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Somatic Cell and Molecular Genetics

, Volume 22, Issue 6, pp 453–460 | Cite as

Construction of a functional cDNA clone of the hamsterERCC2 DNA repair and transcription gene

  • Saloumeh Kadkhodayan
  • Edmund P. Salazar
  • Jane E. Lamerdin
  • Christine A. Weber
Article

Abstract

The complete hamsterERCC2 cDNA was constructed in a plasmid vector from clones of three overlapping reverse transcribed/polymerase chain reaction amplified fragments using unique restriction enzyme recognition sites within the regions of overlap. This complete cDNA insert was then cloned into a mammalian expression vector, pcD2E, and tested for function by the ability to confer UV resistance to theERCC2 mutant CHO cell line UV5. Site-specific mutagenesis was used to introduce the G347→A and G1844→A changes resulting in the Cys 116→Tyr and Gly615→Glu mutations previously identified in UV5 and UVL-13 (also anERCC2 mutant CHO cell line), respectively. The 116Tyr and 615Glu plasmids each failed to confer UV resistance to UV5 or UVL-13 cells, respectively, demonstrating that the changes identified are indeed the causative mutations in UV5 and UVL-13.

Keywords

Restriction Enzyme Expression Vector Transcription Gene cDNA Clone Recognition Site 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Plenum Publishing Corporation 1996

Authors and Affiliations

  • Saloumeh Kadkhodayan
    • 1
  • Edmund P. Salazar
    • 1
  • Jane E. Lamerdin
    • 1
  • Christine A. Weber
    • 1
  1. 1.Biology and Biotechnology Research ProgramLawrence Livermore National LaboratoryLivermore

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