Mouse transgenes in human cells detect specific base substitutions.
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.
Schaff
D A
DA
Department of Anatomy and Cell Biology, University of Cincinnati College of Medicine, OH 45267-0521.
Jarrett
R A
RA
Dlouhy
S R
SR
Ponniah
S
S
Stockelman
M
M
Stambrook
P J
PJ
Tischfield
J A
JA
D000228:Adenine Phosphoribosyltransferase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D005796:Genes; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009153:Mutagens; D010957:Plasmids; D015183:Restriction Mapping; D014162:Transfection
D009153:Mutagens; D000228:Adenine Phosphoribosyltransferase