共查询到20条相似文献,搜索用时 15 毫秒
1.
Dobrovolsky VN McGarrity LJ VonTungeln LS Mittelstaedt RA Morris SM Beland FA Heflich RH 《Mutation research》2005,570(2):227-235
The first step in the activation of the anti-retroviral nucleoside analogue azidothymidine (AZT) involves its conversion to a 5′-monophosphate. In this study, we have evaluated the role of cytosolic thymidine kinase (Tk), the major enzyme involved in phosphorylating thymidine and its analogues, in the nuclear DNA damage produced by AZT in neonatal mice. Tk+/+, Tk+/− and Tk−/− mice were treated intraperitoneally with 200 mg/kg/day of AZT on postnatal days 1 through 8, and micronuclei were measured in peripheral blood 24 h after the last dose. AZT treatment increased the micronucleus (MN) frequencies to similar extents in both the reticulocytes (RETs) and normochromatic erythrocytes (NCEs) of Tk+/+ and Tk+/− mice; AZT did not increase the frequency of micronucleated RETs (MN-RETs) or micronucleated NCEs (MN-NCEs) in Tk−/− mice. Unexpectedly, neonatal Tk−/− mice treated with the vehicle had significantly elevated MN frequencies for both RETs and NCEs relative to Tk+/+ and Tk+/− mice (e.g., 3.4% MN-RETs and 4.8% MN-NCEs in Tk−/− mice versus 0.7 and 0.6% MN-RETs and MN-NCEs in neonatal Tk+/+ mice). Additional assays performed on untreated Tk−/− mice showed that elevated spontaneous MN frequencies persisted until at least 20 weeks of age, which approaches the average lifespan of Tk−/− mice. These results indicate that metabolism by Tk is necessary for the genotoxicity of AZT in neonatal mice; however, the genotoxicity of AZT is not altered by reducing the Tk gene dose by half. The elevated spontaneous MN frequencies in Tk−/− mice suggest the presence of an endogenous genotoxic activity in these mice. 相似文献
2.
A new mouse model (Mutatect) that permits detection of mutations at the hprt (hypoxanthine phosphoribosyltransferase) locus is described. It is highly sensitive to detection of mutants induced by clastogenic agents such as ionizing radiation. MN-11 cells are grown as a subcutaneous tumour in C57BL/6 mice for a period of 2 weeks, during which time they can be exposed to mutagenic treatments. Cells taken from the animal are cultured ex vivo and 6-thioguanine (6-TG)-resistant mutant clones can be readily identified and scored. This model system may have special utility for detecting multi-locus deletion events (chromosomal mutations) induced by high LET forms of radiation that might be encountered in space. 相似文献
3.
Mohmmed A Dasaradhi PV Bhatnagar RK Chauhan VS Malhotra P 《Biochemical and biophysical research communications》2003,309(3):506-511
RNA interference (RNAi) has emerged as a specific and efficient tool to silence gene expression in a variety of organisms and cell lines. An important prospect for RNAi technology is its possible application in the treatment of diseases using short interfering RNAs (siRNAs). However, the effect of siRNAs in adult animals and their potential to treat or prevent diseases are yet to be fully investigated. The main goal of the present study is to find out whether it was possible to carry out RNAi on circulating malaria parasite in vivo. To trigger RNAi in mouse malaria parasite, we used siRNAs corresponding to cysteine protease genes of Plasmodium berghei (berghepain-1 & 2). Intravenous injections of berghepains' siRNAs in infected animal resulted in characteristic enlargement of food vacuole in circulating parasites. Protein analysis of these treated parasites showed substantial accumulation of hemoglobin, which is reminiscent of the effect observed upon treating Plasmodium falciparum with different cysteine protease inhibitors. Parasites treated with berghepain 1 & 2 siRNAs showed marked reduction in the levels of their cognate mRNAs, thereby suggesting specific inhibition of berghepains' gene expression in vivo. We also observed the generation of approximately 25 nt RNA species from berghepains' mRNAs in the treated parasites, which is a characteristic of an RNAi phenomenon. These results thus provide evidence that beyond its value for validation of gene functions, RNAi may provide a new approach for disease therapy. 相似文献
4.
5.
Although chemicals usually induce very similar frequencies of mutations in transgenes and endogenous genes in vivo when given acutely, chronic exposure to N-ethyl-N-nitrosourea (ENU) produced a more complex pattern in which the endogenous locus was spared many mutations. Here, we demonstrate that the effect is neither ENU-specific nor locus-specific, and thus, may be important in the extrapolations of risk assessment and in understanding mutational mechanisms. During chronic mutagen exposure, mutations at the transgene accumulate linearly with time, i.e. in direct proportion to the dose received. In contrast, mutations at the endogenous gene are much less frequent than those of the transgene early in the exposure period and the accumulation is not linear with time, but rather accelerates as the exposure continues. Previous comparisons involved the endogenous Dlb-1 locus and the lacI transgene from the Big BlueMouse in the small intestine. These experiments involved the Dlb-1 locus and the lacZ transgene from the MutaMouse in the small intestine and the hprt locus and the lacZ transgene in splenocytes. Comparisons were made in both tissues after acute and chronic exposures to ENU, the original mutagen, and in the small intestine after exposures to benzo(a)pyrene. All comparisons showed that during chronic exposures mutations at the transgene accumulate linearly with the increasing duration of exposure, whereas induced mutations of the endogenous gene initially accumulate at a slower rate. Thus, the difference in mutational response observed during low chronic treatment is not unique to a particular transgene, endogenous gene, tissue, or mutagen used, but may be a general phenomenon of such genes. 相似文献
6.
7.
S Wakasugi T Iwanaga T Inomoto T Tengan S Maeda M Uehira K Araki J Miyazaki K Eto K Shimada 《Developmental genetics》1988,9(3):203-212
We have created a transgenic mouse which showed an autosomal dominant mutation of facial development. This facial malformation was characterized by a short snout and a twisted upper jaw. All offspring showing the dysmorphic phenotype carried the injected gene. In order to analyze the primary cause of this mutation, newborn mice and embryos were examined. The outcome was that the malformation of nasal and premaxillary bone was not the primary defect but was a secondary event. The primary cause of this dysmorphism was a developmental defect in the first branchial arch. Genomic DNA fragments flanking the insertion site of this mutant mouse were cloned. Using these fragments, we have assigned the integration site to chromosome 13. The gene responsible for a previously reported mutant mouse, one which also has a short snout, is also reported to be on chromosome 13. In the fragments flanking the insertion site of the transgenic mouse, at least one fragment was highly conserved in mammals. These results indicate that this malformation is due to the insertional disruption of a host gene. However, the possibility that this mutation is caused by an inappropriate expression of the injected gene still remains to be investigated. 相似文献
8.
Genetic toxicology assays that rely on S9 microsomal mixes are subject to artifacts related to the generation of mutagenic metabolites by acidic pHs, variation in individual isolations of microsomes and the failure of subcellular fractions to faithfully produce metabolites generated in intact cells. We have developed a gene mutation assay utilizing the human hepatoma cell line HepG2, which has been shown to metabolize a broad spectrum of promutagens. Optimal conditions for assaying the induction of 6-thioguanine-resistant mutants in this cell line include: 1) growth of colonies for three weeks on lethally irradiated feeder layers of 106 thioguanine-resistant HepG2 cells (average plating efficiency = 60–80%); 2) a thioguanine concentration in selection dishes of 10–4M with a maximum seeding density of 2.5 × 105 cells per 100 mm culture dish; and 3) a minimum expression time of 6 days. In addition to ultraviolet light C (254 nm), a cytochrome P450 (cyclophosphamide)-dependent and a cytochrome P448 (aflatoxin B1)-dependent promutagen were shown to induce cytotoxicity and mutations in this test system. The present studies, therefore, suggest that the HepG2 cell line may be useful for a variety of assays in genetic toxicology.Abbreviations HAT
hypoxanthine, aminopterin, thymidine
- TG
6-thioguanine 相似文献
9.
In the course of large-scale mutagenesis studies, we discovered a mutant that provides a new mouse model for human autosomal recessive polycystic kidney disease. Animals homozygous for this mutation, T(2;10)67Gso, present evidence of grossly cystic renal and hepatic tissue at birth and a limited survival time of 3-4 days. The recessively expressed phenotype is associated with inheritance of a reciprocal translocation involving mouse chromosomes 2 and 10. Here we describe the pathology and phenotype of this new mutation. The mapping of the chromosomal breakpoint to the 1.0-cM critical region defined for another mouse autosomal recessive polycystic kidney disease model, juvenile congenital polycystic kidney disease (jcpk), led us to undertake the complementation testing that confirmed T(2;10)67Gso and jcpk are allelic. Because of the strong resemblance between the phenotype associated with these mouse mutations and early childhood polycystic kidney disease, and because of advantages offered by reciprocal translocations for gene mapping and cloning, T(2;10)67Gso should prove a valuable asset for studies concerning this fatal disease. 相似文献
10.
11.
12.
Potassium bromate (KBrO(3)) induces DNA damage and tumors in mice and rats, but is a relatively weak mutagen in microbial assays and the in vitro mammalian Hprt assay. Concern that there may be a human health risk associated with bromate, a disinfectant by-product of ozonation, has accompanied the increasing use of ozonation as an alternative to chlorination for treatment of drinking water. In this study, we have evaluated the mutagenicity of KBrO(3) and sodium bromate (NaBrO(3)) in the Tk gene of mouse lymphoma cells. In contrast to the weak mutagenic activity seen in the previous studies, bromate induced a mutant frequency of over 100 x 10(-6) at 0.6mM with minimal cytotoxicity (70-80% survival) and over 1300 x 10(-6) at 3mM ( approximately 10% survival). The increase in the Tk mutant frequency was primarily due to the induction of small colony of Tk mutants. Loss of heterozygosity (LOH) analysis of 384 mutants from control and 2.7 mM KBrO(3)-treated cells showed that almost all (99%) bromate-induced mutants resulted from LOH, whereas in the control cultures 77% of the Tk mutants were LOH. Our results suggest that bromate is a potent mutagen in the Tk gene of mouse lymphoma cells, and the mechanism of action primarily involves LOH. The ability of the mouse lymphoma assay to detect a wider array of mutational events than the microbial or V79 Hprt assays may account for the potent mutagenic response. 相似文献
13.
14.
15.
Ehninger D Han S Shilyansky C Zhou Y Li W Kwiatkowski DJ Ramesh V Silva AJ 《Nature medicine》2008,14(8):843-848
Tuberous sclerosis is a single-gene disorder caused by heterozygous mutations in the TSC1 (9q34) or TSC2 (16p13.3) gene and is frequently associated with mental retardation, autism and epilepsy. Even individuals with tuberous sclerosis and a normal intelligence quotient (approximately 50%) are commonly affected with specific neuropsychological problems, including long-term and working memory deficits. Here we report that mice with a heterozygous, inactivating mutation in the Tsc2 gene (Tsc2(+/-) mice) show deficits in learning and memory. Cognitive deficits in Tsc2(+/-) mice emerged in the absence of neuropathology and seizures, demonstrating that other disease mechanisms are involved. We show that hyperactive hippocampal mammalian target of rapamycin (mTOR) signaling led to abnormal long-term potentiation in the CA1 region of the hippocampus and consequently to deficits in hippocampal-dependent learning. These deficits included impairments in two spatial learning tasks and in contextual discrimination. Notably, we show that a brief treatment with the mTOR inhibitor rapamycin in adult mice rescues not only the synaptic plasticity, but also the behavioral deficits in this animal model of tuberous sclerosis. The results presented here reveal a biological basis for some of the cognitive deficits associated with tuberous sclerosis, and they show that treatment with mTOR antagonists ameliorates cognitive dysfunction in a mouse model of this disorder. 相似文献
16.
Osteochondrodystrophy (ocd) is a new autosomal recessive mouse mutation characterized by a short, slightly domed head, reduced body size, disproportionately shortened long bones of the legs, supination of the forefeet, and short thickened tail. Histologically, the epiphyses are thinner than normal. The columnar organization of the proliferative zone of cartilage is disorderly, with pleomorphic and occasionally necrotic chondrocytes. Osteochondrodystrophy has been mapped to a position near the centromere of mouse chromosome (Chr) 19. 相似文献
17.
The chubby (cby) mutant is a previously undescribed skeletal mutation in the mouse. Breeding experiments showed that cby is a recessive mutant with complete viability, full penetrance and fertility in both sexes. Tests for allelism showed that the cby mutant is genetically unlike the somewhat similar mutants, stumpy (stm), pituitary dwarf (dw), spondylometaphyseal chondrodysplasia (smc), brachymorphic (bm), and achondroplasia (cn). The defects seem to occur mainly in growth cartilage. Microradiography revealed increased height of the epiphyseal growth plate and irregular bone trabeculae in the metaphysis. Light microscopy showed disturbed columnar organization of proliferative chondrocytes and pronounced signs of cellular disintegration. The hypertrophic zone, however, contained normally shaped chondrocytes arranged in regular columns. In spite of the normal cellular hypertrophy the pattern of cartilage mineralization was disturbed. The electron microscopy studies revealed very high amounts of matrix vesicles and numerous larger membrane coated structures in the extracellular matrix. Biochemical analysis of the affected growth cartilage revealed a slightly modified pattern of proteoglycan subpopulations and considerably longer chondroitin sulfate chains when compared with controls. From the present study it can be concluded that the cby mutant is a genetically and morphologically distinct condition with characteristic, somewhat rickets-like stigmata. The pathogenetic mechanism underlying the condition remains to be clarified. 相似文献
18.
The BALB/cGa mouse strain and its descendants, now called the SELH/Bc strain, have produced two waves of high frequency of spontaneous heritable mutations. One of these, the recessive lidgap-Gates (lg(Ga)) mutation, causes the same open-eyelids-at-birth phenotype as the gene knockout mutations of Map3k1 and co-maps to distal Chr 13. The lg(Ga) mutation is demonstrated to be a 27.5-kb deletion of exons 2-9 in the Map3k1 gene, the first spontaneous mutant allele described at this locus. The lg(Ga) mutation is consistent with a pattern suggesting that the waves of mutation in BALB/cGa and its descendants tend to be large deletions or ETn insertions, whose elevated rate of occurrence is due to an unknown mechanism. 相似文献
19.
Mittelstaedt RA Von Tungeln LS Shaddock JG Dobrovolsky VN Beland FA Heflich RH 《Mutation research》2004,547(1-2):63-69
Mother-to-child transmission of the human immunodeficiency virus (HIV) is reduced by perinatal treatment with the antiretroviral nucleoside analogue 3'-azido-3'-deoxythymidine (AZT, zidovudine). AZT, however, is genotoxic and carcinogenic in mice when administered either transplacentally or neonatally, suggesting a possible cancer risk for children later in life. In a previous study we found that treating B6C3F1/Tk(+/-) mice on postnatal days 1 through 8 with intraperitoneal injections of 200 mg AZT per kg body weight per day significantly increased spleen lymphocyte mutant frequencies in the autosomal Tk gene. Allele-specific PCR of Tk mutants from treated mice indicated that 61% had lost the Tk(+) allele (loss of heterozygosity; LOH), compared with 35% of Tk mutants from control mice, a difference that was significant. In the present study, Tk mutant lymphocyte clones were analyzed further using polymorphic microsatellite markers that flank the Tk gene along the length of mouse chromosome 11. The analysis indicated that allele-loss mutations in control mice were due to either total chromosome loss, mitotic recombination, or both. The pattern of marker loss in mutants from AZT-treated mice differed significantly from the control mice and was consistent with chromosome loss, mitotic recombination, interstitial deletion, gene conversion, and an unusual discontinuous LOH. The results indicate that AZT induced a unique pattern of mutations in the Tk gene of mice and that the major mechanisms of mutation by AZT involved deletion and recombination. 相似文献