New vectors in fission yeast: application for cloning the his2 gene

Abstract

We describe a new Escherichia coli vector (pON5) that allows positive selection for recombinant clones. In this plasmid, the bla gene from pBR322 is permanently active, whereas the neo gene from transposon Tn5 is repressed by the cI-encoded λ. repressor. When DNA is inserted into the Bc/I or HindIII restriction sites situated within the cI gene, the neo gene becomes transcribed from the λ P R promoter. We have also made a Schizosaccharomyces pombe derivative of pON5 (= pON163) by introducing the fission yeast arsl and ura4 + sequences. We show that this plasmid is capable of transforming Sc. pombe ura4 strains, as well as ura3 strains of the distantly related budding yeast Saccharomyces cerevisiae. We have used pON163 for the construction of two fission yeast genomic libraries. From these gene banks clones were isolated that were able to complement fission yeast his2 mutants. Such plasmids could also rescue his4C mutants of Sa. cerevisiae, defective in the histidinol dehydrogenase activity of the multifunctional HIS4 gene product. Finally, we describe the plasmid pDW232 which is useful for functional analysis of fission yeast genes. It is a pGEM3 derivative adapted to fission yeast, carrying multiple cloning sites between the T7 and SP6 promoters, together with arsl and ura4 + from Sc. pombe.