Low-copy episomal vector pFY20 and high-saturation coverage genomic libraries for the fission yeast Schizosaccharomyces pombe.
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.
Wahls
Wayne P
WP
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA.
Davidson
Mari K
MK
D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D018628:Gene Dosage; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D008540:Meiosis / Q000235:genetics; D008938:Mitosis / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012568:Schizosaccharomyces / Q000235:genetics*
D004271:DNA, Fungal