Mutagenicity testing on non-selectively cloned Chinese hamster ovary cells with the protein-mapping method

Zeindl, E; Sperling, Karl; Klose, Joachim

doi:10.1016/0165-1161(82)90020-6

Cited by 7 publications

(1 citation statement)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In mutagenesis pilot studies using two-dimensional PAGE, Zeindl et al (15) detected two structural mutations in 26 clones derived from Chinese hamster ovary cells treated with methylnitrosourea. The total number of locus tests in that study was estimated to be 33,800.…”

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.

View full text Add to dashboard Cite

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 ...

show abstract

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.

View full text Add to dashboard Cite

show abstract

Systematic analysis of the total proteins of a mammalian organism: Principles, problems and implications for sequencing the human genome

Klose

1989

Electrophoresis

View full text Add to dashboard Cite

High-resolution two-dimensional electrophoresis (2-DE) has reached a technological level that allows us to resolve most of the numerous unknown protein species of a mammalian organism if appropriate strategies are used. We will discuss the problems of classification and characterization of proteins and propose a systematic approach to the analysis of the total protein complex. Both a comprehensive as well as a pragmatic approach towards systematic analysis have been considered. A "complex protein database" is suggested and considered with regard to various uses. A systematic analysis of the mouse proteins has been started and some of the preliminary results are summarized here. In particular, genetic properties of the proteins were investigated and are presented in order to demonstrate the significance of a systematic analysis of proteins for research and practical application (e.g. mutagenicity testing). A concept is presented for sequencing the coding DNA of mouse and man, starting with a systematic analysis of mouse proteins and then using two recently developed methods - microsequencing of proteins from spots of 2-DE protein patterns, and utilization of the relatively short N-terminal sequences obtained - to produce the corresponding cDNA's of these proteins.

show abstract

Two-Dimensional Electrophoretic Protein Analysis in Human Genetics

Klose¹

1987

Human Genetics

View full text Add to dashboard Cite

Mutagenicity testing on non-selectively cloned Chinese hamster ovary cells with the protein-mapping method

Cited by 7 publications

References 15 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.

Systematic analysis of the total proteins of a mammalian organism: Principles, problems and implications for sequencing the human genome

Two-Dimensional Electrophoretic Protein Analysis in Human Genetics

Contact Info

Product

Resources

About