Extremely simple, rapid and highly efficient transformation method for the yeast Saccharomyces cerevisiae using glutathione and early log phase cells

JOURNAL OFBIOSCIENCE ANDBIOENGINEERMG Vol. 94, No. 2, 166-171.2002

Extremely Simple, Rapid, and Highly Efficient Transformation Method for the Yeast Saccharumyces cerevisiae Using ~lutathione and Early Log Phase Cells YOSHIYUKI HAYAMA,’ YASUKI FUKUDA,* SHIGEYUKI KAWAI,’ WATARU HASHIMOTO,’ AND KOUSAKU MURATA’* Department of Basic and Applied Molecular Biotechnology, Graduate School ofAgricuIture, Kyoto University Wji, Kyoto 6~I-00~1, Japan~a~ Chu~o Comm~ni~ College, ~izun~~, Cifi 509-~~OI, Japan2 Received 15 April 2002iAccepted 29 May 2002

In the presence of polyethylene glycol (PEG), budding cells of Saccharomyces cererisiue in the early log phase were transformed by exogeneous plasmid DNA without additional specific chemical or physical trea~ents. This capacity of the yeast cells to become competent was strictly dependent on the growth phase, being induced in the early log phase, becoming maximum between the early and mid log phases, and then disappearing rapidly in the mid log phase. The transformation was most efficient at pH 6, and the frequency increased with increasing DNA and cell concentrations. PEGS with average molecular sixes between 1000 and 3500 showed almost the same effects, and were used most efficiently at 35%. The transformation frequency of S. cerevisiae was marked$ enhanced when the oxidized form of glutathione (GSSG), but not the reduced form, was included in the mixture composing early log phase cells, plasmid DNA, and PEG and the transformation system with GSSG could be used as a convenient transformation method for the yeast S. cerevisiae. [Key words: transformation, yeast, Saccharomyces cerevisiae, glutathione, polyethylene glycol] Tr~sfo~ation of the yeast ~acc~aro~yces cerevisiae has been carried out worldwide by alkaii monovalent cation treatment (1). Although the introduction of this method eliminated tedious and intricate procedures and the many complicated steps involved in the protoplast method (2), it still requires both the presence of polyethylene glycol (PEG) and extensive treatment with metal ions at high concentrations to render the cells permeable to plasmid DNAs and to attain the highest transformation frequency, which is realized at the time when the cells begin to lose their viability (1). Because competent cells thus prepared suffer some damage to cell surface structures and/or cellular metabolism, they are unsui~ble for use in studies on the DNA uptake mechanism and other physiological events involved in mediation of the various steps of the transformation process. This is also the case in the transformation of yeast cells by the electroporation method (3), in which cells are subjected to high-voltage electrical pulses to induce competence. Recently, we found that S. cerevisiae cells in the early logarithmic growth phase (early Iog phase) can be transformed with plasmid DNAs in the presence of only polyethylene glycol (PEG), and that the transformation frequency of the system, which we tentatively call “natural transformation” in this article, is high and ofien exceeds that using alkali monovalent cation (LiCl) treatment (1). Furthermore, in the course of studying glutathione-de~cient mu-

tams, we noticed that this riptide, especially its oxidized form (GSSG), was an important biomolecule in determining the transformation frequency. These preliminary results were recently reported (Hayama et al., Abstr. Annu. Meet. Jpn. Sot. Biosci. Biotechnol. Agrochem., p. 288,2002). Gn the basis of the above-mentioned observations, we attempted to establish a novel yeast ~~sfo~ation system comprised of “intact and flawless” cells, that would allow us to study the DNA uptake mechanism in S. cerevisiae cells. MATERIALS AND METHODS Yeast strains and plasmids

Yeast strains S. cerevisiae DKD-

SD-H (u4Ta leu2-3, 112 trpl his3), D13-1A @fATa trpl his3-532 gall) (l), AH-22 (MATa Zeu2-3, 112 his4519 canl) (l), OS1 (LWTa leu2-3, 112 ura3-52 gshl), MT8-1 @fATa ura3-I trpl-1 ade2-1 leu2-3, 112 his3) (4), NA87-1 IA (MATa leu2-3 trpl his3 p-), D308.3 (MATa adel trpl his2 met14 hxkl hxk2 gIk2) (l), YHl (eta trpl ura3 gshl) (5), YH2 (MATa leu2 ura3 gshi) (5), YH3 (MATa leu2 trpl ura3 gshl) (5), and YNN27 (MATa trpl ura3 gaZ2) were used as hosts for plasmid DNAs of YRp7, YEpl3, pAT19, and pDB248 (6). The YRp-type plasmid PAT19 was constructed through insertion of the TYZPI-ARSI segment from pYAB1 (7) into the SspI site of pUC19 (Takara Bio, Otsu). Plasmid DNAs were prepared by the method of Sambrook et al. (8) ffom a lysate of Escherichia coli DH5a (en&f, gvrA96, ~dR~7 (rk-mk+)recA1, retAl, supE44, thi-f , A(lacZYA-argF)U 169, @8OlacZMflS, F, A-) cells habouring the plasmid DNAs, suspended in TE (5.0 mM Tris [pH 7.01 containing 0.5 mM ethylenediaminetetraacetate), and then used for transformation. The presence of plasmid DNA in the yeast

* Corresponding author. e-mail: [email protected] phone: +81-(0)774-38-3766 fax: +81-(0)744-38-3767 166

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TABLE 1. Effects of incubation time, and heat pulse and pH jump treatments on transformation Transformants/lO ug YEpl3 -..___ 15 min 30 min 120 min

Treatment

0 min

-None Heat pulse (42°C 2 min) pH jump (pH 7.O+pH 4.0) @H 7.0jpH9.0)

4 16 -

170 674 -

610 1154 2291 1960

852 2256 -

Tra~fo~ation was carried out as described in Materials and Methods, except that the ~sfo~at~on mixture (3 15 pl) was incubated for various lengths of time (&120min). A sample incubated for 30min was subjected to either heat pulse or pll jump treatment. Heat pulse treatment was performed by immersing the test tubes in water (42°C) for 2 min. pH-jump was achieved by adding 100 nl of 1.OM sodium acetate (pH 4.0) or 15 ul of 1.OM Tris base to the transformation mixture. The transformation frequency represents the average of two independent experiments. transformants was confirmed by agarose gel electrophoresis after the transformation of E. coZi DHSa ceils with DNA extracts prepared from the yeast transformants (1). S. cerevisiae cells pre-grown aerobically at Tr~nsfo~ation 30°C for 16 h in YPD nutrient medium (2.0% glucose, 2.0% bactopeptone, I .O% yeast extract; pH 5.0) were transferred to fresh YPD medium (ZOOmI) to obtain an initial cell concentration of approximately 106/ml, and then incubated krther under the same conditions. In the following transformation procedures, cells, DNAs, and chemicals were prepared in TE. In the early exponential growth phase (A,,,=0.25-0.3; early log phase), which was determined by measuring the absorbance of the culture at 600 nm (A6&, cells were collected, washed once with TE, and then suspended in TE to give a cell concentration of 3 x log/ml. In the case of DKD-SD-H, 0.1 of A6,,0corresponded to 1.26x 1O6cells/ml and this relationship was used to calculate the cell concentrations of haploid strains. The cell suspension (100 pl) was mixed with 1.50 pl of 70% polye~ylene glycol (PE~4000) and 15 pl of 1.O ug/pl plasmid DNA. The entire ~~sfo~ation mixture (3 15 pl), after topping up with TE, was incubated for 30 min at 30°C. Immediately after this incubation, heat pulse or pH junrp treatment could be applied if necessary. The procedures for these treatments are described in the footnote to Table I. The compound solutions (0.05 ml) to be tested for their effects on transformation were included in the transformation mixture specified above. After these procedures, the cells were washed once with TE, spread on plates (1.5% agar) of SD minimal medium (2.0% glucose, 0.67% yeast nitrogen base; pH 5.0) supplemented with nutrients (tryptophan, 40 ug/ml; histidine, 20 ug/ml; methionine, 40 pgml; uracil, 20 &ml) required for their growth, and incubated at 3O’C for 3 d. Colonies appearing on the plates were determined as transformants. The transfo~ation frequency was defned as the number of tr~sfo~~~ per IO pg pl~mid DNA. Tr~sfo~ation of S. cerevisiue DKD5D-H by alkali monovalent cation (LiCl) treatment was performed as described previously (1). Chemicals 2,3-Dihexadecanoyl-sn-glycero-I-phosphochoIine (D-cr-phosphatidylcholine, dipalmitoyi) and polyethylene glycols with various average molecular sizes were purchased from Nacalai Tesque, Kyoto. Reduced and oxidized forms of glutathione were from Kojin, Tokyo. Yeast nitrogen base was obtained from Difco Laboratories, Detroit, MI, USA. RESULTS

Properties

of “natural

transformation”

Although

natural ~~sfo~ation is defined as a genetically programmed physiological state periling the efficient uptake

Time (h) FIG. 1. Detection of a competent state during growth on nutrient medium. Transformation by “natural transformation” and an artificial transformation system was performed as described in Materials and Methods. Filled squares, “Natural transformation”; empty squares, artiticial tr~sf~~ation system (LiCl treatment); circles, growth (Am).

of DNA, and it is distinct from artificial yeast transformation methods involving protoplasts (2), alkali monovalent cation treatment (I), or electroporation (3), in this article we tentatively call our system “natural transformation” to distinguish it from artificial transformation methods. Our standpoint on the use of this term is given in the Discussion section. Growth phase-dependent induction of competence The ability of intact cells of S. cerevisiue DKD-SD-H to incorporate exogeneous plasmid DNA (YEp 13) was investigated in the presence of PE~4000 (Fig. 1). The ability was found to appear in the early log phase, to become maxims between the early and mid log phases, and then to disappear completely in the late log phase on an SD minimal (data not shown) or YPD nutrient medium (Fig. 1). The pattern of competence induction apparently differs horn that of artificial transformation by chemical (LiCl) treatment (I), in which the ability of cells to incorporate plasmid DNA was observed in all the phases examined. The maximum transformation frequency attained using yeast cells in the early log phase exceeded 750 transformants/lO pg plasmid DNA (YEp13), which is about one-half of that obtained with artificial tr~sfo~ation (LiCl treatment) (Fig. 1). However, depending on the early log phase cells prepared, the transfo~ation frequency varied within the range 60~2500 ~ansfo~~ts/lO pg plasmid DNA. Neither the appearance of leucine prototrophs from recipient cells of S. cerevisiae DKD-SD-H due to back-mutation nor a decrease in the viable cell number was detected under the “natural transformation” conditions used (data not shown). The DNA extracts prepared from three of the yeast transformants gave E. cob DH5cx cells ampicillin resistance, indicating that the cells incorporated plasmid YEpl3 through the “natural transformation” process. In fact, DNA analysis of the resultant E. co& DH5a transformants showed the presence of YEpl3, which is absent in the bacterium used as the host (Fig. 2). Some features of ~‘natural transformation ” The fea-

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1 2

tkbp)

3 4

5 6 7

TABLE 2. Effects of diierent types of plasmid DNAs and S. cerevisiae strains on ~sfo~ation

8 9 10 1

Recipient strain DKD-SD-H

Dl3-IA

FIG 2. Detection of plasmids in yeast transformants. DNA from one of the S. cerevisiae transformants was transferred to E. coli DH5a cells, followed by agarose gel electrophoresis. Lane 1, Marker DNAs; lane 2, DNA extract of DH5a; lane 3, YEpt3; lane 4, DNA extract of E. cofi transformant; lane 5, EcoRI-treated I3NA extract used in lane 2; lane 6, EcoRI-treated DNA (YEpl3) in lane 3; lane 7, EcoRI-treated DNA extract used in lane 4; lane 8, PstI-treated DNA extract used in lane 2; lane 9, &&treated DNA (YEpl3) in lane 3; lanelO, PstItreated DNA extract used in lane 4.

B

z %

0

3

4

5

6

7

8

9

0

_./. 105

106

10’

10s

Cell&Ill

PH 7.50

500

250

,

0

’

30

m~13 @g/ml)

100

0

,

2OOa

4000

c

6Oc

PEG (Av. M.W.)

FIG 3. Etfects of pH, molecular weight of PEG, and concentrations of cells and DNA on “natural transformation”. Transformation was carried out, while varying the pH (A), molecular weight of PEG (D), and concentrations of cells(B) and DNA (C) in the standard transformation mixture as described in Materials and Methods, The pH of the ~sfo~ation mixture was adjusted with 0.1 M Trislmaleate. The inset in (D) shows the relative effects of PEG1000 and PEG4000 on the transformation efficiency. The efficiency obtained at 35% PEG4000 was taken as 100%.

tures of “natural transformation” were studied using cells of S. cerevisiue DKD-SD-H in the early log phase. The transformation was most efficient at pH 6 (Fig. 3A). The trans-

formation frequency increased with increasing cell concentration (Fig. 3B), DNA concentration up to 30 pg (Fig. 3C),

AH-22 NA87-11A OS1 MT&l D308.3

Plasmid DNA

Transformants DNA

YEp13 YEpl3 YEp13 YEpl3” YEpl3d pAT19 YRP7 YEpl3 pDB248 YEp13 YEpl3 YEpl3 YRv7

2068 5508 635b 3115 1140 5567 915 1904 1420 25 <5 1040 3763

0 pg

Transformation was carried out as described in Materials and Methods, except that various types of plasmid DNAs and S. cerevisiae strains were used. The ~fo~ation frequency represents the average of two independent experiments. Sb 2,3-Dihexadecanoyl-sn-glycero-I-phosphocholine (PCP) (a, 0.2 mg/ml; b, 0.4 mg/ml) was used in place of PEG4000. The compound dissolved in ethanol was included in the standard ~~sfo~ation mixture, the final ethanoi concentration being 5%. Ethanol at this concentration (5%) showed no effect on the transformation (data not shown). ‘sd YEpl3 linearized by the treatment with BumHI (c) or PvuII (d) was used.

and incubation time (Table 1). Although heat pulse (42”C, 2 min) or pH jump (from pH 7 to 4 or 9) treatment was effective in enhancing the transformation frequency (Table 1), these addition steps were es~ntially ~ecess~ for the “natural transformation” process. Linearized DNA prepared through the digestion of YEpl3 with the restriction enzyme BumHI or PvuII was also incorporated at a frequency comparable with that in the case of non-cut circular YEpI (Table 2), and was present in the yeast transformants as the circular form, as judged by the E. coli DH5a transformation method described above (data not shown). PEGS with average molecular weights between 1000 and 3500 were efficiently utilized for the transformation, and the highest transformation frequency was obtained at 35% for both PEG1000 (av. M.W., 950-1050) and PEG4000 (av. M.W., 270~3500) (Fig. 3D). Metal ions (0.1 M: Li”, Cs+, Rb’, Ca2”,Mg*+,and Mn*+),polysaccharides (0.1%: alginate, xanthan, gellan, mannan, and glucan), poly-amino acids (20 mM: poly&lysine, poly-L-arginine, and poly+glutamate), and polyamines (20 mM: putrescine, spe~i~ne, and spermine) could not replace PEG4000, although 2,3-dihexadecanoyl-sn-glycero- 1-phosphocholine (PCP) (0.2-0.4 mglml) gave a significant number of transformants in the absence of PEG4000 (Table 2). Spe~idine and spermine completely repressed the transformation in the presence of PEG4000 (data not shown). Transformation of various S. cerevisiae strains “Natural ~~sfo~ation” has also been observed in various S. cerevisiae strains and, in the presence of PEG4000 plasmid DNAs of different sizes and replication origins (ars for YRp7 and pAT19; 2 pm DNA for YEpl3 and pDB248 [6]) were incorporated by early log phase cells of these strains (Table 2), although the transformation frequency was low

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TABLE 3. Effects of glutathione on transformation Transformants”/lO pg DNA

Recipient strain

Plasmid DNA

None

GSHb

GSSGb

YNN27 DKD-SD-H AH22 NA87-1 IA

pATl9 YEpl3 YEp13 YEpl3

2,410 2,340 793 37

6,120 6,332 7,680 5,655

12,449 10,031 11,324 6,790

YHl YH2 YH3 OS1

pATl9 YEpl3 YEpl3 YEpl3

5 0 4 5

631 273 180 262

12,667 2,310 2,442 1,328

a Average of two independent experiments. b GSH and GSSG were used at 30 mM. Other conditions were as described in Materials and Methods.

for the respiration-de~cient @-) strain NA87-11A and the glutathione biosynthesis-deficient strain OS 1. Effect of glutathione on “natural transformation” Since, as noted above, glutathione-deficient cells of S. cerevisiue failed to incorporate plasmid DNA, the effect of the tripeptide on the “natural transformation” of S. cerevisiue cells was investigated. Transformation of glutathione-deficient cells Glutathione-deficient cells of S. cerevisiue YHI, YH2, YH3, and OS 1 have a mutation in y-glu~ylcysteine synthetase (EC 6.3.2.2) (5) the first enzyme in the glutathione biosynthetic pathway, and contain no detectable reduced (GSH) and oxidized (GSSG) forms of glutathione (data not shown). Under the standard tr~sfo~ation conditions described in Materials and Methods, the transformation frequency of these glutathione-deficient cells (YH2, YH3, and OS1 with plasmid YEpl3; YHl with plasmid pAT19) was extremely low or completely repressed (Table 3). However, these mutant cells of YHI , YH2, YH3, and OS1 were transformed when GSH or GSSG, especially GSSG, was included in the transformation mixture (Table 3). Although the data are not presented here, the existence of plasmid YEpl3 in the YH2 transformants obtained in the presence of GSH or GSSG was confirmed by a method similar to that shown in Fig. 2. The transformation frequency of YH2 increased with increasing GSSG concen~ation up to 30 mM, while that of YH2 in the presence of GSH was low regardless of the concentration (Fig. 4A). Transformation of glutathione-containing cells Haploid (YNN27, D&CD-SD-H,AH22, and NA87-11A) cells of S. cerevisiae in the early log phase contain glutathione (GSH+ GSSG) at approximately l-2 mg per g-dry cells (data not shown). The transformation frequency of these strains was determined in the absence and presence of GSH or GSSG (Table 3). Even in the absence of GSH and GSSG haploid cells in the early log phase were transformed in the presence of PEG4000. However, addition of GSSG to the mixture markedly increased the transformation frequencies of all the strains tested, although the effect of GSH was low compared with that of GSSG (Table 3). As observed in the case of glutathione-deficient YH2 cells (Fig. 4A), the transformation frequency of YNN27 cells increased with increasing concentration of GSH or GSSG especially GSSG up to 30 mM (Fig. 4B). E&Sect of the incubation time and glututhione

on transfor-

0

10

to

30

GSH or GSSG (mM)

-.

10

20

30

GSH or GSSG (mM)

FIG 4. Effects of GSH and GSSG concentrations on transformation of glutathione-deticient and glutathione-containing cells. Early log phase cells of the glu~~ione-de~cient strain YHZ {A) and glutathione-containing strain YNN27 (B) were transformed with plasmids YEpl3 and pATl9, respectively, in the presence of GSH (empty circles) or GSSG (tilled circles) at the concentrations indicated.

A

B

Time (minf

Time (min)

FIG 5. Effect of incubation time on the transformation frequency of glutathione-containing cells in the presence and absence of GSSG. The transformation of glutathione-containing strains YNN27 (A) and DKD-5D-H (B) with plasmids pATl9 and YEpl3, respectively, was carried out in the presence (tilled circles) and absence (empty circles) of GSSG (30 mM) for the times indicated.

mation of g~utathione-containing

cells Early log phase celIs of S. cerevisiae YNN27 were simultaneously mixed with plasmid pAT19 and PEG4000 in the presence and absence of 30mM GSSG and then incubated for various lengths of time to transform the cells (Fig. 5A). In the presence of GSSG, the tr~sfo~ation frequency increased with the incubation time, and by 60min had reached more than 15,000 transformants/lO pg DNA. Almost the same results were obtained in the case of DKD-SD-H cells with plasmid YEpl3 (Fig. SB). Two of the tr~sfo~an~ obtained in the presence of GSSG required tryptophan and histidine for growth on SD minimal medium, indicating that the require-

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ment of DKD-SD-H cells for leucine was complemented by the plasmid YEpl3. In fact, the DNA extract prepared from one of the transformants changed E. coli DHSa cells from ampicillin-sensitive to ampicillin-resistant, and the presence of plasmid YEpl3 was continned in ampicillin-resistant E. coli DH5cr cells (data not shown). DISCUSSION We have demonstrated the occurrence of a natural transformation-like phenomenon in budding cells of S. cerevisiae. The major question regarding this demons~ation is whether the ~~fo~ation observed is “natural” or “artiticial”. The requirement for PEG (Fig. 3D) causes some ambiguity. However, the following facts (i)-(iii) support the presence of a novel transformation system distinct from artificial systems, i.e., protoplast (2), alkali monovalent cation treatment (1), and electroporation (3) methods. (i) The competence indu~ion is strictly dependent on the growth phase (early log growth phase), as has been indicated in the case of naturally transformable bacteria (9-12), and the pattern of competence development apparently differs from that in the case of artificial transformation by LiCl treatment (Fig. 1). (ii) Growth phase-dependent incorporation of plasmid DNA is commonly observed among strains of 5‘.cerevisiue (Tables 2 and 3). (iii) The function of PEG in the transformation of yeast cells is partially facilitated by 2, 3-dihexadecanoyl-sn-glycero- 1-phosphocholine (PCP), a major component of the yeast cell membrane (Table 2). These features, especially (i) and (iii) prompted us to label the transformation observed “natural ~~sfo~ation~ as explained at the beginning of the Results section. Although this reasoning remains speculative, our finding of a natural transformationlike phenomenon may indicate that natural competence is widespread among microbial species. As to the function of PEG (or PCP) in “natural transfo~ation~ two possibilities can be considered. One is that PEG (or PCP) provides the conditions for competence development by yeast cells, as soil or plants do for competence development in the case of transformable bacteria (9, 13, 14). That is, in the observed transformation system of S. cerevisiae,the induction of a competent state depends only on the physiological state of the cells exposed to PEG (or PCP). The other possibili~ is that the polymer (or PCP) induces conformational changes in DNA so that it can pass through DNA-permeable channels present on the surface of yeast cells. Of these two possibilities, at present the latter seems reasonable with respect to the function of PEG since incubation of PEG with DNA induces drastic conformational changes in DNA (data not shown). We have shown that (i) haploid cells of the yeast S. cerevisiue in the early log phase are transformed with plasmid DNAs in the presence of PEG (or PCP) and (ii) glutathione (GSH and GSSG), especially GSSG enhances the transformation frequency of these early log phase cells. These findings indicate that the DNA uptake system is induced only in yeast cells in the early log phase and requires glutathione (GSH or GSSG) for it to function. The limited effect of GSH is presumably due to the low permeability of the tripeptide with these cells. The few transformants of glutathi-

50 pI 6 x I@/rnI Eariy tog phase ceils -

15 @ 1.0 &.d Plasmid DNA

-

150 pl 70% PEWOOo

-So@

O.lZMGSSG

t blcubation (pH 6-7,309=, S-10 mint (Heat pulse or pH jump, optional) FIG 6. Practical “natural transformation” method for S. cerevisiue. For transformation, early log phase cells of S. cerevisiue aresimultaneously mixed with olasmid DNA. PEG4000. and GSSG and then incubate; for several nknutes. After ~u~ub~io~,.heat pulse (42Y!, 2 min) or pH jump (addition of acid or base) can be applied with effect if required. The volumes and concentrations of cells and chemicals will vary depending on the yeast strain used.

one-deficient mutants that appeared in the absence of both GSH and GSSG (Table 3) may have been due to intracellular glu~~ione ac~ulated during growth on YPD nutrient medium. In fact, when we performed the experiments with SD minimal medium-grown cells, no transformants of YHl were obtained (data not shown). Although the role of glutathione in the “natural transformation” described here will be significant in elucidating the uptake mechanism of plasmid DNAs by early log phase yeast cells, this is not the purpose of the present study. Our finding on the function of GSH and GSSG enabled us to establish a highly convenient transformation method for S. cerevisiuecells. The “natural transformation” conditions obtained were amended and readjusted so that anyone can perform the ~~sfo~ation experiments easily. The practical procedures are illustrated in Fig. 6. All that needs to be done is to prepare early log phase cells and mix them with a plasmid DNA, PEG4000, and GSSG, followed by incubation for several minutes (at least 5-10 min), preferably at pH 6-7 and 30°C. PEG4000 and GSSG are used at 35% and more than 10 mM (Fig. 4A, B), respectively. The cells and plasmid DNAs are used at 1OS/mland 50 &ml, respectively, although the transformation frequency increases with increasing cell concentration. When cells of S. cerevisiue YNN27 and DKD-SD-H were transformed with plasmids pAT19 and YEp13, respectively, under the preferred conditions (GSSG, 30mM) specified in Fig. 6, approximately 50~1000 (after ~cubation for t0 min) and 10,00~20,000 (after incubation for 60 min) transformants were constantly and reproducibly obtained (data not shown). A heat pulse at 42°C for 2 min or a pH jump from pH 7 to pH 4 or 9 obtained by adding either an acid or base, respectively, is effective in enhancing the transformation frequency, but these ad~tional treatments are not crucial for the “natural transformation” reported here, and a sufficiently high transformation frequency is obtained without them. Transformation methods thus far developed can be classitied into the following three categories based on the method of treating the yeast cells: biological (enzyme treatment; protoplast fo~ation~ (2), chemical (metal reagent) (l), and physical (electrical treatment; electroporation) (3). Although these methods have their own features, the “natural transformation” presented here is distinctive in that it facilitates study of the DNA uptake mechanism and other physio-

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TRANSFORMATION

logical events underlying the transformation process, since the yeast cells employed in the “natural transformation” are “intact and flawless.” In addition, the extremely simple, time-saving procedures (Fig. 6) and high transformation frequency promise that the “natural transformation” method described here will be used for yeast transformation in place of the alkali cation treatment method that we developed previously (1).

6.

7.

ACKNOWLEDGMENTS 8. We wish to thank Dr. A. Kawado, Gekkeikan Institute, Kyoto, for providing us with plasmid pAT19 and OS1 strain, and Dr. Y. Ohtake, Asahi Brewery, Tokyo, for yeast strains YHl, YH2, YH3, and YNN27. We also thank Dr. Y. Oshima, professor emeritus of Osaka University, for DKD-SD-H strain. This study was supported in part by the Bio-oriented Technology Research Advancement Institution (BRAIN).

9.

10.

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