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Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites.

Publication ,  Journal Article
Tamura, H; Kohchi, C; Yamada, R; Ikeda, T; Koiwai, O; Patterson, E; Keene, JD; Okada, K; Kjeldsen, E; Nishikawa, K
Published in: Nucleic Acids Res
January 11, 1991

Camptothecin (CPT), a plant alkaloid with antitumor activity, is a specific inhibitor of eukaryotic DNA topoisomerase I. We have previously isolated and characterized a CPT-resistant topoisomerase I isolated from a CPT-resistant human leukemia cell line, CPT-K5. cDNA clones of topoisomerase I were isolated from the CPT-resistant and the parental CPT-sensitive cell lines, respectively. Sequencing of the clones identified two mutations in the cDNA isolated from the resistant cells, which cause amino acid changes from aspartic acid to glycine at residues 533 and 583 of the parental topoisomerase I. When the CPT-K5 topoisomerase I was expressed in E. coli as a fusion protein with Staphylococcal Protein A fragment, the activity was resistant to CPT at a dose level up to 125 microM, whereas the parental fusion protein was sensitive to CPT as low as 1 microM. The resistance index (greater than 125) of the CPT-K5 fusion topoisomerase I is similar to that of the native CPT-K5 topoisomerase I. These results indicate that either or both of the two amino acid changes identified in the mutant enzyme is responsible for the resistance to CPT.

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Published In

Nucleic Acids Res

DOI

ISSN

0305-1048

Publication Date

January 11, 1991

Volume

19

Issue

1

Start / End Page

69 / 75

Location

England

Related Subject Headings

  • Tumor Cells, Cultured
  • Transcription, Genetic
  • Restriction Mapping
  • Recombinant Fusion Proteins
  • Protein Conformation
  • Mutation
  • Molecular Sequence Data
  • Humans
  • Gene Expression
  • Escherichia coli
 

Citation

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Tamura, H., Kohchi, C., Yamada, R., Ikeda, T., Koiwai, O., Patterson, E., … Nishikawa, K. (1991). Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites. Nucleic Acids Res, 19(1), 69–75. https://doi.org/10.1093/nar/19.1.69
Tamura, H., C. Kohchi, R. Yamada, T. Ikeda, O. Koiwai, E. Patterson, J. D. Keene, K. Okada, E. Kjeldsen, and K. Nishikawa. “Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites.Nucleic Acids Res 19, no. 1 (January 11, 1991): 69–75. https://doi.org/10.1093/nar/19.1.69.
Tamura H, Kohchi C, Yamada R, Ikeda T, Koiwai O, Patterson E, et al. Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites. Nucleic Acids Res. 1991 Jan 11;19(1):69–75.
Tamura, H., et al. “Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites.Nucleic Acids Res, vol. 19, no. 1, Jan. 1991, pp. 69–75. Pubmed, doi:10.1093/nar/19.1.69.
Tamura H, Kohchi C, Yamada R, Ikeda T, Koiwai O, Patterson E, Keene JD, Okada K, Kjeldsen E, Nishikawa K. Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites. Nucleic Acids Res. 1991 Jan 11;19(1):69–75.
Journal cover image

Published In

Nucleic Acids Res

DOI

ISSN

0305-1048

Publication Date

January 11, 1991

Volume

19

Issue

1

Start / End Page

69 / 75

Location

England

Related Subject Headings

  • Tumor Cells, Cultured
  • Transcription, Genetic
  • Restriction Mapping
  • Recombinant Fusion Proteins
  • Protein Conformation
  • Mutation
  • Molecular Sequence Data
  • Humans
  • Gene Expression
  • Escherichia coli