YEAST
VOL.
8: 1007-1 0 14 ( 1992)
RNA Delivery in Saccharomyces cerevisiae Using Electroporation JEFFREY G . EVERETT AND DANIEL R. GALLIE* Department of Biochemistry, University of California, Riverside, California 92521-0129, U.S.A.
Received 5 February 1992; accepted 5 May 1992
An efficient delivery method for introducing in vitro synthesized RNA into yeast has been developed using electroporation. Spheroplast preparation, electroporation, and subsequent expression analysis can be accomplished within a single day. The use of introduced mRNA constructs avoids any complications due to nuclear regulation and is particularly suited for cytoplasmic regulatory studies. Moreover, this technique is useful for introducing those RNAs that cannot be made in vivo, such as poly(A)- mRNAs or RNAs with base modifications. We demonstrate that the Escherichia coli GUS gene and the firefly Luc gene are both excellent reporter genes for RNA electroporation. KEY WORDS - Electroporation; spheroplasts; mRNA; translation.
INTRODUCTION The cytoplasmic regulation of gene expression has received increased attention in recent years. In order to assess directly the in vivo impact of regulatory elements on the translational efficiency or stability of an mRNA in higher eukaryotes, a variety of methods which deliver RNA directly to the cytoplasm have been developed including calcium phosphate-mediated uptake (Kleinschmidt and Pederson, 1990), liposome-mediated transfection (Malone et al., 1989) and electroporation (Gallie et al., 1987, 1989; Potter, 1988). These methods have the advantage of being rapid and efficient. Similar studies in yeast, however, have been limited to DNA-based constructs, either as episomes or as genomically-integrated genes. Cytoplasmic regulatory investigations, therefore, may be unnecessarily complicated by changes in transcription, premRNA processing, or nucleocytoplasmic transport. Moreover, certain types of analyses have been impossible to carry out in vivo in yeast. For example, uncapped or poly(A)- mRNAs cannot be generated in vivo and as a result, it has not been possible to measure quantitatively their regulatory impact. In vitro translation lysates have been developed for yeast (Altmann et al., 1989; Blum et al., 1989; Gasior et al., 1979), however, lysates derived from higher eukaryotes have not reflected fully the in vivo contribution of regulatory elements such as the cap (Darzynkiewicz et al., 1988). and poly(A) tail *Addressee for correspondence. 0749-503X/92/12100748 $09.00 0 1992 by John Wiley & Sons Ltd
(Munroe and Jacobson, 1990a). For studies focusing on post-transcriptional regulation in yeast, a RNA delivery system would provide a more direct analysis of cytoplasmic regulation. Recently, RNA delivery into yeast spheroplasts using polyethylene glycol (PEG) was reported (Russell et al., 1991). This approach proved useful in analysing the effect of various 5'-leader sequences on translation. Electroporation has been used as an alternative delivery method for the introduction of nucleic acids into a wide range of prokaryotes (Miller et al., 1988) and eukaryotes (Potter, 1988). DNA (Becker and Guarente, 1991), protein (Uno et al., 1988) and small molecules (Bartoletti et al., 1989; Weaver et al., 1988) have been delivered into intact yeast, as has mRNA into plant (Gallie et al., 1987, 1989) and animal cells (Gallie et al., 1991; Gallie and Walbot, 1990) using electroporation. By manipulation of the electroporation parameters, e.g., the voltage, capacitance, resistance and wave function, a greater level of control over the amount of RNA delivered can be achieved compared to PEG-mediated delivery. In this report, we describe the delivery of in vitro synthesized mRNA constructs into yeast using electroporation and how this technique can be used to focus directly on cytoplasmic regulation. MATERIALS AND METHODS Yeast strains, media and RNA constructs Strain CRY1 MATa canl-100 ade2-1 his3-11,15 leu2-3,112 trpl-1 ura3-I, a derivative of W303a, was
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J. G. EVERETT AND D. R. GALLIE
generated by R. Fuller and C. Brenner and provided by A. Sachs. Saccharomyces cerevisiae was grown on standard YPD medium (1 % yeast extract, 2% Bacto peptone, 2% glucose). The T7-based Luc and P-glucuronidase (GUS) constructs are illustrated in Figure 1 and have been described previously (Gallie et al., 1991). In vitro transcription was carried out as described (Melton et al., 1984) using 40 mM-TrisHCl, pH 7.5, 60 mM-MgCl,, 20 mM-spermidine, 100 mM-NaC1,lOO pg/ml BSA, 0.5 Mm each of ATP, CTP, UTP, plus 170 p~ GTP, 1.6 mM m’GpppG, 100 mM-dithiothreitol, 0.5 u/pl T7 RNA polymerase (New England Biolabs). 0.3 u/pl RNasin (Promega) was included in all reactions to inhibit RNase activity. Relative levels of RNA were quantitated using denaturing formaldehyde-agarose gel analysis (Schenborn and Mierendorf, 1985).
LUC
GUS m7GpppG 17 b
69 b A 2 5
GUS m7GpppG
17 b
n
69 b
A
GUS
Figure 1. Schematic illustration of the mRNA constructs used. mRNAs were synthesized in vitro from T7-based vectors that had been linearized immediately downstream of the poly(A) tract with DraI prior to transcription.
Spheroplast and electroporation conditions
Cells from 15 ml mid-log culture were harvested and resuspended in 5ml sterile yeast spheroplast buffer A (50 mM-Tris, pH 7.5, 1 mM-MgCl,, 30 mMdithiothreitol, 15 mM-P-mercaptoethanol, 1 M-sorbitol). One ml of 1 mg/ml Zymolyase (100 T, ICN), which was dissolved in buffer A and centrifuged briefly, was added to the cells. The cells were placed in Petri plates and shaken gently at 30°C for 30 min. The spheroplasts were harvested and washed twice in 5ml buffer A taking care to resuspend the spheroplasts gently. Following the second wash, the spheroplasts were resuspended in 1 ml of buffer A,
which was then added to 10ml of YPD-sorbitol medium (YPD medium supplemented with 1 Msorbitol). The spheroplasts were allowed to recover by gentle shaking at 30°C for 90 min. The spheroplasts were harvested and washed twice with 5ml sterile 1 M-sorbitol in order to remove any salts which impair efficient electroporation. The spheroplasts were resuspended in 1 M-sorbitol to yield a final concentration of 1 x 10s/ml. The yeast (180 pl) was aliquoted into microfuge tubes and placed on ice. The mRNA to be delivered (typically 1 pg) was placed in a second microfuge tube and kept on ice. The spheroplasts were mixed immediately before electroporation by transferring the yeast to the tube containing the mRNA and then into a standard plastic microcuvette. Electroporation was carried out using the Genezapper (IBI). Optimal electroporation conditions were 800 V, 21 pF, 1000 R. Immediately following electroporation, 0.5 ml YPD-sorbitol medium was added to the electroporation cuvette and the electrodes (0.2cm gap) were rinsed in the cuvette containing the spheroplast-YPD-sorbitol mixture. The spheroplasts were transferred to a test tube and incubated at 30°C for 16 h. Although translation usually ceased within 4-6 h following mRNA delivery as a result of mRNA degradation, a 16-h incubation was chosen as it proved convenient to carry out the enzyme assays on the day following the electroporation. The GUS and luciferase (LUC) enzymes are both stable in yeast at 30°C over this time period. The electroporation of each mRNA construct was carried out in triplicate and each cell extract was assayed in duplicate. The resulting average is reported for each experiment. Luciferase and P-glucuronidaseassays
Luciferase assays were performed essentially as described (de Wet et al., 1987). Following incubation, the spheroplasts were harvested and resuspended in 300 p1 LUC assay buffer (25 mM-Tricine, pH 7-5, 15 mM-MgCl,, 7 mM-P-mercaptoethanol), sonicated for 5 s, and the cell debris was pelleted. 1&100 p1 of cell extract was added to LUC assay buffer containing 2 mM-ATP. Luciferase activity (photons produced) was measured following the injection of 100 pl 0.5 mM-luciferin in LUC assay buffer using a Monolight 20 10 Luminometer (Analytical Luminescence Laboratory). Enzyme activity is measured in light units per milligram protein. Protein was determined using the BioRad Protein assay kit.
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For GUS assays (Jefferson, 1987), the sphero=1 plasts were resuspended in 500 p1 GUS assay buffer 18 (50 mM-sodium phosphate, pH 7.0, 10 mM-EDTA, * 16 10 mM-P-mercaptoethanol), sonicated for 5 s, and the cell debris was pelleted. To 250 p1 cell extract was added 4-methylumbelliferyl P-D-glucuronide (Sigma Co.) to a final concentration of 1 mM in a total volume of 500 p1; this was incubated at 37°C. 150 p1 aliquots were removed at 30, 60 and 90 min 6 and added to 850 p1 0.2 M-sodium carbonate in order to stop the reaction. The fluorescence of these samples was then measured using a TKO 100 0 ' 1 1 1 1 1 1 1 1 1 1 Fluorometer (Hoefer Scientific Instruments). GUS 300 400 500 600 700 800 900 1000 11001200 1300 1400 specific activity, defined as nmoles of product Volts (4-methylumbelliferone) produced per minute per milligram protein, was measured from the kinetics Figure 2. Voltage as a parameter in yeast electroporation of the reaction and the protein concentration. efficiency. Spheroplasts were prepared and electroporation was
2
RESULTS AND DISCUSSION
2ol
carried out as described in Materials and Methods. One pg LucA, mRNA was used for electroporation at each voltage tested. Luciferase activity was used as a measure of electroporation efficiency.
Parameters f o r eficient electroporation
In order to deliver the mRNA successfully, it was necessary to subject the yeast to partial digestion with Zymolyase. No detectable LUC expression resulted from the electroporation of nonspheroplasted yeast with Luc mRNA (see Figure 1 for mRNA constructs used in this study). Luciferase expression increased with digestion time up to 90 min, the longest digestion period examined. Yeast digested for only I 5 min retained their characteristic morphology but upon electroporation, the resulting level of LUC expression was 60% of that for a 90min digestion. The partially spheroplasted yeast did still require osmotic support, suggesting that the integrity of the cell wall had been compromised. These data indicate that it is not necessary to remove the entire cell wall in order to achieve competency for electroporation. Spheroplasts generated from strain CRY 1 were electroporated with 1 pg Luc-A,, mRNA and the parameters of voltage (Figure 2), capacitance (Table 1) and resistance (Table 2) were examined. The level of LUC expression continued to increase up to 750 V, after which it plateaued up to 1000V and decreased at higher voltages. Expression also increased with increasing capacitance and was highest at 21 pF, which is the limit imposed by the instrument. Maximal expression was observed at the highest resistance employed (1000 Q). From these data, the optimal electroporation conditions chosen were 800 V, 21 pF, 1000 Q. No LUC activity was detected in a spheroplast/Luc-A, mRNA mixture that had not undergone electroporation.
Table 1. Effect of capacitance on luciferase (LUC) expression. Capacitance* (PF) 7 14 21
LUC Activity? (light units/mg protein) 8 358 010 16801 190 20 232 200
*Electroporationconditions:800 V and 800 R. 1 pg capped Luc-A, rnRNA used.
Table 2. Effect of resistance on luciferase (LUC) expression. Resistance* (Q)
100 200 400 600 800 1000
LUC Activity? (light units/mg protein) 2 546 000 9 133 880 14 743 370 15 204 500 18 042 510 23 535 590
*Electroporationconditions:800 V and 21 pF. mRNA used.
t 1 pg capped Luc-A,
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J. G. EVERETT AND D. R. GALLIE
Eflect of osmoticum, cell age, carrier RNA, level of input mRNA and ionic strength on electroporation eficiency and expression The impact of the osmoticum was analysed by preparing spheroplasts as described in Materials and Methods but using yeast spheroplast buffer containing either 0.25, 0.5, 0.75 or 1 M-sorbitol. Spheroplasts prepared at each sorbitol concentration were electroporated in sorbitol of equivalent concentration. Table 3 demonstrates that LUC expression increased as the osmoticum increased up to 1 M-sorbitol. Only with the use of the 0.25 Msorbitol medium was the yield of spheroplasts reduced as a result of cell lysis. These results agree with earlier findings demonstrating that cellular function in spheroplasts, as measured by RNA synthesis, is reduced by sorbitol concentrations less than 0.9 M (Hutchison and Hartwell, 1967).
to competition with Luc mRNA during uptake or to interference with yeast tRNA charging. As a result, carrier RNA is not included in our electroporations. Table 4. Effect of carrier RNA on electroporation efficiency. Carrier RNA (MI
LUC Activity* (light units/mg protein)
~~
7 653 968 7 596 484 9 336 274 7 437 053 8 439 332 1 406 906
0 30 60 80 100 200 *1 pg capped Luc-A,, mRNA used.
Table 3. Effect of osmoticum on electroporation efficiency. Sorbitol
concentration (MI
LUC Activity* (light units/mg protein)
0.25 0.50 0.75 1 .o
28 266 3 749 253 1 1 553 815 32 724 149
* 1 pg capped Luc-A,, mRNA used Cell age also influenced translational activity. Late-log phase cells electroporated with Luc-A,, mRNA were only 52% as active (12 129 600 light units/mg protein) as mid-log cells (23 523 790 light units/mg protein) and stationary-phase cells were just 3.6% as active (846 370 light units/mg protein) as mid-log phase cells. Examination of the cell concentration as a parameter in electroporation determined that expression increased with cell concentration (data not shown; the range tested was 1 x I06/ml to 5 x 108/ml). Carrier mRNA had no beneficial effect on mRNA uptake. Increasing amounts of Escherichia coli tRNA were mixed with 1 pg Luc-A,, mRNA immediately before electroporation. No effect on LUC expression was observed up to 100 pg carrier RNA (Table 4); however, higher amounts of carrier RNA resulted in lower LUC expression. The detrimental effect of the carrier RNA may have been due
The dose-response of yeast electroporated with Luc-A,, mRNA was tested to determine the linear range of translation. A linear response was observed over most of the range examined (0.5-1 5 pg), with only a slight decrease in linearity at the higher levels of mRNA (Figure 3). 1 pg mRNA is typically used for electroporation, which falls well within the linear range and thereby does not overwhelm the translational machinery. Previous studies have demonstrated that up to 70% of the yeast population subjected to electroporation can be successfully transformed (Weaver et al., 1988).
v)
E! 210
0
I
I
2.5
5
I 7.5
I
I
10
12.5
I
15
Luc-ASO mRNA ( pg) Figure 3. Dose-response analysis of RNA electroporation in spheroplasts. From 0.5 to 15pg Luc-A, mRNA was electroporatedinto yeast in a constant volume.
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RNA DELIVERY IN SACCHAROMYCES CEREVISIAE
The volume of the sample had a small influence on the level of expression. A constant number of cells were resuspended in varying volumes of 1 Msorbitol and electroporated with 1 pg Luc-A,, mRNA. Expression decreased when the volume was greater than 200 p1 (Table 5). This could be a result of the increase in volume or a decrease in Luc mRNA concentration. We have chosen a volume of 200 pl because of the greater reproducibility between samples. Table 5. Effect of volume on electroporation efficiency. Cell volume
(I4
LUC Activity* (light units/mg protein)
100
150 200 250 300 350 * I pg capped Luc-A,, mRNA used.
10 746 440 10 772 500 10 371 758 6 412 090 4 087 480 5 522 960
Table 6. Effect of salt on electroporation of yeast. Percentage of electroporation buffer made up by lowsalt buffer* 100 89 60 45 25 0
LUC Activity? (light units/mg protein) 24 576 76 946 190 125 297 094 637 146 2 594 124
*Compositionof low-salt buffer is described in the text. tl pg capped Luc-A, mRNA used.
was observed in samples incubated up to 1 min, the longest time tested (data not shown). Normally, only 5 s is required to mix the yeast with the mRNA, transfer to the cuvette, and electroporate. In order to establish the error associated with RNA electroporation in yeast, ten replicates from the same batch of spheroplasts were electroporated at 21 pF, 800 V, 800 R with 1 pg capped Luc-A,, mRNA and allowed to incubate as described. The mean and standard deviation was calculated to be 18 669 166f515 377.
Ionic strength of the electroporation buffer can significantly alter electroporation efficiency. Very low salt conditions are required in electroporation of prokaryotes (Miller et al., 1988). In contrast, electroporation of higher eukaryotes used lower The ejiect of a cap, the leader sequence, and a voltages and salts are commonly part of the electro- poly(A) tail on expression of electroporated mRNA Virtually all mRNAs of higher and lower poration buffer (Callis et al., 1987; Fromm et al., 1987). To determine the effect of the salt concen- eukaryotes contain a cap and poly(A) tail. Both of tration on electroporation, yeast were electro- these mRNA elements are essential for efficient porated in buffer that varied in ionic strength by translation. The cap serves as the binding site for altering the ratio of a low-salt buffer (10 mM-Tris, eukaryotic initiation factor 4E, which is an importpH 7.5, 1 rnM-MgCl,, 10 mM-CaCl,, 10 mM-KCl, ant regulatory point in the initiation of translation 10 mM-NaC1, 1 M-sorbitol) and a 1 M-sorbitol (Altmann et al., 1987; Rhoads, 1988; Sonenberg, solution, thereby maintaining a constant sorbitol 1988).The poly(A) tail serves as the binding site for concentration. Expression increased as the ionic the poly(A) binding (PAB) protein. PAB protein strength of the electroporation buffer decreased has been implicated in promoting 60s ribosomal (Table 6), demonstrating the detrimental effect of subunit binding to the 48s initiation complex in yeast (Munroe and Jacobson, 1990b; Sachs and ions on electroporation efficiency in yeast. The presence of RNase activity in a cell prep- Davis, 1989).Moreover, we have demonstrated that aration can have a significant impact on the a cap and a poly(A) tail interact synergistically to efficiency of delivery during RNA electroporation regulate translational efficiency (Gallie, 1991). as well as on reproducibility. We tested our spheroUsing RNA electroporation, the quantification plast preparation for extracellular RNase activity of the regulatory impact of a cap or poly(A) tail on by mixing Luc mRNA with the spheroplasts and translation can be addressed directly. The GUS gene allowing the cell-luc mRNA mixture to incubate at has gained widespread use in plant studies due to the room temperature for varying amounts of time low endogenous activity of GUS in most plant before electroporating. No detectable difference species. Recently, the use of the GUS gene as a
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J. G. EVERETT AND D. R. GALLIE
Q Figure 4. The impact of cap, poly(A) tail, and the tobacco mosaic virus mRNA leader (Q) on the translation of P-glucuronidase (GUS) mRNA in yeast. 1 pg of each mRNA construct was used for the electroporation, GUS specific activityis indicated above each bar and is expressed as nmol/min per mg protein.
reporter gene in yeast was demonstrated (Schmitz
12 A residues but, as a consequence of its size, it has a packing density of one PAB protein molecule per 25A residues (Sachs er al., 1987). The GUS constructs terminating in either a poly(A),, or poly(A),, tail, therefore, might be expected to accommodate greater regulatory control by a poly(A) tail than one or two molecules of PAB protein, respectively. are Luc mRNA constructs (Gallie et al., 1991) The fact that translation from the poly(A),, conand therefore it represents a more responsive struct was higher than that from the poly(A),, reporter gene for measuring poly(A) tail-mediated construct suggests not only that the presence of a poly(A) tail is important for efficient translation regulation. Addition of a cap increased GUS expression by in yeast, but also that the length is a regulatory six-fold (Figure 4),a result in good agreement with parameter. The untranslated leader (called R) from tobacco the observations made by Russell et al. (1991), in which capped and uncapped Luc mRNAs were mosaic virus enhances the translational efficiency of introduced into yeast spheroplasts using PEG. The chimeric mRNA constructs in plants, and to a lesser addition of a poly(A),, tail to GUS mRNA extent, in animal species (Gallie et al., 1987, 1989; increased expression by 11I-fold (Figure 4). The Gallie and Walbot, 1990). It has been reported average poly(A) tail length for yeast mRNA in the recently that Q enhanced translation in a yeast in cytoplasm, however, is approximately 50 A residues vitro lysate (Altmann et al., 1990). In order to (Sachs and Davis, 1989). When the poly(A) tail was examine whether R could enhance translation in doubled in length to 50 A residues, GUS expression vivo in yeast, GUS-A,, mRNA, containing either R increased an additional 75%. The PAB protein of or the same 17-base control leader used for all the yeast has a minimum binding site requirement of poly(A)+ constructs (Figure l), was translated in et al., 1990). No detectable GUS activity was measured in our S. cerevisiae strains under the conditions used for the E. coli GUS assay. Moreover, the translation of GUS mRNA constructs is under
RNA DELIVERY IN SACCHAROMYCES CEREVISIAE
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electroporated yeast. R did not enhance translation de Wet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R. and Subramani, S . (1987). Firefly luciferasegene: strucin yeast (Figure 4). In fact, expression from the Rture and expression in mammalian cells. Mol. Cell. Biol. containing GUS-A,, mRNA was consistently lower 7,725-737. than that observed for the control GUS-A,, conFromm, M. E., Callis, J., Taylor, L. P. and Walbot, V. struct. These data demonstrate that the ability of (1987). Electroporation of DNA and RNA into plant this leader to enhance translation is highly species protoplasts. Methods Enzymol. 153,351-366. dependent. Moreover, the in vivo results demon- Gallie, D. R. (1991). The cap and poly(A) tail function strate the importance of using an in vivo assay syssynergistically to regulate mRNA translational tem to investigate translational regulation. With the efficiency. Genes and Devel. 5,2108-2 116. development of an efficient delivery system for the Gallie, D. R., Feder, J. N., Schimke, R. T. and Walbot, V. (1991). Post-transcriptional regulation in higher introduction of mRNA into yeast, the cytoplasmic eukaryotes: the role of the reporter gene in controlling regulation of gene expression can now be directly expression. Mol. Gen. Genet. 228,258-264. studied in vivo. ACKNOWLEDGEMENTS The authors thank Marilyn Kobayashi for technical assistance. This work was supported in part by a grant from the University of California Systemwide Biotechnology Research and Education Program. REFERENCES Altmann, M., Blum, S., Wilson, T. M. A. andTrachsel, H. (1990). The 5’-leader sequence of tobacco mosaic virus RNA mediates initiation-factor-4E-independent, but still initiation-factor-4A-dependenttranslation in yeast extracts. Gene 91, 127-129. Altmann, M., Handschin, C. and Trachsel, H. (1987). mRNA cap-binding protein: cloning of the gene encoding protein synthesis initiation factor eIF-4E from Saccharomyces cerevisiae. Mol. Cell. Biol. 7, 998-1 003. Altmann, M., Sonenberg, N. and Trachsel, H. (1989). Translation in Saccharomyces cerevisiae:initiation factor 4E-dependent cell-free system. Mol. Cell. Biol. 9, 4467-4472. Bartoletti, D. C., Harrison, G. I. and Weaver, J. C. (1989). The number of molecules taken up by electroporated cells: quantitative determination. FEBS Letters 256, 4-10. Becker, D. M. and Guarente, L. (1991). High-efficiency transformation of yeast by electroporation. Methods Enz. 194,182-187. Blum, S., Mueller, M., Schmid, R., Linder, P. and Traschsel, H. (1989). Saccharomyces cerevisiae: initiation factor 4A-dependent cell-free system. Proc. Natl. Acad. Sci. USA 86,6043-6046. Callis, J., Fromm, M. and Walbot, V. (1987). Expression of mRNA electroporated into plant and animal cells. Nucl. Acids. Res. 15,5823-583 1. Darzynkiewicz, E., Stepinski, J., Ekiel, I., Jin, Y., Haber, D., Sijuwade, T. and Tahara, S. M. (1988). P-globin or m2,2*337G differ in mRNAs capped with m7G, intrinsic translation efficiency. Nucl. Acids. Res. 16, 8953-8962. m23237G
Gallie, D. R., Lucas, W. J. and Walbot, V. (1989). Visualizing mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and translation. The Plant Cell 1,301-31 1. Gallie, D. R., Sleat, D. E., Watts, J. W., Turner, P. C. and Wilson, T. M. A. (1987). The gene transcripts in vitro and in vivo. Nucl. Acids Res. 15,3257-3273. Gallie, D. R. and Walbot, V. (1990). RNA pseudoknot domain of tobacco mosaic virus can functionally substitute for a poly(A) tail in plant and animal cells. Genes and Devel. 4,1149-1 157. Gasior, E., Herrera, F., Sadnik, I., McLaughlin, C. S. and Moldave, K. (1979). The preparation and characterization of a cell-free system from Saccharomyces cerevisiae that translates natural messenger ribonucleic acid. J . Biol. Chem. 254,3965-3969. Hutchison, H. T. and Hartwell, L. H. (1967). Macromolecule synthesis in yeast spheroplasts. J. Bacteriol. 94,1697-1705. Jefferson, R. A. (1987). Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5, 387-405. Kleinschmidt, A. M. and Pederson, T. (1990). RNA processing and ribonucleoprotein assembly studied in vivo by RNA transfection. Proc. Natl. Acad. Sci. USA 87,1283-1287. Malone, R. W., Felgner, P. L. and Verma, I. M. (1989). Cationic liposome-mediated RNA transfection. Proc. Natl. Acad. Sci. USA 86,6077-608 1. Melton, D. A., Kreig, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K. and Green, M. R. (1984). Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucl. Acids Res. 12, 7035-7056. Miller, J. F., Dower, W. J. and Tompkins, L. S. (1988). High-voltage electroporation of bacteria: genetic transformation of Campylobacter jejuni with plasmid DNA. Proc. Natl. Acad. Sci. USA 85,856860. Munroe, D. and Jacobson, A. (1990a). mRNA poly(A) tail, a 3’ enhancer of translational initiation., Mol. Cell. Biol.10,344-3455. Munroe, D. and Jacobson, A. (1990b). Tales of poly(A): a review. Gene91,151-158.
1014 Potter, H. (1988). Electroporation in biology: methods, applications, and instrumentation. Anal. Biochem. 174, 361-373. Rhoads, R. E. (1988). Cap recognition and the entry of mRNA into the protein synthesis initiation cycle. TIBS 13,52-56. Russell, P. J., Hambridge, S. J. and Kirkegaard, K. (1991). Direct introduction and transient expression of capped and non-capped RNA in Saccharomyces cerevisiae. Nucl. Acids. Res. 19,4949-4953. Sachs, A. B. and Davis, R. W. (1989). The poly(A) binding protein is required for poly(A) shortening and 60s ribosomal subunit-dependent translation initiation. Cell58,857-867. Sachs, A. B., Davis, R. W. and Kornberg, R. D. (1987). A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability. Mol. Cell. Biol. 7,3268-3276. Schenborn, E. T. and Mierendorf, R. C . (1985). A novel transcription property of SP6 and T7 RNA polymerase:
J. G. EVERETT A N D D. R. GALLIE
dependence on template structure. Nucl. Acids Res. 13, 622345236. Schmitz, U. K., Lonsdale, D. M. and Jefferson, R. A. (1990). Application of the fl-glucuronidase gene fusion system to Saccharomyces cerevisiae. Current Genetics 17,261-264. Sonenberg, N. (1988). Cap-binding proteins ofeukaryotic messenger RNA: functions in initiation and control of translation. Prog. Nucleic Acid Res. Mol. Biol. 35, 173-207. Uno, I., Fukami, K., Kato, H., Takenawa, T. and Ishikawa, T. (1988). Essential role for phosphatidylinositol 4,s-bisphosphate in yeast cell proliferation. Nature 333, 188-190. Weaver, J. C., Harrison, G. I., Bliss, J. G., Mourant, J. R. and Powell, K. T. (1988). Electroporation: high frequency of occurrence of a transient high-permeability state in erythrocytes and intact yeast. FEBS Letters 229,30-34.
VOL.
8: 1007-1 0 14 ( 1992)
RNA Delivery in Saccharomyces cerevisiae Using Electroporation JEFFREY G . EVERETT AND DANIEL R. GALLIE* Department of Biochemistry, University of California, Riverside, California 92521-0129, U.S.A.
Received 5 February 1992; accepted 5 May 1992
An efficient delivery method for introducing in vitro synthesized RNA into yeast has been developed using electroporation. Spheroplast preparation, electroporation, and subsequent expression analysis can be accomplished within a single day. The use of introduced mRNA constructs avoids any complications due to nuclear regulation and is particularly suited for cytoplasmic regulatory studies. Moreover, this technique is useful for introducing those RNAs that cannot be made in vivo, such as poly(A)- mRNAs or RNAs with base modifications. We demonstrate that the Escherichia coli GUS gene and the firefly Luc gene are both excellent reporter genes for RNA electroporation. KEY WORDS - Electroporation; spheroplasts; mRNA; translation.
INTRODUCTION The cytoplasmic regulation of gene expression has received increased attention in recent years. In order to assess directly the in vivo impact of regulatory elements on the translational efficiency or stability of an mRNA in higher eukaryotes, a variety of methods which deliver RNA directly to the cytoplasm have been developed including calcium phosphate-mediated uptake (Kleinschmidt and Pederson, 1990), liposome-mediated transfection (Malone et al., 1989) and electroporation (Gallie et al., 1987, 1989; Potter, 1988). These methods have the advantage of being rapid and efficient. Similar studies in yeast, however, have been limited to DNA-based constructs, either as episomes or as genomically-integrated genes. Cytoplasmic regulatory investigations, therefore, may be unnecessarily complicated by changes in transcription, premRNA processing, or nucleocytoplasmic transport. Moreover, certain types of analyses have been impossible to carry out in vivo in yeast. For example, uncapped or poly(A)- mRNAs cannot be generated in vivo and as a result, it has not been possible to measure quantitatively their regulatory impact. In vitro translation lysates have been developed for yeast (Altmann et al., 1989; Blum et al., 1989; Gasior et al., 1979), however, lysates derived from higher eukaryotes have not reflected fully the in vivo contribution of regulatory elements such as the cap (Darzynkiewicz et al., 1988). and poly(A) tail *Addressee for correspondence. 0749-503X/92/12100748 $09.00 0 1992 by John Wiley & Sons Ltd
(Munroe and Jacobson, 1990a). For studies focusing on post-transcriptional regulation in yeast, a RNA delivery system would provide a more direct analysis of cytoplasmic regulation. Recently, RNA delivery into yeast spheroplasts using polyethylene glycol (PEG) was reported (Russell et al., 1991). This approach proved useful in analysing the effect of various 5'-leader sequences on translation. Electroporation has been used as an alternative delivery method for the introduction of nucleic acids into a wide range of prokaryotes (Miller et al., 1988) and eukaryotes (Potter, 1988). DNA (Becker and Guarente, 1991), protein (Uno et al., 1988) and small molecules (Bartoletti et al., 1989; Weaver et al., 1988) have been delivered into intact yeast, as has mRNA into plant (Gallie et al., 1987, 1989) and animal cells (Gallie et al., 1991; Gallie and Walbot, 1990) using electroporation. By manipulation of the electroporation parameters, e.g., the voltage, capacitance, resistance and wave function, a greater level of control over the amount of RNA delivered can be achieved compared to PEG-mediated delivery. In this report, we describe the delivery of in vitro synthesized mRNA constructs into yeast using electroporation and how this technique can be used to focus directly on cytoplasmic regulation. MATERIALS AND METHODS Yeast strains, media and RNA constructs Strain CRY1 MATa canl-100 ade2-1 his3-11,15 leu2-3,112 trpl-1 ura3-I, a derivative of W303a, was
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J. G. EVERETT AND D. R. GALLIE
generated by R. Fuller and C. Brenner and provided by A. Sachs. Saccharomyces cerevisiae was grown on standard YPD medium (1 % yeast extract, 2% Bacto peptone, 2% glucose). The T7-based Luc and P-glucuronidase (GUS) constructs are illustrated in Figure 1 and have been described previously (Gallie et al., 1991). In vitro transcription was carried out as described (Melton et al., 1984) using 40 mM-TrisHCl, pH 7.5, 60 mM-MgCl,, 20 mM-spermidine, 100 mM-NaC1,lOO pg/ml BSA, 0.5 Mm each of ATP, CTP, UTP, plus 170 p~ GTP, 1.6 mM m’GpppG, 100 mM-dithiothreitol, 0.5 u/pl T7 RNA polymerase (New England Biolabs). 0.3 u/pl RNasin (Promega) was included in all reactions to inhibit RNase activity. Relative levels of RNA were quantitated using denaturing formaldehyde-agarose gel analysis (Schenborn and Mierendorf, 1985).
LUC
GUS m7GpppG 17 b
69 b A 2 5
GUS m7GpppG
17 b
n
69 b
A
GUS
Figure 1. Schematic illustration of the mRNA constructs used. mRNAs were synthesized in vitro from T7-based vectors that had been linearized immediately downstream of the poly(A) tract with DraI prior to transcription.
Spheroplast and electroporation conditions
Cells from 15 ml mid-log culture were harvested and resuspended in 5ml sterile yeast spheroplast buffer A (50 mM-Tris, pH 7.5, 1 mM-MgCl,, 30 mMdithiothreitol, 15 mM-P-mercaptoethanol, 1 M-sorbitol). One ml of 1 mg/ml Zymolyase (100 T, ICN), which was dissolved in buffer A and centrifuged briefly, was added to the cells. The cells were placed in Petri plates and shaken gently at 30°C for 30 min. The spheroplasts were harvested and washed twice in 5ml buffer A taking care to resuspend the spheroplasts gently. Following the second wash, the spheroplasts were resuspended in 1 ml of buffer A,
which was then added to 10ml of YPD-sorbitol medium (YPD medium supplemented with 1 Msorbitol). The spheroplasts were allowed to recover by gentle shaking at 30°C for 90 min. The spheroplasts were harvested and washed twice with 5ml sterile 1 M-sorbitol in order to remove any salts which impair efficient electroporation. The spheroplasts were resuspended in 1 M-sorbitol to yield a final concentration of 1 x 10s/ml. The yeast (180 pl) was aliquoted into microfuge tubes and placed on ice. The mRNA to be delivered (typically 1 pg) was placed in a second microfuge tube and kept on ice. The spheroplasts were mixed immediately before electroporation by transferring the yeast to the tube containing the mRNA and then into a standard plastic microcuvette. Electroporation was carried out using the Genezapper (IBI). Optimal electroporation conditions were 800 V, 21 pF, 1000 R. Immediately following electroporation, 0.5 ml YPD-sorbitol medium was added to the electroporation cuvette and the electrodes (0.2cm gap) were rinsed in the cuvette containing the spheroplast-YPD-sorbitol mixture. The spheroplasts were transferred to a test tube and incubated at 30°C for 16 h. Although translation usually ceased within 4-6 h following mRNA delivery as a result of mRNA degradation, a 16-h incubation was chosen as it proved convenient to carry out the enzyme assays on the day following the electroporation. The GUS and luciferase (LUC) enzymes are both stable in yeast at 30°C over this time period. The electroporation of each mRNA construct was carried out in triplicate and each cell extract was assayed in duplicate. The resulting average is reported for each experiment. Luciferase and P-glucuronidaseassays
Luciferase assays were performed essentially as described (de Wet et al., 1987). Following incubation, the spheroplasts were harvested and resuspended in 300 p1 LUC assay buffer (25 mM-Tricine, pH 7-5, 15 mM-MgCl,, 7 mM-P-mercaptoethanol), sonicated for 5 s, and the cell debris was pelleted. 1&100 p1 of cell extract was added to LUC assay buffer containing 2 mM-ATP. Luciferase activity (photons produced) was measured following the injection of 100 pl 0.5 mM-luciferin in LUC assay buffer using a Monolight 20 10 Luminometer (Analytical Luminescence Laboratory). Enzyme activity is measured in light units per milligram protein. Protein was determined using the BioRad Protein assay kit.
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For GUS assays (Jefferson, 1987), the sphero=1 plasts were resuspended in 500 p1 GUS assay buffer 18 (50 mM-sodium phosphate, pH 7.0, 10 mM-EDTA, * 16 10 mM-P-mercaptoethanol), sonicated for 5 s, and the cell debris was pelleted. To 250 p1 cell extract was added 4-methylumbelliferyl P-D-glucuronide (Sigma Co.) to a final concentration of 1 mM in a total volume of 500 p1; this was incubated at 37°C. 150 p1 aliquots were removed at 30, 60 and 90 min 6 and added to 850 p1 0.2 M-sodium carbonate in order to stop the reaction. The fluorescence of these samples was then measured using a TKO 100 0 ' 1 1 1 1 1 1 1 1 1 1 Fluorometer (Hoefer Scientific Instruments). GUS 300 400 500 600 700 800 900 1000 11001200 1300 1400 specific activity, defined as nmoles of product Volts (4-methylumbelliferone) produced per minute per milligram protein, was measured from the kinetics Figure 2. Voltage as a parameter in yeast electroporation of the reaction and the protein concentration. efficiency. Spheroplasts were prepared and electroporation was
2
RESULTS AND DISCUSSION
2ol
carried out as described in Materials and Methods. One pg LucA, mRNA was used for electroporation at each voltage tested. Luciferase activity was used as a measure of electroporation efficiency.
Parameters f o r eficient electroporation
In order to deliver the mRNA successfully, it was necessary to subject the yeast to partial digestion with Zymolyase. No detectable LUC expression resulted from the electroporation of nonspheroplasted yeast with Luc mRNA (see Figure 1 for mRNA constructs used in this study). Luciferase expression increased with digestion time up to 90 min, the longest digestion period examined. Yeast digested for only I 5 min retained their characteristic morphology but upon electroporation, the resulting level of LUC expression was 60% of that for a 90min digestion. The partially spheroplasted yeast did still require osmotic support, suggesting that the integrity of the cell wall had been compromised. These data indicate that it is not necessary to remove the entire cell wall in order to achieve competency for electroporation. Spheroplasts generated from strain CRY 1 were electroporated with 1 pg Luc-A,, mRNA and the parameters of voltage (Figure 2), capacitance (Table 1) and resistance (Table 2) were examined. The level of LUC expression continued to increase up to 750 V, after which it plateaued up to 1000V and decreased at higher voltages. Expression also increased with increasing capacitance and was highest at 21 pF, which is the limit imposed by the instrument. Maximal expression was observed at the highest resistance employed (1000 Q). From these data, the optimal electroporation conditions chosen were 800 V, 21 pF, 1000 Q. No LUC activity was detected in a spheroplast/Luc-A, mRNA mixture that had not undergone electroporation.
Table 1. Effect of capacitance on luciferase (LUC) expression. Capacitance* (PF) 7 14 21
LUC Activity? (light units/mg protein) 8 358 010 16801 190 20 232 200
*Electroporationconditions:800 V and 800 R. 1 pg capped Luc-A, rnRNA used.
Table 2. Effect of resistance on luciferase (LUC) expression. Resistance* (Q)
100 200 400 600 800 1000
LUC Activity? (light units/mg protein) 2 546 000 9 133 880 14 743 370 15 204 500 18 042 510 23 535 590
*Electroporationconditions:800 V and 21 pF. mRNA used.
t 1 pg capped Luc-A,
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Eflect of osmoticum, cell age, carrier RNA, level of input mRNA and ionic strength on electroporation eficiency and expression The impact of the osmoticum was analysed by preparing spheroplasts as described in Materials and Methods but using yeast spheroplast buffer containing either 0.25, 0.5, 0.75 or 1 M-sorbitol. Spheroplasts prepared at each sorbitol concentration were electroporated in sorbitol of equivalent concentration. Table 3 demonstrates that LUC expression increased as the osmoticum increased up to 1 M-sorbitol. Only with the use of the 0.25 Msorbitol medium was the yield of spheroplasts reduced as a result of cell lysis. These results agree with earlier findings demonstrating that cellular function in spheroplasts, as measured by RNA synthesis, is reduced by sorbitol concentrations less than 0.9 M (Hutchison and Hartwell, 1967).
to competition with Luc mRNA during uptake or to interference with yeast tRNA charging. As a result, carrier RNA is not included in our electroporations. Table 4. Effect of carrier RNA on electroporation efficiency. Carrier RNA (MI
LUC Activity* (light units/mg protein)
~~
7 653 968 7 596 484 9 336 274 7 437 053 8 439 332 1 406 906
0 30 60 80 100 200 *1 pg capped Luc-A,, mRNA used.
Table 3. Effect of osmoticum on electroporation efficiency. Sorbitol
concentration (MI
LUC Activity* (light units/mg protein)
0.25 0.50 0.75 1 .o
28 266 3 749 253 1 1 553 815 32 724 149
* 1 pg capped Luc-A,, mRNA used Cell age also influenced translational activity. Late-log phase cells electroporated with Luc-A,, mRNA were only 52% as active (12 129 600 light units/mg protein) as mid-log cells (23 523 790 light units/mg protein) and stationary-phase cells were just 3.6% as active (846 370 light units/mg protein) as mid-log phase cells. Examination of the cell concentration as a parameter in electroporation determined that expression increased with cell concentration (data not shown; the range tested was 1 x I06/ml to 5 x 108/ml). Carrier mRNA had no beneficial effect on mRNA uptake. Increasing amounts of Escherichia coli tRNA were mixed with 1 pg Luc-A,, mRNA immediately before electroporation. No effect on LUC expression was observed up to 100 pg carrier RNA (Table 4); however, higher amounts of carrier RNA resulted in lower LUC expression. The detrimental effect of the carrier RNA may have been due
The dose-response of yeast electroporated with Luc-A,, mRNA was tested to determine the linear range of translation. A linear response was observed over most of the range examined (0.5-1 5 pg), with only a slight decrease in linearity at the higher levels of mRNA (Figure 3). 1 pg mRNA is typically used for electroporation, which falls well within the linear range and thereby does not overwhelm the translational machinery. Previous studies have demonstrated that up to 70% of the yeast population subjected to electroporation can be successfully transformed (Weaver et al., 1988).
v)
E! 210
0
I
I
2.5
5
I 7.5
I
I
10
12.5
I
15
Luc-ASO mRNA ( pg) Figure 3. Dose-response analysis of RNA electroporation in spheroplasts. From 0.5 to 15pg Luc-A, mRNA was electroporatedinto yeast in a constant volume.
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RNA DELIVERY IN SACCHAROMYCES CEREVISIAE
The volume of the sample had a small influence on the level of expression. A constant number of cells were resuspended in varying volumes of 1 Msorbitol and electroporated with 1 pg Luc-A,, mRNA. Expression decreased when the volume was greater than 200 p1 (Table 5). This could be a result of the increase in volume or a decrease in Luc mRNA concentration. We have chosen a volume of 200 pl because of the greater reproducibility between samples. Table 5. Effect of volume on electroporation efficiency. Cell volume
(I4
LUC Activity* (light units/mg protein)
100
150 200 250 300 350 * I pg capped Luc-A,, mRNA used.
10 746 440 10 772 500 10 371 758 6 412 090 4 087 480 5 522 960
Table 6. Effect of salt on electroporation of yeast. Percentage of electroporation buffer made up by lowsalt buffer* 100 89 60 45 25 0
LUC Activity? (light units/mg protein) 24 576 76 946 190 125 297 094 637 146 2 594 124
*Compositionof low-salt buffer is described in the text. tl pg capped Luc-A, mRNA used.
was observed in samples incubated up to 1 min, the longest time tested (data not shown). Normally, only 5 s is required to mix the yeast with the mRNA, transfer to the cuvette, and electroporate. In order to establish the error associated with RNA electroporation in yeast, ten replicates from the same batch of spheroplasts were electroporated at 21 pF, 800 V, 800 R with 1 pg capped Luc-A,, mRNA and allowed to incubate as described. The mean and standard deviation was calculated to be 18 669 166f515 377.
Ionic strength of the electroporation buffer can significantly alter electroporation efficiency. Very low salt conditions are required in electroporation of prokaryotes (Miller et al., 1988). In contrast, electroporation of higher eukaryotes used lower The ejiect of a cap, the leader sequence, and a voltages and salts are commonly part of the electro- poly(A) tail on expression of electroporated mRNA Virtually all mRNAs of higher and lower poration buffer (Callis et al., 1987; Fromm et al., 1987). To determine the effect of the salt concen- eukaryotes contain a cap and poly(A) tail. Both of tration on electroporation, yeast were electro- these mRNA elements are essential for efficient porated in buffer that varied in ionic strength by translation. The cap serves as the binding site for altering the ratio of a low-salt buffer (10 mM-Tris, eukaryotic initiation factor 4E, which is an importpH 7.5, 1 rnM-MgCl,, 10 mM-CaCl,, 10 mM-KCl, ant regulatory point in the initiation of translation 10 mM-NaC1, 1 M-sorbitol) and a 1 M-sorbitol (Altmann et al., 1987; Rhoads, 1988; Sonenberg, solution, thereby maintaining a constant sorbitol 1988).The poly(A) tail serves as the binding site for concentration. Expression increased as the ionic the poly(A) binding (PAB) protein. PAB protein strength of the electroporation buffer decreased has been implicated in promoting 60s ribosomal (Table 6), demonstrating the detrimental effect of subunit binding to the 48s initiation complex in yeast (Munroe and Jacobson, 1990b; Sachs and ions on electroporation efficiency in yeast. The presence of RNase activity in a cell prep- Davis, 1989).Moreover, we have demonstrated that aration can have a significant impact on the a cap and a poly(A) tail interact synergistically to efficiency of delivery during RNA electroporation regulate translational efficiency (Gallie, 1991). as well as on reproducibility. We tested our spheroUsing RNA electroporation, the quantification plast preparation for extracellular RNase activity of the regulatory impact of a cap or poly(A) tail on by mixing Luc mRNA with the spheroplasts and translation can be addressed directly. The GUS gene allowing the cell-luc mRNA mixture to incubate at has gained widespread use in plant studies due to the room temperature for varying amounts of time low endogenous activity of GUS in most plant before electroporating. No detectable difference species. Recently, the use of the GUS gene as a
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J. G. EVERETT AND D. R. GALLIE
Q Figure 4. The impact of cap, poly(A) tail, and the tobacco mosaic virus mRNA leader (Q) on the translation of P-glucuronidase (GUS) mRNA in yeast. 1 pg of each mRNA construct was used for the electroporation, GUS specific activityis indicated above each bar and is expressed as nmol/min per mg protein.
reporter gene in yeast was demonstrated (Schmitz
12 A residues but, as a consequence of its size, it has a packing density of one PAB protein molecule per 25A residues (Sachs er al., 1987). The GUS constructs terminating in either a poly(A),, or poly(A),, tail, therefore, might be expected to accommodate greater regulatory control by a poly(A) tail than one or two molecules of PAB protein, respectively. are Luc mRNA constructs (Gallie et al., 1991) The fact that translation from the poly(A),, conand therefore it represents a more responsive struct was higher than that from the poly(A),, reporter gene for measuring poly(A) tail-mediated construct suggests not only that the presence of a poly(A) tail is important for efficient translation regulation. Addition of a cap increased GUS expression by in yeast, but also that the length is a regulatory six-fold (Figure 4),a result in good agreement with parameter. The untranslated leader (called R) from tobacco the observations made by Russell et al. (1991), in which capped and uncapped Luc mRNAs were mosaic virus enhances the translational efficiency of introduced into yeast spheroplasts using PEG. The chimeric mRNA constructs in plants, and to a lesser addition of a poly(A),, tail to GUS mRNA extent, in animal species (Gallie et al., 1987, 1989; increased expression by 11I-fold (Figure 4). The Gallie and Walbot, 1990). It has been reported average poly(A) tail length for yeast mRNA in the recently that Q enhanced translation in a yeast in cytoplasm, however, is approximately 50 A residues vitro lysate (Altmann et al., 1990). In order to (Sachs and Davis, 1989). When the poly(A) tail was examine whether R could enhance translation in doubled in length to 50 A residues, GUS expression vivo in yeast, GUS-A,, mRNA, containing either R increased an additional 75%. The PAB protein of or the same 17-base control leader used for all the yeast has a minimum binding site requirement of poly(A)+ constructs (Figure l), was translated in et al., 1990). No detectable GUS activity was measured in our S. cerevisiae strains under the conditions used for the E. coli GUS assay. Moreover, the translation of GUS mRNA constructs is under
RNA DELIVERY IN SACCHAROMYCES CEREVISIAE
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electroporated yeast. R did not enhance translation de Wet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R. and Subramani, S . (1987). Firefly luciferasegene: strucin yeast (Figure 4). In fact, expression from the Rture and expression in mammalian cells. Mol. Cell. Biol. containing GUS-A,, mRNA was consistently lower 7,725-737. than that observed for the control GUS-A,, conFromm, M. E., Callis, J., Taylor, L. P. and Walbot, V. struct. These data demonstrate that the ability of (1987). Electroporation of DNA and RNA into plant this leader to enhance translation is highly species protoplasts. Methods Enzymol. 153,351-366. dependent. Moreover, the in vivo results demon- Gallie, D. R. (1991). The cap and poly(A) tail function strate the importance of using an in vivo assay syssynergistically to regulate mRNA translational tem to investigate translational regulation. With the efficiency. Genes and Devel. 5,2108-2 116. development of an efficient delivery system for the Gallie, D. R., Feder, J. N., Schimke, R. T. and Walbot, V. (1991). Post-transcriptional regulation in higher introduction of mRNA into yeast, the cytoplasmic eukaryotes: the role of the reporter gene in controlling regulation of gene expression can now be directly expression. Mol. Gen. Genet. 228,258-264. studied in vivo. ACKNOWLEDGEMENTS The authors thank Marilyn Kobayashi for technical assistance. This work was supported in part by a grant from the University of California Systemwide Biotechnology Research and Education Program. REFERENCES Altmann, M., Blum, S., Wilson, T. M. A. andTrachsel, H. (1990). The 5’-leader sequence of tobacco mosaic virus RNA mediates initiation-factor-4E-independent, but still initiation-factor-4A-dependenttranslation in yeast extracts. Gene 91, 127-129. Altmann, M., Handschin, C. and Trachsel, H. (1987). mRNA cap-binding protein: cloning of the gene encoding protein synthesis initiation factor eIF-4E from Saccharomyces cerevisiae. Mol. Cell. Biol. 7, 998-1 003. Altmann, M., Sonenberg, N. and Trachsel, H. (1989). Translation in Saccharomyces cerevisiae:initiation factor 4E-dependent cell-free system. Mol. Cell. Biol. 9, 4467-4472. Bartoletti, D. C., Harrison, G. I. and Weaver, J. C. (1989). The number of molecules taken up by electroporated cells: quantitative determination. FEBS Letters 256, 4-10. Becker, D. M. and Guarente, L. (1991). High-efficiency transformation of yeast by electroporation. Methods Enz. 194,182-187. Blum, S., Mueller, M., Schmid, R., Linder, P. and Traschsel, H. (1989). Saccharomyces cerevisiae: initiation factor 4A-dependent cell-free system. Proc. Natl. Acad. Sci. USA 86,6043-6046. Callis, J., Fromm, M. and Walbot, V. (1987). Expression of mRNA electroporated into plant and animal cells. Nucl. Acids. Res. 15,5823-583 1. Darzynkiewicz, E., Stepinski, J., Ekiel, I., Jin, Y., Haber, D., Sijuwade, T. and Tahara, S. M. (1988). P-globin or m2,2*337G differ in mRNAs capped with m7G, intrinsic translation efficiency. Nucl. Acids. Res. 16, 8953-8962. m23237G
Gallie, D. R., Lucas, W. J. and Walbot, V. (1989). Visualizing mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and translation. The Plant Cell 1,301-31 1. Gallie, D. R., Sleat, D. E., Watts, J. W., Turner, P. C. and Wilson, T. M. A. (1987). The gene transcripts in vitro and in vivo. Nucl. Acids Res. 15,3257-3273. Gallie, D. R. and Walbot, V. (1990). RNA pseudoknot domain of tobacco mosaic virus can functionally substitute for a poly(A) tail in plant and animal cells. Genes and Devel. 4,1149-1 157. Gasior, E., Herrera, F., Sadnik, I., McLaughlin, C. S. and Moldave, K. (1979). The preparation and characterization of a cell-free system from Saccharomyces cerevisiae that translates natural messenger ribonucleic acid. J . Biol. Chem. 254,3965-3969. Hutchison, H. T. and Hartwell, L. H. (1967). Macromolecule synthesis in yeast spheroplasts. J. Bacteriol. 94,1697-1705. Jefferson, R. A. (1987). Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5, 387-405. Kleinschmidt, A. M. and Pederson, T. (1990). RNA processing and ribonucleoprotein assembly studied in vivo by RNA transfection. Proc. Natl. Acad. Sci. USA 87,1283-1287. Malone, R. W., Felgner, P. L. and Verma, I. M. (1989). Cationic liposome-mediated RNA transfection. Proc. Natl. Acad. Sci. USA 86,6077-608 1. Melton, D. A., Kreig, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K. and Green, M. R. (1984). Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucl. Acids Res. 12, 7035-7056. Miller, J. F., Dower, W. J. and Tompkins, L. S. (1988). High-voltage electroporation of bacteria: genetic transformation of Campylobacter jejuni with plasmid DNA. Proc. Natl. Acad. Sci. USA 85,856860. Munroe, D. and Jacobson, A. (1990a). mRNA poly(A) tail, a 3’ enhancer of translational initiation., Mol. Cell. Biol.10,344-3455. Munroe, D. and Jacobson, A. (1990b). Tales of poly(A): a review. Gene91,151-158.
1014 Potter, H. (1988). Electroporation in biology: methods, applications, and instrumentation. Anal. Biochem. 174, 361-373. Rhoads, R. E. (1988). Cap recognition and the entry of mRNA into the protein synthesis initiation cycle. TIBS 13,52-56. Russell, P. J., Hambridge, S. J. and Kirkegaard, K. (1991). Direct introduction and transient expression of capped and non-capped RNA in Saccharomyces cerevisiae. Nucl. Acids. Res. 19,4949-4953. Sachs, A. B. and Davis, R. W. (1989). The poly(A) binding protein is required for poly(A) shortening and 60s ribosomal subunit-dependent translation initiation. Cell58,857-867. Sachs, A. B., Davis, R. W. and Kornberg, R. D. (1987). A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability. Mol. Cell. Biol. 7,3268-3276. Schenborn, E. T. and Mierendorf, R. C . (1985). A novel transcription property of SP6 and T7 RNA polymerase:
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