Specific Lack of the Hypermodified Nucleoside, Queuosine, in Hepatoma Mitochondria! Aspartate Transfer RNA and Its Possible Biological Significance1

[CANCER RESEARCH 44, 1167-1171, March 1984]

Erika Randerath,2 Hari P. Agrawal, and Kurt Randerath

Department of Pharmacology, Baylor College of Medicine, Texas Medical Center, Houston, Texas 77030

ABSTRACT of this tRNA was found to be GUC; i.e., a guanosine rather than the expected queuosine, 7-(j5-j[(1S,4S,5fl)-4,5-dihydroxy-2-cy- Tumor nucleic acids have frequently been found to be deficient clopenten-1-yl]amino)methyli)-7-deazaguanosine (13), occupied in methylated and other modified nucleotides. In particular, cy- its "wobble" position. The question thus arose whether this lack toplasmic transfer RNAs (tRNAs) from various neoplasms par of queuosine was a general feature of mammalian mt tRNAs or tially lack the hypermodified nucleoside queuosine, a modification a tumor-specific abnormality. This hypermodified nucleoside and specific for anticodons of histidine-, tyrosine-, asparagine-, and its sugar derivatives, mannosylqueuosine and galactosylqueu- aspartic acid-accepting tRNAs. Using aspartate tRNA as an osine, which normally occur in cytoplasmic tRNAs specific for example, we show here that liver mitochondria contain tRNA histidine, tyrosine, asparagine, and aspartic acid, have been fully modified with respect to queuosine, while the corresponding found partially lacking in tumors and transformed cells, as evi tRNA from mitochondria of Morris hepatoma 5123D completely denced by changes in column Chromatographie profiles (see Ref. lacks this constituent. The sequences of these tRNAs, which 19 for review), transglycosylase assay (13), and direct sequence were determined by a highly sensitive 32P-postlabeling procedure analysis (11, 26). This work revealed the partial replacement of entailing the direct identification of each position of the polynu- queuosine by guanosine in the anticodon of cytoplasmic tRNAAsn cleotide chains, were found to be pGAGAUAUUrrTAGUAAAAU- from Walker 256 carcinosarcoma (26) and in cytoplasmic tRNAAsp AAUUACAiÂ¿AACCUUQ(G)UCAAGGUUAAGUUAUAGACUUAA- from rat ascites hepatoma (11), while the corresponding liver AUCUAUAUAUCUUACCAoH. Lack of queuosine in the hepatoma tRNAs had queuosine and the mannose derivative of queuosine, mitochondrial tRNA may be due to the inavailability of queuine respectively. Since no mammalian mt tRNA containing queuosine in the hepatoma mitochondria for incorporation into tRNA or had been sequenced thus far, we have now isolated an aspartate to inhibition of the modifying enzyme, tRNA (guanine)- mt tRNA from rat liver and here report its sequence. In contrast transglycosylase, in the tumor. Taking into account results to its counterpart from hepatoma, the anticodon sequence of of others indicating a possible involvement of the queuosine the liver mt tRNA was found to read QUC, indicating the lack of modification in differentiation of eukaryotic cells, we hypothesize queuosine in the tumor mt tRNA to be a tumor-specific abnor that the queuosine defect may develop at an early stage of mality. carcinogenesis (I.e., during the promotion phase) and be directly involved in abnormalities of mitochondria which have been ob served frequently in transformed cells and tumors. MATERIALS AND METHODS

The sources of materials used in this study have been indicated INTRODUCTION previously (9, 23). Liver mitochondria were prepared from female Buffalo rats as described (6). To isolate mt tRNA*50, total nucleic acids were Lack of modified constituents in tumor cytoplasmic tRNAs extracted in the presence of phenol from mitochondrial pellets (6) and was first demonstrated about 10 years ago (17). Since then, a fractionated on DEAE-cellulose to obtain crude mt tRNA (25). mt tRNA*3" number of individual cytoplasmic tRNA species of neoplastic was purified from the latter by 3 successive polyacrylamide gel electro- origin have been shown to lack specific modified nucleotides phoreses [(a) a 6%:15% stacked gel at 4Â°,(b) an 18%, 3.5 M urea gel (Ref. 18 and the references quoted therein), and bulk mt tRNA3 at 4Â°,and (c) a 20%, 7 M urea gel at 40-42Â°; acrylamide:methylene from Morris hepatomas was also reported to be undermodified bis(acrylamide), 30:1; all gels were run at pH 8.3]. RNA was extracted (6). It was of interest to determine the basis of the latter obser after each step from mÃ©thylÃ¨neblue-stained gel bands as described earlier (20). Since the band patterns on these gels closely resembled vation at the nucleotide sequence level. In connection with other those observed during the isolation of hepatoma mt tRNA*3" (2), the work on mt tRNAs (1, 16, 21), we had isolated and sequenced location of liver mt tRNAAspwas inferred from the earlier experiments, mt previously an aspartate tRNA from the mitochondria of a trans- tRNA*^ was sequenced by a PEI-cellulose thin-layer readout procedure plantable rat tumor, Morris hepatoma 5123D (2). The anticodon (9, 23). To ensure the reproducibility of the sequence data, additional gel electrophoretic and Chromatographie runs were performed (2). RNAs 1This work was supported by USPHS Grants CA 13591 and CA 10893 (P6) from the 3 additional minor bands (2) on the second gel were also awarded by the National Cancer Institute. electrophoresed further on a third gel. These RNA bands were extracted 2 To whom requests for reprints should be addressed. for partial sequence analysis (see below). 3 The abbreviations used are: mt tRNA, mitochondrial tRNA; tRNA*", asparagine-accepting tRNA; tRNA**, aspartic acid-accepting tRNA; PEI-cellulose, poly- ethylenelmine-cellulose; tRNAÃ¢fe, aspartic acid-accepting tRNA in which the anti codon is QUC (Q, queuosine; U, undine, C, cytidine); tRNAT", tyrosine-accepting RESULTS tRNA; tRNA"*, histidine-accepting tRNA; Q-tRNA, tRNA containing queuosine in Purification of Rat Liver mt tRNAAsp.In analogy to hepatoma the first position of the anticodon. Received September 15,1983; accepted December 1,1983. mt tRNA (2), liver mt tRNA was fractionated into 10 bands on

MARCH 1984 1167

the first polyacrylamide gel, where mt tRNAAsp traveled with Â»OH C-70 Band 3 (the fastest band being Band 1). On the second gel, this c C A band was further resolved into 4 components. The mt tRNAAsp pS- U pG-U A-U A-U band was not further resolved on the third gel. About 3.5 Â¿Â¿gof 6 - C-6S G-C-SS mt tRNAAspwere obtained from 100 g of liver. A-U A-U 5-U-A 5-U-A â€¢*-w Â«I ' . A - U 60 5,'A Sequence Analysis. The sequence of rat liver mt tRNAatk v i5 . P U-*UAUCUA A .U-AÃšAUCUAAÃ• was deduced by the thin-layer readout procedure of Gupta and 'AAAAUGmlAu **AAyGn,'AU AUAA U AÂ»UUAC , MÂ«5 Randerath (9), as illustrated in Fig. 1. The RNA was partially * 20 * V- u digested by a single-hit cleavage technique, followed by 32P 25 -A - uVS A-U A-U labeling, gel electrophoresis, contact transfer to PEI-cellulose C-G C-G C-G C-G thin layers, and in situ RNase T2 digestion of the fragments (9, U A-35 U A- 55 23). 32P-labeled 5'-terminal nucleoside 3',5'-bisphosphates were 30-U A 30 U A 0 y C identified by PEI-cellulose thin-layer chromatography in 1.2 M ammonium formate, pH 3.5, and 0.55 M ammonium sulfate MH 5I23D mt Rat liver mt solutions, respectively, as described previously (9), except that the formate system was run under conditions of tank saturation Chart 1. The nucleotide sequences of mt tRNA** from Morris hepatoma 51 23D on PEI-cellulose sheets prepared in the laboratory (23). ano rat liver arranged in the clover-leaf form. A, adenosine; U, uridine; C, cytidine; G, guanosine; ^, pseudouridine; 0. queuosine; pG, phosphoguanosine; mM, 1- In Fig. 1, the formate readout of positions 1 to 70 of the tRNA methyladenosine. is shown. Distinct termini were obtained from all positions, except position 9 which gave 1-methyladenosine and N6-methyladeno- in this RNA of the hypermodified nucleoside queuosine, which sine, due to partial conversion of 1-methyladenosine to A/6- had been found previously only in cytoplasmic and bacterial methyladenosine under the experimental conditions. As shown tRNAs (13). As shown by a comparison of this RNA with the in Fig. 1, the RNA contains 3 modified nucleosides, 1-methylad corresponding mt tRNA from Morris hepatoma 5123D (2), queu enosine, pseudouridine, and queuosine in positions 9, 24, and osine is replaced in the tumor RNA by guanosine in the "wobble" 31, respectively. The hypermodified nucleotide, queuosine3',5'- position of the anticodon, with the rest of the sequence being bisphosphate, showed identical Chromatographie behavior in identical to that of the corresponding liver tRNA. This observation formate and sulfate solvents to that of queuosine 3',5'-bisphos- raises several questions. phate obtained from Escherichia coli tRNATyr (9). Thus, liver mt (a) What is the molecular mechanism(s) underlying the lack of tRNAAsp appears to contain queuosine not further modified by queuosine in tumor tRNA in general and in tumor mt tRNA in mannosylation or galactosylation. The mannosylated and galac- particular? tosylated derivatives would migrate more slowly on PEI-cellulose (o) What is the effect of this deficiency on tRNA functions? than the parent compound, due to additional hydrogen bonding (c) How does this deficiency arise during carcinogenesis, and of the hexose hydroxyls to the Chromatographie medium. what may be its significance for this process? A comparison of the thin-layer readouts of the anticodon (d) Is the queuine-queuosine deficiency involved in the main regions (positions 27 to 37) of mt tRNAAspfrom Morris hepatoma tenance of neoplastic properties? 5123D and rat liver in the 2 solvents is shown in Fig. 2. The In the following discussion, we will try to deal briefly with each readouts differ in position 31, indicating guanosine for the tumor of these points. For a general review of structure, biosynthesis, RNA and queuosine for the liver RNA. These results show that and functions of queuosine in tRNA, the reader is referred to a the "wobble" position of liver mt tRNAAspis fully modified, while recent comprehensive review by Nishimura (13). its hepatoma counterpart completely lacks this modification. A The queuosine modification of tRNA, entailing the insertion, comparison of the complete readouts of the 2 RNAs revealed no by a specific tRNA (guanine)transglycosylase, of queuine base additional differences. Their nucleotide sequences, arranged in into the first position of the anticodon in exchange for guanine the cloverleaf form, are shown in Chart 1. The RNAs have been (13), is unique among all other known enzymatic nucleic acid designated as aspartate tRNAs on the basis of their anticodon modifications, which (except for the synthesis of pseudouridine) triplets GUC and QUC, respectively, and the established codon do not involve glycosidic bond cleavage. Queuine is presumably recognition pattern of mammalian mt tRNAs (3). introduced into liver mt tRNAAspby this same unusual mechanism Attempts to Detect Additional Liver and Hepatoma mt subsequent to transcription of the RNA from mitochondrial DNA. tRNAA>pSpecies. A few tRNA species comigrating with tRNAAsp Furthermore, since queuine is not synthesized in mammalian (Band 3) on the first polyacrylamide gel were isolated as outlined cells, being derived mostly from dietary sources (24), it must be in "Materials and Methods" and subjected to sequence analysis. imported into the mitochondria. Taking into account the small Partial fragments of intermediate chain lengths were analyzed size of the mitochondrial genome (3), we expect the transglyco- primarily to establish anticodon sequences for proper assignment sylase to be coded for by nuclear DNA and to be imported into of amino acid specificity. Neither these tRNAs nor additional the mitochondria in analogy to other mt tRNA modifying enzymes tRNAs isolated from Bands 2 and 4 of the first gel exhibited (10). Since, in yeast, a single tRNA (guanosine-A/2,A/2)dimeth- aspartic acid anticodons. These results show that both liver and yltransferase and a single tRNA (uridine-5)-methyltransferase hepatoma contain only single mt tRNAAspspecies. have been shown to modify both cytoplasmic and mt tRNA (10), mt tRNAAsp is presumably modified by the same tRNA (guan-

DISCUSSION ine)transglycosylase that inserts queuine into cytoplasmic tRNAs in exchange for guanine. The absence of the mannose modifi In this paper, we report the nucleotide sequence of aspartate cation of queuosine in mt tRNAAsp suggests that the mannosyl- tRNA from rat liver mitochondria and demonstrate the presence transferase or its substrate, GDP mannose (13), is not imported

1168 CANCER RESEARCH VOL. 44

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1984 American Association for Cancer Research. Lack of Queuosine in Hepatoma mt tRNA into mitochondria or that mt tRNAAsp does not contain the structural features required for mannosylation (5). A H. Lack of queuosine in rat ascites hepatoma cytoplasmic tRNAAspmay be due to queuine deficiency in the tumor cells or inhibition of the transglycosylase by a tumor-specific factor, rather than to decreased levels or altered specificity of the transglycosylase (13). Queuine deficiency in tumor cells may be caused by increased transcription of tRNA, proceeding at a faster rate than that at which queuine enters the cell (8). This would be in agreement with the high turnover of tRNA observed generally H,cr XH in neoplasia (4, 22). While it is not known whether Morris hepa toma 5123D cytoplasmic tRNAAsp is deficient in queuosine, al tered column Chromatographie profiles of cytoplasmic tRNATyr and tRNAHis from this tumor and of cytoplasmic tRNAAsp from many neoplastic tissues (reviewed in Ref. 19), as well as the deficiency of queuosine in cytoplasmic tRNA from Morris hepa toma 7794A (13), strongly suggest this to be the case. Therefore, the lack of queuosine in hepatoma mt tRNAAsp is presumably due to the same causes implicated in the modification defect of cytoplasmic tRNA, i.e., inavailability of sufficient queuine or inhi bition of the transglycosylase. Currently, one can only speculate about the functional signifi cance of queuosine in mt tRNAAsp and the consequences of its lack. Recent results (13, 29, 30) imply that queuosine is involved in certain regulatory functions, in addition to modulating the rate Chart 2. Comparison of the structure of queuine (B and D) with the structures and extent of aminoacylation. A regulatory phenomenon involv of the tumor promoters (27,31,32) teleocidin A (A ) and 12-O-tetradecanoylphorbol- ing queuosine, observed in bacteria, might also hold for mito 13-acetate (C), respectively. Structural features shared by Compounds A and B and Compounds C and D, respectively, are boxed. The circled hydroxy groups chondria. Thus, the lower viability of an E. coli mutant lacking appear common to each of the 3 compounds. tRNAs containing queuosine when compared with wild type (13), suggests the possibility that the lack of this modification in mt tRNA might be related to the reduced numbers of mitochondria than normal amounts because of high tRNA turnover in cancer in cancer cells (14). cells, may play a role. Whether queuine-queuosine deficiency is While normal adult mammalian tissues appear to contain fully involved in the expression and maintenance of neoplastic prop modified Q-tRNAs, queuosine-deficient tRNAs have been found erties is unknown. However, irrespective of the underlying mech- in a variety of neoplasms (including human hepatomas) (13), in anism(s), the possibility that cellular queuine deficiency and the certain cultured cells (such as undifferentiated Friend erythroleu- consequent queuosine deficiency of cytoplasmic and mt tRNA kemic cells), in insects during certain phases of their life cycles may contribute to cellular dedifferentiation in tumorigenesis and (33), and in fetal and regenerating rat liver [see Nishimura's the maintenance of a dedifferentiated state in neoplasia deserves review (13)]. Differentiation appears invariably accompanied by further investigation. an increase in Q-tRNAs (12, 28). This process is inhibited by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (28), and ACKNOWLEDGMENTS various other experiments indicate opposite effects of tumor promoters and queuine (13). As shown in Chart 2, common We would like to acknowledge the generous gift of the late Dr. Harold P. Morris structural features of these compounds exist that would be in supplying the hepatoma used in this investigation. consistent with a competition of the tumor promoters with queuine for the same cellular receptor(s), thereby conceivably REFERENCES leading to a queuine-queuosine deficiency state and inhibition of 1. Agrawal, H. P., Gupta, R. C., Randerath, K., and Randerath, E. The sequence differentiation at an early stage of carcinogenesis. of mitochondrial arginine tRNA (anticodon UCG) from a transplantable rat tumor, Morris hepatoma 5123D. FEBS Lett., 130: 287-290, 1981. In connection with the present investigation, queuine and/or 2. Agrawal, H. P., Randerath, K., and Randerath, E. Tumor mitochondrial transfer the queuosine modification of mt tRNA may conceivably provide ribonucleic acids: the nucleotide sequence of Morris hepatoma 5123D mito a link between the state of cellular differentiation and mitochon- chondrial tRNASS;. Nucleic Acids Res., 9: 2535-2541,1981. 3. Anderson, S., Bankier, A. T., Barrell, B. G., de Bruijn, M. H. L, Coulson, A. drial development and function, processes which are known to R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, be tightly coupled (15). Thus, the mitochondria! queuine-queu P. H., Smith, A. J. H., Staden, R., and Young, I. G. Sequence organization of osine deficiency might be directly involved in abnormalities of the human mitochondrial genome. Nature (Lond.), 290: 457-465, 1981. 4. Borek, E., Baliga, B. S., Gehrke, C. W., Kuo, C. W., Belman, S., Troll, W., and mitochondria which have been frequently observed in trans Waalkes, T. P. High turnover rate of transfer RNA in tumor tissue. Cancer formed cells and tumors (14). Res.. 37.: 3362-3366,1977. 5. Carbon, P., Haumont, E., Foumier, M.. deHenau, S., and Grosjean, H. Site- The maintenance, in neoplastic cells, of queuosine deficiency directed in vitro replacement of nucleosides in the anticodon loop of tRNA: in tRNA is probably related to altered growth patterns induced application to the study of structural requirements for queuine insertase during carcinogenesis, involving increased transcription of tRNA activity. EMBO J., 2:1093-1097,1983. 6. Chia, L. S. Y., Morris, H. P., Randerath, K., and Randerath, E. Base compo (4, 22). In addition, inhibition of the transglycosylation reaction, sition studies on mitochondrial 4S RNA from rat liver and Morris hepatomas for example, by 7-methylguanine (7), which is released in greater 5123D and 7777. Biochim. Biophys. Acta, 425: 49-62, 1976.

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7. Elliott, M. S., and Trewyn, R. W. Queuine hypomodification of tRNA induced 3483, 1980. by 7-methylguanine. Biochem. Biophys. Res. Commun., 704: 326-332, 1982. 21. Randerath, K., Agrawal, H. P., and Randerath, E. Tumor mitochondrial transfer 8. Farkas, W. R. Effect of diet on the queuosine family of tRNA's of germ-free RNA's: the nucleotide sequence of mitochondrial tRNAtjXo from Morris hepa mice. J. Biol. Chem., 255: 6832-6835, 1980. toma 5123D. Biochem. Biophys. Res. Commun., 700: 732-737,1981. 9. Gupta, R. C., and Randerath, K. Rapid print-readout technique for sequencing 22. Randerath, K., Agrawal, H. P., and Randerath, E. tRNA alterations in cancer. of RNA's containing modified nucleotides. Nucleic Acids Res., 6: 3443-3458, Recent Results Cancer Res., 84: 103-120, 1983. 1979. 23. Randerath, K., Gupta, R. C., and Randerath, E. 3H and Å“P derivative methods 10. Hopper, A. K., Furukawa, A. H., Pham, H. D., and Martin, N. C. Defects in for base composition and sequence analysis of RNA. Methods Enzymol., 65: modification of cytoplasmic and mitochondrial transfer RNA's are caused by 638-680,1980. single nuclear mutations. Cell, 28: 543-550, 1982. 24. Reyniers, J. P., Pleasants, J. R., Wostman, B. S., Katze, J. R., and Farkas, 11. Kuchino, Y., Shindo-Okada, N., Ando, N., Watanabe, S., and Nishimura, S. W. R. Administration of exogenous queuine is essential for the biosynthesis Nucleotide sequences of two aspartic acid tRNA's from rat liver and rat ascites of the queuosine-containing tRNA's in the mouse. J. Biol. Chem., 256: 11591 - hepatoma. J. Biol. Chem., 256: 9059-9062,1981. 11594,1981. 12. Lin, V. K., Farkas, W. R., and Agris, P. F. Specific changes in Q-ribonucleoside 25. Roe, B. A. Studies on human tRNA 1. The rapid and large scale isolation and containing transfer RNA species during Friend leukemia cell erythroid differ partial fractionation of placenta and liver tRNA. Nucleic Acids Res., 2: 21-42, entiation. Nucleic Acids Res., 8: 3481-3489, 1980. 1975. 13. Nishimura, S. Structure, biosynthesis, and function of queuosine in transfer 26. Roe, B. A., Stankiewicz, A. F., Rizi, H. L., Weisz, C., DiLauro, M. N., Pike, D., RNA. Prog. Nucleic Acid Res. Mol. Biol., 28: 49-73, 1983. Chen, C. Y., and Chen, E. Y. Comparison of rat liver and Walker 256 carcinosarcoma tRNA's. Nucleic Acids Res., 6: 673-688,1979. 14. Pedersen, P. L. Tumor mitochondria and the bioenergetics of cancer cells. Prog. Exp. Tumor Res., 22: 190-274,1978. 27. Schmidt, R., Adolf, W., Marston, A., Roeser, H., Sorg, B., Fujiki, H., Sugimura, 15. Pollak, J. K., and Sutton, R. The differentiation of animal mitochondria during T., Moore, R. E., and Hecker, E. Inhibition of specific binding of [3H]phorbol- development. Trends Biochem. Sci., 5: 23-27, 1980. 12,13-dipropionate to an epidermal fraction by certain irritants and irritant 16. Randerath, E., Agrawal, H. P., and Randerath, K. Rat liver mitochondrial lysine promoters of mouse skin. Carcinogenesis (Lond.), 4: 77-81, 1983. tRNA (anticodon U'UU) contains a rudimentary D-arm and 2 hypermodified 28. Shindo-Okada, N., Terada, M., and Nishimura, S. Changes in amount of hypo- nucleotides in its anticodon loop. Biochem. Biophys. Res. Commun., 703: modified tRNA having guanine in place of queuine during erythroid differentia 739-744,1981. tion of murine erythroleukemia cells. Eur. J. Biochem., 775: 423-428, 1981. 17. Randerath, E., Chia, L. S. Y., Morris, H. P., and Randerath, K. Transfer RNA 29. Singhal, R. P. Queuine: an addendum. Prog. Nucleic Acid Res. Mol. Biol., 28: base composition studies in Morris hepatomas and rat liver. Cancer Res., 34: 75-80, 1983. 643-653,1974. 30. Singhal, R. P., and Vakharia, V. N. The role of queuine in the aminoacylation 18. Randerath, E., Gopalakrishnan, A. S., Gupta, R. C., Agrawal, H. P., and of mammalian aspartate transfer RNAs. Nucleic Acids Res., 77: 4257-4272, Randerath, K. Lack of a specific ribose methylation at guanosine 17 in Morris 1983. hepatoma 5123D tRNAgu. Cancer Res., 47: 2863-2867, 1981. 31. Sugimura, T. Potent tumor promoters other than phorbol ester and their 19. Randerath, E., Gopalakrishnan, A. S., and Randerath, K. Transfer RNA in significance. Gann, 73: 499-507, 1982. hepatomas. In: H. P. Morris and W. E. Criss (eds.), Morris Hepatomas, 32. Umezawa, K., Weinstein, I. B., Horowitz, A., Fujiki, H., Matsushima, T., and Mechanisms of Regulation, pp. 517-564. New York: Plenum Publishing Corp., Sugimura, T. Similarity of teleocidin B and phorbol ester tumor promoters in 1978. effects on membrane receptors. Nature (Lond.), 290: 411-413, 1981. 20. Randerath, E., Gupta, R. C., Moms, H. P., and Randerath, K. Isolation and 33. White, B. N., Tener, G. M., Holden, J., and Suzuki, D. T. Activity of a transfer sequence analysis of two major leucine transfer ribonucleic acids (anticodon RNA modifying enzyme during the development of Drosophila and its relation Mm-A-A) from a rat tumor, Morris hepatoma 5123D. Biochemistry, 79: 3476- ship to the su(s) locus. J. Mol. Biol., 74: 635-651,1973.

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cc cc

Y UU U G3I GG

AÂ«oA A4sA A â€¢9Â°A A

A f ? \ .â€¢-â€¢- ruu \ \/MÃ•Ã•\/M uu y \ / uu/ uuu Â§ Â§ cc

C CTO AA \ G3IT GG

Fig. 1. PEI-cellulose thin-layer readout (9,23) of positions 1 to 70 of rat liver mt tRNA**. The solvent was 1.2 M ammonium formate, pH 3.5. The autoradiograms display 5'-Å“P-labeled nucleoside 3',5'-bisphosphates released from the 5'-termini Fig. 2. PEI-cellulose thin-layer readouts of the anticodon regions (positions 27 to 37) of mt tRNA**0 from Morris hepatoma 5123D (left) and from rat liver (right) of S'-^P-labeled fragments of decreasing chain length (left to right). The readout in 1.2 M ammonium forniate, pH 3.5 (fop), and 0.55 M ammonium sulfate (bottom), is a composite of 3 separate chromatograms and was derived from a total of about respectively. C, cytidine; A, adenosine; U, undine; G, guanosine, 0, queuosine. 1 fig of purified tRNA. A, adenosine; G, guanosine; U, undine; mM, 1-methyladenosine; C, cytidine;

MARCH 1984 1171

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1984 American Association for Cancer Research. Specific Lack of the Hypermodified Nucleoside, Queuosine, in Hepatoma Mitochondrial Aspartate Transfer RNA and Its Possible Biological Significance

Erika Randerath, Hari P. Agrawal and Kurt Randerath

Cancer Res 1984;44:1167-1171.

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