Construction of human XRCC1 minigenes that fully correct the CHO DNA repair mutant EM9.
The human gene that corrects the DNA repair defect of the CHO cell mutant EM9 is designated XRCC1 and is the first human gene to be cloned that has an established role in DNA strand-break repair. In this study, either an XRCC1 cosmid genomic fragment or synthetic oligonucleotides were ligated to an incomplete XRCC1 cDNA to generate two full-length XRCC1 cDNA constructs. The ability of these minigene constructs to restore normal levels of sister chromatid exchange (SCE) to EM9 upon transfection was demonstrated, and the transfectants grew at normal rates in selective medium that is fully toxic to EM9 cells. Constructs in which the XRCC1 open reading frame (ORF) was transcribed from the SV40 early promoter or the genomic XRCC1 native promoter were compared in their efficiency of correction. EM9 transfectants derived from the SV40 promoter displayed fewer SCEs and lower sensitivity to CldUrd than either AA8 wild-type cells or transfectants containing the ORF transcribed from the native promoter.
Caldecott
K W
KW
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94551-9900.
Tucker
J D
JD
Thompson
L H
LH
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D003360:Cosmids / Q000235:genetics; D006224:Cricetinae; D004260:DNA Repair / Q000235:genetics*; D005796:Genes / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D012854:Sister Chromatid Exchange / Q000235:genetics*; D014162:Transfection / Q000235:genetics
D009838:Oligodeoxyribonucleotides