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Purification and Properties of the Major Phenylalanyl Transfer RNA Species in Drug-resistant Ehrlich Tumor Cells¹

Department of Biochemistry, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77025

Reverse-phase chromatography profiles of the tRNA's of normal mouse liver and also the Ehrlich ascites cells revealed the presence of three isoaccepting phenylalanyl transfer RNA (tRNA^Phe) species. The first two peaks eluted from these columns, tRNA and tRNA, were small and were followed by a third and major peak designated tRNA. Ehrlich tumor cells with an acquired resistance to nitrogen mustard, however, exhibited an altered chromatographic profile with minor tRNA and tRNA peaks and an exceptionally large tRNA area. This tRNA from the tumor cells as well as the major tRNA from normal mouse liver was isolated and purified to 80% or higher. Coding properties, kinetic studies of aminoacylation, and the nucleoside composition of the tRNA^Phe species were ascertained. The initial velocity of the aminoacylation reaction of the Ehrlich cell tRNA was lower and the K_m was slightly higher than the liver tRNA. This provides some indication that the two transfer RNA's may have a different nucleotide sequence and/or modified bases in the recognition sites of the aminoacyl synthetases. RNase T₁ digests of the two labeled tRNA^Phe's with subsequent chromatography revealed that both may have the same nucleotide sequence from the 3'-terminal to the first guanosine residue. Other findings indicated similar codon responses, the appearance of 3'-methyl-cytidine, and an increase in 5'-methyl-cytidine and dihydrouracil in the predominant tRNA of the resistant Ehrlich tumor cells.

¹ Supported by grants from the American Cancer Society (P-550) and the Robert A. Welch Foundation (G-035).

² Present address: Department of Biochemistry, Kurume University School of Medicine, Kurume, Japan 830.

³ To whom requests for reprints should be sent.

Received 3/20/74. Accepted 8/22/74.