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Title: Mouse transgenes in human cells detect specific base substitutions.

Authors: Schaff, D A; Jarrett, R A; Dlouhy, S R; Ponniah, S; Stockelman, M; Stambrook, P J; Tischfield, J A

Published In Proc Natl Acad Sci U S A, (1990 Nov)

Abstract: We describe a system of transgenic human cell lines that detects and identifies specific point mutations at defined positions within a gene. The target transgenome is a mouse adenine phosphoribosyltransferase (APRT) gene rendered nonfunctional by introduction of a substitution at either of two bases that comprise a splice acceptor site. Reversion at a mutated site results in the expression of wild-type mouse APRT and consequent growth of APRT+ transgenic cell colonies. Site-specific reversion to wild-type sequence is confirmed by regeneration of a previously destroyed diagnostic Pst I site. Two independent cell clones, each with mutant transgenomes bearing an A----G transition, exhibited an up to 7500-fold, dose-dependent induction of reversion following treatment with ethyl methanesulfonate. Treatment of these clones with 2-aminopurine resulted in no induction of revertants. In contrast, another transgenic cell clone, bearing a G----A transition, reverted as a consequence of 2-aminopurine, but not ethyl methanesulfonate, treatment. These data confirm for human cells the proposed mechanisms of action of these mutagens and provide evidence for the utility of our site-specific reversion method for mutagenesis studies.

PubMed ID: 2236079 Exiting the NIEHS site

MeSH Terms: Adenine Phosphoribosyltransferase/genetics*; Animals; Base Sequence; Genes*; Genetic Engineering/methods*; Humans; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed*; Mutagens; Plasmids; Restriction Mapping; Transfection

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