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Complementation of the UV-sensitive phenotype of a xeroderma pigmentosum human cell line by transfection with a cDNA done library (~e~ trans~er/DNA repair) TAL TEITZ*, TOVA NAIMAN~, SOFIA S. ~VISSA~*, SA~ BA~*, HI~OTO OKAYAMA~, AND DAN "~p~menl of B~h~m~s~, ~ G~vrgv S. %Vi~ Faculty o~ Li(~ Sc~ance~, TeI-AvD Univvr~W. Tel-Aviv 6~78. Is~d; ~d ~o~ National lnsfi[at~ of Mcn1~ Health, Belhesda, MD 2020S Communicated by YueI Wal Kan, August 20, 1987 (receieed for reWew March I0. 1987) ABSTRACT In previous work, a xeroderma pigmentosum cell line belonging to complementafion group C was established by transformation with orlgln-defective simian virus 40. We now report the complementatlon of the UV sensitivity of this cell line by gene transfer. A human eDNA clone library constructed in a mammalian expression vector, and itself incorporated in a k phage vector, was introduced into the cells a~ a calcium phosphate precipitate. Following selection to O418 resistance, provided by the nee gene of the vector, transform- ants were selected for IYg resistance. Twenty-one cell clones were obtained with UV-resistancc levels typical of normal human fibrohlasts, All transformants contained vector DNA sequences in their nuclei. Upon further propagation in the absence of selection for G418 resistance, about half of the primary transformants remained UV-reslstant. Secondary trausformants were generated by transfection with a partial digest nf total chromosomal DNA from one of these stable transformants. This resulted in 15 G418-resistant clones, 2 of which exhibited a UV-reslstant phenotype. The other primary clones lost UV resistance rapidly when subcultured in the absence of G418. Importantly, several retained UV resistance under G418 selection pressure. The acquisition of UV re~is- lance by secondary transformants derived by trans£eetion of DNA from a stable primary transformant, and the linkage between G418 and UV resistances in the unstable primary transformants, strongly suggests that the transformants ac- qulred UV resistance through DNA-mediated gene transfer and not by reversion. Xerodcrma pigmcntosum (XP) is an autosomal recessive human disease manifested as an extreme sensitivity to UV light resulting in a very high incidence of skin cancer and, in many patients, neurological abnormalitics. Cells from XP patients are defective in the excision repair of pydmidine rimers in DNA and are extremely sensitive to killing by UV radiation. Nine eomplementation groups (A-I) defective in excision repair have been defined, whereas the XP "variant" appears to have normal levels of excision repair but is impaired in postreplication repair (1). The molecular basis for the different XP mutations has not been characterized-- neither genes nor gene products have been identified. Stable corrections of the UV sensitivity of simian virus 40 (SV40)- transformed XP cell lines ofcomplementation group A (XP-A cell lines) by transfeetion with normal human DNA (2) or following fusion with either x-ray-irradiated Chinese hamster ovary (CHO) cells (3) or microcells containing a single human chromosome (4) were reported. Transient complementation of exeidon-repair defects of XP primary cell lines was achieved either by microinjeeting mRNA from normal human The publieation costs of th~s aai:Ie were defrayed in l~art by pzgc charge ~aymcnt. This z~cle must therefore be hereb~ marked "adveet~semenl" in accvrdanee with 18 U.S.C. ~1734 sc!ely to ina~cate thls fuel cells into XP-A and XP-G cells (5) or by introducing protein extracts from normal human cells into XP-A cells (6, 7) or into ceils of all other XP complementatlon groups (8). Another approach for isolation of human excision-repair genes has been the stable complementation of excision-deficient cells produced in the laboratory. This ted to molecular cloning (9, 10) of a human gene (ERCCI) that complements group-2 CHO mutants, and its mapping to human chromo- some 19 (10-12). The relationship of this gene, if any, to human DNA-repair genes defective in XP is under investi- gation. One of the major obstacles in experiments aiming at stable eomplementation of XP cells is the scarcity of immortalized XP cell lines, Therefore, using for transfection an SV40 DNA fragment that contains a defective origin of replication but a functional gene encoding large tumor (T) antigen, we immor- talized two XP-C cell lines (13). These cell lines retain the high sensitivity to UV irradiation and, unlike many of the SV40-transformed human fibroblasts, show very few chro- mosomal changes, although the transfeeting SV40 DNA is integrated into cellular DNA sequences. We now report the eomplementation by gene transfer of the UV sensitivity of one of these established XP-C cell lines. METHODS Cell Culture, Transfections, and Selection of Transform- ants. The XP-C recipient cell line GbI2096-SV3 had been described (13). This cell line and all cells derived from it by transfeetion, as well as GM637 (SV40-transformed human fibroblasts), were maintained in Dulbeceo's modified Eagle's medium,containing penicillin, streptomycin, gentamicin, and 10% fet~,l bovine serum (Biological Industries, Beth Haemek, Israel). Cells were routinely subcultured every 3-4 days. Tmnsfeetion with the recombinant phage hNMT-peD- GM637 was as described (14) with the single exception that the "expression period" following washing of the phage particles was extended to 2 days. Then the ce|Is were trypsinized and replated at 1:5 dilution. Selection for G418 resistance was instituted 12-16 hr later by the addition of antibiotic G418 (GIBCO} at 400 gg/ml (15, 16). About 2Vz weeks later, G41S-resistant (G418g) colonies were trypsht- ized and replated. The next day the cells were subjected to a l.lV-irradiation protocol, employing 4~'/m2, four times at 1- to 2-day intervals. All operations were conducted in gold light (500-700 nm). Two weeks following the last UV irradiation, the surviving colonies were picked and grown to mass culture for further characterization. During the UV selection and Abbreviations: XP, xer~derraa p[gmentosum; XP-x cell line, XP cell line of complementation group x; SV4O, simian virus 40; G418~, G41g-reslstant; UV~. UV-resistant. STo whom reprint requests should be zddressed. 40008985