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FP5

ECCO Résumé de rapport

Project ID: QLG2-CT-1999-00454
Financé au titre de: FP5-LIFE QUALITY
Pays: Germany

Basic study of cell division genes

Juergens' group focused on the map-based cloning of genes that are deficient in cytokinesis. A total of 38 putative cytokinesis mutants that have been analysed. Complementation tests define 6 different genes. Two of the genes, KNOLLE and KEULE, were previously isolated by map-based cloning. Mapping populations have been established for 4 additional genes.

The KIS gene and 4 PILZ group genes have been isolated by map-based cloning and shown to encode proteins required for microtubule formation and cell division but not for actin filaments and cell growth (Steinborn et al., 2002). Specifically, KIS encodes tubulin-folding cofactor A. The mutant kis has a strong trichome phenotype indicating a defect in proper cell division. The HIK gene has also been isolated by map-based cloning and shown to encode a plant-specific cell cycle-regulated kinesin motor protein involved in phragmoplast microtubule dynamics (Strompen et al., 2002).

The RUK gene has been mapped to a 250 kb interval in the top arm of chromosome 5. Fine mapping of RUK was impeded by low recombination frequency within the relevant interval. New mapping populations have been generated for two different alleles with the aim to identify more relevant recombinants in a different genetic background. In addition, cosmid and transformation-competent BAC clones from the critical genomic interval have been transformed into ruk/ RUK plants and will be further analysed for rescuing activity. The RUK gene encodes a ca. 150kDa protein that appears to be plant-specific and, except for an N-terminal putative kinase domain, has no distinctive domain features. Experiments have been initiated (i) to localise a myc-tagged version of the protein subcellularly and (ii) to examine whether cell cycle-regulated expression of the RUK protein rescues ruk mutant embryos.

To identify a KNOLLE-interacting synaptobrevin (Syb), representatives of 4 subgroups of Syb coding sequences have been tested for their ability to interact with KNOLLE in a GST-fusion pull-down assay. One candidate, AtSyb4, interacts strongly with KNOLLE in the presence of a KNOLLE-interacting t-SNARE, the SNAP25 homologue SNAP33, which has been shown to co-localise with KNOLLE during cell-plate formation.

Recombinant AtSyb4 protein has been used to generate rabbit polyclonal antiserum, which, however, needs to be purified for immuno-localisation studies. Screening of a T-DNA insertion library has yielded syb4 candidate lines, which will be further characterised genetically and phenotypically. Screening of a T-DNA insertion library has yielded two syb4 candidate lines. In both lines, the insertions are poorly transmitted, suggesting that SYB4 is required for gametophyte development. The mutants are being characterised genetically and phenotypically in more detail. The mutant line is gametophyte lethal.

Contact

Gerd JUERGENS, (Head of unit)
Tél.: 49-7071-29-78887
Fax: 49-7071-295797
E-mail