Dominant Lethals Research Articles

The Japanese medaka, Oryzias latipes, as a new model organism for studying environmental germ-cell mutagenesis.

The effects of genotoxic substances on ecosystems should be assessed using various test systems with multiple genetic end points. The most widely used test system has been the specific-locus test developed by W.L. Russell, using the mouse. We are developing a new, nonmammalian test system using the Japanese medaka, Oryzias latipes. We have examined 625,926 embryos that correspond to 1,586,649 loci. In the medaka test system, four genetic end points are evaluated: dominant lethals, total mutations, viable mutations, and malformations. Because the medaka is an oviparous experimental animal, we were able to determine that approximately 90% of spontaneous as well as gamma-ray-induced total mutants died during development, irrespective of spermatogenesis stages at the time of exposure. Exposure of sperm and spermatids to ethylnitrosourea (ENU) also resulted in embryonic death of approximately 90% of total mutants. In sharp contrast, approximately 90% of total mutants recovered from ENU-exposed spermatogonia became viable mutants. These results indicate that the quantitative relationship between induction of specific-locus mutations and dominant lethals remains the same among spermatogenesis stages for gamma-rays, while it is biased excessively to the induction of specific-locus mutations in ENU-exposed spermatogonia. Thus, the assessment should integrate at least two factors, agent-specific and species-specific effects.

Dominant lethal effects after inhalation exposure to 1,3-butadiene

Two independent dominant lethal experiments were performed using different protocols with respect to strains of mice, inhalation exposure duration of 1,3-butadiene, and mating regimen. The short communication summarizes the results of the experiments and compares the induced dominant lethality according to the formula published by Ehling in 1978. Despite the differences in methodology the results are in close agreement. The sum of the dominant lethal effects observed during the first three mating weeks after 1 week of butadiene exposure in one experiment (23.1%) is surprisingly similar to the dominant lethal effect observed during 1 week of mating after 10 weeks of butadiene exposure (28.1%) in the other experiment. The results of the two independent experiments strengthen the conclusion that butadiene is a germ cell mutagen. Furthermore, the results indicate that the effect observed after 10 weeks of exposure is representative of the last three treatment weeks, i.e. treated spermatozoa and spermatids.

Dominant Lethals Induced by Dithane M-45 in Silkworm Bombyx mori

Dithane M-45, a ethylenebis(dithiocarbamate) fungicide widely used in agriculture, including moriculture and sericulture, was tested for its efficacy in inducing dominant lethals in Bombyx mori. A polyvoltine race of Pure Mysore was used for the studies and the topical application method was employed. After treatment with sublethal concentrations of 5, 10, and 20 g/50 larvae, the results have revealed that all of the concentrations could induce significant dominant lethals in a dose-dependent manner compared to controls. The results are discussed in the light of precautionary measures in the use of Dithane M-45 in the sericulture industry.

An assessment of the genetic toxicity of atrazine: Relevance to human health and environmental effects

The genetic toxicity of atrazine, a member of the s-triazine herbicides, was reviewed with the objective of classifying the chemical. Atrazine has been subjected to a broad range of genetic tests with predominantly negative results. Some publications, specifically those measuring dominant lethality in mice and bone marrow clastogenicity in rodents, reported conflicting results across two or more independent tests. Two approaches were employed to evaluate and interpret the results. The first approach attempts to classify each type of genetic endpoint as positive or negative and resolve test conflicts by critical assessment of the study and detailed data. This is the more traditional “expert judgment” approach to hazard assessment. The second approach employs a computer-assisted weight-of-evidence method of data analysis. This approach does not require resolution of conflicts but uses all data sets to arrive at a classification of hazard. The first approach was able to resolve some conflicts but not all. Use of the “expert judgement” results in an equivocal conclusion and classification. Use of the weight-of-evidence method resulted in a conclusion that atrazine does not pose a mutagenic hazard. The weight-of-evidence scheme is proposed to be a more practical and relevant approach for assessing complex data sets.

Functional interactions between the gene tetanic and the Shaker gene complex of Drosophila.

Different phenotypes associated with the tetanic (tta) mutation such as appendage contraction, maternal effect and low viability and fertility are enhanced by one extra dose of the Shaker gene complex (ShC). The tta mutation is lethal with two extra doses of ShC. In addition, tta embryos have a defective nervous system. In this paper, I analyse the interaction between tta and ShC to gain insight into their relationship. Aneuploid analysis suggests that the lethality is due to an interaction of the tta mutation with the maternal effect (ME) region of this gene complex. Mutations in the ME region of ShC partially suppress this interaction. Trans-heterozygous combinations of MEI[l(1)305] and MEIII [l(1)459] mutations causes dominant lethality in a tta background. Trans-heterozygous combinations of an MEII[l(1)1359] mutation with the cited MEI and MEIII mutations are lethal in a tta background. Double mutant combinations and gene dosage experiments, suggest that tta also interacts with the viable (V) region of ShC. These specific genetic interactions indicate that tta and the ME and V regions of ShC are functionally related. These results, together with the previous electrophysiological, molecular and biochemical studies on these mutants suggest an interaction at the protein level. Thus, in the case of the V region, the tta gene product may modulate the activity of the K+ channels encoded in this region. Furthermore, the extreme dosage sensitivity of the interaction between tta and ShC suggests a stoichiometric requirement for the different gene products involved, which might be physically associated and form heteromultimers.

Functional relationships between genes of the Shaker gene complex of Drosophila.

Different mutations belonging to the HLI and HLII complementation groups of the haplolethal (HL) region of the Shaker complex (ShC) are described. The HLI complementation group includes viable (hdp), recessive lethals [l(1)1614], semidominant lethals [l(1)8384] and dominant lethals [l(1)5051, l(1)9916, l(1)13193], lack-of-function alleles that affect nervous system, cuticle and muscle development. The HLI complementation group encodes troponin I. HLII lack-of-function mutations [l(1)174 and l(1)4058] affect nervous system development. The semidominant lethal HLI mutation l(1)8384 shows differential complementation with other mutations in the ME and HL regions of ShC. Thus, heterozygous combinations of l(1)8384 with ME mutations l(1)162 and l(1)387 are poorly viable. The same phenomenon is observed for heterozygotes of l(1)8384 with HL mutations l(1)1199, l(1)2288 and l(1)3014. These specific interactions indicate the existence of functional relationships among the genetic elements of ShC. The implications for the understanding of the functional organization of ShC are discussed.

Developmental Abnormalities Due to Exposure of Mouse Paternal Germ Cells, Preimplantation Embryos, and Organogenic Embryos to Acrylamide

ABSTRACT The developmental toxicity of acrylamide was investigated after paternal germ cells and preimplantation and organogenic embryos were exposed to the agent. In the first experiment, ICR male mice were injected intraperitoneally with single doses of 62.5 or 125 mg/kg acrylamide or daily doses of 50 mg/kg for 5 days. They were mated with untreated virgin ICR female mice on days 1–21 and 64–80 after the last injection. The sperms involved in fertilization during these two periods were postmeiotic germ cells and spermatogonial stem cells, respectively, at the time of acrylamide treatment. The uterine contents were examined on day 18 of gestation for dominant lethal effects, and the fetuses were examined for external malformation. Acrylamide exposure during the postmeiotic cell or spermatogonial stem cell stage caused no significant increases in the incidence of abnormal fetuses. Dominant lethals, however, were clearly induced when the germ cells had been postmeiotic at the time of acrylamide exposure. In the second experiment, ICR mice were injected intraperitoneally with a single dose of 125 mg/kg acrylamide on day 0, 1, 2, or 3 of gestation. The uterine contents were examined on day 18 of gestation. Acrylamide treatment on day 0 of gestation caused a significant increase in the incidence of malformed fetuses, while treatment on day 1, 2, or 3 of gestation failed to cause an increase in malformation. Polydactyly was the most common type of abnormality. In the third experiment, pregnant mice were treated with three daily doses of 50 or 100 mg/kg acrylamide on days 6–8 or 9–11 of gestation, respectively. There was no significant difference between the incidence of malformed fetuses in the control and acrylamide‐treated groups. These experiments demonstrate the vulnerability of preimplantation embryos to the toxic effects of acrylamide, while paternal germ cells and the organogenic embryos are resistant to the induction of fetal malformations.

On the genetic background of the adaptive response to X-rays in Drosophila melanogaster.

The effects of a low dose (0.1-20 mGy) preirradiation with X-rays followed by a higher dose (2 Gy) of the same radiation on the recovery of the genetic damage induced as dominant lethals in mature oocytes (stage 14) of different strains of Drosophila melanogaster were investigated. The response was shown to be dependent on the genotype of the flies tested, since lower frequencies of dominant lethals (DL) were only obtained in strains carrying the white mutation. Based on these observations experiments to locate the genetic factor responsible for the adaptive response (AR) were performed. This factor was found to be in a specific region of the X-chromosome. Additional experiments were carried out to give information on the minimal dose required to induce the AR. The results showed that the lowest dose needed is 0.2 mGy. Increasing the conditioning X-ray dose had no influence on the response.

Cytogenetic and germ cell effects of phosphine inhalation by rodents: II. Subacute exposures to rats and mice.

Phosphine (PH3) is a highly toxic grain fumigant to which there is significant human workplace exposure. To determine the in vivo cytogenetic effects of inhalation of PH3, male F344/N rats and B6C3F1 mice were exposed to target concentrations of 0, 1.25, 2.5, or 5 ppm PH3 for 6 hr/day for 9 days over an 11-day period. Approximately 20 hr after the termination of exposures, blood was removed from the mice and rats by cardiac puncture and the lymphocytes cultured for analyses of sister chromatid exchanges and chromosome aberrations in rats and mice, and micronuclei (MN) in cytochalasin B-induced binucleated lymphocytes from mice. In addition, bone marrow (rats) and peripheral blood (mice) smears were made for the analysis of MN in polychromatic and normochromatic erythrocytes. No significant increase in any of the cytogenetic endpoints was found at any of the concentrations examined. These results indicate that concentrations of PH3 up to 5 ppm are not genotoxic to rodents when administered by inhalation for 9 days during an 11-day period as measured by several cytogenetic assays. To evaluate the effects of PH3 on male germ cells, a dominant lethal test was conducted in male mice exposed to 5 ppm PH3 for 10 days over a 12-day period and mated to groups of untreated females (2 females/male) on each of 6 consecutive 4-day mating intervals. None of the 6 groups of females exhibited a significant increase in percent resorptions. These results indicate that exposure to 5 ppm PH3 by inhalation does not induce dominant lethality in male mouse germ cells at steps in spermatogenesis ranging from late differentiating spermatogonia/early primary spermatocytes through mature sperm.

Effects of anoxia during gamma irradiation on face fly fertility and viability (Diptera: Muscidae).

Irradiating 6-d-old Musca autumnalis De Geer pupae in nitrogen with 500-2,500 rads did not affect adult emergence, female fecundity, or male competitiveness. Male and female fertility decreased with increased dose, reaching about 1% at 2,500 rads. Egg to adult survival upon backcrossing irradiated males or females to unirradiated flies was only 7% at 1,500 rads and 0.7% at 2,000 rads. Doses of up to 2,000 rads did not affect male survival, but males treated with 2,500 rads had reduced survival rates. These results are better than those with irradiation in air and indicate that irradiation with 1,800-2,000 rads in nitrogen would maximize dominant lethality and minimize fitness loss.

Induction of an adaptive response to dominant lethality and to chromosome damage of mouse germ cells by low dose radiation

In the present paper, dominant lethal mutations, chromosome aberrations in spermatocytes and reciprocal translocations in stem spermatogonia were analyzed after whole body exposure of mice to X-radiation. Results both from chromosome aberrations in spermatocytes and for reciprocal translocations in spermatogonia showed that pre-exposure to low doses up to 200 mGy could induce a significant dose-dependent reduction in adapted mice compared to the non-adapted mice; the lower the adaptive dose, the greater the reduction. For dominant lethal mutations, it was found that spermatogonia (both stem cells and differentiated cells) and spermatocytes adapted to 50 mGy X-rays could show an adaptive response, but spermatids and spermatozoa could not.

Characterization of yeast plasma membrane H(+)-ATPase mutant pma1-A135V and its revertants

An A135V substitution in the first transmembrane segment of the yeast plasma membrane H(+)-ATPase (PMA1) confers cellular resistance to hygromycin B, exhibits growth sensitivity to low external pH, and results in a defective enzyme that hydrolyzes ATP at 33% of wild type level. The importance of the A135 residue was probed genetically by analysis involving both site-directed mutagenesis and randomly generated second-site intragenic suppressor mutations. No other amino acid at position 135 gave either the wild type phenotype or the normal enzyme activity of A135. Substitutions with the bulkier amino acid residues A135L, A135I, and A135F produced more severe cellular phenotypes than the original A135V mutation. The substitution of the smaller side chain residue Gly was also a mutant, although not as severe as the A135V mutant. The introduction of a bulky Trp or a polar Ser residue produced dominant lethality, while charged amino acids produced recessive lethality. Reduced rates of proton transport measured by acidification of the medium by whole cells correlate closely with the severity of cellular phenotype. Some of the mutant enzymes exhibit an apparent instability in vitro. Thus, the localized structure around A135 is highly constrained. The cellular sensitivity to low external pH of the A135V mutant was used to select intragenic revertants. Most full revertants (low pHR, HygS) restored A135, but second-site mutations in putative transmembrane segments 2 (V146I and V157F) and 4 (L327V) were also observed. Two partial revertants (low pHR, HygR) have secondary mutations at S660C or a double change at F611L-S660F in the putative ATP binding domain. These results provide additional evidence for functional coupling between the cytoplasmic domain catalyzing ATP hydrolysis and transmembrane helices 1 and 2.

Studies on mutagen sensitive strains of Drosophila melanogaster. XI. Survival (dominant lethality) after X-irradiation and relation to recessive lethals and translocations

Muller-5 males of Drosophila melanogaster were irradiated in N 2 or O 2 and mated to excision repair deficient, post-replication repair deficient ( mei-9 a, mei-41 D5, mus101 D1, mus201 D1, mus302 D1, mus306 D1 and mus308 D2 ) or repair proficient females. The surviving fraction (dominant lethality) was estimated in the F 1 and used to reassess existing recessive lethal and translocation data. The surviving fraction was found to decrease if repair deficient females were used (maternal effect). The dose-effect curves are often biphasic with a steeper slope at low doses than at high ( ≧5 Gy ) doses of X-rays. The high dose part of the curve is sensitive to oxygenation during irradiation and is affected significantly by the mutants with low fertility ( mei-9, mus101 and mus302). The low dose component is not sensitive to oxygenation during irradiation and seems influenced by all seven repair deficient mutants. The sensitivity of the high dose part to oxygenation suggests that this part is related mainly to DNA break damage, while in the low dose part base damage seems more important. Existing recessive lethal and translocation data were plotted against the surviving fraction for a reassessment. In excision repair deficient mutants translocation induction is lower compared to repair proficient flies at the same level of survival (i.e., dominant lethality). Likewise in post-replication repair deficient mutants induction of recessive lethals is decreased. However the frequency of respectively induced recessive lethals and translocations obtained at the same level of X-rays was the same in repair deficient and proficient backgrounds. It is concluded that genetic damage recovered in a repair deficient background is likely to be qualitatively different even if the frequency of the damage induced by a given dose is not altered.

Detection of γ-ray-induced DNA damages in malformed dominant lethal embryos of the Japanese medaka ( Oryzias latipes) using AP-PCR fingerprinting

Adult male fish of the medaka HNI strain exposed to 9.5 Gy or 19 Gy (0.95 Gy/min) of γ-rays were mated with non-irradiated female fish of the Hd-rR strain. Genomic DNA was prepared from malformed individual embryos which were expected to be dominant lethal and used for AP-PCR fingerprinting. By the use of a part of the T3 promoter sequence (20 mer), which to our knowledge, is not found in the medaka genome as an arbitrary primer, we found polymorphisms in genomic fingerprints which could distinguish the parental strains. On the other hand, we found that the fingerprints of F 1 hybrids were the sum of those of their parents. Based on those findings, we analyzed the fingerprints of genomic DNA of each severely malformed embryo, because we expect that radiation-induced genomic damages resulting in severe malformation and eventually in dominant lethals should be detected as changes in paternal fingerprints of F 1 hybrids. Indeed, we succeeded in detecting changes in genomic DNA as loss of some paternal bands in fingerprints of malformed embryos. One of 10 malformed embryos obtained from 9.5 Gy γ-irradiated males had lost one band of the paternal origin and 4 of 12 malformed embryos obtained from 19 Gy γ-irradiated males had lost 5 bands. These results indicated a possibility that quantitative as well as qualitative estimation of γ-ray-induced DNA damages can be made by this method which does not require the functional selection based on a specific target gene.

Effect of low temperature on radiation-induced genetic damage in Drosophila melanogaster: response of motile sperm and late spermatids

The response of fully mature motile sperm and late spermatids when challenged with X-radiation at 0°C has been studied in sex-linked recessive lethals, II–III translocations and dominant lethality experiments. At 0°C a significant increase in both mutagenic and clastogenic damage was detected compared to that obtained at 24°C. Furthermore, the results of experiments performed with different postirradiation temperatures demonstrate that the low temperature during irradiation was the sole factor responsible for the observed increase. In the recessive lethal and translocation tests the response of late spermatids was higher than that shown by motile spermatozoa. As a whole, the results, which are rather similar to data reported on the effect of irradiation in oxygen of the same cell stages, suggest that the low temperature acted as a dose-modifying factor.

Effects of gamma-radiation on mature pupae of maize borer, Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae)

Mature (5- to 6-day-old) male and female pupae of C. partellus were irradiated at 5 to 20 kilo-Roentgen (kR) and 5 to 10 kR respectively. Emergence of normal appearing moths, pupal periods and adult longevities remained statistically unaffected. Fecundity of normal (nonirradiated) females mated with males irradiated as pupae was nonsignificantly different from control, but fecundity of females irradiated as pupae at 10 kR was significantly reduced when they were mated with normal mates. Dominant lethals of varying intensities were induced at 5–20 kR. However, induction of dominant lethality was sex- as well as dose-dependent. Radiation doses inducing 100% dominant lethals in sperm (20 kR) and ova (5 kR) did not mitigate the mating ability of males and receptivity of females. However, receptivity of females was reduced significantly when they were irradiated as pupae at 10 kR. Rates of embryonation in F1 eggs were significantly reduced when normal females were crossed with males irradiated as pupae at 20 kR, but similar effect was observed at 10 kR in reversed crosses. Embryonic development was delayed in eggs obtained from crosses, where male or female parent was irradiated in pupal stage, but in both cases most of the dominant lethals acted late in embryonic development.

Analysis of dominant-negative mutations of the Caenorhabditis elegans let-60 ras gene.

The let-60 gene of Caenorhabditis elegans controls the choice between vulval and hypodermal differentiation in response to an inductive signal from the gonad. let-60 encodes a ras protein that acts downstream of the let-23 receptor tyrosine kinase in a signal transduction pathway. Dominant-negative mutations of let-60 [let-60(dn)] cause a reduction of the gene activity in let-60(dn)/+ heterozygotes and a vulva-less mutant phenotype. We have found that nine let-60(dn) mutations cause replacements of conserved residues. Four are in two novel positions; others are in positions known previously to cause dominant-negative mutations in mammalian cells. The locations of these lesions suggest that they disrupt the ability of the ras protein to bind guanine nucleotides. Four let-60(dn) mutant genes were introduced into wild-type animals in the form of extrachromosomal arrays and were found to generate three dominant phenotypes--lethality, vulva-less, or multivulva--depending on gene dose and alleles. The dominant lethality caused by high-dose transgenic let-60(dn) genes suggests a toxic effect of these mutant genes in early development. The dominant-negative effects of these mutations in heterozygotes are likely to be caused by competition between let-60(dn) and let-60(+) protein for a positive regulator. All let-60(dn) mutations interfere with let-60(+) activity, but some alleles have partial constitutive activity, suggesting that the ability to interact with the activator is separable from the ability to exert a physiological effect (stimulation of vulval differentiation). These dn mutations might be useful for interfering with ras-mediated signal transduction pathways in other multicellular organisms.

Site-directed mutagenesis of the yeast actin gene: a test for actin function in vivo.

The yeast Saccharomyces cerevisiae has a single actin gene, ACT1, whose protein product is essential for cell viability. To study the structure-function relationship of this evolutionarily highly conserved protein, we have introduced into the gene several mutations leading to substitutions of amino acids that, by chemical crosslinking experiments, have previously been identified as potential sites for the interaction of actin with several actin-binding proteins and of actin monomers in filaments. The in vitro mutated actin genes were used to replace one chromosomal ACT1 allele in diploid cells. From diploid transformants, haploids that solely depended on mutant actins were isolated and their phenotypic alterations studied. The replacement of the N-terminal acidic residues (Asp2 and Glu4) with valine was functionally neutral. Substitutions of Asp11 led to dominant lethality. Substitutions of Lys191, Lys336, Trp356, Lys373 and Cys374 were without observable effect on cell growth, proliferation and morphology. Deletion of the C-terminal end, Lys-Cys-Phe-COOH, was lethal, whereas successive removal of the C-terminal Phe375 or Cys374 and Phe375 resulted in temperature sensitivity. At the nonpermissive temperature, the mutant cells were characterized by an increase in size, a tendency to lyse and significant alterations of the actin cytoskeleton.

A search for strain differences in response of mice to mutagenesis by thio-TEPA

After treatment of mice with thio-TEPA Malashenko and colleagues found differences among inbred strains in yield of dominant lethals and of chromosome aberrations in bone marrow, which they attributed to genes affecting repair. An attempt was made to confirm this work by comparing yields of dominant lethals in different strains of females mated to the same strain of males. However, no differences were found, all strain combinations giving 42–49% dominant lethals after a dose of 2 mg/kg thio-TEPA to late spermatids. Thus, the existence of genetic differences in repair of thio-TEPA induced lesions between strains CBA and C57BL/6J and between C3H/He and 101/H is not confirmed. Possible reasons for the discrepant results are discussed.

Mutational Analysis of CDC42Sc, a Saccharomyces cerevisiae Gene That Encodes a Putative GTP-Binding Protein Involved in the Control of Cell Polarity

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site. The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains. Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds. Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells. The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other. This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein. The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts. In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.