YEAST VOL. 10 141-149 (1994)

Selectable Marker Replacement in Saccharomyces cerevisiae MARC VIDAL* AND RICHARD F. GABERt

Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, U. S. A .

Received 9 July 1993; accepted 30 July 1993

Selectable markers integrated by the ‘gamma’deletion method (Sikorski and Hieter, 1989) can be efficiently replaced in vivo with other markers by transformation with homologous plasmids. Transformation frequencies in experiments designed to replace original selectable markers with an alternate marker were high and molecular analysis confirmed that all transformants that exhibited the expected phenotypes (loss of the original prototrophy and gain of the alternate prototrophy) resulted from homologous recombination between plasmid sequences at the target locus. This technique involves no plasmid construction and greatly facilitates the generation of yeast cells containing multiple gene disruptions.

KEY WORDS - Saccharomyces cerevisiae; selectable marker; transformation.

INTRODUCTION In Saccharomyces cerevisiae and other yeasts, gene replacement by homologous recombination can be used to generate deletion-mutant strains suitable for studying the function of a given gene. The starting wild-type strain usually carries an auxotrophic mutation so that gene replacement by a selectable marker can be performed directly. Classically, gene function studies involved the disruption of a single gene. However, recent studies of gene redundancy andor gene inter- actions have given rise to the need for efficient methods to construct multiple gene deletions. The study of cyclin function in cell division cycle control, for example, required the construction of a strain containing three gene deletions and the insertion of a conditionally expressed gene (clnlA, cin2A, cln3A, GAL1::CLNl; Richardson et al., 1989). Cloning of human and Drosophila cyclin homologues by functional complementa- tion required that this strain carry additional auxotrophies to allow transformation with cDNA libraries (Leopold and O’Farrell, 1991). Estab- lishment of gene interactions between the cyclins

?To whom correspondence should be addressed. *Present address: Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, U.S.A.

ccc 0749-503x/94/02014149 0 1994 by John Wiley & Sons Ltd

and .the S W14-S W16 transcription factors involved strains containing up to four deletions (Nasmyth and Dirick, 1991; Ogas et al., 1991). Similarly, detection of the function of a sixth glucose transporter gene in S. cerevisiae required the deletion-disruption of five other glucose transporter genes: SNF3 HXTl HXT2 HXT3 and HXT4 (KO et al., 1993). In order to perform such sophisticated genetic analyses it is crucial to be able to conveniently generate sets of versatile deletion-mutant strains with multiple combinations of selectable markers.

An efficient method to generate deietions was described by Sikorski and Hieter (1989) and is commonly known as the ‘gamma’ deletion method. In addition, a set of yeast strains contain- ing multiple non-revertible auxotrophic mutations (ura3-52, trplAl, ieu2A1, and his3A200) were constructed to take advantage of widely used selectable markers (Sikorski and Hieter, 1989). Corresponding integrative, centromeric and multi- copy vectors containing the URA 3, TRPl , LEU2 or HIS3 selectable markers are now available (Christianson et al., 1992; Sikorski and Hieter, 1989). The ‘gamma’ method allows one-step replacement of a genomic locus yielding an inte- grated copy of the selectable marker flanked by plasmid sequences (Figure 1). Using the integrative

142 M. V D A L AND R. F. GABER

Figure 1. Selectable marker replacement scheme. After ‘gamma’ deletiondisruption of a target gene by the initial selectable marker flanked with plasmid sequences (thin lines), an alternate selectable marker can be introduced by homologous recombination or gene conversion. Tube lines represent homologous genomic sequences used for ‘gamma’ deletion; bold lines represent flanking genomic sequences.

Table 1. Strains

R757 M398* M469 M476 M537 M771 M774 M7 16t M718 M836 M925

MATa ura3-52 his4-15 lys9 MATa ura3-52 trplAl his3A2OO leu2Al trklA MATa ura3-52 trplAl his3A200 l d A 1 trklA trk2A::HISJ MATa urs3-52 trplAl hWA2200 l e d A l trklA rpd1A::TRPl MATa ura3-52 trplAl his3A2200 l d A 1 M A Ta ura3-52 trpl A1 his3A2200 leu2A1 trkl A rpd3A:: HIS3 MATa ura3-52 trplAl his3A2200 leu2Al trklA rpa3A:: URA3 MATa ura3-167 trplA1 his3A200 leu2A1 pho2A::LEU2 MATa ura3-167 trplAl his3A2200 l edA1 pho2A::LEUZ rpd1A::TRPl MATa ura3-52 trplAl his3A.2200 leu2AI trklA rpd1A::TRPl rp&A::HIS3 MATa ura3-52 trplAl his3A2200 leu2A1 trklA rp&A::LEU2

+All of the urd-52 his3A2200 leu2Al strains used in this study were derived from those described in Sikorsky er a/. (1989). tDerived from YH40.P2 (phoZA::LECJ2), provided by G. Berben.

plasmids, constructions can be generated in vitro and transformed back into yeast to generate gene disruptions containing the URA3, HIs3, TRPl or LUE2 selectable markers.

We show that any selectable marker introduced by the ‘gamma’ method can be easily replaced in vivo by any of the selectable markers described above (Figure 1). Transformation of a ‘gamma’

SELECTABLE MARKER REPLACEMENT 143

Table 2. Frequencies of selectable marker replacement*

pRS303 pRS304 pRS305 pRS306 Strain Target locus (HIS3) (TRPI) (LEU2) (URA3)

A M771 M774 M925

B M476 M469

C

M836

M718

M716

rpd3A:: HIS3 rpd3A:: URA3 rpd3A:: LEU2

rpdl A:: TRPI trk2A::HIS3

rpd3A::HIS3 rpdl A:: TRPI

rpdl A:: TRPI, pho2A::LEU2

pho2A:: LE U2

- 56/60 57/60

15/15 -

-

8319%

o/isq

70/73* 54/57 56/60

-

23/24

-

-

0110

34/37 31/32 89/95 -

- 87/90

23/24 1912 1 ND ND

49/55? 3 6/40 $

4815211

- 0124

*Expressed as the number of transformants in which the rpd3-associated prototrophic phenotype was lost per total number of transformants tested. ?Of the 49 that lost an initial prototrophic phenotype, 24 became His- and 25 became Trp -, indicating replacement of the original selectable markers at the rpd3 anbd rpdl loci, respectively. $Of the 36 that lost an initial prototrophic phenotype, 17 became His- and 18 were Trp- , indicating replacement of the original selectable markers at the rpd3 anbd rpdl loci, respectively. $83 lost the rpdl-associated Trp+ phenotype; none of the 96 transformants tested lost the pho2- associated Leu+ phenotype. (148 lost the rpdl-associated Trp+ phenotype; none of the 52 transformants tested lost the pho2- associated Leu’ phenotype. peletion of PH02 (G. Berben, pers. comm.) was performed by the ‘one-step deletion’ method (Rothstein, 1983) and therefore contains no integrated plasmid sequences.

deleted recipient containing an initial selectable marker with any of the related linearized integra- tive plasmids generates, in 95% of the transfor- mants, cells in which the initial selectable marker has been replaced by the new marker. Southern blot analysis indicates that this replacement occurs by homologous recombination or gene conversion. In the exceptional transformants (5%), integration of the new selectable marker seems to occur either by gene conversion or homologous recombination at the chromosomal locus of the selectable marker, or through an integrative recombination event at the ‘gamma’ deletion locus. These exceptional transformants are easily detected phenotypically since they retain the initial prototrophic pheno- type.

Selectable marker replacement techniques facili- tate the construction of versatile yeast deletion- mutant strains since fewer in vitro constructions

are required to generate numerous combinations of selectable markers.

MATERIALS AND METHODS The yeast strains used in this study are listed in Table 1. The plasmids used are described in Sikorski and Hieter (1989) and were kindly pro- vided by the authors. Growth conditions, genetic crosses and drop-out media were as described in Sherman et al. (1986). Yeast transformation was performed by the cation method described by Ito et al. (1983) with three pg of purified plasmid DNA. The pRS plasmids were linearized with BamHI prior to transformation in yeast (Maniatis et al., 1982). Southern blot analysis was performed as described (Ausubel et al., 1989) using radio- labelled (Feinberg and Vogelstein, 1983) fragments that contained selectable markers from the pRS

144 M. VIDAL AND R. 1:. <;ABI<R - eel

d B s

His + - + - - + + His + - + - - + + Trp + - - + + + + T r p + - - + + + +

HIS3 Probe TRPI Probe Figure 221

plasniids (Sikorski and Hieter, 1989). The relevant restriction fragments containing either the URA3, T R P I . LEU2 or HIS3 probes extended from PstI An initial gamma deletion mutalion in RPD3 to A p I . XhriI to HinDIII, EcoRI to Chi, and (rpdA::HIS3) a non-essential gene required for Hii7DIII to PstI, respectively, on the appropriate both maximal repression and activation of niany pRS plasmid. The washing conditions of the genes in S. crre~isiae (Vidal and Gaber, 1991). Southern blots were stringent (O.I '%, SSC, O.I'%l served as a convenient locus to demonstrate select- SDS, 65°C) (Maniatis rt d., 1982). able marker replacement. Transformations in

RESULTS AND DISCUSSION

Sl:l,lK’l’ABl,l; MARKER REPLACEMEN-I 145

His + - + - + + His + - + - + + L e u + - - + + + Leu + - - + + +

HIS3 Probe LEU2 Probe Figiiir 2b.

strain M I 7 1 (ut.tr3-52 ttplA1 liis3A200 Ieu2A1 trlil At.pdA:: H l S 3 ; Table 1) were performed with linearized integrative plasmids pRS304 ( T R P l ) , pRS30S (LEU?) and pRS306 (URA3) . Lineariz- ation was achieved by digestion at a unique site within the multiple cloning site of these plasmids. As expected, transformation efficiencies were ap- proximately 100-fold higher when the plasmids were linearized compared to experiments using non-linearized plasmids (data not shown).

Approximately 95’X of the transibt-mants ob- tained with the linearized integrative plasmids exhibited loss of the original His’ phenotype. suggesting that the HIS3 selectable marker had been eliminated (Table 2A). Southern blot analysis of genomic DNA isolated from such His trans- formants confirmed both the absence of I 1 I S . i sequences (Figure 2A, B and C ; lelt panels) and the presence of the newly integrated selectable marker ( Figure 2A, B and C; right panels). Thus, all o f the

146 M . VIDAL AND R . F. CABER

His

Ura

d d ? 3 s ? 3 e s s

+ - + - + + - - + +

HIS3 Probe

2 r; E 3

nr m

3

l?

E m

H i s + - + - + Ura + - - + +

URA3 Probe Figure 2c

Figure 2. Southern blot analysis of transformants obtained from selectable marker replacement strategies. This experiment involved recombination events in strains containing the initial selectable marker HIS3 and alternate selectable markers T R P l (A). LEU2 (B) or URA3 (C) . In A, B and C, left and right panels show identical genomic DNA digested with BgflI and Clul, electrophoresed under identical conditions, and subsequently hybridized with the indicated probes. Top and bottom panels show the relevent genotypes and growth phenotypes, respectively. In each case, the first three lanes are controls showing hybridization patterns for strain R757 (HIS3, ura3-52, T R P l and LEU2; lane I ) , M398 (his3A200, ura3-52, i rp lA l and IeuZAl; lane 2) and M771 (hi.v3A200. ura3-52, t rp lAl , leuZA1 and rpd3A;;HISS; lane 3). The remaining lanes show hybridization patterns for transformants after selectable marker replacement into M771. Hybridization patterns confirmed loss of the initial selectable marker in the His transformants and integration of T R P l at rpd3A;;HIS3 (A) and of LEU2 and URA3 at their respective loci (B,C) in the His’

transformants

SELECTABLE MARKER REPLACEMENT 147

Table 3. Deletion method comparison

Stability of deletion/

disruption

Presence of Selectable selectable marker marker replacement

Integrative disruption No One-step disruption Yes Integration-excision Yes hisG:: URA3::hisG Yes ‘Gamma’ deletion Yes

Yes * Yes No No No

Optional No Yes Yes

Selectable marker

elimination Reference

* Shortle et al. (1982) No Rothstein (1983) Yes Winston et al. (1983) Yes Alani et al. (1987) Yes Sikorski and Hieter (1989)

*Although formally possible, no published reports available.

transformants that exhibited a Trp’ His -, Leu+ His- or Ura+ His- (depending on which plas- mid was used) had undergone selectable marker replacement.

A minority of the transformants (approximately 5%) obtained in these experiments remained His+ (Table 2A). Southern blot analysis revealed that the structure of the rpd3A::HIS3 locus in these transformants remained unchanged (Figure 2A, B and C ; left panels). Two classes of recombination events were represented among these exceptional transformants. When pRS305 (LEU2) and pRS306 (URA3) were used, the exceptional trans- formants were generated by recombination events at their respective homologous chromosomal loci, i.e. LEU2 ( Figure 2B, right panel) or URA3 (Figure 2C, right panel). Because the ura3-52 and leu261 loci in strain M771 contain genomic se- quences homologous to the URA3 and LEU2 sequences of pRS305 and pRS306 plasmids used in these experiments (Sikorski and Hieter, 1989), the His+ Ura+ or His+ Leu+ transformants probably arose as a result of gene conversion or homologous recombination events at the ura3-52 and leu2A1 loci. In the two His+ Trp+ transformants analysed, integration seemed to have occurred at the rpd3A::HIS3 locus (Figure 2A, right panel), pre- sumably by a single recombination event between integrated and undigested plasmid sequences. This integration was confirmed in genetic crosses be- tween the MATa His+ Trp+ strains and control MATa wild-type His - Trp - strains; no recombi- nation was observed between the HIS3 and TRPl markers in 20 tetrads.

Similar transformation efficiencies and frequen- cies of selectable marker replacement were ob- tained when the initial marker at the rpd3 ‘gamma’ deletion locus was TRPl (rpdl A:: TRPl , strain

M476, Table 2B) or URA3 (rpd3A::URA3, strain M774, Table 2A). Selectable marker replacement is not limited to a single round of substitution. After replacement of rpd3A:: HIS3 by rpd3A::LEU2, the rpd3A::LEU2 mutation could be replaced with any of the other markers used in this study (strain M925, Table 2A). Selectable marker replacement at other loci (trk2A::HIS3, strain M469, and rpdlA::TRPl, strain M476, Table 2B) was also performed to show that this method can be generally used.

In a strain containing a double deletion of the RPD3 and RPDl genes (rpd3A::HIS3 rpdl A:: TRPl , strain M836) with both selectable markers flanked by identical plasmid sequences, the marker replacement events occurred at each locus with similar frequencies, as revealed by the number of transformants which had lost one or the other of the initial prototrophies. No transfor- mants were recovered in which both initial pro- totrophies were simultaneously lost, suggesting that the frequency of such double events is rela- tively low.

The ‘one-step gene-disruption’ method de- scribed by Rothstein (1983) allows replacement of chromosomal sequences with a selectable marker that is flanked directly with native genomic se- quences and contains no plasmid sequences. We used such a deletion allele of the BAS2lPH02 gene (pho2A::LEU2, strain M716, Table 1) to demon- strate that the presence of flanking plasmid se- quences are required for selectable marker replacement to occur. Only a few transformants were obtained after transformation with the linear- ized integrative plasmids and none of them lost the initial prototrophy. Similarly, in strain M718 con- taining both a one-step deletion (pho2A::LEU2) and a ‘gamma’ deletion (rpdlA:: T R P l ) mutation,

148 M. VIDAL AND R. F. GABER

selectable marker replacement occurred only at the RPDl locus, giving rise to Leu+ Trp- transformants exclusively (Table 2C).

Five basic methods have been described to gen- erate deletion alleles of yeast genes (Table 3 and references therein). These methods are based on different integration schemes and result in different deletion alleles. In the process of investigating the function of a particular gene, several experiments might have to be performed that require different deletion alleles. For some uses, e.g., mapping pur- poses, or synthetic lethality experiments, the pres- ence of a selectable marker at the mutant locus is required. In other cases a deletion allele might be required in which the initial selectable marker can either be replaced by an alternative marker or simply eliminated. For example, a mutation at a particular locus may be necessary to generate the appropriate background for the expression of genes carried on plasmids containing the same initial selectable marker. The choice of a deletion method is not trivial since these differences can restrict the subsequent experimental strategies or necessitate laborious strain constructions by recombination.

The integrative disruption method (Shortle et al., 1982) can introduce a selectable marker into a targeted locus and since it results in a duplication of flanking sequences, could in principle be used for selectable marker replacement. However, the disrupted allele generated by this method reverts at high frequency due to the presence of repeated sequences after integration (reviewed in Ausubel et al., 1989). The one-step gene-deletion method (Rothstein 1983) does not allow selectable marker replacement since no plasmid sequences remain following the integration event. The integration- excision method (Winston et al., 1983) creates a deleted allele in which the selectable marker is automatically eliminated and therefore is not con- venient for mapping or synthetic lethality experi- ments which benefit from having the locus marked genetically. Another method that contains the ad- vantages of the one-step deletion and integration- excision methods was developed by Alani et al. (1987). Although this method results in deletion/ disruption alleles from which the original URA3 selectable marker can be eliminated, URA3 cannot be replaced with an alternative selectable marker. On the other hand, as summarized in Table 3, the ‘gamma’ deletion method (Sikorski and Hieter, 1989) is suitable for all purposes. This method generates a non-revertible genetically marked

deletion allele that can be efficiently replaced. In theory, selectable markers contained in ‘gamma’ deletion mutations can also be eliminated. For example, transformation of a recipient containing a URA3-marked ‘gamma’ deletion mutation with a related plasmid (linearized at the poly cloning site) followed by selection for 5-fluoro-orotic acid resis- tance (Boeke et al., 1987) should result in elimina- tion of the resident URA3-containing plasmid and its replacement by the unmarked donor plasmid.

In summary, selectable marker replacement, facilitating the construction of strains with mul- tiple gene disruptions is becoming increasingly important as functionally redundant members of gene families are discovered and as the number of genes of unknown function, identified through large genomic sequencing projects, increases. Ver- satile sets of yeast strains can be conveniently generated by transformation when systematic use is made of yeast strains containing multiple non- revertible auxotrophic mutations corresponding to a given set of standard vectors (Sikorski and Hieter, 1989) and when these vectors are used in integrative plasmid constructions to generate deletions or disruptions by the ‘gamma’ deletion method (Sikorski and Hieter, 1989).

ACKNOWLEDGEMENTS This work has been supported by grants from the National Science Foundation (DCB-8711346 and DCB-8657 150) and the National Institutes of Health (GM45739). M.V. is a recipient of the Belgian ‘Fonds National de la Recherche Scientifique’.

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