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An analysis of Q and Q∗ containing tRNAs during the development of Lucilia sericata, Musca domestica and Tenebrio molitor
Insect Biochem., Vol. 9, pp. 375 to 378. © Pergamon Press Ltd. 1979. Printed in Great Britain.
0020-1700/79/0701-0375 $02.00/0
AN ANALYSIS OF Q AND Q* CONTAINING tRNAs DURING THE DEVELOPMENT OF LUCILIA SERICATA, MUSCA DOMESTICA AND TENEBRIO MOLITOR BRADLEY N. WHITE and NORMANJ. LASSAM* The Group in Eukaryotic Molecular Biology and Evolution, Department of Biology, Queen's University, Kingston, Ontario, Canada (Received 24 October 1978)
Abstract--The chromatographic profiles of the family of tRNAs containing the hypermodifiednucleoside Q and Q* in the first position of their anticodons were examined in the third instar larvae and adults of Lucilia sericata and Musca domestica. The absolute amounts of these tRNAs remained constant during development relative to phenylalanine tRNA. In M. domestica only Q containing forms were found in both larvae and adults while in L. sericata, the larvae contained a significantportion of the Q lacking form. In Tenebrio molitor the Q lacking form of tyrosine tRNA appeared to be only significant in pupae. The developmentalpatterns of Q biosynthesisin the Diptera therefore range from almost entirely Q containing in M. domestica to entirely Q lacking in Drosophila raised under certain conditions. This suggests that this modification to the anticodon is not playing a common regulatory function in the development of these insects. Key Word Index: tRNA, nucleoside Q and Q*, Musca domestica, Lucilia sericata, Tenebrio molitor
INTRODUCTION DURING the development of Drosophila melanogaster there are marked changes in the family of tRNAs which contain the hypermodified nucleoside Q or Q* (WHITE et al., 1973a, b). The structure of Q has been shown to be a 7-(4, 5-cis-dehydroxy-l-cyclopenten-3yl-aminomethyl)-7-diazaguanosine (KASAI et al., 1975), whereas Q* is even further modified, having either a mannose or galactose residue at the 4 position of the cyclopenten diol (KASAI et al., 1976). Only tyrosine, histidine, asparagine or aspartic acid tRNAs have been found to contain Q or Q* and these nucleosides are always located in the first (5') position of the anticodon. These four tRNAs form a natural family in that they respond to codons of the variety XA c. Although the function of the hypermodification is not understood there is evidence that Q containing tRNAs preferentially bind to XAU or XAC codons (HARADA and NISHIMURA, 1972). In Drosophila, prior to pupation, the major forms of aspartic acid, asparagine, tyrosine and histidine tRNAs are those lacking Q or Q* while after pupation the forms containing Q are produced. There is considerable evidence that the 6 forms (containing Q or Q*) and ~ forms (lacking Q or Q*) (WHITEe t al., 1973a) are in fact transcribed from the same genes and are chromatographically distinct only because of the degree of post-transcriptional modification; these chromatographically distinct but genetically identical tRNAs have been termed homogenic (WHITE et al., 1973a). The developmental changes observed in Drosophila prompted an investigation into the universality of the . . . . . . . . . . . . . . . . . . .
process in other holometabolous insects, especially other Diptera. The family of Q-containing tRNAs and phenylalanine tRNA as a control were therefore examined in M u s c a domestica, Lucilia sericata "and Tenebrio molitor.
MATERIALS AND METHODS Materials.
Uniformly [14C]-labelled-aminoacids were obtained from New England Nuclear Corporation and Amersham/Searle (L-aspartic acid--203 mCi/mmol; L-histidine--302 mCi/mmol; L-phenylalanine~72 mCi/mmol; L-tyrosine-441 mCi/mmol; and L-asparagine--179 mCi/mmol). Aquasol was obtained from New England Corp. Adogen 464, a trialkylmethylammonium chloride with the predominant chain length of the alkyl groups being Cs-Clo, was a gift from Ashland Chemical Co., Columbus, OH. Plaskon CTFE 2300 powder was a gift from Allied Chemical Corp., Morristown, NY. Growth of insects Musca domestica and Lucilia sericata larvae were reared on
beef meat at 25°C and relative humidity of 50%. Late third instar larvae and adults between 1 and 2 weeks old were used for tRNA extractions. Tenebrio molitor larvae were obtained from Carolina Biological Supply Co., Burlington, NC and reared at 29°C according to the method of PATTERSON(1957). Under these conditions the pupal period lasted for 6 days with adults emergingon the seventh. Large larvae, late pupae and 1-2 week old adults were used for tRNA extractions. Isolation of' tRNA and preparation oJ aminoacyl-tRNA synthetases The methods used are described fully elsewhere (WHITEet al., 1973a) and are modificationsof the procedures described by TWARDZIKet al. (1971). Aminoacylation of tRNA.
* Present Address: Department of Biology, McMaster Transfer RNA was aminoacylated at 22°C in a final University, Hamilton, Ontario, Canada. reaction volume of 0.2 ml. Each ml of the reaction mixture 375
376
BRADLEYN. WHITEANDNORMANJ. LASSAM
contained: Tris-HCl, 50 ,umoles (pH 7.5 or 8.0); 2mercaptoethanol, 5 #moles; 19 unlabelled amino acids, 50 /~moles of each; MgC1z, ATP and [14C] amino acid, (6--50 nmoles) in the amounts reported previously (WHITE and TENER, 1973), 1--5 A260 units of tRNA and enough crude aminoacyl-tRNA synthetases to completely charge all the tRNA in less than 20 min. Preparation of labelled aminoacyl-tRNA Reaction mixtures for the preparation of [14C]-labelled aminoacyl-tRNA were increased proportionately from those described above to a total reaction volume of 1 ml. The reaction mixtures were incubated for 30 min at 22~C and applied to DEAE-cellulose columns as described by YANG and NOVELLI(1968). The aminoacyl-tRNA prepared in this way was stored at -20'~C until used. Reversed-phase chromatography of aminoaeyl-tRNA The RPC-5 system of PEARSONet al., (1971) was used. The Plaskon CTFE was coated with Adogen 464 (WmxE et al., 1973a). The 0.9 cm i.d. × 14 cm columns were developed at 22 or 37°C with a linear 100 ml NaC1 gradient (details of the NaC1 gradients are shown in the Figure legends) containing 0.01 M MgC12, 0.01 M sodium acetate (pH 4.5) and 1 mM 2-mercaptoethan01. Two hundred 0.5 ml fractions were collected and radioactivity was determined by the addition of five volumes of Aquasol and counting in,a Packard Tri-carb scintillation counter (efficiency of 76~o for [14C] under these conditions). These columns are so reproducible that peak height is used as an approximate measure of the amounts of the homogeneic forms (WHITEet al., 1973a). RESULTS AND DISCUSSION
the situation found in Drosophila except the relative amounts differ. In Drosophila there is only Q lacking (7) forms in third instar larvae and then up to equal amounts of Q containing (6) in aged adults (WHITE et al., 1973a). Therefore in both Drosophila and L. sericata there is an increase in Q containing histidine t R N A s from third instar larvae to adults while in M. domestica there is only the Q containing form at both stages. An almost identical pattern is observed for the tyrosine t R N A s (Fig. 2) as would be expected if the family of Q containing t R N A s were being modified as a group. The asparagine t R N A s behave similarly except there are two species which both undergo this change in L. sericata (Fig. 3). The presence of several asparagine t R N A s is similar to the situation in Drosophila where there are three major species (WHITE et al., 1973a). There is essentially only one aspartic acid t R N A in both L. sericata and M. domestica at both larval and adult stages. The major form has been shown to contain Q* in L. sericata by its insensitivity to periodate modification and sensitivity to C N B r (White, 1974a; WOSNICK and WHITE, 1978). The markedly reduced amount of the Q* lacking t R N A in L. sericata is also consistent with the situation in Drosophila where at both larval and adult stages the Aspr form is much more reduced than the 7 forms of tyrosine asparagine or histidine t R N A s (WHITE et al., 1973a). This appears to be due to increased transcription of the aspartic acid t R N A genes after pupation when only Q forms seem to be produced. There are two chromatographically distinct phenylalanine t R N A s in both flies which remain in approximately equal proportions in the larvae and adults (Fig. 5). In Drosophila only one chromatographically distinct phenylalanyl-tRNA is resolvable (WHITE and TENER, 1973).
Musca domestica and Lucilia sericata As was found with Drosophila (WHITE et al., 1973a), the amount of the Q containing t R N A s relative to phenylalanine t R N A remained constant during development (Table l). Therefore the chromatographic changes of the Q containing and Q lacking forms are proportional in nature. Tenebrio molitor The chromatographic profiles of asparagine, Because of the concerted proportional changes aspartic acid, histidine tyrosine and phenylalanine observed in the Q containing family only tyrosine t R N A s for third instar larvae and a d u l t s ' o f M. t R N A s and phenylalanine t R N A s as a control, were domestica and L. sericata are shown in Figs. 1-5. For examined in T. molitor. The changes in tyrosine t R N A M. domestica only one major peak of [~4C]-histidyl during development were less clear than with the t R N A could be resolved in both third instar larvae and Diptera with a probable increase in the Q-lacking form adults while in L. sericata a second major component that decreased in the adult was observed. By use of in the pupae compared with larvae and adults (Fig. 6). The two phenylalanine t R N A s remained constant periodate modification and C N B r treatment it has been previously shown that the earlier eluting peak of during development as they did with the Diptera. In all insects examined the total amount of the Q L. sericata contained Q (WHITE, 1974a) while the later containing t R N A s remain fairly constant relative to one lacked it. This developmental change is similar to Table 1. Amino acid acceptor activity from third instar larvae and adults of M. domestica and L. sericata His Source of tRNA
Tyr
Asp AsH (pmol/A z6o unit)*
Phe
His
Tyr Asp AsH (pmol/pmol Phe)t
M. domestica
larvae adults
29.2 + 0.3 20.4 + 0.1 46.0 + 0.3 30.8 + 0.1 32.4 + 0.2 33.8 _ 0.4 24.4 _ 0.2 55.9 4- 0.4 34.1 4- 0.2 37.5 4- 0.5
0.90 0.90
0.63 0.65
1.42 1.49
0.95 0.91
L. sericata
larvae adults
33.0 4- 0.2 23.5 _ 0.4 51.2 + 0.4 34.0 + 0.3 35.1 + 0.3 36.8 + 0.3 26.4 + 0.1 57.8 + 0.1 37.5 _ 0.I 38.3 ___ 0.3
0.94 0.96
0.67 0.69
1.46 1.51
0.97 0.98
* Mean of five determinations + S.E.M. t Because of differing levels of contaminating rRNA in the tRNA preparations, the results are more easily compared by:mean pmol amino acid/Az6 o mean pmol Phe/A2o o
Insect tRNAs d
C
C
J
16-
S 1412IO-
X
E ~-~4-
\
2-
gs
ds
I
105 65
~s
377
6
76? 5o X4-
L ~65
85
~6s ,~5
B5
165
s~
12S
gs /s
15 g5 75 ¢5 Fraction no.
Fraction no.
Fig. 1. RPC-5 chromatography of []4C]-histidyl-tRNA. Elution was by a 100 ml linear gradient from 0.50-0.65 M NaCI at 22°C: (a) M. domestica larvae; (b) M. domestica adults; (c) L. sericata larvae; (d) L. sericata adults.
C 8-
6-
x
E 2-
85
105
85
75
~5
L_
A
It5
Fraction n o
Fig. 2. RPC-5 chromatography of [~4C]-tyrosyl-tRNA. Elution was bY a 100 ml linear gradient from 0.55-0.70 M NaCI at 37°C: (a), (b), (c), (d) as in Fig. 1.
105
g5 ~5 £5
Fig. 4. RPC-5 chromatography of [x4C]-aspartyl-tRNA. Elution was by a 100 ml linear gradient from 0.50-0.65 M NaCI at 37°C: (a), (b), (c), (d) as in Fig. 1. phenylalanine during development. Unlike Drosophila both L. sericata and M. domestica have the majority of their Q family tRNAs in the Q containing form in the larval stages. It has been found that both mutations (WroTE et al., 1973a; WHITE, 1974b) and growth conditions (WosN]CK and WroTE, 1977) can effect the proportions of the Q containing form to the Q lacking form. The fact that adult Drosophila can be produced with either entirely Q containing or Q lacking tRNAs poses an intriguing problem as to the function of Q. It is one of the most highly modified bases known and the only one with modification of the purine skeleton; it occupies the first position of the anticodon and is presumably of importance to translation. Although little is known of the biosynthesis of Q it is clearly complex requiring several enzymes. There is a general correlation that Q-lacking forms appear more prevalent when there is rapid growth such as in larvae of Drosophila and L. sericata or in rich growth conditions and high temperatures (WosNICK and WHITE, 1977). However, this in no way helps explain the function of Q or how Drosophila in particular can develop without it.
a
2,1
85
b
C
4-
•
E
1 2"
gs r~
5~ 75
is
75 9'5
515
75
~J5
Fraction no.
Fig. 3. RPC-5chromatographyof[14C]-asparaginyl-tRNA. Elution was by a 100 ml linear gradient from 0.50-0.60 M NaCI at 22°C: (a), (b), (c), (d) as in Fig. 1.
,15 135
,~s
,~5
,~5 ,~5 ,15
J
I:]5
155
Fraction no
Fig. 5. RPC-5 chromatography of [14C]-phenylalanyltRNA. Elution was by a 100 ml linear gradient from 0.50-0.65 M NaCI at 37°C: (a), (b), (c), (d), as in Fig. 1.
378
BRADLEY N. WHITE AND NORMAN J. LASSAM a
b
io.
86-
A
4~
d
C
80
100 120
x E 24-
HARADA R. and NISHIMURA S. (1972) Possible anticodon sequences of tRNA Hi~, tRNA ASh, and tRNA ASp from Escherichia coli B. Universal presence of nucleoside Q in the first positions of the anticodons of these transfer ribonucleic acids. Biochemistry 11, 301-308. KASAI H., OHASHI Z., HARADA F., N1SH1MURA S., OPPENHEIMER N. J., CRAINP. F., LIEHRJ. G., VON MINDEN D. A. and McCLOSKEY J. A. (1975) Structure of the modified nucleoside Q isolated from Escherichia coli transfer ribonucleic acid, 7-(4, 5-cis-dihydroxy-1cyclopenten-3-yl-aminomethyl)-7-diazaguanine. Biochemistry 14, 4198-4208. KASAI H., NAKANISHIK., MACFARLANE R. D., TORGERSON D. F., OHASHI Z., McCLOSKEY J. S., GROSS H. J. and
O..
-lO
201612B-
J
6'O do
;60
,oo
Fraction
no.
Fig. 6. RPC-5 chromatography of T. molitor [14C]-tyrosyl and [~4C]-phenylalanyl-tRNAs. Elution was by a 100 ml linear gradient from 0.55 to 7.0 M NaC1 at 37°C: (a) larval [14C]-tyrosyl-tRNA: (b) pupal [14C]-tyrosyl-tRNA; (c) adult p4C]-tyrosol-tRNA; (d) pupal [14C]-phenylalanyl-tRNA.
There is an indication that Q metabolism may be related to the guanylation reaction first observed with r a b b i t reticulocytes (FARKAS, and SINGH, 1973). Drosophila t R N A is a substrate for the reticulocyte guanylating enzyme a n d guanylating activity has been found in extracts from adult Drosophila (WosNIcK et al., 1978; McKINNON et al., 1978). This guanylation reaction appears to be the m o s t promising a p p r o a c h to the study of the biosynthesis of Q and hence to the function of the changes in Q t R N A s during development.
Acknowledgements--This work was supported by the National Research Council of Canada.
REFERENCES FARKAS W. R. and SINGH R. D. (1973) Guanylation of transfer ribonucleic acid by a cell-free by lysate of rabbit reticulocytes. J. biol. Chem. 248, 7780-7785.
NISH1MURA S. (1976) The structure of Q* Nucleoside isolated from rabbit liver transfer ribonucleic acid. J. Am. Chem. Soc. 98, 5044-5046. MCKINNON R. D., WOSNICK M. A. and WHITE B. N. (1978) The role of the guanine insertion enzyme in Q biosynthesis in Drosophila melanogaster. Nucl. Acids"Res. 5, 4856--4876. PATTERSON P. (1957) Quantitative and qualitative changes observed in the free alpha amino nitrogen fraction of Tenebrio molitor. J. molec. Biol. 65, 729-735. PEARSON R. L., WEISS J. F. and KELMERS A. D. (1971) Improved separation of transfer RNAs on polychlorotrifluoroethylene-supported reverse-phase chromatography columns. Biochim. biophys. Acta 228, 770-774. TWARDZ1K D. R., GRELL E. H. and JACOBSON K. B. (1971) Mechanism of suppression in Drosophila: A change in tyrosine transfer RNA. J. molec. Biol. 57, 231-245. WHITE B. N., TENER G. M., HOLDEN J. and SuzuKI D. T. (1973a) Activity of a transfer RNA modifying enzyme during the development of Drosophila and its relationship to the su(s) locus. J. molec. Biol. 74, 635-651. WHITE B. N., TUNER G. M., HOLOEN J. and SUZUKI D. T. (1973b) Analysis of tRNSs during the development of Drosophila. Devl Biol. 33, 185-195. WHITE B. N. and TUNER G. M. (1973l Chromatography of Drosophila tRNA on BD-cellulose. Can. J. Bioehem. 51, 896-902. WroTE B. N. (1974a) Chromatographic changes in specific tRNAs after reaction with cyanogen bromide and sodium periodate. Biochim biophys. Acta 353, 283-291. WHITE B. N. (1947b) An analysis of tRNAs in five minutes and two suppressors. Drosoph. InJl SPry. 51, 58-59. WOSNICK M. A. and WHITE B. N. (1977) A doubtful relationship between tyrosine tRNA and suppression of the vermilion mutant in Drosophila. Nucl. Acids Res. 4, 3919-3930. WOSNlCK M. A. and WHITE B. N, (1978) Purification and nucleoside composition of a Q*-containing aspartic acid tRNA from Drosophila. Bioehem. biophys. Res. Commun. 81, 1131-1138. WOSNICK M. A., McKINNON R. D. and WroTE B. N. (1978l Guanylation of Drosophila tRNA in heterologous and homologous systems. Can. Fed. Biol. Soc. Proc. 21, 61. YANG W. K. and NOVELLIG. P. (1968) Isoaccepting tRNAs in mouse plasma cell tumors that synthesize different myeloma proteins. Biochem. biophys. Res. Commun. 31, 534-539.
0020-1700/79/0701-0375 $02.00/0
AN ANALYSIS OF Q AND Q* CONTAINING tRNAs DURING THE DEVELOPMENT OF LUCILIA SERICATA, MUSCA DOMESTICA AND TENEBRIO MOLITOR BRADLEY N. WHITE and NORMANJ. LASSAM* The Group in Eukaryotic Molecular Biology and Evolution, Department of Biology, Queen's University, Kingston, Ontario, Canada (Received 24 October 1978)
Abstract--The chromatographic profiles of the family of tRNAs containing the hypermodifiednucleoside Q and Q* in the first position of their anticodons were examined in the third instar larvae and adults of Lucilia sericata and Musca domestica. The absolute amounts of these tRNAs remained constant during development relative to phenylalanine tRNA. In M. domestica only Q containing forms were found in both larvae and adults while in L. sericata, the larvae contained a significantportion of the Q lacking form. In Tenebrio molitor the Q lacking form of tyrosine tRNA appeared to be only significant in pupae. The developmentalpatterns of Q biosynthesisin the Diptera therefore range from almost entirely Q containing in M. domestica to entirely Q lacking in Drosophila raised under certain conditions. This suggests that this modification to the anticodon is not playing a common regulatory function in the development of these insects. Key Word Index: tRNA, nucleoside Q and Q*, Musca domestica, Lucilia sericata, Tenebrio molitor
INTRODUCTION DURING the development of Drosophila melanogaster there are marked changes in the family of tRNAs which contain the hypermodified nucleoside Q or Q* (WHITE et al., 1973a, b). The structure of Q has been shown to be a 7-(4, 5-cis-dehydroxy-l-cyclopenten-3yl-aminomethyl)-7-diazaguanosine (KASAI et al., 1975), whereas Q* is even further modified, having either a mannose or galactose residue at the 4 position of the cyclopenten diol (KASAI et al., 1976). Only tyrosine, histidine, asparagine or aspartic acid tRNAs have been found to contain Q or Q* and these nucleosides are always located in the first (5') position of the anticodon. These four tRNAs form a natural family in that they respond to codons of the variety XA c. Although the function of the hypermodification is not understood there is evidence that Q containing tRNAs preferentially bind to XAU or XAC codons (HARADA and NISHIMURA, 1972). In Drosophila, prior to pupation, the major forms of aspartic acid, asparagine, tyrosine and histidine tRNAs are those lacking Q or Q* while after pupation the forms containing Q are produced. There is considerable evidence that the 6 forms (containing Q or Q*) and ~ forms (lacking Q or Q*) (WHITEe t al., 1973a) are in fact transcribed from the same genes and are chromatographically distinct only because of the degree of post-transcriptional modification; these chromatographically distinct but genetically identical tRNAs have been termed homogenic (WHITE et al., 1973a). The developmental changes observed in Drosophila prompted an investigation into the universality of the . . . . . . . . . . . . . . . . . . .
process in other holometabolous insects, especially other Diptera. The family of Q-containing tRNAs and phenylalanine tRNA as a control were therefore examined in M u s c a domestica, Lucilia sericata "and Tenebrio molitor.
MATERIALS AND METHODS Materials.
Uniformly [14C]-labelled-aminoacids were obtained from New England Nuclear Corporation and Amersham/Searle (L-aspartic acid--203 mCi/mmol; L-histidine--302 mCi/mmol; L-phenylalanine~72 mCi/mmol; L-tyrosine-441 mCi/mmol; and L-asparagine--179 mCi/mmol). Aquasol was obtained from New England Corp. Adogen 464, a trialkylmethylammonium chloride with the predominant chain length of the alkyl groups being Cs-Clo, was a gift from Ashland Chemical Co., Columbus, OH. Plaskon CTFE 2300 powder was a gift from Allied Chemical Corp., Morristown, NY. Growth of insects Musca domestica and Lucilia sericata larvae were reared on
beef meat at 25°C and relative humidity of 50%. Late third instar larvae and adults between 1 and 2 weeks old were used for tRNA extractions. Tenebrio molitor larvae were obtained from Carolina Biological Supply Co., Burlington, NC and reared at 29°C according to the method of PATTERSON(1957). Under these conditions the pupal period lasted for 6 days with adults emergingon the seventh. Large larvae, late pupae and 1-2 week old adults were used for tRNA extractions. Isolation of' tRNA and preparation oJ aminoacyl-tRNA synthetases The methods used are described fully elsewhere (WHITEet al., 1973a) and are modificationsof the procedures described by TWARDZIKet al. (1971). Aminoacylation of tRNA.
* Present Address: Department of Biology, McMaster Transfer RNA was aminoacylated at 22°C in a final University, Hamilton, Ontario, Canada. reaction volume of 0.2 ml. Each ml of the reaction mixture 375
376
BRADLEYN. WHITEANDNORMANJ. LASSAM
contained: Tris-HCl, 50 ,umoles (pH 7.5 or 8.0); 2mercaptoethanol, 5 #moles; 19 unlabelled amino acids, 50 /~moles of each; MgC1z, ATP and [14C] amino acid, (6--50 nmoles) in the amounts reported previously (WHITE and TENER, 1973), 1--5 A260 units of tRNA and enough crude aminoacyl-tRNA synthetases to completely charge all the tRNA in less than 20 min. Preparation of labelled aminoacyl-tRNA Reaction mixtures for the preparation of [14C]-labelled aminoacyl-tRNA were increased proportionately from those described above to a total reaction volume of 1 ml. The reaction mixtures were incubated for 30 min at 22~C and applied to DEAE-cellulose columns as described by YANG and NOVELLI(1968). The aminoacyl-tRNA prepared in this way was stored at -20'~C until used. Reversed-phase chromatography of aminoaeyl-tRNA The RPC-5 system of PEARSONet al., (1971) was used. The Plaskon CTFE was coated with Adogen 464 (WmxE et al., 1973a). The 0.9 cm i.d. × 14 cm columns were developed at 22 or 37°C with a linear 100 ml NaC1 gradient (details of the NaC1 gradients are shown in the Figure legends) containing 0.01 M MgC12, 0.01 M sodium acetate (pH 4.5) and 1 mM 2-mercaptoethan01. Two hundred 0.5 ml fractions were collected and radioactivity was determined by the addition of five volumes of Aquasol and counting in,a Packard Tri-carb scintillation counter (efficiency of 76~o for [14C] under these conditions). These columns are so reproducible that peak height is used as an approximate measure of the amounts of the homogeneic forms (WHITEet al., 1973a). RESULTS AND DISCUSSION
the situation found in Drosophila except the relative amounts differ. In Drosophila there is only Q lacking (7) forms in third instar larvae and then up to equal amounts of Q containing (6) in aged adults (WHITE et al., 1973a). Therefore in both Drosophila and L. sericata there is an increase in Q containing histidine t R N A s from third instar larvae to adults while in M. domestica there is only the Q containing form at both stages. An almost identical pattern is observed for the tyrosine t R N A s (Fig. 2) as would be expected if the family of Q containing t R N A s were being modified as a group. The asparagine t R N A s behave similarly except there are two species which both undergo this change in L. sericata (Fig. 3). The presence of several asparagine t R N A s is similar to the situation in Drosophila where there are three major species (WHITE et al., 1973a). There is essentially only one aspartic acid t R N A in both L. sericata and M. domestica at both larval and adult stages. The major form has been shown to contain Q* in L. sericata by its insensitivity to periodate modification and sensitivity to C N B r (White, 1974a; WOSNICK and WHITE, 1978). The markedly reduced amount of the Q* lacking t R N A in L. sericata is also consistent with the situation in Drosophila where at both larval and adult stages the Aspr form is much more reduced than the 7 forms of tyrosine asparagine or histidine t R N A s (WHITE et al., 1973a). This appears to be due to increased transcription of the aspartic acid t R N A genes after pupation when only Q forms seem to be produced. There are two chromatographically distinct phenylalanine t R N A s in both flies which remain in approximately equal proportions in the larvae and adults (Fig. 5). In Drosophila only one chromatographically distinct phenylalanyl-tRNA is resolvable (WHITE and TENER, 1973).
Musca domestica and Lucilia sericata As was found with Drosophila (WHITE et al., 1973a), the amount of the Q containing t R N A s relative to phenylalanine t R N A remained constant during development (Table l). Therefore the chromatographic changes of the Q containing and Q lacking forms are proportional in nature. Tenebrio molitor The chromatographic profiles of asparagine, Because of the concerted proportional changes aspartic acid, histidine tyrosine and phenylalanine observed in the Q containing family only tyrosine t R N A s for third instar larvae and a d u l t s ' o f M. t R N A s and phenylalanine t R N A s as a control, were domestica and L. sericata are shown in Figs. 1-5. For examined in T. molitor. The changes in tyrosine t R N A M. domestica only one major peak of [~4C]-histidyl during development were less clear than with the t R N A could be resolved in both third instar larvae and Diptera with a probable increase in the Q-lacking form adults while in L. sericata a second major component that decreased in the adult was observed. By use of in the pupae compared with larvae and adults (Fig. 6). The two phenylalanine t R N A s remained constant periodate modification and C N B r treatment it has been previously shown that the earlier eluting peak of during development as they did with the Diptera. In all insects examined the total amount of the Q L. sericata contained Q (WHITE, 1974a) while the later containing t R N A s remain fairly constant relative to one lacked it. This developmental change is similar to Table 1. Amino acid acceptor activity from third instar larvae and adults of M. domestica and L. sericata His Source of tRNA
Tyr
Asp AsH (pmol/A z6o unit)*
Phe
His
Tyr Asp AsH (pmol/pmol Phe)t
M. domestica
larvae adults
29.2 + 0.3 20.4 + 0.1 46.0 + 0.3 30.8 + 0.1 32.4 + 0.2 33.8 _ 0.4 24.4 _ 0.2 55.9 4- 0.4 34.1 4- 0.2 37.5 4- 0.5
0.90 0.90
0.63 0.65
1.42 1.49
0.95 0.91
L. sericata
larvae adults
33.0 4- 0.2 23.5 _ 0.4 51.2 + 0.4 34.0 + 0.3 35.1 + 0.3 36.8 + 0.3 26.4 + 0.1 57.8 + 0.1 37.5 _ 0.I 38.3 ___ 0.3
0.94 0.96
0.67 0.69
1.46 1.51
0.97 0.98
* Mean of five determinations + S.E.M. t Because of differing levels of contaminating rRNA in the tRNA preparations, the results are more easily compared by:mean pmol amino acid/Az6 o mean pmol Phe/A2o o
Insect tRNAs d
C
C
J
16-
S 1412IO-
X
E ~-~4-
\
2-
gs
ds
I
105 65
~s
377
6
76? 5o X4-
L ~65
85
~6s ,~5
B5
165
s~
12S
gs /s
15 g5 75 ¢5 Fraction no.
Fraction no.
Fig. 1. RPC-5 chromatography of []4C]-histidyl-tRNA. Elution was by a 100 ml linear gradient from 0.50-0.65 M NaCI at 22°C: (a) M. domestica larvae; (b) M. domestica adults; (c) L. sericata larvae; (d) L. sericata adults.
C 8-
6-
x
E 2-
85
105
85
75
~5
L_
A
It5
Fraction n o
Fig. 2. RPC-5 chromatography of [~4C]-tyrosyl-tRNA. Elution was bY a 100 ml linear gradient from 0.55-0.70 M NaCI at 37°C: (a), (b), (c), (d) as in Fig. 1.
105
g5 ~5 £5
Fig. 4. RPC-5 chromatography of [x4C]-aspartyl-tRNA. Elution was by a 100 ml linear gradient from 0.50-0.65 M NaCI at 37°C: (a), (b), (c), (d) as in Fig. 1. phenylalanine during development. Unlike Drosophila both L. sericata and M. domestica have the majority of their Q family tRNAs in the Q containing form in the larval stages. It has been found that both mutations (WroTE et al., 1973a; WHITE, 1974b) and growth conditions (WosN]CK and WroTE, 1977) can effect the proportions of the Q containing form to the Q lacking form. The fact that adult Drosophila can be produced with either entirely Q containing or Q lacking tRNAs poses an intriguing problem as to the function of Q. It is one of the most highly modified bases known and the only one with modification of the purine skeleton; it occupies the first position of the anticodon and is presumably of importance to translation. Although little is known of the biosynthesis of Q it is clearly complex requiring several enzymes. There is a general correlation that Q-lacking forms appear more prevalent when there is rapid growth such as in larvae of Drosophila and L. sericata or in rich growth conditions and high temperatures (WosNICK and WHITE, 1977). However, this in no way helps explain the function of Q or how Drosophila in particular can develop without it.
a
2,1
85
b
C
4-
•
E
1 2"
gs r~
5~ 75
is
75 9'5
515
75
~J5
Fraction no.
Fig. 3. RPC-5chromatographyof[14C]-asparaginyl-tRNA. Elution was by a 100 ml linear gradient from 0.50-0.60 M NaCI at 22°C: (a), (b), (c), (d) as in Fig. 1.
,15 135
,~s
,~5
,~5 ,~5 ,15
J
I:]5
155
Fraction no
Fig. 5. RPC-5 chromatography of [14C]-phenylalanyltRNA. Elution was by a 100 ml linear gradient from 0.50-0.65 M NaCI at 37°C: (a), (b), (c), (d), as in Fig. 1.
378
BRADLEY N. WHITE AND NORMAN J. LASSAM a
b
io.
86-
A
4~
d
C
80
100 120
x E 24-
HARADA R. and NISHIMURA S. (1972) Possible anticodon sequences of tRNA Hi~, tRNA ASh, and tRNA ASp from Escherichia coli B. Universal presence of nucleoside Q in the first positions of the anticodons of these transfer ribonucleic acids. Biochemistry 11, 301-308. KASAI H., OHASHI Z., HARADA F., N1SH1MURA S., OPPENHEIMER N. J., CRAINP. F., LIEHRJ. G., VON MINDEN D. A. and McCLOSKEY J. A. (1975) Structure of the modified nucleoside Q isolated from Escherichia coli transfer ribonucleic acid, 7-(4, 5-cis-dihydroxy-1cyclopenten-3-yl-aminomethyl)-7-diazaguanine. Biochemistry 14, 4198-4208. KASAI H., NAKANISHIK., MACFARLANE R. D., TORGERSON D. F., OHASHI Z., McCLOSKEY J. S., GROSS H. J. and
O..
-lO
201612B-
J
6'O do
;60
,oo
Fraction
no.
Fig. 6. RPC-5 chromatography of T. molitor [14C]-tyrosyl and [~4C]-phenylalanyl-tRNAs. Elution was by a 100 ml linear gradient from 0.55 to 7.0 M NaC1 at 37°C: (a) larval [14C]-tyrosyl-tRNA: (b) pupal [14C]-tyrosyl-tRNA; (c) adult p4C]-tyrosol-tRNA; (d) pupal [14C]-phenylalanyl-tRNA.
There is an indication that Q metabolism may be related to the guanylation reaction first observed with r a b b i t reticulocytes (FARKAS, and SINGH, 1973). Drosophila t R N A is a substrate for the reticulocyte guanylating enzyme a n d guanylating activity has been found in extracts from adult Drosophila (WosNIcK et al., 1978; McKINNON et al., 1978). This guanylation reaction appears to be the m o s t promising a p p r o a c h to the study of the biosynthesis of Q and hence to the function of the changes in Q t R N A s during development.
Acknowledgements--This work was supported by the National Research Council of Canada.
REFERENCES FARKAS W. R. and SINGH R. D. (1973) Guanylation of transfer ribonucleic acid by a cell-free by lysate of rabbit reticulocytes. J. biol. Chem. 248, 7780-7785.
NISH1MURA S. (1976) The structure of Q* Nucleoside isolated from rabbit liver transfer ribonucleic acid. J. Am. Chem. Soc. 98, 5044-5046. MCKINNON R. D., WOSNICK M. A. and WHITE B. N. (1978) The role of the guanine insertion enzyme in Q biosynthesis in Drosophila melanogaster. Nucl. Acids"Res. 5, 4856--4876. PATTERSON P. (1957) Quantitative and qualitative changes observed in the free alpha amino nitrogen fraction of Tenebrio molitor. J. molec. Biol. 65, 729-735. PEARSON R. L., WEISS J. F. and KELMERS A. D. (1971) Improved separation of transfer RNAs on polychlorotrifluoroethylene-supported reverse-phase chromatography columns. Biochim. biophys. Acta 228, 770-774. TWARDZ1K D. R., GRELL E. H. and JACOBSON K. B. (1971) Mechanism of suppression in Drosophila: A change in tyrosine transfer RNA. J. molec. Biol. 57, 231-245. WHITE B. N., TENER G. M., HOLDEN J. and SuzuKI D. T. (1973a) Activity of a transfer RNA modifying enzyme during the development of Drosophila and its relationship to the su(s) locus. J. molec. Biol. 74, 635-651. WHITE B. N., TUNER G. M., HOLOEN J. and SUZUKI D. T. (1973b) Analysis of tRNSs during the development of Drosophila. Devl Biol. 33, 185-195. WHITE B. N. and TUNER G. M. (1973l Chromatography of Drosophila tRNA on BD-cellulose. Can. J. Bioehem. 51, 896-902. WroTE B. N. (1974a) Chromatographic changes in specific tRNAs after reaction with cyanogen bromide and sodium periodate. Biochim biophys. Acta 353, 283-291. WHITE B. N. (1947b) An analysis of tRNAs in five minutes and two suppressors. Drosoph. InJl SPry. 51, 58-59. WOSNICK M. A. and WHITE B. N. (1977) A doubtful relationship between tyrosine tRNA and suppression of the vermilion mutant in Drosophila. Nucl. Acids Res. 4, 3919-3930. WOSNlCK M. A. and WHITE B. N, (1978) Purification and nucleoside composition of a Q*-containing aspartic acid tRNA from Drosophila. Bioehem. biophys. Res. Commun. 81, 1131-1138. WOSNICK M. A., McKINNON R. D. and WroTE B. N. (1978l Guanylation of Drosophila tRNA in heterologous and homologous systems. Can. Fed. Biol. Soc. Proc. 21, 61. YANG W. K. and NOVELLIG. P. (1968) Isoaccepting tRNAs in mouse plasma cell tumors that synthesize different myeloma proteins. Biochem. biophys. Res. Commun. 31, 534-539.