The use of two-dimensional electrophoresis to detect mutations induced in mouse spermatogonia by ethylnitrosourea

Marshall, Richard; Raj, A. Sudharsan; Grant, Francis James; Heddle, John A.

doi:10.1139/g83-070

Cited by 23 publications

(5 citation statements)

References 18 publications

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“…The number of spots scored is not given. Marshall et al (17) detected two structural mutations in two-dimensional preparations of the livers ofthe offspring of N-ethyl-N-nitrosourea-treated male mice. Sixtyseven offspring were examined, resulting in a total of =46,000 locus tests.…”

Section: Discussionmentioning

confidence: 99%

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

Hanash

Boehnke

Chu

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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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 ...

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Section: Discussionmentioning

confidence: 99%

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

Hanash

Boehnke

Chu

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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“…Susan Lewis and co-workers (Johnson & Lewis, 1981;Lewis et al, 1991Lewis et al, , 1992 then Marshall et al (1983) and Peters, Ball and Andrews (1986) developed a sort of 'electrophoretic multiple loci test' and in so doing identified many new mutations. Susan Lewis and co-workers (Johnson & Lewis, 1981;Lewis et al, 1991Lewis et al, , 1992 then Marshall et al (1983) and Peters, Ball and Andrews (1986) developed a sort of 'electrophoretic multiple loci test' and in so doing identified many new mutations.…”

Section: The Induction Of New Mutations In Specific Regions Of the Momentioning

confidence: 99%

Chemical mutagenesis of the mouse genome: an overview

Gu�net

2004

Genetica

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The careful comparison of the phenotypic variations generated by different alleles at a given locus, including of course, those alleles with a deleterious effect, is often an important source of information for the understanding of gene functions. In fact, every time it is possible to match a specific alteration observed at the genomic level with a particular pathology, it is possible to establish a relationship between a gene and its function. When considered from this point of view, the production of new mutations by experimental mutagenesis appears as an alternative to the strategy of in vitro gene invalidation by homologous recombination in embryonic stem (ES) cells, with the advantage that experimental mutagenesis does not require any previous knowledge of the gene structure at the molecular level. Homologous recombination in ES cells is a 'gene driven' approach, in which mutant alleles are produced for those genes that we already know. Experimental mutagenesis, on the contrary, is a 'phenotype driven' approach, in which unknown genes are identified based on phenotypic changes. Also, while homologous recombination in ES cells requires a rather sophisticated technology, mutagenesis is simple to achieve but relies greatly on the efficiency of the mutagenic treatment as well as on the use of an accurate protocol for phenotyping. In this review, we will address a few comments about the different techniques that can be used for the induction of point mutations in the mouse germ line with special emphasis on chemical mutagenesis. We will also discuss the limitations of experimental mutagenesis and the necessity to look for alternative ways for the discovery of new genes and gene functions in the mouse.

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“…Methods are also available for studying mutant offspring at the biochemical level in animal model systems, e.g., changes in protein charge [Marshall et al, 1983;Johnson and Lewis, 19811 and enzyme activity [Feuers et al, 1983;Bishop and Feuers, 19821. Although the number of chemicals tested in these systems is limited, such methodology has the potential for being automated and shows promise of incorporation into future testing schemes.…”

Section: Methods For Detection Of Inherited Mutations In Animals Genementioning

confidence: 99%

Human mutagens: Evidence from paternal exposure?

Narod

Douglas²,

Nestmann³

et al. 1988

Environ and Mol Mutagen

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The importance of inherited mutations as a cause of human disease has been established clearly through examples of well-defined genetic anomalies, such as Down syndrome and retinoblastoma. Furthermore, it is suspected that environmental contaminants induce mutations resulting in increased risk for such defects in subsequent generations of persons exposed. The present lack of direct evidence for induced inherited genetic disorders in human beings hampers the development of risk estimation techniques for extrapolation from animal models. The most extensive prospective epidemiologic studies of inherited genetic effects have involved survivors of atomic bomb detonations and patients treated with cancer chemotherapy. In neither case has a significant elevation in inherited genetic effects or cancer been detected in the offspring of exposed individuals. Epidemiologic studies of subjects receiving chronic exposure may be confounded by the effect of maternal exposure during pregnancy. Consideration of only paternal exposure can minimize the confounding influence of teratogenicity, enhancing the resolving power of studies for inherited effects. Using this approach, retrospective (case-control) studies of childhood cancer patients have provided limited but suggestive evidence for inheritance of induced effects. Endpoints, such as congenital malformations and spontaneous abortion following paternal exposure, can also be considered as indicators of heritable mutagenic effects. For example, there is limited evidence suggesting that paternal exposure to anaesthetic gases may cause miscarriage and congenital abnormalities as a result of induced male germ cell mutations. By comparing male-exposure endpoints for which there are human data, as described above, with parallel or similar animal endpoints, such as dominant lethal, inherited cancer and "male teratogenic" effects, it is possible that suitable models for extrapolating to human risk can be developed. In order to establish a clearer relationship between induced mutation and genetic disease, the current surveillance systems should be expanded to include endpoints relevant to genetic study. The relaxation of regulations regarding access to census data could improve the chances of documenting such an association.

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The use of two-dimensional electrophoresis to detect mutations induced in mouse spermatogonia by ethylnitrosourea

Cited by 23 publications

References 18 publications

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

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

Chemical mutagenesis of the mouse genome: an overview

Human mutagens: Evidence from paternal exposure?

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