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1.
Genetics ; 119(3): 693-703, 1988 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-3402732

RESUMO

A subclone of a human diploid lymphoblastoid cell line, TK-6, with consistently high cloning efficiency has been used to estimate the rates of somatic mutations on the basis of protein variation detected by two-dimensional polyacrylamide gel electrophoresis. A panel of 267 polypeptide spots per gel was screened, representing the products of approximately 263 unselected loci. The rate of human somatic mutation in vitro was estimated by measuring the proportion of protein variants among cell clones isolated at various times during continuous exponential growth of a TK-6 cell population. Three mutants of spontaneous origin were observed, giving an estimated spontaneous rate of 6 x 10(-8) electrophoretic mutations per allele per cell generation (i.e., 1.2 x 10(-7) per locus per cell generation). Following treatment of cells with N-ethyl-N-nitrosourea, a total of 74 confirmed variants at 54 loci were identified among 1143 clones analyzed (approximately 601,000 allele tests). The induced variants include 65 electromorphs which exhibit altered isoelectric charge and/or apparent molecular weight and nine nullimorphs for each of which a gene product was not detected at its usual location on the gel. The induced frequency for these 65 structural gene mutants is 1.1 x 10(-4) per allele. An excess of structural gene mutations at ten known polymorphic loci and repeat mutations at these and other loci suggest nonrandomness of mutation in human somatic cells. Nullimorphs occurring at three heterozygous loci in TK-6 cells may be caused by genetic processes other than structural gene mutation.


Assuntos
Modelos Genéticos , Mutação , Proteínas/genética , Linhagem Celular , Linhagem Celular Transformada , Eletroforese em Gel de Poliacrilamida , Etilnitrosoureia/farmacologia , Humanos , Cinética , Proteínas/isolamento & purificação
2.
Proteins ; 2(1): 13-9, 1987.
Artigo em Inglês | MEDLINE | ID: mdl-3328857

RESUMO

The feasibility of detecting human somatic structural gene mutations by two dimensional electrophoresis has been investigated. A lymphoblastoid cell line was grown as a mass culture in the presence of ethylnitrosourea, after which cells were regrown as single cell clones. A total of 257 polypeptide spots were analyzed in gels derived from 186 clones. Four structural mutations were detected by visual analysis of the gels. Computer analysis of gels corresponding to the mutant clones was also undertaken. At a spot size threshold of 200 spots to be matched using a computer algorithm, all four mutant polypeptides were detected. These results indicate the usefulness of the two-dimensional approach for mutagenesis studies at the protein level.


Assuntos
Genes , Mutação , Células Clonais , Eletroforese em Gel de Poliacrilamida , Técnicas Genéticas , Humanos , Linfócitos , Computação Matemática , Proteínas/genética , Proteínas/isolamento & purificação
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