Nonrandom distribution of structural mutants in ethylnitrosourea-treated cultured human lymphoblastoid cells.

Abstract

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 ...

Development of a possible nonmammalian test system for radiation-induced germ-cell mutagenesis using a fish, the Japanese medaka (Oryzias latipes).

“…Nevertheless, the calculated DD for three spermatogenic stages for these two genetic endpoints coincided well with each other; 1-3 rad for sperm, 5-6 rad for spermatids, and 30-40 rad for spermatogonia. Hanash et al (26) have reported that in a human somatic cell system the loci that support genetic variation are more mutable than the monomorphic loci. The test system we developed depends on the existence of genetic variation at the loci employed.…”

Spectrum of ENU‐induced mutations in phenotype‐driven and gene‐driven screens in the mouse

“…The whole genome of Arabidopsis thaliana appeared equally susceptible to ethyl methanesulfonate-induced mutations, but mutations were mainly G:C to A:T changes, with a strong bias with respect to the neighboring base pairs [Greene et al, 2003]. Finally, nonrandom distribution of protein structural mutants was found in an experimental system using ENU-treated human lymphoblastoid cells [Hanash et al, 1988].…”

A Genetic Screen to Isolate <em>Toxoplasma gondii</em> Host-cell Egress Mutants

“…However, ENU preferentially targets AT base pairs 25 , which we validated in Toxoplasma (Farrell, Marth, Gubbels et al, manuscript submitted). Since the frequency of A/T is lower in amino acid coding sequence than non-coding sequence, the majority of mutations induced by ENU will be in non-coding sequence.…”