Yeast Cloning Vectors and Genes

Victoria Lundblad1

1 University of California, Berkeley, Berkeley, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 13.4
DOI:  10.1002/0471142727.mb1304s21
Online Posting Date:  May, 2001
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Abstract

This unit describes some of the most commonly used yeast vectors, as well as the cloned yeast genes that form the basis for these plasmids. Yeast vectors can be grouped into five general classes, based on their mode of replication in yeast: YIp, YRp, YCp, YEp, and YLp plasmids. With the exception of the YLp plasmids (yeast linear plasmids), all of these plasmids can be maintained in E. coli as well as in S. cerevisiae and thus are referred to as shuttle vectors. The nomenclature of different classes of yeast vectors, as well as details about their mode of replication in yeast are discussed.

     
 
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Table of Contents

  • Section II: Yeast Vectors
  • Plasmid Nomenclature
  • Maps of Selected Plasmids and Genes
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Figures

  •  FigureFigure 13.4.1 YIp5. YIp5 contains the 1.1-kb HindIII URA3 gene cloned into the AvaI site of pBR322 via the addition of poly(dG-dC) tails (Struhl et al., 1979). Since this plasmid does not contain a yeast origin of replication, transformants occur by integration into the yeast genome at the URA3 locus; the frequency of transformation can be increased by linearization of the plasmid within the URA3 insert. The complete nucleotide sequence is available from the Vecbase database (file name: Vecbase.Yip5) and a detailed restriction map can be found in the New England Biolabs catalog.

    The URA3 gene encodes orotidine-5¢-phosphate (OMP) decarboxylase, a 267-amino-acid protein required for uracil biosynthesis. The map shown is the gene from strain +D4, which can be expressed in E. coli without an external bacterial promoter (Rose et al., 1984). Loss of URA3+ function can be directly selected using 5-FOA: ura3– cells are resistant to 5-FOA, whereas 5-FOA is toxic to cells synthesizing the URA3 gene product (Boeke et al., 1984). This negative selection has been exploited in a variety of gene replacement schemes, discussed in UNIT 13.10. The URA3 gene can also complement mutations in the pyrF gene in E. coli using strain DB6656 (pyrF::Mu, lacZam trpam hsrk– hsmk+; Bach et al., 1979).
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  •  FigureFigure 13.4.2 YRp7. This plasmid contains the 1453-bp EcoRI TRP1 ARS1 fragment from S. cerevisiae inserted into the EcoRI site of pBR322 (Struhl et al., 1979).

    The TRP1 RI circle is a derivative of YRp7 containing only the 1453-bp TRP1 ARS1 EcoRI fragment. This plasmid is mitotically and meiotically unstable, but is present in 100 to 200 copies per plasmid-bearing cell in both cir+ and ciro strains.

    A genomic plasmid bank has been constructed by inserting size-selected Sau3A partial fragments into the BamHI site of YRp7 (Nasmyth and Reed, 1980).

    The TRP1 ARS1 1453-bp EcoRI fragment contains both the TRP1 gene, encoding N-(5¢-phosphoribosyl)-anthranilate isomerase, and the autonomous replication sequence ARS1. The intact TRP1 gene can complement mutations in the trpC gene of E. coli, using E. coli JA300 (thr1 leuB6 thi1 thyA trpC1117 hsrk– hsmk– strr; Tschumper and Carbon, 1982). The chromosomal replicator ARS1 lies between positions 615 and 1453 (on a HindIII-EcoRI fragment) and is composed of three domains. Domain A contains an 11-bp core sequence (position 857 to 867) consisting of a consensus sequence found in many other ARS elements and which is essential for ARS1 function. Domains B and C flank Domain A and are relatively AT-rich regions that contribute to, but are not essential, for ARS function.
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  •  FigureFigure 13.4.3 YEp24. YEp24 has the 2.2-kb EcoRI fragment of the B form of the 2 µm plasmid and the 1.1-kb HindIII URA3 gene inserted into the EcoRI and HindIII sites, respectively, of pBR322 (Botstein et al., 1979). The expression of the tetr gene is variable among different isolates of this plasmid. YEp24 is mitotically stable in cir+ strains at a copy number of about 20 but is unstable in ciro strains. The complete sequence of YEp24 is available from the Vecbase database (file name: Vecbase.Yep24) and a detailed restriction map can be found in the New England Biolabs catalog.
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  •  FigureFigure 13.4.4 2µm plasmid. The 2µm circle is a naturally occurring DNA plasmid found in almost all strains of S. cerevisiae, with a copy number of ~20 to 80. The plasmid exists in two different forms, A and B (the former is shown above), due to intra-molecular recombination between two perfect 599-bp inverted repeats. Strains that carry this plasmid are called cir+; strains missing the plasmid cir0 have been identified or isolated (UNIT 13.9). It is extremely stable mitotically, with a spontaneous loss rate in haploid cells of 10–4 per generation; during meiosis the plasmid is transmitted to all four spore products. The plasmid has been completely sequenced (Hartley and Donelson, 1980) and the sequence is available from GenBank (Plant: yscplasm).
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  •  FigureFigure 13.4.5 YCp50. This vector is a derivative of YIp5 and YCp19. The EcoRI site of YCpl9 was removed (producing an unsequenced deletion of about 190 bp) and a PvuII-HindIII fragment (containing CEN4 and ARS1) from this derivative was cloned into the PvuII site of YIp5, with loss of the PvuII site (Rose et al., 1987). Due to the presence of the CEN element, this plasmid exists in low copy in yeast (1 to 2 copies/cell) and is mitotically stable (<1%loss per cell per generation). This plasmid has not been completely sequenced; a more complete restriction map is available in Rose et al., 1987.

    A set of genomic plasmid banks using YCp50 and size-selected DNA fragments has been constructed (Rose et al., 1987). These plasmid banks provide an alternative to genomic libraries constructed in high-copy-number vectors, useful when isolating genes that would be lethal in yeast when present in high copy.

    Both the CEN3 (Fig. 13.4.7) and CEN4 (above) sequences were identified based on their ability to confer mitotic stability and proper meiotic segregation to autonomously replicating plasmids (Fitzgerald-Hayes et al., 1982; Mann and Davis, 1986). Nucleotide sequence comparison combined with functional analysis has shown that centromeres contain three conserved structural elements. Elements I and III show the highest degree of sequence conservation between different centromeres, and are separated by an extremely AT-rich region of about 90 bp, designated Element II. Full CEN4 activity is contained within the 850-bp PvuII-HpaI fragment (which contains Elements I, II, and III), although the adjacent 905-bp HpaI-EcoRI fragment also confers some mitotic stability to unstable ARS-containing plasmids (Mann and Davis, 1986).
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  •  FigureFigure 13.4.6 pYAC3. pYAC vectors are used to clone very large fragments of exogenous DNA onto artificial linear chromosomes, which can be stably maintained in yeast. This vector, which can be propagated as a circular plasmid in E. coli, contains a unique cloning site in the SUP4 gene (an ochre-suppressing allele of a tyrosine tRNA), as well as ARS1 and CEN4 elements, required for stable single-copy propagation of the artificial chromosome. The TEL sequences are derived from Tetrahymena telomeres and have been shown to function as telomeres in yeast. To clone an insert, pYAC3 is digested with BamHI (which cuts adjacent to the telomere sequences) and SnaBI; the resulting vector arms (containing either TRP1, ARS1, and CEN4 or URA3) are ligated to insert fragments with SnaBI-compatible ends. The resulting ligation products are transformed into a ura3– trp1– ade2-1 yeast strain, using the spheroplast protocol, selecting for Ura+ and subsequently screening for Trp+ (to insure that both vector arms are present). Transformants can be further screened for the presence of inserts in the middle of the SUP4 gene by using a color assay: colonies in which the ade2-1 ochre mutation is suppressed by SUP4 are white, whereas inactivation of the suppressor results in red colonies.

    The pYAC vector shown above is one of a collection of three plasmids, each with a different cloning site inserted into the SUP4 gene: pYAC4 and pYAC5 contain EcoRI and NotI sites, respectively, in place of the SnaBI site found in pYAC3. Selected restriction sites (not necessarily unique) are shown for pYAC3, as well as sites that have been destroyed in the process of plasmid construction. The SUP4 gene is shown as a wavy line. For more detailed discussion of the cloning protocol, as well as details of the construction of this vector, see Burke et al., 1987.

    The HIS3 gene encodes imidazoleglycerolphosphate (IGP) dehydratase, which catalyzes a step in the histidine biosynthetic pathway. This 1822-bp fragment also contains a portion of two other genes: pet56, required for mitochondrial function, and ded1, required for cell viability (Struhl, 1985). Mutations in the hisB gene of E. coli can be complemented by the cloned HIS3 yeast gene, using E. coli BA1 (thr1 leuB6 trpC1117 hisB463 Tn10::near hisB thi1 thyA hsrk– hsmk–strr; Murray et al., 1986).
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  •  FigureFigure 13.4.7 The LEU2 gene encodes -isopropylmalate (-IPM) dehydrogenase, which catalyzes the third step in leucine biosynthesis (Andreadis et al., 1982). Unlike several other yeast genes involved in amino acid biosynthesis, LEU2 (and LEU1, which is coordinately regulated with LEU2) is under specific amino acid control: gene expression is repressed by elevated concentrations of leucine. The leu2-d allele is a deletion of the 5¢-flanking region of the LEU2 message which leaves only 29 bp preceding the LEU2 intiation codon; this derivative of the LEU2 gene, when present on a YEp plasmid, requires a very high plasmid copy number to give a Leu+ phenotype and has been used to cure cir+ strains of the endogenous 2µm plasmid (see UNIT 13.9). Also contained in this 2230-bp XhoI-SalI fragment are 95 bases of the 330-nucleotide element. Although this element diverges in sequence from other elements, when the entire 2230-bp fragment is used as a probe of genomic yeast DNA elements present elsewhere in the genome will be detected at a low level. The cloned LEU2 gene can complement mutations in the leuB6 gene of E. coli using the strain JA300 (thr1 leuB6 thi1 thyA trpC1117 hsrk– hsmk– strr; Tschumper and Carbon, 1982).

    The LYS2 gene is the structural gene for -aminoadipate reductase, which catalyzes an essential step in lysine biosynthesis. The gene, which has not yet been sequenced, is present on a 4.6-kb EcoRI-HindIII genomic fragment, and gives rise to a 4.2-kb LYS2 transcript, which is under general amino acid control (Eibel and Philippsen, 1983; Barnes and Thorner, 1986). Much larger genomic fragments (up to 15.7 kb) containing the LYS2 gene have been isolated, providing a large variety of restriction sites flanking the gene for cloning purposes. As with URA3, a positive selection for lys2– mutants exists: such mutants can be selected on medium containing -aminoadipatic acid and lysine, with a spontaneous frequency of 10–5 to 10–6. Because the pathways for lysine biosynthesis in bacteria and fungi are not the same, no E. coli mutations can be complemented by the cloned LYS2 gene.

    See the legend to Figure 13.4.5 for a discussion of CEN3.
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Literature Cited

Literature Cited
    Andreadis, A., Hsu, Y.-P., Kohlhaw, G.B., and Schimmel, P. 1982. Nucleotide sequence of yeast LEU2 shows 5¢ noncoding region has sequences cognate to leucine. Cell 31:319-325.
    Bach, M.L. LaCroute, F., and Botstein, D. 1979. Evidence for transcriptional regulation of orotidine-5¢-phosphate decarboxylase in yeast by hybridization of mRNA to the yeast structural gene cloned in E. coli. Proc. Natl. Acad. Sci. U.S.A. 76:386-390.
    Barnes, D.A. and Thorner, J. 1986. Genetic manipulation of Saccharomyces cerevisiae by use of the LYS2 gene. Molec. Cell. Biol. 6:2828-2838.
    Boeke, J., LaCroute, F., and Fink, G.R. 1984. A positive selection for mutants lacking orotidine-5¢-phosphate decarboxlyase activity in yeast: 5-fluoroorotic acid resistance. Mol. Gen. Genet. 197:345-346.
    Botstein, D., Falco, S.C., Stewart, S.E., Brennan, M., Scherer, S., Stinchcomb, D.T., Struhl, K., and Davis, R.W. 1979. Sterile host yeasts (SHY): A eukaryotic system of biological containment for recombinant DNA experiments. Gene 8:17-24.
    Burke, D.T., Carle, G.F., and Olson, M.V. 1987. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236:806-812.
    Clarke, L. and Carbon, J. 1978. Functional expression of cloned yeast DNA in Escherichia coli: Specific complementation of argininosuccinate lyase (argH) mutations. J. Mol. Biol. 120:517-532.
    Eibel, H. and Philippsen, P. 1983. Identification of the cloned S. cerevisiae LYS2 gene by an integrative transformation approach. Mol. Gen. Genet. 191:66-73.
    Elledge, S.J. and Davis, R.W. 1988. A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. In press.
    Fitzgerald-Hayes, M., Clarke, L., and Carbon, J. 1982. Nucleotide sequence comparisons and functional analysis of yeast centromere DNAs. Cell 29:235-244.
    Hartley, J.L. and Donelson, J.E. 1980. Nucleotide sequence of the yeast plasmid. Nature 286:860-865.
    Hill, J.E., Myers, A.M., Koerner, T.J., and Tzagoloff, A. 1986. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2:163-167.
    Ma, H., Kunes, S., Schatz, P.J., and Botstein, D. 1987. Plasmid construction by homologous recombination in yeast. Gene 58:201-216.
    Mann, C. and Davis, R.W. 1986. Structure and sequence of the centromeric DNA of chromosome 4 in Saccharomyces cerevisiae. Mol. Cell. Biol. 6:241-245.
    Murray, A.W., Schultes, N.P., and Szostak, J.W. 1986. Chromosome length controls mitotic chromosome segregation in yeast. Cell 45:529-536.
    Nasmyth, K.A. and Reed, S.I. 1980. Isolation of genes by complementation in yeast: Molecular cloning of a cell cycle gene. Proc. Natl. Acad. Sci. U.S.A. 77:2119-2123.
    Parent, S.A., Fenimore, C.M., and Bostian, K.A. 1985. Vector systems for the expression, analysis and cloning of DNA sequences in S. cerevisiae. Yeast 1:83-138.
    Pouwels, P.H., Enger-Valk, B.E., and Brammar, W.J. 1985. Cloning Vectors: A Laboratory Manual. Elsevier Science Publishing, Amsterdam.
    Rose, M., Grisaffi, P., and Botstein, D. 1984. Structure and function of the yeast URA3 gene: expression in Escherichia coli. Gene 29:133-124.
    Rose, M.D., Novick, P., Thomas, J.H., Botstein, D., and Fink, G.R. 1987. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 60:237-243.
    Struhl, K. 1985. Nucleotide sequence and transcriptional mapping of the yeast pet56-his3-ded1 gene region. Nucl. Acids Res. 13:8587-8601.
    Struhl, K., Stinchcomb, D.T., Scherer, S., and Davis, R.W. 1979. High-frequency transformation of yeast: Autonomous replication of hybrid DNA molecules. Proc. Natl. Acad. Sci. U.S.A. 76:1035-1039.
    Tschumper, G. and Carbon, J. 1980. Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene. Gene 10:157-166.
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