RESUMO
The resistance to 5,5,5-trifluoro-DL-leucine, encoded by the dominant allele LEU4-1, was used as a selectable marker to transform laboratory and natural Saccharomyces cerevisiae strains by the lithium acetate procedure. Results of transformation of S. cerevisiae laboratory and wine natural strains showed that trifluoroleucine resistance is a very effective selection marker and can be widely used to transform prototrophic S. cerevisiae strains. The LEU4-1 gene could also be exploited to improve wine flavour, as indicated by the higher isoamyl alcohol content of the transformants compared to the parental strains.
Assuntos
Leucina/análogos & derivados , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/efeitos dos fármacos , Transformação Genética , 2-Isopropilmalato Sintase/genética , Resistência Microbiana a Medicamentos/genética , Fermentação , Proteínas Fúngicas/genética , Genes Dominantes , Marcadores Genéticos , Leucina/metabolismo , Leucina/farmacologia , Pentanóis/metabolismo , Plasmídeos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Vinho/microbiologiaRESUMO
Seven spontaneous Saccharomyces cerevisiae mutants that express dominant resistance to 5,5,5-trifluoro-DL-leucine have been characterised at the molecular level. The gene responsible for the resistance was cloned from one of the mutants (FSC2.4). Determination of its nucleotide sequence showed that it was an allele of LEU4 (LEU4-1), the gene that encodes alpha-isopropyl malate synthase I (alpha-IPM synthase I), and that the mutation involved a codon deletion localised close to the 3' end of the LEU4 ORF. Six different point mutations--four transitions and two transversions--were found in the remaining mutants. Alpha-IPM synthase activity was found to be insensitive to feedback inhibition by leucine in five of the strains. In the other two the enzyme was resistant to Zn2+-mediated inactivation by Coenzyme A, a previously postulated control mechanism in energy metabolism; as far as we know, this represents the first direct in vivo evidence for this mechanism. The seven mutations define a region, the R-region, involved in both leucine feedback inhibition and in Zn2+-mediated inactivation by CoA. Deletion experiments involving the R-region showed that it is also necessary for enzyme activity.
