Preparation of a cell-free translation system from PC12 cells

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Neurochemical

Research,

Vo/. 21, No. 7, 1996, pp. 801-807

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Preparation of a Cell-Free Translation System from PC12 Cells* Makoto Shibutani,! Eugene Kim,! Philip Lazarovici,l Mari Oshima,! and Gordon Gurofp,2

(Accepted January

9, 1996)

The postmitochondrial fraction (S10) contains the cellular components essential for translation, and a high-salt wash (HSW) of the ribosomes is enriched in eukaryotic initiation factors. This report describes the preparation of a cell-free translation system utilizing an SI 0 extract from PC 12 cells. The products synthesized from either firefly luciferase mRNA or PC12 cell poly(A) RNAs in the PCll-SlO extract were increased by the addition of the HSW from PC12 cells. Increases in the translation of luciferase mRNA by the addition of PCI2-HSW were dose-dependent and also dependent on the time of incubation. The translation of human epidermal growth factor receptor (hEGFR) mRNA could also be detected in the PCll-SlO extract translation system by immunoprecipitation. N-linked glycosylation of the translation products also was observed. The efficiency of translation was altered by the addition of Mg2+or K+, and optimization of the concentrations of these ions was necessary for each mRNA. The translation system made from PC12 cells, then, is capable of the synthesis of proteins of relatively high molecular weight and should be useful for analyzing mechanisms of translational control during proliferation and differentiation of cells from a neuronal lineage.

KEY WORDS:

PCI2; translation;

SIO; epidermal

INTRODUCTION

which interact with specific sequences and/or structures in the untranslated region of mRNAs (5). However, translational involvement in the process of neuronal differentiation has not been extensively studied. The nerve growth factor (NGF)-responsive rat pheochromocytoma cell line PC12 is a generally-accepted model system for the study of neuronal differentiation (6,7). Several NGF-induced phenomena in PC12 cells, i.e., the down-regulation of the epidermal growth factor receptor (EGFR) and of c-neu, the regeneration of neurites, appear to involve translational regulation (8-10). The rabbit reticulocyte lysate has been widely used

It has become increasingly clear that translational controls contribute to the overall regulation of cell growth, viral infection, and heat shock, possibly through modulation of the activity of translation factors and related proteins, i.e., eukaryotic initiation factors (eIFs), eukaryotic elongation factors (eEFs), ribosomal proteins (1--4). Furthermore, the translation of individual gene products is known to be regulated by repressor proteins I

Section on Growth Factors, National Human Development, land 20892.

Institute of Child Health and

National Institutes offIealth,

Bethesda, Mary-

2 Address reprint requests to: Or. Gordon Guroff, Section on Growth Factors, National Institute of Child Health and Human Development, National Institutes of Health, Building 49, Room 5A64, Bethesda, Maryland 20892. Telephone: (301) 496-4751; Fax: (301) 402-2079. * Special issue dedicated

growth factor receptor; luciferase.

Abbreviations:

HSW,

high-salt

wash;

hEGFR,

human

epidermal

growth factor receptor; NGF, nerve growth factor; SIO, postmitochondrial fraction; OTT, dithiothreitol; SOS, sodium dodecyl sulfate; elF, eukaryotic initiation factor; eEF, eukaryotic elongation factor

to Dr. Hans Thoenen.

801 0364-3190/96/0700-0801$09.50/0 @ 1996 Plenum Publishing Corporation

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Fig. 1. In vitro translation of luciferase mRNA and of PCI2-poly(A) RNAs in the rabbit reticulocyte lysate and in the PCI2-SI0 extract After incubation of the 12.S III translation mixtures at 30°C, 2.S III of translation products were re suspended in 12.S III of SDS sample buffer and the proteins were resolved on 7.5% SDS-polyacrylamide gels. A: Rabbit reticulocyte lysate. The translation mixture was incubated for I h with SOO ng of luciferase mRNA (lane 1) or SOOng of PCI2 cell poly(A) RNA (lane 2). The arrowhead shows a major band among the translation products migrating at 62 kDa. Band C: Effects of incubation period and HSW addition on the level of protein synthesis in a PC12 cell-S I 0 extract translation system. The incubation periods for translation were I h (B) and 18 h (C), respectively. Translation was performed by the addition of SOOng of luciferase mRNA (lanes 1 and 4) or of 500 ng ofpoly(A) RNAs (lanes 2 and 5). Lanes 3 and 6 were control incubations lacking mRNA. Translation mixtures in lanes 1-3 contained 20% volume of dialysis buffer and lanes 4-6 contained 20% dialyzed HSW (2.0 A26o/ml) from PC12 cells.

as a mammalian cell-free translation system because of its high efficiency. However, the postmitochondrial fractions (S10) of different cell lines have also been utilized for translation (11,12). Another preparation, the high-salt wash (HSW) of ribosomes is known to be rich in initiation factors (13). It would be useful to establish a translation system from PC12 cells for the investigation of the translational regulation associated with neuronal differentiation. In this study we describe such a cell-fi;ee translation system from PC12 cells and detail its ability to translate the mRNA for the human EGFR, an mRNA chosen for study because of the possibility that the down-regulation of the EGFR by NGF is under translational control (8).

min at 1,600 g and the mitochondrial fraction was subsequently removed by centrifugation for 20 min at 7,800 g. This S10 extract (90100 A26o1ml)was treated with micrococcal nuclease (Pharmacia) in the presence of calcium chloride at 20°C for 15 min, according to a previously published method (1S). The S10 extract was adjusted to 10% glycerol and stored in small portions at -70°C. Preparation

of High Salt Wash (HSW) from

PC12 Cells. The

isolation of HSW from PC12 cells was performed by the method of Morley and Hershey (13), modified to include the addition of 1 IlM okadaic acid to the hypotonic buffer. The SI 0 extract was centrifuged at 541,000 g for 20min at 4°C and the ribosomal pellet was suspended at a concentration of 22S-250 A26o1mlin hypotonic buffer. KCI was added to a final concentration of 0.5 M and the suspension was stirred for IS min at 4°C, Following centrifugation for 20 min at S41,000 g, the upper two-thirds of the supematant was removed and dialyzed for 4 h against 5 mM Tris, pH 7.5, 100 mM KCI, 0.05 mM EDTA, 1 mM DTT, and 5% glycerol. After dialysis, the preparation was treated with micrococcal nuclease as described above and frozen at -70°C in small portions.

EXPERIMENTAL PROCEDURE Preparation ofS10 Extractfrom PC12 Cells. An S10 extract from PC12 cells was prepared according to the procedure described by Molla et al. (14). PC12 cells were harvested and washed twice with Earle's balanced salt solution (GIBCO-BRL). The cell pellet was resuspended in 1.5 volumes of a hypotonic buffer (10 mM HEPESKOH, pH 7.4, 10 mM potassium acetate, 1.5 mM magnesium acetate, 2.S mM dithiothreitol (DTT), 1/1000 volume of protease inhibitor mixture). The protease inhibitor mixture contained 20 mM leupeptin, 200 mM aminoethylbenzenesulfonyl fluoride, 2 mg/ml aprotinin, IM benzamidine, and 1 mM pepstatin, all products of CalBiochem. Cells were kept on ice for 10 min and then disrupted at 4°C with 25 strokes in a Dounce homogenizer. Nuclei were removed by centrifugation for 5

RNA Preparation.

Poly(A) RNAs from PCI2 cells were isolated

using the FastTrack mRNA isolation kit (Invitrogen). Capped human epidermal growth factor receptor (hEGFR) mRNA was synthesized using the SP-6 promoter of plasmid pSPER-7, kindly provided by Dr. Glenn Merlino, as described by Clark et al. (16). Firefly luciferase mRNA (uncapped) was provided by Promega. In Vitro Translation. Rabbit reticulocyte translation incu6iiti~n mixtures contained 50% volume of nuclease-treateci reticulocyte lysate (Promega) and the following additions: 20 IlM amino acid mixture minus methionine, 0.8 mCi/ml L-[35S]-methionine (Amersham; 1200 Ci/mmol), 20 mM KCI, 0.8 units/ill RNasin ribonuclease inhibitor, 1/1000 volume of protease inhibitor mixture, and in vitro transcribed mRNAs or poly(A) RNAs from PCI2 cells. The total reaction volumes for the translation of the poly(A) RNAs, hEGFR mRNA, and luciferase mRNA in the

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Translation System from PC12 Cells

803

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sodium citrate, 50 mM sodium phosphate buffer, pH 6.5, 25 mM sodium chloride, 0.05% SDS, and 1/1000 volume of protease inhibitor mixture. After the addition of 20 mM EDT A, 1% j3-mercaptoethanol, and 0.5% N-octylglucoside, the incubation mixtures were treated with 0.4 U of N-glycosidase F from Flavobacterium meningosepticum (Boehringer-Mannheim, Gennany) for 3 h at 37°C. The total reaction

-116

- 97

volume of the glycosidase incubation was 15 111. Reactions were stopped by the addition of 2x SDS sample buffer. Apoferritin (Sigma; 5 I1g) was used as a positive control for the N-glycosidase treatment. Electrophoresis and Autoradiography. Either immunoprecipitates from the translation of hEGFR or total incubations from the translation

- 66

of poly(A) RNAs or luciferase mRNA were resuspended

- 45 1

2

3

RESUL TS AND DISCUSSION

4

Fig. 2. Effect of HSW on the translation of luciferase mRNA in the PC12-SIO extract system. The 12.5 III reaction mixture contained 500 ng of luciferase mRNA and increasing amounts of HSW. Lane I, dialysis buffer; lanes 2-4, HSW, 0.5 A26o1ml, 1.0 A26o1ml, and 2.0 A26o1ml, respectively. Densitometric analysis of the luciferase peak yielded relative values of: lane I, lOO; lane 2, 170; lane 3, 380; lane 4, 610. The samples were incubated at 30°C for 18 h. The arrowhead shows a major band among the translation products migrating at 62 kDa.

reticulocyte lysate were 12.5 Ill, lOO Ill, and 12.5 Ill, respectively. The translation reactions were incubated for I h at 30°C. The PCI2-SIO

extract translation

system contained

40% volume

of SIO extract from PCI2 cells with the following additions: I mM ATP, 50 I1M GTP, la mM creatine phosphate, 50 I1g/ml creatine phosphokinase, 0.5 mM DTT, 50 I1g/ml calf liver tRNAs, 20 I1M amino acid mixture minus methionine, 0.8 mCi/ml L-[35S]methionine (1200 Ci/mmol), 0.8 unit/Ill RNasin ribonuclease inhibitor, 22 mM HEPESKOH, pH 7.4, 240 I1M spermidine, 1/1000 volume of protease inhibitor mixture, and in vitro transcribed mRNA. Magnesium acetate and potassium acetate solutions were added, where appropriate, to raise the concentrations of magnesium or potassium above those contributed from the PC 12-S 10 extract itself. The total reaction volumes of the SIO extract translation

of the poly(A)

RNAs, hEGFR

mRNA,

and

luciferase mRNA were 12.5 Ill, 100 III and 12.5 Ill, respectively. The translation reactions were incubated for either I h or 18 h at 30°C. Immunoprecipitation.

To detect hEGFR translation products,

im-

munoprecipitation was performed using either protein A-agarose or protein G-agarose, according to the method described previously (8). Both polyclonal 1210 antibody (8) and monoclonal 6FI (Medical and Biological Lab. Co., Ltd., Japan) were used. The 1210 antibody was raised against 12 amino acids of the intracellular domain of hEGFR. The 6FI antibody was also made against the intracellular domain (amino acids 648-1186) of recombinant hEGFR. To validate the immunoprecipitation ofhEGFR, preincubation for peptide competition was performed before immunoprecipitation with anti-peptide antibody 1210. The antibody solution was preincubated with 5 volumes of a solution containing peptide antigen (I mg/ml) for 30 min at room temperature. Glycosidase Treatment. Immunoprecipitated agarose beads were denatured at 37°C for I h in an incubation

in 2x SDS

sample buffer and the proteins resolved by electrophoresis on 7.5% SDS-polyacrylamide gels. The protein products were detected by autoradiography.

buffer containing

25 mM

The PC12 cell line has been used extensively for the studies of neuronal differentiation, because NGF induces these cells to acquire a number of features characteristic of mature sympathetic neurons. Thus, treatment with NGF causes the cells to stop dividing, become excitable, and extend neurites (6,17). They will, in addition, synapse with appropriate muscle cells in culture (18). The large number of changes that the cells undergo upon NGF treatment fall into two groups (19). There are a series of rapid, membrane-based events that are independent of RNA synthesis and there are other, longerterm alterations that require gene expression. It has been recognized in recent years that mRNA translation is an important control point in growth factorinduced gene expression (1). Because NGF treatment has been shown to cause changes in the phosphorylation, and, presumably also in the function, of eIF-4E, eEF-2, and the S6 protein of the ribosomes (10,20,21), all elements of the translation mechanism, NGF-induced alterations in the characteristics of translation would not be surprising. Indeed, several NGF-induced phenomena in PC12 cells are thought to be controlled at this level (8,9). Accordingly, it seemed useful to establish a suitable assay system utilizing PC12 cells for the investigation of the translational control associated with NGF-ind].lced neuronal differentiation. Fig. 1 shows the products of the translation of firefly luciferase mRNA and poly(A) RNAs from PCl2 cells in both rabbit reticulocyte lysate and PC12-SlO extract systems. Firefly luciferase is a monomeric, 62 kDa protein that does not require posttranslational modification for enzymatic activity (22). In the rabbit reticulocyte system a single major band of about 62 kDa was synthesized in vitro in response to the addition of luciferase mRNA (Fig. lA). This band is comparable to that seen

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30

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90 120

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K+(mM)

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30

Mg2+(mM)

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Fig. 3. Determination of the optimum additional salt concentrations for the translation of luciferase mRNA in the PCI2-SI0 extract system. Both magnesium acetate and potassium acetate were added to the translation mixture in addition to those contributed by the PC 12-S 10 extract itself. Translation was programmed by the addition of luciferase mRNA to the 12.5 III reaction mixture, which was then incubated at 30°C for I h. A: potassium curve; B: magnesium curve. The arrowhead shows a major band among the translation products migrating at 62 kDa.

Rabbit reticulocyte

PC I2-S 10 extract

lysate

kDa

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138kDa7 134 kDa

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Fig. 4. Immunoprecipitation of the translation products of hEFGR mRNA in both rabbit'reticulocyte lysates and the PCI2-SI0 extract system in the presence of the peptide antigen. The total reaction mixture was 100 Ill. The samples were incubated at 30°C for 1 h. The translation products were immunoprecipitated with either polyclonal 1210 antibody (lanes 1-3 and 5-7) or monoclonal6FI antibody (lanes 4 and 8). The immunoprecipitates were resolved on 7.5% SDS-polyacrylamide gels. Preincubation for peptide competition before immunoprecipitation was done for 30 min at room temperature with the anti-peptide 1210 antibody (lanes 3 and 7). Translation was programmed by the addition of 250 ng of hEGFR mRNA (lanes 2-4 and 6-8). Lanes I and 5 were control incubations without the addition of mRNA.

in a previous report (23). A similar band was also observed in the S10 extract translation system (Fig 1B, IC), although the intensity of the signal produced by the S10 extract was much less than that seen in the reticulocyte system. Poly(A) RNAs from PC12 cells gave rise to a series of translation products in both systems and, again, the bands representing these products were less intense when synthesized by the S10 extract. These bands were not characterized further. In a HeLa cell S10 extract, the synthesis of poliovirus peptides requires a longer incubation period than that needed for comparable translation in the reticulocyte system (14). Figures 1B and IC show that a longer incubation period with the PC 12-S10 extract also increases the amounts of translation products. The initiation step is a major point of translational regulation, and the modulation of the activity of thei:iFs by phosphorylation has received considerable attention (1,3,4). Taking this into consideration, a HSW was prepared from PC12 cells, in the presence of 1 IlM okadaic acid to inhibit endogenous phosphatase activities, and added to the S10 extract translation system. The addition of the HSW increased the level of translation of both

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Translation System from PCl2 Cells

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160 kDa'-., 138 kDa ,.,-!

134 kDa

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1 Fig. 5. N-Linked deglycosylation of the immunoprecipitates from the translation of hEGFR mRNA in the PCI2-SI0 extract system. Translation was programmed by the addition of 250 ng of hEGFR mRNA to a 100 J.!l translation mixture. After incubation at 30°C for I h, the translation products were immunoprecipitated and the immunoprecipitated agarose beads were denatured at 37°C for I h in incubation buffer. After denaturation, the incubation mixtures were either treated with 0.4 U of N-glycosidase F (lane 3) or incubated with buffer alone (lane 2) at 37°C for 3 h in the presence of EDTA, l3-mercaptoethanol, . and N-octylglucoside. The total reaction mixture for the enzymatic digestion was 15 J.!l. The reaction mixtures were then diluted with 15 J.!Iof SDS sample buffer. Lane I: immunoprecipitated translation products from a rabbit reticulocyte lysate. Immunoprecipitation was performed with 6Fl antibody.

luciferase and poly(A) RNAs (Figs. IB and lC). As shown in Fig. 2, the effect of additional HSW to the SI 0 traction appeared to be dose-dependent. Translation lysates contain a number of components that are necessary for their high protein synthesis activity. The lysates can also be inhibited by a wide range of compounds and conditions. It has been reported that different Mg2+concentrations are optimal for different initiation sites, and that termination can be suppressed to some extent by slightly superoptimum Mg2+concentrations (24,25). Optimum K+concentrations also differ depending on the mRNA used (25). The optimum concentrations of Mg2+ and K+ were determined for the translation of luciferase from uncapped mRNA in the SIO extract (Fig. 3A and 3B). The efficiency of translation changed with the addition of Mg2+or K+. Optimum additional concentrations of magnesium acetate and potassium acetate were found to be 0.4 mM and 0 mM, respectively. To further test the in vitro systems, capped mRNA encoding hEGFR was translated both in the rabbit reticulocyte lysate and in the PC12-SIO extract, and the prod-

ucts were immunoprecipitated with either of two different anti-EGFR antibodies (Fig. 4). Both of the antiEGFR antibodies recognized the same protein bands in each translation system. In the rabbit reticulocyte lysate, there were many immunoprecipitated bands and the molecular weight of the uppermost band was 138 kDa. These findings are consistent with those reported previously (16). In contrast, in the PC12-SIO system there were two major bands of immunoprecipitated protein migrating at 134 and 160 kDa. The specificity of the immunoprecipitation was validated in both translation systems by demonstrating a reduction in the immunoprecipitated protein bands by preincubation of the antibody with the peptide antigen (Fig. 4, lanes 3 and 7). Clark et al. (16) indicated that the many bands that appeared as a result of the translation of hEGFR in the rabbit reticulocyte lysate are most likely due to premature chain termination during translation. Compared with the rabbit reticulocyte lysate, the PC 12-S10 extract showed no bands other than the two major ones. Addition of the HSW to the rabbit reticulocyte lysate significantly decreased the smaller bands resulting trom the translation (data not shown). These data would suggest that the rabbit reticulocyte lysate is lacking a component or components essential for completion of the translation process, or contains insufficient amounts of such components, that the PC 12-S 10 extract contains adequate amounts, and that these components are especially enriched in the HSW. The mature human EGFR is composed of a single polypeptide chain of 170 kDa, with about 40 kDa of that made up of N-linked carbohydrate as a result of posttranslational modification (26-28). It is also known to be subject to phosphorylation (28). The unmodified precursor and the mature core polypeptides have predicted molecular weights of 134.3 and 131.4 kDa, respectively (26). It is possible that the 160 kDa band seen in the SIO system is due to such posttranslational modifications. The autoradiogram shown in Fig. 5 depicts the translation products after N-glycosidase F digestion of immunoprecipitates from an SI 0 extract programmed with hEGFR mRNA. The upper 160 kDa band representing a protein immunoprecipitated from the untteated sample clearly has disappeared trom the N-glycosidasetTeated sample and probably has shifted to the position of the lower band (134 kDa). This result strongly suggests that the upper band seen following translation in the PC 12-S10 extract is due to the increase in the molecular weight of this receptor protein caused by Nlinked glycosylation of the translation product. It is not clear why the uppermost band (138 kDa) immunoprecipitated trom the rabbit reticulocyte system is slightly

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hEGFR mRNA(ng) 250

K+(mM) 0 0 30 30 Mg2+(mM) 0.4 0.8 0.4 0.8

0

10

50 250 1250 kDa

1

2

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4

5

kDa

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1200 I -116

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Fig. 6. Dose dependency of hEGFR mRNA and determination of the optimal salt concentrations in the PCI2-SlO extract system. The translation was performed at 30°C for I h in a 100 III reaction mixture. Immunoprecipitation was done with 6FI antibody. A: Translation of increasing amounts of hEGFR mRNA in the PCl2-SlO extract system. Lane I contains translation products immunoprecipitated from a rabbit reticulocyte lysate. B: Effect of added magnesium or potassium. Both magnesium acetate and potassium acetate were added to the translation mixture in addition to the concentration contributed trom the PCI2-S 10 extract itself. Translation was programmed by the addition of 250 ng of hEGFR mRNA.

larger than the lower band seen in the 8 10 extract. The rabbit reticulocyte lysate has been shown to have the ability to catalyze posttranslational modifications such as phosphorylation, acetylation, and O-linked glycosylation in the translation of some specific proteins (29,30). It is reasonable to suggest that the increased molecular weight is due to some posttranslational modification in the rabbit reticulocyte lysate as well as in the PC 12-810 extract. An increase in the amount of mRNA added to the incubation containing PC 12-810 extract led to a parallel increase in the amount of radioactive translation product immunoprecipitated (Fig. 6A). The efficiency of hEGFR translation was also changed by the addition of Mg2+or K+,the optimum additional concentration of magnesium acetate and potassium acetate for the translation of hEGFR in the 810 system was 0.4 mM and 30 mM, respectively (Fig. 6B). In summary, we report the preparation of a PCI2 cell-derived 810 extract translation system. The disadvantages of this system are its low efficiency of translation and the apparent need to optimize the ionic concentrations for each individual mRNA. The advantages of this preparation are that it comes from a neuronal cell type, that it allows the translation of rather large complete proteins, that it permits posttranslational modifications not seen with the reticulocyte system, and

that it will lend itself to the fractionation and identification of the stimulatory components of the high salt wash. This system -/ should prove useful for the detection of alterations in translational control produced by growth factors.

ACKNOWLEDGMENT The authors are grateful for the continued interest and advice of Dr. Glenn Medino. We also would like to express our appreciation to Dr. Greg Beckler of Promega for his help in the use of translation systems and his careful reading of this manuscript.

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control in mammalian cells.

4. Merrick, W. C. 1992. Mechanism and regulation of eukaryotic protein synthesis. Microbiol. Rev. 56:291-315. 5. Standart, N., and Jackson, R. J. 1994. Regulation of translation by specific protein/mRNA interactions. Biochimie 76:867-879. 6. Greene, L. A., and Tischler, A. S. 1976. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells

,.. ->

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807

Translation System from PC12 Cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73:2424-2428. 7. Goodman, R., Chandler, C., and Herschman, H. R. 1979. Pheochromocytoma cell lines as models of neuronal differentiation. Pages 653-669, in Sato, G., and Ross, R. (eds.) Hormones and Cell Culture, Vol. 6, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 8. Oshima, M., Weiss, L., Dougall, W. C., Greene, M. I., and Guroff, G. 1995. Down-regulation of c-neu receptors by nerve growth factor in PC12 cells. 1. Neurochem. 65:427--433. 9. Twiss, J. L., and Shooter, E. M. 1995. Nerve growth factor promotes neurite regeneration in PC12 cells by translational control. J. Neurochem. 64:550-557. 10. Frederickson, R. M., Mushynski, W. E., and Sonenberg, N. 1992. Phosphorylation of translational initiation factor eIF-4E is induced in a ras-dependent manner during nerve .growth factor-mediated PC12 cell differentiation. Mol. Cell. BioI. 12:1239-1247. 11. Brown, B. A., and Ehrenfeld, E. 1979. Translation of poliovirus RNA in vitro: changes in cleavage pattern and initiation sites by ribosomal salt wash. Virology 97:396--405. 12. Teerink, H., Kasperaitis, M. A. M., Moor, C. H. D., Voorma, H. 0., and Thomas, A. A. M. 1994. Translation initiation on the insulin-like growth factor II leader 1 is developmentally regulated. Biochem. J. 303:547-553. 13. Morley, S. J., and Hershey, J. W. B. 1990. A fractionated reticulocyte lysate retains high efficiency for protein synthesis. Biochimie 72:259-264. 14. Molla, A., Paul, A. V., and Wimmer, E. 1991. Cell-free de novo synthesis of poliovirus. Science 254: 1647-1651. 15. Pelham, H. R. B., and Jackson, R. 1. 1976. An efficient mRNAdependent translation system from reticulocyte lysate. Eur. 1. Biochem. 67:247-256. 16. Clark, A. J. L., Beguinot, L., Ishii, S., Ma, D. P., Roe, B. A., Merlino, G. T., and Pastan, I. 1986. Synthesis of epidermal growth factor (EGF) receptor in vitro using SP6 RNA polymerase-transcribed template mRNA. Biochim. Biophys. Acta 867:244-251. 17. Dichter, M. A., Tischler, A. S., and Greene, L. A. 1977. Nerve growth factor induced increase in electrical excitability and acetylcholine sensitivity of a rat pheochromocytoma cell line. Nature 268:501-504.

18. Schubert, D., Heinemann, S., and Kidokoro, Y. 1977. Cholinergic metabolism and synapse formation by a rat nerve cell line. Proc. Natl. Acad. Sci. USA 74:2579-2583. 19. Bradshaw, R. A. 1978. Nerve growth factor. Annu. Rev. Biochem. 47:191-216. 20. Koizumi, S., Ryazanov, A., Hama, R., Chen, H-C., and Guroff, G. 1989. Identification of Nsp100 as elongation factor 2 (EF-2). FEBS Lett. 253:55-58. 21. Ha1egoua, S., and Patrick, J. 1980. Nerve growth factor mediates phosphorylation of specific proteins. Cell 22:571-581. 22. de Wet, J. R., Wood, K. V., DeLuca, M., He1inski, D. R., and Subramani, S. 1987. Firefly luciferase gene: Structure and expression in mammalian cells. Mol. Cell. BioI. 7:725-737. 23. Wood, K. V., de Wet, J. R., Dewji, N., and DeLuca, M. 1984. Synthesis of active firefly luciferase by in vitro translation of RNA obtained from adult lanterns. Biochem. Biophys. Res. Commun. 124:592-596. 24. Pelham, H. R. B. 1978. Leaky AUG termination mosaic virus mRNA. Nature 272:469--471.

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25. Jackson, R. 1., and Hunt, T. 1983. Preparation and use of nucleasetreated rabbit reticulocyte Iysates for the translation of eukaryotic messenger RNA. Meth. Enzymol. 96:50-74. 26. Ullrich, A., Coussens, L., Hayflick, 1. S., Dull, T. J., Gray, A., Tarn, A. W., Lee, J., Yarden, Y, Libermann, T. A., Sch1essinger, 1., Downward, J., Mayes, E. L. V., Whittle, N., Waterfield, M. D., and Seeburg, P. H. 1984. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 309:418-425. 27. Lane, M. D., Ronnett, G., Slieker, L. J., Kohanski, R. A., and Olson, T. L. 1985. Post-translational processing and activation of insulin and EGF proreceptors. Biochimie 67:1069-1080. 28. Hernandez-Sotomayor, S. M. T., and Carpenter, G. 1992. Epidermal growth factor receptor: element of intracellular communication. J. Membrane BioI. 128:81-89. 29. Gibbs, P. E. M., Zouzias, D. c., and Freedberg, 1. M. 1985. Differential post-translational modification of human type I keratins synthesized in a rabbit reticulocyte cell-free system. Biochim. Biophys. Acta 824:247-255. 30. Starr, C. M., and Hanover, 1. A. 1990. Glycosylation of nuclear protein p62. Reticulocyte lysate catalyzes O-linked N-acety1g1ucosamine addition in vitro. J. BioI. Chem. 265:6868-6873.

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