An improved method for yeast 2μm plasmid curing

Gone, 88 (1990) 241-245

241

Elsevier GENE 03456

Short Communications An improved method for yeast 2pro plasmid curing (Saccharomyces cerevisiae; recombinant DNA; plasmid transformation; Southern-blot analysis; incompatibility; su]fometuron methyl resistance)

Wei Xiao and Gerald H. Rank Department of Biology, University of Saskatchewan. Saskatoon, Sask. (S7N OWO Canada) Received by J. Marmur: 29 October 1989 Accepted: 4 December 1989

SUMMARY

NMR 1-410, a dominant resistance marker, was cloned into the FLP gone of 2 pm DNA to produce the chimeric YEp vector pWX823B. Selection for SMRI-410 at high concentrations of sulfometuron methyl maintained pWX823 at high copy number and resulted in the rapid and efficient loss of native 2 pm DNA. Using this protocol approximately 15~o ofthe cells monitored showed loss of 2 pm DNA. The curing methodology is more efficient and convenient than previous methods and has the added advantage of being applicable to wild-type prototrophic cells.

INTRODUCTION

Most Saccharomyces yeasts contain an endogenous 2 pm plasmid at about 60 copies per cell (for review see Futcher, 1988). 2 #m DNA does not contribute any known phonetype to the host. Rather, 2 pm-free cells ([ctr ° ]) often exhibit a selective advantage over isogonic [cir ÷ ] strains (Futcher and Cox, 1983; Mead et al., 1986). The [cir ° ] strains may also be useful with binary vector systems for the stable maintenance and high-level gone expression of 2 pro-based chimeric plasmids (Blanc et al., 1979). Several different methods have been applied to eliminate 2 #m DNA from Correspondence to: Dr. W. Xiao, Department of Biology, University of

Saskatchewan, Saskatoon, Sask. (STN 0W0 Canada) Tel. (306)9664440; Fax (306)966-4461.

Abbreviations: Ap, ampicillin; bp, base pair(s); ilv, lie + Val; kb, 1000 bp; nt, nucleotide(s); ori, origin of DNA replication; R, resistant; s, sensitive; SD, Bacto yeast nitrogen base (6.7 mg per ml)/giucose (20 mg per ml); SM, snlfometuron methyl; wt, wild type; YPD, Bacto yeast extract (10 mg per ml)/Baeto peptone (20 mg per ml)/glucose (20 mg per ml); [ ], denotes plasmid-carrier state. 0378-1119/90/$03.50 © 1990Elsevier Science Publishers B.V. (Biomedical Division)

host cells. Under continuous growth, spontaneous [cir ° ] can be isolated at very low frequency due in part to a selective advantage of [cir ° ] (Futcher and Cox, 1983; Mead et al., 1986). However, the 2 #m plasmid is efficientlytransmitted during the sexual cycle (Further ot al., 1988), which limits the usefulness of this method. Cells harboring pJDB219-derived 2 #m chimeric plasmids, when growing in Leu-deficient medium, often resulted in subcultures devoid of endogenous 2 #m DNA (Dobson et al., 1980; Toh-E and Wickner, 1981; Erhart and Hollenbcrg, 1981). The defective leu2-d gone in these plasmids resulted in a high copy number of chimeric plasmids, which was responsible for 2 pm plasmid loss (Erhardt and Hollenbcrg, 1983; Futcher and Cox, 1984). Harford and Peeters 0987) developed a protocol to select for tip- from successive culture of 2 pm transformants. Although this method accommodates different selectable markers (e.g., LEU2, TRPI and URA3), it requires a much longer time and effort to screen for tip- progenies. In addition, neither of the methods can be applied to wt strains and tend to produce unwanted mutants or revertants from the original strains at high frequency (Erhart and Hollenberg, 1981; Mead et al.,

242 1987; G. Arndt, personal commanication and our own observations). In this communication, we describe a more general and convenient method to cure endogenous 2 ~m circle DlqA from wt yeast strains.

EXPERIMENTAL AND DISCUSSION

(a) Plasmid construction To cure wt yeast strains ofendogenous 2 #m plasmid, we chose yeast S M R i as a selectable marker. S M R I is an allele o f l L V 2 that confers resistance to the herbicide SM (Falco and Dumas, 1985). SMRI-410 contains a single point mutation at nt 574 of the IL V2-coding region, resulting in a Pro ~ Ser substitution (Yadav et al., 1986). This gene can be used as a selectable marker to transform wt laboratory strains and industrial Saccharomyces yeasts (Casey et al., 1988; Xiao and Rank, 1989). We made a 2 #m chimeric plasmid by inserting the 3.3-kb BamHI-Xhol SMRI-410 fragment from pWX509 (Casey et al., 1988) between B a m H I and SalI sites of pRIT12309 (Harford and Pecters, 1987). The resulting plasmid, pWX823B, contains the entire 2 # m DNA with S M R I -410 + pBR327 inserted at FLP (Fig. 1).

(b) Method for 2/~m plasmid curing Yeast cells were transformed (Ito etal., 1983) by pWX823B and plated on SD-supplelnented minim~d medium + SM. The transformants were streaked onto SD + 50 #g SM/ml medium, incubated for three days at 30°C, and the resultant cells were used to inoculate 5 ml of YPD liquid medium. Cultures were allowed to replicate (30°C, 150 rpm overnight twice) for a total of 20-30 generations. A 10-#1 sample was diluted and plated onto YPD at approx. 200 colonies per plate. These colonies were replicated onto YPD and SD + SM medium. SM s colonies on YPD medium were screened by colony or blot hybridization with a 2 #m specific probe under single-copy detection conditions. Probe-negative colonies were thus free of both endogenous and chimeric 2 #m plasmids. An ilv2 strain WX2-2d (mat¢~, ura3-52, trpl, ilv2-Al, GAL + ) and a wt ILV2 strata a ade4 HR (mata, tvpl, ade4) were used to examine 2 #m curing efficiency. After nonselective (YPD liquid)culture, 40-80% cells became SMS; among these approx. 15% were [cir ° ] (Fig. 2; Table I). The [cir ° ] status of probe-negative lines was further confirmed by transformation with pWX823B. Southern analysis of transformants showed only the B form of pWX823, which defined the tip- genotype resulting from loss ofnative 2 #m DNA (Harford and Peeters, 1987) and ruled out the possibility of cells harboring a low copy number of 2 pm plasmid.

(c) Dynamics of 2 #at plasmld loss An initial a ade4 HR[pWX823] transformant was

pWX823B 12.6kb

Fig. I. A diagram of plasmid pWX823B, pWX823B was constructed by insertion of SMRI.410 (Falco and Dumas, 1985) into pRITI2309 (Hat'ford and Peeters, 1987). it consists of the complete 2 #m sequence, SMRI.410 as a resistance-sclectable marker in yeast and pBR327 (single line). The FLP gene is inactivated by the $MRI.410 + pBR327 insertion. However, FLP protein provided in warts by native 2 pm DNA induces site-specific recombination at FRT sites (blackened box) and thus equilibrates pWX823B and pWX823A, collectively called pWX823. Restriction cleavage sites: A, AvaI; E, EcolU; H, H/ndilI; He, H/nell; P, PslI; X, Xbal.

cultured on SD minimal medium with different concentrations of SM. Total yeast DNA was extracted after a 3-day culture, digested with Clal and subjected to Southern-blot analysis. Clal cleaves both pWX823 (12.6 kb) and 2 #m circle (6.3 kb) once, whereas S M R I resides in a 8.7-kb ClaI chromosomal fragment (Xiao and Rank, 1989) that can be easily iden*.ified. Probing with ILI,'2 gave a direct measure of pWX823 copy number per genome using the nuclear ILV2 gene as reference. Use of the 2 #m DNA probe gave the relative copy number of pWX823 and endogenous 2 #m DNA. Fig. 3 summarizes these data. The copy number of chimeric plasmid (pWX823) increased with increased SM concentration - - similar to ieu2-d transformants under selective pressure (Erhart and Hoilenberg, 1983; Futcher and Cox, 1984). The ratio ofpWX823 to endogenous 2 #m also increased. It was estimated that 2 #m circle molecules per cell decreased from 37 at 10 #g SM/ml to 18 at 50 #g SM/mi. Consistent with this estimation is the observation that a ade4 HR[pWX823] selected on SD + 10 #g SM/ml, and WX2-2d[pWX823] selected on SD-ilv, had slightly increased SM s after nonselective culture but fewer [cir ° ] colonies than when grown in SD + 50 #g SM/ml (Table I). The unchanged cumulative copy number of 2 #m plus pWX823B at 10, 25 and 50 #g SM/ml (62, 64, 63/cc11,

243

M

A

B 0

13

D -9( Fig. 2, Screening ol [cir ° ] subcultures. Yeast strains a ade4 HR (panels A, B) and WX2-2d (panels C, D) were transformed with pWX823B and the curing procedure was as dt~cribed in section h. Yeast DNA was isolated from SMs colonies by a modification ofthe method of Holrman and Winston (1987y Cells from the t~st !)late were directly transferred to an eppeodorf tube containing 0.2 ml lysis buffer, 0.1 ml phenol, 0.1 ml chloroform and 0.2 8 acid-washed glass beads. After vortexing (top speed, 2 rain) and microcentrifuBation (13000 rpm, 5 rain), the supernntant was transferred to a new tube. DNA was precipitated by 2 vols, of cold ethanol and resuspeoded in sucrose Ioadin8 buffer. Southern transfer and hybridization conditions have been described before (Xian and Rank, 1989). 2 #m probe was made from pRIT 12309(Harford and Peeters, 1987)~Over 40 samples can be put into one mini-gel. (Panels A and C) Photographs of nthidium-bromide-stained (0.1%) agarnse gel; (panels B and D) autoradiographs of x-ray film after Southern hybridization, Lane M contained pWX823B as control, Arrowheads point to the monomer form of 2/~m circle.

TABLE I 2/~m DNA curing efficiency by pWX823B Strain B

a ade4 HR

pWX823B Selective media

Screeningb of SM s

Identification of [dr ° ] c

Number of

SM s

Number of

[ctr°]

colonies

(%)

samples

(%)

(ILV2)

SD + 50/A8 SM/ml SD + 10/*8 SM/ml

196 196

70.0 79.1

48 48

12.5 0

WX2-2d (ilv2-4 ! )

SD + 50 pg SM/ml SD-ilv

194 194

41.2 46.9

$6 56

16.1 3.6

B Strains are described in section a. b SM s subcultures were obtained by somatic segregation of pWX823 in YPD medium and screened by colony replication. e Identification of [cir °] subcultures was by Southern-blot analysis.

respectively, Fig. 3) suggests incompatibility between chimeric 2 pm and endogenous 2 pm plasmids (Gerbaud and Guerineau, 1980). This is probably due to 2 #m circle

autoregulation controlled by REPI and REP2 (Som et al., 1988).

244 2,5

50

2.0

4O ¢D

U I "~ %'~,XS I't

aberrant 2Fm-null segregants. This interpretation is strengthened by the observation of a large natural variation in 2 #m copy number (Futcher and Cox, 1984) and the anticipation that SM selection favors S M R I - 2 pm during plasmid partitioning.

.

ACKNOWLEDGEMENTS

~

"O

20 "~

1.0

/ 10

(~5

0

i

i

I

i

'

10

20

30

40

50

SM (~lml) Fig. 3. The effect of SM concentration on plasmid copy level. Strain a ade4 HR[pWX823] was cultured on different concentrations of SM media and the total DNA was subject m Southern-blot analysis with ILV2 and pRITI2309 as probes. ILV2 probe was prepared as before (Xiao and Rank, 1989). The x-ray autoradiograph was analysed on a dansitometer (Molecular Dynamics, 300A Computing Densitometer) to determine DNA copy number by the method of Jayaram et al. (1983). Nuclear !£V2 and plasmid-borne SMRI have the same length of homology to the ILV2 probe. An adjustment was made to accommodate the different lengths of homolog~ to pRIT 12309 of pWX823 (9.6 kb) and 2pro (6.3kb). [], pWX823/cell; @, endogenous 2Fro/cell; O, pWX823/2 Fm ratio.

The authors thank Dr. M.N. Harford for plasmid pRITI2309 and Ms. M. Anderson for densitometry analysis. This work was supported by the National Science and Engineering Research Council of Canada. REFERENCES Blanc, H., Gerbaud, C., Slonimski, P.P. and GueHneau, M.: Stable yeast transformation with chimeric plasmids using a 2 Fm-circular DNAless strain as a recipient. Mol. Gen. Genet. 176 (1979) 335-342. Casey, G.P., Xiao, W. and Rank, G.H.: A convenient dominant selection marker for gene transfer in industrial strains of Saccharomyces yeast: SMRI encoded resistance to the herbicide sulfometuron methyl. J. Inst. Brew. 94 (1988) 93-97. Dobson, MJ., Futcber, A.B. and Cox, B.S,: Loss of 2 #m DNA from Saccharomyces cerev[siae transformed with the chimaeric plasmid pJDB219. Curr. Genet. 2 (1980) 201-205. Erhart, E. and Hollenberg, C.P.: Curing ofSaccharomyce$ cerevislae2 ~m DNA by transformation, CulT. Genet. 3 (1981) 83-89. Erhart, E. and Holienberg, C.P.: The presence of a defective LEU2 gane on 2 p DNA recombinant plasmids of Saccharomyces cerevlslae is responsible for curing and high copy number, J. Bacteriol. 156 (1983)

625-635.

(d) Conclusions

The protocol presented here enables curing wt yeast strains ofendogenous 2 ~tm DNA with a high efficiencyand in a short time period - - approx, ten days from yeast transformation to [cir ° ] subcultures, This rapid procedure will minimize the accumulation of deleterious mutations found in other curing protocols (Erhart and Hollenberg, 1981; Mead et al., 1987). SM concentration can be conveniently varied from strain to strain as required for stringent selection. Although colony hybridization can be used to identify [cir ° ] subcultures, Southern or dot-blot analyses gave us more consistent results. The high efficiency of 2 Fm curing using pWX823B in conjunction with rapid yeast DNA preparation (Fig. 2, legend) makes this analysis practical for screening of [cir ° ] isolates. Yeast 2 Fm circle has an inefficient partition system; stable maintenance of 2 pm relies on mechanisms including F L P - F R T site-specific recombination and sexual transmission (Futcher, 1988). At a higher SM concentration, increased chimeric 2 Fm (pWX823) reduces the endogenous 2 Fm leveldue to plasmid incompatibility (Gerbaud and Guerineau, 1980)and/or via 2 Fm autoregulation (Som et al., 1988). These phenomena increase the probability of

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4 (1988) 27-40. Futcher, A.B. and Cox, B.S.: Maintenance of the 2 Fm circle plusmid in populations of Saecharomyces cerevisiae. J. Bactariol. 154 (1983) 612-622. Futcher, A.B. and Cox, B.S.: Copy number and the stability of 2Fro circle-based artificial plasmids of $accharomyces cerevtsiae. J. Bacteriol. 157 (1984) 283-290. Futcher, B., Reid, E. and Hickey, D.A.: Maintenance of the 2 Fm circle plasmid of $accharomyces ceeevisiae by sexual transmission: an example of a selfish DNA. Genetics ! 18 (1988) 411-415. Gerbaud, C and Guerineau, M.: 2 ~m plasmid copy number in different yeast strains and repartition of endogenous and 2 gm chimeric pinsraids in transformed strains. Cm'r. Genet. I (1980) 219-228. Harford, M.N. and Peeters, M.: Curing of endogenous 2 micron DNA in yeast by recombinant vectors. Curr. Genet. 11 (1987) 315-319. HolTman, C.S. and Winston, F.: A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformations of Escherichia coll. Gene 57 (1987) 267-272. lto, H., Fukuda, Y., Murata, K. and Kimura, A.: Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153 (1983) 163-168. Jayaram, M., Li, Y. and Broach, J.R.: The yeast plasmid 2p circle encodes components required for its high copy propagation. Cell 34 (1983) 95-104.

245 Mead, DJ., Gardner, D.CJ. and Oliver, S.G.: The yeast 2# plasmid: strategies for the survival of a selfish DNA. Mol. Gen. Genet. 20~ (1986) 41"/-421. Mead, DJ., Gardner, D.CJ. and Oliver, S.G.: Pbenotypic differences ~ between induced and spontaneous 2/~-plasmid-free segresants el; Saccharomyces cerevisiae. CNtrr.Genet. I ! (1987) 415-418. Sore, T., Armstrons, K.A., Volkert, F.C. and Broach, J.R.: Autoregulation of 2/Am circle gene expression provides a model for maintenance of stable plasmid copy levels. Cell 52 (1988) 27-37,

Toh-E, A. and Wickner, R.B.: Curing of the 2tJ DNA plasmid from SaccAaromyces ¢er~sfae. J. Bactcriol. 145 (198t) 1421-1424. Xiao, W. and Rank, G H.: The construction of recombinant industrial yeasts free ofbacterial sequences by ~ e d 8ene replacement into a nonessential region oftbe genome. Gone 76 (1989) 99-107. Yadav, N., McDevitt, R.E., Benard, S. and Falco, S.C.: Singleamino acid substitutions in the enzyme acetolaetate synthase confer resistance to the herbicide sulfometuron methyl. Prec. Natl. Acad. Sci. USA 83 (1986) 4418-4422.