Construction and characterization of centromeric, episomal and GFP-containing vectors for Saccharomyces cerevisiae prototrophic strains.
A set of shuttle yeast vectors containing the dominant selectable markers KanMX4 or HphMX4 cassettes, conferring resistance to geneticin and hygromycin B, respectively, was constructed. Dominant selectable markers are useful for genetic manipulation of natural, wine and industrial strains which do not contain any auxotrophic markers as well as of strains which cannot grow on synthetic mineral medium. Vectors were characterized by (i) copy number, (ii) mitotic stability both in selective and non-selective conditions, (iii) the efficiency and frequency of transformations, (iv) optimal adaptation times in non-selective media, (v) optimum conditions for transformation of various laboratory, commercial and wine strains, and (vi) expression level of an inserted gene. Furthermore we produced GFP-containing vectors that can be used for protein subcellular localization in prototrophic strains.
Baruffini
Enrico
E
Department of Genetics, Biology of Microorganisms, Anthropology, Evolution, University of Parma, Viale Usberti 11/A, 43100 Parma, Italy.
Serafini
Fausta
F
Lodi
Tiziana
T
D000935:Antifungal Agents / Q000494:pharmacology; D015152:Blotting, Northern; D003001:Cloning, Molecular / Q000379:methods; D004351:Drug Resistance / Q000235:genetics; D018628:Gene Dosage; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D042822:Genomic Instability; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D012641:Selection, Genetic; D014162:Transfection / Q000379:methods; D014920:Wine
D000935:Antifungal Agents; D049452:Green Fluorescent Proteins