A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast

Gene. 25 (1983) 333-341

333

Elsevier GENE 885

A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast (Transformation; polyethylene glycol; Escherichia coil; Saccharomyces cerevisiae; recombinant DNA; protoplasts; CaCI2)

Robert J. Klebe, June V. Harriss, Z. Dave Sharp and Michael G. Douglas Department of Anatomy, The Universityof TexaX Health Science Center, San Antonio, TX 78284 (U. S.A .) Tel. (512) 691-6543 and Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX 78284 (U.S.A.) Tel. (512) 691-6851 (Received May 23rd, 1983) (Revision received July 18th, 1983) (Accepted July 20th, 1983)

SUMMARY

Polyethylene glycol (PEG) can induce genetic transformation in both bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) without cell wall removal. PEG-mediated transformation of E. coil is technically simple and yields transformants with an efficiency of 106--107 transformants/pg DNA. Detailed analysis of the parameters involved in PEG-mediated transformation of E. cell reveals basic differences between the PEG and standard CaCI2 methods for transformation of E. colt. PEG-mediated transformation of yeast is far simpler than existing protoplast methods and is comparable in efficiency. The new methods described here for PEG-mediated genetic transformation may prove to be of general utility in performing genetic transformation in a wide variety of organisms.

INTRODUCTION

Many genetically important species are not capable, under natural conditions, of undergoing genetic transformation (Venema, 1979). However, protoplasts of E. cell (Young and Sinsheimer, 1967) and several other bacterial species (Chang and

Abbreviations: LB, Luria broth; NB, 0.15 M NaCI + 10raM bicine, pH 8.35; PBS, Dulbecco's phosphate-buffered saline (see MATERIALS AND METHODS, section d); PEG, polyethylene glycol; PEG-1000, PEG of average Mr 1000; SAM, 0.5 M sorbitol + 20 mM MgCI2 + 100 rnM K" acetate, pH 5.4; SBEG, 1 M sorbitol + 10 mM bieine, pH 8.35 + 3% ethylene glycol; TE, 10 mM Tris, pH 8.0, + 1 mM EDTA. 0378-1119/83/$03.00 © 1983 Elsevier Science Publishers

Cohen, 1979; Suarez and Chater, 1980; Brown and Carlton, 1980) can undergo DNA-mediated genetic transformation and transfection. Similarly, removal of the yeast cell wall by digestion with zymolyase permits successful DNA uptake (Beggs, 1978; Hinnen et al., 1978). Other methods permitting transformation of incompetent organisms include CaCI2 treatment (Mandel and Higa, 1970), osmotic shock (Taketo and Kumo, 1969a), freezing and thawing (Mackal et al.,1964),growth in hypertonie medium (Taketo and Kumo, 1969b),penicillintreatment (Taketo and Kumo, 1969a), and exposure to basic polymers (Bvnzinger, 1977; Osowiecki and Sk.linska, 1974). Protoplast formation generally results in higher

334

yields of transformants//~g DNA than methods employing intact or partially intact cells (Chang and Cohen, 1979). However, due to its simplicity and relatively high yield of transformants, the CaCI2 method of Mandel and Higa (1970) has become a standard method. Recent improvements of the CaCI2 method involve increasing the time of exposure to CaCI2 (Dagert and Ehrlich, 1979), the addition of rubidium chloride (Kushner, 1978), the inclusion of dithiothreitol, hexamine cobalt (III), and DMSO plus a freeze-thaw protocol (Hanahan, 1983), and optimization of conditions (Norgard et al., 1978). Unfortunately, techniques equivalent to the E. coli CaCI2 method do not exist for genetic transformation in many other genetically important species. In this report, a procedure employing polyethylene glycol is described which yields large numbers of transformants in both prokaryotic and eukaryotic organisms without cell wall digestion.

MATERIALS AND METHODS

(a) Preparation of polyethylene glycol (PEG) PEG-1000 obtained from several sources was found to vary in consistency from a hard, waxy solid to a soft jelly and also often contained acidic impurifles. To avoid this lot-to-lot variability, PEG-1000 was purified before use. PEG-1000 was purified by preparing a 50~ (w/v) solution of PEG-1000 in benzene from which the PEG was precipitated by the addition, with stirring, of 1 vol. ofisooetane (2,2,4-trimethylpentane). After cooling to 6°C for 1 h or more, the oil which separated upon addition of isooctane solidified into a wax-like cake. The solvent was discarded and the solidified material was redissolved in the volume of benzene used initially. The PEG was re-precipitated by the addition, with stirring, of 5 vols. of ethyl ether. Following 4 h or more at 6°C, the precipitate was collected on a sintered glass funnel (course grade), washed with ethyl ether, and dried under high vacuum following removal of the majority of the ether via house vacuum. The resulting material was afree flowing, white powder which was stored in a sealed container. The purified material did not lose weight upon further lyophilization and a 50~ solu-

tion of purified PEG-1000 was approx, pH 5.2. Purification of PEGs of other M r values could be carried out in a fashion analogous to that of PEG-1000 except (a)PEG-200 and PEG-600 formed an oil which did not solidify upon isooctane precipitation; (b) PEG-200 did not solidify after precipitation with ether, and (c)PEG-3000 and PEG-6000 required gentle heating to dissolve in benzene. PEG solutions should be sterilized by membrane filtration rather than steam autoclaving. Membrane filtration was carried out with an 0.2-#m filter (Amicon, Lexington, MA). While a 50~o PEG-1000 solution can be membrane filtered without technical difficulties, the high viscosity of PEGs with higher Mr will increase fdtration time considerably. During the pH adjustment of viscous PEG solutions, it was found that NaOH and HCI solutions formed droplets which required considerable time to dissolve completely. Hence, the accurate pH adjustment of viscous PEG solutions presents technical difficulties which can be avoided by allowing sufficient time for acid or base to completely dissolve. We noted that basic PEG solutions, in particular, gradually decrease in pH during storage at room temperature. This finding is probably attributable to adsorption of atmospheric CO2. Thus, rechecking of the pH of PEG solutions is recommended (a)just after membrane filter sterilization of freshly prepared solutions and (b) following prolonged storage.

(b) Reagents All chemicals were of reagent grade. Following chloramphenicol amplification (Clewell, 1972), plasmid DNA was twice purified by CsCl-ethidium bromide density gradient centrifugation (Radloff et al., 1967). Plasmid DNA was stored at - 7 0 c C in TE.

(c) E. coil and yeast strains The following E. coli strains were employed: RR1 (Bolivar et al., 1977); HB101 (Boyer and RoulandDussoix, 1969); M94 (Meselson and Yuan, 1968) and ED8767 (Grosveld et al., 1982). Yeast strain RC-5 (Broach et al., 1979) was studied.

(d) Genetic transformation of E. coil LB broth (Maniatis et al., 1982) was employed for the culture of the E. coli strains studied (Table I); LB

335

broth containing 1.5~o Bacto-agar (Difco, Detroit, MI) and 50 #g/ml ampicillin was used for the selection of pBR322 transformants. Cultures were inoculated with a 1 : 50 dilution of an overnight starter culture, maintained at 36.5°C, and harvested 4h later when absorbance ofA6oo = 0.6 to 0.7 had been achieved. 8 ml of the log phase culture was washed with 5 ml of SAM by centrifugation for 5 min at 7000 x g at 20°C. The pellet was resuspended in 0.2 ml SAM. Alter 5 min, 1 to 20 #I ofplasmid DNA was added at 22°C. DNA was diluted in TE in siliconized glass tubes. After 10 min, 1.5 ml of 25~ PEG-1000 + 100 mM succinate, adjusted to pH 4.4 with NaOH, was added and the preparation was shaken several times during a 5-rain incubation period at 22°C. The action of PEG was stopped by the addition of 5 ml of PBS (8.0 g NaCI, 0.2 g KC1, 0.1 g MgC12, 0.1g CaC12, 1.15g Na2HPO4, 0.2g KH2PO 4 adjusted to 1 1 and pH 7.2). The bacteria were washed once with PBS and resuspended in 5 ml LB for 30 rain at 37°C prior to being plated on selective medium. The results presented represent averages of duplicate determinations. Comparable results were obtained in two or more separate trials conducted on different occasions. The procedures given above were arrived at by selecting optimal conditions for each parameter in the procedure (Figs. 1-6) and re-optimizing all other parameters after the change of any one parameter. Using this procedure for E. cog strain RR1, transformants were recovered with an efficiency which averaged 1.96 x 106 transformants//~g DNA (Tables I and II, Figs. 1-6).

(e) Genetic transformation of yeast A partially optimized procedure t'or yeast transfermarion is presented below which will be compared in the text to the method described for E. coll. YPD broth (Sherman et al., 1979) was used in the routine culture of yeast strain RC-5, which is a leucine auxotroph (Broach et al., 1979). Cultures were inoculated with a 1 : 25 dilution of an overnight starter culture, maintained at 30°C, and harvested once the cell density had reached A60o = 0.6. Log-phase yeast (10 ml) was washed with 5 ml of SBEG by centrifugation at 1000 x g for 3 rain at 22 ° C. All subsequent incubations were carded out at 30°C. The pellet was resuspended in 0.2 ml SBEG;

and after 5 min, 1 to 20 #1 of YEp13 plasmid DNA (Broach et al., 1979) was added. After 10 rain, the preparation was placed in a - 7 0 0 C freezer for 10 rain (or longer) and thawed by rapid agitation in a 37°C water bath. 1.5ml of 4 0 ~ PEG1000 + 200 mM bicine, pH 8.35 (stored frozen) was added; and after a 1-h incubation, the ceils were washed with 2 ml of NB. Following eentrifugation, the pellet was resuspended in 1 ml of NB and plated on selective medium. Leu + transformants were selected on a synthetic medium containing yeast nitrogen base, glucose, a mixture of amino acids lacking leucine and 3 ~ agar (Sherman et al., 1979). Cultures were incubated at 30°C; transformants were countable in 2-3 days. Under the conditions described above, 200-1000 transformants were recovered per pg YEp 13 plasmid DNA. Yeast protoplast methods yield 103-104 transformants/#g DNA (Struhl et al., 1979; Broach et al., 1979). When YEp 13 DNA was used to transform E. cell, transformants were recovered with only 1.5~ the efficiency of several other vectors (not shown). Hence, the somewhat reduced transformation efficiency of the PEG method vs. protoplast methods for yeast may be attributable, at least in part, to the inefficiency of the YEpl3 vector employed in the studies described here.

(f) Sources of technical problems Probably the chief source of technical difficulties is the proper preparation and maintenance of PEG solutions. Such problems are easily avoided by observing the precautions noted in section a which concerns the preparation of PEG solutions. A second major source of variability in the yield of transformants is the method of removing PEG. The rapid mixing of wash solution (NB or PBS) with PEG-treated organisms results in a 3-fold or more reduction in the yield of transformants. Optimal yields oftransformants are obtained by slow addition of the wash solution to PEG followed by gentle inversion of the tube to yield a homogeneous solution. Since PEG-mediated transformation of both E. coli and yeast is markedly inhibited at temperatures below 15 °C, the temperature of centrifuge rotors should be approx. 200C before use.

336 RESULTS

too1

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.-- 90 80 70

(a) General overview The protocols for PEG-mediated genetic transformarion of both E. coli and yeast have been described in MATERIALS AND METHODS, sections d and e. In the following section, a detailed analysis of parameters involved in the E. coil system will be presented. While the transformation procedures employed with E. coil and yeast differ in several regards, the two procedures are fundamentally quite similar. In essence, two basic steps are involved. First, cells are treated with D N A under hypertonic conditions. Second, PEG-1000 treatment is required to complete the transformation event. Incorporation of a freeze-thaw step enhances transformation of yeast by about three-fold. The efficiency of PEG-mediated genetic transformation obtained with several E. coil strains is given in Table I. Differences in transformability between E. coil strains were noted with both the P E G and CaCI 2 procedures (Table I). Strain RR1 transformed readily with both the P E G and CaCI2 procedures while other strains yielded large numbers oftransformants with one or both methods. RR1 has been shown to be more easily transformed with the CaCI 2 method particularly when plasmids with inserts are employed (Peacock et al., 1981). While the PEG-mediated transformation method for E. coli is comparable in efficiency and technical simplicity to the standard CaCI 2 method (Mandel and Higa,

TABLE I Efficiency of genetic transformation Transformation of several E. coli strains with pBR322 was carried out with the PEG method described in MATERIALS AND METHODS, section d. All strains were grown in LB broth for 4 h and adjusted to cell densities equivalent to strain RR1 (A 600 = 0.6--0.7). The results indicate that the efficiency of PEGmediated transformation depends on the strain studied (100~, relative transformation = 1.34 x 106 transformants/#g pBR322 plasmid DNA). E.. coli strain

~, Relative transformation

RRI HB 101 M94 ED8767

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Fig. 1. Effectof cell densityon transformationefficiency.E. coil was cultured in LB broth using a 1 : 50 dilution of an overnight culture as an inoculum (see MATERIALS AND METHODS, section d). Bacteria were harvested at various intervals and the cell density (open circles) and transformationefficiency(closed circles) were determined.Transformationefficiencywas optimal just as cells entered stationary phase and decreased sharply in stationary phase cells. In this initial study, 40~ PEG-1000and SAM at pH 4.7 were employedinstead of 25~o PEG-1000and SAM at pH 5.4 in the optimizedprocedure(100~ relativetransformation = 0.91 ×' 106 transformants/#g DNA).

1970), the P E G procedure for yeast has several advantages over the current yeast methodology which involves the use of protoplasts (Beggs, 1978; Hinnen et al., 1978). In the following discussion, parameters of PEG-mediated genetic transformation of both E. coli and yeast will be described.

(b) Culture conditions The optimal cell density for transformation was determined by assaying cells for transformation competence at various intervals during the culture cycle (Fig. 1). Optimal transformation efficiencies were obtained with late log phase E. coli; in contrast, middle log phase cells are most transformable with the CaC12 method (Norgard et al., 1978). Transformarion efficiency dropped precipitously in stationary phase cells. Starting with a 1 : 5 0 dilution of a saturated overnight culture as inoculum, the optimal cell density for transformation was obtained at 4 h for E. coil

(c) DNA concentration The effect of plasmid D N A concentration on efficiency of transformation was studied by treating

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Fig. 2. Effect of DNA concentration on transformation efficiency. Using optimized conditions described in the MATERIALS AND METHODS, section d, E. coil was transformed with pBR322 plasmid DNA. Optimal transformation efficiency was obtained with 0.6pg pBR322 DNA (2.56 x 106 transformants//ag DNA).

cells with increasing amounts of pBR322 plasrnid DNA (Fig. 2). When less than 0.1 #g of DNA was applied, the number of transformants increased linearly with increasing DNA concentration. The number of transformants recovered per #g DNA decreased at high DNA concentrations (Fig. 2). DNA adsorption was carried out in the smallest volume technically practical (0.2 ml SAM for E. colO. The yield of transformants was found to decrease progressively as the volume of SAM increased. When seven plasmid DNAs other than pBR322 were studied, large numbers of transformants were recovered (not shown). (d) Effect of tonicity Prior to DNA treatment, cells were washed and resuspended in a DNA absorption solution which contains sorbitol (Fig~ 3), a buffer (Fig. 4), and a divalent cation (Fig. 5). For both E. coli and S. cerevisiae, the efficiency of PEG-mediated transformarion was found to increase with increasing sorbitol concentration with 0.5 M sorbitol being the optimal concentration for E. coli (Fig. 3). A concentrationdependent increase in the yield of transformants is also observed if sorbitol is replaced by sucrose, NaC1, KCI, LiCI or NH,CI.

Fig. 3. Effect oftonicity on transformation efficiency. Cells were washed and resuspended in a SAM solution containing varying amounts of sorbitol. The yield of transformants was optimal at 0.5 M sorbitol (100% relative transformation -- 5.56 × 106 transformants/pg DNA).

(e) Effect of pH The SAM and PEG solutions employed in PEGmediated transformation ofE. coli were studied over a wide pH range. While the initial wash solution (SAM) was effective over a rather broad pH range (Fig. 4), PEG treatment was found to be effective only between pH 3.25 to 5.0 (Fig. 4). A marked difference was observed in the pH optima for PEG-mediated transformation of E. cog and yeast. While the PEG solution for E. coli had an acidic pH optimum, a PEG solution of pH 8.3 resulted in the highest yield of transformants for S. g, I0090 80 7O 60 o> 50 _o 40 30 20 IO C 2.0

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Fig. 4. The pH optima for PEG-mediated genetic transformation were studied. In the case of the SAM solution (open circles) used for washing and DNA treatment, a broad range of pH was found to be effective. The optimum pH for the PEG-1000 solution (closed circles) was determined to be pH 4.4 (100~ relative transformation 9.28 x 106 transformants//~g DNA for the SAM solution and 1.72 x 106 transformants/pg DNA for the PEG solution).

338

cerevisiae, Even though the solutions used for transformation of E. coil and S. cerevisiae were quite similar, no transformants were recovered when the E. coli solutions were used to transform S. cerevisiae or vice versa. As noted below, the DNA adsorption solution (SAM) employed with E. coil contains divalent cations, whereas the DNA adsorption solution (SBEG) for S. cerevisiae lacks divalent cations. Even if divalent cations are added to SBEG when this solution is used to transform E. coil, no transformants are recovered, The observations above indicate that optimal PEG-mediated transformation of a new organism may require somewhat different conditions than those employed for either E. coil or S. cerevisiae.

(f) Divalent cation requirement While no obvious divalent cation requirement is observed during yeast transformation, a marked increase in E. coli transformation is observed in the presence of Mg2+, in particular. While the now standard E. coil transformation procedure of Mandel and Higa (1970) is most effective (i) at divalent cation concentrations of 30 mM or more and (ii) in the presence of Ca 2+, in particular, the PEGmediated procedure described here for E. coil requires low concentrations of divalent cations and •-

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Mga + ion is most effective (Fig. 5). In addition to Mg2 +, several other divalent cations were found to be effective in promoting genetic transformation. Mg2+, Mn 2 +, and Sr 2 + were found to be 2 to 3 times as active as Ca 2 + or Ba 2 +.

(g) PEG concentration PEG-1000 promoted transformation of E. coil at concentrations greater than 20% with an optimal concentration occurring at 25 % (Fig. 6). PEG-600, PEG-3000, and PEG-6000, purified as described in MATERIALS AND METHODS, section a, produced transformants with an efficiency of, respectively, 5%, 61%, and 30%, relative to PEG-1000 (100%). Ethylene glycol and PEG-200 yielded no transformants.

(h) Effects of temperature With the exception of the freeze-thaw step in yeast transformation, incubation at all stages of the procedure at 22°C for E. coli and 30°C for S. cerevisiae produced optimal yields of transformants. It is interesting to note that, while DNA treatment at 4 °C is required in the CaCl2 procedure for E. coil (Mandel and Higa, 1970; Hanahan, 1983), DNA treatment at 6 ° C, rather than 22 ° C, greatly decreased the yield of transformants for both E. coil and yeast when the PEG procedure was employed. Treatment at 6 °C was more inhibitory during exposure to DNA than during PEG treatment. When transformation is carried out at either 6°C or 37°C

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:~ 4o Fig. 5. The divalent cation dependence of PEG-mediated transformation of E. coil, The indicated concentrations of divalent cations were employed in SAM during the initial wash and DNA adsorption steps, Transformation efficiency increased with increasing concentrations of Mga+ and Ca 2+ (as well as Ba2+, Sra+, and Mn 2+ (not shown)). A decrease in transformants rcvovered occurred at the 30-100 mM concentration range of divalent cations (100 mM Ca 2+ is optimal in the standard CaCI2 method of Higa and Mandel, 1970) (100% relative transformation ffi 1.27 x l0 s transformants/p,g DNA),

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Percent PEG Fig. 6. The effect of PEG concentration on the yield of E. coli transformants. 25% PEG was twice as effective as other active PEG concentrations in four separate trials (100 % relative transformation ffi2.75 x l0s transformants/~DNA),

339

with E. coli,lessthan 5 ~ ofthe optimum number of transformants are recovered. (i) Time requirement for PEG treatment

While a 10 min treatment with D N A was optimal for both E. coil and yeast, the time required for the transformation-promoting action of P E G was different for each organism. P E G treatment for 2 rain produced optimal yields of E. coli transformants; however, a 1 h P E G treatment was required for optimal transformation of yeast. (j) Requirements for PEG-mediated genetic transformation

In the preceding sections, an analysis of parameters involved in PEG-mediated transformation (Figs. I-6) was presented. Table II indicates the relative importance of each step in the procedure. The omission of D N A or P E G completely abolished the yieldof transformants and a marked decrease in transformants isnoted when eithersorbitolor M g 2 + was omitted from the S A M solution(Table II).Ifthe finalwash by centrifugationwasdeleted,76 ~ of the control number of transformants were recovered. The omission of the lastcentrifugationstep streamlinesthe overallprocedure without a great reduction in efficiency.

TABLE II Effect of deletion ofvarious steps in PEG-mediatedtransformation Transformation was carriedout as describedin MATERIALS AND METHODS, section d, exceptfor the deletion of individual components or steps in the procedure. In the case of the " - final centrifugation" entry, cells treated with PEG were diluted with PBS; however, such cells were immediately diluted in LB without the final centrifugation step (100% relative transformation = I.I x l06 transformants/pg DNA). Component or step omitted

% Relative transformationE, coli

Control -sorbitol in SAM - DNA - PEG- 1000 -Mg a+ in SAM - final centrifugation

100 4 0 0 0.05 76

DISCUSSION

Both E. coli and the yeast, S. cerevisiae, can be genetically transformed by quite similar procedures which employ polyethylene glycol. The transformation efficiency obtained with the PEG procedure described here for E. coli is comparable in efficiency to the standard CaCI2methods for E. coli transformation (Mandel and Higa, 1970). While methods involving protoplasted yeast yield 103-104 transformants/#g DNA (Struhl et al., 1979; Broach et al., 1979), the PEG procedure described here for yeast yields 200-1000 transformants/#g DNA without cell wall digestion. Thus, the PEG procedure for yeast is the method of choice when extremely large numbers of transformants are not required. The efficiency of the PEG-mediated transformationmethod for E. cell reported here iscomparable to thatobtained with the CaCI2 method. Transformation efficiencies of 106-107 transformants/#g D N A have been observed with the CaCl2 method (Dagert and Ehrlich, 1979; Kushner, 1978; Norgard et al.,1978) while the P E G method has an average efficiency of 1.96 x 106 transformants/#g D N A (Tables I and If, Figs. I-6). The similarityof the PEG-mediated methods reported here for E. celland yeast indicatesthat PEGmediated genetic transformation may become a simple, general method applicableto a wide variety of organisms. P E G has been demonstrated to induce genetic transformation following protoplast formation in several microbial species (Chang and Cohen, 1979; Suarez and Chater, 1980; Brown and Carlton, 1980; Hinnen et al.,1978).The P E G procedure described here does not require cell wall removal but is similar in some respects to previously described procedures for the PEG-mediated transformation of protoplasts. A very efficient PEG-mediated transformation method has recently been described forB..mbt/l/s protoplasts (Chang and Cohen, 1979). which is similar to the P E G procedure described here for intact E. coil cells. The basic difference between the P E G methods for B. ~btilis protoplasts and intact E. coli appears to be the p H at which the procedures are performed, i.e., p H 6.5 for B. ~btilis protoplasts (Chang and Cohen, 1979) vs. p H 4.4 for intact E. coli (Fig.4). While the procedures for PEG-mediated genetic transformation orE. coli and yeast are basically quite

340

similar, PEG-induced transformation differs in many fundamental respects from other transformation procedures described in the literature. In the ease of E. coil, two types of transformation procedures have been described; namely (a)the now standard CaC12 method of Mandel and Higa (1970) and Co) methods involving protoplasts (Young and Sinsheimer, 1967; Chang and Cohen, 1979). The PEG procedure described here differs from the CaC12 method in several notable respects. While the CaCI2 method requires treatment ofE. coli at 4 ° C for 1 h or more (Dagert and Ehrlich, 1979), the PEG method functions optimally at 220C and is markedly inhibited at 6°C. The CaCI2 method yields optimal results when Ca 2 + concentration is 30 mM or higher (Norgard et al., 1978); in contrast, the PEG method for E. eoli requires lower concentrations of divalent cations, and is actually inhibited by the higher concentrations (100 raM) of divalent cations (Fig. 5) which produce optimal results in the CaCI2 method (Norgard et al., 1978). It is also noteworthy that, of the divalent cations tested, Ca 2 ÷ is the most effective in the CaCI2 procedure (Norgard et al., 1978; Weston et al., 1981), whereas Mg2+ is the divalent cation of choice in the PEG procedure (Fig. 5). As noted earlier, the PEG method for yeast transformation has no obvious divalent cation requirement. Recently, a similar method for transforming intact yeast, employing alkali cations and PEG, has been reported which also does not require divalent cations (Ito et al., 1983). The transformation methods for E. coil also differ in that the pH optimum for the PEG and CaCI2 methods are pH 4.4 and pH 7.5, respectively (Fig. 4; Norgard et al., 1978). The E. coil and yeast PEG procedures require hypertonie (0.5 M) conditions during DNA adsorption (Fig. 3) while optimal results are obtained with the CaC12 procedure when 0.1 M (or less) CaCI2 is employed (Norgard et al., 1978). Since many parameters of the PEG and CaCI2 procedures differ, the mechanisms of transformation involved probably differ as well. The PEG method may provide a means of transforming several E. cell strains and other species which fail to transform with the CaCI2 method.

ACKNOWLEDGEMENTS

Our thanks to Dr. Barbara Bachmann and the E. coli Genetic Stock Center for the gift of certain

strains used in this study. We wish to thank Ms. Sharon Ray for aid in preparation of the manuscript. This study was supported, in part, by grants from the National March of Dimes, the National Cancer Institute (CA33074), and the National Science Foundation (PCM-8218137).

ADDENDUM

We have recently optimized a high-efficiency PEG-mediated procedure for infecting human HeLa ceils with infectious poliovirus RNA. The procedure for introducing nucleic acids into mammalian cells is similar to the yeast procedure described here except that (a) 1.25 M KCI is substituted for 1 M sorbitol, (b) 30 mM CaC12 is used, and (c) the time of PEG1000 exposure is reduced to 2 rain.

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