XERODERMA-PIGMENTOSUM VARIANT - GENERATION AND CHARACTERIZATION OF FIBROBLASTIC CELL-LINES TRANSFORMED WITH SV40 LARGE T-ANTIGEN

Xeroderma pigmentosum variant (XPV) fibroblasts from the XP4BE strain (CRL1162) were transformed with the SV40 large T antigen with the purp ose of generating immortalized cell lines that are defective in postre plication repair (PRR). Two transformation and selection protocols wer e used and at least two independent clones were obtained, which behave d in culture as immortal cell lines, Fingerprinting analyses were used to demonstrate their origin from XP4BE cells and to compare their gen etic profiles. These cell lines were shown to be hypersensitive to kil ling by uv when compared to SV40-transformed fibroblasts derived from foreskins of normal neonates, One of the XPV transformed cell lines (C Tag) was characterized further as a potential source of cell-free extr acts with capability for catalyzing the T antigen-dependent in vitro r eplication of plasmid DNA carrying the SV40 origin of replication. In this assay system, CTag extracts were shown to be as active as those p roduced from HeLa cells. In vitro replication of uv-damaged plasmid DN A by protein extracts from PRR-defective (XPV) and PRR-proficient cell s might allow the identification and characterization of protein facto rs that contribute to normal replication of uv-damaged DNA. Ultraviole t irradiation of plasmid DNA templates caused dose-dependent inhibitio n of in vitro replication by both HeLa and CTag extracts. (C) 1995 Aca demic Press, Inc.