Genes of human mitochondrial tRNALeu(UUR) (mtRNALeu(UUR)) and its mutant (mtRNALeu(M)) were synthesized and inserted into the plasmid pGEM-9Zf(-) respectively. E.coli JM 109 was transformed by the recombinant plasmids containing the target genes. The mtRNALeu(UUR) and mtRNALeu(M) were expressed up to 19.10% and 17.76% of total small RNA respectively. They were purified to 54% homogeneity by DEAE-sepharose-CL4B column chromatography and finally repurified by 15% PAGE/urea. Their kinetic parameters for E.coli LeuRS were measured. The results showed that the value of kcal/ Km of mtRNALeu(M) was about one fifth of that of mtRNALeu(UUR) and indicated the leucine acceptability of mtRNALeu(M) was much lower than that of mtRNALeu(UUR).
- Gene cloning
- Human mitochondrial tRNA
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Environmental Science(all)
- Agricultural and Biological Sciences(all)