A System of Shuttle Vectors and Yeast Host Strains Designed for Efficient Manipulation of DNA in Saccharomyces ceratisiae

A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cereuisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trpl , leu2 and ura3 mutations. A set of YCp and YIP vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HZS3, T R P l , LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast. P LASMID cloning vectors that can “shuttle” DNA between yeast and bacteria are fundamental tools in the molecular genetic analysis of Saccharomyces cerevisiae. They are used extensively to manipulate cloned yeast genes and to define cis-acting DNA elements such as promoters and autonomously replicating sequences. Of the several types of shuttle vectors that have been created (PARENT, FENIMORE and BOSTIAN 1985), the Yeast Integrating Plasmid (YIP) and Yeast Centromere Plasmid (YCp) types seem to have the most general applicability. However, despite their broad utility and routine usage, most YIP and YCp vectors in common use today remain the original prototypes, plasmids that were not designed for cloning efficiency or versatility. Cloning in these prototypes (YCp50, YIp5, etc.) is hampered by the availability of few useful cloning sites, the lack of a visual screen for recombinants, the limited variety of yeast selectable markers, and the relatively low copy number of pBR322 replicons. Improved yeast vectors have been constructed (BALDARI and CESARENI 1985; HILL et al. 1986; ELLEDGE and DAVIS 1988), but to date no uniform set of multipurpose YIP and YCp vectors has been made that overcomes all of these drawbacks. In contrast to yeast vectors, Escherichia coli cloning vectors have been systematically modified and have evolved into efficient, multipurpose cloning vehicles (BALBAS et al. 1986). Perhaps the most sophisticated of these is the phagemid pBLUESCRIPT (Stratagene). It contains an f l phage origin of DNA replication for the in vivo production of single stranded (ss) DNA and T3/T7 phage promoters for the in vitro production of RNA. It also contains a large polylinker region and a bacterial gene that allows a color screen for recombinants so that standard cloning is greatly simplified. Genetics 122: 19-27 (May, 1989) In an effort to incorporate these advanced features into yeast cloning vectors, we have constructed a set of YIP and YCp vectors based on the backbone of pBLUESCRIPT. These new vectors are small (56 kb), contain many unique cloning sites, replicate to high copy number in bacteria, and offer a choice of four yeast selectable markers. The vectors make cloning efficient in that most DNA manipulations (sequencing, unidirectional deletions, mutagenesis, etc.) can be performed in the same plasmid that is introduced into yeast. In addition, we have made yeast strains that contain nonrevertible (deletion) auxotrophic mutations that can serve as host to any or all of these plasmids. Together, the vectors and yeast strains provide the basic materials for a wide variety of molecular genetic manipulations in S. cerevisiae. MATERIALS AND METHODS Bacterial strains and media: Escherichia coli strains DH5a [F-, endA1, hsdR17(rk-mk+), supE44, thi-1, X, recA, gyrA96, r e l A l , A(argF-laczya), U169, 080 lac ZhM151, from Bethesda Research Laboratories; and MH 1066 [ AlacX74, hsr-, rpsl, pyrETn5, leuB600, trpC9830, galE, galK], from MIKE HALL via J. Boeke (Johns Hopkins University); were used as plasmid hosts. Recombinant plasmids containing the yeast URA3, TRPl, or LEU2 genes were selected in MH 1066 by complementation on M9 medium with uracil (20 Fg/ml), tryptophan (30 rg/ml), or leucine (40 Pg/ml) added as required. Yeast transformations and media: Yeast transformations were performed by the LiAc procedure of ITO et al. (1 983). Media components were from Difco or Sigma. Yeast media were as described by SHERMAN, FINK and LAWRENCE (1979) and HIETER et al. (1 985a). DNA manipulations: Restriction enzymes and DNA polymerases were purchased from Boehringer Mannheim and used as instructed. T4 DNA ligase was purchased from New England Biolabs. Standard recombinant DNA techniques 20 R. S. Sikorski and P. Hieter