Two-dimensional polyacrylamide gel electrophoresis has been used to detect somatic cell gene mutations altering protein structure, following ethylnitrosourea treatment of cultured human lymphoblastoid cells. A total of 267 polypeptides encoded by 263 loci were scored in a series of 1143 lymphoblastoid clones. Sixty-five electrophoretic mutants were detected at a total of 49 loci. Sixteen of the 65 mutations were phenotypically repeat mutations, occurring at 11 loci. Furthermore, structural mutations occurred more frequently at loci known to be polymorphic. These results provide evidence that the mutations that are detectable at the protein level by two-dimensional polyacrylamide gel electrophoresis do not occur at random and that their frequency is greater among polymorphic loci.The question of how random mutational events are distributed throughout the genome of higher eukaryotes is not only of broad evolutionary interest but also has practical relevance in experimental mammals and humans; also of broad interest is the question of what are the potential errors in extrapolating from mutation rates at a relatively few loci to the entire genome? Because two-dimensional polyacrylamide gel electrophoresis permits the clear visualization of hundreds of cellular polypeptides (1), to monitor human populations for changing mutation rates, we have been exploring the potential of two-dimensional PAGE coupled with silver staining for polypeptide localization (2). N-Ethyl-N-nitrosourea has been shown to be a very potent mutagen in the mouse specific-locus test, leading to its use as a model chemical mutagen (3). To determine the usefulness and limitations of two-dimensional PAGE for mutation studies, we have used a human diploid lymphoblastoid cell line (TK-6) mutagenized with N-ethyl-N-nitrosourea (4). Evidence is presented for the nonrandom distribution of structural gene mutations among the 263 scored loci encoding proteins.
MATERIALS AND METHODSExperimental Design. The human lymphoblastoid cell line TK-6 was kindly provided by William Thilly (5). A subclone with a high plating efficiency of -70% was isolated. The subclone could be maintained in exponenti gowth fpr Etn indefinite period as a proliferating mass culture. The experimental design consisted of treating a mass culture with either N-ethyl-N-nitrosourea at 50 ,g/ml for 40 min or five successive daily exposures to the mutagen at 10 ,ug/ml. The former treatment resulted in 95% cell killing, and the latter resulted in 60% cell killing. Single-cell clones were isolated at various times following treatment and individually cultured, until a population size of 8-12 x 106 cells was obtained. Harvested cells were either pelleted for two-dimensional PAGE analysis or stored frozen in liquid nitrogen for subsequent propagation and repeated analysis. Control clones were isolated from an untreated mass culture under conditions comparable to those for treated clones.Two-Dimensional Electrophoresis. Cell pellets were solubilized by addition of 40 A.l of a lysis buffer ...