The use of the mouse APRT gene in cultured human cells to study point mutations

Abstract

Site-specific mutagenesis, gene transfer, and somatic cell selection have been used to develop a method for the identification of site- and sequence-specific point mutationsinduced in a mammalian cell transgenic system. This method utilizes a mouse adenine phosphoribosyltransferase [APRT] gene containing a specific point mutation in an intronjexon splice recognition sequence, which disrupts mRNA processing and destroys a diagnostic Pst I restriction site. This construct was introduced by cotransfection,with the bacterial n&Qr gene into HTD-114, a non-spontaneously-reverting, Aprt- human fibrosarcoma-derived cell line. The construct s·tudied contains I an A to G transition which is revertible with ethyl methanesulfonate [EMS], a mutagen known to produce G to A transitions in eukaryotes and prokaryotes. Exposing two independently derived cell lines containing the construct to EMS concentrations ranging from 0 to 200 ugjml (corresponding-to 100 to 0.8 percent survival) produced a frequency of reversion to the Aprt+ phenotype of 10-7 to 10-2 .