Analysis of point mutations induced by ultraviolet light in human cells.
Mutations induced in cultured human cells by 254-nm UV light were analyzed within exon 3 of the hypoxanthine guanine phosphoribosyl transferase (HPRT) gene. Five large independent cultures of human lymphoblastoid cells, line TK6, were exposed to 4 J/m2 of 254-nm UV light and mutants at the HPRT locus were selected en masse by 6-thioguanine (6TG) resistance. Exon 3 of the HPRT gene was amplified from the mutant cells by polymerase chain reaction (PCR) using modified T7 DNA polymerase. Denaturing gradient gel electrophoresis (DGGE) was used to separate the mutant sequences from the wild type as mutant/wild-type heteroduplexes. Individual mutant bands were isolated from the gel and the nature of the mutations was determined by direct sequencing. Eight predominant mutations were detected in the 184-bp exon 3 sequence. Of these, 3 transition, including 2 G-C to A-T and 1 A-T to G-C and 2 A-T to C-G transversions, appeared in all 5 UV-treated cultures but not in untreated cultures and were thus considered to be mutational hotspots. These observations are similar in nature to those previously reported in bacterial and rodent cells. A single G deletion, a tandem substitution of CpT for TpA, and a tandem triple substitution of GpGpA for ApApG were also observed but in only 2, 2 and 3 of the 5 UV-treated cultures, respectively. Numerical analysis of the mutant fractions of these 8 mutations indicated that each of them was distributed as a set of non-random and independent events, i.e., a mutational hotspot.
Keohavong
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Center for Environmental Health Sciences, Whitaker College of Health Sciences and Technology, Cambridge, MA 02139.
Liu
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Thilly
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D000483:Alleles; D001345:Autoradiography; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D006358:Hot Temperature; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007945:Leukemia, Lymphoid / Q000473:pathology; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D013866:Thioguanine / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000528:radiation effects*; D014466:Ultraviolet Rays
D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine