Skip to main content
SpringerLink
Log in

Gene cloning, expression and purification of human mitochondrial tRNALeu(UUR) and its mutant

  • Published:
Science in China Series C: Life Sciences Aims and scope Submit manuscript

Abstract

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 forE.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).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Anderson, S., Bankier, A. T., Barrell, B. G. et al., Sequence and organization of the human mitochondrial gene, Nature, 1981, 290(9): 457.

    Article  PubMed  CAS  Google Scholar 

  2. Wang, X. M., Yang, Y. S., The mutation of human mitochondrial tRNAand diseases, Chemistry of Life, 1999, 19(4): 171.

    CAS  Google Scholar 

  3. Goto, Y., Nonaka, I., Horai, S., A mutation in the tRNALeu(UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies, Nature, 1990, 348(13): 651.

    Article  PubMed  CAS  Google Scholar 

  4. Schapira, A. H. V., Mitochondrial disorders, Current Opinion in Genetics and Development, 1993, 3: 457.

    Article  PubMed  CAS  Google Scholar 

  5. Moraes, C. T., Ricci, E., Bonilla, W. et al., The mitochondrial tRNALeu(UUR) mutation in mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode (MELAS): genetic, biochemical and morphological correlations in skeletal muscle, Am. J. Hum. Genet., 1992, 50: 934.

    PubMed  CAS  Google Scholar 

  6. Chen, L., Jin, Y. X., Wang, D. B. et al., Species-specific identity elements of tRNATrp, Prog. Natl. Sci., 2000, 10(3): 192.

    CAS  Google Scholar 

  7. Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 1989, 556–558.

    Google Scholar 

  8. Merri, C. R., Goldman, D., Keuren, M. L. V., Gel protein stains: silver stain, Methods Enzymol, 1984, 104: 441.

    Article  Google Scholar 

  9. Li, Y., Wang, E., Wang, Y., Overproduction and purification ofEscherichia coli tRNAleu, Science in China, Ser. C, 1998, 41(3): 225.

    Article  CAS  Google Scholar 

  10. Hess, J. F., Parisi, M. A., Bennett Clayton, D. A., Impairment of mitochondrial transcription termination by a point mutation associated with the MELAS subgroup of mitochondrial encephalomyopathies, Nature, 1991, 352: 236.

    Article  Google Scholar 

  11. Yousufzai, A. Y. K., Bradford, M. W., Shrago, E. et al., Characterization of the adenine nucleotide translocase of pancreatic islet mitochondria, FEBS Lett., 1982, 137: 201.

    Article  Google Scholar 

  12. Welsh, N., Paabo, S., Welsh, M., Decreased mitochondrial gene expression in isolated islet of rats injected neonatally with streptozotocin, Diabetologia, 1991, 34: 626.

    Article  PubMed  CAS  Google Scholar 

  13. Chen, L., Jin, Y. X., Wang, D. B., Mitochondrial tRNATrpzs imply an eubacterial origin, Acta Biochimica et Biophysica Sinica, 2000, 32(2): 100.

    PubMed  CAS  Google Scholar 

  14. Gray, M. W., Origin and evolution of mitochondrial DNA, Annual. Rev. Cell Biol., 1989, 5: 25.

    CAS  Google Scholar 

  15. Anderson, S., Bankier, A. T., Barrell, B. G. et al., Sequence and organization of the human mitochondrial genome, Nature, 1981, 290: 457.

    Article  PubMed  CAS  Google Scholar 

  16. Anderson, S., De Bruijn, M. H., Coulson, A. R. et al., Complete sequence of bovine mitochondrial DNA conversed features of the mammalian mitochondrial genome, J. Mol. Biol., 1982, 156(4): 683.

    Article  PubMed  CAS  Google Scholar 

  17. Bibb, M. J., Van Etten, R. A., Wright, C. T. et al., Sequence and gene organization of mouse mitochondrial DNA, Cell, 1981, 26(2): 167.

    Article  PubMed  CAS  Google Scholar 

  18. Gada, G., Pepe, G., De Candia, G. et al., The complete nucleotide sequence of theRattus norvegiens mitochondrial genome: cryptic signals revealed by comparative analysis between vertebrates, J. Mol. Evol., 1989, 28(6): 497.

    Article  Google Scholar 

  19. Desjardins, P., Morais, R., Sequence and gene organization of the chicken mitochondrial genome—A novel gene order in higher vertebrates, J. Mol. Biol., 1990, 212(4): 599.

    Article  PubMed  CAS  Google Scholar 

  20. Roe, B. A., Ma, D. P., Wilson, R. K. et al., The complete nucleotide sequence of theXenopus laevis mitochondrial genome, J. Biol. Chem., 1985, 260(17): 9759.

    PubMed  CAS  Google Scholar 

  21. Clary, D. O., Wolstenholme, D. R., The mitochondrial DNA molecule ofDrosophila Yakuba: nucleotide sequence, gene organization, and genetic code, J. Mol. Evol., 1985, 22(3): 252.

    Article  PubMed  CAS  Google Scholar 

  22. Garesse, R., Drosophila melanogaster mitochondrial DNA: gene organization and evolutionary considerations, Genetics, 1988, 118(4): 649.

    PubMed  CAS  Google Scholar 

  23. Cantalor, P., Rofberti, M., Rainaldi, G. et al., The complete nucleotide sequence, gene organization and genetic code of the mitochondrial genome of paracentrotus lividus, J. Biol. Chem., 1989, 264(19): 10965.

    Google Scholar 

  24. Jacobs, H. T., Elliott, D. J., Math, V. B. et al., Nucleotide sequence and gene organization of sea urchin mitochondrial DNA, J. Mol. Biol., 1988, 202(2): 185.

    Article  PubMed  CAS  Google Scholar 

  25. Rich, A., RajBhandary, U. I., Transfer RNA: molecular structure, sequence, and properties, Annu. Rev. Biochem., 1976, 45: 805.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Chinese Academy of Sciences, 200031, Shanghai, China

    Weiguo Han, Li Chen, Jing Liu, Youxin Jin & Debao Wang

  2. Department of Biochemistry, Shanghai Medical University, 200032, Shanghai, China

    Weiguo Han & Xiliang Zha

Authors
  1. Weiguo Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Li Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jing Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiliang Zha
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Youxin Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Debao Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Youxin Jin.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Han, W., Chen, L., Liu, J. et al. Gene cloning, expression and purification of human mitochondrial tRNALeu(UUR) and its mutant. Sci. China Ser. C.-Life Sci. 44, 113–120 (2001). https://doi.org/10.1007/BF02879315

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02879315

Keywords

Search

Navigation