Mutation Rate and Predicted Phenotypic Target Sizes in Ethylnitrosourea-Treated Mice

Abstract

Chemical mutagenesis of the mouse is ongoing in several centers around the world, with varying estimates of mutation rate and number of sites mutable to phenotype. To address these questions, we sequenced ‫6.9ف‬ Mb of DNA from G 1 progeny of ethylnitrosourea-treated mice in a large, broad-spectrum screen. We identified 10 mutations at eight unique sites, including six nonsynonymous coding substitutions. This calibrates the nucleotide mutation rate for two mutagenesis centers, implies significance criteria for positional cloning efforts, and provides working estimates of effective genetic target sizes for selected phenotypes.M UTAGENESIS experiments provide both novel direct measurement of the nucleotide substitution rate (Beier 2000;Kile et al. 2003). In particular, comparison mutations for biological studies and the means to estimate genomic parameters of the traits under of the nucleotide change rate to the rate of recovered phenotypic deviants should allow calculation of the efstudy. For example, the effective number of genes or the genome-wide target size for a particular phenotypic fective genome target size mutable to a given phenotype, without making assumptions about the distribution of muclass are of interest both for understanding the genetic architecture of a trait and for assessing the saturation table sites among distinct genes. Direct ascertainment of induced nucleotide changes also provides immediate acof mutable sites within a given screen. Estimates of these parameters depend on knowing the mutation rate cess to mutations of potential biological interest. Accurate measurement of the substitution rate is additionally within the experiment. Mutation rates, first defined by Muller, classically have been estimated by specific locus useful in evaluating candidate polymorphisms in positional cloning efforts. Brown and colleagues have pretests for failure to complement phenotype of a known mutation (Muller 1928; Russell 1951; Russell and viously estimated the rate of nucleotide change in a large chemical mutagenesis using heteroduplex detection by Montgomery 1982). Such estimates can be biased by the nature of the chosen gene (or genes) and the generdenaturing HPLC as a surrogate for sequence (Coghill et al. 2002). These results may be difficult to apply more ality of phenotypes caused by its mutation. Inferring the nucleotide substitution rate from specific locus data generally because few mutations were observed (and the detection rate was not established), the potential therefore requires assumptions about the fraction of sites mutable to the tested phenotype and is open to subbias from sampling only three loci is unclear, and the mutagenesis protocol and strain choice are substantially stantial locus-specific bias. For many applications, the specific locus mutation rate is a sufficient term by itself, different from those used in other centers. Here we use DNA sequencing at 84 sentinel sites across 51 autosomal irrespective of the underlying nucleotide change rate. Indeed, classical esti...

Generation of N-Ethyl-N-nitrosourea (ENU) Diabetes Models in Mice Demonstrates Genotype-specific Action of Glucokinase Activators

“…The dose of ENU used has been reported to produce a mutation rate between 2.6 ϫ 10 Ϫ7 and 1.04 ϫ 10 Ϫ6 mutations/base pair in B6 mice (23)(24)(25)(26)(27). As has been argued previously (28), such a mutation rate would produce multiple mutations in a single mouse, but the mutations would be spaced widely enough that they would sort independently during meiosis.…”

Choline Kinase β Mutant Mice Exhibit Reduced Phosphocholine, Elevated Osteoclast Activity, and Low Bone Mass

“…Mice-To identify novel genes regulating bone homeostasis, we employed an ENU-induced mutagenesis approach (32). Radiographic screening of several ENU-induced mutant mouse lines identified a mutant mouse line, named flipper (flp/ flp), that exhibited a systemic reduction in bone density as demonstrated in the hind limb and tail compared with wild type littermate controls at 2 months of age (Fig.…”

Second-generation high-throughput forward genetic screen in mice to isolate subtle behavioral mutants

“…The initial estimate of mutation rates determined by Russell and colleagues (50) using specific locus mouse tester strain was 1/700. Using traditional sequencing as well as next-generation sequencing efforts, the mutation rate has been estimated at ≈1.4 per million base pair for C57BL/6J at the dose of ENU used in this project (51)(52)(53). Because 1.5% of the 2.5-Gb mouse genome is coding and ≈70% of coding mutations would be nonsynonymous (51), we expect 37 nonsynonymous mutations per haploid genome [(2.5 × ) × 0.015 × 0.7].…”