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Nucleotide sequence of three isoaccepting lysine tRNAs from rabbit liver and SV40-transformed mouse fibroblasts.

The lysine isoacceptor tRNAs differ in two aspects from the majority of the other mammalian tRNA species: they do not contain ribosylthymine (T) in loop IV, and a 'new' lysine tRNA, which is practically absent in non-dividing tissue, appears at elevated levels in proliferating cells. We have therefore purified the three major isoaccepting lysine tRNAs from rabbit liver and the 'new' lysine tRNA isolated from SV40-transformed mouse fibroblasts, and determined their nucleotide sequences. Our basic findings are as follows. a) The three major lysine tRNAs (species 1, 2 and 3) from rabbit liver contain 2'-O-methylribosylthymine (Tm) in place of T. tRNA1Lys and tRNA2Lys differ only by a single base pair in the middle of the anticodon stem; the anticodon sequence C-U-U is followed by N-threonyl-adenosine (t6A). TRNA3Lys has the anticodon S-U-U and contains two highly modified thionucleosides, S (shown to be 2-thio-5-carboxymethyl-uridine methyl ester) and a further modified derivative of t6 A (2-methyl-thio-N6-threonyl-adenosine) on the 3' side of the anticodon. tRNA3Lys differs in 14 and 16 positions, respectively, from the other two isoacceptors. b) Protein synthesis in vitro, using synthetic polynucleotides of defined sequence, showed that tRNA2Lys with anticodon C-U-U recognized A-A-G only, whereas tRNA3Lys, which contains thio-nucleotides in and next to the anticodon, decodes both lysine codons A-A-G and A-A-A, but with a preference for A-A-A. In a globin-mRNA-translating cell-free system from ascites cells, both lysine tRNAs donated lysine into globin. The rate and extent of lysine incorporation, however, was higher with tRNA2Lys than with tRNA3Lys, in agreement with the fact that alpha-globin and beta-globin mRNAs contain more A-A-G than A-A-A- codons for lysine. c) A comparison of the nucleotide sequences of lysine tRNA species 1, 2 and 3 from rabbit liver, with that of the 'new' tRNA4Lys from transformed and rapidly dividing cells showed that this tRNA is not the product of a new gene or group of genes, but is an undermodified tRNA derived exclusively from tRNA2Lys. Of the two dihydrouridines present in tRNA2Lys, one is found as U in tRNA4Lys; the purine next to the anticodon is as yet unidentified but is known not be t6 A. In addition we have found U, T and psi besides Tm as the first nucleoside in loop IV.

Animals

Modified nucleosides of Bacillus subtilis transfer ribonucleic acids.

An analysis of the kinds and amounts of minor nucleosides of transfer ribonucleic acids (tRNA's) from Bacillus subtilis 168 trpC2 is presented. Identification and quantitation were accomplished using ion exclusion chromatography, thin-layer and paper chromatography, and ultraviolet absorption properties. Nucleosides and their amount in moles per 80 residues are as follows: guanosine (25.7), cytidine (22.0), adenosine (15.2), uridine (13.1), 5-methyluridine (0.98), pseudouridine (1.54), 1-methyladenosine (0.15), N6-methyladenosine (0.01), 7-methyladenosine (0.10), 2-methyladenosine (0.03), 7-methylguanosine (0.20), N2-methylguanosine (0.14), 1-methylguanosine (0.14), a methylated pyrimidine (0.17), a methylated derivative of N6-(delta 2-isopentenyl)adenosine (0.02), ribose methylated nucleosides (0.02), 4-thiouridine (0.12), 2-thio-5-(N-methylaminomethyl) (0.09), and an unknown thionucleoside (0.12). Although the composition is similar to that of Escherichia coli in the proportion of major nucleosides, the content of pseudouridine and 5-methyluridine, and the degree of base and ribose methylation, the composition is more similar to that of the tRNA's of yeast and higher organisms in its lower degree of thiolation, the presence of significant amounts of 1-methyladenosine, and the low levels of 2-methyladenosine and 6-methyladenosine. Therefore, the nucleoside composition of B. subtilis presents some different aspects from those usually given as characteristic for bacterial tRNA's. It is not known whether these differences are due to variation between bacterial species in general or related to the process of differentiation.

Adenosine

Chemotherapy of advanced measurable colon and rectal carcinoma with oral 5-fluorouracil, alone or in combination with cyclophosphamide or 6-thioguanine, with intravenous 5-fluorouracil or beta-2'-deoxythioguanosine or with oral 3(4-methyl-cyclohexyl)-1(2-chlorethyl)-1-nitrosourea: a Phase II-III study of the Eastern Cooperative Oncology Group (EST 4273).

In a randomized multi-institutional trial of the Eastern Cooperative Oncology Group, 316 patients with advanced measurable colorectal adenocarcinoma were treated with a weekly schedule of 5-fluorouracil given orally and intravenously with oral-5-fluorouracil in combination with cyclophosphamide or 6-thioguanine, or with oral Methyl CCNU administered once every eight weeks. On failure or progression, 133 protocol patients crossed-over to a secondary therapy, while 116 other patients previously treated with 5-fluorouracil off protocol were randomized to treatment with Methyl CCNU or B-2'-deoxythioguanosine. Response rates among patients who had received no prior chemotherapy were 18% to oral 5-FU, 15% to intravenous 5-FU and to MeCCNU, 12% to 5-FU and 6-thioguanine and 5% to cyclophosphamide and 5-FU, with little activity (3% response rate) in crossover or previously treated patients. Treatment with 5-FU, particularly oral 5-FU was associated with the least drug-related toxicity. Hematologic toxicity was greatest with Methyl CCNU, but was no more frequent in previously treated than in untreated patients. A tendency toward cumulative bone marrow depression was noted. 5-FU was effective only in ambulatory patients, whereas responses among non-ambulatory patients were seen only in the group treated with Methyl-CCNU.

Adenocarcinoma

Clinical studies of beta-thioguanine deoxyriboside alone and in combination with arabinosyl cytosine.

Beta-thioguanine deoxyriboside (betaTGdR) is a purine nucleoside derivative which was studied alone or in combination with arabinosyl cytosine (Ara-C) in patients with solid tumors and acute leukemia. No significant responses were observed in 22 patients with solid tumors. The response rate with betaTGdR alone in acute leukemia was 26% and in combination with Ara-C was 24%. Responses were generally of short duration. Toxicity included myelosuppression, nausea, stomatitis, hyperpigmentation, photosensitivity, and liver function abnormalities.

Acute Disease

Methionine metabolism in apple tissue.

A metabolic intermediate isolated from apple tissue fed either methionine or 5'-methylthioadenosine has been tentatively identified as a methionine-pyridoxal Schiff base. The formation of this compound is discussed in relation to ethylene biosynthesis.

Adenosine

5-methylthioribose 1-phosphate: a product of partially purified, rat liver 5'-methylthioadenosine phosphorylase activity.

5'-Methylthioadenosine phosphorylase from rat liver has been purified 112-fold. A molecular weight of 90 000 for the enzyme was estimated from gel filtration on Sephadex G-150. The Km for 5'-methylthioadenosine was 4.7 . 10(-7) M, while the Km for phosphate was 2 . 10(-4) M. The products of the reaction were isolated and identified as adenine and 5-methylthioribose 1-phosphate. In addition to 5'-methylthioadenosine the nucleoside analogues 5'-ethylthioadenosine and 5'-n-propylthioadenosine also served as substrates for the enzyme. The 7-deaza analogue 5'-methylthiotubercidin was found to be an inhibitor of the reaction, but was inactive as a substrate.

Adenosine