Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
  • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
  • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

• the present invention relates to a cell-free translation system.
• the invention relates to a cell-free translation system derived from cell extracts, that reproduces the synergism that occurs during translation in vivo between the cap and the poly (A) tail structures of the mRNA.
• Translational control is an important mechanism for the regulation of gene expression during a variety of biological processes, including cellular metabolism (Hentze & Kuhn, (1996) P.N.A.S. USA 93: 8175-8182), cell differentiation (Ostareck et al., 1997, Cell. 89: 597-606) and embryonic development (Wickens et al., 1996, in Translation Control, Eds. Hershey, J.W.B., Mathews, M.B. and Sonenberg, N. (Cold Spring Harbour Lab. Press, Plainview, NY, pp. 411-450). Failure to regulate translation properly often leads to disease.
• a cell-free translation system derived from animal cells that is capable of reproducing this synergism would be of great value for a number of reasons. Firstly, this would facilitate and enhance the validity of basic research into the factors and mechanisms involved in translational control in higher eukaryotes, including the regulation of translation by cis- and trans- acting factors, the molecular basis of synergism, mRNA stability and polyadenylation. Second, species-specific differences in translational mechanisms could be evaluated. Such a system would also allow the improvement of large-scale protein synthesis, both with respect to the efficiency of translation and to the fidelity of post-translational modification.
• a method for the in vitro translation of a ribonucleic acid template comprising incubating a cell extract of a multicellular eukaryote with said ribonucleic acid template under conditions such that translation of the RNA template to produce its encoded protein by one or more components in the cell extract occurs and the amount of the encoded protein thus produced is greater than the total of (a) the amount of the encoded protein that is produced under said conditions when the ribonucleic acid template has a 5' cap but no 3' poly A tail, plus (b) the amount of the encoded protein that is produced under said conditions when the ribonucleic acid template has a 3' poly A tail but no 5' cap.
• the cell extract may be an animal cell extract, such as a mammalian cell, or an insect cell. Specific examples include cell extracts of Drosophila, C. elegans, rodent, rabbit (for example, reticulocyte cells), human (for example HeLa cells) and primate cells.
• the cells may be embryonic or adult or from a particular tissue such as the ovaries.
• the cell extract is a Drosophila cell extract, most preferably a Drosophila embryo or ovary cell extract.
• the extract exhibits properties that were previously unforeseen, such as the capability to reproduce the synergism that occurs during translation in vivo between the cap and the poly(A) tail structures of an mRNA molecule, and the capability to perform mRNA polyadenylation.
• the procedure is simple and allows the preparation of large amounts of extract at low cost. This makes the system useful in both basic research and in commercial applications as is discussed in more detail below.
• RNA templates suitable for use in the methods of in vitro translation of the invention may be recombinant or can be purified native templates.
• the poly(A) tail of the RNA template can be encoded in a DNA coding sequence which can be transcribed to generate an RNA template with a poly(A) tail of defined length.
• An alternative method of generating the poly(A) tail is the use of a poly(A) polymerase in an in vitro reaction to add the tail to the template as a post- transcriptional modification.
• the 5' cap portion may be added co-transcriptionally to the RNA template by the RNA polymerase.
• a suitable protocol can be found in "Protocol and Applications Guide”; Promega, 2nd edition, p62. If the template is a purified native RNA template, the cap structure will already be in place.
• a method for the preparation of a Drosophila embryo extract comprising the steps of a) dechorionating Drosophila embryos in an aqueous isotonic buffer comprising detergent and bleach; b) washing the embryos; c) homogenizing the embryos to produce a homogenate; d) centrifuging the homogenate; and e) recovering non-pelleted material from the centrifuged homogenate.
• This cell extract is particularly suited for use in the method of the first aspect of the invention.
• the age of the embryo is not crucial to the method of the invention, although embryos of between the ages of 1 hour and 12 hours of development are preferred.
• the embryos Before preparation of extract, the embryos should preferably be collected in a sieve and washed extensively with water to remove debris from the culture medium in which they have been laid.
• the embryos For preparation of extract, the embryos are washed with an aqueous buffer, preferably with fresh EW buffer (0.7% NaCl, 0.04% Triton X-100), and poured into a suitable vessel. An excess of buffer is then added and the embryos allowed to settle. At this stage, floating embryos should be discarded.
• fresh buffer For dechorionation, fresh buffer should be added and the embryo suspension agitated, preferably using a spinning magnet or other suitable device. In all steps for the preparation of cell extract, ethanol- based buffers should be avoided.
• bleach is added to the embryo suspension, preferably at a concentration of about 3% by volume and the solution incubated for an appropriate time. Preferably, incubation is for between 1 and 9 minutes, most preferably 3 minutes.
• Dechorionation may be carried out at a temperature of between 18 and 37°C. Preferably, dechorionation is carried out at about 25°C.
• embryos are preferably transferred to a sieve and washed extensively with water. Dechorionated embryos are then transferred to a suitable vessel (such as a beaker or cylinder) and buffer such as DE buffer (lOmM HEPES pH 7.4, 5mM DTT) added. Floating embryos are discarded. Preferably, about 1 volume (with respect to the volume of settled embryos) of fresh buffer containing protease inhibitors is added to prevent proteolysis of components of the extract.
• a suitable vessel such as a beaker or cylinder
• buffer such as DE buffer (lOmM HEPES pH 7.4, 5mM DTT) added. Floating embryos are discarded.
• Floating embryos are discarded.
• about 1 volume (with respect to the volume of settled embryos) of fresh buffer containing protease inhibitors is added to prevent proteolysis of components of the extract.
• Embryos are then homogenised.
• homogenisation is carried out at about 4°C.
• a Potter-Elvehjem homogeniser is used at about 1500 rpm (about 20 strokes).
• the homogenate should preferably be kept on ice to minimise proteolysis.
• the homogenate is preferably centrifuged, ideally in an ultracentrifuge at 24,000 rpm (40,000g)/TLS-55 rotor/4°C/20 minutes.
• the clear interphase is taken, ideally by puncturing the tube with a syringe, and is transferred to a Falcon tube.
• Glycerol may then be added to the extract, preferably to a 10% final concentration, and the extract aliquoted, frozen in liquid nitrogen and kept at -70°C for future use.
• Drosophila ovaries may also be used to produce a cell extract.
• the procedure is as follows: adult female flies are manually dissected to obtain the ovaries, which should be placed in an eppendorf tube on ice in an aqueous medium, for example PBS. Ovaries are then allowed to settle, before transferring the ovaries slowly from an isotonic medium to a hypotonic medium.
• the invention also provides a method for the preparation of a mammalian cell extract comprising the steps of a) collecting cells by centrifugation; b) washing the cells; c) resuspending the cells; d) homogenising the cells to produce a homogenate; e) centrifuging the homogenate and f) recovering non-pelleted material from the centrifuged homogenate.
• This cell extract is well-suited to use in the method of the first aspect of the invention.
• the cells Before preparation of extract, the cells should be grown in culture, preferably at exponential growth. Suspension cultures grown in, for example Jocklik's medium supplemented with 5% newborn bovine serum at 37°C, are suitable. The cells may be collected from culture by centrifugation, for example, by harvesting at 700g for 15 minutes. For preparation of extract, the cells should be washed with phosphate buffered saline at 4°C, before resuspension in a suitable ice cold hypotonic buffer such as a buffer containing containing Hepes 10 mM. PH 7.6. KOAc 10 mM. Mg(OAc) 2 0.5 mM and Dithiothreitol 5 mM. Reduced amounts of DTT are not recommended and addition of protease inhibitors is optional.
• the cells are homogenised.
• homogenisation is carried out at about 4°C.
• the cells are homogenised using between 10 and 30 strokes of a Dounce homogeniser (pestle type B).
• the homogenate should preferably be kept on ice to avoid proteolysis.
• the homogenate is then centrifuged, ideally at 14000g for 5 minutes at 4°C. A longer centrifugation is not recommended. Supernatant fluid is divided into aliquots frozen in liquid nitrogen and stored at -80° C. Addition of glycerol is not necessary.
• the extract of Drosophila embryos or ovaries, or of mammalian cells produced according to the methods of the second aspect of the invention.
• a method for the in vitro translation of a ribonucleic acid template comprising the steps of adding a ribonucleic acid template to a translation mix in the presence of the embryo, ovary or mammalian cell extract of the third aspect of the invention to form a reaction mix and incubating the reaction mix for at least 90 minutes at between 18°C and 28°C.
• the reaction mix is incubated for at least 90 minutes at 25°C.
• reaction mix a solution of components necessary for in vitro translation to occur.
• Such components may include any of the following: spermidine, amino acids, creatine phosphate, creatine kinase, dithiothreitol (DTT), buffer, Mg(OAc) 2 , KOAc, tRNA, cell extract and RNA template.
• DTT dithiothreitol
• Mg(OAc) 2 Mg(OAc) 2
• KOAc tRNA
• cell extract RNA template
• the buffer is HEPES buffer, although any buffer with a low concentration of salt (below lOmM) may be used.
• HEPES buffer is most preferably used at a final concentration of between 16 and 24mM.
• final concentration means the concentration of the component that is present in the in vitro translation mix. 5 These concentrations are optimal for the translation reaction to take place.
• said tRNA is calf liver tRNA, at a final concentration of below 150 ⁇ g/ml, most preferably about lOO ⁇ g/ml.
• the final concentration of spermidine is preferably about 0.1 mM.
• final concentration is about 60 ⁇ M.
• concentration can be in the range 17-23mM. It is shown herein (see Figure 6) that it is preferable to use fresh or newly thawed creatine phosphate. The level of translation doubles when fresh creatine phosphate is used. Creatine kinase should be used at a concentration of about 0.08mg/ml.
• concentrations of DTT, Mg(OAc) 2 and KOAc should be optimised for each !5 individual mRNA template, as will be appreciated by those skilled in the art.
• ideal concentrations are DTT, 1.2mM; Mg(OAc) 2 , 0.6mM; and KOAc, 60mM.
• ideal concentrations are DTT, OmM; Mg(OAc) 2 , 0.4mM; and KOAc, 30mM.
• final concentration of Mg(OAc) 2 can be in the range 0-3mM.
• KOAc can range between 0 and 200mM.
• DTT can range between 0-4mM.
• Embryo or ovary extract should preferably be used at a concentration of about 40% by volume.
• the final concentration of spermidine is preferably about 0.1 mM.
• final concentration is about lOO ⁇ M.
• creatine phosphate the concentration can be in the range 17-23mM.
• Creatine kinase should be used at a concentration of about 0.05mg/ml.
• concentrations of Mg(OAc) 2 and KOAc should be optimised for each individual mRNA template, as will be appreciated by those skilled in the art. For example, suitable concentrations may be along the following lines; Mg(OAc) 2 , 2.5mM; KOAc, 50mM.
• final concentration of Mg(OAc) can be in the range 0-3mM.
• KOAc can range between 0 and 200mM.
• DTT can range between 0-4mM.
• Mammalian cell extract should preferably be used at a concentration of about 40% by volume.
• RNA template concentration will vary depending upon the RNA type. The skilled artisan will appreciate that optimisation of the system may be easily performed for a particular RNA species of choice using routine procedures. For example, for the species c-luc-a (coding for firefly luciferase) a concentration of about 3.2ng/ ⁇ l is optimal using Drosophila embryo extract.
• the mRNA template should preferably be capped and should contain a poly (A) tail of more than 31 adenine nucleotides (more preferably 70-100 adenines).
• test reactions were performed with M1414WT (bgal) [this codes for the ⁇ -galactosidase enzyme and contains 5' and 3' UTR sequences from Drosophila oskar mRNA], c-Luc-a and Luc mRNA (Promega).
• Reaction mix prepared in this fashion contributes to the advantageous features of the method of this aspect of the invention, namely reproducing the synergy between the 5' cap and 3' poly (A) tail of the RNA molecule for translation.
• yeasts do possess enzymes that are capable of effecting the post-translational modification of proteins, the type and extent of modification tends to differ from that found in higher eukaryotes. Consequently, the Drosophila system of the present invention generates proteins modified similarly to the state in which they are found in vivo.
• the incubation step is preferably performed for sufficient time to allow the translation reaction to proceed to the extent desired. Preferably, the incubation step is performed for at least about 90 minutes.
• the in vitro translation reaction is performed in a final volume of about 12.5 microlitres.
• Table 2 the volume of stock concentrations is given in order to make up a reaction volume of 12.5 microlitres.
• reaction mix For HeLa cell extract, the reaction mix may be as follows:
• a fifth aspect of the invention there is provided the use of cell extract according to the third aspect of the invention in a method of in vitro translation of a ribonucleic acid template, such as the methods of the first and fourth aspects of the invention.
• the method reproduces the synergism exhibited in vivo between the 5' cap and 3' poly (A) tail of the ribonucleic acid template.
• cell extract according to the third aspect of the invention or method of first or the fourth aspects of the invention to screen for compounds that either a) decrease the translation, polyadenylation and/or stability of all mRNA species, or of specific mRNAs; b) potentiate the translation, polyadenylation and/or stability of all mRNA species, or of specific mRNAs; or c) decrease or potentiate the synergism between the 5' cap and the 3' poly(A) tail of the mRNA.
• Suitable compounds may interact (either directly or indirectly) with only one of the 5' cap or poly(A) structures.
• compounds identified in such a screen have an effect on translational control in higher eukaryotes, in particular insects.
• This screening method has advantages over prior art methods because the synergy exhibited between cap and poly(A) structures mimics the features of in vivo translation and allows the screening in an in vitro system for modifiers effecting translation that are significantly more likely to function in vivo.
• Suitable compounds may be natural or synthetic, for example, an aptamer, or a peptide and may be useful for therapy of diseases and for the control of plagues and diseases caused by insects, for example malaria and plant pestilence.
• the compound may that interact with 5' cap or 3' poly (A) structures of an mRNA template or affect in some way the synergism between the 5' cap and 3' poly (A) tail of an mRNA template.
• compounds may be specific for a certain group of mRNA templates, or for mRNA templates within a certain species.
• an eighth aspect of the invention there is provided the use of the cell extract according to the third aspect of the invention or method of the first or fourth aspects of the invention in research into the regulation and function of post- transcriptional mRNA modification or post-translational protein modification.
• kits for the analysis of in vitro translation, polyadenylation and/or stability of ribonucleic acid should contain cell extract according to the third aspect of the invention along with stock solutions of all the other components of the translation mix in appropriate quantities.
• the kit will therefore in addition comprise any one or all of spermidine, amino acids, creatine phosphate, creatine kinase, optionally dithiothreitol (DTT), buffer, Mg(OAc) 2 , KOAc, tRNA, and RNA template.
• the buffer is HEPES buffer
• the tRNA is calf liver tRNA and ideally, the creatine phosphate is made fresh.
• creatine phosphate may be supplied in powdered form in the kit for dissolution in distilled water as and when required for use.
• an RNA template will not be supplied as part of the kit, since in the main part, it is envisaged that the kit will be purchased for the purposes of experimental research.
• a control RNA template may be included in the kit so that a user can ensure that the translation reaction is proceeding appropriately.
• comparison with a control RNA template may be used to optimise the concentration of test RNA, DTT, Mg(OAc) 2 and KOAc that is to be used.
• the kit preferably also contains appropriate instructions to enable a user to perform the in vitro translation reaction appropriately.
• Figure 1 shows comparative results of translation levels and levels of synergy found between 5' cap and 3' poly(A) tail of an RNA template.
• Figure 2 is an expanded scale of the results of Figure 1.
• Figure 3 shows optimisation of Mg 2+ concentration.
• Figure 4 shows optimisation of K + concentration.
• Figure 5 shows optimisation of creatine kinase concentration.
• Figure 6 shows how percentage translation is improved by using fresh creatine phosphate.
• Figure 7 shows optimisation of creatine phosphate concentration.
• Figure 8 shows optimisation of spermidine concentration.
• Figure 9 shows optimisation of tRNA concentration.
• Figure 10 shows optimisation of temperature, amino acid concentration and DTT concentration using c-luc-a RNA.
• Figure 11 shows the time course of translation of CAT mRNAs using the HeLa cell extract.
• Figure 12 shows Northern blot analysis of the transcripts during the translation assay. Bars represent radioactive intensity of each messenger measured by a phosphoimager.
• Graph (A) shows the four messengers at different times, from 0 to 150 minutes.
• Graph (B) shows the Cap and the Cap-pA transcripts from 0 to 90 min.
• Figure 13 shows time course of translation of the Luc messengers. Efficiency of translation is represented by light emission measured by a luminometer.
• Graph (B) shows the data represented in Graph (A) with the y axis maximum at 20000 luminescence instead of 550000.
• Extracts from lh30', 3h, 6h and 12h embryos were prepared with similar results.
• Embryos were laid by 2-3 day old adult Drosophila flies on agar-apple juice plates (2.9% Agar; 30% Apple Juice; 4.4% Rubensirup; 0.25% Nipagin). The plates, spread with some fly food (220ml deionized water, 1.4ml Propionic acid; 150g dry yeast), were left overnight for embryo laying).
• Embryos from 16 agar-apple juice plates were collected in a pile of sieves (the first with a cut-off size of an adult fly, the second with a cut-off size of a fly appendix [e.g.: a leg, a head or an antenna], the last with a cut-off size of a single embryo) and washed extensively (-5-10 minutes) with tap water to remove debris coming from the plates.
• embryos were subsequently washed with freshly prepared isotonic EW buffer (0.7% NaCl, 0.04% TritonX-100). To do this, embryos were transferred to a 500ml cylinder containing EW buffer, and were allowed to settle for -3-5 minutes (roughly the time needed for 90% of the embryos to pellet by gravity) and were then washed twice with 500ml EW Buffer. Floating embryos were eliminated by suction.
• EW buffer 0.7% NaCl, 0.04% TritonX-100
• Embryos were dechorionated in the 500 ml cylinder at room temperature ( ⁇ 20-25°C) with 260ml of EWB (0.7% NaCl, 0.04% TritonX-100, 3% Sodium Hypochlorite) for 3 minutes under vigorous agitation provided by a magnetic stirrer.
• EWB 0.7% NaCl, 0.04% TritonX-100, 3% Sodium Hypochlorite
• Sodium Hypochlorite was from Sigma, Thomas Chemikalien, or U.S. CHLOROX. No significant change in the translation efficiency of the extract was noted for different bleach types.
• Dechorionated embryos were transferred back to the sieves, and were vigorously and extensively washed with tap water (by flushing a strong stream of water for about 5-10 minutes).
• Washed embryos were settled by gravity twice with 100 ml of DE buffer (lOmM HEPES pH 7.4, 5mM DTT) in a 100ml cylinder. Floating embryos were discarded, and an equivalent of one volume (with respect to the settled embryos) of DEI buffer (lOmM HEPES pH 7.4, 5mM DTT, lx COMPLETE-Protease Inhibitors from Boehringer Mannheim cat# 1697498) was added.
• Embryos in DEI buffer were homogenised in a cold room ( ⁇ 4°C) by 20 strokes of a Potter-Elvehjem homogeniser at 1500 rpm and the homogenate was kept on ice.
• the homogenate was spun in a table-top ultracentrifuge (Beckmann) at 24000 rpm (40000x g) in a TLS-55 rotor at 4 C for 20 minutes.
• the clear aqueous interphase was taken by puncturing the tube with a syringe, and was transferred to a Falcon tube. Glycerol was added to 10% final, and the extract was aliquoted, flash-frozen in liquid nitrogen and stored at -70°C.
• reaction is performed in a final volume of 12.5 ⁇ l, containing the following mix (a):
• the reaction was incubated at 25°C for 90 min.
• a master mix should be prepared for as many samples as it is required.
• the mRNA template should be capped and should contain a poly(A) tail of more than 31 A's (usually 70-100 A's), although mRNAs without a poly(A) tail are also translated albeit at lower efficiency.
• a curve of translation with different amounts of RNA should be performed to use an amount in the linear range of translation.
• M1414WT RNA encodes for the ⁇ -galactosidase enzyme (bgal) and contains 5' and 3' UTR sequences from Drosophila oskar mRNA.
• c-Luc-a encodes for the firefly luciferase enzyme.
• mLuc-a encodes for the firefly luciferase enzyme, and contains the 5' UTR of Drosophila msl-2 mRNA.
• c-CAT-a encodes for the chloramphenicol acetyl transferase enzyme. All RNAs are capped and polyadenylated.
• Spermidine is from SIGMA, cat# S-0381.
• Amino acids were obtained from Promega (cat #L4461), or can be self-made from a SIGMA kit (cat# LAA-21).
• Creatine Kinase is from Boehringer Mannheim (cat# 126969). The stock solution is prepared at 10 mg/ml in 50% glycerol, 20 mM HEPES-KOH pH 7,4. Using this protocol, together with the extract described in Example l,high levels of translation and synergy can be obtained, compared to other methods (see Figure 1).
• Example 1 The method of Example 1 was followed (not that of Scott et al), and one concentration value was varied for each experiment. Luc RNA from Promega was used for these experiments, except where otherwise stated.
• Figure 3 shows optimisation of Mg concentration. Optimum concentration was found to be 0.3mM.
• Figure 4 shows optimisation of K + concentration. Optimum concentration was found to be 74mM for this system.
• Figure 5 shows optimisation of creatine kinase concentration. Optimum concentration was found to be 0.08mg/ml for this system.
• Figure 6 shows how % translation could be improved more than two times by using fresh creatine phosphate.
• Figure 7 shows optimisation of creatine phosphate concentration. Optimum concentration was found to be 23mM for this system.
• Figure 8 shows optimisation of spermidine concentration. Optimum concentration was found to be O.lmM for the c-luc-a RNA system.
• Figure 9 shows optimisation of tRNA concentration. Optimum concentration was found to be 1 OO ⁇ g/ml for the luc RNA system.
• Figure 10 shows optimisation of temperature, amino acid concentration and DTT concentration using c-luc-a RNA.
• Optimum temperature was found to be 25°C.
• Optimum amino acids concentration was found to be 60 ⁇ M.
• Optimum DTT concentration was found to be OmM.
• Drosophila embryo extracts were prepared in parallel either according to the method described in Scott et al. (1979) or to the method of the invention. These extracts were assayed for translation using either the conditions described in Scott et al. (1979) or the conditions described above.
• the Scott protovol consisted in dechorionating the emryos in a solution of 50% ethanol/50% chlorox for 1 m in under agitation, washing the embryos 5 times in PBS after dechorionation using a table-top centrifuge at top speed to obtain the embryo pellet and homogenising the embryo pellet in lOmM HEPES pH7.4, 6mM ⁇ -mercaptoethanol.
• the extracts obtained using this method were used in an in vitro translation reaction using either the conditions described in Example 2.1, those described by Scott et al (1979) or those described by Scott with an increased time of translation.
• the RNA used for translation encoded the firefly luciferase and contained either a cap (c), a poly(A) (a), or both (c-a).
• the results indicate that by using our extract in our in vitro translation conditions a high degree if translation and synergism was obtained. When the Scott extract was used under Scott's in vitro translation conditions, no synergism was obtained and a very low level of translation was observed.
• HeLa cells were maintained at exponential growth in suspension cultures at 37° C in Jocklik's Medium supplemented with 5% newborn bovine serum. Approximately 204 litres of cells at densities of 3-6 x 10 5 cells/ml were harvested by centrifugation at 700 g for 15 minutes and washed three times with phosphate buffered saline (PBS) at 4° C.
• PBS phosphate buffered saline
• Pelleted cells were resuspended in 1 volume of ice-cold hypotonic buffer containing Hepes 10 mM, pH 7.6; KOAc 10 mM; Mg(Oac) 2 0.5 mM; and Dithiothreitol 5 mM. Reduced amounts of DTT are not recommended and addition of protease inhibitors is optional.
• Incubation mixtures contain: untreated extract 40% of the final volume
• Reaction mixtures typically at a final volume of 12.5 ⁇ l were incubated at 37° C for 90 minutes.
• transcripts coding for the reporter enzymes CAT (Preiss. T. & Hentze. M. W. 1998 Nature 392:516-520) and firefly luciferase. Luc, (Iizuka. N. et al. 1994 Mol. Cell. Biol. 14:7322-7330) were tested in the translation assay and the functional half life of each transcript has been determined to be as follows: - 5' capped (Cap, m7Gppp) 40 minutes - 5' capped and polyadenylated (pA, 98 adenines) 55 minutes
• Figure 8 shows a northern blot analysis of the transcripts during the translation assay. Bars represent radioactive intensity of each messenger measured by a phosphoimager. Graph (A) shows the four messengers at different times, from 0 to 150 min. Graph (B) shows the Cap and the Cap-pA transcripts from 0 to 90 min.
• Figure 9 shows the time course of translation of the Luc messengers. Efficiency of translation is represented by light emission measured by a luminometer.
• Graph (B) shows the data represented in graph (A) with the y axis maximum at 20000 luminescence instead of 550000.

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