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FIG. 1. C h r o m a t o g r a p h y o f [ 3 H ] a m i n o a c y l a t e d tRNAL_% o n S e p h a r o s e u s i n g a d e c r e a s i n g salt g r a d i e n t . See t e x t for d e t a i l s .

tionated E. coli K12 tRNA is LeutRNA2 L~u, LeutRNA5 Leu, LeutRNA3 L~u, LeutRNA1Leu, and LeutRNA4 Leu 1, It is important to note that this method works as described here only when the columns are operated at 4 ° and at a p H value of 4.5.

[16] Transfer R N A Isoacceptors in Cultured Chinese Hamster Ovary Cells1 By

ARNOLD E. HAMPEL 2 and BRUCE RUEFER

Multiplicity of tRNA species for a single amino acid is a commonly observed phenomenon. 3 Multiple tRNA isoacceptors are required for many amino acids because of degeneracy of the genetic code; 4 however, METHODS 1N ENZYMOLOGY, VOL. LIX

Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181959-0

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the number of isoacceptors often seen is far greater than that required by the code. Because of this observation, it has often been suggested that tRNA is involved in roles other than classical protein synthesis? For several mammalian tRNAs there has been preservation of identical base sequences between species. The pattern of communality of tRNA species in mammals argues for the selection of a single versatile model mammalian system to completely catalog the isoacceptor profiles as the first step in systematically defining all their cellular functions. The Chinese hamster ovary (CHO) cell line is especially well suited for this purpose because it is easily grown in a partially defined media and amenable to the study of a variety of cellular and genetic problems. 6 We have shown that multiple isoacceptors exist for all CHO tRNAs except tRNA Trp, which has a single isoaccepting species. A total of 77 tRNA isoaccepting species in CHO cells are found by the RPC-5 chromatographic method, which represents a minimal number with more likely to be present. Variations between tRNA isoacceptors specific for a given amino acid would be expected to be due to both structural differences and the degree of base modification. Methods Cell Culture. A hypodiploid (modal chromosome number of 21) line of Chinese hamster ovary cells 7 is grown in Ham's F-10 medium supplemented with 15% calf serum (prepared in our laboratory), 100 units of penicillin, and 100/zg of streptomycin per milliliter. Cells are grown in 4-liter spinner flasks (Bellco) and harvested during logarithmic growth at a density of 3 to 5 x 10~ celis/ml by the addition of 500 ml of 0.25 M sucrose (-20 °) to the suspension and centrifuged (4 °) at 1000 g for 10 rain in 250-ml polypropylene centrifuge bottles (Nalge) and resuspended in cold 0.25 M sucrose. Cells (109) are washed once and resuspended in 9 ml of buffer A: 0.1 M KCI, 10 mM Tris.HCl (pH 7.5 at 25°), 1 mM MgC12, and 0.1 mM dithiothreitol. 1 This research was supported by N I H Grant GM 19506 and Research Career Development Award I KO4 GM 70424. 2 Work performed in the D e p a r t m e n t s of Biological Sciences and Chemistry, Northern Illinois University, DeKalb, Illinois. :3 R. M. Kathari and M. W. Taylor, J. Chromatogr. 8 6 , 2 8 9 (1973). 4 D. $611, J. Cherayil, D. Jones, R. Faulkner, A. H a m p e l , R. Bock, and H. G. K h o r a n a , Cold Spring Harbor Syrup. Quant. Biol. 32, 51 (1966). 5 U. Littauer and H. Inouye, Annu. Rev. Biochem. 42, 439 (1973). 6 C. R. R i c h m o n d , D. F. Peterson, and P. F. Mullaney, " M a m m a l i a n Cells: Probes and Problems.'" Technical Information Center, U.S. Energy Res. and Devel. Admin., 1975. 7 j. Tjio and T. T. Puck, J. Exp. Med. 108, 259 (1958).

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Crude Aminoacyl-tRNA Synthetase Preparation. A crude aminoacyltRNA synthetase preparation containing activities for all 20 amino acids is prepared from cells fresh or stored frozen at -80 °. The cells (109) in 18 ml of buffer A are broken by the addition of 2 ml of 10% Nonidet P40 (Particle Data Laboratories) with gentle hand mixing for 30 rain on ice. Nuclei and cell debris are removed by centrifuging for 30 min at 1000 gay at 4 °. The supernatant is centrifuged at 145,000 g at 4 ° for 2.5 hr in one A-211 centrifuge tube (International Equipment Co.) through a 1.5ml 34% sucrose pad containing buffer A with KCI at 10 mM (buffer B). The crude supernatant is dialyzed overnight at 4 ° against 2 liters of buffer B with 40% glycerol. The final enzyme preparation containing 50 Az80 units/ml with 7.5 mg of protein per milliliter is stored at -20 °. Protein is determined by a modification of the method of Lowry as described. 8 Preparation of Chinese Hamster Ovary tRNA. Supernatant prepared by removing all ribosomes from broken cells through centrifugation of 109 cells in 20 ml of buffer A, for 2.5 hr through a 1.5-ml 34% sucrose pad at 145,000 g at 4 ° is shaken with an equal volume of phenol for 15 rain at room temperature. The emulsion is centrifuged at 4000 g for 20 min, the aqueous phase is carefully removed, and the preparation is phenol-treated twice more. The final aqueous phase is precipitated with 2 volumes of 100% ethanol and allowed to precipitate overnight at -20 °. This is centrifuged; the pellet is redissolved in buffer A and precipitated twice more with ethanol. The final tRNA pellet is redissolved in buffer B without glycerol. The long period of time involved in the tRNA preparation is adequate for removal of endogenous amino acids acylated to tRNA because of the lability of the aminoacyl-tRNA ester linkage at pH 7.5. This avoids using the high alkaline pH stripping conditions that destroy certain minor bases in tRNA and even produce a limited number of phosphodiester cleavages. The level of charging of tRNA TM, which contains the most stable aminoacyl-tRNA ester bond, is used as a reference. An average preparation accepted 46 pmol of valine/A~60 unit of tRNA, which corresponds to 3.5% of the total heterogeneous tRNA preparation being available for valine aminoacylation. The--CCA termini of these preparations have been shown to be intact by their inability to incorporate ATP when assayed with Escherichia coli ATP, CTP-tRNA nucleotidyltransferase according to Hampel et al. 9 Yeast tRNA is used as a control to verify enzymic activity. 8 A. Hampel and M. D. Enger, J. Mol. Biol. 79, 285 (1973). 9 A. Hampel, A. Saponara, R. Waiters, and M. D. Enger, Biochim. Biophys. Acta 269, 428 (1972).

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Aminoacylation of tRNA. Two A260 units of CHO tRNA are aminoacylated at 37 ° in 1 ml of incubation mix containing 20 mM Tris.HC1, 15 mM MgC12, 0.1 mM EDTA, 5 mM ATP, 0.75 mM CTP, and 200/~1 of crude aminoacyl-tRNA synthetase (50 A280/ml) all at pH 7.5. The incubation mix contains 10-20 tam 14C- or ~H-labeled amino acid, which is about a 100-fold molar excess over tRNA. The other nonradioactive 19 amino acids are present at 10/aM to overcome possible spurious charging by radioactive contaminants and to prevent misacylation of noncognate tRNAs. Enough aminoacyl-tRNA synthetase is present to allow completion of reaction in 10 min. Incubation is terminated after 20 min by the addition of 1/20 volume of 2 M Na acetate (pH 5.0). An equal volume of phenol is added, and the mixture is shaken at room temperature for 6 min; phases are separated by centrifugation at 2000 g for 20 min at 25 °, and the aqueous phase is removed. The phenol step is repeated twice with volume restored by 0.1 M Na acetate, pH 5.0, when necessary; 16 A260 units of carrier yeast tRNA are then added, and the tRNA is washed 3 times by ethanol precipitation. The last tRNA precipitate is lyophilized to dryness and redissolved in 200 /zl of the respective RPC-5 starting buffer. RPC-5 Chromatography. Reversed-phase chromatography is done using method B of the RPC-5 system described by Pearson et al.l° The Plaskon 2300 CTFE powder (polychlorotrifloroethylene) used was a generous gift from Allied Chemical Corp., Morristown, New Jersey. The powder (300 g) is mixed at very low speed in a Waring blender with 12 ml of Tricaprylylmonomethyl-ammonium chloride (Ashland Chemical Company, Columbus, Ohio) dissolved in 450 ml of chloroform. After blending 1-2 hr, dry nitrogen gas is blown through the mix while blending until the chloroform is driven off. The average particle size of the resulting resin is 10/xm. Chromatography is carried out using a modification of the high-pressure system of Kelmers and Heatherly.11 Columns, 0.64 × 20 cm, are poured in a 25 ° water-jacketed LC-6M-13 high-pressure glass column (Laboratory Data Control) using a 1:1 slurry in starting buffer and maintaining a flow rate of near 2 ml/min with pressure up to 500 psi from a Milton Roy Model 396 minipump. Sample (200/xl) is added and gradient changes are made with an 8-part sample injection valve (SV-8031 Laboratory Data Control). Elution is at 500 psi with 100-ml linear gradients of various NaC1 concentrations (examples are given in figure legends) in 10 mM MgCI2, ~0R. Pearson, J. Weiss, and A. D. Kelmers, Biochim. Biophys. Acta 228, 770 (1971). 11 A. D. Kelmers and D. E. Heatherly, Anal. Biochem. 44, 486 (1971).

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0.4 mM dithiothreitol, and 10 mM Na acetate, pH 4.5. Temperature is maintained at 25 ° using a Lauda constant-temperature bath. Fractions are collected at 30-sec intervals with pressures near 500 psi and flow rates near 1.85 ml/min. All flow rates are normalized to this rate. The column void volume is tube 7, w h e r e all free amino acids are eluted, and gradients are started at tube 10. Effluent is monitored at 260 nm using a l-mm flow cell in a Hitachi 124 spectrophotometer. All samples (approximately 0.9 ml) are collected directly in scintillation vials with 14C radioactivity determined by liquid scintillation counting using 10 ml of Bray's solution lz for ~4C single-label samples or 10 ml of aquasol (New England Nuclear) for a4C, 3H double-label samples. Identification of Multiple I s o a c c e p t o r s

Asp, Asn, His, and Tyr Specific" tRNA Isoacceptors. Figure 1 shows the isoacceptor profiles for tRNAs specific for Asp, Asn, His, and Tyr. tRNA ASp shows five isoacceptor peaks. Peaks 1, 2, 3, and 5 are small, and peak 4, the major peak, constitutes most of the tRNA A~p. tRNA ash contains a very minor peak 1, the largest 2, and another major peak 3. A significant shoulder is present on peak 3 representing a later-eluting peak 4. tRNA ~ contains three major peaks with an additional very small early-eluting peak. The isoacceptor profiles of tRNA A~p from CHO cells correspond to pattern II of Gallagher et aI.13 This pattern is most commonly found in rapidly growing cells rather than in more quiescent cells and is characterized by a larger peak 4. By analogy with SV40-transformed 3T3 cells '4 the minor peaks 1 and 3 would be expected to contain the modified G base Q, 7-(4,5 cis-dihydroxy-l-cyclopenten-3-ylaminomethyl)-7-deazaguanosine, is while peaks 2 and 4 would contain the unmodified G. Q base is found as the 5'-terminal base of the anticodon of certain isoacceptors specific for Asp, Asn, His, and Tyr. These tRNAs correspond to the codons -Ac U with Q preferentially recognizing U. Glu, Gin, and Lys Specific tRNA Isoacceptors. Figure 2 shows the isoacceptor profiles for Glu, Gin, and Lys specific tRNAs, tRNA C~u contains three peaks, the major species peak 1 eluting quite early at fraction 22 followed by a smaller peak 2 and another major peak 3. tRNA c~" contains two major peaks, tRNA Lys has four characteristic 12G. A. Bray, Anal. Biochem. 1, 279 (1960). 13R. E. Gallagher, R. C. Ting, and R. Gallo, Biochim. Biophys. Acta 272, 568 (1972). 14j. R. Katze, Biochim. Biophys. Acta 383, 131 (1975). 15H. Kasai, Z. Ohashi, F. Harada, S. Nishimura, W. Oppenheimer, P. Crain, J. Liehr, D. von Minden, and J. McCloskey, Biochemistry 14, 4198 (1975).

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Transfer RNA isoacceptors in cultured Chinese hamster ovary cells.

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