DEVELOPMENTAL
BIOLOGY
@,268-277(1976)
A Reexamination of the Leucine tRNAs and the Leucyl-tRNA Synthetase in Developing Tenebrio molitor NORMAN J. LASSAM, HARVEY Department
LERER,
of Biology, Queen’s University,
AND
BRADLEY
Kingston,
Ontario,
N. WHITE Canada
Accepted October 16, 1975 The translational control mechanism previously proposed for the synthesis of adult cuticular proteins in Tenebrio molitor is dependent upon the appearance of a major, novel leucine tRNA and a change in leucyl-tRNA synthetase activity just prior to adult emergence. The properties of the leucyl-tRNA synthetase extracted from pupae were reexamined. Under optimal aminoacylation conditions, no new leucine isoaccepting tRNAs were observed during development. However, under suboptimal conditions, a differential charging of the leucine tRNA species was noted. The chromatographic profiles of leucyl-tRNAs aminoacylated in vivo in both early and late pupae were found to be the same and were identical to the profiles obtained by charging tRNAs in vitro. Previous evidence for a translational control system operating in Tenebrio is discussed in relation to these results.
al., 1975). No significant differences were found. However, under suboptimal conditions, differential extents of aminoacylation of the leucine tRNAs in vitro were observed. Examination of the leucyltRNAs charged in vivo confirmed the results obtained in vitro in that no new leutine tRNA was detectable.
INTRODUCTION
Extensive studies on the mealworm Tenebrio molitor have indicated that the synthesis of adult cuticular proteins is under translational control (Ilan et al., 1966; Ilan et al., 1970; Ilan and Ilan, 1975a). This control mechanism appeared to be mediated by a new or modified leucyl-tRNA synthetase and a major novel leucine tRNA species, the synthesis of which seemed to be under the control of juvenile hormone (Ilan et al., 1972). Changes in aminoacyl-tRNAs during the development of Drosophila have been observed (White et al., 1973a,b; Grigliatti et al., 1974) but have not been correlated with any developmental control mechanism. The Tenebrio system appeared to be ideally suited for a detailed examination of a control mechanism mediated by a tRNA. We soon found that the conditions previously reported (Ilan et al., 1970) for the aminoacylation of leucine tRNAs were far from optimal and therefore we reexamined the properties of the leucyl-tRNA synthetase. Using improved aminoacylation conditions, the leucyl-tRNAs from the different developmental stages of Tenebrio were compared as reported before (Lassam et
MATERIALS
268 Copyright 6 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
METHODS
Materials. Labeled amino acids and Aquasol were obtained from New England Nuclear Corp. Adogen 464, a trialkylmethylammonium chloride with the predominant chain length of the alkyl groups being C8-Co, was a gift from Ashland Chemical Co., Columbua, Ohio. Plaskon CTFE2300 powder was a gift from Allied Chemical Corp., Morristown, N. J. Growth of Tenebrio. T. molitor larvae were obtained from Carolina Biological Supply Co., Burlington, N. C., and reared at 29°C according to the method of Patterson (1957). Under these conditions the pupal period lasted for 6 days, with adults emerging on the seventh. Isolation of tRNA. Transfer RNA was extracted with phenol directly from whole organisms by the methods described previously for Drosophila (White et al., 1973a).
LASSAM,
LERER
AND
WHITE
Leucine tRNAs
Preparation of aminoacyl-tRNA synthetases. Aminoacyl-tRNA synthetases were normally prepared from the 105,OOOg (90 min) supernatant by the methods described previously for Drosophila (White et al., 1973a), with the addition that 1 mM phenylthiourea was added to all of the extraction buffers. A “microsomal wash” aminoacyl-tRNA synthetase prepared according to the method of Ilan (1968) was also used. of tRNA in witro. Aminoacylation Transfer RNA was aminoacylated at 30°C in a final reaction volume of 0.2 ml. For leucine, each milliliter of the reaction mixture contained: Tris-HCl, 50 pmoles (pH 7.5); 2-mercaptoethanol, 5 pmoles; MgC12, 5 pmoles; ATP, 8 pmoles; [14Clamino acid, 50 pmoles; KCl, 10 pmoles; the aminoacyltRNA synthetase preparation contained 1 mg of protein and tRNA, 0.3-0.8 mg. For phenylalanine and tyrosine, the conditions were the same except that each milliliter of reaction mixture contained: MgCl*, 10 pmoles; ATP, pmoles; [14Clamino acid, 25 Fmoles; KCl, 50 pmoles. The rate of the aminoacylation reaction was followed by pipeting aliquots on Whatman 3MM filter paper disks and removing acid-soluble radioactive amino acids (Bollum, 1959). Initial rates of reaction were determined by stopping the reaction at 2 min by the addition of cold 10% TCA and collecting the precipitate on Millipore filters. Aminoacyl-tRNA for reversed-phase chromatography was isolated from the components of the reaction mixture by DEAE-cellulose chromatography as described by Yang and Novelli (1968). Aminoacylation of tRNA in uiuo. Approximately 25 pupae were injected with 5 &i (in approximately 5 ~1) each of l3 Hlleucine (50 Ci/pmole) and left 3-5 min. The pupae were then homogenized in equal volumes of 88% phenol and a buffer containing 0.25 M NaCl, 0.01 M MgCl*, 0.001 M 2-mercaptoethanol, and 0.01 M sodium acetate, pH 4.5. The aqueous
in Developing
Tenebrio
269
phase was removed and the phenol was reextracted with an equal volume of buffer: The pooled aqueous phases were applied to a DEAE-cellulose column equilibrated with the same buffer. The column was washed with this buffer and the [3 Hlleucyl-tRNA was eluted with the buffer containing 0.75 M NaCl. This material was diluted to 0.375 M NaCl and ap plied to the RPC-5 columns. Reversed-phase chromatography of aminoacyl-tRNA. The RPC-5 system of Pearson, Weiss, and Kelmers (1971) was used. The Plaskon CTFE was coated with Adogen 464 as described elsewhere (White et al., 1973a). The 0.9 x 14-cm columns were developed at 37°C with a linear loo-ml NaCl gradient containing 0.01 M MgC&, 0.01 M sodium acetate (pH 4.5), and 1 n&f X-mercaptoethanol. The 1.25 x 50-cm columns were developed at 37°C with a linear 500-ml NaCl gradient. Radioactivity was determined in the fractions by the addition of 5 volumes of Aquasol and counting in a scintillation counter. RESULTS
Properties of the leucyl-tRNA synthetase from last-day pupae; apparent K, for leutine. The effect of varying the concentration of [I4 Clleucine on the formation of 114Clleucyl-tRNA catalyzed by the synthetase was studied (Fig. 1). From a Lineweaver-Burk plot of the data the apparent K, for leucine was found to 22 x lo-” M. This compares with an apparent K, of 2 x lo-” M of phenylalanine for the Tenebrio phenylalanyl-tRNA synthetase (unpublished results). Magnesium and ATP optima. The effect of varying the ATP concentration at constant MgCl, (5 mM) on the initial rate of [14Clleucyl-tRNA formation is shown in Fig. 2. A sharp peak at 8 mM ATP was found. The effect of varying the MgCl, concentration at constant ATP (8 m&f) on the initial rate of [I4 Clleucyl-tRNA formation is also shown in Fig. 2. Again a relatively sharp peak at 5 mM MgCl, was found.
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DEVELOPMENTAL BIOLOGY
Monovalent cation requirement. The effect of varying the concentration of KCl, NaCl, and NH&l on the initial rate of [l* Clleucyl-tRNA formation is shown in
FIG. 1. Variation of [Wlleucine concentration in the esterification reaction. The assay conditions were as described under “Methods” except that varying concentrations of [Wlleucine were used. Reactions were terminated after 2 min with 10% TCA and the precipitate collected on Millipore tilters. The reaction velocity V is expressed as cpm incorporated/2-min reaction time; substrate concentration (S) is expressed as micromoles [“C]leucine.
VOLUME 49, 1976
Fig. 3. A broad optimum of 8-20 mM KC1 was found. Sodium chloride was not as effective as KCl, while NH4Cl markedly stimulated the initial rate at relatively low concentrations. pH optimum. The effect of varying the pH of Tris-HCl buffer on the initial rate of 11*Clleucyl-tRNA formation gave the optimum of pH 8.5, as found by Ilan et al. (1975). Acceptance of [‘*Clamino acids by tRNA from the developmental stages. The quantitative levels of tyrosine, phenylalanine, and leucine tRNAs at the different developmental stages is shown in Table 1. Relative to tyrosine and phenylalanine, there is little change in the acceptance of leucine . RPC-5 chromatography of late puusing different pal[‘* Clleucyl-tRNAs preparations of early and late pupal synthetases. Profiles of leucyl-tRNAs from late pupae are shown in Fig. 4. No major
"1
2j5
-r
20
10
I
20
--I
40
do
IdO
mM FIG. 2. Effect of ATP and MgCl, concentration on the initial rate of [Wlleucyl-tRNA formation. The assay conditions were as described under “Methods” except that varying ATP or MgCl, concentrations were used. Reactions were terminated after 2 min with 10% TCA and the precipitate collected on Millipore filters. m-m, ATP; O-O, M&l,.
FIG. 3. Effect of monovalent cation concentration on the initial rate of [Wlleucyl-tRNA formation. The assay conditions were as described under “Methods” except that varying concentrations of KCl, NaCl, and NH&l were used. Reactions were terminated after 2 min with 10% TCA and the precipitate collected on Millipore filters. O-O, NH&l; A-A, NaCl; D-M, KCl.
Lassa~,
TABLE
LERER
AND WHITE
Leucine tRNAs
1
ACCEPTANCE OF AMINO ACIDS BY tRNA FROM DIFFERENT DEVELOPMENTAL STAGES” Amino
acid
Picomoles of amino acid accepted per AIM) unit of total tRNA Larval
Firstday pupal
Last&Y pupal
55.3 26.3
55.1 25.4
Ratio of first-day pupal:lastday pupal
44.3 17.7
Tenebrio
271
min, and the 13Hlleucyl-tRNA was extracted as described in Materials and Methods. The chromatographic profiles obtained are virtually identical and are the same as the [‘4C]leucyl-tRNAs aminoacylated in vitro (Fig. 7).
Adult
DISCUSSION
Leucine
in Developing
38.1 15.2
1.07 1.02
Phenylalanine Tyrosine 19.3 24.7 22.7 15.8 1.09 a The aminoacylation conditions are described in “Methods.” The aminoacyl-tRNA synthetase preparation used was from last-day pupae.
qualitative or quantitative differences were observed using the different synthetase preparations. Rates and extent of [14C]leucyl-tRNA formation under limiting conditions. As the concentration of [14Clleucine in the reaction mixture was reduced below 25 @, the rate and extent of aminoacylation dropped markedly (Fig. 5a). A reduction in the amount of enzyme resulted in a similar decrease in the rate and extent of [I4 Clleucyl-tRNA formation (Fig. 5b). Suboptimal cation and ATP conditions also significantly decreased the rate and extent of [I4 Clleucyl-tRNA formation (Fig. 52). RPC-5 chromatography of [I4 C]leucyltRNAs charged under suboptimal conditions. Because the extent of [I4 ClleucyltRNA formation was markedly reduced under various suboptimal conditions, it was of interest to determine whether this affected all of the leucine tRNAs equally. The chromatographic profiles of [I4 Clleucyl-tRNAs aminoacylated under various suboptimal conditions are shown in Fig. 6. Transfer RNAIL”” and tRNAZLeU are aminoacylated relatively poorly when compared to tRNAsk”. RPC-5 chromatography of [3H]leucyltRNAs aminoacylated in vivo. First- and last-day pupae as well as larvae and adults were injected with [3H]leucine and left 3 -5
The potential of a translational control mechanism mediated by tRNA has been recognized for a long time (Ames and Hartman, 1965; Stent, 1965). Studies on the mealworm T. molitor appeared to provide the clearest demonstration of such a control mechanism in a eukaryotic system (Ilan, 1966,1969; Ilan et al., 1970,1972; Ilan and Ilan, 1973, 1974, 1975a,b). These reports indicated that the mRNAs for adult cuticular proteins were synthesized in early pupae but were not translated until the late pupal stage (several days later) when a major new leucine tRNA and a new or modified leucyl-tRNA synthetase appeared. Indirect examination by chromatography of RNAse T1 digests of labeled leucyltRNAs suggested that the new tRNA was the major leucine isoacceptor in late pupae (Ilan and Ilan, 1975a). Previous studies (Ilan et al., 1970) also indicated that there was a substantial increase in total leucine tRNAs during pupal development. Using the optimized aminoacylating conditions described here, no significant changes were found during development in the amount of leucine tRNA relative to phenylalanine or tyrosine. The possibility that the previously reported change in leucine tRNA may be qualitative rather than quantitative was investigated by RPC-5 chromatography (Lassam et al., 1975). No significant changes were found in the individual leucine isoaccepting tRNAs. Even if a new leucine tRNA cochromatographed with a preexisting one, a quantitative change would be expected in the profile. No such change was observed. Quantitative analysis of tRNAs requires complete and rapid aminoacylation. Sub-
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DEVELOPMENTAL
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VOLUME
a
49,
1976
b 40-
G ci 0; 5
3
1
I’ 80
120
Fraction
140
no.
FIG. 4. Chromatography of late-pupal [W]leucyl-tRNAs aminoacylated with different synthetase preparations. Elution was by loo-ml linear gradients from 0.55 to 0.675 M NaCl on jacketed columns (13 x 0.9 cm). The flow rate was 15 ml/hr with 0.5-ml fractions being collected. (a) Late pupal aminoacyl-tRNA synthetase prepared from 105,OOOgsupernatant as described in “Methods.” (b) Late pupal aminoacyl-tRNA synthetase prepared from “microsomal wash” (Ilan 1968). (cl Early pupal aminoacyl-tRNA synthetase prepared from the 105,OOOgsupernatant.
optimal aminoacylation conditions result in incomplete charging with the tRNAIL’” less and tRNAzL”” becoming substantially charged than tRNASL”“. If this situation did arise during the in vitro translation of mRNA, an apparent translational control mechanism could result. However, the inability of the mRNAs to be translated would be due not to the absence of specific tRNAs but to the poor aminoacylation of these tRNAs.
The translational control mechanism proposed for the synthesis of adult cuticular proteins of Tenebrio is dependent not only on a novel tRNA appearing in late pupae but also on the appearance of a new or modified leucyl-tRNA synthetase. This synthetase has been reported to be identical to the one present in early pupae with the exception that only the synthetase from late pupae can aminoacylate the new major tRNA (Ilan and Ilan, 1975a). The
Leucine tRNAs
LASSAM, LERER AND WHITE
a
50pM .
,*/(-•-*
in Developing
273
Tenebrio
1-D075mghl .e= r;, 0 I / 0.50
;3-
mgml
l
.
;
.
2 I
/
d y
2-
; E-
.
3 “J
0 25m9ld
l-
E
.
I k.
min
min
properties of the 1eucyLtRNA synthetase used in these experiments were compared with those reported for the purified Tenebrio leucyl-tRNA synthetase (Ilan and Ilan, 1975a). They were found to be considerably different in several respects. The optimum Mg:ATP ratio was reported to be 2.5 (Ilan and Ilan, 1975a). The present study found the optimum ratio to be 0.63, which compares favorably with those previously described for Drosophila (White and Tener, 1973) and mammalian erythrocyte (Smith and McNamara, 1971) leucyltRNA synthetase systems. The apparent K, for leucine was found to be 22 @, which is 10 times lower than that reported
FIG. 5. Effect of various conditions on the rates and extent of [Wlleucyl-tRNA formation. The assay conditions were as described under “Methods” except where otherwise indicated. The rate of aminoacylation was followed by the method of Bollum (1959). (a) Effect of varying the concentration of [Wlleucine. (b) Effect of varying the concentration of the aminoacyl-tRNA synthetases. (c) m----m, complete; A-A, minus KCl; O-O, 10 mM MgCl, and 4 mkf ATP; O---Cl,10 n&f MgCl,, 4 mkf ATP, and 50 mM imidazole pH 7.0.
for the purified enzyme (Ilan and Ilan, 1975a). No significant differences were observed in leucyl-tRNA synthetases extracted from different developmental stages. A possible explanation for the inconsistencies between the data presented here for the leucyl-tRNA synthetase and those previously published (Ilan and Ilan, 1975a) may be due to differences in the method of preparing the enzyme extracts. Ilan and Ilan (1975a) purified the enzyme from the microsomal pellet while the enzyme used in this study was extracted from the 105,OOOg supernatant. We found that while the properties of enzyme from the
274
DEVELOPMENTAL
BIOL~Y
microsomal pellet were the same as that extracted from the supernatant, the supernatant extraction gave a much greater yield.
VOLUME
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1976
The chromatographic profiles of the [3Hlleucyl-tRNAs aminoacylated in viuo are the same as those obtained by in vitro charging of tRNA. This provides good evi-
b 30-
20
(j
16
16
12
a-
/
a0
Fraction
I
I
120
I
I
160
no.
6. Chromatography of [W]leucyl-tRNAs aminoacylated under suboptimal conditions. (a) Complete. (b) 10 n&f MgCl,, 4 m&f ATP, and 50 mM imidazole pH 7.0. (c) 2 @f [“Clleucine. (d) 0.25 mg/ml aminoacyltRNA synthetase preparation. FIG.
LASSAM, LERER AND WHITE
Leucine tRNAs
15
in Developing
Tenebrio
275
b
10
5-
,-5 z m k
15
d
cb 4 X E: Ii
10
cj
5
Fraction
no.
FIG. 7. Chromatography of [3Hlleucyl-tRNAs aminoacylated in viuo. Pupae were injected with [3Hlleucine and the [3H]leucyl-tRNA extracted as described in the “Methods.” Elution was by 500-ml linear gradients with 0.55-0.675 M NaCl on jacketed columns (50 x 1.25 cm). The flow rate was 75 ml/hr with 2.5ml fractions being collected. (a) Larval; (b) first-day pupal; (c) last-day pupal (pharate adult); (d) adult.
dence that the procedures for the isolation of tRNA and aminoacyl-tRNA synthetases as well as the assay conditions for the aminoacylation of the tRNA in vitro yield results that truly reflect the situation in vivo. Experiments analyzing the effects of actinomycin D on the development and RNA synthesis of Tenebrio (Ilan et al., 1966) have implied that the mRNAs for adult cuticular proteins are synthesized
early in the pupal stage but not translated until later in pupal development. However, as injection of actinomycin D into pupae inhibited only 51-67% of labeled uridine incorporation into RNA (Ilan et al., 19661, it cannot be concluded that the synthesis of mRNA coding for the adult cuticular proteins was blocked. It has been claimed by Ilan et al. (1968; 19741, apparently on the basis of published analyses on the beetle Agrianome spini-
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DEVELOPMENTAL BIOLOGY
collis (Hackman and Goldberg, 19581, that adult Tenebrio cuticules are high in tyrosine (20%), whereas pupal cuticles have “low tyrosine” content. This difference has been utilized (Ilan et al., 1970) as an index of the synthesis of adult cuticular proteins in vitro. It appears from the more recent work of Pate1 (1972) as well as Anderson et al. (1973) that adult cuticular proteins have in fact less tyrosine than pupal cuticle, thus invalidating the use of high tyrosine content as an index of adult cuticular protein synthesis. The translational control mechanism that has been proposed for Tenebrio (Ilan, 1969; Ilan et al., 1970, 1972; Ilan and Ilan 1974, 1975a,b) rests on three major points: (1) the synthesis of the mRNAs for adult cuticular proteins early in pupation, (2) a high tyrosine content of the adult cuticular proteins that can be used as an index of the synthesis of these proteins, and (3) the appearance of a new leucine tRNA and a new or modified leucyl-tRNA synthetase late in pupation. It would appear from the data and discussion presented here that the proposed mechanism of translational control of adult cuticular proteins in Tenebrio molitor must be seriously questioned. REFERENCES AMES, B., AND HARTMAN, P. (1963). The his operon. Cold Spring Harb. Symp. Quant. Biol. 28, 349355. ANDERSON, S. O., CHASE, A. M., AND WILLIS, J. H. (1973). The amino acid composition of cuticles from Tenebrio molitor with special reference to the action of juvenile hormone. Insect Biochem. 3, 171-180. BOLLUM, R. G. (1959). Thermal conversion of nonpriming deoxyribonucleic acid to primer. J. Biol. Chem. 234, 2133-2734. GRIGLIATTI, T. A., WHITE, B. N., TENER, G. M., KAUFMAN, T. C., HOLDEN, J. H., AND SUZUKI, D. T. (1974). Studies on the tRNA genes of Drosophila. Cold Spring Harbor Symp. Quant. Biol. 38, 461-474. HACKMAN, R. H., AND GOLDBERG, M. (1958). Proteins of the larval cuticle of Agrianome spinicollis (Coleoptera). J. Insect Phys. 2, 221-231. ILAN, J., ILAN, J., AND QUASTEL, J. H. (1966). Effects
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of actinomycin D on nucleic acid metabolism and protein biosynthesis during metamorphosis of Tenebrio molitor. Biochem. J. 100, 441-447. ILAN, J. (1968). Amino acid incorporation and aminoacyl transfer in an insect pupal system. J. Biol. Chem. 243, 58594866. ILAN, J. (1969). The role of tRNA in translational control of specific mRNA during insect metamorphosis. Cold Spring Harb. Symp. Quant. Biol. 34, 787-791. ILAN, J., ILAN, J., AND PATEL, N. G. (1970). Mechanism of gene expression in Tenebrio molitor. Juvenile hormone determination of translational control through transfer ribonucleic acid and enzyme. J. Biol. Chem. 245, 1275-1281. ILAN, J., ILAN, J., AND PATEL, N. G. (1972). Regulation of messenger RNA translation on mediated by juvenile hormone, In “Insect Juvenile Hormones Chemistry and Action” (J. J. Menn and M. Beroza, eds.), pp. 43-68. Academic Press, New York. ILAN, J., AND ILAN, J. (1973). Protein synthesis and insect morphogenesis. Ann. Rev. Ent. 18, 167-181. ILAN, J., AND ILAN, J. (1974). Protein synthesis in insects, In “Physiology of Insecta” (M. Rockstein, ed.), Vol. 4, pp. 355-422. Academic Press, New York. ILAN, J., AND ILAN, J. (1975a). Similarities in properties and a functional difference in purified leucyl-tRNA synthetase isolated from two developmental stages of Tenebrio molitor. Develop. Biol. 42, 64-74. ILAN, J., AND ILAN, J. (1975b). Regulation of messenger RNA translation during insect development. Curr. Top. Develop. Biol. 9, (in press). LASSAM, N. J., LERER, H., AND WHITE, B. N. (1975). Translational control in the mealworm, Tenebrio molitor. Nature (London) 256, 734-735. PATEL, N. G. (1972). Hormonal regulation of protein synthesis in insects. In “Molecular Genetic Mechanism in Development and Aging” (M. Rockstein and G. T. Baber, eds.), pp. 145-198. Academic Press, New York. PATTERSON, P. (1957). Quantitative and qualitative changes observed in the free alpha amino nitrogen fraction of Tenebrio molitor. J. Mol. Biol. 65, 729735. PEARSON, R. L., WEISS, J. F., AND KELMERS, A. D. (1971). Improved separation of transfer RNA’s on polychlorotrifluoroethylene - supported reversedphase chromatography columns. Biochem. Riophys. Acta 228, 770-774. SMITH, D. W. E., AND MCNAMARA, A. L. (1971). Specialization of rabbit reticulocyte transfer RNA content for hemoglobin synthesis. Science 171, 571-579. STENT, G. (1964). The operon. Science 144, 816-820. WHITE, B. N., TENER, G. M., HOLDEN, J., AND Su-
LASSAM,
LERER
AND
WHITE
Leucine tRNAs
D. T. (1973a). Activity of a transfer RNA modifying enzyme during the development ofDrosophilu and its relationship to the sub) locus. J. Mol. Biol. 74, 635-651. WHITE, B. N., ANDTENER, G. M. (1973). Chromatography of Drosophila tRNA on BD-cellulose. ZUKI,
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Canad. J. Biochem. 51, 896-902. W., AND NOVELLI, G. D. (1968). Isoaccepting tRNAs in mouse plasma cell tumors that synthesize different myeloma protein. Biochem. Biophys. Res. Commun. 31, 534-539.
YANG,
BIOLOGY
@,268-277(1976)
A Reexamination of the Leucine tRNAs and the Leucyl-tRNA Synthetase in Developing Tenebrio molitor NORMAN J. LASSAM, HARVEY Department
LERER,
of Biology, Queen’s University,
AND
BRADLEY
Kingston,
Ontario,
N. WHITE Canada
Accepted October 16, 1975 The translational control mechanism previously proposed for the synthesis of adult cuticular proteins in Tenebrio molitor is dependent upon the appearance of a major, novel leucine tRNA and a change in leucyl-tRNA synthetase activity just prior to adult emergence. The properties of the leucyl-tRNA synthetase extracted from pupae were reexamined. Under optimal aminoacylation conditions, no new leucine isoaccepting tRNAs were observed during development. However, under suboptimal conditions, a differential charging of the leucine tRNA species was noted. The chromatographic profiles of leucyl-tRNAs aminoacylated in vivo in both early and late pupae were found to be the same and were identical to the profiles obtained by charging tRNAs in vitro. Previous evidence for a translational control system operating in Tenebrio is discussed in relation to these results.
al., 1975). No significant differences were found. However, under suboptimal conditions, differential extents of aminoacylation of the leucine tRNAs in vitro were observed. Examination of the leucyltRNAs charged in vivo confirmed the results obtained in vitro in that no new leutine tRNA was detectable.
INTRODUCTION
Extensive studies on the mealworm Tenebrio molitor have indicated that the synthesis of adult cuticular proteins is under translational control (Ilan et al., 1966; Ilan et al., 1970; Ilan and Ilan, 1975a). This control mechanism appeared to be mediated by a new or modified leucyl-tRNA synthetase and a major novel leucine tRNA species, the synthesis of which seemed to be under the control of juvenile hormone (Ilan et al., 1972). Changes in aminoacyl-tRNAs during the development of Drosophila have been observed (White et al., 1973a,b; Grigliatti et al., 1974) but have not been correlated with any developmental control mechanism. The Tenebrio system appeared to be ideally suited for a detailed examination of a control mechanism mediated by a tRNA. We soon found that the conditions previously reported (Ilan et al., 1970) for the aminoacylation of leucine tRNAs were far from optimal and therefore we reexamined the properties of the leucyl-tRNA synthetase. Using improved aminoacylation conditions, the leucyl-tRNAs from the different developmental stages of Tenebrio were compared as reported before (Lassam et
MATERIALS
268 Copyright 6 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
METHODS
Materials. Labeled amino acids and Aquasol were obtained from New England Nuclear Corp. Adogen 464, a trialkylmethylammonium chloride with the predominant chain length of the alkyl groups being C8-Co, was a gift from Ashland Chemical Co., Columbua, Ohio. Plaskon CTFE2300 powder was a gift from Allied Chemical Corp., Morristown, N. J. Growth of Tenebrio. T. molitor larvae were obtained from Carolina Biological Supply Co., Burlington, N. C., and reared at 29°C according to the method of Patterson (1957). Under these conditions the pupal period lasted for 6 days, with adults emerging on the seventh. Isolation of tRNA. Transfer RNA was extracted with phenol directly from whole organisms by the methods described previously for Drosophila (White et al., 1973a).
LASSAM,
LERER
AND
WHITE
Leucine tRNAs
Preparation of aminoacyl-tRNA synthetases. Aminoacyl-tRNA synthetases were normally prepared from the 105,OOOg (90 min) supernatant by the methods described previously for Drosophila (White et al., 1973a), with the addition that 1 mM phenylthiourea was added to all of the extraction buffers. A “microsomal wash” aminoacyl-tRNA synthetase prepared according to the method of Ilan (1968) was also used. of tRNA in witro. Aminoacylation Transfer RNA was aminoacylated at 30°C in a final reaction volume of 0.2 ml. For leucine, each milliliter of the reaction mixture contained: Tris-HCl, 50 pmoles (pH 7.5); 2-mercaptoethanol, 5 pmoles; MgC12, 5 pmoles; ATP, 8 pmoles; [14Clamino acid, 50 pmoles; KCl, 10 pmoles; the aminoacyltRNA synthetase preparation contained 1 mg of protein and tRNA, 0.3-0.8 mg. For phenylalanine and tyrosine, the conditions were the same except that each milliliter of reaction mixture contained: MgCl*, 10 pmoles; ATP, pmoles; [14Clamino acid, 25 Fmoles; KCl, 50 pmoles. The rate of the aminoacylation reaction was followed by pipeting aliquots on Whatman 3MM filter paper disks and removing acid-soluble radioactive amino acids (Bollum, 1959). Initial rates of reaction were determined by stopping the reaction at 2 min by the addition of cold 10% TCA and collecting the precipitate on Millipore filters. Aminoacyl-tRNA for reversed-phase chromatography was isolated from the components of the reaction mixture by DEAE-cellulose chromatography as described by Yang and Novelli (1968). Aminoacylation of tRNA in uiuo. Approximately 25 pupae were injected with 5 &i (in approximately 5 ~1) each of l3 Hlleucine (50 Ci/pmole) and left 3-5 min. The pupae were then homogenized in equal volumes of 88% phenol and a buffer containing 0.25 M NaCl, 0.01 M MgCl*, 0.001 M 2-mercaptoethanol, and 0.01 M sodium acetate, pH 4.5. The aqueous
in Developing
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269
phase was removed and the phenol was reextracted with an equal volume of buffer: The pooled aqueous phases were applied to a DEAE-cellulose column equilibrated with the same buffer. The column was washed with this buffer and the [3 Hlleucyl-tRNA was eluted with the buffer containing 0.75 M NaCl. This material was diluted to 0.375 M NaCl and ap plied to the RPC-5 columns. Reversed-phase chromatography of aminoacyl-tRNA. The RPC-5 system of Pearson, Weiss, and Kelmers (1971) was used. The Plaskon CTFE was coated with Adogen 464 as described elsewhere (White et al., 1973a). The 0.9 x 14-cm columns were developed at 37°C with a linear loo-ml NaCl gradient containing 0.01 M MgC&, 0.01 M sodium acetate (pH 4.5), and 1 n&f X-mercaptoethanol. The 1.25 x 50-cm columns were developed at 37°C with a linear 500-ml NaCl gradient. Radioactivity was determined in the fractions by the addition of 5 volumes of Aquasol and counting in a scintillation counter. RESULTS
Properties of the leucyl-tRNA synthetase from last-day pupae; apparent K, for leutine. The effect of varying the concentration of [I4 Clleucine on the formation of 114Clleucyl-tRNA catalyzed by the synthetase was studied (Fig. 1). From a Lineweaver-Burk plot of the data the apparent K, for leucine was found to 22 x lo-” M. This compares with an apparent K, of 2 x lo-” M of phenylalanine for the Tenebrio phenylalanyl-tRNA synthetase (unpublished results). Magnesium and ATP optima. The effect of varying the ATP concentration at constant MgCl, (5 mM) on the initial rate of [14Clleucyl-tRNA formation is shown in Fig. 2. A sharp peak at 8 mM ATP was found. The effect of varying the MgCl, concentration at constant ATP (8 m&f) on the initial rate of [I4 Clleucyl-tRNA formation is also shown in Fig. 2. Again a relatively sharp peak at 5 mM MgCl, was found.
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Monovalent cation requirement. The effect of varying the concentration of KCl, NaCl, and NH&l on the initial rate of [l* Clleucyl-tRNA formation is shown in
FIG. 1. Variation of [Wlleucine concentration in the esterification reaction. The assay conditions were as described under “Methods” except that varying concentrations of [Wlleucine were used. Reactions were terminated after 2 min with 10% TCA and the precipitate collected on Millipore tilters. The reaction velocity V is expressed as cpm incorporated/2-min reaction time; substrate concentration (S) is expressed as micromoles [“C]leucine.
VOLUME 49, 1976
Fig. 3. A broad optimum of 8-20 mM KC1 was found. Sodium chloride was not as effective as KCl, while NH4Cl markedly stimulated the initial rate at relatively low concentrations. pH optimum. The effect of varying the pH of Tris-HCl buffer on the initial rate of 11*Clleucyl-tRNA formation gave the optimum of pH 8.5, as found by Ilan et al. (1975). Acceptance of [‘*Clamino acids by tRNA from the developmental stages. The quantitative levels of tyrosine, phenylalanine, and leucine tRNAs at the different developmental stages is shown in Table 1. Relative to tyrosine and phenylalanine, there is little change in the acceptance of leucine . RPC-5 chromatography of late puusing different pal[‘* Clleucyl-tRNAs preparations of early and late pupal synthetases. Profiles of leucyl-tRNAs from late pupae are shown in Fig. 4. No major
"1
2j5
-r
20
10
I
20
--I
40
do
IdO
mM FIG. 2. Effect of ATP and MgCl, concentration on the initial rate of [Wlleucyl-tRNA formation. The assay conditions were as described under “Methods” except that varying ATP or MgCl, concentrations were used. Reactions were terminated after 2 min with 10% TCA and the precipitate collected on Millipore filters. m-m, ATP; O-O, M&l,.
FIG. 3. Effect of monovalent cation concentration on the initial rate of [Wlleucyl-tRNA formation. The assay conditions were as described under “Methods” except that varying concentrations of KCl, NaCl, and NH&l were used. Reactions were terminated after 2 min with 10% TCA and the precipitate collected on Millipore filters. O-O, NH&l; A-A, NaCl; D-M, KCl.
Lassa~,
TABLE
LERER
AND WHITE
Leucine tRNAs
1
ACCEPTANCE OF AMINO ACIDS BY tRNA FROM DIFFERENT DEVELOPMENTAL STAGES” Amino
acid
Picomoles of amino acid accepted per AIM) unit of total tRNA Larval
Firstday pupal
Last&Y pupal
55.3 26.3
55.1 25.4
Ratio of first-day pupal:lastday pupal
44.3 17.7
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271
min, and the 13Hlleucyl-tRNA was extracted as described in Materials and Methods. The chromatographic profiles obtained are virtually identical and are the same as the [‘4C]leucyl-tRNAs aminoacylated in vitro (Fig. 7).
Adult
DISCUSSION
Leucine
in Developing
38.1 15.2
1.07 1.02
Phenylalanine Tyrosine 19.3 24.7 22.7 15.8 1.09 a The aminoacylation conditions are described in “Methods.” The aminoacyl-tRNA synthetase preparation used was from last-day pupae.
qualitative or quantitative differences were observed using the different synthetase preparations. Rates and extent of [14C]leucyl-tRNA formation under limiting conditions. As the concentration of [14Clleucine in the reaction mixture was reduced below 25 @, the rate and extent of aminoacylation dropped markedly (Fig. 5a). A reduction in the amount of enzyme resulted in a similar decrease in the rate and extent of [I4 Clleucyl-tRNA formation (Fig. 5b). Suboptimal cation and ATP conditions also significantly decreased the rate and extent of [I4 Clleucyl-tRNA formation (Fig. 52). RPC-5 chromatography of [I4 C]leucyltRNAs charged under suboptimal conditions. Because the extent of [I4 ClleucyltRNA formation was markedly reduced under various suboptimal conditions, it was of interest to determine whether this affected all of the leucine tRNAs equally. The chromatographic profiles of [I4 Clleucyl-tRNAs aminoacylated under various suboptimal conditions are shown in Fig. 6. Transfer RNAIL”” and tRNAZLeU are aminoacylated relatively poorly when compared to tRNAsk”. RPC-5 chromatography of [3H]leucyltRNAs aminoacylated in vivo. First- and last-day pupae as well as larvae and adults were injected with [3H]leucine and left 3 -5
The potential of a translational control mechanism mediated by tRNA has been recognized for a long time (Ames and Hartman, 1965; Stent, 1965). Studies on the mealworm T. molitor appeared to provide the clearest demonstration of such a control mechanism in a eukaryotic system (Ilan, 1966,1969; Ilan et al., 1970,1972; Ilan and Ilan, 1973, 1974, 1975a,b). These reports indicated that the mRNAs for adult cuticular proteins were synthesized in early pupae but were not translated until the late pupal stage (several days later) when a major new leucine tRNA and a new or modified leucyl-tRNA synthetase appeared. Indirect examination by chromatography of RNAse T1 digests of labeled leucyltRNAs suggested that the new tRNA was the major leucine isoacceptor in late pupae (Ilan and Ilan, 1975a). Previous studies (Ilan et al., 1970) also indicated that there was a substantial increase in total leucine tRNAs during pupal development. Using the optimized aminoacylating conditions described here, no significant changes were found during development in the amount of leucine tRNA relative to phenylalanine or tyrosine. The possibility that the previously reported change in leucine tRNA may be qualitative rather than quantitative was investigated by RPC-5 chromatography (Lassam et al., 1975). No significant changes were found in the individual leucine isoaccepting tRNAs. Even if a new leucine tRNA cochromatographed with a preexisting one, a quantitative change would be expected in the profile. No such change was observed. Quantitative analysis of tRNAs requires complete and rapid aminoacylation. Sub-
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DEVELOPMENTAL
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VOLUME
a
49,
1976
b 40-
G ci 0; 5
3
1
I’ 80
120
Fraction
140
no.
FIG. 4. Chromatography of late-pupal [W]leucyl-tRNAs aminoacylated with different synthetase preparations. Elution was by loo-ml linear gradients from 0.55 to 0.675 M NaCl on jacketed columns (13 x 0.9 cm). The flow rate was 15 ml/hr with 0.5-ml fractions being collected. (a) Late pupal aminoacyl-tRNA synthetase prepared from 105,OOOgsupernatant as described in “Methods.” (b) Late pupal aminoacyl-tRNA synthetase prepared from “microsomal wash” (Ilan 1968). (cl Early pupal aminoacyl-tRNA synthetase prepared from the 105,OOOgsupernatant.
optimal aminoacylation conditions result in incomplete charging with the tRNAIL’” less and tRNAzL”” becoming substantially charged than tRNASL”“. If this situation did arise during the in vitro translation of mRNA, an apparent translational control mechanism could result. However, the inability of the mRNAs to be translated would be due not to the absence of specific tRNAs but to the poor aminoacylation of these tRNAs.
The translational control mechanism proposed for the synthesis of adult cuticular proteins of Tenebrio is dependent not only on a novel tRNA appearing in late pupae but also on the appearance of a new or modified leucyl-tRNA synthetase. This synthetase has been reported to be identical to the one present in early pupae with the exception that only the synthetase from late pupae can aminoacylate the new major tRNA (Ilan and Ilan, 1975a). The
Leucine tRNAs
LASSAM, LERER AND WHITE
a
50pM .
,*/(-•-*
in Developing
273
Tenebrio
1-D075mghl .e= r;, 0 I / 0.50
;3-
mgml
l
.
;
.
2 I
/
d y
2-
; E-
.
3 “J
0 25m9ld
l-
E
.
I k.
min
min
properties of the 1eucyLtRNA synthetase used in these experiments were compared with those reported for the purified Tenebrio leucyl-tRNA synthetase (Ilan and Ilan, 1975a). They were found to be considerably different in several respects. The optimum Mg:ATP ratio was reported to be 2.5 (Ilan and Ilan, 1975a). The present study found the optimum ratio to be 0.63, which compares favorably with those previously described for Drosophila (White and Tener, 1973) and mammalian erythrocyte (Smith and McNamara, 1971) leucyltRNA synthetase systems. The apparent K, for leucine was found to be 22 @, which is 10 times lower than that reported
FIG. 5. Effect of various conditions on the rates and extent of [Wlleucyl-tRNA formation. The assay conditions were as described under “Methods” except where otherwise indicated. The rate of aminoacylation was followed by the method of Bollum (1959). (a) Effect of varying the concentration of [Wlleucine. (b) Effect of varying the concentration of the aminoacyl-tRNA synthetases. (c) m----m, complete; A-A, minus KCl; O-O, 10 mM MgCl, and 4 mkf ATP; O---Cl,10 n&f MgCl,, 4 mkf ATP, and 50 mM imidazole pH 7.0.
for the purified enzyme (Ilan and Ilan, 1975a). No significant differences were observed in leucyl-tRNA synthetases extracted from different developmental stages. A possible explanation for the inconsistencies between the data presented here for the leucyl-tRNA synthetase and those previously published (Ilan and Ilan, 1975a) may be due to differences in the method of preparing the enzyme extracts. Ilan and Ilan (1975a) purified the enzyme from the microsomal pellet while the enzyme used in this study was extracted from the 105,OOOg supernatant. We found that while the properties of enzyme from the
274
DEVELOPMENTAL
BIOL~Y
microsomal pellet were the same as that extracted from the supernatant, the supernatant extraction gave a much greater yield.
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1976
The chromatographic profiles of the [3Hlleucyl-tRNAs aminoacylated in viuo are the same as those obtained by in vitro charging of tRNA. This provides good evi-
b 30-
20
(j
16
16
12
a-
/
a0
Fraction
I
I
120
I
I
160
no.
6. Chromatography of [W]leucyl-tRNAs aminoacylated under suboptimal conditions. (a) Complete. (b) 10 n&f MgCl,, 4 m&f ATP, and 50 mM imidazole pH 7.0. (c) 2 @f [“Clleucine. (d) 0.25 mg/ml aminoacyltRNA synthetase preparation. FIG.
LASSAM, LERER AND WHITE
Leucine tRNAs
15
in Developing
Tenebrio
275
b
10
5-
,-5 z m k
15
d
cb 4 X E: Ii
10
cj
5
Fraction
no.
FIG. 7. Chromatography of [3Hlleucyl-tRNAs aminoacylated in viuo. Pupae were injected with [3Hlleucine and the [3H]leucyl-tRNA extracted as described in the “Methods.” Elution was by 500-ml linear gradients with 0.55-0.675 M NaCl on jacketed columns (50 x 1.25 cm). The flow rate was 75 ml/hr with 2.5ml fractions being collected. (a) Larval; (b) first-day pupal; (c) last-day pupal (pharate adult); (d) adult.
dence that the procedures for the isolation of tRNA and aminoacyl-tRNA synthetases as well as the assay conditions for the aminoacylation of the tRNA in vitro yield results that truly reflect the situation in vivo. Experiments analyzing the effects of actinomycin D on the development and RNA synthesis of Tenebrio (Ilan et al., 1966) have implied that the mRNAs for adult cuticular proteins are synthesized
early in the pupal stage but not translated until later in pupal development. However, as injection of actinomycin D into pupae inhibited only 51-67% of labeled uridine incorporation into RNA (Ilan et al., 19661, it cannot be concluded that the synthesis of mRNA coding for the adult cuticular proteins was blocked. It has been claimed by Ilan et al. (1968; 19741, apparently on the basis of published analyses on the beetle Agrianome spini-
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DEVELOPMENTAL BIOLOGY
collis (Hackman and Goldberg, 19581, that adult Tenebrio cuticules are high in tyrosine (20%), whereas pupal cuticles have “low tyrosine” content. This difference has been utilized (Ilan et al., 1970) as an index of the synthesis of adult cuticular proteins in vitro. It appears from the more recent work of Pate1 (1972) as well as Anderson et al. (1973) that adult cuticular proteins have in fact less tyrosine than pupal cuticle, thus invalidating the use of high tyrosine content as an index of adult cuticular protein synthesis. The translational control mechanism that has been proposed for Tenebrio (Ilan, 1969; Ilan et al., 1970, 1972; Ilan and Ilan 1974, 1975a,b) rests on three major points: (1) the synthesis of the mRNAs for adult cuticular proteins early in pupation, (2) a high tyrosine content of the adult cuticular proteins that can be used as an index of the synthesis of these proteins, and (3) the appearance of a new leucine tRNA and a new or modified leucyl-tRNA synthetase late in pupation. It would appear from the data and discussion presented here that the proposed mechanism of translational control of adult cuticular proteins in Tenebrio molitor must be seriously questioned. REFERENCES AMES, B., AND HARTMAN, P. (1963). The his operon. Cold Spring Harb. Symp. Quant. Biol. 28, 349355. ANDERSON, S. O., CHASE, A. M., AND WILLIS, J. H. (1973). The amino acid composition of cuticles from Tenebrio molitor with special reference to the action of juvenile hormone. Insect Biochem. 3, 171-180. BOLLUM, R. G. (1959). Thermal conversion of nonpriming deoxyribonucleic acid to primer. J. Biol. Chem. 234, 2133-2734. GRIGLIATTI, T. A., WHITE, B. N., TENER, G. M., KAUFMAN, T. C., HOLDEN, J. H., AND SUZUKI, D. T. (1974). Studies on the tRNA genes of Drosophila. Cold Spring Harbor Symp. Quant. Biol. 38, 461-474. HACKMAN, R. H., AND GOLDBERG, M. (1958). Proteins of the larval cuticle of Agrianome spinicollis (Coleoptera). J. Insect Phys. 2, 221-231. ILAN, J., ILAN, J., AND QUASTEL, J. H. (1966). Effects
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of actinomycin D on nucleic acid metabolism and protein biosynthesis during metamorphosis of Tenebrio molitor. Biochem. J. 100, 441-447. ILAN, J. (1968). Amino acid incorporation and aminoacyl transfer in an insect pupal system. J. Biol. Chem. 243, 58594866. ILAN, J. (1969). The role of tRNA in translational control of specific mRNA during insect metamorphosis. Cold Spring Harb. Symp. Quant. Biol. 34, 787-791. ILAN, J., ILAN, J., AND PATEL, N. G. (1970). Mechanism of gene expression in Tenebrio molitor. Juvenile hormone determination of translational control through transfer ribonucleic acid and enzyme. J. Biol. Chem. 245, 1275-1281. ILAN, J., ILAN, J., AND PATEL, N. G. (1972). Regulation of messenger RNA translation on mediated by juvenile hormone, In “Insect Juvenile Hormones Chemistry and Action” (J. J. Menn and M. Beroza, eds.), pp. 43-68. Academic Press, New York. ILAN, J., AND ILAN, J. (1973). Protein synthesis and insect morphogenesis. Ann. Rev. Ent. 18, 167-181. ILAN, J., AND ILAN, J. (1974). Protein synthesis in insects, In “Physiology of Insecta” (M. Rockstein, ed.), Vol. 4, pp. 355-422. Academic Press, New York. ILAN, J., AND ILAN, J. (1975a). Similarities in properties and a functional difference in purified leucyl-tRNA synthetase isolated from two developmental stages of Tenebrio molitor. Develop. Biol. 42, 64-74. ILAN, J., AND ILAN, J. (1975b). Regulation of messenger RNA translation during insect development. Curr. Top. Develop. Biol. 9, (in press). LASSAM, N. J., LERER, H., AND WHITE, B. N. (1975). Translational control in the mealworm, Tenebrio molitor. Nature (London) 256, 734-735. PATEL, N. G. (1972). Hormonal regulation of protein synthesis in insects. In “Molecular Genetic Mechanism in Development and Aging” (M. Rockstein and G. T. Baber, eds.), pp. 145-198. Academic Press, New York. PATTERSON, P. (1957). Quantitative and qualitative changes observed in the free alpha amino nitrogen fraction of Tenebrio molitor. J. Mol. Biol. 65, 729735. PEARSON, R. L., WEISS, J. F., AND KELMERS, A. D. (1971). Improved separation of transfer RNA’s on polychlorotrifluoroethylene - supported reversedphase chromatography columns. Biochem. Riophys. Acta 228, 770-774. SMITH, D. W. E., AND MCNAMARA, A. L. (1971). Specialization of rabbit reticulocyte transfer RNA content for hemoglobin synthesis. Science 171, 571-579. STENT, G. (1964). The operon. Science 144, 816-820. WHITE, B. N., TENER, G. M., HOLDEN, J., AND Su-
LASSAM,
LERER
AND
WHITE
Leucine tRNAs
D. T. (1973a). Activity of a transfer RNA modifying enzyme during the development ofDrosophilu and its relationship to the sub) locus. J. Mol. Biol. 74, 635-651. WHITE, B. N., ANDTENER, G. M. (1973). Chromatography of Drosophila tRNA on BD-cellulose. ZUKI,
in Developing
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Canad. J. Biochem. 51, 896-902. W., AND NOVELLI, G. D. (1968). Isoaccepting tRNAs in mouse plasma cell tumors that synthesize different myeloma protein. Biochem. Biophys. Res. Commun. 31, 534-539.
YANG,
