Molecular cloning of the human nucleotide-excision-repair gene ERCC4.
ERCC4 was previously identified in somatic cell hybrids as a human gene that corrects the nucleotide-excision-repair deficiency in mutant hamster cells. The cloning strategy for ERCC4 involved transfection of the repair-deficient hamster cell line UV41 with a human sCos-1 cosmid library derived from chromosome 16. Enhanced UV resistance was seen with one cosmid-library transformant and two secondary transformants of UV41. Cosmid clones carrying a functional ERCC4 gene were isolated from a library of a secondary transformant by selecting in Escherichia coli for expression of a linked neomycin-resistance gene that was present in the sCos-1 vector. The cosmids mapped to 16p13.13-p13.2, the location assigned to ERCC4 by using somatic cell hybrids. Upon transfection into UV41, six cosmid clones gave partial correction ranging from 30% to 64%, although all appeared to contain the complete gene. The capacity for in vitro excision of thymine dimers from a plasmid by transformant cell extracts correlated qualitatively with enhanced UV resistance.
Thompson
L H
LH
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94551-0808.
Brookman
K W
KW
Weber
C A
CA
Salazar
E P
EP
Reardon
J T
JT
Sancar
A
A
Deng
Z
Z
Siciliano
M J
MJ
D000818:Animals; D002460:Cell Line; D002885:Chromosomes, Human, Pair 16; D003001:Cloning, Molecular; D003360:Cosmids; D006224:Cricetinae; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D008040:Genetic Linkage; D006801:Humans; D006822:Hybrid Cells; D009355:Neomycin / Q000494:pharmacology; D014162:Transfection; D014170:Transformation, Genetic
D004268:DNA-Binding Proteins; C095888:xeroderma pigmentosum group F protein; D009355:Neomycin