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108 _

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The wheat germ extract in vitro translation system has been used widely for faithfuland efficient translation of viral and eukaryotic messenger RNAs in a heterologouscell-free system (1-9). With respect to the yield oftranslation products, the wheat germextract is less efficient than most reticulocyte lysate cell-free systems (see Chapters106, 107, and 109). There are advantages, however, of using wheat germ extracts:

1. The in vivo competition of mRNAs for translation is more accurately represented, makingthe wheat germ system preferable for studying regulation of translation (1).

2. Particularly low levels of endogenous mRNA and the endogenous nuclease activityobviate the requirement for treatment with a calcium-activated nuclease. There is, therefore, less disruption of the in vivo situation and contamination with calcium ions is lessharmful. The identification of all sizes of exogenous mRNA-directed translation productsis facilitated because of the low levels of endogenous mRNA present.

3. There is no posttranslational modification of translation products; primary products aretherefore investigated, although processing may be achieved by the addition of microsomal membranes to the translation reaction.

4. The ionic conditions of the reaction may be altered to optimize the translation of large orsmall RNAs (2) Note

Translational activity is optimized by the incorporation of an energy-generating system of ATP, GTP, creatine phosphate, and creatine kinase (3). Wheat germ is inexpensive and commercially available Note 2); preparation of the extract is rapid andsimple, resulting in high yields. Wheat germ extract cell-free system kits are also commercially available.

.Components of the wheat germ in vitro translation system are heat-labile and mustbe stored in aliquots of convenient volumes at -70C. Freeze-thaw cycles must beminimized. Sterile techniques are used throughout. RNase contamination is preventedby heat-sterilization (250C, 8 h) of glassware and tips, and so on, or by diethylpyrocarbonate treatment of glassware, followed by thorough rinsing of equipment insterile distilled water.

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8921. Wheat germ extract: This is prepared essentially as described by Roberts and Paterson (4).The procedure must be carried out at 4C, preferably in plastic containers since initiationfactors stick to glass. Fresh wheat germ (approx 5 g) Note 2) is ground with an equalweight of sand and 28 mL of 20 HEPES, pH 7.6, 100 KCl, 1 magnesiumacetate, 2 CaCI and 6 2-mercaptoethanol, added gradually. This mixture is thencentrifuged at for 10 min at 2C, pH 6.5. This pH prevents the release of endogenous mRNA from polysomes and therefore removes the requirement for a preincubationto allow polysome formation (4,5). The supernatant (S-28) is then separated from endogenous amino acids and plant pigments that are inhibitory to translation, by chromatographythrough Sephadex G-25 (coarse) in 20 HEPES, pH 7.6, 120 KCl, 5 magnesiumacetate, and 6 2-mercaptoethanol. Reverse chromatography will prevent the loss ofamino acids. Fractions of more than 20 A260 nm/mL are pooled before being storedin aliquots at a concentration of approx 100 A260 nrn/mL, at -70C. The extract remainstranslationally active for a year or more.

2. L-[3H]- or L-[35S]-amino acids: 10-50 flCi of an appropriate amino acid (abundant in theprotein[s] of interest) is added to the reaction to allow detection of translation products.Convenient specific activities are 140 Ci/mmol tritiated, or 1 Ci/mmol [35S]-amino acids,respectively Note 3). Aqueous solutions should be used since ethanol, salts, detergents, and various solvents interfere with translation. Ethanol should be removed by lyophilization and the effects on translation of other solutions should be determined prior totheir use. [35S]-labeled amino acids must be stored in small aliquots at -70C where theyremain stable for up to 6 mo, after which time sulfoxide products of degradation inhibittranslation.

3. Messenger RNA: The extraction of both total and polyadenylated RNA has been describedby a number of authors Total RNA (1.5 or 150 polyadenylatedRNA (in sterile distilled water) are convenient stock concentrations. RNA is stable formore than a year at -70C. Contamination with potassium Note phenol, and ethanol must be prevented by 70% (v/v) ethanol washes, chloroform:butanol (4: 1) extractions,and lyophilization respectively.4. lOX energy mix: 10 ATP, 200 GTP, 80 creatine phosphate. Potassium saltsof the nucleotide triphosphates should be used and the final pH adjusted (if necessary) to7.4-7.6 with sodium hydroxide.

5. 0.5-1.0 M potassium acetate Note 25 magnesium acetate.6. 20 dithiothreitol.7. 0.6-1.2 spermine or 4.0-8.0 spermidine Note 4).8. 0.2 M HEPES, pH 7.4-7.6 Note 5).9. 200-500 creatine kinase Note 6).

All preparations are carried out on ice. After use, components are quick-frozen on dryice. Reactions are carried out in sterile plastic microfuge tubes.

1. Mix the following solutions (all components are ~ ) 5 of energy mix, 5 ofpotassium and magnesium acetate, 5 of dithiothreitol, 5 of HEPES, 5 of spermine, 10 of 0.3-8.0 mRNA in dH Note 7), 10 of wheat germ extract,10 of creatine kinase (0.8-1.0 A260 U), and 5 of creatine kinase. If a number ofincubations are to be made, a master mix of the first five solutions may be prepared and25 aliquoted into each reaction tube. Creatine kinase is added last to ensure that noenergy is wasted. The solutions are mixed by tapping the tube or by gentle vortexing.Microsomal membranes (0.5 A260 U) may be added before the creatine kinase to detectcotranslational modification of translation products Note 8).

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8932. Incubate at 28C for 1h Note 9). The reaction is terminated by placing the tubes at 4C.3. Incorporation of radioactive amino acids into mRNA-derived translation products isdetected by TCA precipitation of an aliquot ofthe reaction Chapter 107 and NoteIncorporation of radioactivity into translation products is generally not as well-stimulatedby mRNA added to wheat germ extracts as it is in described reticulocyte lysates.4. The remaining in vitro translation products may be analyzed further by standard techniques, including tryptic mapping and ion-exchange chromatography, but specific products may be analyzed by immunoprecipitation followed by SDS-polyacrylamide gelelectrophoresis.

1. Wheat germ extract translational activity is particularly sensitive to variation in the concentration of potassium ions. At concentrations lower than 70 small mRNAs arepreferentially translated, whereas larger mRNAs are translated at potassium acetate concentrations of 70 or greater (2,5). Polypeptides of up to 200 kDa are synthesizedunder correct ionic conditions (9). Furthermore, chloride ions appear to inhibit translationsuch that potassium acetate should preferably be used (5). In this context, residual potassium should be removed from RNA preparations, by 70% (v/v) ethanol washes.

2. Inherent translational activity varies with the batch of wheat germ. Israeli mills (for example,"Bar-Rav" Mill, Tel Aviv) supply wheat germ, the extracts of which are usually active.

3. Most of the endogenous amino acids are removed by chromatography through SephadexG-25 (coarse) Chapter 31). Depending on the batch of wheat germ extract, addition ofamino acids (to 25 and/or tRNA (to 58 /lg/mL) may be necessary to optimize translational activity. Wheat germ extract is particularly sensitive to amino acid starvation; useof radioactive amino acids at specific activities greater than those suggested may result ininhibition of translation because of amino acid starvation.

4. The use of either spermine or spermidine generally stimulates translation, and is essentialfor the synthesis of larger polypeptides (5), probably by stabilizing longer mRNAs. Omission of either compound will increase the optimum magnesium acetate concentration to4.0-4.3

5. HEPES has been shown to buffer the wheat germ extract in vitro translation system moreeffectively than Tris-acetate (4). Use of the latter will alter the optimum potassium andmagnesium concentration.6. Commercial preparations of creatine kinase differ with respect to the levels of nucleasecontamination. This must be considered when larger amounts of the enzyme are to be used.

7. Heating of large mRNAs at 70C for I min followed by rapid cooling on ice increases theefficiency of their translation in wheat germ extract in vitro translation systems.8. Cotranslational processing of translation products may be detected by the addition of dogpancreas microsomal membranes to the translation incubation. They may be prepared asdescribed by Jackson and Blobel (12) or may be ordered with a commercial translation kit.Microsomal membranes should be stored in aliquots of approx 5 A260 nmU in 20 HEPES,pH 7.5, at -70C. Repeated freezing and thawing must be avoided.

9. mRNA-stimulated incorporation of radioactive amino acids into translation products islinear, after a 5 min lag, for 50 min and is complete after 90 min. The system is labile attemperatures >30C; optimum activity is achieved at 25-30C depending on the batch ofwheat germ extract. An incubation temperature of 28C is generally used.10. In order to obtain maximum translational activity, it is necessary to determine the optimafor each preparation of wheat germ extract; mRNA concentration, potassium and magnesium concentrations, and incubation temperature. Take into account the concentration ofsalts in the wheat germ extract column eluate.

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8941. Steward, A. G., Lloyd, M., and Arnstein, H. R. V. (1977) Maintenance of the ratio of

ex and globin synthesis in rabbit reticulocytes. Eur. Biochem. 80,453-459.2. Benveniste, K., Wilczek, J., Ruggieri, and Stern, R. (1976) Translation of collagenmessenger RNA in a system derived from wheat germ. Biochemistry 15, 830-835.3. Huntner, A. R., Farrell, P. J., Jackson, R. J., and Hunt, T. (1977) The role of polyaminesin cell-free protein in the wheat germ system. Eur. Biochem. 75, 149-157.

4. Roberts, B. E. and Paterson, B. M. (1973) Efficient translation of tobacco mosaic virusRNA and rabbit globin 9S RNA in a cell-free system from commercial wheat germ.Proc. Natl. Acad. Sci. USA 70, 2330-2334.

5. Davies, J. W., Aalbers, M. J., Stuik, E. J., and van Kammen, (1977) Translation ofcowpea mosaic RNA in cell-free extract from wheat germ. FEBS Lett. 77,265-269.

6. Boedtker, H., Frischauf, M., and Lehrach, H. (1976) Isolation and translation of calvaria procollagen messenger ribonucleic acids. Biochemistry 15, 4765-4770.

7. Patrinou-Georgoulas, M. and John, H. A. (1977) The genes and mRNA coding for thetheory chains of chick embryonic skeletal myosin. Cell 12, 491-499.8. Larkins, B. Jones, R. and Tsai, C. Y. (1976) Isolation and in vitro translation ofzein messenger ribonucleic acid. Biochemistry 15, 5506-5511.

9. Schroder, J., Betz, B., and Hahlbrock, (1976) Light-induced enzyme synthesis in cellsuspension cultures of petroselinum. Eur. Biochem. 67,527-541.

10. Pelham, H. R. B. and Jackson, R. J. (1976) An efficient mRNA-dependent translationsystem from reticulocyte lysates. Eur. Biochem. 67,247-256.

11. Darnbrough, C. H., Legon, S., Hunt, T., and Jackson, R. J. (1973) Initiation of proteinsynthesis: evidence for messenger RNA-independent binding ofmethionyl-transfer RNAto the 40S ribosomal subunit. Mol. Bioi. 76,379-403.12. Jackson, R. C. and Blobel, G. (1977) Post-translational cleavage of presecretory proteinswith an extract of rough microsomes, from dog pancreas, with signal peptidase activity.Proc. Natl. Acad. Sci. USA 74, 5598-5602.