Induction of adenine salvage in mouse cell lines deficient in adenine phosphoribosyltransferase.

Abstract

, manuscript in preparation). Adenine salvage was examined in two APRT pseudorevertant cell lines, their two APRT homozygous deficient parental cell lines, and a genotypic APRT revertant cell line (i.e., one with measurable APRT activity and DAP sensitivity). Adenine accumulation was observed in both revertant phenotypes and was demonstrated by high-performance liquid chromotography to be linked with adenine metabolism. The ability to salvage adenine declined substantially in the pseudorevertant cell lines when they were removed from selective media containing inhibitors of de novo 5'-AMP synthesis (alanosine and azaserine); for one pseudorevertant cell line this decline was accelerated by the addition of DAP to the medium. The readdition of alanosine or azaserine to the growth medium of the pseudorevertant lines induced adenine salvage to its previous levels. An APRT-like cross-reacting material was found in the pseudorevertant cell lines, although its relationship to adenine salvage is unknown. A low level of constitutive adenine salvage was found in the parental APRT-deficient lines, and it was also possible to induce adenine salvage in these cell lines. These findings suggest a novel regulatory mechanism for adenine salvage.It has been demonstrated by fluctuation analysis (15) and replica plating (12) that in bacteria, mutations occur spontaneously in the absence of selective pressure. These observations have served as the basis for mutational analysis at many mammalian loci, using various selective systems that identify but do not induce mutant and revertant genotypes (5, 7, 24). The toxic purine analog 2'6'-diaminopurine (DAP) is routinely used for the selection of adenine phosphoribosyltransferase (APRT)-deficient cell lines, and it has been demonstrated that such cells can be identified (1) and selected (2) in the absence of DAP-containing medium. APRT catalyzes the conversion of adenine to 5'-AMP (16), and it is the enzyme responsible for the accumulation of adenine against a concentration gradient (i.e., adenine salvage) (3,26). It is therefore possible to select for reversion at the APRT locus by blocking de novo synthesis of 5'-IMP with azaserine (7) or the conversion of 5'-IMP to 5'-AMP with alanosine (5) and forcing cells to salvage adenine for survival. Reversion at the APRT locus is usually linked to reacquisition of enzymatic activity (4,25). Although the molecular bases underlying forward mutations at the APRT locus have been partially elucidated (20,22,23), detailed studies concerning the nature of reversion at this locus are still lacking.In other work, we will describe the isolation of differentiated mouse cell lines selected from homozygous APRTdeficient parentals for their ability to salvage adenine. Suprisingly, most of these lines were deficient in detectable APRT activity and are therefore termed APRT pseudorevertants (

Microhomology-assisted scarless genome editing in human iPSCs

“…We phenotyped edited clones by testing their resistance to 2,6-diaminopurine (DAP, Supplementary Fig. 1 and 15b ), a toxic purine analog 35 . Parental 1383D6 and homozygous APRT Silent/Silent mutants displayed severe drug sensitivity to 10 µg/mL DAP treatment, with nearly complete cell killing within just 48 h (Supplementary Fig.…”

Defining a genotoxic profile with mouse embryonic stem cells

“…102 Aprt is a member of the adenine salvage pathway; therefore, cells disrupted for Aprt function can be selected in 2 0 6 0 -diaminopurine (DAP) while cells that maintain Aprt function can be selected in alanosine or azaserine. 104 These LOH reporter ES cells are heterozygous for Aprt and were derived from a 128XC3HF1 cross. Therefore, a series of PCR reactions can identify each chromosome so the investigator can distinguish the potential events that cause LOH (loss of Aprt function).…”