Service Communautaire d'Information sur la Recherche et le Développement - CORDIS

Tou genes cloned into yeast strains

To check whether the expression and activity of ToMO is achievable in a simple eukaryotic organism, as a preparative step for the transformation of arbuscular-mycorrhizal fungi with tou genes, the cloning of touABCDEF genes for the transformation of Saccharomyces cerevisiae has been performed. tou genes have been preliminary cloned from a vector carrying all the six structural genes into modified prokaryotic vectors.

ToMO subunits have been expressed either as wild-type or as histidine-tagged proteins in Escherichia coli; policlonal antibodies against ToMO proteins have been produced by inoculation in rabbit, in order to be used for western blot analyses. tou genes have been subsequently cloned into centromeric yeast expression vectors, and two yeast strains have been transformed to achieve the expression of wild-type and recombinant ToMO proteins.

By western blot we checked the expression of ToMO subunits; the expression of recombinant ToMOHisA,E,D,F was detected using commercial antibodies, while the polyclonal antibodies above mentioned have been used for the wild-type ToMO D,F proteins.

While there is wealth of publications concerning the cloning and expression of genes belonging to complex organisms such as eukaryotes into simpler models, the converse is scarcely documented. The difficulties experienced during the experiments reflect the higher complexity of the eukaryotic system. In any case, to our knowledge, this is the first example of the cloning and expression of such a complex enzymatic system in eukaryotes.

More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/

Reported by

Università degli Studi del Piemonte Orientale "Amedeo Avogadro"
Dipartimento di Scienze dell'Ambiente e della Vita, Via Bellini 25/g
15100 Alessandria
Italy