Transfer RNA methylase activity in normal and dystrophic chicken muscle

Adult-dystrophic chicken muscle had 30% higher tRNA methylase activity and 42% higher tRNA methylating capacity than normal-adult chicken muscle. Eighty percent of the tRNA methylase activity of the dystrophic muscle resulted in the synthesis of N²-methylguanine, and 9% in the formation of N²,N²-dimethylguanine. From adult-normal muscle extracts, 33% of the tRNA methylase activity was due to the synthesis of N²-methylguanine, and 45% to the formation of N²,N²-dimethylguanine. Eight other methylated bases accounted for 5–15% of the enzyme activity in both tissues. Dialyzed and nondialyzed adult-normal muscle extracts had equivalent tRNA methylase activity. However, the dialyzed extracts synthesized 22% more N²-methylguanine and 18% less N²,N²-dimethylguanine than the nondialyzed extracts. Dialysis had no effect on the tRNA methylase activity or tRNA methylation pattern produced by adult-dystrophic muscle.     

Age-dependent changes in the specificity of tRNA methyltransferases in the cerebellum of the icteric and nonicteric Gunn rat

The activity of tRNA methyltransferases present in the cerebellum of 6- and 21-day-old nonicteric and icteric Gunn rats was compared using purifiedE. coli tRNAs as substrates. At 6 days the tRNA methyltransferases of the icteric animals were significantly more effective in methylating tRNA^Glu ₂ and tRNA^Phe than were those of their nonicteric counterparts. This relationship reversed itself at 21 days. The action of the tRNA methyltransferases from the 6-day-old icteric animals led to higher proportions of 1-methyladenine in tRNA^Glu ₂ and tRNA^Phe than were obtained using the corresponding enzymes of the nonicteric animals. The proportion ofN ²-methylguanine was also higher, yet only in tRNA^fMet and not in tRNA^Phe. The study reveals much more extensive fluctuations in the activity and in the substrate recognition specificity among the cerebellar tRNA methyltransferases of the icteric than among those of the nonicteric controls during the crucial 6–21 day period of cerebellar development.     

In vitro methylation of rat liver tRNA-synthesis of 3-methylcytosine and 1-methylhypoxanthine

Rat liver methylating enzymes could methylate tRNA extracted from the livers of rats treated with 35 mg/100 g L-ethionine 19 h prior to sacrifice. 1-methylhypoxanthine and 3-methylcytosine were among the methylated bases synthesized $\text{in vitro}$. The synthesis of 3-methylcytosine was dependent on the presence of Mg⁺⁺ although this ion inhibited the overall methylation of the tRNA.     

New archaeal methyltransferases forming 1-methyladenosine or 1-methyladenosine and 1-methylguanosine at position 9 of tRNA

Two archaeal tRNA methyltransferases belonging to the SPOUT superfamily and displaying unexpected activities are identified. These enzymes are orthologous to the yeast Trm10p methyltransferase, which catalyses the formation of 1-methylguanosine at position 9 of tRNA. In contrast, the Trm10p orthologue from the crenarchaeon Sulfolobus acidocaldarius forms 1-methyladenosine at the same position. Even more surprisingly, the Trm10p orthologue from the euryarchaeon Thermococcus kodakaraensis methylates the N¹-atom of either adenosine or guanosine at position 9 in different tRNAs. This is to our knowledge the first example of a tRNA methyltransferase with a broadened nucleoside recognition capability. The evolution of tRNA methyltransferases methylating the N¹ atom of a purine residue is discussed.     

Transfer ribonucleic acid methylase activity in adult and fœtal rat liver

Foetal rat liver extracts were found to have higher tRNA methylase activities than corresponding extracts of adult liver. When the specific activities were expressed per mg of liver or per mg of protein, the foetal tRNA methylating enzymes were respectively 2.5 and 6 times higher than those of adult livers.The presence of an inhibitor in adult liver can be excluded, since the same recoveries of total tRNA methylase activity were obtained after partial purification of both adult and foetal liver extracts: yields were close to 100 per cent.The apparent Km's for the substrates in the methylating reactions were the same when tRNA methylases from either adult or foetal liver were used: values were 0.2 μM for Escherichia coli tRNA and 2.1 μM for S-adenosyl-l-methionine.After T₁-T₂ ribonuclease digestion of an in vitro methylated tRNA, similar methyl nucleotide patterns were observed in foetal and adult enzymatic extracts.It is concluded that the same tRNA methylase pool is present in adult and foetal liver. In addition, it is hypothesized that the different reaction rates exhibited by these enzymes might be due to the tRNA functional requirements rather than to the presence of a tRNA methylase inhibitor.     

N2-guanine specific transfer RNA methyltransferase II from rat liver

An enzyme was purified from rat liver and leukemic rat spleen which methylates guanosine residues in tRNA to N²-methylguanosine. By sequence analysis of bulk E. coli tRNA methylated with crude extracts it was shown that the enzyme is responsible for about 50% of total m²G formed invitro. The extent of methylation of a number of homogenous tRNA species was measured using the purified enzyme from both sources. Among tested E. coli tRNAs only tRNA^Arg, tRNA^Phe, and tRNA^Val yielded significantly more m²G than the bulk tRNA. The K_m for tRNA^Arg in the methylation reaction with enzymes from either tissue was 7.8 × 10⁻⁷ M as compared to the value 1 × 10⁻⁵ M obtained for the bulk tRNA. In a pancreatic RNase digest of bulk tRNA as well as of pure tRNA^Arg, tRNA^Phe, and tRNA^Val, A-m²G-Cp was found to be the only sequence methylated. Thus, the mammalian methyltransferase specifically recognizes the guanylate residue at position 10 from the 5′-end contained in a sequence (s⁴)U-A-G-Cp. Furthermore, there is no change between the enzyme from normal liver and leukemic spleen in the affinity for tRNA, the methylating capacity, and tRNA site and sequence recognition specificity.     

Exonuclease 1 (Exo1) is required for activating response to SN1 DNA methylating agents

S_N1 DNA methylating agents are genotoxic agents that methylate numerous nucleophilic centers within DNA including the O⁶ position of guanine (O⁶meG). Methylation of this extracyclic oxygen forces mispairing with thymine during DNA replication. The mismatch repair (MMR) system recognizes these O⁶meG:T mispairs and is required to activate DNA damage response (DDR). Exonuclease I (EXO1) is a key component of MMR by resecting the damaged strand; however, whether EXO1 is required to activate MMR-dependent DDR remains unknown. Here we show that knockdown of the mouse ortholog (mExo1) in mouse embryonic fibroblasts (MEFs) results in decreased G2/M checkpoint response, limited effects on cell proliferation, and increased cell viability following exposure to the S_N1 methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), establishing a phenotype paralleling MMR deficiency. MNNG treatment induced formation of γ-H2AX foci with which EXO1 co-localized in MEFs, but mExo1-depleted MEFs displayed a significant diminishment of γ-H2AX foci formation. mExo1 depletion also reduced MSH2 association with DNA duplexes containing G:T mismatches in vitro, decreased MSH2 association with alkylated chromatin in vivo, and abrogated MNNG-induced MSH2/CHK1 interaction. To determine if nuclease activity is required to activate DDR we stably overexpressed a nuclease defective form of human EXO1 (hEXO1) in mExo1-depleted MEFs. These experiments indicated that expression of wildtype and catalytically null hEXO1 was able to restore normal response to MNNG. This study indicates that EXO1 is required to activate MMR-dependent DDR in response to S_N1 methylating agents; however, this function of EXO1 is independent of its nucleolytic activity.     

Clustering of transfer RNA genes of Xenopus laevis

The arrangement of the reiterated DNA sequences complementary to transfer RNA has been studied in Xenopus laevis. Prehybridization of denatured DNA with an excess of unfractionated tRNA results in a small but well-defined increase in the buoyant density of fragments which contain sequences homologous to tRNA. The density increase is smaller than that found for 5 S DNA, but is the same or nearly so for all tRNA coding sequences examined. These results indicate that the majority of tRNA genes are clustered together with spacer DNA, the average size of which is estimated to be approximately 0.5 × 10⁶ daltons (native) DNA.In high molecular weight native DNA preparations, the sequences homologous to unfractionated tRNA, tRNA^Val, tRNA₁^Met and tRNA₂^Met band in CsCl at 1.707, 1.702, 1.708 and 1.711 g cm^?3, respectively. The mean buoyant densities are constant at all molecular weights examined but they do not correspond to the base compositions of the complementary tRNA species. These results indicate that isocoding genes are linked to spacer DNA in separate and extensive gene clusters, and that the different clusters contain different spacer DNA sequences. These clusters form well-defined cryptic DNA satellites which are potentially separable from each other as well as from other chromosomal DNA.     

Overproduction and purification of Escherichia coli tRNALeu

Chemically synthesized genes encodingEscherichia coli tRNA ₁ ^Leu and tRNA ₂ ^Leu were ligated into the plasmid pTrc99B. then transformed intoEscherichia coli MT102, respectively. The positive transformants, named MT-Leu1 and MT-Leu2, were confirmed by DNA sequencing, and the conditions of cultivation for the two transformants were optimized. As a result, leucinc accepting activity of their total tRNA reached 810 and 560 pmol/A₂₆₀, respectively: the content of tRNA ₁ ^Leu was 50% of total tRNA from MT-Leu1, while that of tRNA ₂ ^Leu was 30% of total tRNA from MT-Leu2. Both tRNA^Leus from their rotal tRNs were fractionated to 1 600 pmol/A₂₆₀ after DEAE-Sepharose and BD-cellulose column chromatography. The accurate kinetic constants of aminoacylation of the two isoacceptors of tRNA^Leu catalyzed by leucyl-tRNA synthetase were determined. Project supported by the National Natural Science Foundation of China (Grant No. 39570164).     

Mature methyl-deficient tRNA isolated from a mammary adenocarcinoma

A transplantable rat tumor, mammary adenocarcinoma 13762, accumulates tRNA which can be methylated in vitro by mammalian tRNA (adenine-1) methyltransferase. This unusual ability of the tumor RNA to serve as substrate for a homologous tRNA methylating enzyme is correlated with unusually low levels of the A₅₈-specific adenine-1 methyltransferase. The nature of the methyl-accepting RNA has been examined by separating tumor tRNA on two-dimensional polyacrylamide gels. Comparisons of ethidium bromide-stained gels of tumor vs. liver tRNA show no significant quantitative differences and no accumulation of novel tRNAs or precursor tRNAs in adenocarcinoma RNA. Two-dimensional separations of tumor RNA after in vitro [¹⁴C]methylation using purified adenine-1 methyltransferase indicate that about 25% of the tRNA species are strongly methyl-accepting RNAs. Identification of six of the tRNAs separated on two-dimensional gels has been carried out by hybridization of cloned tRNA genes to Northern blots. Three of these, tRNA^Lys3, tRNA^Gln and tRNA^Met_i, are among the adenocarcinoma methyl-accepting RNAs. The other three RNAs, all of which are leucine-specific tRNAs, show no methyl-accepting properties. Our results suggest that low levels of a tRNA methyltransferase in the adenocarcinoma cause selected species of tRNA to escape the normal A₅₈ methylation, resulting in the appearance of several mature tRNAs which are deficient in 1-methyladenine. The methyl-accepting tRNAs from the tumor appear as ethidium bromide-stained spots of similar intensity to those seen for RNA from rat liver; therefore, methyladenine deficiency does not seem to impair processing of these tRNAs.     

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. ^{1, 2} It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. ² A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, ³Dictyostelium discoideum^4,5 and Drosophila. ⁶ Our recent work has shown that Ehmeth methylates tRNA^Asp, and this finding indicates that this enzyme has a dual DNA/tRNA^Asp methyltransferase activity. ⁷ This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. ⁸ The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.Download video file.^{(77M, mov)}     

Target recognition,RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA)

Although the DNA methyltransferase 2 family is highly conserved during evolution and recent reports suggested a dual specificity with stronger activity on transfer RNA (tRNA) than DNA substrates, the biological function is still obscure. We show that the Dictyostelium discoideum Dnmt2-homologue DnmA is an active tRNA methyltransferase that modifies C38 in tRNA^Asp(GUC) in vitro and in vivo. By an ultraviolet-crosslinking and immunoprecipitation approach, we identified further DnmA targets. This revealed specific tRNA fragments bound by the enzyme and identified tRNA^Glu(CUC/UUC) and tRNA^Gly(GCC) as new but weaker substrates for both human Dnmt2 and DnmA in vitro but apparently not in vivo. Dnmt2 enzymes form transient covalent complexes with their substrates. The dynamics of complex formation and complex resolution reflect methylation efficiency in vitro. Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress. However, dnmA expression only partially correlated with tRNA methylation in vivo. Strikingly, dnmA expression in the laboratory strain AX2 was significantly lower than in the NC4 parent strain. As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation.     

Methyl-deficient mammalian transfer RNA: II. Homologous methylation in vitro of liver tRNA from normal and ethionine-fed rats: ethionine effect on 5-methyl-cytidine synthesis in vivo

Following hydroxyapatite chromatography, rat liver tRNA methylase activity was assayed with liver tRNA from normal rats and with methyl-deficient liver tRNA from ethionine-fed rats. The difference in homologous methylation between normal and methyl-deficient tRNA was maximal in certain fractions in presence of cadaverine, and much less in presence of Mg⁺⁺ or Mg⁺⁺ plus cadaverine. These methylase fractions, which contained endogenous tRNA, were used for preparative homologous methylation of added normal and methyl-deficient tRNA in presence of 30 mM cadaverine. The ¹⁴C-methylated tRNA was digested with RNase T₂ and the resulting methylated mononucleotides were characterized and quantitated after twodimensional thinlayer chromatography and autoradiography. The major products of homologous tRNA methylation were m⁵C and m¹A. However, the methylase fraction used here did not catalyze the formation of m⁶₂A with m⁶₂A-deficient tRNA as substrate.- In addition to the previously described, analytically detectable m⁶₂A-deficiency, a partial m⁵C-deficiency was demonstrated in liver tRNA from ethionine-fed rats by measuring the methylacceptance in vitro. In presence of cadaverine, with the methylase fraction used here, methyl-deficient tRNA from ethionine-fed rats was a twofold more efficient methyl-acceptor in vitro than normal liver tRNA, while endogenous tRNA isolated from the methylase fraction was a threefold more efficient methyl-acceptor than normal liver tRNA. Homologous methylation of normal tRNA, as observed here, has not been described before.     

DNA repair in personalized brain cancer therapy with temozolomide and nitrosoureas

Alkylating agents have been used since the 60ties in brain cancer chemotherapy. Their target is the DNA and, although the DNA of normal and cancer cells is damaged unselectively, they exert tumor-specific killing effects because of downregulation of some DNA repair activities in cancer cells. Agents exhibiting methylating properties (temozolomide, procarbazine, dacarbazine, streptozotocine) induce at least 12 different DNA lesions. These are repaired by damage reversal mechanisms involving the alkyltransferase MGMT and the alkB homologous protein ALKBH2, and through base excision repair (BER). There is a strong correlation between the MGMT expression level and therapeutic response in high-grade malignant glioma, supporting the notion that O⁶-methylguanine and, for nitrosoureas, O⁶-chloroethylguanine are the most relevant toxic damages at therapeutically relevant doses. Since MGMT has a significant impact on the outcome of anti-cancer therapy, it is a predictive marker of the effectiveness of methylating anticancer drugs, and clinical trials are underway aimed at assessing the influence of MGMT inhibition on the therapeutic success. Other DNA repair factors involved in methylating drug resistance are mismatch repair, DNA double-strand break (DSB) repair by homologous recombination (HR) and DSB signaling. Base excision repair and ALKBH2 might also contribute to alkylating drug resistance and their downregulation may have an impact on drug sensitivity notably in cells expressing a high amount of MGMT and at high doses of temozolomide, but the importance in a therapeutic setting remains to be shown. MGMT is frequently downregulated in cancer cells (up to 40% in glioblastomas), which is due to CpG promoter methylation. Astrocytoma (grade III) are frequently mutated in isocitrate dehydrogenase (IDH1). These tumors show a surprisingly good therapeutic response. IDH1 mutation has an impact on ALKBH2 activity thus influencing DNA repair. A master switch between survival and death is p53, which often retains transactivation activity (wildtype) in malignant glioma. The role of p53 in regulating survival via DNA repair and the routes of death are discussed and conclusions as to cancer therapeutic options were drawn.     

Unmasking a killer: DNA O6-methylguanine and the cytotoxicity of methylating agents

Methylating agents are potent carcinogens that are mutagenic and cytotoxic towards bacteria and mammalian cells. Their effects can be ascribed to an ability to modify DNA covalently. Pioneering studies of the chemical reactivity of methylating agents towards DNA components and their effectiveness as animal carcinogens identified O⁶-methylguanine (O⁶meG) as a potentially important DNA lesion. Subsequent analysis of the effects of methylating carcinogens in bacteria and cultured mammalian cells — including the discovery of the inducible adaptive response to alkylating agents in Escherichia coli — have defined the contributions of O⁶meG and other methylated DNA bases to the biological effects of these chemicals. More recently, the role of O⁶meG in killing mammalian cells has been revealed by the lethal interaction between persistent DNA O⁶meG and the mismatch repair pathway. Here, we briefly review the results which led to the identification of the biological consequences of persistent DNA O⁶meG. We consider the possible consequences for a human cell of chronic exposure to low levels of a methylating agent. Such exposure may increase the probability that the cell's mismatch repair pathway becomes inactive. Loss of mismatch repair predisposes the cell to mutation induction, not only through uncorrected replication errors but also by methylating agents and other mutagens.     

Transfer RNA Bound to MnmH Protein Is Enriched with Geranylated tRNA – A Possible Intermediate in Its Selenation?

The wobble nucleoside 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U) is present in bacterial tRNAs specific for Lys and Glu and 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U) in tRNA specific for Gln. The sulfur of (c)mnm⁵s²U may be exchanged by selenium (Se)–a reaction catalyzed by the selenophosphate-dependent tRNA 2-selenouridine synthase encoded by the mnmH (ybbB, selU, sufY) gene. The MnmH protein has a rhodanese domain containing one catalytic Cys (C97) and a P-loop domain containing a Walker A motif, which is a potential nucleotide binding site. We have earlier isolated a mutant of Salmonella enterica, serovar Typhimurium with an alteration in the rhodanese domain of the MnmH protein (G67E) mediating the formation of modified nucleosides having a geranyl (ge)-group (C₁₀H₁₇-fragment) attached to the s² group of mnm⁵s²U and of cmnm⁵s²U in tRNA. To further characterize the structural requirements to increase the geranylation activity, we here report the analysis of 39 independently isolated mutants catalyzing the formation of mnm⁵ges²U. All these mutants have amino acid substitutions in the rhodanese domain demonstrating that this domain is pivotal to increase the geranylation activity. The wild type form of MnmH⁺ also possesses geranyltransferase activity in vitro although only a small amount of the geranyl derivatives of (c)mnm⁵s²U is detected in vivo. The selenation activity in vivo has an absolute requirement for the catalytic Cys97 in the rhodanese domain whereas the geranylation activity does not. Clearly, MnmH has two distinct enzymatic activities for which the rhodanese domain is pivotal. An intact Walker motif in the P-loop domain is required for the geranylation activity implying that it is the binding site for geranylpyrophosphate (GePP), which is the donor molecule in vitro in the geranyltransfer reaction. Purified MnmH from wild type and from the MnmH(G67E) mutant have bound tRNA, which is enriched with geranylated tRNA. This in conjunction with earlier published data, suggests that this bound geranylated tRNA may be an intermediate in the selenation of the tRNA.     

Transfer RNA analysis during the reproductive cycle of a freshwater teleost,H. fossilis

Transfer RNA was analyzed qualitatively as well as quantitatively from ovaries of the fresh water teleostHeteropneustes fossilis for twelve months. The tRNA samples were found to be pure and devoid of any high molecular weight RNA or DNA contaminations. The quantity of tRNA as well as its biological activity, assayed byin vitro aminoacylation using homologous aminoacyl tRNA synthetases, were found to be higher during resting and preparatory (pre-vitellogenic) phases, i.e. from November to March, as compared to vitellogenic and spawning phases of the fish, i.e. from April to October. The highest tRNA pool and its activity was found in the month of February, which coincides with the early preparatory phase. The results indicate that the accumulation of active tRNA starts in the resting phase. Such an accumulation of tRNA may be a part of the enrichment of mature eggs with complete translational machinery before ovulation in order to cope with the high rate of protein synthesis after fertilization.Abbreviations aaRS aminoacyl tRNA synthetase - [¹⁴C] APH [¹⁴C]-algal protein hydrolysate - ATP adenosine triphosphate - DTT dithiothreitol - EDTA ethylene diamine tetra acetic acid - GSI gonado somatic index - TCA trichloroacetic acid - tRNA transfer RNA     

Chloroethylating and methylating dual function antineoplastic agents display superior cytotoxicity against repair proficient tumor cells

Two new agents based upon the structure of the clinically active prodrug laromustine were synthesized. These agents, 2-(2-chloroethyl)-N-methyl-1,2-bis(methylsulfonyl)-N-nitrosohydrazinecarboxamide (1) and N-(2-chloroethyl)-2-methyl-1,2-bis(methylsulfonyl)-N-nitrosohydrazinecarboxamide (2), were designed to retain the potent chloroethylating and DNA cross-linking functions of laromustine, and gain the ability to methylate DNA at the O-6 position of guanine, while lacking the carbamoylating activity of laromustine. The methylating arm was introduced with the intent of depleting the DNA repair protein O⁶-alkylguanine-DNA alkyltransferase (AGT). Compound 1 is markedly more cytotoxic than laromustine in both AGT minus EMT6 mouse mammary carcinoma cells and high AGT expressing DU145 human prostate carcinoma cells. DNA cross-linking studies indicated that its cross-linking efficiency is nearly identical to its predicted active decomposition product, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE), which is also produced by laromustine. AGT ablation studies in DU145 cells demonstrated that 1 can efficiently deplete AGT. Studies assaying methanol and 2-chloroethanol production as a consequence of the methylation and chloroethylation of water by 1 and 2 confirmed their ability to function as methylating and chloroethylating agents and provided insights into the superior activity of 1.     

Trace 5-Methylaminomethyl-2-selenouridine in bovine tRNA and the selenouridine synthase activity in bovine liver

We measured the amount of Se in bovine liver tRNA. tRNA was chromatographed on a BD-cellulose column and Se-rich tRNA was eluted from the column in front of a main tRNA peak. There was 0.3 mmol Se/mol of tRNA and this level is about one tenth that of Escherichia coli tRNA. This suggests the presence of an enzyme that modifies tRNA with Se in bovine liver. We isolated the activity of this enzyme (selenouridine synthase) by chromatography of bovine liver extracts on a DEAE-cellulose column. ATP and selenophosphate synthetase, as well as selenouridine synthase and tRNA, were necessary for the reaction. ⁷⁵Se was used to label the reaction products, which were analyzed by TLC after digestion with ribonuclease T₂. The position of the ⁷⁵ Se-nucleotide on a TLC plate was identical to that of the Se-nucleotide, 5-methylaminomethyl-2-seleno-Up, prepared from ⁷⁵Se-tRNA in E. coli.