Development of a genetic transformation system using new selectable markers for fission yeast Schizosaccharomyces pombe

We describe the development of a new transformation system, using multiple auxotrophic marker genes, for the fission yeast Schizosaccharomyces pombe. We developed three new auxotrophic marker genes (arg12(+), tyr1(+) and ade7(+)) and generated a new host strain, YF043, by Cre-loxP-mediated gene disruption. YF043 possessed six mutated biosynthetic genes (leu1-32, ura4-M190T, arg12::loxP, tyr1::loxP, ade7::loxP and his2::loxP). The combination of this host strain and the new selectable markers can be used for gene disruption using the same preexisting transformation systems. In addition, Sz. pombe vectors were constructed, containing selectable marker genes that complement the auxotrophies of YF043. These new vectors are available for gene disruption and heterologous protein expression in strain YF043. The new Sz. pombe host strain will be a useful tool for molecular genetic studies of Sz. pombe where multiple recombinant modifications or multiple mutations are needed.     

A set of genetic markers for the chromosomes of the imperfect yeast Arxula adeninivorans

The nuclear genome of the anamorphic yeast Arxula adeninivorans was analysed by benomyl-induced haploidization of parasexual hybrids marked with 32 auxotrophic mutations and pulsed field gel electrophoresis followed by DNA hybridization. Twenty-seven genes have been arranged into four linkage groups by haploidization, 15 genes belong to group 1, six to group 2, and three each to groups 3 and 4. Five genes could be localized by DNA hybridization on three out of four separated chromosomes. The gene LYS2 of the largest linkage group 1 and the 25S rDNA were identified on the largest chromosome, the GAA and the TEF1 gene on chromosome 2, and the ILV1 gene of linkage group 4 on the smallest chromosome.     

A novel kanamycin/G418 resistance marker for direct selection of transformants in Escherichia coli and different yeast species

We have developed a set of cloning vectors possessing a modified Tn903 kanamycin resistance gene that enables the selection of both kanamycin‐resistant transformants in Escherichia coli and G418‐resistant transformants in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha and Pichia pastoris. Expression of this gene in yeast is controlled by the H. polymorpha glyceraldehyde‐3‐phosphate dehydrogenase promoter, while expression in E. coli is governed by an upstream E. coli lacZ promoter. Applicability of the vectors for gene disruption in H. polymorpha and S. cerevisiae was demonstrated by inactivation of the HpMAL1 and URA3 genes, respectively. One of the vectors possesses a H. polymorpha ARS allowing plasmid maintenance in an episomal state. The small size of the vectors (2–2.5 kb) makes them convenient for routine DNA cloning. In addition, we report a novel approach for construction of gene disruption cassettes. Copyright © 2009 John Wiley & Sons, Ltd.