- Email: [email protected]
Development of anin VitroTranslation System from Wheat Germ That Is Dependent upon the Addition of Eukaryotic Initiation Factor 2
140
NOTES & TIPS
ear with respect to protein concentration (data not shown). Moreover, the decrease in band intensity during step 7 was uniform for different proteins. The reduction in background color after ammoniacal staining has been suggested using Kodak Rapid Fix solution (7), but unless controlled with methanol, this procedure could completely destain the gel, including the protein bands (4). Our method is much simpler, specific, and faster and it results in complete clearing of the background not achieved in earlier methods (4). This staining procedure can also be used to silver stain the polymers of ADP-ribose resolved on 20% polyacrylamide sequencing gel as described earlier (8). We achieved the clearance of the background color without affecting the intensity of the poly(ADP-ribose) bands. REFERENCES 1. Merril, C. R. (1990) Methods Enzymol. 182, 477–488. 2. Ansorge, W. (1985) J. Biochem. Biophys. Methods 11, 13–20. 3. Oakley, B. R., Kirsch, D. R., and Morris, N. R. (1980) Anal. Biochem. 105, 361–363. 4. Wray, W., Boulikas, T., Wray, V. P., and Hancock, R. (1981) Anal. Biochem. 118, 197–203. 5. Laemmli, U. K. (1970) Nature 227, 680–685. 6. Hochstrasser, D. F., Patchornik, A., and Merril, C. R. (1988) Anal. Biochem. 173, 412–423. 7. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1992) Short Protocols in Molecular Biology, Wiley, New York. 8. Shah, G. M., Poirier, D., Duchaine, C., Brochu, G., Desnoyers, S., Lagueux, J., Verreault, A., Hoflack, J. C., Kirkland, J. B., and Poirier, G. G. (1995) Anal. Biochem. 227, 1–13.
Development of an in Vitro Translation System from Wheat Germ That Is Dependent upon the Addition of Eukaryotic Initiation Factor 2 Lisa A. Benkowski,1 Joanne M. Ravel, and Karen S. Browning2 Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712 Received August 7, 1995
At least eight factors (eIF-1,3 eIF-2, eIF-3, eIF-4A, eIF-4B, eIF-4C, eIF-4F, and eIF-5) are required for the
initiation of translation of eukaryotic mRNAs (1). It has been of interest to determine which of these factors, if any, play a role in the preferential translation of one mRNA over another. In vitro protein synthesis systems were developed that were deficient in initiation factors eIF-4C (2), eIF-4A, eIF-4B, eIF-4F, or eIF-3 (3–5). Using these deficient systems we were able to show that mRNAs differ in the amounts of eIF-4A, eIF-4B, and eIF-4F (3–5), but not eIF-3 (3–5) or eIF-4C (2) required for half-maximal translation, indicating that eIF-4A, eIF-4B, and eIF-4F play a role in the preferential translation of one mRNA over another. The work of several investigators has suggested that competition of mRNAs for eIF-2 plays a role in the regulation of protein synthesis. DiSegni et al. (6) showed that translational competition between a- and b-globin mRNA can be relieved by addition of highly purified eIF-2. Later work indicated that competition for eIF-2 is also involved in the regulation of Mengovirus RNA translation (7). Furthermore, Scheper et al. (8) showed that encephalomyocarditis virus RNA effectively competes with globin mRNA for eIF-2/2B in a rabbit reticulocyte translation assay. These competition studies, performed in crude extracts, suggested that there are differences in the concentrations of eIF-2 required for efficient translation. Currently there are no mRNAdependent, in vitro assay systems from wheat germ or rabbit reticulocytes that are dependent upon the addition of eIF-2 because these assay systems contain small amounts of crude fractions. These crude fractions are required as a source of aminoacyl-tRNA synthetases, elongation factors, and termination factors, but also contain eIF-2 (9,10). The amount of an ammonium sulfate fraction (40–70%) added to the wheat germ assay system contains sufficient amounts of eIF-2 to obtain optimal translation (10). The only in vitro assay currently available for measuring the activity of eIF-2 is based on the ability of eIF-2 to form a ternary complex formation with GTP and [35S]Met-tRNAi . This assay does not measure the overall activity of eIF-2 in the initiation process. In this report we describe the development an in vitro translation system from wheat germ which is dependent upon the addition of eIF-2 and can be used to determine whether there are significant differences in the concentration of eIF-2 required for the translation of different mRNAs.
1
Present address: Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599. 2 To whom correspondence should be addressed. Fax: 512-4718696. E-mail: [email protected]. 3 Abbreviations used: eIF-, eukaryotic initiation factor; STNV, satellite tobacco necrosis virus; AMV, alfalfa mosaic virus; Hepes, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; 40–70 ASF, 40–70% ammonium sulfate fraction; PGA, protein G effluent. ANALYTICAL BIOCHEMISTRY
MATERIALS AND METHODS
Materials. Wheat germ extracts, high salt-washed ribosomes, 40–70% ammonium sulfate fraction (40–70 ASF), and highly purified preparations of wheat germ eIF-2 (10), eIF-4A (10), eIF-4C (2), eIF-4B (11), eIF-3 (10), and eIF-4F (12) were prepared as described. Rabbit antibodies to wheat germ eIF-2 were prepared as
232, 140–143 (1995)
0003-2697/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ m4485$$nat
11-06-95 11:53:28
abnt
AP-Anal Bio
NOTES & TIPS
described previously (13). Goat anti-rabbit IgG (H / L) conjugated to horseradish peroxidase was obtained from Kirkegaard & Perry Laboratories; ABTS was obtained from Sigma; and microtiter plates were obtained from Nunc. Protein G gel was from Pierce Chemicals. Plasmids containing cDNA for mRNAs were constructed as described previously: STNV RNA (4), AMV RNA 4 (3), barley a-amylase (14), oat avenin (15), and oat globulin (15). Plasmids containing oat avenin and globulin cDNAs were generously provided by Brian Larkins, University of Arizona. Preparation of mRNAs by in vitro transcription. Capped mRNAs (AMV RNA 4, barley a-amylase, avenin, globulin) and uncapped STNV RNA were prepared by in vitro transcription of the linearized plasmids as described previously except that [a-32P]UTP (1–100 cpm/pmol) was used instead of [a-32P]ATP (3,4). The transcribed mRNAs migrated as single bands on 4% polyacrylamide–8 M urea gels (data not shown). It was determined that §70% of the capped transcription products contained a 5* cap structure (3, data not shown). Preparation of a protein GreIF-2 antibody affinity column. A 1-ml column of protein G gel was equilibrated with 5 ml binding buffer (50 mM sodium acetate, pH 5.0) at room temperature. Rabbit serum containing antibodies raised to wheat germ eIF-2 was brought to 50% saturation by the addition of solid ammonium sulfate. The precipitated proteins were collected by centrifugation, resuspended in 5 ml of buffer B-40 (20 mM Hepes/KOH, pH 7.6, 1 mM DTT, 0.1 mM EDTA, 10% glycerol, 40 mM KCl) and dialyzed at 47C against buffer B-40. The precipitated proteins containing antibodies to eIF-2 (0.5 ml containing 22 mg of protein) were diluted 1:1 with binding buffer, applied to the column, and allowed to adsorb to the column for 1 h at room temperature. The column was then washed with 6–10 ml of binding buffer and the effluent was collected. The effluent was analyzed for eIF-2 antibodies by ELISA (13); approximately 50% of the eIF-2 antibodies applied to the column were retained on the Protein G column. Chromatography of wheat germ 40–70 ASF on protein GreIF-2 antibody affinity column. The protein Pro GreIF-2 affinity column was cooled to 47C and washed with buffer B-40 until the pH of the effluent was 7.6. The 40–70 ASF (2 ml) was passed slowly (Ç4 ml/hr) through the column followed by 2 ml buffer B40. The effluent was collected in 0.5-ml fractions, assayed for eIF-2 by ELISA as described below, and stored at 0707C in small aliquots. The effluent was given the designation, 40–70 PGA. ELISA. The ELISA was performed as described (13). To quantitate the amount of eIF-2 antibodies in serum or antibody preparations, 20 ng of eIF-2 was placed in the wells of the microtiter plate and appro-
/ m4485$$nat
11-06-95 11:53:28
abnt
141
priate dilutions of samples containing antibody to eIF2 were made. To quantitate the amount of eIF-2 in a sample, appropriate dilutions of the samples containing eIF-2 were made and compared to a standard curve of highly purified eIF-2 (1–10 ng) in the presence of a 1/1000 dilution of the eIF-2 antibody (13). In vitro translation system from wheat germ dependent upon the addition of eIF-2. The reaction mixture (3) contained in 100 ml: 24 mM Hepes/KOH, pH 7.6, 2.4 mM DTT, 0.1 mM spermine, 2.5 mM Mg(OAc)2 , 40 mM KCl, 90 mM KOAc, 1 mM ATP, 0.2 mM GTP, 7.8 mM creatine phosphate, 3 mg of creatine kinase, 34 mM [14C]leucine (200 cpm/pmol), 50 mM each of 19 amino acids, 0.75 A260 units of wheat germ tRNA, 2.5–3 A260 units of salt-washed ribosomes, indicated amounts of 40–70 ASF or 40–70 PGA, 6 mg eIF-4C, 5 mg eIF-3, 3 mg eIF4F, 20 mg eIF-4A, 1.2 mg eIF-4B, and 5 pmol mRNA. The reaction mixture was incubated for 30 min at 277C and the amount of [14C]leucine incorporated into hot trichloroacetic acid precipitable material was determined as previously described (10). To determine the concentration of eIF-2 required to obtain half-maximal rate of translation (C0.5), eIF-2 was added in rate-limiting amounts. The C0.5 value was calculated directly from a dose–response curve after correcting for the amount of eIF-2 remaining in the 40–70 PGA (õ0.3 mg). The values reported are an average of two or more experiments. RESULTS AND DISCUSSION
To obtain an in vitro translation system dependent upon the addition of eIF-2, it was necessary to reduce the amount of eIF-2 in the 40–70 ASF (Ç1000 mg/ml) while retaining the other necessary components. This was achieved by the passage of the 40–70 ASF through a protein GreIF-2 antibody column. Analysis of the effluent from the protein GreIF-2 antibody column by ELISA showed that the concentration of eIF-2 in the effluent was Ç50 mg/ml or about 20-fold less than that in the 40–70 ASF. The data in Fig. 1 show the effects of eIF-2 on STNV RNA directed polypeptide synthesis when 40–70 ASF or 40–70 PGA was added to the translation system. As shown in Fig. 1A, translation was dependent upon the addition of 40–70 ASF and very little stimulation was obtained upon the addition of eIF-2. Based on the concentration of eIF-2 in the 40–70 ASF estimated by ELISA (1000 mg/ml), addition of as little as 3 ml of 40– 70 ASF would supply 3 mg of eIF-2. The data in Fig. 1B show that very little translation was obtained when the effluent from the PGA column was used in the absence of eIF-2 and the addition of 3 mg of eIF-2 stimulated translation approximately sixfold. Higher levels of the 40–70 PGA (12 ml) appeared to be toxic. Also, the maximal amount of translation obtained with 40–
AP-Anal Bio
142
NOTES & TIPS
FIG. 1. Ability of 40–70 ASF and 40–70 PGA to support polypeptide synthesis in the absence and presence of eIF-2. The standard reaction mixture contained 5 pmol of STNV RNA and 40–70 ASF (A) or 40–70 PGA (B) as indicated in the absence (l) or presence (s) of 3 mg eIF2. (C) The standard reaction mixture contained 10 pmol of STNV RNA, 6 ml of 40–70 PGA, and eIF-2 as indicated.
70 PGA and eIF-2 was only about 50% of that obtained with untreated 40–70 ASF. It is not known whether the 40–70 PGA contains a toxic component (i.e., a component that leached from the protein G column) or whether a stimulatory factor is also removed by the protein GreIF-2 antibody column. A typical response to eIF-2 obtained in the translation system containing 40–70 PGA and STNV RNA is shown in Fig. 1C. The concentrations of eIF-2 required for half-maximal translation (C0.5) for several mRNAs were determined as described under Materials and Methods and the results are given in Table 1. The C0.5 values for eIF4F determined as described previously (3) are given for comparison purposes. The concentration of eIF-2 required for half-maximal translation of the five
TABLE 1
Comparison of the Concentrations of eIF-2 and eIF-4F Required for Half-Maximal Rate of Translation Concentration (Co.5)a (nM) mRNA
eIF-2b
eIF-4Fc
AMV4 STNV a-Amylase Avenin Globulin
60 60 90 60 90
6 4 17 3 7
a The concentrations required to obtain half-maximal rate of translation were determined as described (3,4), assuming the following Mr: eIF-2 (130,000) and eIF-4F (330,000). b The reaction mixture contained 6 ml of the 40–70 PGA and 5 pmol of the indicated mRNA. c The reaction mixture contained 5 ml of 40–70 ASF and 5 pmol of the indicated mRNA.
/ m4485$$nat
11-06-95 11:53:28
abnt
mRNAs tested differed by no more than 1.5-fold. We have also previously reported the amounts of eIF-2 required for the translation of rabbit a- and b-globin mRNA to be 49 and 44 nM, respectively (5). The concentration of eIF-2 present in a wheat germ S30 translation system, estimated by ELISA, is Ç400 nM (13). This amount of eIF-2 is 4-fold higher than the highest concentration of eIF-2 required for half-maximal translation of the mRNAs tested. In contrast, the concentration of eIF-4F required for half-maximal translation of these mRNAs differed by as much as 7-fold. The concentration of eIF-4F in a wheat germ S30 translation system is only Ç20 nM which is about the same concentration required for half-maximal translation of barley a-amylase and therefore could be rate-limiting for this particular mRNA. The method described here, removing a specific protein from a crude extract using an antibody affinity column, allows the development of an assay dependent upon a single component of a complicated, multicomponent enzyme system (e.g., protein synthesis). This method may be applied to other multicomponent systems where removal of a single component would be advantageous. The eIF-2-dependent translation system described in this paper was used to measure the amount of eIF-2 required for the initiation of several mRNAs. These preliminary findings strongly suggest that the concentration of eIF-2 is not rate-limiting in wheat germ and does not play a role in the preferential initiation of the mRNAs tested. Acknowledgments. We thank Sandra Lax and Anneke Metz for their excellent technical assistance. This work supported by a grant from the National Science Foundation to J.M.R. and K.S.B. (DMB9105487).
AP-Anal Bio
NOTES & TIPS
REFERENCES 1. Merrick, W. C. (1994) Biochimie 76, 822–830. 2. Timmer, R. T., Lax, S. R., Hughes, D. L., Merrick, W. C., Ravel, J. M., and Browning, K. S. (1993) J. Biol. Chem. 268, 24863– 24867. 3. Fletcher, L., Corbin, S. D., Browning, K. S., and Ravel, J. M. (1990) J. Biol. Chem. 265, 19582–19587. 4. Timmer, R. T., Benkowski, L. A., Schodin, D., Lax, S. R., Metz, A. M., Ravel, J. M., and Browning, K. S. (1993) J. Biol. Chem. 268, 9504–9510. 5. Timmer, R. T., Benkowski, L. A., Ravel, J. M., and Browning, K. S. (1995) Biochem. Biophys. Res. Commun. 210, 370–377. 6. DiSegni, G., Rosen, H., and Kaempfer, R. (1979) Biochemistry 18, 2847–2854. 7. Rosen, H., DiSegni, G., and Kaempfer, R. (1982) J. Biol. Chem. 257, 946–952. 8. Scheper, G. C., Thomas, A. A. M., and Voorma, H. O. (1991) Biochim. Biophys. Acta 1089, 220–226. 9. Crystal, R. G., Elson, N. A., and Anderson, W. F. (1974) Methods Enzymol. 30, 101–127. 10. Lax, S. R., Lauer, S. J., Browning, K. S., and Ravel, J. M. (1986) Methods Enzymol. 118, 109–128. 11. Browning, K. S., Maia, D. M., Lax, S. R., and Ravel, J. M. (1987) J. Biol. Chem. 262, 538–541. 12. Browning, K. S., Webster, C., Roberts, J. K. N., and Ravel, J. M. (1992) J. Biol. Chem. 267, 10096–10100. 13. Browning, K. S., Humphreys, J., Hobbs, W., Smith, G. B., and Ravel, J. M. (1990) J. Biol. Chem. 265, 17967–17973. 14. Browning, K. S., Lax, S. R., Humphreys, J., Ravel, J. M., Jobling, S. A., and Gehrke, L. (1988) J. Biol. Chem. 263, 9630–9634. 15. Boyer, S. K., Shotwell, M. A., and Larkins, B. A. (1992) J. Biol. Chem. 267, 17449–17457.
A Reagent for both Visualization and Chemiluminescence Detection of Horseradish Peroxidase-Tagged DNA Probes Bibijana Cercek, Keith Roby, and Martin Siaw Diagnostic Systems Group, Beckman Instruments, Inc., Brea, California 92621 Received August 15, 1995
Although chemiluminescent detection provides a rapid and highly sensitive means for quantitation of DNA probes, the signal cannot be measured again, and unless photographed, no permanent record remains (1, 4). Hence, visualization by staining of DNA probe bands would provide a useful adjunct to such a method especially for application in medical diagnostics (2). We describe the application of a borax buffer-based signal reagent, originally developed for horseradish ANALYTICAL BIOCHEMISTRY
peroxidase (HRP)1-enhanced chemiluminescence measurement (1, 3, 4), to the detection of streptavidinlinked HRP tagged with biotinylated DNA probes on positively charged and neutral nylon membranes by direct visualization. This signal reagent also provides the option of chemiluminescence detection, generally measured within the first 60 s, is retained (1, 4). Experimental conditions and the formulation of the signal reagent were as described previously (1). The signal reagent based on borax buffer was evaluated since it was previously shown (4) that the borax buffer based formulation gives greatly improved, very low, reagent background signals at the detection limit of the chemiluminometer and optimal signal to background ratios, as well as rapid signal rise to steady-state and prolonged steady-state signal emission when compared to all the many other buffers investigated in HRP paraiodophenol-enhanced chemiluminescence measurement (4). As can be seen in Table 1, the method allows a detection limit of 11 ng biotinylated DNA/slot blot and up to 1 mg biotinylated DNA was slot blotted and readily visualized. Visualization at the detection limit is possible within less than 1 min with the 101 signal reagent. However, the colored stain lasts only up to 24 h. Using the 11 signal reagent, visualization is possible within 10 to 15 min after signal reagent addition, and the yellow–brown color of the stained bands is stable for at least 4 weeks at room temperature in the dark. The background was unstained and colorless, under all conditions investigated, even 4 weeks after exposure to the signal reagent. No difference in either intensity or time to full color development was observed using either the Tropix negatively charged or the Millipore positively charged nylon membranes for slot blotting the biotinylated DNA. By a process of component elimination (Table 1), it is evident that luminol is not requisite for rapid, specific color development and acceptable detection sensitivity. Although omission of the para-iodophenol enhancer from the signal reagent adversely affects both the color development time and the detection limits, excluding perborate or streptavidin–HRP totally abolishes the color development reaction. The absolute requirement for perborate and HRP suggests a free radical mediated reaction mechanism, possibly analogous to that of the multi-step chemiluminescenceenhanced reaction itself (3). The effect of enhancers other than para-iodophenol on the color and/or intensity of the deposited colored product has yet to be determined and would be of interest in establishing the role of the enhancer in the color development process. Furthermore, the nature of the colored product deposited is at present unknown. However, thermal or photochemical degradation of luminol solutions results in a 1
Abbreviation used: HRP, horseradish peroxidase.
232, 143–144 (1995)
0003-2697/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ m4485$$nat
11-06-95 11:53:28
abnt
143
AP-Anal Bio
NOTES & TIPS
ear with respect to protein concentration (data not shown). Moreover, the decrease in band intensity during step 7 was uniform for different proteins. The reduction in background color after ammoniacal staining has been suggested using Kodak Rapid Fix solution (7), but unless controlled with methanol, this procedure could completely destain the gel, including the protein bands (4). Our method is much simpler, specific, and faster and it results in complete clearing of the background not achieved in earlier methods (4). This staining procedure can also be used to silver stain the polymers of ADP-ribose resolved on 20% polyacrylamide sequencing gel as described earlier (8). We achieved the clearance of the background color without affecting the intensity of the poly(ADP-ribose) bands. REFERENCES 1. Merril, C. R. (1990) Methods Enzymol. 182, 477–488. 2. Ansorge, W. (1985) J. Biochem. Biophys. Methods 11, 13–20. 3. Oakley, B. R., Kirsch, D. R., and Morris, N. R. (1980) Anal. Biochem. 105, 361–363. 4. Wray, W., Boulikas, T., Wray, V. P., and Hancock, R. (1981) Anal. Biochem. 118, 197–203. 5. Laemmli, U. K. (1970) Nature 227, 680–685. 6. Hochstrasser, D. F., Patchornik, A., and Merril, C. R. (1988) Anal. Biochem. 173, 412–423. 7. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1992) Short Protocols in Molecular Biology, Wiley, New York. 8. Shah, G. M., Poirier, D., Duchaine, C., Brochu, G., Desnoyers, S., Lagueux, J., Verreault, A., Hoflack, J. C., Kirkland, J. B., and Poirier, G. G. (1995) Anal. Biochem. 227, 1–13.
Development of an in Vitro Translation System from Wheat Germ That Is Dependent upon the Addition of Eukaryotic Initiation Factor 2 Lisa A. Benkowski,1 Joanne M. Ravel, and Karen S. Browning2 Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712 Received August 7, 1995
At least eight factors (eIF-1,3 eIF-2, eIF-3, eIF-4A, eIF-4B, eIF-4C, eIF-4F, and eIF-5) are required for the
initiation of translation of eukaryotic mRNAs (1). It has been of interest to determine which of these factors, if any, play a role in the preferential translation of one mRNA over another. In vitro protein synthesis systems were developed that were deficient in initiation factors eIF-4C (2), eIF-4A, eIF-4B, eIF-4F, or eIF-3 (3–5). Using these deficient systems we were able to show that mRNAs differ in the amounts of eIF-4A, eIF-4B, and eIF-4F (3–5), but not eIF-3 (3–5) or eIF-4C (2) required for half-maximal translation, indicating that eIF-4A, eIF-4B, and eIF-4F play a role in the preferential translation of one mRNA over another. The work of several investigators has suggested that competition of mRNAs for eIF-2 plays a role in the regulation of protein synthesis. DiSegni et al. (6) showed that translational competition between a- and b-globin mRNA can be relieved by addition of highly purified eIF-2. Later work indicated that competition for eIF-2 is also involved in the regulation of Mengovirus RNA translation (7). Furthermore, Scheper et al. (8) showed that encephalomyocarditis virus RNA effectively competes with globin mRNA for eIF-2/2B in a rabbit reticulocyte translation assay. These competition studies, performed in crude extracts, suggested that there are differences in the concentrations of eIF-2 required for efficient translation. Currently there are no mRNAdependent, in vitro assay systems from wheat germ or rabbit reticulocytes that are dependent upon the addition of eIF-2 because these assay systems contain small amounts of crude fractions. These crude fractions are required as a source of aminoacyl-tRNA synthetases, elongation factors, and termination factors, but also contain eIF-2 (9,10). The amount of an ammonium sulfate fraction (40–70%) added to the wheat germ assay system contains sufficient amounts of eIF-2 to obtain optimal translation (10). The only in vitro assay currently available for measuring the activity of eIF-2 is based on the ability of eIF-2 to form a ternary complex formation with GTP and [35S]Met-tRNAi . This assay does not measure the overall activity of eIF-2 in the initiation process. In this report we describe the development an in vitro translation system from wheat germ which is dependent upon the addition of eIF-2 and can be used to determine whether there are significant differences in the concentration of eIF-2 required for the translation of different mRNAs.
1
Present address: Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599. 2 To whom correspondence should be addressed. Fax: 512-4718696. E-mail: [email protected]. 3 Abbreviations used: eIF-, eukaryotic initiation factor; STNV, satellite tobacco necrosis virus; AMV, alfalfa mosaic virus; Hepes, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; 40–70 ASF, 40–70% ammonium sulfate fraction; PGA, protein G effluent. ANALYTICAL BIOCHEMISTRY
MATERIALS AND METHODS
Materials. Wheat germ extracts, high salt-washed ribosomes, 40–70% ammonium sulfate fraction (40–70 ASF), and highly purified preparations of wheat germ eIF-2 (10), eIF-4A (10), eIF-4C (2), eIF-4B (11), eIF-3 (10), and eIF-4F (12) were prepared as described. Rabbit antibodies to wheat germ eIF-2 were prepared as
232, 140–143 (1995)
0003-2697/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ m4485$$nat
11-06-95 11:53:28
abnt
AP-Anal Bio
NOTES & TIPS
described previously (13). Goat anti-rabbit IgG (H / L) conjugated to horseradish peroxidase was obtained from Kirkegaard & Perry Laboratories; ABTS was obtained from Sigma; and microtiter plates were obtained from Nunc. Protein G gel was from Pierce Chemicals. Plasmids containing cDNA for mRNAs were constructed as described previously: STNV RNA (4), AMV RNA 4 (3), barley a-amylase (14), oat avenin (15), and oat globulin (15). Plasmids containing oat avenin and globulin cDNAs were generously provided by Brian Larkins, University of Arizona. Preparation of mRNAs by in vitro transcription. Capped mRNAs (AMV RNA 4, barley a-amylase, avenin, globulin) and uncapped STNV RNA were prepared by in vitro transcription of the linearized plasmids as described previously except that [a-32P]UTP (1–100 cpm/pmol) was used instead of [a-32P]ATP (3,4). The transcribed mRNAs migrated as single bands on 4% polyacrylamide–8 M urea gels (data not shown). It was determined that §70% of the capped transcription products contained a 5* cap structure (3, data not shown). Preparation of a protein GreIF-2 antibody affinity column. A 1-ml column of protein G gel was equilibrated with 5 ml binding buffer (50 mM sodium acetate, pH 5.0) at room temperature. Rabbit serum containing antibodies raised to wheat germ eIF-2 was brought to 50% saturation by the addition of solid ammonium sulfate. The precipitated proteins were collected by centrifugation, resuspended in 5 ml of buffer B-40 (20 mM Hepes/KOH, pH 7.6, 1 mM DTT, 0.1 mM EDTA, 10% glycerol, 40 mM KCl) and dialyzed at 47C against buffer B-40. The precipitated proteins containing antibodies to eIF-2 (0.5 ml containing 22 mg of protein) were diluted 1:1 with binding buffer, applied to the column, and allowed to adsorb to the column for 1 h at room temperature. The column was then washed with 6–10 ml of binding buffer and the effluent was collected. The effluent was analyzed for eIF-2 antibodies by ELISA (13); approximately 50% of the eIF-2 antibodies applied to the column were retained on the Protein G column. Chromatography of wheat germ 40–70 ASF on protein GreIF-2 antibody affinity column. The protein Pro GreIF-2 affinity column was cooled to 47C and washed with buffer B-40 until the pH of the effluent was 7.6. The 40–70 ASF (2 ml) was passed slowly (Ç4 ml/hr) through the column followed by 2 ml buffer B40. The effluent was collected in 0.5-ml fractions, assayed for eIF-2 by ELISA as described below, and stored at 0707C in small aliquots. The effluent was given the designation, 40–70 PGA. ELISA. The ELISA was performed as described (13). To quantitate the amount of eIF-2 antibodies in serum or antibody preparations, 20 ng of eIF-2 was placed in the wells of the microtiter plate and appro-
/ m4485$$nat
11-06-95 11:53:28
abnt
141
priate dilutions of samples containing antibody to eIF2 were made. To quantitate the amount of eIF-2 in a sample, appropriate dilutions of the samples containing eIF-2 were made and compared to a standard curve of highly purified eIF-2 (1–10 ng) in the presence of a 1/1000 dilution of the eIF-2 antibody (13). In vitro translation system from wheat germ dependent upon the addition of eIF-2. The reaction mixture (3) contained in 100 ml: 24 mM Hepes/KOH, pH 7.6, 2.4 mM DTT, 0.1 mM spermine, 2.5 mM Mg(OAc)2 , 40 mM KCl, 90 mM KOAc, 1 mM ATP, 0.2 mM GTP, 7.8 mM creatine phosphate, 3 mg of creatine kinase, 34 mM [14C]leucine (200 cpm/pmol), 50 mM each of 19 amino acids, 0.75 A260 units of wheat germ tRNA, 2.5–3 A260 units of salt-washed ribosomes, indicated amounts of 40–70 ASF or 40–70 PGA, 6 mg eIF-4C, 5 mg eIF-3, 3 mg eIF4F, 20 mg eIF-4A, 1.2 mg eIF-4B, and 5 pmol mRNA. The reaction mixture was incubated for 30 min at 277C and the amount of [14C]leucine incorporated into hot trichloroacetic acid precipitable material was determined as previously described (10). To determine the concentration of eIF-2 required to obtain half-maximal rate of translation (C0.5), eIF-2 was added in rate-limiting amounts. The C0.5 value was calculated directly from a dose–response curve after correcting for the amount of eIF-2 remaining in the 40–70 PGA (õ0.3 mg). The values reported are an average of two or more experiments. RESULTS AND DISCUSSION
To obtain an in vitro translation system dependent upon the addition of eIF-2, it was necessary to reduce the amount of eIF-2 in the 40–70 ASF (Ç1000 mg/ml) while retaining the other necessary components. This was achieved by the passage of the 40–70 ASF through a protein GreIF-2 antibody column. Analysis of the effluent from the protein GreIF-2 antibody column by ELISA showed that the concentration of eIF-2 in the effluent was Ç50 mg/ml or about 20-fold less than that in the 40–70 ASF. The data in Fig. 1 show the effects of eIF-2 on STNV RNA directed polypeptide synthesis when 40–70 ASF or 40–70 PGA was added to the translation system. As shown in Fig. 1A, translation was dependent upon the addition of 40–70 ASF and very little stimulation was obtained upon the addition of eIF-2. Based on the concentration of eIF-2 in the 40–70 ASF estimated by ELISA (1000 mg/ml), addition of as little as 3 ml of 40– 70 ASF would supply 3 mg of eIF-2. The data in Fig. 1B show that very little translation was obtained when the effluent from the PGA column was used in the absence of eIF-2 and the addition of 3 mg of eIF-2 stimulated translation approximately sixfold. Higher levels of the 40–70 PGA (12 ml) appeared to be toxic. Also, the maximal amount of translation obtained with 40–
AP-Anal Bio
142
NOTES & TIPS
FIG. 1. Ability of 40–70 ASF and 40–70 PGA to support polypeptide synthesis in the absence and presence of eIF-2. The standard reaction mixture contained 5 pmol of STNV RNA and 40–70 ASF (A) or 40–70 PGA (B) as indicated in the absence (l) or presence (s) of 3 mg eIF2. (C) The standard reaction mixture contained 10 pmol of STNV RNA, 6 ml of 40–70 PGA, and eIF-2 as indicated.
70 PGA and eIF-2 was only about 50% of that obtained with untreated 40–70 ASF. It is not known whether the 40–70 PGA contains a toxic component (i.e., a component that leached from the protein G column) or whether a stimulatory factor is also removed by the protein GreIF-2 antibody column. A typical response to eIF-2 obtained in the translation system containing 40–70 PGA and STNV RNA is shown in Fig. 1C. The concentrations of eIF-2 required for half-maximal translation (C0.5) for several mRNAs were determined as described under Materials and Methods and the results are given in Table 1. The C0.5 values for eIF4F determined as described previously (3) are given for comparison purposes. The concentration of eIF-2 required for half-maximal translation of the five
TABLE 1
Comparison of the Concentrations of eIF-2 and eIF-4F Required for Half-Maximal Rate of Translation Concentration (Co.5)a (nM) mRNA
eIF-2b
eIF-4Fc
AMV4 STNV a-Amylase Avenin Globulin
60 60 90 60 90
6 4 17 3 7
a The concentrations required to obtain half-maximal rate of translation were determined as described (3,4), assuming the following Mr: eIF-2 (130,000) and eIF-4F (330,000). b The reaction mixture contained 6 ml of the 40–70 PGA and 5 pmol of the indicated mRNA. c The reaction mixture contained 5 ml of 40–70 ASF and 5 pmol of the indicated mRNA.
/ m4485$$nat
11-06-95 11:53:28
abnt
mRNAs tested differed by no more than 1.5-fold. We have also previously reported the amounts of eIF-2 required for the translation of rabbit a- and b-globin mRNA to be 49 and 44 nM, respectively (5). The concentration of eIF-2 present in a wheat germ S30 translation system, estimated by ELISA, is Ç400 nM (13). This amount of eIF-2 is 4-fold higher than the highest concentration of eIF-2 required for half-maximal translation of the mRNAs tested. In contrast, the concentration of eIF-4F required for half-maximal translation of these mRNAs differed by as much as 7-fold. The concentration of eIF-4F in a wheat germ S30 translation system is only Ç20 nM which is about the same concentration required for half-maximal translation of barley a-amylase and therefore could be rate-limiting for this particular mRNA. The method described here, removing a specific protein from a crude extract using an antibody affinity column, allows the development of an assay dependent upon a single component of a complicated, multicomponent enzyme system (e.g., protein synthesis). This method may be applied to other multicomponent systems where removal of a single component would be advantageous. The eIF-2-dependent translation system described in this paper was used to measure the amount of eIF-2 required for the initiation of several mRNAs. These preliminary findings strongly suggest that the concentration of eIF-2 is not rate-limiting in wheat germ and does not play a role in the preferential initiation of the mRNAs tested. Acknowledgments. We thank Sandra Lax and Anneke Metz for their excellent technical assistance. This work supported by a grant from the National Science Foundation to J.M.R. and K.S.B. (DMB9105487).
AP-Anal Bio
NOTES & TIPS
REFERENCES 1. Merrick, W. C. (1994) Biochimie 76, 822–830. 2. Timmer, R. T., Lax, S. R., Hughes, D. L., Merrick, W. C., Ravel, J. M., and Browning, K. S. (1993) J. Biol. Chem. 268, 24863– 24867. 3. Fletcher, L., Corbin, S. D., Browning, K. S., and Ravel, J. M. (1990) J. Biol. Chem. 265, 19582–19587. 4. Timmer, R. T., Benkowski, L. A., Schodin, D., Lax, S. R., Metz, A. M., Ravel, J. M., and Browning, K. S. (1993) J. Biol. Chem. 268, 9504–9510. 5. Timmer, R. T., Benkowski, L. A., Ravel, J. M., and Browning, K. S. (1995) Biochem. Biophys. Res. Commun. 210, 370–377. 6. DiSegni, G., Rosen, H., and Kaempfer, R. (1979) Biochemistry 18, 2847–2854. 7. Rosen, H., DiSegni, G., and Kaempfer, R. (1982) J. Biol. Chem. 257, 946–952. 8. Scheper, G. C., Thomas, A. A. M., and Voorma, H. O. (1991) Biochim. Biophys. Acta 1089, 220–226. 9. Crystal, R. G., Elson, N. A., and Anderson, W. F. (1974) Methods Enzymol. 30, 101–127. 10. Lax, S. R., Lauer, S. J., Browning, K. S., and Ravel, J. M. (1986) Methods Enzymol. 118, 109–128. 11. Browning, K. S., Maia, D. M., Lax, S. R., and Ravel, J. M. (1987) J. Biol. Chem. 262, 538–541. 12. Browning, K. S., Webster, C., Roberts, J. K. N., and Ravel, J. M. (1992) J. Biol. Chem. 267, 10096–10100. 13. Browning, K. S., Humphreys, J., Hobbs, W., Smith, G. B., and Ravel, J. M. (1990) J. Biol. Chem. 265, 17967–17973. 14. Browning, K. S., Lax, S. R., Humphreys, J., Ravel, J. M., Jobling, S. A., and Gehrke, L. (1988) J. Biol. Chem. 263, 9630–9634. 15. Boyer, S. K., Shotwell, M. A., and Larkins, B. A. (1992) J. Biol. Chem. 267, 17449–17457.
A Reagent for both Visualization and Chemiluminescence Detection of Horseradish Peroxidase-Tagged DNA Probes Bibijana Cercek, Keith Roby, and Martin Siaw Diagnostic Systems Group, Beckman Instruments, Inc., Brea, California 92621 Received August 15, 1995
Although chemiluminescent detection provides a rapid and highly sensitive means for quantitation of DNA probes, the signal cannot be measured again, and unless photographed, no permanent record remains (1, 4). Hence, visualization by staining of DNA probe bands would provide a useful adjunct to such a method especially for application in medical diagnostics (2). We describe the application of a borax buffer-based signal reagent, originally developed for horseradish ANALYTICAL BIOCHEMISTRY
peroxidase (HRP)1-enhanced chemiluminescence measurement (1, 3, 4), to the detection of streptavidinlinked HRP tagged with biotinylated DNA probes on positively charged and neutral nylon membranes by direct visualization. This signal reagent also provides the option of chemiluminescence detection, generally measured within the first 60 s, is retained (1, 4). Experimental conditions and the formulation of the signal reagent were as described previously (1). The signal reagent based on borax buffer was evaluated since it was previously shown (4) that the borax buffer based formulation gives greatly improved, very low, reagent background signals at the detection limit of the chemiluminometer and optimal signal to background ratios, as well as rapid signal rise to steady-state and prolonged steady-state signal emission when compared to all the many other buffers investigated in HRP paraiodophenol-enhanced chemiluminescence measurement (4). As can be seen in Table 1, the method allows a detection limit of 11 ng biotinylated DNA/slot blot and up to 1 mg biotinylated DNA was slot blotted and readily visualized. Visualization at the detection limit is possible within less than 1 min with the 101 signal reagent. However, the colored stain lasts only up to 24 h. Using the 11 signal reagent, visualization is possible within 10 to 15 min after signal reagent addition, and the yellow–brown color of the stained bands is stable for at least 4 weeks at room temperature in the dark. The background was unstained and colorless, under all conditions investigated, even 4 weeks after exposure to the signal reagent. No difference in either intensity or time to full color development was observed using either the Tropix negatively charged or the Millipore positively charged nylon membranes for slot blotting the biotinylated DNA. By a process of component elimination (Table 1), it is evident that luminol is not requisite for rapid, specific color development and acceptable detection sensitivity. Although omission of the para-iodophenol enhancer from the signal reagent adversely affects both the color development time and the detection limits, excluding perborate or streptavidin–HRP totally abolishes the color development reaction. The absolute requirement for perborate and HRP suggests a free radical mediated reaction mechanism, possibly analogous to that of the multi-step chemiluminescenceenhanced reaction itself (3). The effect of enhancers other than para-iodophenol on the color and/or intensity of the deposited colored product has yet to be determined and would be of interest in establishing the role of the enhancer in the color development process. Furthermore, the nature of the colored product deposited is at present unknown. However, thermal or photochemical degradation of luminol solutions results in a 1
Abbreviation used: HRP, horseradish peroxidase.
232, 143–144 (1995)
0003-2697/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ m4485$$nat
11-06-95 11:53:28
abnt
143
AP-Anal Bio