Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene.
The mutagen-sensitive CHO line irs1SF was previously isolated on the basis of hypersensitivity to ionizing radiation and was found to be chromosomally unstable as well as cross-sensitive to diverse kinds of DNA-damaging agents. The analysis of somatic cell hybrids formed between irs1SF and human lymphocytes implicated a human gene (defined as XRCC3; x-ray repair cross-complementing), which partially restored mitomycin C resistance to the mutant. A functional cDNA that confers mitomycin C resistance was transferred to irs1SF cells by transforming them with an expression cDNA library and obtaining primary and secondary transformants. Functional cDNA clones were recovered from a cosmid library prepared from a secondary transformant. Transformants also showed partial correction of sensitivity to cisplatin and gamma-rays, efficient correction of chromosomal instability, and substantially improved plating efficiency and growth rate. The XRCC3 cDNA insert is approximately 2.5 kb and detects an approximately 3.0-kb mRNA on Northern blots. The cDNA was mapped by fluorescence in situ hybridization to human chromosome 14q32.3, which was consistent with the chromosome concordance data of two independent hybrid clone panels.
Tebbs
R S
RS
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, CA 94551-0808, USA.
Zhao
Y
Y
Tucker
J D
JD
Scheerer
J B
JB
Siciliano
M J
MJ
Hwang
M
M
Liu
N
N
Legerski
R J
RJ
Thompson
L H
LH
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D002522:Chlorocebus aethiops; D002874:Chromosome Mapping; D002883:Chromosomes, Human, Pair 14; D002945:Cisplatin / Q000633:toxicity; D003001:Cloning, Molecular; D003360:Cosmids; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004351:Drug Resistance / Q000235:genetics; D005720:Gamma Rays; D015723:Gene Library; D006801:Humans; D006822:Hybrid Cells; D017404:In Situ Hybridization, Fluorescence; D008214:Lymphocytes / Q000378:metabolism; D016685:Mitomycin / Q000633:toxicity; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D014960:X Chromosome / Q000187:drug effects* / Q000528:radiation effects
D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D016685:Mitomycin; D002945:Cisplatin