THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 36, Issue of September 9, pp. 22902-22906, 1994 Printed in U.S.A.

Primary and Higher Order Structures of Nematode (Ascaris suum) Mitochondrial tRNAs Lacking Either the T or D Stem*

(Received for publication, May 3, 1994, and in revised form, June 28, 1994)

Yoh-ichi Watanabe, Hiromichi Tsuruis, Takuya Ueda, Rieko Furushimag, Shinzaburo Takamiyas, Kiyoshi Kitan, Kazuya Nishikawall, and Kimitsuna Watanabe** From the Department of Chemistry and Biotechnology, Faculty of Engineering, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, the $Department of Pathology and the §Department of Parasitology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113, the IDepartment of Parasitology, Institute of Medical Science, University of Tokyo, Shiroganedai, Minato-ku, Tokyo 108, and the IDepartment of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 227, Japan

By fractionation using polyacrylamide gel electro- phoresis and/or a preparative hybrid selection method employing solid-phase DNA probes, we prepared and characterized mitochondrial tRNAs from the body wall muscle of Ascaris mum, all of which are thought to lack either the T stem or the D stem from their gene se- quences (Okimoto, R., and Wolstenholme, D. R. (1990) EMBO J. 10, 340543411). Some of the partially purified tRNAs were appreciably aminoacylated with an extract of A. suum mitochondria. The three species sequenced had CCA sequence at their 3'-ends, and tRNAMet had 5-formylcytidine at the anticodon first position, a new modified nucleoside found at the same position of bovine mitochondrial tRNAMet (Moriya, J., Yokogawa, T., Wakita, K., Ueda, T., Nishikawa, K., Crain, P. E, Hashizume, T., Pomerantz, S. C., McCloskey, J. A., Kawai, G., Hayashi, N., Yokoyama, S., and Watanabe, K. (1994) Biochemistry 33,2234-2239). Enzymatic probing of these tRNAs supported the secondary structural model pro- posed by Okimoto and Wolstenholme in the reference cited above. Chemical probing of tRNAPhe demonstrated the existence of tertiary interactions between the (T arm-variable loop)-replacement loop and the D arm. The results suggest that these tertiary interactions enable the bizarre tRNAs of nematode mitochondria to main- tain an L-shape-like structure in order to function in the nematode mitochondrial translation system.

Whereas all prokaryotic and eukaryotic cytoplasm tRNAs have a common cloverleaf structure (l), mitochondrial (mt)' tRNAs encoded on metazoan mt DNA have been found to pos- sess quite unusual secondary structures as far as can be in- ferred from their gene sequences (2, 3); in particular, they ex-

priority areas (to K. K. and K. W.) and a grant-in-aid (to K. K.) from the * This work was supported by grants-in-aid for scientific research on

Ministry of Education, Science, and Culture of Japan, the Human Fron- tier Science Program Organization (to K. W.), and a Japan Society for the Promotion of Science fellowship for Japanese junior scientists (to Y. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted

028746, and 028747. to the G'enBankTM/EMBL Data Bank with accession nunbeds) 028745,

2111 (ext. 7216); Fax: 81-3-5800-6950. ** To whom reprint requests should be addressed. Tel.: 81-3-3812-

The abbreviations used are: mt, mitochondrial; TV-replacement loop or TVR loop, (T arm-variable loop)-replacement loop; DR loop, D arm- replacement loop; f5C, 5-formylcytidine; PA, P-threoninocarbonyla- denosine; m'A, l-methyladenosine; m6A, 6-methyladenosine; m2G, 2-methylguanosine; m'G, l-methylguanosine.

hibit considerable variations in both the size and the sequence of the D and T arms. The structures of some of the mt tRNAs from higher animals have been characterized (4-10).

An extreme case is seen in the metazoan mt tRNA genes of the nematodes Ascaris suum and Caenorhabditis elegans (11, 12). Sequence determination of these mt DNAs has revealed that in a total of 22 tRNA genes encoded on the nematode genome, there is no tRNA gene with the common cloverleaf secondary structure. Whereas two putative serine tRNA genes lack the D stem like tRNASe'(GCU)s of other metazoan mito- chondria (1,4-7), the other 20 putative tRNAgenes all lack the T stem. This is also the case in another nematode, Meloidogyne javanica (13). These putative mt tRNA genes have a loop com- posed of 4-12 nucleotides in place of the usual T arm, which corresponds to the original T arm and variable loop and which is thus called the (T arm-variable loop)-replacement loop (TV- replacement loop) (11).

In small RNA fractions derived from A. s u u m and C. elegans, Okimoto and Wolstenholme (14) detected stable transcripts de- rived from some of the putative mt tRNA genes, which was confirmed by Northern blot analysis. However, there have been no reports providing a structural analysis of these mt tRNAs (including the direct sequencing and identification of their modified nucleosides) as well as a functional analysis.

In this paper, we make a direct sequence analysis, and probe the structure and aminoacylation activity of some of the mt tRNAs purified from the parasitic nematode, A. suum.

MATERIALS AND METHODS Chemicals and En~ymes-[y-~*PlATP (110 TBq/mmol) and [5'-32P1cy-

tidine-3',5'-diphosphate (110 TBq/mmol) or uniformly 14C-labeled L-amino acids (6.7-18.0 GBqImmol) were obtained from Amersham or DuPont NEN. Modifying enzymes and nucleases used were from the same suppliers as described (9,lO). Escherichia coli tRNALy*, an in vitro transcript of bovine mt tRNAser(GCU), and bovine mt tRNAMet were kindly provided by N. Hayashi, Y. Yotsumoto, and J. Moriya, respec- tively, of our laboratory. Other chemicals used were of analytical or biochemical grade.

Oligodeozynucleotides-Oligodeoxynucleotides were synthesized by a DNA synthesizer (model 381A or 391, Applied Biosystems) as de- scribed (151, deprotected, and purified on denaturing polyacrylamide gels. For the preparation of solid-phase DNA probes, synthetic oli- gomers with an aminohexyl linker (Aminolink 2, Applied Biosystems) at their 5"termini were synthesized, deprotected, and desalted by a fast desalting column (Pharmacia Biotech Inc.).

Preparation of Total tRNA from A. suum-Body wall muscle from A. suum (purchased from a local slaughterhouse in Tokyo) was prepared as described (16), frozen or lyophilized, and stored at -20 "C. Preparation of tRNA from the body wall muscle was performed according to the method reported by Ueda et al. (5). The yield of total tRNA was 1 AZ60 unit (about 40 pg) from 0.5-1 g (wet weight) of A. suum body wall muscle.

Fractionation ofmt tRNA by Preparative Gel Electrophoresis-Total tRNA was fractionated by denaturing gels. After electrophoresis, the gel

22902

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Structure of Nematode Mitochondrial tRNAs 22903

a b

1 2

63nt

FIG. 1. a, polyacrylamide gel electrophoresis of total tRNA fraction of A. suum. Lane 1, size markers. 76 nt, E. coli tRNALy*; 63 nt, in vitro transcript of bovine mt tRNAS"'(GCU) gene. Lane 2,A. mum total tRNA fraction. b, fractionation of total tRNA fraction of A. suum by polyac- rylamide gel electrophoresis. The composition of the gel was 15% poly- acrylamide, 7 M urea, and 10% glycerol. After electrophoresis, the gel was stained with 0.02% (wh) toluidine blue.

was stained with 0.02% (w/v) toluidine blue, and the bands containing mt tRNAs were excised (see "Results"). tRNAs were eluted and precipi- tated with ethanol.

Purification of Individual mt tRNA by the Hybrid Selection Method Using Solid-phase DNA Probes-The preparation of solid-phase DNA probes and preparative hybrid selection of tRNAs were carried out as described (17). The sequence of the probe for tRNAPhE is 5'-XCACTC- CTAACATTTCCCACTTCTTCAAAG-3' (complementary to the 3'-end 30 nucleotides of tRNAPh' gene ( l l ) , where X is the aminohexyl linker).

Sequence Determination of tRNA-tRNAs obtained as described above were further purified by gel electrophoresis and sequenced as described (18, 19). Nucleotide composition analysis was carried out as described (20) using two-dimensional TLC (19, 20). For comparison of an unknown nucleoside of A. suum mt tRNAhfet with 5-formylcytidine (PC), 5'4abeled nucleotides were prepared from both bovine and A. suum tRNA% using two-dimensional TLC (19, 20). The nucleotides corresponding to PC were compared with respect to their mobilities on two-dimensional TLCs (19, 20).

Preparation of A. mum mt Extract-Preparation of the mt fraction from body wall muscle of A. suum was performed as described without the step of proteinase treatment (16). The mt fraction was treated as described previously (7) without the steps of precipitation with ammo- nium sulfate and column chromatographies. The supernatant of ultra- centrifugation was dialyzed extensively against a buffer consistingof 30 mM Tris-HCI, pH 7.5, 5 mM MgCl,, 6 mM P-mercaptoethanol, 10% (v/v) glycerol, and 10 PM phenylmethylsulfonyl chloride a t 4 "C. The solution was mixed with glycerol (final concentration, 50% (v/v)), and stored at -20 "C.

Aminoacylation of tRNA-Aminoacylation of tRNAs was performed as described (7). In the standard conditions, the reaction mixture (20 111) contained tRNA (0.01 A,,, unit) and mt extract (0.8 mg/ml protein) (21) and was incubated a t 37 "C for 10 min.

Enzymatic Probing of mt tRNAs-3'- or 5'-end-labeled tRNAs were prepared as described (22, 23) and purified by denaturing gel electro- phoresis. Enzymatic probing was performed as described (9).

Chemical Probing of mt tRNA-Chemical modification of the 3'-end- labeled tRNAPhc with dimethyl sulfate or diethyl pyrocarbonate and the detection of modified nucleotides were performed as described (10,24).

RESULTS Detection ofA. suum mt tRNAs by Gel Electrophoresis-Body

wall muscle ofA. suum was used as a source of tRNAs for large scale preparation. When the total tRNA fraction was analyzed by denaturing polyacrylamide gel electrophoresis, several bands appeared in the region corresponding to small RNAs (about 50-60 nucleotides long as estimated from size markers) (Fig. la) . To confirm that these RNAs were of mt DNA origin, these bands were subjected to Northern blot hybridization us-

ing DNA oligomers complementary to parts of mt tRNA gene sequences, with the result that at least eight species (Arg, His, Leu(GAA), Lys, Met, Phe, Ser(UCU), and Ser(UGA)) of mt tRNA were detected (data not shown).

Preparation and Characterization of mt tRNAs-The above hybridization analysis showed that mt tRNAs, including even tRNA1*YS, with the longest size among the mt tRNAs as inferred from their putative gene sequences (121, could be well sepa- rated from the cytoplasmic tRNA fraction. We thus carried out large scale fractionation of the mt tRNAs by gel electrophoresis, which resulted in the 10 fractions shown in Fig. lb. From 100 A,,, units of the total tRNA fraction, 0.4-2.5 A,,, units of tRNA was recovered in each fraction.

From partial sequencing of these tRNA fractions it was de- duced that among the fractions shown in Fig. lb, fraction l contained only tRNASer(UCU), whereas fraction 10 contained tRNRMeL and tRNALys (data not shown). All of the other fractions contained more than two species of mt tRNAs; 16 species of mt tRNAs were finally detected in these fractions (data not shown). Together with the results of the hybridization analy- ses, we thus identified a t least 18 species of tRNAs derived from the putative mt tRNA genes, the undetected species being Leu(UAG), Gln, Thr, and Asn-specific tRNAs. Furthermore, some of these fractions (e.g. 6 for Phe and Asp, 10 for Met) accepted appreciable amounts (10-100 pmoVA,,, unit of tRNA) of the respective '*C-amino acid with the mt extract with good reproducibility (data not shown).

Final purification of these tRNAs for sequencing was per- formed by the above mentioned gel electrophoresis andor a preparative hybrid selection using a solid-phase DNA probe (17). In particular, tRNAPhe could be purified only by using the latter method, because it was difficult to exclude the other contaminated mt tRNAs by gel electrophoresis. Thus, we could obtain tRNAMet, tRNAS"'(UCU), and tRNAPhe in sufficient amounts for sequencing and structural analysis.

Sequence Determination of tRNAMe', tRNAse7UCU), and tRNAphe-Fig. 2 shows the sequences of these three tRNAs determined by a combination of the postlabeling methods (18, 19).

First, we confirmed directly that A. suum mt tRNAs have 3'-CCA sequences common to all tRNAs, although they are not encoded on the mt DNA. The post-transcriptional addition of the CCA sequence to the 3'-ends of mt tRNAs in A. suum and C. elegans has already been proposed by Okimoto and Wolsten- holme as judged from an estimation of the length of mt tRNAs by gel electrophoresis (14).

We have identified two different species of tRNAS"'(UCU) with the shortest known chain lengths (54 nucleotides) (l), having either G or A at their 5' termini (Fig. 3a). The former species with G' is identical to that of the mt genome DNA reported so far (11, 12), although the relative content of these two species appears to be roughly equivalent. This heterogene- ity is probably caused by the polymorphism of A. suum mt DNA?

tRNASer(UCU) has two kinds of modified nucleosides, Vr and t6A. These modifications are probably partial, although a quan- titative analysis has not been carried out because of the limited amount of purified tRNA species. On the other hand, tRNA"' (Fig. 3b) has four kinds of modified nucleosides, Vr, mlA (or m'A, probably converted from m'A during purification and analysis of tRNA), m2G, and a novel nucleoside a t position 34. This was sensitive to RNase CL3 and had mobilities on two- dimensional TLC identical to those of PC, which was recently identified by us as the novel nucleoside found at the anticodon

2Y. Watanabe, K. Kita, T. Ueda, and K. Watanabe, unpublished observation.

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22904 Structure of Nematode Mitochondrial tRNAs

tRNA*YUCU) (a), tRNA"'" ( b ) , and FIG. 2. Nucleotide sequences of

tRNAPhe (c ) . The letters in parentheses show residues with partial modification or putative base conversion during se- quencing analysis (in the case of m6A in tRNA""). The black and open triangles indicate the cleavage sites with RNase T2 and RNase V1, respectively (the cleavage strengths are shown by their sizes) as de- duced from the results in Fig. 3.

a - N G ?$ Nc

acceptor

DR loop anticodon

- acceptor stem DR loop anticodon stem anticodon loop anticodon stem

loop anticodon stem

T stem

T loop

- - -

T stem

-

acceptor stem

c

T stem

T loop

T stem

acceptor stem

a A 4 c 4

A- G-U: E4

A- U C-G

t6A (A)

b

b - NGT2 V I Nc 5 10 5 10

r- 5___ acceDtor stem

acceptor stem

N R Imp

anticodon

antcodon stem

loop anticodon stem

D stem

D loop

D stem

acceptw stem

N R loop anticodon

antlcodon stem

loop anticodon stem D stem

D loop

D stem

acceptor stem

C

acceptor

N R loop stem

anticodon stem anticodon

antlcodon stem

loop

D stem

D l q

D sten

acceptc stem

G U U

J J U

C

D A - U G-Ua

U' mlG \ - GN T2 V1 NC

51050 13510 510

acceptor

TVR loop stem

anticodon stem anticodon loop anticodon stem

D stem

D Imp

D stem

, stem acceptor

FIG. 3. Enzymatic probing of A. suum mt tRNAsr(UCU) (a), tRNAMet (b) , and tRNAPhe (c). In a and 6 , the labeled tRNAs were reacted with 0.002 unit of RNase T2 and 0.007 unit of RNase V1 for 5 or 10 min a t 37 "C. In c, the labeled tRNAs were reacted with 0.0005 (indicated as 5 in the figure), 0.0035 (35), or 0.005 (50) unit of RNase T2 and 0.007 (indicated as 7), 0.035 (35), or 0.05 (50) unit of RNase VI for 10 min-, G , and N are intact tRNA, alkaline ladder, and digestion with RNase T1, respectively. Nc, digestion with Neurospora crassa endonuclease under denaturing conditions for 5 min in a and b, and for both 5 and 10 min in c, with the lanes being marked as 5 or 10, respectively, as size markers. Autoradiograms are shown only in the case of 3'4abeled tRNA (a), or 5"labeled tRNA (b and c), although the experiments were carried out using both 5'- and 3"labeled tRNAs. All of the data are summarized in the putative secondary structures shown in Fig. 2.

first position of bovine mt tRNAMet (25). Thus, nematode mt probing technique using RNase T2 specific for single-strand tRNA"" also possesses PC at the anticodon first position. regions (26), and RNase V1 specific for double-strand regions tRNAPh" was purified by a preparative hybrid selection method, (26) (Fig. 3; the summarized results are shown in Fig. 2). RNase and its sequence is shown in Fig. 3c, including modified nucleo- T2 preferentially cleaved the anticodon loops, the CCA termini, sides of q, m'A, and m'G. and D loops in all the tRNAs. It is intriguing that the extra loop

Structural Probing of mt tRNAs-To elucidate the higher was sensitive in tRNASe'(UCU) having the usual T stem, order structures of these tRNAs, we adapted the enzymatic whereas the 3"halves of the TV-replacement loop were sensi-

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FIG. 4. Chemical Drobine of the 3‘-

Structure of Nematode Mitochondrial tRNAs

a C - N S D D - N S D D - N S D D A(N-7) G(N-7) C(N-3)

612612 _612612 1

labeled A. suum mttRNAPKC. a, autora- diogram of the electrophoresed gel.-, in- cubated without reagents; N , native condition (with 10 mh1 MgCl, at 37 “C); SD, semidenaturing condition (with 1 mw EDTA at 37 “C); D, denaturing condition (with 1 mlrr EDTAat 90 “C). The numbers show reaction time periods in minutes. b, summary of the results shown on the sec- ondary structure of tRNA”’” and proposed tertiary interactions. Itulic letters indi- cate residues unable to be probed because of chemical reactivity or the electro- phoretic resolution. Residues that are re- active under the native condition are circled, and those that are not reactive under the native condition but reactive under semidenaturing or denaturing con- ditions are boxed. Residues with ellipses are those that are reactive under the na- tive condition with moderate reactivity. The dotted lines show the tertiary inter- actions described under “Discussion.”

tive in tRNAMct and tRNAPh“. RNase V1 actually cleaved the stem regions that were inferred from the gene sequences. The results essentially support the secondary structural model pro- posed by Wolstenholme et al. (11).

We also applied a chemical probing technique to tRNAphe using dimethyl sulfate and diethyl pyrocarbonate, which moni- tors N-3 of cytidine and N-7 of purine bases (26) (Fig. 4u). All of the A and C residues in the stem regions in the putative sec- ondary structure except A6’ forming a mismatch with G6 were protected against the reagents under the native condition. G(N-7) in the stem regions (except GIo, GZR, and G43) was reac- tive, probably because of the stacking irregularity (26). These results also supported the above mentioned secondary struc- tural model. It is noticeable that some residues in the loop regions are protected from the reagents under the native con- dition, which strongly suggests the involvement of these resi- dues in the tertiary interactions (see “Discussion”).

DISCUSSION

We adopted preparative selective hybridization using solid- phase DNA probes (17) for the purification of mt tRNAs. This method is very powerful, especially for the purification of tRNA whose content is very low, because its high specificity enables the number of purification steps to be reduced. We demon- strated that an individual mt tRNA could be obtained by this method in a quantity sufficient for biochemical studies, even from samples that are not usually large enough.

The results from sequencing the 3‘-ends of nematode mt tRNAs showed the presence of the CCA sequence, which is indispensable for peptidyl transfer on ribosomes (27). This was confirmed by the observation that tRNAs partially purified by gel electrophoresis could accept the cognate amino acids with the mt extract, which also suggests that these tRNAs maintain a structure capable of functioning in the nematode mt transla- tional system.

A modified cytidine was found at the wobble position of A. suum mt tRNAMe‘. From an analysis of the nematode mt DNA sequence, not only AUG but also AUA is thought to be trans- lated as methionine (12,28). The DNA sequence data of various metazoan mitochondria (except echinoderms (28) and cnidaria (3)) suggest that AUA specifies methionine, rather than isoleu-

acceptor stem

D stem D loop D stem anticodon stem

anticodon loop

anticodon stem

22905

b A 76

TV-replacement loop

35

acceptor stem

cine. However, since the tRNAMet gene with the TAT anticodon is not present in metazoan mt DNA, except that of the blue mussel (29), and there is no evidence of the import of any cytoplasmic tRNA into mitochondria in these animals (30), the transcript from the tRNAMC‘ gene with the CAT anticodon should read the AUA codon. As discussed in a previous report (251, PC found at the anticodon first position of bovine mt tRNAM“‘ may read the AUAcodon. It is intriguing that the same modified nucleoside exists in the same position of A. suum mt tRNAMet, suggesting a common functional role between mam- malian and nematode mt tRNAMe% in the decoding of the AUA codon. Since it has been reported that the anticodon of mos- quito mt tRNAMet is unmodified CAU (31), in insect mitochon- dria, the AUA codon may be decoded by tRNAMe’ with the anticodon CAU by a different mechanism(s1 from those of nem- atode or mammalian mitochondria.

mlA was found at position 9 of tRNAM“‘ and tRNAPhe. All metazoan mt tRNAs, whose sequences have been determined at the RNA level except for tRNA%, possess 1-methyl purine derivatives at this position insofar as the position is occupied by a purine residue (1).

The enzymatic probing data ofA. suum mt tRNAPhe, tRNAMe‘, and tRNASer(UCU) are consistent with the putative secondary structure deduced from the gene sequence (11, 12). In the loop regions of the putative secondary structures of tRNAPhc and tRNAMe‘, phosphodiester bonds of Ll-L4 (according to the num- bering system of Ref. 12) in the TV-replacement loop were less sensitive toward RNase T2 than those of the other residues in the TV-replacement loop. The residues a t positions Ll-L4 were also protected from dimethyl sulfate or diethyl pyrocarbonate under the native Condition. These results suggest the existence of some interactions between these residues and the other re- gions. Based on the conservation of these residues (in particu- lar, L2 and L3 are highly conserved as purines (12)) and resi- dues in the D arm in various nematode mt tRNAs, Okimoto and Wolstenholme have proposed the presence of tertiary interac- tions between the D arm and the TV-replacement loop (14).

The sequence comparison of the nematode mt tRNA genes showed that residues at 15 and L4 were conserved enough to form an AI5-T” or TI5-A” base pair. In usual tRNAs, the terti- ary interaction between purine a t position 15 and its comple-

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22906 Structure of Nematode Mitochondrial tRNAs

mentary pyrimidine at position 49 is always present (32-34). Based on the tertiary interactions between the D arm and the variable loop in usual tRNAs and the experimental results in this study, it is speculated that tertiary base pairs such as

tRNAPhe ofA. s u m mitochondria, as shown by the dotted lines in Fig. 4b. These interactions, which probably correspond, re- spectively, to U8-A14, A9-AZ3, G10-G45, G22-G46, Gi5-C4*, and GZ6- AM in yeast tRNAPhe (351, will enable nematode mt tRNAs to maintain an L-shape-like tertiary structure similar to that of usual tRNAs, which will be helpful for the tRNAs to maintain a mutual distance and orientation between two functional do- mains, the anticodon and 3'-CCA terminus, that are the same as those of the usual tRNAs. Recently, Nureki et al . (36) re- ported that E. coli tRNA1Ie in vitro transcript lacking the T stem could be efficiently aminoacylated by E. coli isoleucyl-tRNA synthetase. They proposed a tertiary structural model for this tRNA mutant possessing the tertiary interactions present in the original tRNA1le, which serve to maintain the L-shape-like structure necessary for aminoacylation (36). Together with these results and the above mentioned structural implications, even nematode mt tRNAs with their bizarre cloverleaf struc- ture are presumably able to function in the nematode mt trans- lation system.

U8-Al4 mlA9-A23 ~ 1 0 - ~ L 2 A22-AL3 Ul5-AL4 , , and AZ6-GL1 exist in

Acknowledgments-We thank our colleagues, T. Yokogawa and S. Yoshinari (Ehime University) for technical comments and T. Kuramochi and T. Kuroda for technical assistance. Our thanks are also due to Prof. K. Miura (Gakushuin University) for encouragement and Prof. D. R. Wolstenholme (University of Utah) for the mt DNA sequence informa- tion before its publication.

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WatanabeY Watanabe, H Tsurui, T Ueda, R Furushima, S Takamiya, K Kita, K Nishikawa and K

tRNAs lacking either the T or D stem.Primary and higher order structures of nematode (Ascaris suum) mitochondrial

1994, 269:22902-22906.J. Biol. Chem. 

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