Study on the biosynthesis of the modified uridine derivatives at the first position of the anticodon of tRNA

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1. Bacillus subtilis was cultivated in IOL of a medium of Antibiotic Medium 3 120 times. Crude tRNA was extracted from B. subtilis cells with 88% phenol and purified by a combination of several column chromatography. Glycine(2 kinds),lysine, threonine, valine accepting tRNAs were purified.2. In order to change the anticodon nucleotides with unmodified oligonucleotide, each tRNA molecule was digested with RNase TI, RNase A or nuclease Sl. 3'- and 5' half molecules were purified by polyacrylamide gel electrophoresis.3. RNase H was very effective to obtain the specific half molecule of tRNA. The enzyme can split in RNA strand in the double strand region of RNA=DNA. The chimeric oligomers which is complimentary to the nucleotide sequence of stem and loop region of anticodon were synthesized. The phosphodiester bond between permanent U34 and C33 was specifically hydrolyzed.4. 5'-Half molecules from glycine, lysine, threonine tRNAs were ligated with the unmodified oligonucleotide, [32P]pUp, … More [32P]pUUU or [32P]pUA by RNA ligase. After 3'-terminal phosphate was eliminated by PMase, if any, they were annealed with corresponding 3'-half molecule. Two halves were ligated again by RNA ligase to give a whole tRNA molecule which contains unmodified anticodon nucleotide.5. Reconstructed tRNA molecules were incubated with B. subtilis S-100 or S-30 fraction. After incubation, tRNA was precipitated by alcohol and hydrolyzed with nuclease Pl. The modification of uridylic acid in the first position of the anticodon, which contains [32P] at the 5' phosphate was checked by thin layer chromatography. None of the reconstructed tRNA molecule was modified at the first nucleotide in the anticodon by crude enzyme fraction. It may be difficult to modify isotope-labeled tRNA as a substrate because of the low concentration of a substrate or cosubstrate.6. We have not obtained a much progress in this project in these two years. But, we hope to continue this research project to get an anticipated results. Less