Characterization and analysis of Chinese hamster ovary cell ERCC1 mutant alleles.
We isolated and characterized the ERCC1 coding sequence from three Chinese hamster ovary (CHO) parental (CHO-AA8, CHO-AT3-2 and CHO-9) and 10 ERCC1 mutant cell lines. Two general classes of mutations were observed: two mutant cell lines exhibited nucleotide additions or deletions to produce frameshift mutations and seven mutant cell lines exhibited point mutations that resulted in transitions or transversions, including nonsense mutations and mutations that generated intron/exon splicing errors. One mutant (UV201) which had been provisionally assigned to ERCC1 complementation group 1 (CG1) had no detectable mutation in its coding sequence. Of the nine ERCC1 mutant alleles characterized two mutations were identified in the XpA binding region of the Ercc1 protein; no mutations were found in the N-terminal portion of the Ercc1 protein. Results of Northern hybridization analysis showed that the relative levels of ERCC1 mRNA differed significantly both among the parental cell lines and among the mutant cell lines derived from each parental cell line. Western analysis with a CHO Ercc1-specific antibody detected Ercc1 protein in each of the parental cell lines and also in UV201. The marked reduction in Ercc1 protein levels observed in all the other mutants examined supports the hypothesis that ERCC1 mutations may destabilize this polypeptide.
Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville 78957, USA.
R L
RL
Rolig
M P
MP
Lowery
G M
GM
Adair
R S
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Nairn
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells / Q000502:physiology*; D006224:Cricetinae; D004268:DNA-Binding Proteins; D004720:Endonucleases; D005786:Gene Expression Regulation; D014644:Genetic Variation; D008969:Molecular Sequence Data; D009154:Mutation; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D004268:DNA-Binding Proteins; D011506:Proteins; D004720:Endonucleases