Low‐copy episomal vector pFY20 and high‐saturation coverage genomic libraries for the fission yeast Schizosaccharomyces pombe

Abstract

In fission yeast, as in many organisms, episomally replicating plasmid DNA molecules can be used for a wide variety of applications. However, replicating plasmids described previously are each propagated at a high copy number per cell. Plasmid fission yeast twenty (pFY20) contains the ura4(+) gene for positive and negative selection, an origin of replication (ars1) and a stability element (stb). Although this plasmid does not have a centromere, it is propagated with a copy number of about two plasmids per haploid genome equivalent and it is transmitted with relatively high fidelity in mitosis and meiosis. This low-copy vector is useful for screens and mutational studies where overexpression (e.g. from high copy plasmids) is undesirable. We therefore constructed multiple partial-digest, size-fractionated, fission yeast genomic DNA libraries in pFY20 and in the cloning vector pBluescript KS(+). These libraries have sufficient complexity (average of 2100 genome equivalents each) for saturation screening by complementation, plasmid shuffle or hybridization.