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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 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. ---------------------------------------------------------------------------------------------------------------------------------- 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 Page 42 sur 56
<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 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) ---------------------------------------------------------------------------------------------------------------------------------- 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. ---------------------------------------------------------------------------------------------------------------------------------- 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 Page 43 sur 56
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