A new mouse insertional mutation that causes sensorineural deafness and vestibular defects.

This article describes a new recessive insertional mutation in the transgenic line TgN2742Rpw that causes deafness and circling behavior in mice. Histologic analysis revealed virtually complete loss of the cochlear neuroepithelium (the organ of Corti) in adult mutant mice. In association with the neuroepithelial changes, there is a dramatic reduction of the cochlear nerve supply. Adult mutants also show morphological defects of the vestibular apparatus, including degeneration of the saccular neuroepithelium and occasional malformation of utricular otoconia. Audiometric evaluations demonstrated that the mice displaying the circling phenotype are completely deaf. Molecular analysis of this mutant line revealed that the transgenic insertion occurred without creating a large deletion of the host DNA sequences. The mutant locus was mapped to a region on mouse chromosome 10, where other spontaneous, recessive mutations causing deafness in mice have been mapped.

Induced mouse chromosomal rearrangements as tools for identifying critical developmental genes and pathways.

Due to the rapid advances that have been made in molecular and genetic technology during the past decade, the genes associated with a large number of human hereditary diseases have been isolated and analyzed in detail. These cloned genes provide new tools for research geared toward a better understanding of normal human development, and also of the many ways that basic, essential morphologic pathways can be disturbed. Chromosomal rearrangements, especially deletions and translocations, have been especially beneficial in the mapping and isolation of human disease genes because of their visibility on both the cytogenetic and molecular levels. However, these useful types of mutations occur with low frequency in the human population. Chromosomal rearrangements can be induced relatively easily in mice, and several large, independent collections of translocation and deletion mutants have been generated in the course of risk-assessment and mutagenesis studies over the past several decades. Combined with new molecular technologies, these collections of mutant animals provide a means of gaining ready access to genes associated with developmental defects including craniofacial abnormalities, hydrocephaly, skeletal deformities, and complex neurologic disorders. As an illustration of this approach, we briefly review our progress in the study of three mutations associated with defects in palate development, juvenile growth, fitness and sterility, and neurologic development in mice, respectively.

Generation and characterization of heritable reciprocal translocations in mice.

Reciprocal translocations have provided crucial tools for the localization of genes associated with a variety of human cancers and hereditary diseases. Although heritable translocations are relatively rare in humans, they can be easily induced in mice through exposure of male germ cells at specific spermatogenic stages to different types of radiation and chemicals. Mutagenesis schemes that produce translocations at high frequencies in the progeny of treated males are summarized, and the use of these valuable mutations for analyzing developmental consequences of partial aneuploidy, for identification of mutant genes, and for other purposes is reviewed. Preliminary studies of a large collection of translocation mutants, including several stocks that display dominantly or recessively inherited phenotypes caused by the disruption of critical genes are described. These combined studies demonstrate that several mutagenesis protocols can be used to generate easily mapped, novel mouse mutations with high efficiency and highlight the unique value of reciprocal translocations as tools for gaining access to the biological functions of mammalian genes.

Dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis induced in male mouse germ cells by glycidamide, a metabolite of acrylamide.

The hypothesis that acrylamide induces dominant lethal mutations and heritable translocations in male mice, not through direct adduction, but by conversion to the reactive epoxide, glycidamide, was investigated. Three studies, namely, induction of dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis in spermatids, which were conducted earlier in this laboratory for acrylamide, were also performed for glycidamide to determine its mutagenic properties and to compare responses. Results of these studies are consistent with the proposal that in vivo conversion to glycidamide is responsible for the mutagenicity of acrylamide in male mice.

Dominant lethal and heritable translocation tests with chlorambucil and melphalan in male mice.

Chemicals used in the treatment of cancer include several that are potent mutagens in a range of in vitro and in vivo assays. For some, genetic effects have also been demonstrated in humans, detected as chromosomal aberrations in peripheral lymphocytes. Because (1) many of these agents are confirmed mutagens, (2) humans are exposed to them in relatively high doses, and (3) an increasing number of early cancer victims are surviving to reproductive age, it is important that information be available on the genetic and reproductive hazards associated with exposure to these agents. Chlorambucil and melphalan are structurally related chemicals that are included in our efforts to identify and assess such hazards among cancer chemotherapy agents. To date, both have been reported to induce specific locus mutations in germ cells of male mice (Russell et al., 1989; Russel et al., 1992b) and melphalan is one of very few chemicals shown to induce such mutations in spermatogonial stem cells. More recently, both chemicals were found to have strong reproductive effects in female mice (Bishop and Generoso, 1995, in preparation). In the present studies, these chemicals were tested for the induction of dominant lethal mutations and heritable translocations in male mice. Both chemicals were found to have reproductive effects attributable to cytotoxicity in specific male germ cell stages and to induce dominant lethal mutations and heritable translocations in postmeiotic germ cells, particularly in mid to early stage spermatids. Thus, relatively extensive data are now available for assessing the genetic and reproductive hazards that may result from therapeutic exposures to these chemicals.

Complementation analyses for 45 mutations encompassing the pink-eyed dilution (p) locus of the mouse.

The homozygous and heterozygous phenotypes are described and characterized for 45 new pink-eyed dilution (p) locus mutations, most of them radiation-induced, that affect survival at various stages of mouse development. Cytogenetically detectable aberrations were found in three of the new p mutations (large deletion, inversion, translocation), with band 7C involved in each case. The complementation map developed from the study of 810 types of compound heterozygotes identifies five functional units: jls and jlm (two distinct juvenile-fitness functions, the latter associated with neuromuscular defects), pl-1 and pl-2 (associated with early-postimplantation and preimplantation death, respectively), and nl [neonatal lethality associated with cleft palate (the frequency of rare "escapers" from this defect varied with the genotype)]. Orientation of these units relative to genetic markers is as follows: centromere, Gas-2, pl-1, jls, jlm p, nl (equatable to cp 1 = Gabrb3); pl-2 probably resides in the c-deletion complex. pl-1 does not mask preimplantation lethals between Gas2 and p; and no genes affecting survival are located between p and cp1. The alleles specifying mottling or darker pigment (generically, pm and px, respectively) probably do not represent deletions of p-coding sequences but could be small rearrangements involving proximal regulatory elements.

Candidate gene associated with a mutation causing recessive polycystic kidney disease in mice.

A line of transgenic mice was generated that contains an insertional mutation causing a phenotype similar to human autosomal recessive polycystic kidney disease. Homozygotes displayed a complex phenotype that included bilateral polycystic kidneys and an unusual liver lesion. The mutant locus was cloned and characterized through use of the transgene as a molecular marker. Additionally, a candidate polycystic kidney disease (PKD) gene was identified whose structure and expression are directly associated with the mutant locus. A complementary DNA derived from this gene predicted a peptide containing a motif that was originally identified in several genes involved in cell cycle control.

Cytogenetic analysis of malformed mouse fetuses derived from balanced translocation heterozygotes.

Reciprocal translocations are readily induced by various physical and chemical mutagens in certain germ-cell stages. Carriers of balanced reciprocal translocations generally exhibit no abnormal phenotypes, except for occasional male sterility, but about half (on average) of their progeny carry grossly unbalanced chromosome complements and die prenatally, so that the carriers are said to be 'semisterile'. Since death of such progeny generally occurs in very early embryonic stages, it would be of minor importance in an analogous human situation. Several types of unbalanced segregants, however, survive to late gestational or even to postnatal stages and are often malformed. Recently, it was determined in this laboratory that over one half of the male carriers of methylene-bisacrylamide-induced translocations, sired litters that had late-dying and/or malformed fetuses (Rutledge et al., 1990). Five high-anomaly translocation stocks derived from that study and four derived from studies with other mutagens were analyzed cytogenetically to determine (1) the chromosomes and breakpoints involved, (2) the nature of chromosome imbalance in malformed fetuses, and (3) the types of meiotic segregation that produce late-surviving unbalanced segregants. Cytogenetic analysis of the 9 translocation stocks revealed 18 breakpoints located in 12 chromosomes. Each translocation had at least one breakpoint located near the centromere or the telomere. All translocations produced abnormal fetuses that were partially monosomic for a very short terminal chromosome segment, and partially trisomic for a segment that can be of various lengths, 2-10 times as long as the monosomic segment. In 6 stocks, these abnormal fetuses arose by adjacent-1 or alternate segregation; in the other three they arose by adjacent-2 segregation. In addition, tertiary trisomy by 3-1 missegregation was also observed in two of the stocks.

Molecular characterization of a pericentric inversion in mouse chromosome 8 implicates telomeres as promoters of meiotic recombination.

A "hot spot" of meiotic recombination has been found in males on murine chromosome 8 using nonisotopic hybridization of a series of probes to mitotic and meiotic chromosomes. The sequences responsible for this enhanced recombination are the telomeric repeats. Mice both normal and hetero- or homozygous for a pericentric inversion, In(8)1 Rl, were analyzed. The inversion subdivides chromosome 8 into three discreet regions: (1) a fraction of the micro "short arm" that contains 30-150 kb of telomeric sequences and only about one-fifth of the contiguous minor-satellite sequences (approximately 200 kb); (2) the inverted region; and (3) the noninverted distal two-thirds of the chromosome. In 70 spermatocytes from inversion heterozygotes, examined by electron microscopy, synapsis of the inverted region was complete but entirely nonhomologous. Nonhomologous synapsis persists from initiation of synaptonemal complex formation in zygonema/early pachynema until dissolution in late pachynema. This nonhomologous synapsis also suppresses crossing over within the inverted segment. The opportunity for proximal homologous recombination is thus restricted to the roughly 250 kb segment located between the short-arm break and the end of the bivalent. Nonetheless, an extreme proximal chiasma was observed in 11% of the heterozygous chromosome-8 bivalents, 34% of the normal 8 bivalents and 35% of the homozygous inversion 8 bivalents from spermatocyte preparations. Since in the normal chromosomes all minor satellite sequences are adjacent to the telomere, while in the inversion chromosomes most of these sequences are transposed to an interstitial position without a corresponding shift in chiasma position, the minor-satellite sequences can be ruled out as promoters of recombination.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic, cytogenetic, and molecular analyses of mutations induced by melphalan demonstrate high frequencies of heritable deletions and other rearrangements from exposure of postspermatogonial stages of the mouse.

Specific-locus experiments have previously shown melphalan to be mutagenic in all male germ-cell stages tested and particularly so in early spermatids. All but 2 of 24 specific-locus mutations recovered were tested genetically, cytogenetically, and/or molecularly. At least 12 of 15 tested mutations recovered from postspermatogonial stages but only 1 of 7 mutations recovered from stem-cell or differentiating spermatogonia gave evidence of being deletions or other rearrangements. Melphalan-induced mutations, thus, confirm the pattern of dependence of mutation structure on germ-cell stage that had been shown earlier for other chemicals. Results of the present investigation illustrate the capabilities of combined genetic, cytogenetic, and molecular analyses for characterizing the nature of specific-locus mutations. Fine-structure molecular mapping of long regions surrounding specific loci has been greatly facilitated by the availability of genetic reagents (particularly, deletion complexes) generated in specific-locus experiments over the course of decades. Reciprocally, this mapping permits increasingly detailed characterization of the nature of lesions induced by mutagenic exposures of germ cells, adding great powers for qualitative analysis of mutations to the specific-locus test. Cytogenetic and genetic investigations also provide evidence on lesion type, especially for loci at which mutations cannot yet be analyzed molecularly. Melphalan, like chlorambucil, can generate many mutations, a high proportion of which are deletions and other rearrangements, making this chemical valuable for generating mutations (at any locus) amenable to molecular access.

Induction of specific-locus mutations in male germ cells of the mouse by acrylamide monomer.

Acrylamide monomer (AA), injected into male mice at the maximum tolerated dose of 5 x 50 mg/kg (24-h intervals), significantly increased the specific-locus mutation rate in certain poststem-cell stages of spermatogenesis, but not in spermatogonial stem cells. Germ-cell stages in which the treatment induced dominant lethals--namely, exposed spermatozoa and late spermatids (number of surviving offspring only 3% and 27%, respectively, of those in concurrent controls)--jointly yielded the highest frequency of specific-locus mutations. AA thus conforms to Pattern 1 in our earlier classification of chemicals according to the spermatogenic stage at which they elicit maximum response (Russell et al., 1990). No specific-locus mutations were observed among 17,112 offspring derived from exposed spermatogonial stem cells, a result which rules out (at the 5% significance level) an induced mutation rate greater than 2.3 times the historical control rate. A sustained high productivity in matings made for several months following week 3 indicates that there is no significant spermatogonial killing and that cell selection is presumably not the explanation for the negative result. On the basis of genetic and/or cytogenetic evidence, the mutations induced postmeiotically by AA were 'large lesions' (multi-locus), while one of 2 recovered from exposure of differentiating spermatogonia is probably a small lesion. An earlier survey of mammalian mutagenesis results led us to conclude that, regardless of the classification of a chemical according to the stage at which it elicits its maximum response, the nature of mutations is determined by the germ-cell stage in which they are induced (Russell et al., 1990). The AA results on lesion size and on distribution of mutations among the loci fit the general pattern.

Molecular and genetic characterization of a radiation-induced structural rearrangement in mouse chromosome 2 causing mutations at the limb deformity and agouti loci.

Molecular characterization of mutations in the mouse, particularly those involving agent-induced major structural alterations, is proving to be useful for correlating the structure and expression of individual genes with their function in the whole organism. Here we present the characterization of a radiation-induced mutation that simultaneously generated distinct alleles of both the limb deformity (ld) and agouti (a) loci, two developmentally important regions of chromosome 2 normally separated by 20 centimorgans. Cytogenetic analysis revealed that an interstitial segment of chromosome 17 (17B- 17C; or, possibly, 17A2-17B) had been translocated into the distal end of chromosome 2, resulting in a smaller-than-normal chromosome 17 (designated 17del) and a larger form of chromosome 2 (designated 2(17). Additionally, a large interstitial segment of the 2(17) chromosome, immediately adjacent and proximal to the insertion site, did not match bands 2E4-2H1 at corresponding positions on a normal chromosome 2. Molecular analysis detected a DNA rearrangement in which a portion of the ld locus was joined to sequences normally tightly linked to the a locus. This result, along with the genetic and cytogenetic data, suggests that the alleles of ld and a in this radiation-induced mutation, designated ldIn2 and ajIn2, were associated with DNA breaks caused by an inversion of an interstitial segment in the 2(17) chromosome.

Increased incidence of developmental anomalies among descendants of carriers of methylenebisacrylamide-induced balanced reciprocal translocations.

N,N'-Methylenebisacrylamide (MBA), a dimer of the monomeric acrylamide, was studied for induction of clastogenic effects in germ cells of male mice. It was found to be effective in inducing dominant-lethal mutations and heritable translocations in maturing sperm. The semisterile translocation carriers and their normal counterparts were used to determine the health impact of transmitted chromosomal rearrangements through anatomical analysis of their immediate descendants in utero. As expected, semisterility resulted primarily from embryonic death during the periimplantation stages presumably caused by sperm segregants with unbalanced chromosome complement fertilizing some of the eggs. Among conceptuses that survived to mid- and late-gestation stages, there was an increased incidence of developmental anomalies including fetal death and phenotypic defects. These anomalies are assumed to be caused by certain types of unbalanced segregants that are compatible with survival beyond the periimplantation period. This class of unbalanced segregants represent in humans a major health problem to the mother and her conceptus.

Genetic and molecular analysis of chlorambucil-induced germ-line mutations in the mouse.

Eighteen variants recovered from specific locus mutation rate experiments involving the mutagen chlorambucil were subjected to several genetic and molecular analyses. Most mutations were found to be homozygous lethal. Because lethality is often presumptive evidence for multilocus-deletion events, 10 mutations were analyzed by Southern blot analysis with probes at, or closely linked to, several of the specific locus test markers, namely, albino (c), brown (b), and dilute (d). All eight mutations (two c; three b; two d; and one dilute-short ear [Df(d se)]) that arose in post-spermatogonial germ cells were deleted for DNA sequences. No evidence for deletion of two d-se region probes was obtained for the remaining two d mutations that arose in stem-cell spermatogonia. Six of the primary mutants also produced low litter sizes ("semisterility"). Karyotypic analysis has, to date, confirmed the presence of reciprocal translocations in four of the six. The high frequency of deletions and translocations among the mutations induced in post-spermatogonial stages by chlorambucil, combined with its overall high efficiency in inducing mutations in these stages, should make chlorambucil mutagenesis useful for generating experimentally valuable germ-line deletions throughout the mouse genome.

Correlation between meiotic behavior and breakpoints with respect to G-bands in two X-4 mouse translocations: T(X;4)7R1 and T(X;4)8R1.

The meiotic synaptic behavior of male mice heterozygous for one of two X-4 translocations was examined to test a recently advanced hypothesis (Ashley, 1988) suggesting that it is possible to predict the synaptic behavior (nonhomologous vs. homologous) and recombinational parameters (suppression vs. nonsuppression of crossing-over) of a chromosome aberration from mitotic G-band breakpoint data. The hypothesis was based on prior observations of synaptic behavior in a series of X-autosome translocations in mice. The breakpoints of the translocation T(X;4)7R1 are both in G-light bands. As predicted by the hypothesis, synapsis was restricted to homology. In contrast, one breakpoint of the translocation T(X;4)8R1 lies in a "stippled" band of the standard diagrams of Nesbitt and Francke (1981). As predicted (Ashley, 1988), "stippled" bands are shown here to synapse nonhomologously, i.e., they behave as "G-dark." The linkage data, as they relate to the synaptic data and the predictions of the hypothesis, are also discussed.

Concentration-response curves for ethylene-oxide-induced heritable translocations and dominant lethal mutations.

Male mice were subjected to repeated inhalation exposures to different concentrations (165, 204, 250, or 300 ppm) of ethylene oxide (EtO) during an 8.5-week period. Transmitted clastogenic effects of these exposures were measured in terms of induction of dominant lethal mutations and heritable translocations. The concentration-response curves for both endpoints are not linear but are markedly concave upward. Significant increases in dominant lethals were detected at all concentrations, except the lowest one. In comparison, the incidences of heritable translocations were significantly increased at all concentrations.

Chlorambucil effectively induces deletion mutations in mouse germ cells.

The chemotherapeutic agent chlorambucil was found to be more effective than x-rays or any chemical investigated to date in inducing high yields of mouse germ-line mutations that appear to be deletions or other structural changes. Induction of mutations involving seven specific loci was studied after exposures of various male germ-cell stages to chlorambucil at 10-25 mg/kg. A total of 60,750 offspring was scored. Mutation rates in spermatogonial stem cells were not significantly increased over control values; this negative result is not attributable to selective elimination of mutant cells. Mutations were, however, clearly induced in treated post-stem-cell stages, among which marked variations in mutational response were found. Maximum yield occurred after exposure of early spermatids, with approximately 1% of all offspring carrying a specific-locus mutation in the 10 mg/kg group. The stage-response pattern for chlorambucil differs from that of all other chemicals investigated to date in the specific-locus test. Thus far, all but one of the tested mutations induced by chlorambucil in post-stem-cell stages have been proved deletions or other structural changes by genetic, cytogenetic, and/or molecular criteria. Deletion mutations have recently been useful for molecular mapping and for structure-function correlations of genomic regions. For generating presumed large-lesion germ-line mutations at highest frequencies, chlorambucil may be the mutagen of choice.

Fetal anomalies produced subsequent to treatment of zygotes with ethylene oxide or ethyl methanesulfonate are not likely due to the usual genetic causes.

Earlier studies in this laboratory revealed that ethylene oxide (EtO) or ethyl methanesulfonate (EMS) induced high frequencies of midgestation and late fetal deaths, and of malformations among some of the surviving fetuses, when female mice were exposed at the time of fertilization of their eggs or during the early pronuclear stage of the zygote. Effects of the two mutagens are virtually identical. Thus, in investigating the mechanisms responsible for the dramatic effects in the early pronuclear zygotes, the two compounds were used interchangeably in the experiments. First, a reciprocal zygote-transfer study was conducted in order to determine whether the effect is directly on the zygotes or indirectly through maternal toxicity. And second, cytogenetic analyses of pronuclear metaphases, early cleavage embryos, and midgestation fetuses were carried out. The zygote transplantation experiment rules out maternal toxicity as a factor in the fetal maldevelopment. Together with the strict stage specificity observed in the earlier studies, this result points to a genetic cause for the abnormalities. However, the cytogenetic studies failed to show structural or numerical chromosome aberrations. Since intragenic base changes and deletions may also be ruled out, it appears that the lesions in question induced in zygotes by the two mutagens are different from conventional ones and, therefore, could be a novel one in experimental mammalian mutagenesis. Alternatively, the mechanism could involve a non-mutational 'imprinting' process that caused changes in gene expression.

No evidence found for induction of dominant lethal mutations and heritable translocations in male mice by calcium cyclamate.

Calcium cyclamate, an artificial sweetener, was studied for its effectiveness in inducing transmissible chromosomal aberrations in germ cells of male mice. Both the dominant-lethal and the heritable translocation tests were carried out following daily treatment (on weekdays) of males by oral intubation with the maximum tolerated dose for 6 weeks. Calcium cyclamate is negative in both tests; therefore, there is no evidence of induced chromosome breakage and exchange.

Mouse specific-locus test for the induction of heritable gene mutations by dibromochloropropane (DBCP).

The nematocide DBCP (1,2-dibromo-3-chloropropane) produced negative results in a specific-locus test for gene-mutation induction in the germline of male (101 X C3H)F1 mice, most of which were treated with 5 daily intraperitoneal injections of 80 mg/kg (total exposure, 400 mg/kg); a few received lower exposures. For treated spermatogonial stem cells, the finding of 2 mutations among 39519 offspring--a rate almost identical to the control rate--rules out (at the 5% significance level) an induced mutation frequency greater than 2.0 times the historical control rate. From treated poststem-cell stages, no mutants were found among 6240 offspring, ruling out (at the 5% significance level) a multiple of 8.0 times the control for these cell types. A multiple rearrangement (7 chromosomes involved in 3 translocations) found in one of the mutants probably arose as a postmeiotic event not associated with the DBCP treatment. The fertility of DBCP-treated males was not disturbed, in keeping with the absence of germ-cell toxicity and dominant lethals found by other investigators in these mice, and in contrast to results in certain other species. While the treated (101 X C3H)F1 mice are Ah-responsive, other findings make it questionable whether biotransformation of DBCP to reactive intermediates is accomplished via the Ah-receptor system.