A novel Escherichia coli-derived mutation detected with the Big Blue cII mutant selectable assay.
Transgenic mouse mutation detection systems allow investigation of the origins and mechanisms of mutation associated with exogenous and endogenous mutagen exposures in individual tissues and cell types. In the past, selection assays for transgenic mutants have been contaminated with nonmurine-derived mutations and assay validation is critical to ensure murine in vivo origins of mutations. This is critical in studies of spontaneous mutations and extrapolation to endogenous mammalian genes. Herein, we provide one measure of the contribution of Escherichia coli (E. coli)-derived mutations to the Big Blue(R) cII transgene mutant selection assay. We report the first direct evidence of an E. coli-derived cII mutation identified among mutations recovered in the cII selective assay. An E. coli transposable 5 (Tn5) element IS50R inverted repeat (1,534 bp) was identified at base pair 414 in the cII transgene and the insertion generated a 9 bp target site duplication typical of this type of transposition. The bacterial transposition occurred only once in the assay of 25 x 10(6) plaque forming units and sequencing of 1,177 cII mutants. The observed frequency of this type of mutation is 4 x 10(-8) in retrieved lambda phage and 8.5 x 10(-4) in harvested cII mutants and thus a very rare occurrence in typical analyses of spontaneous in vivo mutations. Given that the frequency of transposition is equal to, or an order of magnitude higher, than the frequency of point mutations in E. coli, this article provides excellent validation for the murine origins of mutations detected using the cII mutant selection assay.
Ward
Tonya L
TL
Department of Biology, The University of Western Ontario, London Ontario N6A 5B7, Canada.
Prtenjaca
Anita
A
Hill
Kathleen A
KA
D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D055029:Inverted Repeat Sequences; D051379:Mice; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests / Q000379:methods*; D009154:Mutation