New and Redesigned pRS Plasmid Shuttle Vectors for Genetic Manipulation of Saccharomycescerevisiae

Abstract

We have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in the budding yeast Saccharomyces cerevisiae and the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes HIS2 and ADE1, respectively. Our pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed to increase the availability of unique restriction sites within their polylinker regions. Hence, our pRSII plasmids are a complete set of integrating, centromere and 2μ episomal plasmids with the biosynthetic marker genes ADE2, HIS3, TRP1, LEU2, URA3, HIS2, and ADE1 and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, we have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin, and bialaphos. Finally, in the process of generating the new plasmids, we have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using our updated sequences, we constructed pRS plasmid backbones with a unique restriction site for inserting new markers to facilitate future expansion of the pRS series.

Keywords: Saccharomyces cerevisiae; auxotrophic marker; drug resistance marker; plasmid shuttle vector; polylinker/multiple cloning site.

Figure 1

Features of new S. cerevisiae HIS2-marked plasmid shuttle vectors. (A) Restriction maps of the integrating plasmids pRS306H2 (Chee and Haase 2010) and pRSII309. (B) Episomal plasmids pRSII319 (CEN) and pRSII329 (2μ). Although the features of each plasmid are drawn to scale, the size of the maps are not scaled according to plasmid size. Aside from the two NdeI sites highlighted in red for pRS306H2, only unique restriction sites are shown and isoschizomers are indicated.

Figure 2

Features of existing S. cerevisiae ADE2 and new ADE1-marked plasmid shuttle vectors. (A) Restriction maps of pRS402 built using existing GenBank (left) and experimentally determined (right) sequence data. A previously undocumented 163-base pair insertion indicated in dark purple; this insertion is a nearly identical repeat of 163 nucleotides 3′ of the ADE2 marker and hence carries an extra pRS reverse primer binding site (highlighted). This repeat was removed to generate the pRS backbone plasmid pRS40BglII (B) that was subsequently used to construct pRSII402 and pRSII408. (C) Restriction maps of pRSII408, pRSII418, and pRSII428. Unique restriction sites are shown in black, and non-unique BglII and NdeI sites are shown in red; isoschizomers are also indicated.

Figure 3

Schematic diagrams of the prototrophic biosynthetic marker genes found in pRSII series plasmids. Restriction sites in each marker that were targeted for removal before incorporation into pRSII series plasmids are indicated, as are the restriction sites that immediately flank the ADE2, LEU2, ADE1, and HIS2 markers within the pRS or pRSII plasmids. The ORF in each marker is indicated by a block arrow. A complete list of pRSII plasmids is found in Table 1, and the oligonucleotides used for site-directed mutagenesis of restriction sites are found in Table S2. The BamHI site found in the ADE1 genomic sequence was previously removed from the ADE1 allele (Nagley et al. 1988) used to generate pRSII408. The BglII site found in the ADE2 genomic sequence was also previously removed (Stotz and Linder 1990) from the ADE2 allele used to generate pRSII402. Although the ApaI site in URA3 overlaps with a dcm methylation site, plasmid DNA isolated from DH5α dcm⁼ bacteria is still cleaved at this site by ApaI. The gene diagrams shown are drawn to scale.

Figure 4

Features of pRS400 and plasmids derived from it carrying new dominant drug resistance MX4 cassettes that can be amplified by PCR for gene disruption/deletion in yeast. (A) Restriction maps of pRS400 with kanMX4 cassette for G418 resistance, based on existing GenBank (left) and experimentally derived (right) nucleotide sequences. The orientation of the kanMX4 cassette is inverted in the Genbank sequence. (B) New MX4 plasmids derived from pRS400. Top, pRS40B with bleMX4 cassette for phleomycin resistance (left) and pRS40H with hphMX4 cassette for hygromycin B resistance (right). Bottom, pRS40N with natMX4 cassette for nourseothricin resistance (left) and pRS40P with patMX4 cassette for bialaphos resistance (right). Unique restriction sites are shown in black, whereas the NcoI sites we found to be non-unique in the patMX4 cassette (File S1) are shown in red; isoschizomers are also indicated.