombination products were frequently lost. Some 

chromosomal recombination substrates survived in the F1 plants producing the chimeric 

F1 plants for the excision products. They became stabilized in the F2 generation after the 

cre::FLP gene segregated out. These observations may indicate that the efficiency of sitespecific 

recombination is affected by plant developmental stages with site-specific 

recombination being more prevalent in the developing embryos. Both FLP and Cre 

activities were comparable, producing clean and precise chromosomal excision products 

in maize. The crossing strategy proved to be suitable for genetic engineering of maize 

using the FLP or Cre site-specific recombination systems. 

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P - 14. In search of a role for Dss1 in the model plant Arabidopsis thaliana 

Emeline Dubois, Fabien Delacote, Marie-Pascale Doutriaux. Institut de Biotechnologie des 

Plantes-UMR8618, Université Paris XI-CNRS, 91405 Orsay, France 

In human, mutations of the BRCA2 gene are responsible for a hereditary form of breast 

cancer predisposition (1,2). One important role of Brca2 is to act on homologous 

recombination by loading Rad51 onto ssDNA at the site of the double strand breaks. 

Dss1 is a partner of Brca2 in human and Ustilago maydis (3,4). In Ustilago, Dss1 is 

essential to the Brca2 function in somatic cells (5). In addition, Dss1 was shown to belong 

to the 26S proteasome in human and yeast (6,7), whereas there is no Brca2 in yeast. Two 

isoforms of Brca2 and two isoforms of Dss1 are encoded for in the Arabidopsis thaliana 

genome. A potential role for AtDss1 in the proteasome has been revealed by the 

functional complementation of sem1 (the Dss1 ortholog) mutants in yeast. We are now 

examining the role of Dss1 in relation to Brca2 in Arabidopsis. One of the two Brca2 

isoforms interacts with both AtDss1 isoforms, whereas the other one interacts with only 

one (8). RNAi constructs have been established to inactivate Brca2 or Dss1 constitutively 

(p35S) or at meiosis (pDMC1). Plants transformed with the RNAi/BRCA2 constructs were 

sterile, due to aberrant meiosis (9). Plants transformed with the RNAi/DSS1 constructs 

are fertile. Thus, a role of AtDss1 at meiosis seems to be excluded. To further investigate 

the role of Brca2 and Dss1 in somatic DNA repair, we have transformed plants containing 

recombination substrates (10), with both RNAi constructs (under the control of the 

constitutive p35S promoter). Those plants are now being evaluated in terms of their 

sensitivity to genotoxic stresses and for their recombination rate. 1. Wooster et al (1995) 

Identification of the breast cancer susceptibility gene BRCA2. Nature 378: 789-792 2. 

Scully , Livingston (2000) In search of the tumour-suppressor functions of BRCA1 and 

BRCA2. Nature 408: 429-432 3. Marston, et al (1999) Interaction between the product of 

the breast cancer susceptibility gene BRCA2 and DSS1, a protein functionally conserved 

from yeast to mammals. Mol. Cell. Biol. 19 4633-42. 4. Kojic et al (2003) The BRCA2interacting 

protein DSS1 is vital for DNA repair, recombination, and genome stability in 

Ustilago maydis. Mol. Cell. 12:1043-9. 5. Kojic et al (2005) Brh2-Dss1 interplay enables 

properly controlled recombination in Ustilago maydis.Mol Cell Biol. 25:2547-57. 6. 

Funakoshi et al (2004) Sem1, the yeast ortholog of a human BRCA2-binding protein, is a 

component of the proteasome regulatory particle that enhances proteasome stability. J 

Cell Sci. 117:6447-54. 7. Krogan, et al (2004) Proteasome involvement in the repair of 

DNA double-strand breaks. Mol. Cell. 16 : 1027-34. 8. Dray et al (2006) Interaction 

between Arabidopsis Brca2 and Its Partners Rad51, Dmc1, and Dss1. Plant Physiol. 

140:1059-69.. 9. Siaud et al (2004) Brca2 is involved in meiosis in Arabidopsis thaliana as 

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suggested by its interaction with Dmc1. EMBO J 23:1392-1401 10. Molinier et al (2004) 

Interchromatid and Interhomolog Recombination in Arabidopsis thaliana. The Plant Cell, 

16 : 342-35 

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P - 15. AtMUS81 and its Genetic Interactions with RecQ-Helicases in Arabidopsis 

thaliana 

Frank Hartung, Stefanie Suer, Andreas Braun, Tina Bergmann, Holger Puchta. Botanical 

Institute II, University of Karlsruhe, 76128 Karlsruhe, Germany 

The endonuclease MUS81 has been shown in a variety of organisms to be involved in DNA 

repair in mitotic and meiotic cells. Homologues of the MUS81 gene exist in the genomes 

of all eukaryotes, pointing to a conserved role of the protein. However, the biological role 

and meiotic significance of MUS81 varies between different eukaryotes. For example, 

while loss of the gene results in strongly impaired fertility in S. cerevisiae and nearly 

complete sterility in S. pombe, it is not essential for meiosis in mammals. We identified a 

structural and functional homologue (AtMUS81/At4g30870) in the genome of 

Arabidopsis thaliana and determined the full-length cDNA of this gene, using RACE 

technology. Analysing two independent T-DNA insertion mutant lines of AtMUS81, we 

found that they are sensitive to the mutagens MMS and MMC but not to bleomycin. In 

contrast to yeast, no meiotic defect of Atmus81-1 and 2 was detectable resulting in a 

normal fertility. Crosses of Atmus81-1 with a hyperrecombinogenic mutant of the 

AtRECQ4A helicase resulted in synthetic lethality in the double mutant, whereas other 

AtRECQ genes tested did not show this effect. Thus, the nuclease AtMUS81 and the 

helicase AtRECQ4A seem to be involved in two alternative pathways of resolution of 

replicative DNA structures in somatic cells and the knock out of both pathways leads to 

plant lethality. 

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P - 16. Meiotic recombination frequency analysis in Arabidopsis thaliana mutants 

Veena Hedatale, Tony Connors, Tom Gerats, Janny Peters. Plant Genetics, Radboud 

University, 6525 ED Nijmegen, India 

Using sequence data from cDNA AFLP-based transcript profiles in Petunia undergoing 

male meiosis (Cnudde et al., 2006), we are trying to understand the roles of the 

corresponding Arabidopsis genes in homologous recombination. In order to estimate 

recombination frequency in meiotic mutants, we are employing a previously described 

fluorescent seed-based assay (Melamed-Bessudo et al, 2005). In addition to the available 

tester line from Melamed-Bessudo and colleagues, four new tester lines were developed, 

consisting of GFP and RFP markers linked in cis on three different chromosomes. In the 

future we plan to generate meiotic testers for all five Arabidopsis chromosomes. The 

available tester lines were crossed to several mutants to monitor the effect of meiotic 

genes on meiotic recombination. From the F2 population plants heterozygous for the 

fluorescent markers and homozygous for the mutant or wild-type locus were selected. By 

backcrossing the selected plants to a male sterile line, the recombination rate in the GFP- 

RFP marker interval of these plants can be compared to determine the effect of the 

mutation on recombination frequency. References: 1. Cnudde F, Hedatale V, de Jong H, 

Pierson ES, Rainey DY, Zabeau M, Weterings K, Gerats T, Peters JL (2006), Changes in gene 

expression during male meiosis in Petunia hybrida, Chromosome Res. 14:919-932. 2. 

Melamed-Bessudo C, Yehuda E, Stuitje AR, Levy AA. (2005), A new seed-based assay for 

meiotic recombination in Arabidopsis thaliana, Plant J 43:458-466. 

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P - 17. Functional analysis of the OsEZ polycomb group gene in rice 

Doolyi Kim, Myungsuk Jung, Insun Yoon, Yeonhee Lee, Seokcheol Suh. Cell and Genetics, 

National Institute of Agricultural Biotechnology, 441-707 Suwaon, South Korea 

The epigenetic control of gene expression is equally important in plants. Genetic and 

molecular analyses in recent years have started to illuminate how products of these 

multiple genes interact to initiate seed development. PcG proteins are known to form 

multimeric complexes that modulate gene expression by changing higher order 

chromatin structure. In this study, EZ gene was isolated and characterized from rice. A 

full-length EZ cDNA was obtained by reverse transcriptase (RT)-PCR. EZ gene is producted 

to encode a protein of 895 amino acid with an estimated molecular 99.8 kDa. The EZ 

protein contains a characteristic SET domain. To identified interaction of EZ gene with 

polycomb group protein, we also present evidence, from yeast two-hybrid experiment, of 

physical interaction of EZ and FIE proteins, as would be expected in a polycomb type 

system. Also, the EZ cDNA was inserted into binary vector pMJ-Cytc. The vectors 

described above were introduced into Agrobacterium tumefaciens strain LBA4404 to 

obtain a transgenic plants. We have obtained transgenic lines from rice. The phenotype of 

transgenic plants were investigated for functional characterization of EZ gene. 

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P - 18. A Novel Model for Chloroplast Replication Mechanism in Green Plants 

Neeraja Krishnan, Basuthkar Rao. Department of Biological Sciences, Tata Institute of 

Fundamental Research, 400005 Mumbai, India 

A Novel Model for Chloroplast Replication Mechanism in Green Plants Neeraja M. 

Krishnan*, Basuthkar J. Rao Correspondence Address: B-202, Department of iological 

Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai – 

400 005 INDIA Email Address: neeraja@tifr.res.in, bjrao@tifr.res.in Abstract Chloroplast 

replication mechanism, despite 30 years of work, is still sketchy and not well understood. 

The initial proposition of rolling circle, bi-directional replication mechanism in tobacco 

and pea chloroplast genomes is now being questioned by the new hypothesis of 

homologous recombination-mediated replication based on complex, branched multimeric 

forms of these molecules. We address this issue of uni- vs. bi-directional replication by 

analyzing nucleotide composition in the regions between known replication origins, with 

an aim of revealing any cytosine to thymine deamination gradients. These gradients 

typically result from accumulation of deaminations over the extent of singlestrandedness 

experienced by the genome and can, therefore, be used as a strong 

signature of single-strandedness. Such analyses can thus, throw more light on the singlestrandedness 

levels experienced by these regions during replication. Fine-mapping of 

replication origins on tobacco chloroplast genome revealed two pairs of replication 

origins (A which folds into a simple linear hair-pin form, and B with a complex D-Loop 

like form), one pair on each inverted repeat. We found homologues of these replication 

origins on other Viridiplantae chloroplast genomes, using NCBI pair-wise BLAST tool. Our 

linear regression analyses on the nucleotide compositions of the non-coding regions and 

the synonymous third codon position of the coding regions, between any two replication 

origins, reveal existence of C to T deamination-type mutation gradients. We find 

increasing gradients for C to T deaminations in the regions interspersed between the 

complex origins B on either inverted repeat, only on one strand, suggesting unidirectional 

replication. These C to T gradients were however, found for both the strands 

in the region between origins A and B, on each inverted repeat. This indicates singlestrandedness 

on both strands, suggesting bi-directional replication. The larger region 

between the pair of origins, A on each inverted repeat again reveals increasing C to T 

deaminations only on one strand, suggesting uni-directional replication. We summarize 

and present these results in the form of a model for replication mechanism in chloroplast 

genomes. 

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P - 19. Majority of DNA double strand breaks are in plant genome rapidly 

removed independently of NHEJ repair pathways. 

Jaroslav Kozak, Chris West, Charles White, Karel J Angelis . Molec. Farming and DNA 

Repair, Inst. Experimental Botany, 160 00 Praha 6, Czech Republic 

Plants developed during evolution repair capacity to protect genome integrity against 

environmental genotoxines like UV or ionizing radiation. Among DNA lesions induced by 

radiation, the most lethal are double strand breaks (DSB) when only few, if unrepaired, 

can cause cell death. Repair of DSB induced in plant DNA by ionizing radiation or 

radiomimetic mutagens has been shown earlier. Several repair mechanisms were 

proposed for DSB elimination and nonhomologous joining of DNA ends (NHEJ) suggested 

as the major repair pathway in mammals and higher plants. However, the role of 

individual genes on the kinetic and extent of DSB repair in genomic DNA have not been 

established yet. Here we show that genes of NHEJ pathway do not have key role in overall 

repair of DSB and other repair pathway that quickly removes majority of DSB is active in 

higher plants. DSB detected by electrophoretic single cell (comet) assay under neutral 

conditions are in NHEJ pathway mutants AtLig4 and AtKu80 repaired faster (t½ = 3.5 and 

3.4 minutes respectively) than in w.t. Arabidopsis (t½ = 5 min) and AtLig1a (t½ = 9 min), 

which was tested as possible alternative ligase to complete DSB sealing. Also 

chromosome maintenance proteins AtMim and AtBru1 (t½ = 50 and 15 min. respectively) 

were found to have dramatic effect on kinetics of overall DSB repair. Our results 

demonstrate that role of individual genes, if mutated, could be substituted by other 

genes, e.g. Lig4 in ligation step by Lig1. We also show dominant role of genes involved in 

structural maintenance of DNA to repair DSB. 

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P - 20. Zinc finger transcription factors to discover Arabidopsis mutants with 

enhanced homologous recombination 

Beatrice Lindhout, Johan Pinas, Paul Hooykaas, Bert van der Zaal. Molecular and 

developmental genetics, Institute of Biology, Leiden University, 2333 AL Leiden, 

Netherlands 

A library of genes for zinc finger artificial transcription factors (ZF-ATF) was generated by 

fusion of DNA sequences encoding three-fingered Cys2His2 ZF domains to the VP16 

activation domain and put under control of the promoter of the ribosomal protein gene 

RPS5A of Arabidopsis thaliana. After introduction of this library into an Arabidopsis 

homologous recombination (HR) indicator line, we selected primary transformants 

exhibiting multiple somatic recombination events. After PCR-mediated rescue of ZF 

sequences, reconstituted ZF-ATFs were reintroduced in the target line. In this manner, a 

ZF-ATF was identified that led to a 200 to 1000-fold increase in somatic HR, also in an 

independent second target line. A mutant plant line, expressing the HR-inducing ZF-ATF 

exhibited increased resistance to the DNA-damaging agent bleomycin and was more 

sensitive to methyl methanesulfonate (MMS), a combination of traits not described 

before. Our results demonstrate that the use of ZF-ATF pools is highly rewarding when 

screening for novel dominant phenotypes in Arabidopsis. 

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P - 21. Addressing the role of chromatin structure on gene targeting rates 

Marie Mirouze, Jon Reinders, Jerzy Paszkowski. Laboratory of Plant Genetics, Université de 

Genève Sciences III, 1211 Genève, Switzerland 

Addressing the role of chromatin structure on gene targeting rates Marie Mirouze, Jon 

Reinders and Jerzy Paszkowski Laboratory of Plant Genetics, University of Geneva, 

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Sciences III, Quai Ernest-Ansermet 30, CH-1211 Genève, Switzerland We describe an 

experimental system which is currently under development in our laboratory to address 

to which extent epigenetic modifications affect gene targeting rates in Arabidopsis 

thaliana. For this we use PPOX gene encoding protoporphyrinogen oxidase, an enzyme 

involved in chlorophyll biosynthesis. Inhibition of PPOX by the herbicide Butafenacil 

results in rapid plant death due to oxidative stress. However, the combination of two 

mutations in PPOX gene confers resistance towards Butafenacil. It was shown that the 

introduction of these mutations to the chromosomal copy of PPOX gene by gene targeting 

is possible and can be used to assess gene targeting frequencies. We first examined 

epigenetic marks at the PPOX locus. DNA methylation analyses indicate that in the wild 

type plants PPOX locus is associated with substantial levels of DNA methylation, 

therefore, using mutants impaired in maintenance of DNA methylation may alter the 

epigenetic status of this locus. We developed an EpiRILs population comprising F8 lines 

from a cross between Col WT and met1-3, a mutant affected in a methyltransferase 

responsible for CpG methylation maintenance. These EpiRILs have the same genetic Col 

background, but differ in their cytosine methylation profiles. We will describe the interest 

of using this epigenetic material to assess the role of chromatin structure in homologous 

recombination rates. This research is supported by the European Community in the frame 

of the TAGIP project (No. 018785). 

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P - 22. Genetic modification of rice plants by using Zinc Finger technology. 

Keishi Osakabe, Kiyomi Abe, Masaki Endo, Hiroaki Saika, Seiichi Toki. Division of Plant 

Sciences, National Institute of Agrobiological Sciences, 305-8602 Tsukuba, Ibaraki, Japan 

Zinc finger nucleases (ZFNs) are synthetic nucleases consisting of a zinc finger DNAbinding 

domain fused to the cleavage domain of the restriction endonuclease, Fok I. ZFNs 

can be used to introduce double-strand DNA breaks (DSBs) in specific DNA sequences, 

and promote site-specific homologous recombination (gene targeting) and targeted 

genome modification at the desired genomic loci including deletion- and insertion-type 

mutations. To apply this technology to the genetic modification of rice, we made a 

system to identify potential zinc-finger target sites in a gene of interest with the FASTA 

program based on the finding by Barbas's group (1). We already established the efficient 

transformation system of rice (2) and the gene targeting system with the rice acetolactate 

synthase (OsALS) gene locus (3). Thus, we chose the OsALS gene for the initial gene 

targeting experiment with ZFNs. We identified a ZFN recognition sequence at the Cterminal 

region of OsALS using our searching system, and designed ZFNs (ZFNosals_L 

and ZFNosals_R). We also confirmed that the combination of ZFNosals_L and ZFNosals_R 

could digest its recognition site on the OsALS gene in in vitro. We are further confirming 

digestion in in vivo by the ZFNs, and testing whether the introduction of the ZFNs can 

increase the efficiency of gene targeting at the OsALS gene locus. References 1. Mandell, 

J.G. and Barbas III, C.F. (2006) Nucleic Acids Res., 34, W516-W523. 2. Toki, S., et al. (2006) 

Plant J., 46, 969-976. 3. Endo, M., et al. (2006) 8th International Congress of Plant 

Molecular Biology, Book of Abstracts, p153. 

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P - 23. Two unlinked double-strand breaks can induce reciprocal exchanges in 

plant genomes via homologous recombination and non-homologous endjoining 

Michael Pacher, Waltraud Schmidt-Puchta, Holger Puchta. Molecular Biology & 

Biochmeistry of plants / Prof. Puchta, University of Karlsruhe / Institute of Botany II, 76128 

Karlsruhe, Germany 

Using the rare-cutting endonuclease I-SceI we were able to demonstrate before that the 

repair of a single DSB in a plant genome can be mutagenic due to insertions and 

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deletions. However, during replication or due to irradiation several breaks might be 

induced simultaneously. To analyze the mutagenic potential of such a situation we 

established an experimental system in tobacco harboring two unlinked transgenes each 

carrying an I-SceI site. After transient expression of I-SceI a kanamycin resistance marker 

could be restored by joining two previously unlinked broken ends, either by homologous 

recombination (HR) or by non-homologous end-joining (NHEJ). Indeed, we were able to 

recover HR and NHEJ events with similar frequencies. Despite of the fact that no selection 

was applied for joining the two other ends, the respective linkage could be detected in 

most cases tested, demonstrating that the respective exchanges were reciprocal. The 

frequencies obtained indicate that DSB-induced translocation is up to two orders of 

magnitude more frequent in somatic cells than ectopic gene conversion. Thus, DSB 

induced reciprocal exchanges might play a significant role in plant genome evolution. The 

technique applied in this study may also be useful for the controlled exchange of 

unlinked sequences in plant genomes. 

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P - 24. ATM-mediated transcriptional and developmental responses to ?-rays in 

Arabidopsis 

Lilian Ricaud, Caroline Proux, Jean-Pierre Renou, Olivier Pichon, Sylvain Fochesato, Phiippe 

Ortet, Marie-Hélène Montané. Direction des Sciences du vivant, CEA, 13108 St Paul-lez- 

Durance cedex, France 

(1) CEA, DSV, Institut de Biologie Environnementale et de Biotechnologie (iBEB), Service de 

biologie végétale et de microbiologie environnementales (SBVME), F-13108 Saint Paul-lez- 

Durance, France. (2) Unité de Recherche en Génomique Végétale, INRA, 2 rue Gaston 

Crémieux, F-91057 Evry, France (3) Present address: Plate-forme puces à ADN, Genopole, 

Institut Pasteur, 28 rue du Docteur Roux, F-75724 Paris cedex 15, France *E-mail: mariehelene.montane@cea.fr 

ATM (Ataxia Telangiectasia Mutated) is an essential checkpoint 

kinase that signals DNA double-strand breaks in eukaryotes. Its depletion causes meiotic 

and somatic defects in Arabidopsis and progressive motor impairment accompanied by 

several cell deficiencies in patients with ataxia telangiectasia (AT). To obtain a 

comprehensive view of the ATM pathway in plants, we performed a time-course analysis 

of seedling responses by combining confocal laser scanning microscopy studies of root 

development and genome-wide expression profiling of wild-type (WT) and homozygous 

ATM-deficient mutants challenged with a dose of ?-rays (IR) that is sublethal for WT 

plants. Early morphologic defects in meristematic stem cells indicated that AtATM, an 

Arabidopsis homolog of the human ATM gene, is essential for maintaining the quiescent 

center and controlling the differentiation of initial cells after exposure to IR. Results of 

several microarray experiments performed with whole seedlings and roots up to 5 h post- 

IR, which were compiled in a single table; sequence and function homology searches; 

import of cell cycling, and mutant-constitutive expression characteristics; and a 

simplified functional classification system were used to identify novel genes. The 

transcription burst was almost exclusively AtATM-dependent or weakly AtATRdependent, 

and followed two major trends of expression in atm: (i)-loss or severe 

attenuation and delay, and (ii)-inverse and/or stochastic, as well as specific, enabling one 

to distinguish IR/ATM pathway constituents. The hundreds of radiomodulated genes 

identified were not a random collection, but belonged to functional pathways such as 

those of the cell cycle; cell death and repair; DNA 3R; and transcription; translation; and 

signaling, indicating the strong cell reprogramming and double-strand break abrogation 

functions of ATM checkpoints. Accordingly, genes in all functional classes were either 

down or up-regulated concomitantly with downregulation of chromatin deacetylases or 

upregulation of acetylases and methylases, respectively. Determining the early 

transcriptional indicators of prolonged S-G2 phases that coincided with cell proliferation 

delay, or an anticipated subsequent auxin increase, accelerated cell differentiation or 

death, was used to link IR-regulated hallmark functions and tissue phenotypes after IR. 

Our data provide a large resource for studies on the interaction between plant 

checkpoints of the cell cycle, development, hormone response, and DNA repair functions, 

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because IR-induced transcriptional changes partially overlap with the response to 

environmental stress. Putative connections of ATM to stem cell maintenance pathways, 

and to non-DSB/DSBs repair mechanisms in plants after IR are discussed. 

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P - 25. Cellular and molecular understanding of plant DNA damage response 

through ATM, ATR, H2AX, E2F and RNR proteins. 

Hélène Roa, Julien Lang, Lenin Sanchez-Calderon, Ondrej Smetana, Frédéric Lincker, 

Chamseddine Mediouni, Guy Houlné. plant cellular biology, IBMP/CNRS UPR2357 

Strasbourg, 67084 Strasbourg, France 

Plants are continuously exposed to high levels of genotoxic stresses which can induce 

DNA damage, and according to its could lead to cellular death. So through evolution, 

plants have fixed efficient DNA repair mechanisms. Among key factors involved in DNA 

repair, Ribonucleotide reductase has to be highly regulated for providing the cell with 

dNTPs. In Arabidopsis the 3 AtRNR2 encoding the small R2 subunit present specific 

profile of gene induction, R2-3b is significantly induced in response to IR or the 

radiomimetic bleomycin (ATM-dependency) but not upon HU contrarily to R2-3 and R2-5 

genes. These genes presented E2F-elements in their promoters and as described in 

tobacco, we demonstrate that E2F factor plays an important role to drive their gene 

induction upon DNA damage in Arabidopsis (replicative stress or DSB) under the control 

of ATM, ATR or alternative pathways. In addition GFP-NtE2F protein fusion presents a 

nuclear localization in both tobacco and Arabidopsis cells and forms some clear and 

discrete nuclear foci when these cells are submitted to bleomycin. Most of these NtE2F 

nuclear foci colocalized with Atgamma H2AX, a marker of DSBs, and their number was 

increasing in a time-dependent manner upon BLM treatment. Such NtE2F foci formation is 

ATM-dependent and occurs with a higher density in the root meristem. Interestingly, 

increasing time of BLM treatment leads also to increased cell death concomitant with 

strong gene induction of particular PCD marker genes. Works is in progress to 

understand the role of E2F in DNA repair and PCD cellular processes. 

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P - 26. CHARACTERIZATION OF ARABIDOPSIS EXONUCLEASE 1 

Max Roessler, Karel Riha , Austrian Academy of Sceinces, Gregor Mendel Institute, 1030 

Vienna, Austria 

Exonuclease 1 (EXO1) is a multitasking nuclease, with 5' - 3' exonuclease activity, which is 

present in virtually all eukaryotic organism from yeast to mammals. Studies performed 

mainly in yeast have demonstrated that EXO1 is involved in mismatch DNA repair (MMR). 

It is also implicated in the resection of DNA double strand breaks, homologous 

recombination, telomere maintenance, meiosis, and DNA replication. However, these 

functions of EXO1 are less well understood. We found that the Arabidopsis genome 

encodes two paralogous proteins, which we refer to as AtEXO1A and AtEXO1B. 

Arabidopsis is the only known organism containing two putative EXO1 genes. The 

homology between the AtEXO1A and AtEXO1B proteins is limited to the N-terminal 

nuclease domain suggesting that the proteins have evolved different functions. 

Supporting this idea we found that expression levels of EXO1A and EXO1B transcripts 

differes in various tissues. We characterized mutant lines harbouring T-DNA insertions in 

the AtEXO1A and AtEXO1B genes. These lines showed no sensitivity to DNA damage 

inducing agents (Mitomycin C, Menadion, Hydroxy Urea, Methyl-Methane Sulfonate, 

Bleocin). Further experiments are underway to examine the function of the EXO1 

paralogues at telomeres, in MMR and in homologous recombination (HR). 

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P - 27. Relationships between genetic and physical maps in bread wheat: 

Recombination studies on chromosome 3B. 

cyril saintenac, pierre sourdille. UMR 1095 Amelioration et Sante des plantes, INRA, 63000 

Clermont-Ferrand, France 

Recombination and population history seems to play an important role at several levels 

of the gene evolution dynamics (duplications, deletions, mutations. It is admitted that 

recombination increases the sequence divergence rate (polymorphism) as well as the 

deletion-duplication rate. However, the balance between these two phenomena remains 

poorly understood. In wheat, we recently showed that recombination varies at the 

chromosome scale where it increases gradually with the distance from the centromere 

with some local variations. However, we still have the idea neither of the distribution of 

the recombination along the chromosome nor of the relationships between its intensity 

and gene evolution as well as their polymorphism. Recent progresses in wheat genomics 

have made possible the elaboration of the first chromosome-specific BAC library (Safar et 

al, 2004, Plant J., 39: 960-968) from the wheat chromosome 3B. We recently started to 

build the physical map of this chromosome by fingerprinting and contiguing the 68,000 

BAC clones from this library. The physical map is now made of about 2,000 contigs 

covering more than 85% of the chromosome. The aims of the PhD will be to use this 

resource to study the recombination at different levels: 1- At the level of the whole 

chromosome, the recombination gradient will be studied by refining the position of the 

break-points observed using deletion lines. The relationships between genetic and 

physical distances will be precised by: (1) increasing the number of deletion lines 

evaluated; (2) using the genetically anchored contigs within a deletion bin. We will also 

evaluate the influence of the length of the chromosome arm on the recombination by 

studying segregating populations issued from the cross between the deletion lines and a 

normal cultivar (Renan). 2- At the level of a deletion bin known to be gene-rich (bin 3BL7, 

~200 Mb) and where the recombination is frequent, we will increase the density of 

markers (1/5Mb). We will then be able to compare the recombination in gene-rich (contigs 

with at least one EST assigned) and gene-poor (contigs without any EST assigned) regions. 

Moreover we want to know if the distribution of recombination is conserved between two 

different segregating populations. 3- At the level of a sequenced region. In the course of 

the IWGSC, it was planned to establish a physical contig spanning the region covering 

several resistance loci (FHB, Rph7, Sr2, Sn2) and to define a Minimal Tiling Path (MTP) that 

will be completely sequenced and analysed (estimated to be between 14 and 23 Mb). This 

region covers between 6 and 12 cM according the segregating populations studied and is 

thus highly recombinogenic. At the present time, 60 markers have been identified in the 

region and it is planned to map those that are polymorphic and to increase the density of 

markers by using additional ISBPs in order to identify putative recombination hot-spots. 

All the results will bring a new insight in the recombination in wheat which will be 

helpful for further positional cloning of genes of agronomical interest on chromosome 3B 

as well as on other regions of the genome. 

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P - 28. Characterisation of AtTRB1 binding properties 

Iva Santruckova, Ctirad Hofr, Petra Schrumpfova, Jiri Fajkus. Functional Genomics and 

Proteomics, Masaryk University, 625 00 Brno, Czech Republic 

Telomeres play an essential role in maintenance of chromosome integrity. Their 

protective function is dependent on dynamic association of telomeric DNA with histones 

and specific telomeric proteins. Here we report on characterization of AtTRB1 

(At1g49950, 33kDa), a member of plant-specific group of putative telomere-binding 

proteins – the Smh (Single myb histone) family. This family is defined by the presence Nterminal 

Myb-like domain, centrally localized histone H1/H5 domain and a C-terminal 

coiled-coil. In Arabidopsis, the family includes 5 members – AtTRB1-5. Two of them - 

AtTRB2 and AtTRB3 – have been characterized previously in respect of their DNA-protein 

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and protein-protein interactions. The functional importance of AtTRB1 at telomeres 

comes from the results of our recent protein-protein interaction studies which showed 

(using yeast two-hybrid assay and IP) an interaction between AtTRB1 and AtPot1-2, a 

member of AtPot family in Arabidopsis. The focus of this study has been directed 

towards the description of the DNA binding capability of AtTRB1 and its truncated 

variants. AtTRB1 and its fragments were cloned and expressed in bacterial system and 

the purified proteins were used in Electrophoretic Mobility Shift Assay (EMSA) and 

Surface Plasmon Resonance (SPR). It has been shown that myb-like domain of AtTRB1 

preferentially binds telomeric over non-telomeric dsDNA. However, AtTRB1 lacking the 

Myb-like domain also shows preference to telomeric dsDNA. Histone H1/H5 domain 

alone is not able to show any binding preference. These data suggest a positive role of Cterminal 

coiled-coil domain in sequence-specific binding of AtTRB1 to telomeric DNA 

sequence. Our research is supported by Grant Agency of the Czech Republic 

(521/050055), Grant Agency of the Czech Acad. Sci (IAA600040505), Ministry of 

Education (LC LC06004) and institutional funding (MSM0021622415) 

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P - 29. A HYPOMORPHIC ALLELE OF TELOMERASE DELAYS ONSET OF GROWTH 

DEFECTS IN ARABIDOPSIS WITHOUT PREVENTING TELOMERE 

SHORTENING 

Martina Schirato, Barbara Zellinger, Karel Riha , Austrian Academy of sciences - Gregor 

Mendel Institute, 1030 Vienna, Austria 

Disruption of Arabidopsis telomerase reverse transcriptase (TERT) gene leads to a 

gradual telomere shortening and an onset of developmental defects after six generations 

of telomerase deficiency. To investigate whether telomere shortening and occurrence of 

growth abnormalities can be reverted by reintroduction of telomerase, we transformed 

fourth generation (G4) of Arabidopsis tert mutants with a construct expressing TERT 

cDNA from a strong constitutive 35S promoter (35S::TERT). Overexpression of the TERT 

re-established telomerase activity in tert mutants, but failed to rescue telomere 

shortening. In fact, telomeres in tert plants ectopically expressing TERT cDNA shortened 

at a similar rate as telomeres in control tert mutants or in tert lines overexpressing 

catalytically inactive TERT(D860N) construct. However, while the tert and tert 

35S::TERT(D860N) lines started exhibiting developmental defects at G6 and could not be 

propagated beyond G8, the onset of growth defects was significantly delayed in the lines 

complemented with the 35S::TERT construct and they could be propagated for at least 13 

generations. Interestingly, no telomeric DNA could be detected in these plants from G10 

onwards by standard techniques suggesting that chromosome termini contain no or only 

very short arrays of telomeric repeats. These data suggest that the hypomorphic 

35S::TERT allele significantly extends proliferation capacity of cells with critically short 

telomeres. 

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P - 30. Molecular analysis of Phaseolus coccineus PCNA 

Wojciech Strzalka, Anna Kaczmarek, Barbara Naganowska, Alicja Ziemienowicz. Faculty 

of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, 

Poland 

Molecular analysis of Phaseolus coccineus PCNA Strzalka W1, Kaczmarek A2, 

Naganowska B2 and Ziemienowicz A1 1Department of Molecular Genetics, Faculty of 

Biochemistry, Biophysics and Biotechnology Jagiellonian University, Gronostajowa 7, 30- 

387 Kraków, Poland. 2Institute of Plant Genetics, Polish Academy of Science, 

Strzeszynska 34, 60-479 Poznan, Poland Proliferating cell nuclear antigen (PCNA) is one 

of the most conserved proteins present both in animal and plant organisms. PCNA 

functions as a auxiliary factor of DNA polymerase delta and naturally forms a trimeric 

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ring encircling DNA that serves as a platform for binding various proteins involved in 

DNA metabolism. Moreover, it also plays an important role in DNA repair and in 

regulation of the cell cycle. Although the role of PCNA in animal and yeast cells has been 

studied extensively, only few information concerning the role of PCNA in plant cells, 

especially in DNA repair, is currently available. The aim of our study was molecular 

analysis of P. coccineus PCNA. First, by using 5’ and 3’ RACE technique we identified 

cDNA encoding PcPCNA1 ad PcPCNA2. The identity between these two proteins on the 

amino acid level was 54%. Second, we purified the recombinant PcPCNA1 and PcPCNA2 

proteins and compared their biochemical properties. Our results showed that PcPCNA1 

was able to stimulate the activity of human DNA polymerase delta, whereas no 

stimulatory effect was observed for PcPCNA2. Moreover, we observed that PcPCNA1 but 

not PcPCNA2 was (i) present as a trimer in solution, (ii) was able interact with p21 

peptide, and (iii) was recognized by anti-human PCNA antibody PC10. Furthermore, we 

analysed the patterns of PcPCNA1 and PcPCNA2 gene expression during seed germination 

and in the runner bean organs. Real-time RT-PCR analysis showed changes in the 

expression of both genes during germination with maximum levels reached 48-72 h after 

imbibing. The transcripts could be also detected in seed micropylar region, and they were 

present at very low level in roots, stem and leaves of 10 day old plants. Finally, the copy 

number of PcPCNA genes in the genome of P. coccineus and their chromosomal 

localisation was analysed using Southern blotting and PRINS techniques. The 

hybridization pattern suggested the presence of at least two PCNA genes in the runner 

bean genome. On the other hand, the presence of single dot signals observed on 

metaphase chromosomes (PRINS analysis) indicated that both PcPCNA1 and PcPCNA2 

genes were localised most probably in a single locus in a centromeric region of a 

submetacentric chromosome. These results implicate a number of the intriguing 

questions concerning the role of PCNA in plants that we are going to answer in the 

coming future. Moreover, our studies may help us to understand the evolution route from 

bacterial sliding clamp to eukaryotic PCNA. 

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P - 31. DNA damage repair/toleration protein Drt112 may be involved in 

integration of T-DNA into the plant genome 

Wojciech Strzalka, Tomasz Herjan, Monika Kapusniak, Karolina Peplowska, Alicja 

Ziemienowicz. Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian 

University, 30-387 Krakow, Poland 

Agrobacterium tumefaciens, a plant natural pathogen, has an ability of interkingdom 

DNA transfer. In favourable conditions this bacterium is able to transfer a large fragment 

of the resident Ti-plasmid (T-DNA: transferred DNA) to the plant cell, where T-DNA gets 

integrated into the host genome. T-DNA integration into the plant chromosomes occurs 

via illegitimate recombination, involving various pathways of DNA repair. This process 

requires plant factors, such as DNA ligase and other enzymes involved in DNA 

repair/recombination, although the role of bacterial factors, such as VirD2 protein, 

cannot be excluded. VirD2 was shown previously not to act as a ligase for T-DNA 

integration, however it may play a crucial role in recruiting plant factors to the site of T- 

DNA integration. By employing yeast two-hybrid system, we have identified as a VirD2interacting 

factor a precursor of Drt112 protein, that may be involved in T-DNA 

integration. Two cDNA, DRT111 and DRT112, have been previously selected from E. coli 

mutants lacking RuvC endonuclease (recombination resolvase) transformed with 

plasmids expressing Arabidopsis thaliana cDNA library (Pang Q. et al., Nucleic Acid 

Research, 1993, 21(7): 1647-53). Expression of these plant cDNAs increased the resistance 

of the bacterial mutant to DNA damaging factors, indicating involvement of the Drt 

proteins in DNA repair and recombination (Drt: DNA-damage-repair/toleration). By using 

far-Western blotting we have shown that only Drt112 but not Drt111 protein interacted 

with VirD2, suggesting the potential involvement of the Drt112 protein in the T-DNA 

integration process, most likely by resolving T-DNA integration intermediates. Further 

studies aiming to discover the role of Drt proteins in plants will help us to reveal the 

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mechanism of Drt-mediated DNA repair/recombination pathway employed for T-DNA 

integration. 

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P - 32. Positional cloning of the hus2 locus in Arabidopsis thaliana: A new player 

in the cell-cycle arrest response to replication blocks 

Paul Sweeney, Daniel Lynch, Anne Britt, Kevin Culligan. Biochemistry and Molecular 

Biology, University of New Hampshire, 03824 Durham, United States 

Cell-cycle arrest is an integral component of the cellular response to DNA damage in 

eukaryotic cells. Persisting DNA damage can block replication during S-phase, resulting in 

G2-phase arrest. This arrest prevents chromosome loss by blocking cell-cycle progress 

into M-phase with an incompletely replicated genome, and also allows time for repair and 

additional DNA synthesis. ATR, a phosphoinositide (PI)-3-like protein kinase, controls this 

G2/M transition in response to blocked replication. We have shown previously that 

Arabidopsis plants defective in ATR are hypersensitive to the replication-blocking agent 

HU (hydroxyurea), likely due to a G2-arrest defect, and display a short, “hairy” root 

phenotype when grown on HU-containing plates. To further define molecular steps in the 

ATR-dependent cell-cycle response, we developed a genetic screen to isolate Arabidopsis 

mutants that display a similar (atr-like) phenotype when grown on HU plates. One 

resultant mutant, termed hus2 (for HydroxyUrea Sensitive), is hypersensitive to 

replication blocking agents and is defective in G2 arrest, similar to the atr mutant. 

However, complementation studies suggest hus2 is an independent locus from ATR. To 

identify the mutation responsible for the hus2 phenotype, we genetically mapped the 

hus2 locus to the distal end of chromosome 5 between the genetic (CAPS) markers RPS4 

and PDC2. To refine the location of the hus2 mutation between these markers, we 

generated additional (dCAPS) markers and found the hus2 mutation is located within an 

80 Kb region (18,470 Kb to 18,550 Kb), which encodes ~18 putative genes. Of these 18 

genes, none have a characterized or predicted role in the DNA damage response in plants. 

To identify the mutated locus in hus2, we are currently employing a combination of 

complementation (TAC clones), T-DNA knockouts, and sequencing. The results of these 

experiments will be presented. 

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P - 33. Isolation of Rice FOX Lines Tolerance to Ultraviolet-B Stress 

Shinya Takahashi, Tomoko Kuriyama, Takanari Ichikawa, Youichi Kondou, Yukako 

Hasegawa, Mika Kawashima, Shu Muto, Hirohiko Hirochika, Minami Matsui . Plant 

Functional Genomics Research Team, RIKEN Plant Science Center, 230-0045 Yokohama, 

Japan 

To identify large-scale useful rice gene, we are established rice FOX lines using 13,000 

non-redundant rice full length cDNAs to introduce them into Arabidopsis plants. 

Ultraviolet-B (UV-B) radiation can damage plants due to destruct several cell components, 

such as DNA, and reduce gene expressions. In higher plants, there are some protective 

mechanisms such as DNA repair, ROS scavenging system and accumulation of UV-B 

absorbing pigments. To isolate useful rice genes that confer UV-B stress tolerance, we try 

to screen UV-B tolerant mutants from rice FOX lines. We already isolated 49 UV-B tolerant 

mutant candidates in T2 generation from ca. 4,500 lines by root bending assay screening. 

Each isolated mutant contained one or two copies of rice cDNA which may encode cellcycle 

related genes, stress responsive genes, transcription factors and some unknown 

function genes. Now we differentiate the isolated mutants based on phenotypes of root 

growth with or without UV-B irradiation. This research is supported by the Special 

coordination funds for Promoting Science and Technology entitled Rapid identification of 

useful traits using rice full-length cDNAs. 

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P - 34. Towards efficient gene targeting in plants 

Seiichi Toki, Masaki Endo, Ryuya Ohishi, Masaaki Umeda, Kiyomi Abe, Hiroaki Saika, Keishi 

Osakabe. Plant Genetic Engineering Research Unit, National Institute of Agrobiological 

Scienced, Ibaraki 305-8602 Tsukuba, Japan 

Modification of genes by gene targeting (GT) should be the most precise gene 

manipulation technique and direct method for future molecular breeding in plants. In 

order to improve the frequency of GT, we are currently applying several approaches 

including the use of the zinc-finger nucleases, chromatin remodeling factors and cell 

cycle regulators. We are also trying to establish the model gene targeting system in 

Arabidopsis and rice. In this paper we would like to present current status of our study 

and discuss potential use of our study for the practical molecular breeding in plants. 

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P - 35. High-frequency site directed modification of the tobacco SuRB locus 

mediated by zinc-finger nucleases 

Jeffrey A. Townsend, David A. Wright, Ronnie J. Winfrey, Stacey Thibodeau-Beganny, 

Magdalena Eichtinger, J. Keith Joung, Daniel F. Voytas. Genetics, Cell and Developmental 

Biology, Iowa State University, 50011-3650 Ames, Iowa, United States 

Zinc-finger (ZF) proteins can be engineered to recognize unique chromosomal sites. When 

fused to a nuclease domain they make targeted double-strand breaks that stimulate 

homologous recombination between the chromosome and an extrachromosomal DNA 

donor. Here we demonstrate high frequency, site directed modification of the tobacco 

SuRB locus using engineered zinc-finger nucleases. The SuRB gene codes for 

acetohydroxyacid synthase (AHAS) the target enzyme for sulfonylurea and imidazolinone 

herbicides. Single amino acid changes in the enzyme confer herbicide resistance. 

Substitutions at amino acid P196A and S647T confer sulfonylurea and imidazolinone 

resistance respectively, while a W573L substitution confers resistance to both herbicide 

classes. Promotorless, nonfunctional fragments of the SuRB gene that encode these 

substitutions plus a novel restriction site have been cloned and introduced into tobacco 

protoplasts with pairs of zinc-finger nucleases designed to recognize specific SuRB 

sequence. The ZF proteins were generated by selection using a bacterial cell-based twohybrid 

(B2H) scheme. Herbicide resistant colonies occurred at a rate of more than 3% of 

that given by the control, functional SuRB mutants (3% of illegitimate recombination). The 

herbicide resistance profile of the recombinants in all cases matches that expected given 

the donor DNA used. 

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P - 36. Designed zinc finger nucleases as genome editing tools 

Fyodor Urnov, Jeffrey Miller, Russell DeKelver, Erica Moehle, Jeremy Rock, Kenneth Kim, 

Edward Rebar, Philip Gregory, Michael Holmes. Genomics, Sangamo BioSciences, 94804 

Richmond, United States 

Designed zinc finger nucleases (ZFNs) provide a generally applicable solution to a 

fundamental problem in biology: the efficient alteration of genotype in plant and animal 

cells to an investigator-specified allelic form. Work by Jasin et al on the homing 

endonuclease I-SceI (1) and the invention of ZFNs by Chandrasegaran et al (2) provided 

the basis for work by Carroll et al on ZFN-driven homologous recombination in Xenopus 

(3), followed by studies of Porteus and Baltimore on reporter gene targeting in human 

cells (4), which, importantly, have been recapitulated in Arabidopsis by Drews et al (5) 

and in tobacco cells by Voytas et al (6). The transition to editing native loci of one’s 

choosing was enabled by the development of designed zinc finger proteins (7) that allow 

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one to target a DSB to essentially any position in the genome of any cell, and yield rapid, 

selection-free, high efficiency gene correction (8) and gene addition (9) at endogenous 

loci. We will describe recent progress in designed ZFN technology: our development of 

massively parallel tools for studying genome editing, the structure-based redesign of 

ZFNs aimed at ensuring single-locus specificity of the editing process, and the remarkable 

efficiency of single-step endogenous gene disruption we have observed in a broad range 

of cell types. 1. Rouet, P., et al. 1994. Proc Natl Acad Sci U S A 91:6064-8. 2. Kim, Y. G., et 

al. 1996. Proc Natl Acad Sci U S A 93:1156-60. 3. Bibikova, M., et al. 2001. Mol Cell Biol 

21:289-97. 4. Porteus, M. H., and D. Baltimore. 2003. Science 300:763. 5. Lloyd, A., et al. 

2005. Proc Natl Acad Sci U S A 102:2232-7. 6. Wright, D. A., et al. 2005. Plant J 44:693- 

705. 7. Pabo, C. O., et al. 2001. Annu Rev Biochem 70:313-340. 8. Urnov, F. D., et al. 2005. 

Nature 435:646-51. 9. Moehle, E. A., et al. 2007. Proc Natl Acad Sci U S A 104:3055-3060. 

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P - 37. Strategies to modify and monitor gene targeting and meiotic 

recombination in crops. 

Philippe Vain, Vera Thole, Barbara Worland, Katie Mayes, Upendra Devisetty, Chanate 

Malumpong, Sean Mayes. Agricultural and Environmental Sci (Biosciences), University of 

Nottingham, LE12 5RD Loughborough, United Kingdom 

Significant progress has been made recently in perturbing genetic recombination 

processes in model plants, particularly Arabidopsis thaliana and tobacco. While these 

approaches have not yet coalesced into practical and efficient methods to achieve Gene 

Targeting (GT) and to modify meiotic recombination, a firm foundation is being laid. We 

have been looking at opportunities to test promising results in model di- and 

monocotyledonous species, with an ultimate aim to manipulate these processes in crop 

species, particularly wheat. We have developed a deficient GUS-based marker system, 

which could be repaired by GT. As this system does not require plant regeneration, we 

can rapidly examine very large numbers of events. A number of genes involved in 

recombination, particularly in rice have been cloned into expression vectors and we are 

beginning to characterise the effect of these genes in A. thaliana as a first step. The 

system we have developed should allow the same recombination genes to be tested in a 

number of species (A. thaliana, tobacco and rice), allowing rapid follow up of positive 

results from models to crops. We will use the same transgenic lines that have been tested 

for GT effects to assess effects in meiotic recombination, through crossing and 

microsatellite analysis. In addition to this, we are currently screening neutron deletion 

material of wheat to identify deletion of specific homeologues of RAD51 and DMC1 

(which may not cause full sterility in this hexaploid) and intend to compare these with 

characterisation of rice Tos17 insertional inactivation lines for the rice equivalents (where 

two versions of each gene exist). Results to date and our future plans will be presented. 

---------------------------------------------------------------------------------------------------------------------------------- 

P - 38. Reverse Progeny Mapping 

Aat Vogelaart, Johan Schut, Rob Dirks, Cilia Lelivelt. Celbiology, Rijk Zwaan Breeding BV, 

4793 RS Fijnaart, Netherlands 

A method is provided for mapping traits in organisms, in particular in plants. The 

method comprises a) providing a population of SDR-0 organisms, that each arise from 

one member of a population of unreduced cells resulting from second division 

restitution, in particular a population of unreduced spores; b) producing SDR-I progeny 

populations of each of these SDR-0 organisms; c) phenotyping the SDR-I progeny 

populations to identify segregating traits within each SDR-I progeny population; d) if 

segregating progeny is present in a SDR-I progeny population genotyping the 

corresponding SDR-0 organism and comparing the genotype thereof with the genotype of 

the other SDR-0 organism to identify heterozygous chromosomal regions associated with 

Page 47 sur 56


the occurrence of the segregating trait identified in the said SDR-I progeny population. 

This method provides a reliable method for the mapping of complex traits and is a very 

efficient alternative to the development and use of heterogeneous inbred families. WO 

2006/094774. 

---------------------------------------------------------------------------------------------------------------------------------- 

P - 39. A functional analysis of DNA ligases from Arabidopsis thaliana 

Wanda M. Waterworth, Ghzaleh Masnavi, Claire Provost, Paul Sunderland, Christopher E. 

West, Clifford M. Bray. Molecules to Cells, University of Manchester, UK, M13 9PT 

Manchester, United Kingdom Faculty of Life Sciences, University of Manchester, Manchester 

M13 9PT, U.K.; Centre for Plant Sciences, University of Leeds, Leeds, LS2 9JT, UK 

Eukaryotic organisms possess multiple DNA ligases with distinct roles in DNA 

metabolism. In plants, DNA ligases are crucial for the maintenance of nuclear, 

mitochondrial and plastid genome stability and both the levels and their intracellular 

distribution are tightly regulated. The three DNA ligase genes expressed in Arabidopsis 

(DNA ligases I, IV and VI; AtLIG1, AtLIG4 and AtLIG6) have been functionally 

characterized in our laboratory. AtLIG1 has roles in both DNA repair and replication, and 

possibly recombination, and is indispensable for cell viability. AtLIG1 expresses one 

major and two minor mRNA transcripts differing only in the length of the 5’ untranslated 

leader sequences preceding a common ORF. Intracellular targeting of DNA ligase 1 is 

controlled via an evolutionarily conserved posttranscriptional mechanism that involves 

the use of alternative start codons to translate multiple ligase activities from a single 

transcript. A further level of control involves hierarchical dominance of the different 

targeting sequences when more than one targeting sequence is present in the same ligase 

isoform. The maintenance of genomic integrity involves the function of all three DNA 

ligases in Arabidopsis. As with many DNA repair factors, the ligases are likely to form 

dynamic multi protein complexes to repair DNA damage. Ongoing studies are concerned 

with the identification and characterisation of interacting protein partners for AtLig1 and 

the physiological functions and importance of the novel plant specific ligase 6. 

---------------------------------------------------------------------------------------------------------------------------------- 

P - 40. I-SceI-stimulated intrachromosomal homologous recombination in maize 

Sophie Wehrkamp-Richter, Wyatt Paul, Julien Levy, Samuel LeGoff, Jean-Baptiste Laffaire, 

Fabienne Degroote, Pascual Perez, Georges Picard. Gene Function and Maize Traits Group, 

Biogemma, 63000 Clermont-Ferrand, France 

Gene targeting (GT) by homologous recombination (HR) allows the precise modification of 

any gene. GT has become an indispensable tool in yeast and mouse, however, this tool is 

still missing in higher plants since the frequency of homologous recombination in plants 

is too low (10-4 to 10-5) compared to the frequency of illegitimate recombination (NHEJ). 

However, in model plants, induction of a double strand break at the target locus by the 

expression of the meganuclease I-SceI results in a dramatic stimulation of HR. In 

collaboration with Cellectis (who develop meganuclease technologies) we are applying 

these results to maize in order to develop an efficient gene targeting system for a major 

crop plant. We have generated maize plants which contain a reporter of 

intrachromosomal HR and plants with constitutive I-SceI expression. In this assay the 

frequency of HR is estimated by the reconstitution by HR, of GUS in pollen. Preliminary 

data suggests that I-SceI -induced DSBs can stimulate HR in maize. We are now 

developing an inducible meganuclease in order to control HR and develop vectors that 

both measure RH and NHEJ in maize. 

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P - 41. Programmed response to IR-induced genomic stress 

Kaoru Yoshiyama, Clare Robertson, Kevin Culligan, Anne Britt. Plant Biology, University fo 

California, Davis, 95616 Davis, United States 

Cellular responses to DNA damage are many and varied. Many of these responses are 

programmed, and the complete response requires the presence of multiple signal 

transduction pathways. The genetic requirements for these responses can be difficult to 

sort out in mammals, as many defects in damage repair or response lead to the induction 

of apoptosis. Plants, fortunately, are not as exquisitely sensitive to mismanaged damage, 

and so make excellent model systems for the study of the roles of these proteins both in 

response to acute damage, and in the general maintenance of unperturbed mitotic and 

meiotic cells. Whole-genome analysis indicates that plants, unlike yeasts and mammals, 

exhibit a robust transcriptional response to DNA damage that includes many genes 

relevant to DNA repair and cell cycle regulation. However, this response is governed, at 

least in part, by the same signal transduction components that are required for other 

types of damage response in yeasts and mammals. Thus plants may make an excellent 

model system for the genetic analysis of DNA damage response. In this presentation we 

will focus on our efforts, suing both classical genetic and reverse genetic techniques, to 

identify components of the plant DNA damage response pathway. 

---------------------------------------------------------------------------------------------------------------------------------- 

P - 42. Identification of genes required for DNA-damage-induced cell cycle arrest 

in Arabidopsis 

Kaoru Yoshiyama, Anne Britt. Plant Biology, University of California Davis, 95616 Davis, 

United States 

The genome of a living organism is subjected to a wide variety of genotoxic stresses of 

both endogenous and environmental origin. DNA-damage-induced cell cycle checkpoints 

are a crucial component of genome maintenance. These checkpoints sense DNA damage, 

pause the cell division cycle to provide time for repair, and finally release the cell cycle 

from arrest. The mammalian ATM and ATR protein kinases are key proteins involved in 

the cell cycle arrest, and Arabidopsis has ATM and ATR homologs. However, in contrast 

to yeast or mammalian cells, little is known about DNA damage checkpoint in plants. 

AtXPF is a homolog of human repair endonuclease XPF. The xpf mutant of Arabidopsis 

can’t form true leaves after irradiation as seeds, showing sensitivity to gamma-radiation. 

We screened for suppressor mutants in the xpf background using EMS and isolated sog 

(suppressor of gamma radiation) mutants that can form true leaves following radiation, 

suggesting that this cell cycle arrest is due to a signaling mechanism rather than to 

inhibition of cell cycle by physically blocking the cellular machinery. We have observed 

that xpf sog1-1 seeds are hypersensitive to HU (hydroxyurea), an inhibitor of 

ribonucleotide reductase. They could germinate on the HU plates, but after that, they 

were dead. Surprisingly, when the seeds are grown on “no HU” plates for 5 days, and the 

seedlings are transferred to HU plates, the plants can grow as well as wild type. These 

results suggest that SOG might be important during or immediately after germination. We 

reported that in wild-type Arabidopsis, hundreds of genes are induced in response to 

gamma-radiation, and the induction is dependent on ATM. BRCA1, RAD51 and CYCB1;1 

transcriptions increased rapidly and strongly. Interestingly, the transcription inductions 

of these genes were compromised severely in the xpf sog1-1 mutant, suggesting that the 

SOG gene plays an important role in upstream of signal transduction like ATM. (The xpf 

mutation does not affect the induction of the genes.) To identify the SOG gene, we’re 

currently mapping the mutation, and we know that the SOG gene lies in the vicinity of 

CIW12 marker on chromosome I. We are hoping that identifying the function of SOG gene 

is a good way to clarify the mechanism of checkpoint mechanism in plants and, perhaps, 

in animals. 

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Page 50 sur 56

Participants 

---------------------------------------------------------------------------------------------------------------------------------- 

Kiyomi Abe 

National Institute of Agrobiological Sciences 

Plant Genetic Engineering Research Unit 

Japan 

kiyomi@affrc.go.jp 

Olena Alkhimova 

Institute of Molecular Biology and Genetics 

National Academy of Sciences of Ukraine 

Ukraine 

o.g.alkhimova@imbg.org.ua 

Lorinda Anderson 

Colorado State University 

Biology 

United States 

lorinda.anderson@colostate.edu 

Karel J. Angelis 

Inst. Experimental Botany 

Molec. Farming and DNA Repair 

Czech Republic 

angelis@ueb.cas.cz 

Geert Angenon 

Vrije Universiteit Brussel 

Plant Genetics 

Belgium 

Geert.Angenon@vub.ac.be 

Susan Armstrong 

University of Birmingham 

School of Biosciences 

United Kingdom 

s.j.armstrong@bham.ac.uk 


Naomi Avivi-Ragolsky 

Weizmann Institute of Science 

Plant Sciences 

Israel 

lpavivi@wicc.weizmann.ac.il 

Kamakshi Balakrishnan 

Tata Institute of Fundamental Research (TIFR) 

Department of Biological sciences 

India 

kamakshihema@tifr.res.in 

Abdellah Barakate 

University of Dundee 

based at Scottish Crop Research Institute 


abdellah.barakate@scri.ac.uk 

Joke Baute 

VIB - Ghent University 

Plant Systems Biology 

Belgium 

joke.baute@psb.ugent.be 

Dagmar Berenova 

Institute of Experimental botany ASCR 

Laboratory of DNA repair 


berenova@ueb.cas.cz 

Christian Biesgen 

SunGene GmbH 

Molecular Enabling Technologies 

Germany 

Christian.Biesgen@sungene.de 

Dita Bohdanecka 

Institute of experimental botany, 

Academy of sciences 

Molecular farming and DNA Repair Laboratory 


bohdanecka@ueb.cas.cz 

Anne Britt 

University fo California, Davis 

Plant Biology 

United States 

abbritt@ucdavis.edu 

Jitka Brouzdova 

Institute of Experimental Botany 

Laboratory of DNA repair and molecule farm 


brouzdova@ueb.cas.cz 

Paul Bundock 

Keygene N.V. 

Upstream Research 

Netherlands 

paul.bundock@keygene.com 

Tamara Byk-Tennenbaum 

Evogene Ltd 

Israel 

tamara.byk@evogene.com 

Charles Cai 

Dow AgroSciences, LLC. 

Biochemistry and Molecular Biology 

United States 

ccai2@dow.com 

W. Zacheus Cande 

Univeersity of California, Berkeley 

Dept of Molecular and Cell biology 

United States 

zcande@berkeley.edu 

Marie-Edith Chabouté 

IBMP/CNRS UPR2357 Strasbourg 

plant cellular biology 

France 

marie-edith.chaboute@ibmp-ulp.u-strasbg.fr 

Page 51 sur 56

Cyril Charbonnel 

CNRS UMR6547 

Université Blaise Pascal 

France 

cycharbo@univ-bpclermont.fr 

Monique Compier 

Add2X Biosciences BV 

Netherlands 

compier@add2x.com 

Toon Cools 

Ghent University / VIB 


Belgium 

tocoo@psb.ugent.be 

Gregory Copenhaver 

The University of North Carolina at 

Chapel Hill 

Dept. of Biology and The Carolina Center 

for Genome Sciences 

United States 

gcopenhaver@bio.unc.edu 

Kevin Culligan 

University of New Hampshire 

Biochemistry and Molecular Biology 

United States 

k.culligan@unh.edu 

Kathleen D'Halluin 

Bayer BioScience N.V. 

Research 

Belgium 

Kathleen.D'Halluin@bayercropscience.com 

Sylvie De Buck 

VIB, UGent 


Belgium 

sybuc@psb.ugent.be 


Sylvia de Pater 

Leiden University 

Institute Biology Leiden 

Netherlands 

b.s.de.pater@biology.leidenuniv.nl 

PHILIP DEAN 

UNIVERSITY OF LEEDS, UK 

CENTRE FOR PLANT SCIENCES 


bmbpjd@leeds.ac.uk 

Fabien Delacote 

Université paris sud / CNRS 

Institut de biotechnologie des plantes 

UMR8618 

France 

fabien.delacote@u-psud.fr 

Annie Depeiges 

CNRS UMR6547 


France 

annie.depeiges@univ-bpclermont.fr 

Rob Dirks 

Rijk Zwaan Breeding BV 

Biotechnologial laboratory 

Netherlands 

d.van.noort@rijkzwaan.nl 

Marie-Pascale Doutriaux 

CNRS Université Paris XI 

Institut de Biotechnologie des Plantes 

France 

marie-pascale.doutriaux@u-psud.fr 

manuel dubald 

Bayer CropScience 

BioScience 

France 

manuel.dubald1@bayercropscience.com 

Emeline Dubois 

Université Paris XI-CNRS 

Institut de Biotechnologie des Plantes- 

UMR8618 

France 

emeline.dubois@u-psud.fr 

Masaki Endo 


Plant Engineering Research Unit, 

Division of Plant Sciences 

Japan 

mendo@affrc.go.jp 

Yonat Eshchar 



Israel 

yonat.eshchar@wicc.weizmann.ac.il 

Jiri Fajkus 

Masaryk University and Institute of 

Biophysics ASCR 

Functional Genomics and Proteomics 


fajkus@sci.muni.cz 

Chris Franklin 

University of Birmingham 

School of Biosciences 


F.C.H.Franklin@bham.ac.uk 

Maria Gallego 

Universite Blaise Pascal 

CNRS UMR 6547 

France 

megalleg@univ-bpclermont.fr 

Marcelina Garcia Aguilar 

Institute for research and Development (IRD) 

Plant Genome and Development Laboratory 

France 

garciam@mpl.ird.fr 

Pascal Genschik 

Institut de Biologie Moleculaire 

des Plantes CNRS 

France 

Pascal.Genschik@ibmp-ulp.u-strasbg.fr 

Page 52 sur 56

tom gerats 

Radboud University 

IWWR/Plant Genetics 

Netherlands 

t.gerats@science.ru.nl 

Mathilde Grelon 

INRA 

Genetic and Plant Breeding 

France 

grelon@versailles.inra.fr 

Manju Gupta 

Dow AgroSciences 

Plant Genetics and Biotechnology 

United States 

mgupta@dow.com 

James E. Haber 

Brandeis University 

Rosenstiel Basic Medical Sciences Research 

Center 

United States 

haber@brandeis.edu 

Claire Halpin 

University of Dundee 

Plant Research Unit, College of Life Sciences 


c.halpin@dundee.ac.uk 

Frank Hartung 

University of Karlsruhe 

Botanical Institute II 

Germany 

frank.hartung@bio.uka.de 

John Hays 

Oregon State University 

Dept. of Environmental and 

Molecular Toxicology 

United States 

haysj@science.oregonstate.edu 

Veena Hedatale 

Radboud University 


India 

v.hedatale@science.ru.nl 

Barbara Hohn 

Freidrich Meischer Institut 

Switzerland 

barbara.hohn@fmi.ch 


Paul Hooykaas 

Leiden University /Institute of Biology 

Leiden (IBL) 

Molecular and Developmental Genetics 

Netherlands 

p.j.j.hooykaas@biology.leidenuniv.nl 

Shigeru Iida 

National Institute for Basic Biology 

Division of Molecular Genetics 

Japan 

shigiida@nibb.ac.jp 

Doolyi Kim 

National Institute of 

Agricultural Biotechnology 

Cell and Genetics 

South Korea 

dykim@rda.go.kr 

Jaroslav Kozak 

Academy of Sciences of the Czech Republic 

Institute of Experimental Botany, 

Laboratory of DNA repair 


kozak@ueb.cas.cz 

Neeraja Krishnan 

Tata Institute of Fundamental Research 

Department of Biological Sciences 

India 

neeraja@tifr.res.in 

julien lang 

IBMP/CNRS 

department of cellular biology 

France 

Julien.Lang@ibmp-ulp.u-strasbg.fr 

samuel le goff 

CNRS UMR6547 


France 

samuel.le_goff@univ-bpclermont.fr 

Cilia Lelivelt 

Rijk Zwaan Breeding BV 

Celbiology 

Netherlands 

d.van.noort@rijkzwaan.nl 

Avraham Levy 

Weizmann Institute of Sciences 


Israel 

avi.levy@weizmann.ac.il 

Franck Lhuissier 

Keygene 

Upstream research 

Netherlands 

franck.lhuissier@keygene.com 

Michal Lieberman - Lazarovich 


Plant Sciences department 

Israel 

michal.lieberman@weizmann.ac.il 

Beatrice Lindhout 

Institute of Biology, Leiden University 

Molecular and developmental genetics 

Netherlands 

b.i.lindhout@biology.leidenuniv.nl 

L. Alexander Lyznik 

Pioneer Hi-Bred International, Inc 

Crop Genetics Research 

United States 

alex.lyznik@pioneer.com 

Page 53 sur 56

Sean Mayes 

University of Nottingham 

Agricultural and Environmental Sci 

(Biosciences) 


sean.mayes@nottingham.ac.uk 

Cathy Melamed Bessudo 

The Weizmann Institute of Science 

Plant Sciences Department 

Israel 

cathy.bessudo@weizmann.ac.il 

Raphaël Mercier 

INRA 

genetics 

France 

rmercier@versailles.inra.fr 

Marie Mirouze 

Université de Genève Sciences III 

Laboratory of Plant Genetics 

Switzerland 

marie.mirouze@bioveg.unige.ch 

Jon Mitchell 

Dow AgroSciences LLC 

R&D/Dept. of Biochemistry 

and Molecular Biology 

United States 

jcmitchell@dow.com 

Marie-Hélène Montané 

CEA 

Direction des Sciences du vivant 

France 

marie-helene.montane@cea.fr 

Graham Moore 

John Innes Centre 

Crop Genetics 


graham.moore@bbsrc.ac.uk 

Keishi Osakabe 


Division of Plant Sciences 

Japan 

kosakabe@affrc.go.jp 

Michael Pacher 

University of Karlsruhe / Institute of Botany II 

Molecular Biology & Biochmeistry of plants 

Germany 

Michael.Pacher@botanik2.uni-karlsruhe.de 

Frédéric Pâques 

Cellectis S.A. 

France 

paques@cellectis.com 


Jerzy Paszkowski 

Université de Genève 

Switzerland 

Jerzy.Paszkowski@bioveg.unige.ch 

Wyatt PAUL 

Biogemma 

Cereal Functional Analysis Group 


wyatt.paul@biogemma.com 

Janny Peters 

Radboud University Nijmegen 


Netherlands 

jl.peters@science.ru.nl 

Thomas Peterson 

Iowa State University 

Genetics, Development and Cell Biology 

United States 

thomasp@iastate.edu 

Holger Puchta 

University Karlsruhe 

Botany II 

Germany 

holger.puchta@bio.uka.de 

Bernd Reiss 

MPI für Züchtungsforschung 

Department Coupland 

Germany 

reiss@mpiz-koeln.mpg.de 

Karel Riha 

Austrian Academy of Sceinces, 

Gregor Mendel Institute 

Austria 

karel.riha@gmi.oeaw.ac.at 

Eddy Risseeuw 

National Research Council 

Plant Biotechnology Institute 

Canada 

risseeuwe@nrc.ca 

Max Roessler 

Austrian Academy of Sceinces, 


Austria 

max.roessler@gmi.oeaw.ac.at 

Teresa Roldan-Arjona 

Universidad de Cordoba 

Departamento de Genetica 

Spain 

ge2roarm@uco.es 

Cyril Saintenac 

INRA 

UMR 1095 Amelioration et Sante des plantes 

France 

saintena@clermont.inra.fr 

Ayako Sakamoto 

Japan Atomic Energy Agency 

Radiation-Applied Biology 

Japan 

sakamoto.ayako@jaea.go.jp 

Aviva Samach 

Weizmann Institute of sciences 

Dept. of plant sciences 

Israel 

aviva.samach@weizmann.ac.il 

Page 54 sur 56

Iva Santruckova 

Masaryk University 

Functional Genomics and Proteomics 


iva.sant@mail.muni.cz 

Didier Georges Schaefer 

INRA, 

Station de génétique et 

amélioration des plantes 

France 

dschaefer@versailles.inra.fr 

Martina Schirato 

Austrian Academy of sciences 


Austria 

martina.schirato@gmi.oeaw.ac.at 

Peter Schlögelhofer 

Max F. Perutz Laboratories/ 

University of Vienna 

Dept. of Chromosome Biology 

Austria 

peter.schloegelhofer@univie.ac.at 

Ingo Schubert 

Leibniz-Institute of Plant Genetics 

and Crop Plant Research 

Cytogenetics and Genome Analysis 

Germany 

schubert@ipk-gatersleben.de 

Dorothy Shippen 

Texas A&M University 

Biochemistry and Biophysics 

United States 

dshippen@tamu.edu 

Vipula Shukla 

Dow AgroSciences, LLC 

Discovery R&D 

United States 

vkshukla@dow.com 


Marketa Smidkova 

IEB ASCR 

Laboratory of molecular farming 

and DNA repair 


m.smidkova@seznam.cz 

Pierre SOURDILLE 

INRA UMR1095 

Amélioration et Santé des Plantes 

Structure, Function and Evolution 

of Wheat Genomes 

France 

pierre.sourdille@clermont.inra.fr 

Wojciech Strzalka 

Jagiellonian University 

Faculty of Biochemistry, Biophysics 

and Biotechnology 

Poland 

strzalka@if.uj.edu.pl 

Shinya Takahashi 

RIKEN Plant Science Center 

Plant Functional Genomics Research Team 

Japan 

shinya@psc.riken.jp 

Seiichi Toki 

National Institute of Agrobiological Scienced 

Plant Genetic Engineering Research Unit 

Japan 

stoki@affrc.go.jp 

Jeffrey Townsend 


Genetics, Cell and Developmental Biology 

United States 

townend@iastate.edu 

Tzvi Tzfira 

University of Michigan 

Department of Molecular, Cellular, & 

Developmental Biology 

United States 

ttzfira@umich.edu 

Fyodor Urnov 

Sangamo BioSciences 

Genomics 

United States 

furnov@sangamo.com 

Frédéric Van Ex 

Vrije Universiteit Brussel (V.U.B.) 

Laboratory of Plant Genetics 

Belgium 

fvex@vub.ac.be 

Jean-Baptiste VANNIER 

CNRS UMR6547 


France 

jbvannie@univ-bpclermont.fr 

Laurent Vespa 

Texas A&M University 

Biochemistry and Biophysics 

United States 

vespa@tamu.edu 

Corina Belle Villar 

ETH Zurich, Institute of Plant Sciences 

Plant Developmental Biology 

Switzerland 

cvillar@ethz.ch 

Dan Voytas 


Dept. of Genetics, Development & Cell Biology 

United States 

voytas@iastate.edu 

Koichi Watanabe 

Leibniz Institute of Plant Genetics 

and Crop Plant Research 

Germany 

watanabe@ipk-gatersleben.de 

Page 55 sur 56

Wanda Melody Waterworth 

University of Manchester, UK 

Molecules to Cells 


mqbsswmw@manchester.ac.uk 

Sophie Wehrkamp-Richter 

Biogemma 

Gene Function and Maize Traits Group 

France 

sophie.wehrkamp@biogemma.com 

Clifford Weil 

Purdue University 

Dept of Agronomy 

United States 

cweil@purdue.edu 

Chris West 

University of Leeds 

Centre for Plant Sciences 


c.e.west@leeds.ac.uk 


Charles White 

CNRS UMR6547 


France 

chwhite@univ-bpclermont.fr 

Wei Xiao 

University of Saskatchewan 

Department of Microbiology and Immunology 

Canada 

wei.xiao@usask.ca 

Kaoru Yoshiyama 

University of California Davis 

Plant Biology 

United States 

kyoshiyama@ucdavis.edu 

Alicja Ziemienowicz 

Jagiellonian University 

Faculty of Biochemistry, Biophysics 

and Biotechnology 

Poland 

alicja@mol.uj.edu.pl 

Page 56 sur 56

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