Інновації в сучасній освіті

22 ref. doi: 10.1093/nar/gng158International audienc

Identification by mutational analysis of four critical residues in the molybdenum cofactor domain of eukaryotic nitrate reductase

AbstractThe nucleotide sequence of the nitrate reductase (NR) molybdenum cofactor (MoCo) domain was determined in four Nicotiana plumbaginifolia mutants affected in the NR apoenzyme gene. In each case, missense mutations were found in the MoCo domain which affected amino acids that were conserved not only among eukaryotic NRs but also in animal sulfite oxidase sequences. Moreover an abnormal NR molecular mass was observed in three mutants, suggesting that the integrity of the MoCo domain is essential for a proper assembly of holo-NR. These data allowed to pinpoint critical residues in the NR MoCo domain necessary for the enzyme activity but also important for its quaternary structure

Mutation detection using ENDO1: Application to disease diagnostics in humans and TILLING and Eco-TILLING in plants

<p>Abstract</p> <p>Background</p> <p>Most enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA by a mismatch-specific endonuclease at mismatch sites and the analysis of the digestion product on a DNA sequencer. Important limitations of these methods are the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in pool of DNA, the cost of the analysis and the ease by which the technique could be implemented in a standard molecular biology laboratory.</p> <p>Results</p> <p>The co-agroinfiltration of ENDO1 and p19 constructs into <it>N. benthamiana </it>leaves allowed high level of transient expression of a mismatch-specific and sensitive endonuclease, ENDO1 from <it>Arabidopsis thaliana</it>. We demonstrate the broad range of uses of the produced enzyme in detection of mutations. In human, we report the diagnosis of the G1691A mutation in <it>Leiden factor-V </it>gene associated with venous thrombosis and the fingerprinting of HIV-1 quasispecies in patients subjected to antiretroviral treatments. In plants, we report the use of ENDO1 system for detection of mutant alleles of <it>Retinoblastoma</it>-<it>related </it>gene by TILLING in <it>Pisum sativum </it>and discovery of natural sequence variations by Eco-TILLING in <it>Arabidopsis thaliana</it>.</p> <p>Conclusion</p> <p>We introduce a cost-effective tool based on a simplified purification protocol of a mismatch-specific and sensitive endonuclease, ENDO1. Especially, we report the successful applications of ENDO1 in mutation diagnostics in humans, fingerprinting of complex population of viruses, and in TILLING and Eco-TILLING in plants.</p

Molecular analysis of Arabidopsis thaliana transparent testa (tt) genes in Brassica napus

Lotz T, Snowdon R, Horn R, et al. Molecular analysis of Arabidopsis thaliana transparent testa (tt) genes in Brassica napus. In: PLANT BREEDING Opportunities for new cruciferous crops. Rapeseed Congress Proceedings. 2003

GeneFarm, structural and functional annotation of Arabidopsis gene and protein families by a network of experts

Genomic projects heavily depend on genome annotations and are limited by the current deficiencies in the published predictions of gene structure and function. It follows that, improved annotation will allow better data mining of genomes, and more secure planning and design of experiments. The purpose of the GeneFarm project is to obtain homogeneous, reliable, documented and traceable annotations for Arabidopsis nuclear genes and gene products, and to enter them into an added-value database. This re-annotation project is being performed exhaustively on every member of each gene family. Performing a family-wide annotation makes the task easier and more efficient than a gene-by-gene approach since many features obtained for one gene can be extrapolated to some or all the other genes of a family. A complete annotation procedure based on the most efficient prediction tools available is being used by 16 partner laboratories, each contributing annotated families from its field of expertise. A database, named GeneFarm, and an associated user-friendly interface to query the annotations have been developed. More than 3000 genes distributed over 300 families have been annotated and are available at http://genoplante-info.infobiogen.fr/Genefarm/. Furthermore, collaboration with the Swiss Institute of Bioinformatics is underway to integrate the GeneFarm data into the protein knowledgebase Swiss-Prot