Yeast Yeast 2002; 19: 393402. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002 / yea.

841

Yeast Functional Analysis Report

Sets of integrating plasmids and gene disruption cassettes containing improved counter-selection markers designed for repeated use in budding yeast
Rinji Akada*, Isao Hirosawa, Miho Kawahata, Hisashi Hoshida and Yoshinori Nishizawa
Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Yamaguchi University, Tokiwadai, Ube 755-8611, Japan

* Correspondence to: R. Akada, Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611, Japan. E-mail: rinji@po.cc.yamaguchi-u.ac.jp

Abstract
Counter-selection is a useful gene manipulation technique for repeated gene disruptions, gene shufings and gene replacements in yeasts. We developed a novel counter-selection system using a galactose-inducible growth inhibitory sequence (Kawahata et al. 1999. Yeast 15: 110). This counter-selection marker, named GAL10pGIN11, has several advantages over previous counter-selection markers, i.e. use of an inexpensive galactose medium for counter-selection, combined use with any transformation markers for gene introduction, and no requirement of specic mutations in the host strains. The GIN11 sequence, which is a part of an X-element of the subtelomeric regions, contained a conserved autonomously replicating sequence, causing the possibility of inefcient chromosomal integration. We isolated GIN11 mutants that lost the replication activity but retained the growth-inhibitory effect when overexpressed. A mutant GIN11M86 sequence was selected and fused to the CUP1 promoter for the counter-selection on a copper-containing medium. The GALpGIN11M86 and the CUPpGIN11M86 were used for constructing sets of integrating plasmids containing auxotrophic markers involving HIS3, TRP1, LEU2, URA3 or ADE2, or a drug-resistant marker PGKpYAP1. In addition, a set of gene disruption cassettes that contained each of the auxotrophic markers and the GALpGIN11M86, which were anked by direct repeats of a hisG sequence, were constructed. The counter-selectable integrating plasmids and the gene disruption cassettes can allow the markers to be used repeatedly for yeast gene manipulations. Copyright # 2002 John Wiley & Sons, Ltd. Keywords: Saccharomyces cerevisiae; transformation; integration; gene disruption; counter-selection; GIN11; marker recycling

Received: 6 July 2001 Accepted: 8 October 2001

Introduction
The counter-selection method is well known as a gene manipulation technique used for marker recycling, plasmid shufing and gene replacement in yeasts (Rothstein, 1991; Sikorski and Boeke, 1991; Toh-e, 1995; McNabb et al., 1997). The most popular counter-selection system is the loss of the URA3, which is selected on a 5-uoroorotic acid plate (Boeke et al., 1984, 1987; Alani et al., 1987; McNabb et al., 1997; Goldstein et al., 1999). In addition, selections of the loss of LYS2, CAN1, CYH2, TRP, and GAP1 genes on plates containing a-aminoadipic acid, canavanine, cycloheximide,
Copyright # 2002 John Wiley & Sons, Ltd.

5-uoroanthranilic acid and minimal L-proline+ D-histidine medium, respectively, are known (Struhl, 1983; Sikorski and Boeke, 1991; Toyn et al., 2000; Regenberg and Hansen, 2000). These systems require the presence of the respective ura3x, lys2x, can1r, cyh2r, trpx, and gap1x mutations, limiting the use of many strains. Another type of marker recycling system is based on the use of a site-specic recombinase and its target sites (Cregg and Madden, 1989; Roca et al., 1992; Sauer, 1994; Toh-e, 1995; Gu ldener et al., 1996; Storici et al., 1999; Delneri et al., 2000; Steensma and Linde, 2001). A site-specic recombinase efciently excises a marker anked by its target sites, but this system requires additional

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introduction of the plasmid containing the recombinase, and thus requires an additional transformation with another marker besides the marker for gene disruption. To circumvent the limitation of strains and markers usable for counter-selection, we have developed an alternative counter-selection system, which is usable with any transformation markers without any requirement of specic mutations or additional markers (Akada et al., 1997; Kawahata et al., 1999). Overexpression of growth-inhibitory (GIN) sequences from the GAL10 promoter causes growth inhibition in yeast; therefore growth on a galactose medium provides a selection for loss of the sequence. We have selected the GIN11 sequence as the most suitable for the development of a counter-selection system because of its strong effect on growth inhibition, the short DNA length, and the non-protein form of its action. This counter-selection marker has been effectively applied to construct a recombinant brewers yeast that did not have any mutations required for the previous counter-selection systems (Akada et al., 1999). The GIN11 sequence contains a part of the subtelomere X-element, in which a conserved autonomously replicating sequence (ARS) exists. The presence of ARS was apparently problematic when GIN11 was used for a one-step disruption cassette containing tandem repeats of the hisG sequence for marker recycling (Kawahata et al., 1999). Therefore, we have constructed GIN11 mutants that have lost their ARS activity but retained their growth-inhibitory activity in this study. In addition, the only limitation of our system was the use of Gal+ strains for the galactoseinduced overexpression of GIN11; thus Galx strains could not be used. Therefore, we have developed an alternative conditional growth inhibition system, using the CUP1 promoter (Etcheverry, 1990). Based on these improvements, we have constructed sets of counter-selectable integrating plasmids and markerrecycling disruption cassettes that can be applied to most yeast strains.

Standard yeast and E. coli media were used as described (Ausubel et al., 1987; Rose et al., 1990). Yeast cells were grown in YPD medium (2% glucose, 1% yeast extract, 2% polypeptone) or YPGal medium (2% galactose, 1% yeast extract, 2% polypeptone) at 28uC. Synthetic dextrose medium (SD) consisted of 2% glucose, 0.7% yeast nitrogen base without amino acids (aa) and essential nutrients as required. SD-Leu, SD-Ura and SD-Lys were drop-out SD media lacking leucine, uracil, and lysine, respectively, but containing all other essential nutrients. SGLeu and SGUra were synthetic galactose media in which 2% glucose of SDLeu and SDUra was substituted for 2% galactose, respectively. SD+5 aa contained leucine, uracil, adenine, tryptophan and histidine in the SD medium. CuSO4 (0.7 M stock) was mixed with SD media at indicated concentrations described in the text. A 5-uoroorotic acid plate was made as described previously (Boeke et al., 1984). Yeast transformations were performed by the simple lithium acetate method (Chen et al., 1992).

Mutagenesis of GIN11 ARS

The PCR was performed with Taq Plus Long DNA polymerase (Stratagene), according to the manufacturers instructions. pSMA116 (Kawahata et al., 1999) was used as a template. The primers used were GIN11-AC (5k-ANNNNAATAATTATCT TTCAACTTTACAAAATAAAC-3k) and GIN11PAW (5k-pGNNNAGGTGATTTTGGTGTTGAA TTTTCTG-3k), in which N and p indicate any nucleotides and phosphorylated 5k-end, respectively. The PCR cycle was initiated at 95uC for 1 min and followed by 25 cycles of 95uC for 30 s and 65uC for 5 min in a total 25 ml reaction mixture. After the PCR, 20 units of DpnI (New England BioLabs) and 2.5 units of Cloned Pfu DNA polymerase (Stratagene) were added, incubated at 37uC for 30 min, and then incubated at 72uC for 30 min; 2 ml mixture was directly used for a ligation reaction in a total 10 ml mixture with T4 DNA ligase. The ligation mixture was transformed to E. coli.

Materials and methods Strains and media

Escherichia coli strain DH5a was used for the construction and amplication of plasmids. The Saccharomyces cerevisiae strain used was W303-1A (MATa ade2-1 can1-100 ura3-1 leu2-3, 112 trp1-1 his3-11, 15 GAL+).
Copyright # 2002 John Wiley & Sons, Ltd.

Plasmids
The plasmids constructed in this study are listed in Table 1 and shown in Figures 2 and 4. The GIN11M86 fragment was derived from pGG116 (p23M86) that contained two nucleotide changes at the ARS consensus.
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Table 1. Plasmids
Plasmids pGG113 pGG114 pGG115 pGG116 pGG117 pGG119 pCG113 pCG114 pCG115 pCG116 pCG117 PCG119 pGG213 pGG214 pGG215 pGG216 PGG217 pGG215dLYS2 pCUPM86 Selection and counter-selection markers HIS3 GALpGIN11M86 TRP1 GALpGIN11M86 LEU2 GALpGIN11M86 URA3 GALpGIN11M86 ADE2 GALpGIN11M86 PGKpYAP1 GALpGIN11M86 HIS3 CUPpGIN11M86 TRP1 CUPpGIN11M86 LEU2 CUPpGIN11M86 URA3 CUPpGIN11M86 ADE2 CUPpGIN11M86 PGKpYAP1 CUPpGIN11M86 hisGHIS3GALpGIN11M86hisG hisGTRP1GALp-GIN11M86hisG hisGLEU2GALp-GIN11M86hisG hisGURA3GALp-GIN11M86hisG hisGADE2GALp-GIN11M86hisG lys2D::[hisGLEU2GALpGIN11M86hisG] URA3 CUPpGIN11M86 2mm ori Description Integrating plasmid Integrating plasmid Integrating plasmid Integrating plasmid Integrating plasmid Integrating plasmid Integrating plasmid Integrating plasmid Integrating plasmid Integrating plasmid Integrating plasmid Integrating plasmid Marker recycling disruption cassette Marker recycling disruption cassette Marker recycling disruption cassette Marker recycling disruption cassette Marker recycling disruption cassette LYS2 disruption with LEU2 recycling Multicopy plasmid

Construction of pGG113, pGG114, pGG115, pGG117 and pGG119

A 1.8 kb DraIIINotI fragment of pGG116 was subcloned into DraIIINotI cleaved pRS303, pRS304, pRS305 (Sikorski and Hieter, 1989) and pBluescript KS+ (Stratagene), resulting in pGG113, pGG114, pGG115 and pBlueKS+M86, respectively. pGG117 was constructed by inserting a 2.2 kb BglII ADE2 fragment treated with dNTP and Klenow DNA polymerase (hereafter called blunt-ended) from pASZ11 (Stotz and Linder, 1990) to the EcoRV site of the pBlueKS+M86. For pGG119, a 3.1 kb XhoI SalI fragment from YCpPGKpYAP1 (Akada et al., 2002) was inserted into the XhoISalI sites of pBluescript KS+ to form pBlueKS+PGKpYAP1. A 1.8 kb DraIIINotI fragment from pGG116 was inserted into DraIIINotI cleaved pBlueKS+PGKp YAP1 to form pGG119. The PGKpYAP1 contained in the pGG119 is a new drug resistance marker with which transformants can be selected by cerulenin- or cycloheximide-containing plates (Akada et al., 2002).

Construction of pCG113, pCG114, pCG115, pCG116, pCG117 and pCG119

The plasmids containing CUPpGIN11M86 were named pCG plasmids. pRCUP2 bearing 2 mm ori and URA3 was constructed by subcloning a 0.8 kb SmaIClaI fragment from pCUP (Kang et al., 1990) into NaeIClaI cleaved pRS426 (Christianson et al.,
Copyright # 2002 John Wiley & Sons, Ltd.

1992). The GIN11M86 sequence was amplied with KOD DNA polymerase (Toyobo) from pGG116. The primers used were GIN11-SE (5k-GAATTC GTCGACGGATCAATAACAG-3k) and GIN11CE (5k-GAATTCATCGATACTAGATGCACTCA TATC-3k), in which italics indicate additional sequences used for the restriction enzyme digestion. The PCR was initiated at 94uC for 1 min and followed by 25 cycles of 94uC for 20 s, 55uC for 2 s, and 74uC for 30 s. A 0.7 kb SalIClaI fragment of the PCR-amplied GIN11M86 was subcloned into XhoIClaI cleaved pRCUP2 to form pCUPM86. pCG116 was constructed by inserting a 1.1 kb bluntended BamHIClaI fragment containing CUPp GIN11M86 from pCUPM86 into the NaeI site of pRS306 (Sikorski and Hieter, 1989). A 1.5 kb DraIII NotI fragment containing CUPpGIN11M86 from pCG116 was subcloned into DraIIINotI cleaved pRS303, pRS304, pRS305, pGG117, and pBluescript KS+ to form pCG113, pCG114, pCG115, pCG117, and pBlueKS+CG86, respectively. pCG119 was constructed by inserting a 3.1 kb XhoISalI fragment from YcpPGKpYAP1 (Akada et al., 2002) into XhoISalI sites of pBlueKS+CG86.

Construction of gene disruption cassettes designed for repeated use

The plasmid p30M86 containing GALpGIN11M86 anked by a bacterial hisG sequence (Alani et al., 1987) was constructed as follows. A ClaI linker
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(5k-ccatcgatgg-3k) was inserted into the blunt-ended KpnI site of pSMA111 (Kawahata et al., 1999) to form pGU108c-4. Then, a 2.0 kb SacIClaI fragment containing GALpGIN11 from the pGU108c4 was subcloned into SacIClaI cleaved p1-2 (Kawahata et al., 1999) to form p30-3. A 0.34 kb HpaIClaI fragment from the PCR-amplied GIN11M86 was subcloned into HpaIClaI cleaved p30-3 to form p30M86.
pGG213

pGG216

A 1.15 kb blunt-ended HindIII fragment containing URA3 from p1-2 (Kawahata et al., 1999) was inserted into the blunt-ended BamHI site of the p30M86 to form p4aM86. The BamHI linker was inserted into the blunt-ended SpeI site of the p4aM86 to form p4aM86B. A 1.2 kb BglIINotI fragment containing a hisG sequence from p4-1 was subcloned into BamHINotI cleaved p4aM86B to form pGG216.
pGG217

A 1.8 kb BamHI fragment containing HIS3 from the pSK-HIS3, which contained the HIS3 gene in pBluescript SK+ (Stratagene), was inserted into the BamHI site of the p30-3 to form p40-1. A SmaI linker (5k-ccccgggg-3k) was inserted into the bluntended SpeI site of the p40-1 to form p41-1. A 1.6 kb SmaINotI fragment of the p1-2 was subcloned into SmaINotI cleaved p41-1 to form p42-1. A 1.95 kb HpaIDraIII fragment from the p30M86 was subcloned into HpaIDraIII cleaved p42-1 to form pGG213.
pGG214

A 2.1 kb BglII fragment containing ADE2 from pASZ11 was inserted into the BamHI site of the p30M86 to form p32aM86. The BamHI linker was inserted into the SpeI site of the p32aM86 to form p32aM86B. A 1.2 kb BglIINotI fragment from p4-1 was subcloned into BamHINotI cleaved p32aM86B to form pGG217.
pLUCG86

A 1.1 kb HindIII URA3 fragment from p1-2 was inserted into the HindIII site of pCG115 to form pLUCG86.
pGG215-dLYS2

An EcoRI linker (5k-ggaattcc-3k) was inserted into the blunt-ended SpeI site of the p30-3 to form p331. A 0.9 kb BglIIEcoRI fragment containing TRP1 from YCpN1 (Nakayama et al., 1985) was subcloned into BamHIEcoRI cleaved p33-1 to form p34-1. A BamHI linker (5k-cggatccg-3k) was inserted into the blunt-ended EcoRI site of the p34-1 to form p35-1. A 1.2 kb BglIINotI fragment containing a hisG sequence from p4-1 (Kawahata et al., 1999) was subcloned into BamHINotI cleaved p35-1 to form p36-1. A 1.52 kb HpaIBglII fragment containing the hisG sequence and a part of the GIN11M86 sequence from the p30M86 was subcloned into HpaIBglII cleaved p36-1 to form pGG214.
pGG215

A 6.0 kb BglIINotI fragment from pGG215 was subcloned into BglIINotI cleaved p6-1 (Kawahata et al., 1999; Fleig et al., 1986).

Plasmid loss and marker recycling methods

Yeast transformants of the NcoI-cleaved pLUCG86 were grown in YPD medium for 2 days at 28uC and 100 ml 1/1000 diluted culture were spread on SD+5 aa plates containing 0.25, 0.5 and 0.7 mM of copper sulfate. Colonies that appeared on the plate were tested for growth on a SDLeu plate. For loss of the LEU2 marker after gene disruption, Lysx transformants of pGG215-dLYS2 were spread on YPGal plates and grown for 23 days. Colonies that appeared on the plates were tested for the growth on SDLeu selection plates to examine the presence or the absence of the LEU2 marker.

A 2.2 kb HpaI fragment containing LEU2 of YEp13 (Broach et al., 1979) was inserted into the blunt-ended BamHI site of the p30-3 to form p45-1. The BamHI linker was inserted into the blunt-ended SpeI site of the p45-1 to form p46-1. A 1.2 kb BglIINotI fragment containing a hisG sequence from the p4-1 was subcloned into the BamHI-NotI cleaved p46-1 to form p47-1. A 1.52 kb HpaIBglII fragment of the p30M86 was subcloned into HpaIBglII cleaved p47-1 to form pGG215.
Copyright # 2002 John Wiley & Sons, Ltd.

Results Mutagenesis of GIN11 ARS

The plasmid pSMA116 (Kawahata et al., 1999) was amplied with primers containing degenerate nucleotide sequences at the ARS site. After the
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PCR reaction, DpnI restriction enzyme digestion was performed to cleave an adenine-methylated GATC recognition sequence, thus digesting the plasmid DNA template but not the PCR-amplied DNA (Weiner et al., 1994). The amplied DNA was circularized with T4 DNA ligase and transformed to E. coli. Plasmids were extracted from 120 E. coli transformants and transformed into yeast. Among the 120 plasmids, three plasmids, called p23M56, p23M86 and p23M107, showed a very low transformation efciency (03 colonies/plate instead of >1000 in other plasmids). Yeast transformants of the three plasmids did not lose Ura+ markers after cultivation for 2 days in YPD liquid medium. Also, they did not grow on the 5-uoroorotic acid plate, indicating that these transformants were chromosomal integrants. The entire nucleotide sequences of GIN11 from the three plasmids were determined. One or two nucleotide changes occurred only at the ARS site (Table 2) and those mutations were consistent with the ARS-inactivation mutations (Van Houten and Newlon, 1990). These results indicated that the ARS of GIN11 of the three plasmids were inactivated. To determine whether mutant GIN11 containing inactive ARS caused growth inhibition when overexpressed, the yeast transformants containing these plasmids were spotted on an SGUra plate. The results showed that the GIN11 mutants caused growth inhibition on a galactose medium (Figure 1A, data not shown for M56 and M107). A GIN11 mutant containing two nucleotide changes (GIN11M86) derived from p23M86 was selected for further plasmid constructions and hereafter the p23M86 plasmid was renamed pGG116.

strains, we fused the GIN11M86 under the CUP1 promoter and named it CUPpGIN11M86. A multicopy plasmid pCUPM86 and an integrating plasmid pCG116 containing the CUPpGIN11M86 and URA3 were constructed. Yeast transformants of pCUPM86 were tested for the growth-inhibitory effect on an SD medium containing 0.7 mM copper sulfate. The transformants did not grow on the copper medium, indicating that the copperinducible GIN11M86 expression also caused growth inhibition (Figure 1A). The transformants of pCG116 showed the same result (data not shown). When cells transformed with pCUPM86 containing URA3 and CUPpGIN11M86 were grown on non-selective plates containing copper and uracil, cells lost the uracil requirement, as shown by replica-plating on SDUra plates, indicating that plasmid loss can be positively selected on a copper-containing medium. To examine the effectiveness of the positive selection for the loss of chromosomally integrated plasmid, we made the plasmid pLUCG86 containing LEU2, URA3 and CUPpGIN11M86. The pLUCG86 was cleaved with NcoI, which was located within the URA3

A positive selection for plasmid loss on a copper plate

Previous counter-selection using GALpGIN11 can only be applied to Gal+ strains. To apply Galx
Figure 1. Growth inhibition caused by overexpression of an ARS-inactivated GIN11 mutant and its use for plasmid loss selection. (A) Transformants of plasmids containing a mutant GIN11M86 fused to GAL10 (left) and CUP1 (right) promoter were spotted and incubated on SGUra containing galactose and SDLeu+Cu2+ (0.7 mM copper sulfate) plates, respectively. (B) Cells containing pCUPM86 (URA3 and CUPp GIN11M86) or pRS306 were grown on a non-selective SD+5 aa medium containing indicated concentrations of copper for 2 days and then cells were replica-plated on SDUra to examine the presence or the absence of URA3 marker
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Table 2. Sequence of GIN11 ARS mutants

Plasmid pSMA116 wild-type p23M56 p23M86 (pGG116) p23M107
1

Sequence at ARS1 5k-TTTTATGTTTA-3k 5k-TTTTATGTTCA-3k 5k-TTTTATGCTCA-3k 5k-TTTTATGTGTA-3k

Underlines are mutations.

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gene, and transformants were selected on an SD Leu plate. A transformant that grew on SDUra Leu but not on SDUraLeu+0.7 mM Cu2+ was selected. The transformant was grown in liquid YPD medium for 2 days and the diluted cell suspension was spread on SD+5 aa plates containing 0, 0.25, 0.5 or 0.7 mM copper sulfate. Colonies that appeared on the plates were tested for the presence or the absence of the LEU2 marker. By the selection on 0.25, 0.5 and 0.7 mM copper plates, 40.5% (37), 52.7% (150) and 62.5% (200) of colonies (total numbers examined in parentheses) were Leux, respectively, indicating that positive selection for loss of the integrated plasmid could be achieved on the copper plates. Slightly effective counterselection was seen on a plate containing a higher concentration of copper but with over 0.7 mM copper concentration, yeast cells could not grow well because of the toxic effect of copper. YPD containing copper sulfate (0.54.0 mM tested) did not elicit both a toxic effect and copper-inducible growth inhibition, thus YPD+Cu2+ could not be used for the positive selection (data not shown).

examine LYS2 disruption. Among the 37 Leu+ colonies picked, 26 colonies showed Lysx, indicating efcient LYS2 disruption (70.3%) with the hisGLEU2GALpGIN11M86hisG cassette. After disruption, cells were suspended in sterile water, spread on YPGal plates and incubated for 3 days. Among 130 colonies grown on YPGal plates, 127 colonies (97.7%) lost the Leu+ phenotype but retained Lysx, indicating that loss of the LEU2 marker could be positively selected on a YPGal plate after the one-step gene disruption (Figure 3). Predicted loss of the LEU2 and the GALp GIN11M86 by the recombination between the hisG sequences was conrmed by PCR (data not shown). We constructed plasmids containing GALp GIN11M86 and ve transformation markers anked by direct repeats of hisG sequences (Table 1, Figure 4). These cassettes will be useful for repeated one-step gene disruptions.

Discussion
We constructed ARS-inactivated GIN11 mutants. The ARS-inactive GIN11 caused growth inhibition when overexpressed, indicating that ARS activity is not required for the growth-inhibitory effect of GIN11. The GIN11 sequence contains a part of the conserved subtelomeric X-element that is located at most of the chromosomal ends of S. cerevisiae (Zakian, 1989). The function of the subtelomeric sequence and the reason why overexpression of X-element causes growth inhibition have not yet been revealed. The presence of ARS consensus at the subtelomeric regions was also reported in Hansenula polymorpha (Sohn et al., 1999), suggesting that the subtelomeric regions played an important role for chromosomal replication at the ends. In this study, we have improved the GIN11 by inactivating its ARS activity and developed a new counter-selection marker selectable on a copper plate, in addition to the previous galactoseselectable marker. The CUPpGIN11M86 will extend its application to Galx strains, which was the only limitation for the use of GALpGIN11 marker. However, the efciency of counter-selection by the CUPpGIN11M86 marker was 4163%, which was worse than that of the galactose selection with GALpGIN11M86 (y98%). We could not explain this, but part of the reason might be due to that the toxic effect of copper on cells might
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Construction of counter-selectable integrating plasmids

Using the GALpGIN11M86 and the CUPp GIN11M86, integrating plasmids were constructed (Table 1, Figure 2). All the integrating plasmids were conrmed to cause growth inhibition on either galactose or copper plates.

Use of the GALpGIN11M86 for repeated gene disruption

In the previous study (Kawahata et al., 1999), we constructed a plasmid (p6-1) bearing the cassette which consisted of the GALpGIN11 and URA3 anked by direct repeats of hisG sequences (Alani et al., 1987) within the LYS2 gene for the one-step LYS2 disruption. This construct, however, was hardly used for LYS2 disruption, probably due to the presence of the ARS in GIN11 (approximately 0.1% in Kawahata et al., 1999). We constructed a similar disruption cassette, hisGLEU2GALp GIN11M86hisG, without ARS activity. The cassette was inserted into the LYS2 gene, resulting in pGG215dLYS2 (Figure 3). The pGG215dLYS2 was digested with XbaI, and transformed to W3031A. The transformants were selected on an SDLeu plate and tested for growth on an SDLys plate to
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Integrating plasmids and gene disruption cassettes with improved counter-selection markers 399

Figure 2. Counter-selectable integrating plasmids containing galactose-inducible GALpGIN11M86 (pGG series) and copper-inducible CUPpGIN11M86 (pCG series)
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Figure 3. One-step LYS2 disruption by LEU2 and subsequent marker elimination

affect the selection. The counter-selectable integrating plasmids containing ve common auxotrophic markers, i.e. URA3, LEU2, HIS3, ADE2 and TRP1, and a new drug-resistant marker PGKp YAP1 (Akada et al., 2002), will greatly facilitate multiple DNA manipulations in yeast and construction of recombinant industrial yeast strains free of undesirable DNA sequences (Akada et al., 1999). Similar counter-selection markers were reported by using a conditionally inducible CHA1 or MET25 promoter, fused to the PKA3 gene encoding a

cAMP-dependent protein kinase catalytic subunit (Olesen et al., 2000). The efciency of plasmid loss or loop-out by their counter-selectable markers on the selective condition has not been reported; therefore, the effectiveness of the use of CHA1 or MET25 promoter-driven PKA3 for counterselection can not be compared with that of our system. During the course of our study, we chose GIN11 as the best counter-selective marker among 16 growth-inhibitory genes that we isolated (Akada et al., 1997). The criteria for selecting the GIN11

Figure 4. Plasmids containing marker recycling cassette

Copyright # 2002 John Wiley & Sons, Ltd.

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sequence among the genes were: (a) its strong growth-inhibitory activity when overexpressed; (b) no growth-inhibitory effect when not expressed, even though it was carried on a multicopy plasmid; (c) no plasmid loss during incubation in an expression-repressible YPD medium; (d) the efcient plasmid loss during growth on a YPGal medium; (e) short DNA length for plasmid constructions; and (f) no appearance of suppressor mutants (GIN11 does not encode protein, thus the possibility of mutations causing defect of growthinhibitory activity may be lower than proteinencoding sequences). It is noteworthy that the efciency of the plasmid loss was not correlated with the strength of the growth-inhibitory effect of the genes. If overexpression of a gene caused lethality of cells, it seems that the cells would die on a galactose medium, and hence the total number of surviving cells would decrease. In such a case, even if the growth-inhibitory effect was strong, cells that lost the growth-inhibitory gene might not be selected efciently. Moreover, plasmid loss should not occur when the counter-selective marker gene is in a repressed state. The GIN11 passed all these criteria. When GIN11 was overexpressed in a galactose medium, the proliferation of cells containing GALpGIN11 was immediately stopped and growthinhibited cells were still alive after 24 h incubation in a galactose medium (data not shown). Therefore, our GIN11 is not one of the counter-selection markers but the best marker selected from all our candidate genes (Akada et al., 1997). In addition to integrating plasmids, we have constructed disruption cassettes containing GALp GIN11M86 and ve auxotrophic transformation markers anked by tandem repeats of the hisG sequence. This cassette was successfully used for the one-step LYS2 disruption and subsequent marker elimination. In gene disruption cassettes previously constructed with counter-selection markers, disruption markers and available strains were limited (Alani et al., 1987; Szent-Gyorgyi, 1996; McNabb et al., 1997). In another system using site-specic recombinases, available strains were also limited because an additional marker was necessary for retaining a plasmid expressing a recombinase gene in addition to the disruption markers (Roca et al., 1992; Sauer, 1994; Toh-e, 1995; Gu ldener et al., 1996). The disruption cassettes constructed here, however, can be used directly to disrupt any genes in yeast strains bearing ura3, trp1, his3, leu2 or ade2 mutations that are commonly used for laboratory
Copyright # 2002 John Wiley & Sons, Ltd.

strains. If necessary, any type of transformation marker (Wach et al., 1994; Goldstein and McCusker, 1999) can be combined with our counter-selection markers, providing versatile tools for multiple gene manipulations in most yeast strains (Delneri et al., 1999; Winzeler et al., 1999).

Acknowledgements
This work was supported Grant No. 10760053 from The Ministry of Education, Science, and Sports in Japan.

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