Isolation and sequence analysis of two major leucine transfer ribonucleic acids (anticodon Mm-A-A) from a rat tumor, Morris hepatoma 5123D.

Abstract

The nucleotide sequences of two major tRNALeu species (anticodon Mn-A-A)isolated from Morris hepatoma 5123D were determined by a combination of a newly developed thin-layer readout sequencing method [Gupta, R. C., & Randerath, K. (1979) Nucleic Acids Res. 6, 3443-3458] and additional 3H- and 32P-labeled derivative methods entailing chromatographic fingerprinting and base-specific enzymatic cleavages. The nucleotide sequence of the two hepatoma tRNAMm-A-ALeu species, one of which has U and the other of which has A in position 50 at the tip of the long extra arm, is pG-U-C-A-G-m2G-A-U-G-(m2)G-C-(ac4)C-G-A-G-U-G-G-D-C-psi-A-A-G-G-C-m22G-C-C-A-G-A--C-U-Mm-A-A-m1G*-psi-psi-C-U-G-G-L-(psi)U-C-C-G-U- or A-A-U-G-G-A-G-m5C-G-U-G-G-G-T-psi-C-G-m1A-A-U-C-C-C-A-C-U-U-C-U-G-A-C-A-C-C-AOH. These are the first leucine tRNA sequences from higher eukaryotes that have been determined. Noteworthy features of the mammalian leucine tRNAs are the presence of psi in the beta region of the D loop and the occurrence of three unknown hypermodified nucleosides (Mm, m1G*, and L) in positions 35, 38, and 45, respectively. m1G* was converted to m1G by treatment with alkali. Sequencing gels indicated that the parent base of the 2'-O-methylated nucleoside Mm may be a pyrimidine, probably a C derivative, as indicated by the chromatographic behavior of nucleotides containing Mm. The presence of a pyrimidine in the wobble position would be consistent with the antidodon sequence Mm-A-A and the leucine condons U-U-G and U-U-A. The occurrence of a hypermodified nucleoside, L, in the first position of the long extra arm appears unusual; thus far the only modified nucleoside found in this position is Um in eukaryotic serine tRNAs. Since all tRNAs with a long extra arm sequenced to date have a pyrimidine in this position, L is likely to be a pyrimidine, probably a U derivative, as inferred from chromatographic data.