PRS yeast vectors with a LYS2 marker

Abstract

Vol. 36, No. 2 (2004) BioTechniques 213 We constructed two new general purpose cloning vectors for use in Saccharomyces cerevisiae. The new plasmids are pRS307, an integrating (YIp) vector with a LYS2 selectable marker, and pRS327, a multicopy (YEp) vector with a LYS2 marker and a 2-μm origin of replication. These plasmids, along with the previously described pRS317 plasmid, a single copy (YCp) vector with a LYS2 marker and a (centromeric) CEN element, provide a complete set of LYS2 plasmids with the Bluescript® polylinker that complement the widely used existing pRS vectors. Studies in yeast have been facilitated by sets of plasmid vectors with polylinkers that allow easy cloning. The YCplac/YIplac/YEplac set of vectors (1) contain the pUC polylinker (2) and a nutritional marker for selection in yeast. Thus, any fragment inserted into one of these vectors can be easily transferred to other versions, such as YCp, YIp, or YEp, or to a vector with a different selectable marker, either LEU2, TRP1, or URA3. The pRS set of plasmids (3,4) used the polylinker from the Bluescript plasmids (Stratagene, La Jolla, CA, USA) with YCp, YIp, and YEp versions constructed. Subsequently, pRS plasmids with the MET15 and ADE2 markers were constructed (5). Many commonly used yeast strains, including the BY strains (5) used to generate the complete collection of gene knockouts (6) have a lys2 mutation. Although the YCp plasmid with a LYS2 marker, pRS317, has been described (7), YIp and YEp versions have not been available. Here we describe the construction of YIp and YEp vectors with a LYS2 marker with the Bluescript polylinker. The integrating LYS2 plasmid pRS307 was constructed by ligation as follows. pRS317 was cleaved at the ApaLI site common to the pRS vectors, the ends were blunted with the Klenow fragment of DNA polymerase, the DNA was cleaved with EcoRI, and the 5.57-kb ApaLI (blunt)/EcoRI fragment was purified. pRS306 (4) was cleaved with NdeI, the ends were blunted with the Klenow fragment of DNA polymerase, cleaved with EcoRI, and the