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[CANCER RESEARCH 31, 697-700, May 1971] Transfer RNA Specificity in Mammalian Tissues and Codon Responses of Seryl Transfer RNA Dolph Hatfield, Franklin H. Portugal, and Mary Caicuts National Cancer Institute, NIH, Bethesda, Maryland 20014 The possibility that tRNA may play a role in cellular regulation was first suggested by Itano (6) and subsequently by Ames and Hartman (2) and by Stent (13). Although no unique role of tRNA in regulation has thus far been established, changes in tRNA have been observed during phage infection, under varying growth conditions, in differentiation, and in oncogenesis. Presumably, these changes invoke changes in protein biosynthesis. In multicellular organisms, where many metabolic processes are unique to individual tissues, differences in tRNA in different tissues might be expected if these adaptor molecules play a role in regulation. A study was therefore undertaken to examine several aminoacyl-tRNA's in bovine kidney, liver, and brain. tRNA was isolated by 2 methods. In Method 1, tRNA was extracted from whole tissues with buffer and phenol, followed by isolation of the tRNA with diethylaminoethyl cellulose chromatography. In Method 2, particulate cellular matter was first removed, and the tRNA was then extracted with phenol (5). Isolated tRNA was deacylated in 1.8 M Tris, pH 8.0, for l hr at 37°.Aminoacyl-tRNA synthetases were prepared from each tissue (5). Labeled aminoacyl-tRNA's were prepared as shown above (see Chart 1), with the exception of the 1st eluting peak of liver seryl-tRNA and the 2nd eluting peak of brain seryl-tRNA. These peaks have a shoulder in Chart 1 but no shoulder in Chart 2. Additional studies suggest that these differences in elution profiles are not due to method of preparation but to a better resolution obtained in the experiment shown in Chart 1. In any case, the differences in methionyl-, arginyl-, and seryl-tRNA's are not due to mitochondrial tRNA and are present when the tRNA's are prepared by 2 methods. The differences are observed when methionyl-, arginyl-, and seryl-tRNA's of liver are acylated with 3H-labeled amino acid and those of brain with 14C-labeled amino acid, i.e., when the isotopes are reversed. The differences are also observed when these aminoacyl-tRNA's of liver are acylated with an enzyme previously described (5) and applied to the reversed phase Chromatographie system of Kelmers et al. (7). Chromatography of 3 aminoacyl-tRNA's investigated in preparation from brain and those of brain with an enzyme preparation from liver, i.e., when the aminoacyl-tRNA synthetases are reversed. These results and those given above demonstrate that the differences are present in the isolated tRNA of each tissue and are not due to isotope, to tissue-specific aminoacyl-tRNA synthetases or to the tRNA of a subcellular organeile. To determine whether the tissue specificity observed in methionyl-, arginyl-, and seryl-tRNA's of bovine liver and bovine liver and brain are shown in Chart 1. The elution profiles of methionyl-, arginyl-, and seryl-tRNA's are brain also occur in these tissues of another mammal, the elution profiles of these aminoacyl-tRNA's from rabbit liver compared. Significant differences were observed in their elution profiles, and the most pronounced differences were found in the seryl-tRNA's of these tissues. The elution profiles of methionyl-, arginyl-, and seryl-tRNA's of liver and kidney and of phenylalanyl-, lysyl-, and leucyl-tRNA's of liver, and brain were compared (Chart 3). The profiles of liver and brain methionyl-tRNA's (Chart 3, upper graph) are kidney, and brain were similar and were not further investigated. tRNA in mitochrondria is known tobe different from that in cytoplasm. Mitochondria were not removed during preparation of the tRNA used in the above studies. tRNA was therefore prepared from a postmitochrondrial fraction of bovine liver and brain (Method 2). Diethylaminoethyl cellulose chromatography was not used in this procedure, and thus the 2 methods of preparation differ considerably. The tRNA prepared by the latter procedure from liver and brain was acylated in separate experiments with radioactive methionine, arginine, and serine. Their elution profiles are compared in Chart 2. The upper graph shows the profiles of liver and brain methionyl-tRNA's, the middle graph shows arginyl-tRNA's, and the lower graph shows seryl-tRNA's. The differences observed in the elution profiles of each of these aminoacyl-tRNA's are similar to the corresponding profiles comparable to those of bovine liver and brain. Although arginyl-tRNA of liver (middle graph) was resolved into 3 major peaks as compared to 2 in bovine liver, differences between arginyl-tRNA's of the 2 rabbit tissues are evident. The major differences which were observed in the seryl-tRNA's of bovine brain and liver (see Chart 1 and 2) were also present in the corresponding rabbit tissues (Chart 3, lower graph). The elution profiles of chicken liver and brain methionyl-, arginyl-, and seryl-tRNA's are compared in Chart 4. Methionyl-tRNA's (upper graph) were resolved into 4 peaks and quantitative differences were observed. Differences were also observed in the elution profiles of arginyl-tRNA's (middle graph). Differences were observed in the seryl-tRNA's of chicken liver and brain; but these were not as pronounced as those in the corresponding tissues of mammals. The peak of seryl-tRNA in brain which eluted third from the column ran slightly in front of that in liver, suggesting that differences were present in this peak. Additionally, a shoulder is present on Peak III of brain which is not present in liver. The differences observed in chicken liver and brain MAY 1971 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1971 American Association for Cancer Research. 697 D. Hatfield, F. H. Portugal, and M. Caicuts methionyl-, I4C 3H 2500-r5000 3000 6000 1500+3000 / (Beefuï-erl => o o 4000T8000 were also observed when the tRNA was prepared by Method 1 and when the isotopes and the aminoacyl-tRNA synthetases of each tissue were reversed. Several experiments were carried out to determine whether the observed differences are due to changes that occur during isolation. Studies in other laboratories have shown that tRNA may be modified by action of caustic reagents used in tRNA perparation (14), dimer formation (1, 10), loss of CCA terminus (8), and treatment with magnesium which may activate an inactive tRNA (9). Seryl-tRNA of bovine liver and brain, which manifested the most pronounced differences in the present investigation, was selected as a model for these studies. The differences in seryl-tRNA did not appear to arise in preparation of brain and liver tRNA as a result of the step used to deacylate endogenous aminoacyl-tRNA's or as a result Arg-tRNA (BeefBrain [I4C] Arg-tRNA arginyl-, and seryl-tRNA's [I4d Ser-tRNA~, (Beefüver) 2000-1-4000 150 FRACTION NUMBER Chart 1. Comparison of elution profiles of methionyl-, arginyl-, and seryl-tRNA's of bovine liver and brain. Liver tRNA was acylated with liver aminoacyl-tRNA synthetases and l4C-labeled amino acid and compared to brain tRNA acylated with brain aminoacyl-tRNA synthetases and 3H-labeled amino acid. tRNA was prepared by Method of the use of caustic reagents (phenol and ethanol); nor did they appear to be due to dimer formation or loss of CCA terminus. Furthermore, the differences did not appear to be affected by heating brain and liver tRNA at elevated temperatures in the presence or absence of magnesium or by prolonged dialysis against EDTA. Slight changes in the elution profiles of brain and liver seryl-tRNA were observed following incubation of brain tRNA in liver extract and of liver tRNA in brain extract, but these changes were not significant enough to account for the differences observed. 1. '4C 3H 2000 -ri 0000 [3H] Met-tRNA (Rabbit Broin) —,io 2000 -r 4000 1000 - - 5000 o-'t 4000-T- uj 2000 -- - 3 O U O-1 2000 -i- IOOO - - o-Jk 150 300 o-1 150 FRACTION NUMBER Chart 2. Comparison of elution profiles of methionyl-, arginyl-, and seryl-tRNA's of bovine liver and brain. Conditions were the same as those in Chart 1 with the exception that tRNA was prepared by Method 2. 698 3OO FRACTION NUMBER Chart 3. Comparison of elution profiles of methionyl-, arginyl-, and seryl-tRNA's of rabbit liver and brain. Conditions were the same as those in Chart 2. CANCER RESEARCH VOL. 31 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1971 American Association for Cancer Research. tRNA Specificity Table 1 Codon responses of Peak III from chicken liver Assay conditions were slightly modified from those of Nirenberg and Leder (12) as previously described (5). Codons were the gift of Dr. Marshall W. Nirenberg. CodonUCUUCCUCAUCG, UGANonePeak AGC, UAA, UAG, III(A pmoles bound0)0.5540.0220.1140.005 or less (0.153)b 0 Amount of seryl-tRNA bound to ribosomes in presence of codon minus the amount bound in absence of codon. b Amount of seryl-tRNA bound to ribosomes in absence of codon. very slight with UCC (see Table 1). The codon responses of Peaks I to III of bovine liver seryl-tRNA and of Peak III of chicken liver seryl-tRNA were similar to those of guinea pig liver reported by Caskey et al. (4) and to those of chicken liver reported by Bernfield (3) at this symposium. The peak of seryl-tRNA that eluted in the salt wash (Peak IV of liver and Peak VI of brain) responded to UGA and UCU. For resolution of the UGA and UCU response, brain seryl-tRNA was fractionated at a different salt gradient (5), -0.5 and the terminal peak was assayed with UGA, UCU, UCA, UGU, UGC, UGG, UUA, AGA, CGA, and GGA. This peak responded only to UGA. Therefore, a species of seryl-tRNA is present in isolated tRNA of bovine tissues which recognizes specifically the codon UGA. Furthermore, a species of 150 300 seryl-tRNA obtained from rabbit liver and from chicken liver FRACTIONNUMBER Chart 4. Comparison of elution profiles of methionyl-, arginyl-, and recognized UGA (5). seryl-tRNA's of chicken liver and brain. Conditions were the same as Bernfield (3) reported the occurrence of 0-phosphorylseryl-tRNA in rooster liver (see also Ref. l 1); those in Chart 2. this species eluted in a terminal position from benzoylated diethylaminoethyl cellulose and did not respond to any of the Seryl-tRNA of bovine liver and brain was fractionated on a known serine codons. It seems likely that the seryl-tRNA reversed phase Chromatographie column, and the individual which recognizes UGA (seryl-tRNAyGA) is the same species Studies peaks were assayed for codon recognition (5). Four peaks of reported by Bernfield to be 0-phospnorylseryl-tRNA. to elucidate the role of seryl-tRNAUGA in higher organisms, liver seryl-tRNA eluted from the column and were designated, in their order of elution, Peaks I to IV (Peak IV eluted in l M as well as the possibility the serine moiety may undergo further modification, are in progress. NaCl). In a separate experiment, 6 peaks of brain seryl-tRNA eluted from the column and were designated I to VI. Peaks I, III, and IV in liver correspond to Peaks I, IV, and VI, respectively, in brain; Peaks II and V in brain constitute peaks REFERENCES which are not evident in liver. The results of the codon studies have been reported elsewhere (5) and are summarized below. 1. Adams, A., and Zachau, H. G. Serine Specific Transfer Ribonucleic All fractionated peaks were assayed at 0.01 M Mg4*with known serine codons, UCU, UCC, UCA, UCG, AGU, and AGC, and with terminator codons, UGA, UAA, and UAG. Peak I of liver and Peaks I and II of brain responded to AGU and AGC. Peak II of liver and Peak III of brain responded to UCG. Peak III of liver and Peaks IV and V of brain responded to UCU, UCA, and UCC; the level of response was most pronounced with UCU, less with UCA, and very slight with UCC. The corresponding peak (Peak III) of seryl-tRNA in chicken liver responded similarly to these codons; the level of response was most pronounced with UCU, less with UCA, and Acids 15. Some Properties of the Aggregates from Serine Specific Transfer Ribonucleic Acids. European J. Biochem., 5: 556-558, 1968. 2. Ames, B. N., and Hartman, P. E. The Histidine Operon. Cold Spring Harbor Symp. Quant. Biol., 28: 349-356, 1963. 3. Bernfield, M. R., and Mäenpää, P. H. Seryl Transfer RNA Changes during Estrogen-induced Synthesis and a Unique Seryl Transfer RNA Modification. Cancer Res., 31: 684-687, 1971. 4. Caskey, C. T., Beaudet, A., and Nirenberg, M. RNA Codons and Protein Synthesis 15. Dissimilar Responses of Mammalian and Bacterial Transfer RNA Fractions to Messenger RNA Codons. J. Mol. Biol., 37: 99-118, 1968. MAY 1971 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1971 American Association for Cancer Research. 699 D. Hatfield, F. H. Portugal, and M. Caicuts 5. Hatfield, D., and Portugal, F. H. Seryl-tRNA in Mammalian Tissues: Chromatographie Differences in Brain and Liver and a Specific Response to the Codon, UGA. Proc. Nati. Acad. Sei. U. S., 67: 1200-1206, 1970. 6. llano, H. A. The Synthesis and Structure of Abnormal Hemoglobins. In: J. H. P. Jonxis (ed.). Abnormal Hemoglobins in Africa. A Symposium, pp. 3-16. Philadelphia: F. A. Davis Co., 1965. 7. Kelmers, A. D., Novelli, G. D., and Stulberg, M. P. Separation of Transfer Ribonucleic Acids by Reverse Phase Chromatography. J. Biol. Chem., 240: 3979-3983, 1965. 8. Lebowitz, P., Ipata, P. L., Makman, M. H., Richards, H. H., and Cantoni, G. L. Resolution of Cytidine- and Adenosine-terminal Transfer Ribonucleic Acids. Biochemistry, 5: 3617-3625, 1966. 9. Lindahl, T., Adams, A., and Fresco, J. R. Renaturation of Transfer Ribonucleic Acids through Site Binding of Magnesium. Proc. Nati. Acad. Sei. U. S., 55: 941-948, 1966. 10. Loehr, J. S., and Keller, E. B. Dimers of Alanine Transfer RNA with Acceptor Activity. Proc. Nati. Acad. Sci. U. S., 61: 1115-1122, 1968. 11. Mäenpää, P. H., and Bernfield, M. R. A Specific Rooster Liver tRNA Containing Phosphoserine. Federation Proc.,29: 468, 1970. 12. Nirenberg, M., and Leder, P. RNA Codewords and Protein Synthesis. The Effect of Trinucleotides upon the Binding of sRNA to Ribosomes. Science, 145: 1399-1407, 1964. 13. Stent, G. S. The Operon: On its Third Anniversary. Science, 144: 816-820, 1964. 14. Sueoka, N., and Hardy, J. Deproteinization of Cell Extract with Silicic Acid. Arch. Biochem. Biophys., 125: 558-566, 1968. evidence for phosphoseryl-tRNA in yeast or bacterial tRNA acylated either with homologous or with the rat or rooster liver enzyme. Dr. Hatfield: With rat liver, have you been able to obtain a phosphorylating seryl-tRNA enzyme? Dr. Bernfield: We have some evidence for that, but I should mention that it is very difficult to do unless you get reasonably pure tRNA from that peak. It is very difficult to pick up unless you further fractionate that last tRNA peak. Dr. Weinstein: I would like to make a comment on redundancy which I think your data indicate very nicely. As the brain goes through the trouble of making an extra serine tRNA, Peak II which has codon recognition identical to Peak I, and there are, of course, many such examples, normal liver has 3 tyrosyl-tRNA's which have identical codon recognition. I think we are stuck with the problem of redundancy. I wonder what your comments are? Dr. Hatfield: One of the problems here may be that this species really needs another methyl group or something of this nature to become identical to Peak I, and that may be what we are actually separating. Peak II may be a precursor to Peak I. I don't know what to say about tRNA differences at the moment. I don't think anyone knows what to make of them. Dr. Borek: I would just like to be redundant to what Dr. Weinstein said. There are all of these changes in the seryl-tRNA's. Dr. Bernfield finds it in the rooster with enormous doses of hormone. We find it with normal administration of hormone in the ovariectomized uterus. Unfortunately, all we can measure is codon response. Isn't Discussion Dr. Bemfield: Have you looked for phosphoserine? Dr. Hatfield: Yes, we have. We have encountered problems in looking for derivatives of serine, and I don't know why. In the case of JV-acetylserine, we spent a lot of time on this problem. We are able to acetylate the serine but not enzymatically. I think this is just one problem we have encountered there. With regard to phosphoserine, what we have done is to isolate the last peak, and we have tried to phosphorylate the serine. We have not thus far been successful. Dr. Bernfield: I could mention in this regard we have not found any evidence of an acetylserine in the rat and rooster. I should also mention that we were not able to find any 700 this essentially our problem? The codon response is rather limited. There are only 64 of them. Once we could find some in vitro assays which would give us greater latitude than the codon response, I think we might be finding differences in function. Dr. Bock: I would like to comment on the codon recognition pattern where you find that, unlike the normal "wobble hypothesis" patterns, Base A is the 3rd letter of the codon. Ukita has found that type of pattern for a glutamate acceptor tRNA and has shown that a modified 2-thiouridine is the base which recognizes the A. It is possible selectively to destroy the thiouridine bases without harming other RNA components. Thus it may be possible to test whether the tRNA you studied also has a mio base in the anticodon. CANCER RESEARCH VOL. 31 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1971 American Association for Cancer Research. Transfer RNA Specificity in Mammalian Tissues and Codon Responses of Seryl Transfer RNA Dolph Hatfield, Franklin H. Portugal and Mary Caicuts Cancer Res 1971;31:697-700. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/31/5/697.citation Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1971 American Association for Cancer Research.