Construction of a functional cDNA clone of the hamsterERCC2 DNA repair and transcription gene
- 1 November 1996
- journal article
- Published by Springer Nature in Somatic Cell and Molecular Genetics
- Vol. 22 (6) , 453-460
- https://doi.org/10.1007/bf02369437
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
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