Objective Genome sequence data present new avenues to investigate certain diseases. Such studies would be facilitated by the availability of model systems in which to test potential targets. This project is aimed at developing such systems for lysyl-tRNA synthesis, an essential pathway in protein biosynthesis. Several pathogens contain novel enzymes for lysyl-tRNA synthesis that are ideal diagnostic and therapeutic targets. Also, defects in lysyl-tRNA synthesis are responsible for some mitochondrial myopathies. Adder initial in vitro studies, genes encoding the appropriate enzymes will be used to establish Bacillus subtilis tester strains. This will provide a means to readily test predictions from in vitro studies. The final validation will be performed with the pathogens themselves and with human cell lines. The general principles of functional genomics which will be developed in this project can be readily extended to other essential pathways.1. Bacterial class I lysyl-tRNA synthetases (KRS1). Genes encoding KRS1 were cloned from 3 pathogenic Borrelia strains which cause Lyme disease. KRS1 from B. burgdorferi was overproduced and purified for biochemical assays and antibody production;2. Human mitochondrial lysyl-tRNA synthetase.The gene for this enzyme has been cloned by the end of the project. Human cytosolic enzyme, different only by a few amino acids at the N-terminus, has been overexpressed and used for biochemical studies;3. Lysyl-tRNA synthetase-specific diagnostic targets and model inhibitors. The KRS1 encoding gene was validated as a diagnostic target for Lyme disease; KRS2-specific inhibitors were identified biochemically;4. Defined in vitro system for mitochondrial Lys-tRNALys synthesis. Human mitochondrial tRNA (wild-type and variants) have been prepared by in vitro translation of the corresponding cloned genes. Since the wild-type transcript does not fold correctly, a pseudo-wild type sequence has been engineered and validated as reference molecule. Variants have been prepared on the basis of this pseudo-wild-type sequence.Optimal aminoacylation conditions have been defined;5. Molecular basis of MERFF phenotype and of Leigh syndrome. The MERRF syndrome was shown not to be correlated to a defect in the aminoacylation properties of the mutated tRNA in vitro, nor to a structural defect of the tRNA;6. B. subtilis strains expressing foreign lysyl- tRNA synthetase genes;7. Comparative study of lysyl-tRNA synthetase activities in vitro and in vivo;8. Automated diagnosis of Lyme disease. A semi-automated PCR assay was established for the identification of pathogenic Borrelia isolates. Existing antigen-based protocols were validated and adapted to the XNA platform;9. Automated diagnosis of mitochondrial myopathies. A rapid and simple method to identify single-nucleotide polymorphisms in the human mitochondrial tRNA genes was developped. It is based on DNA/DNA hybridization on chips and has been validated with already diagnosed patient samples;10. Interim and final progress reports. All reports were delivered as required;11. Presentations and peer-reviewed publications. Numerous public presentations and publications in peer-reviewed journals served to disseminate project results;12. Differential proteomic maps of healthy and disease-carrying human mitochondria. A comparative proteomic approach of wild-type and MERRF mutated mitochondria allowed to distinguish 40 proteins of mitochondrial location but of cytosolic origin, undergoing significant quantitative variations in MERRF mitochondria. These proteins may be secondary effectors of the initial mutation and their perturbations may contribute to the disease status of the cell. According to their function, quantitative increase of some of the proteins may correspond to defense mechanisms of the cell to the disease;13.The technology for detection of specific antibodies in human serum samples advanced to a highly reproducible level by using the small synthetic peptide antigen as well as natural protein isolates. Using the same biochip surface 12 mitochondrial mutations can be detected. Automation detection has been recently achieved using a commercially available 3dimensional surface. Fields of science natural sciencesbiological sciencesgeneticsDNAsocial sciencessociologyindustrial relationsautomationnatural sciencesbiological sciencesgeneticsmutationnatural scienceschemical sciencesorganic chemistryaminesnatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes Programme(s) FP5-LIFE QUALITY - Specific Programme for research, technological development and demonstration on "Quality of life and management of living resources", 1998-2002 Topic(s) 1.1.1.-8. - Research into genomes and diseases of genetic origin Call for proposal Data not available Funding Scheme CSC - Cost-sharing contracts Coordinator STATENS SERUM INSTITUT EU contribution No data Address 5,Artillerivej 5 2300 KOEPENHAGEN Denmark See on map Total cost No data Participants (4) Sort alphabetically Sort by EU Contribution Expand all Collapse all CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE France EU contribution No data Address Rue Jules Hetzel 9 92190 Meudon See on map Total cost No data CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE France EU contribution No data Address Rue René Descartes 15 67084 STRASBOURG See on map Total cost No data THE PROVOST, FELLOWS AND SCHOLARS OF THE COLLEGE OF THE HOLY AND UNDIVIDED TRINITY OF QUEEN ELIZABETH NEAR DUBLIN HEREINAFTER TRINITY COLLEGE DUBLIN Ireland EU contribution No data Address Trinity College 2 DUBLIN See on map Total cost No data UNIVERSITY OF ULM Germany EU contribution No data Address Steinhoevelstrasse, 9 89075 ULM See on map Total cost No data
