Construction of a functional cDNA clone of the hamsterERCC2 DNA repair and transcription gene
- 33 Downloads
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.
KeywordsRestriction Enzyme Expression Vector Transcription Gene cDNA Clone Recognition Site
Unable to display preview. Download preview PDF.
- 4.Friedberg, E.C., Walker, G.C., and Sicde, W. (1995).DNA Repair and Mutagenesis. American Society for Microbiology Press, Washington, D.C.Google Scholar
- 20.Steffan, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K., and Pease, L.R. (1989).Gene 77:51–59.Google Scholar