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ACCESS Résumé de rapport

Project ID: ICA4-CT-2002-10011
Financé au titre de: FP5-INCO 2
Pays: Spain

DNA microarrays

Metabolic processes, like the whole metabolism have to function within the context of the host and be tightly regulated, so detrimental energy fluxes are avoided in order to not compromise host-fitness and survival. These mechanisms need to link the biodegradation pathway to host physiology by several bridges.

We have designed and used custom DNA arrays to be able of studying as a whole, the links between the cell physiology and a given degradative pathway. We choose genes, which report the status of the cell; sigma factors, heat shock related chaperones, iron-regulated genes, sulfur regulated or nitrogen response elements etc. In one of the arrays, we printed such genes of Pseudomonas along with the well-characterized genes coding for the TOL pathway, which is composed by four transcriptional units. The array of both stress genes and TOL genes was employed to see how the cells behave in the presence of different effector molecules of the pathway and the response of the pathway on mRNA levels.

The second designed array is based on the genome of the 2,4-dichlorophenoxyacetate degrader Cupriavidus necator JMP134. The available complete genome sequence allowed performing the metabolic reconstruction of the aromatic catabolism of this bacterium. This strain is able to use more than 60 aromatic compounds as a sole carbon and energy source, including halo-, and nitro- aromatics, C6-C2, and C6-C3 compounds. About 300 catabolic genes have been identified, encoding the peripheral reactions and the twelve main ring cleavage routes that the in vivo studies predict. We developed a catabolic/physiological 50-mer oligonucleotide microarray containing 348 key probe sequences, including most of the genes involved in catabolic pathways for aromatic compounds and key genes involved in global regulation.

A third designed system is a phylogeny-based DNA array for application to the analysis of microbial population diversity and dynamics in soils and other complex ecosystems. The microbial phylogeny-based DNA array system is dependent upon the sequence differences contained within hyper-variable regions of the rRNA genes. More than 2000 bacteria were evaluated for determining the best regions of the 16S rRNA genes to be used for differentiation and identification. These regions are targeted for PCR-amplification to generate the polynucleotide products to be used as probes (as opposed to using oligonucleotide probes) of approximately 250-300 nucleotides in length. A major advantage of the proposed strategy is the potential for generating the reference DNA or probes, from microbial isolates, from cloned DNA or from total PCR-amplified community DNA/RN, without prior sequence knowledge or dependence upon evolving sequence databases.

PCR-products or genomic DNAs used as reference DNAs are spotted and total community DNA polynucleotide probes are generated by PCR for hybridisation. The level of resolution for such hybridisation probes is approximately 5% sequence difference, corresponding to genus and sub-genus level differentiation. Through a combination of probes with progressively higher resolution levels, the identities of the individual members of a given microbial community can be analysed.

Informations connexes

Reported by

Campus de la Universidad Autonoma de Madrid
28049 MADRID