Neighboring base identity affects N-ethyl-N-nitrosourea-induced mutagenesis in Escherichia coli

This study investigated the influence of different neighboring base contexts on the production of base substitutions generated by N-ethyl-N-nitrosourea (ENU). A set of bacterial strains having all possible bases neighboring an ochre (TAA) nonsense mutation in the tyrA gene of Escherichia coli were employed and true reversions of the nonsense mutation were induced by two separate doses of ENU. Base substitution mutations were investigated by direct sequencing methods. These studies revealed that 1) mutations occurring at 5'-purine-T sites were produced better, on average, than mutations involving 5'-pyrimidine-T sites, and 5'-TT sites contributed the least to the formation of mutations, 2) the order of preference for A:T to G:C transitions was 5'-GT>5'-AT, 5'-CT>5'-TT, and 3) A:T to C:G transversions at the first position of the codon (GAA mutations) were produced best at 5'-AT sites, while A:T to T:A transversions at the third position (TAT mutations) occurred more often at 5'-GT sites. These findings suggest that the occurrence of a specific mutation may reflect the sequence-dependent probability of DNA damage at a particular site as well as factors involving preferential DNA repair or differential base selection by DNA polymerase.     

Analysis of ENU-induced mutations at the Adh locus in Drosophila melanogaster

N-Ethyl-N-nitrosourea (ENU) was used to induce mutations in the Drosophila melanogaster, alcohol dehydrogenase (Adh) gene. Flies were treated with ENU and mated to homozygous intragenic Adh null mutants; Adh null mutations were selected by exposure of the F1 generation to 1-penten-3-ol. Fourteen Adh null mutations were recovered which included 11 from spermatozoa, 2 from oocytes and 1 from a premeiotic spermatocyte. 2 mutations from spermatozoa and 1 of the mutations from oocytes were multilocus deficiencies which included the Adh locus as determined by complementation tests. The remaining 11 intragenic Adh null mutations were sequenced using the Sanger dideoxy method. One Adh null mutation induced in an oocyte was an AT to TA transversion and the mutation induced in a premeiotic spermatocyte was a GC to AT transition, both of which resulted in a single amino acid substitution. The 11 null mutations induced in spermatozoa were a data set in which both the dose of ENU and the treated germ-cell stage were held constant; therefore, only these 11 mutations were used to calculate the mutation frequency and compare the mutations at the Adh locus with those recovered in other studies. The dose of ENU induced a sex-linked recessive lethal frequency approximately 300 times that of the spontaneous frequency; therefore, these mutations were assumed to have been induced by ENU. 2 of the 11 mutations induced in spermatozoa were multilocus deficiencies and 9 were intragenic mutations. 7 of the 9 intragenic mutations were GC to AT transitions which resulted in 5 single amino acid substitutions, 1 premature translation termination codon, and 1 splice site mutation.(ABSTRACT TRUNCATED AT 400 WORDS)     

The induction of forward and reverse specific-locus mutations and dominant cataract mutations in spermatogonia of treated strain DBA/2 mice by ethylnitrosourea

The mutagenic effectiveness of ethylnitrosurea (ENU) was assessed in treated spermatogonia of DBA/2 mice. In a total of 17,515 offspring examined following 160 mg ENU/kg body weight treatment of parental males, 26 forward specific-locus mutations, 2 reverse specific-locus mutations and 9 dominant cataract mutations were recovered. ENU increased the mutation rate to all 3 genetic endpoints. However, ENU was less effective in treated DBA/2 mice than in the standard experimental protocol employing treated hybrid (102 X C3H)F1 male mice. This observed difference for a direct-acting mutagen such as ENU may result from differences in the detoxification of ENU or from differences in the DNA-repair capabilities of strain DBA/2. The first documented reverse mutation of the b allele is reported. The reversion was shown to be due to an AT to GC transition. To date, in addition to the reverse mutation of the b allele, 5 independent ENU-induced mutations recovered in germ cells of the mouse have been molecularly characterized and all have been shown to be base substitutions at an AT site. This is in contrast to the expected mechanism of ENU mutation induction due to O6-ethylguanine adduct formation which results in a GC to AT base-pair substitution and emphasizes the complexities of mutagenesis in germ cells of mammals.     

Analysis of chromosomal rearrangements induced by postmeiotic mutagenesis with ethylnitrosourea in zebrafish

Mutations identified in zebrafish genetic screens allow the dissection of a wide array of problems in vertebrate biology. Most screens have examined mutations induced by treatment of spermatogonial (premeiotic) cells with the chemical mutagen N-ethyl-N-nitrosourea (ENU). Treatment of postmeiotic gametes with ENU induces specific-locus mutations at a higher rate than premeiotic regimens, suggesting that postmeiotic mutagenesis protocols could be useful in some screening strategies. Whereas there is extensive evidence that ENU induces point mutations in premeiotic cells, the range of mutations induced in postmeiotic zebrafish germ cells has been less thoroughly characterized. Here we report the identification and analysis of five mutations induced by postmeiotic ENU treatment. One mutation, snh(st1), is a translocation involving linkage group (LG) 11 and LG 14. The other four mutations, oep(st2), kny(st3), Df(LG 13)(st4), and cyc(st5), are deletions, ranging in size from less than 3 cM to greater than 20 cM. These results show that germ cell stage is an important determinant of the type of mutations induced. The induction of chromosomal rearrangements may account for the elevated frequency of specific-locus mutations observed after treatment of postmeiotic gametes with ENU.     

Efficient Recovery of Enu-Induced Mutations from the Zebrafish Germline

We studied the efficiency with which two chemical mutagens, ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) can induce mutations at different stages of spermatogenesis in zebrafish (Brachydanio rerio). Both EMS and ENU induced mutations at high rates in post-meiotic germ cells, as indicated by the incidence of F(1) progeny mosaic for the albino mutation. For pre-meiotic germ cells, however, only ENU was found to be an effective mutagen, as indicated by the frequencies of non-mosaic mutant progeny at four different pigmentation loci. Several mutagenic regimens that varied in either the number of treatments or the concentration of ENU were studied to achieve an optimal ratio between the mutagenicity and toxicity. For the two most mutagenic regimens: 4 X 1 hr in 3 mM ENU and 6 X 1 hr in 3 mM ENU, the minimum estimate of frequencies of independent mutations per locus per gamete was 0.9-1.3 X 10(-3). We demonstrate that embryonic lethal mutations induced with ENU were transmitted to offspring and that they could be recovered in an F(2) screen. An average frequency of specific-locus mutations of 1.1 X 10(-3) corresponded to approximately 1.7 embryonic lethal mutations per single mutagenized genome. The high rates of mutations achievable with ENU allow for rapid identification of large numbers of genes involved in a variety of aspects of zebrafish development.     

The induction of recessive mutations in mouse primordial germ cells with N-ethyl-N-nitrosourea

A specific-locus test was carried out to examine the mutagenic activity of N-ethyl-N-nitrosourea (ENU) on mouse primordial germ cells (PGC). Embryos of C3H/He mice were treated transplacentally with 30 or 50 mg ENU per kg of maternal body weight on day 8.5, 10.5, or 13.5 of gestation (G8.5 day, G10.5 day, or G13.5 day). Male and female mice that had been treated with ENU in embryonic stages were mated with female or male tester PW mice to detect recessive mutations induced in PGC.

ENU induced recessive mutations at a relatively high rate in PGC at these developmental stages. The most sensitive stage was G10.5 day. On G8.5 day, the induced mutation rate in males and females was not significantly different. Cluster mutations, which originate from the limited number of PGC and cell killing, were more frequently induced at an earlier developmental stage. The induced mutation rate per unit dose of ENU (1 mg/kg) was higher in G8.5 and G10.5 day PGC than in stem-cell spermatogonia. It can be concluded that mouse PGC are more sensitive than stem-cell spermatogonia to the induction of recessive mutations by ENU.     

Estimating the number of coding mutations in genotypic and phenotypic driven <Emphasis Type="Italic">N</Emphasis>-ethyl-<Emphasis Type="Italic">N</Emphasis>-nitrosourea (ENU) screens: revisited

We recently described methods for estimating the number of N-ethyl-N-nitrosourea (ENU)-induced coding mutations in phenotypic and genotypic screens. In this article we revisit these methods, clarifying their application. In particular, we focus on the difference between unconditional and conditional probabilities. We also introduce a website to assist investigators in the application of these equations ().     

DNA adduct formation in mouse testis by ethylating agents: a comparison with germ-cell mutagenesis

DNA adduct formation in various organs of mice was determined after i.p. injection with the ethylating agents N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), and diethyl sulfate (DES). The potency of the 3 chemicals to react either at the O6 position of guanine or at the N-7 position of guanine was related to their potency to induce mutations in the specific-locus assay of the mouse. ENU, which produces relatively high levels of O-alkylations (O6-ethylguanine), is primarily mutagenic in spermatogonia of the mouse, whereas EMS and DES, which produce relatively high levels of N-alkylations (7-ethylguanine) in DNA, are much more mutagenic in post-meiotic stages of male germ cells. The relationship between exposure to ENU and the dose, determined as O6-ethylguanine per nucleotide in testicular DNA, is non-linear. However, the relationship between dose and mutation induction in spermatogonia by ENU appears to be linear, which is expected if O6-ethylguanine is the major mutagenic lesion. The relatively high mutagenic potency of EMS and DES in the late stages of spermatogenesis is probably due to the accumulation of apurinic sites which generate mutations after fertilization. A comparison of mutation induction by ENU in spermatogonia and mutation induction in cultured mammalian cells indicates that about 10 O6-ethylguanine residues were necessary in the coding region of a gene to generate a mutation.     

The paternal genome in mouse zygotes is less sensitive to ENU mutagenesis than the maternal genome

The two parental genomes lie separate within the zygote and may be differentially affected by environmental influences. We have shown earlier (Russell et al., 1988) that the maternal genome within the mouse zygote is exquisitely sensitive to the induction of point mutations by N-ethyl-N-nitrosourea (ENU), and that the initial lesion probably occurs in one strand of the DNA. The present experiment measured specific-locus mutation induction in the paternal genome. Zygotes containing a multiple-recessive maternal genome (a; b; p cch; d se; s) and the corresponding wild-type alleles in the paternal one were exposed to 50 mg ENU/kg in vivo at one of two stages: the presumed times of sperm entry and early pronuclear stage. At weaning age, the resulting mice were examined for mutations at the marked loci as well as for other mutations producing externally visible phenotypes. At the marked loci, one possible mosaic (for b) was observed among 2113 classified offspring that had been treated with ENU as zygotes; this animal failed to transmit a mutation. By contrast, in the reciprocal cross (which tests the maternal genome) we had observed 8 specific-locus mutations (6 of them mosaics) among 1555 offspring that had received the same dose of ENU during sperm entry (and completion of oocyte meiosis II). In the present experiment, we also found one mutation at other loci (two at other loci in the reciprocal cross). The frequency of offspring with small white belly spots was significantly greater in the treated groups (3.5 and 1.9% at the earlier and later stage, respectively) than in the control (1.0%), the excess being almost entirely due to daughters. Genetic tests of a large number of such offspring failed to find a genetic cause. Instead, it appears that this phenotype may be influenced by factors in the intrauterine environment. It is concluded that shortly after sperm entry, the paternal genome of the zygote is less sensitive than the maternal one to the induction of mutations by ENU.     

Mutation analysis using the restriction site mutation (RSM) assay

The restriction site mutation (RSM) assay (see Steingrimsdottir et al. [H. Steingrimsdottir, D. Beare, J. Cole, J.F.M. Leal, T. Kostic, J. Lopez-Barea, G. Dorado, A.R. Lehmann, Development of new molecular procedures for the detection of genetic alteration in man, Mutat. Res. 353 (1996) pp. 109–121] for a review) has been developed as a genotypic mutation detection system capable of identifying mutations occurring in restriction enzyme sites of genomic DNA. Here we will report the steps taken to overcome some of the initial problems of the assay, namely the lack of quantitative data and limited sensitivity, the aim being to achieve a methodology suitable for the study of low dose chemical exposures. Quantitative data was achieved in the RSM assay by the inclusion of an internal standard molecule in the PCR amplification stage, thus allowing the calculation of both spontaneous and induced mutation frequencies. The sensitivity of the assay was increased through the discovery that intron sequences of genomic DNA accumulated more mutations in vivo compared to the exons, presumably due to differential selective pressure within genes [G.J.S. Jenkins, I.deG. Mitchell, J.M. Parry, Enhanced restriction site mutation (RSM) analysis of 1,2-dimethylhydrazine-induced mutations, using endogenous p53 intron sequences, Mutagenesis 12 (1997) pp. 117–123]. This increased sensitivity was examined by applying the RSM assay to analyse the persistence of N-ethyl-N-nitrosourea (ENU)-induced mutations in mice testes. Germ line mutations were sought in testes DNA 3, 10 and 100 days after ENU treatment. Mutations were detected in exons and especially intron regions, the intron mutations were more persistent, still being detected 100 days post-chemical treatment. Assignment of these mutations as ENU induced was complicated in some cases where the spontaneous mutation level was high. This theme of mutation persistence was further investigated by studying the presence of 4-nitroquinoline-1-oxide (4-NQO)-induced DNA mutations in vitro. This study also analysed the relationship between DNA adduct formation and DNA mutation induction by the concurrent RSM analysis and post-labelling analysis of 4-NQO treated human fibroblasts. The results demonstrated that early DNA mutations detected 4 days post-treatment by the RSM assay were probably ex vivo mutations induced by Taq polymerase misincorporation of 4-NQO adducted DNA, due to the maximum levels of 4-NQO adducts being present at this time point. A later mutational peak, after the adduct level had declined, was assumed to be due to DNA sequence changes produced in the fibroblasts by the in vivo processing of DNA adducts.     

High-Resolution Multipoint Linkage-Disequilibrium Mapping in the Context of a Human Genome Sequence

A new method is presented for fine-scale linkage disequilibrium (LD) mapping of a disease mutation; it uses multiple linked single-nucleotide polymorphisms, restriction-fragment-length polymorphisms, or microsatellite markers and incorporates information from an annotated human genome sequence (HGS) and from a human mutation database. The method takes account of population demographic effects, using Markov chain Monte Carlo methods to integrate over the unknown gene genealogy and gene coalescence times. Information about the relative frequency of disease mutations in exons, introns, and other regions, from mutational databases, as well as assumptions about the completeness of the gene annotation, are used with an annotated HGS, to generate a prior probability that a mutation lies at any particular position in a specified region of the genome. This information is updated with information about mutation location, from LD at a set of linked markers in the region, to generate the posterior probability density of the mutation location. The performance of the method is evaluated by simulation and by analysis of a data set for diastrophic dysplasia (DTD) in Finland. The DTD disease gene has been positionally cloned, so the actual location of the mutation is known and can be compared with the position predicted by our method. For the DTD data, the addition of information from an HGS results in disease-gene localization at a resolution that is much higher than that which would be possible by LD mapping alone. In this case, the gene would be found by sequencing a region < or =7 kb in size.     

Focusing forward genetics: a tripartite ENU screen for neurodevelopmental mutations in the mouse

The control of growth, patterning, and differentiation of the mammalian forebrain has a large genetic component, and many human disease loci associated with cortical malformations have been identified. To further understand the genes involved in controlling neural development, we have performed a forward genetic screen in the mouse (Mus musculus) using ENU mutagenesis. We report the results from our ENU screen in which we biased our ascertainment toward mutations affecting neurodevelopment. Our screen had three components: a careful morphological and histological examination of forebrain structure, the inclusion of a retinoic acid response element-lacZ reporter transgene to highlight patterning of the brain, and the use of a genetically sensitizing locus, Lis1/Pafah1b1, to predispose animals to neurodevelopmental defects. We recovered and mapped eight monogenic mutations, seven of which affect neurodevelopment. We have evidence for a causal gene in four of the eight mutations. We describe in detail two of these: a mutation in the planar cell polarity gene scribbled homolog (Drosophila) (Scrib) and a mutation in caspase-3 (Casp3). We find that refining ENU mutagenesis in these ways is an efficient experimental approach and that investigation of the developing mammalian nervous system using forward genetic experiments is highly productive.     

THE EVOLUTIONARY DYNAMICS OF SEXUALLY ANTAGONISTIC MUTATIONS IN PSEUDOAUTOSOMAL REGIONS OF SEX CHROMOSOMES

Sex chromosomes can evolve gene contents that differ from the rest of the genome, as well as larger sex differences in gene expression compared with autosomes. This probably occurs because fully sex‐linked beneficial mutations substitute at different rates from autosomal ones, especially when fitness effects are sexually antagonistic (SA). The evolutionary properties of genes located in the recombining pseudoautosomal region (PAR) of a sex chromosome have not previously been modeled in detail. Such PAR genes differ from classical sex‐linked genes by having two alleles at a locus in both sexes; in contrast to autosomal genes, however, variants can become associated with gender. The evolutionary fates of PAR genes may therefore differ from those of either autosomal or fully sex‐linked genes. Here, we model their evolutionary dynamics by deriving expressions for the selective advantages of PAR gene mutations under different conditions. We show that, unless selection is very strong, the probability of invasion of a population by an SA mutation is usually similar to that of an autosomal mutation, unless there is close linkage to the sex‐determining region. Most PAR genes should thus evolve similarly to autosomal rather than sex‐linked genes, unless recombination is very rare in the PAR.     

Spectra of molecular changes induced in DNA of Drosophila spermatozoa by 1-ethyl-1-nitrosourea and X-rays

Mutations induced in Drosophila spermatozoa at the alcohol dehydrogenase Adh locus by 1-ethyl-1-nitrosourea (ENU) were compared to X-ray-induced mutations using genetic tests for complementation, southern blotting, western blotting and northern blotting. 8 of 10 ENU-induced mutations complemented all known adjacent loci and were presumed to be intragenic. In contrast, 8 of 30 X-ray-induced mutations were intragenic. Southern blot analysis showed that 2 of 7 intragenic mutations induced by X-rays were altered at the Adh locus, whereas all 8 intragenic ENU mutants appeared normal. Western blot analysis showed 4 of 7 intragenic mutants induced by X-rays produced a detectable polypeptide; 1 of the 4 had normal molecular weight and charge. In contrast, 7 of the 8 intragenic mutants induced by ENU produced a polypeptide of normal molecular weight and charge. One ENU and two X-ray-induced mutants, which had normal southern blots and no detectable polypeptide, produced normal molecular weight mRNA by northern blots. The interpretation of these results is that in spermatozoa X-rays induce primarily deletions that either produce deficiencies of the Adh locus or nonsense mutations within the locus, whereas ENU induces primarily missense mutations. This forward mutation assay based on loss of enzymatic activity efficiently recovered a broad spectrum of mutations ranging from missense to intragenic deletions and multi-locus deficiencies. Only 3 of these 40 mutations produced a polypeptide detectable as an electrophoretic variant.     

Induction of specific-locus mutations in female mice by 1-ethyl-1-nitrosourea and procarbazine

Using the specific-locus method, the ability of 1-ethyl-1-nitrosourea (ENU) and procarbazine hydrochloride (procarbazine) to induce gene mutations in mouse oocytes was tested and confirmed. The sensitive stage for the induction of mutations in oocytes with 160 mg/kg of ENU is 2-4 weeks post treatment. The induced mutation frequency in this mating interval was 5.1 X 10(-7) mutations/locus/gamete/mg/kg. The induction of mutations by procarbazine occurred 8-33 weeks after treatment. The induced mutation frequency in this mating interval for the 400 mg/kg group was 0.4 X 10(-7) mutations/locus/gamete/mg/kg. One third of the induced mutations was lethal in homozygous condition in both experiments. ENU and procarbazine have a lower mutational response in oocytes than in stem-cell spermatogonia.     

ENU-induced mutagenesis in grass carp (Ctenopharyngodon idellus) by treating mature sperm

N-ethyl-N-nitrosourea (ENU) mutagenesis is a useful approach for genetic improvement of plants, as well as for inducing functional mutants in animal models including mice and zebrafish. In the present study, mature sperm of grass carp (Ctenopharyngodon idellus) were treated with a range of ENU concentrations for 45 min, and then wild-type eggs were fertilized. The results indicated that the proportion of embryos with morphological abnormalities at segmentation stage or dead fry at hatching stage increased with increasing ENU dose up to 10 mM. Choosing a dose that was mutagenic, but provided adequate numbers of viable fry, an F1 population was generated from 1 mM ENU-treated sperm for screening purposes. The ENU-treated F1 population showed large variations in growth during the first year. A few bigger mutants with morphologically normal were generated, as compared to the controls. Analysis of DNA from 15 F1 ENU-treated individuals for mutations in partial coding regions of igf-2a, igf-2b, mstn-1, mstn-2, fst-1 and fst-2 loci revealed that most ENU-treated point mutations were GC to AT or AT to GC substitution, which led to nonsense, nonsynonymous and synonymous mutations. The average mutation rate at the examined loci was 0.41%. These results indicate that ENU treatment of mature sperm can efficiently induce point mutations in grass carp, which is a potentially useful approach for genetic improvement of these fish.     

Lampe1: an ENU-germline mutation causing spontaneous hepatosteatosis identified through targeted exon-enrichment and next-generation sequencing

Using a small scale ENU mutagenesis approach we identified a recessive germline mutant, designated Lampe1 that exhibited growth retardation and spontaneous hepatosteatosis. Low resolution mapping based on 20 intercrossed Lampe1 mice revealed linkage to a ～14 Mb interval on the distal site of chromosome 11 containing a total of 285 genes. Exons and 50 bp flanking sequences within the critical region were enriched with sequence capture microarrays and subsequently analyzed by next-generation sequencing. Using this approach 98.1 percent of the targeted DNA was covered with a depth of 10 or more reads per nucleotide and 3 homozygote mutations were identified. Two mutations represented intronic nucleotide changes whereas one mutation affected a splice donor site in intron 11-12 of Palmitoyl Acetyl-coenzyme A oxygenase-1 (Acox1), causing skipping of exon 12. Phenotyping of Acox1(Lampe1) mutants revealed a progression from hepatosteatosis to steatohepatitis, and ultimately hepatocellular carcinoma. The current approach provides a highly efficient and affordable method to identify causative mutations induced by ENU mutagenesis and animal models relevant to human pathology.     

A genetic screen for mutations affecting embryonic development in medaka fish (Oryzias latipes)

In a pilot screen, we assayed the efficiency of ethylnitrosourea (ENU) as a chemical mutagen to induce mutations that lead to early embryonic and larval lethal phenotypes in the Japanese medaka fish, Oryzias latipes. ENU acts as a very efficient mutagen inducing mutations at high rates in germ cells. Three repeated treatments of male fish in 3 mM ENU for 1 h results in locus specific mutation rates of 1.1-1.95 x10(-3). Mutagenized males were outcrossed to wild type females and the F1 offspring was used to establish F2 families. F2 siblings were intercrossed and the F3 progeny was scored 24, 48 and 72 h after fertilization for morphological alterations affecting eye development. The presented mutant phenotypes were identified using morphological criteria and occur during early developmental stages of medaka. They are stably inherited in a Mendelian fashion. The high efficiency of ENU to induce mutations in this pilot screen indicates that chemical mutagenesis and screening for morphologically visible phenotypes in medaka fish allows the genetic analysis of specific aspects of vertebrate development complementing the screens performed in other vertebrate model systems.     

Differential effects of low- and high-dose X-rays on N-ethyl-N-nitrosourea-induced mutagenesis in thymocytes of B6C3F1 gpt-delta mice

Carcinogenesis in humans is thought to result from exposure to numerous environmental factors. Little is known, however, about how these different factors work in combination to cause cancer. Because thymic lymphoma is a good model of research for combined exposure, we examined the occurrence of mutations in thymic DNA following exposure of B6C3F1 gpt-delta mice to both ionizing radiation and N-ethyl-N-nitrosourea (ENU). Mice were exposed weekly to whole body X-irradiation (0.2 or 1.0 Gy), ENU (200 ppm) in the drinking water, or X-irradiation followed by ENU treatment. Thereafter, genomic DNA was prepared from the thymus and the number and types of mutations in the reporter transgene gpt was determined. ENU exposure alone increased mutant frequency by 10-fold compared to untreated controls and over 80% of mutants had expanded clonally. X-irradiation alone, at either low or high dose, unexpectedly, reduced mutant frequency. Combined exposure to 0.2 Gy X-rays with ENU dramatically decreased mutant frequency, specifically G:C to A:T and A:T to T:A mutations, compared to ENU treatment alone. In contrast, 1.0 Gy X-rays enhanced mutant frequency by about 30-fold and appeared to accelerate clonal expansion of mutated cells. In conclusion, repeated irradiation with 0.2 Gy X-rays not only reduced background mutation levels, but also suppressed ENU-induced mutations and clonal expansion. In contrast, 1.0 Gy irradiation in combination with ENU accelerated clonal expansion of mutated cells. These results indicate that the mode of the combined mutagenic effect is dose dependent.     

An ENU-induced mutation in the Ankrd11 gene results in an osteopenia-like phenotype in the mouse mutant Yoda.

The mechanisms that regulate bone mass are important in a variety of complex diseases such as osteopenia and osteoporosis. Regulation of bone mass is a polygenic trait and is also influenced by various environmental and lifestyle factors, making analysis of the genetic basis difficult. As an effort toward identifying novel genes involved in regulation of bone mass, N-ethyl-N-nitrosourea (ENU) mutagenesis in mice has been utilized. Here we describe a mouse mutant termed Yoda that was identified in an ENU mutagenesis screen for dominantly acting mutations. Mice heterozygous for the Yoda mutation exhibit craniofacial abnormalities: shortened snouts, wider skulls, and deformed nasal bones, underlined by altered morphology of frontonasal sutures and failure of interfrontal suture to close. A major feature of the mutant is reduced bone mineral density. Homozygosity for the mutation results in embryonic lethality. Positional cloning of the locus identified a missense mutation in a highly conserved region of the ankyrin repeat domain 11 gene (Ankrd11). This gene has not been previously associated with bone metabolism and, thus, identifies a novel genetic regulator of bone homeostasis.