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Mutagenesis
Kinds of Mutagenesis Terms modified by Mutagenesis Selected AbstractsThe taurine transporter: mechanisms of regulationACTA PHYSIOLOGICA, Issue 1-2 2006X. Han Abstract Taurine transport undergoes an adaptive response to changes in taurine availability. Unlike most amino acids, taurine is not metabolized or incorporated into protein but remains free in the intracellular water. Most amino acids are reabsorbed at rates of 98,99%, but reabsorption of taurine may range from 40% to 99.5%. Factors that influence taurine accumulation include ionic environment, electrochemical charge, and post-translational and transcriptional factors. Among these are protein kinase C (PKC) activation and transactivation or repression by proto-oncogenes such as WT1, c-Jun, c-Myb and p53. Renal adaptive regulation of the taurine transporter (TauT) was studied in vivo and in vitro. Site-directed mutagenesis and the oocyte expression system were used to study post-translational regulation of the TauT by PKC. Reporter genes and Northern and Western blots were used to study transcriptional regulation of the taurine transporter gene (TauT). We demonstrated that (i) the body pool of taurine is controlled through renal adaptive regulation of TauT in response to taurine availability; (ii) ionic environment, electrochemical charge, pH, and developmental ontogeny influence renal taurine accumulation; (iii) the fourth segment of TauT is involved in the gating of taurine across the cell membrane, which is controlled by PKC phosphorylation of serine 322 at the post-translational level; (iv) expression of TauT is repressed by the p53 tumour suppressor gene and is transactivated by proto-oncogenes such as WT1, c-Jun, and c-Myb; and (v) over-expression of TauT protects renal cells from cisplatin-induced nephrotoxicity. [source] CLIP-170 interacts with dynactin complex and the APC-binding protein EB1 by different mechanismsCYTOSKELETON, Issue 3 2003Holly V. Goodson Abstract CLIP-170 is a "cytoplasmic linker protein" implicated in endosome-microtubule interactions and in control of microtubule dynamics. CLIP-170 localizes dynamically to growing microtubule plus ends, colocalizing with the dynein activator dynactin and the APC-binding protein EB1. This shared "plus-end tracking" behavior suggests that CLIP-170 might interact with dynactin and/or EB1. We have used site-specific mutagenesis of CLIP-170 and a transfection/colocalization assay to address this question in mammalian tissue culture cells. Our results indicate that CLIP-170 interacts, directly or indirectly, with both dynactin and EB1. We find that the CLIP-170/dynactin interaction is mediated by the second metal binding motif of the CLIP-170 tail. In contrast, the CLIP-170/EB1 interaction requires neither metal binding motif. In addition, our experiments suggest that the CLIP-170 /dynactin interaction occurs via the shoulder/sidearm subcomplex of dynactin and can occur in the cytosol (i.e., it does not require microtubule binding). These results have implications for the targeting of both dynactin and EB1 to microtubule plus ends. Our data suggest that the CLIP-170/dynactin interaction can target dynactin complex to microtubule plus ends, although dynactin likely also targets MT plus ends directly via the microtubule binding motif of the p150Glued subunit. We find that CLIP-170 mutants alter p150Glued localization without affecting EB1, indicating that EB1 can target microtubule plus ends independently of dynactin. Cell Motil. Cytoskeleton 55:156,173, 2003. © 2003 Wiley-Liss, Inc. [source] Mutation in the abcb7 gene causes abnormal iron and fatty acid metabolism in developing medaka fishDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 9 2008Akimitsu Miyake The medaka fish (Oryzias latipes) is an emerging model organism for which a variety of unique developmental mutants have now been generated. Our recent mutagenesis screening of the medaka isolated a unique mutant that develops a fatty liver at larval stages. Positional cloning identified the responsible gene as medaka abcb7. Abcb7, a mitochondrial ABC (ATP binding cassette) half-transporter, has been implicated in iron metabolism. Recently, human Abcb7 was found to be mutated in X-linked sideroblastic anemia with cerebellar ataxia (XLSA/A). The homozygous medaka mutant exhibits abnormal iron metabolism in erythrocytes and accumulation of lipid in the liver. Microarray and in situ hybridization analyses demonstrated that the expression of genes involved in iron and lipid metabolisms are both affected in the mutant liver, suggesting novel roles of Abcb7 in the development of physiologically functional liver. The medaka abcb7 mutant thus could provide insights into the pathogenesis of XLSA/A as well as the normal function of the gene. [source] Designing mouse behavioral tasks relevant to autistic-like behaviors,DEVELOPMENTAL DISABILITIES RESEARCH REVIEW, Issue 4 2004Jacqueline N. Crawley Abstract The importance of genetic factors in autism has prompted the development of mutant mouse models to advance our understanding of biological mechanisms underlying autistic behaviors. Mouse models of human neuropsychiatric diseases are designed to optimize (1) face validity, i.e., resemblance to the human symptoms; (2) construct validity, i.e., similarity to the underlying causes of the disease; and (3) predictive validity, i.e., expected responses to treatments that are effective in the human disease. There is a growing need for mouse behavioral tasks with all three types of validity for modeling the symptoms of autism. We are in the process of designing a set of tasks with face validity for the defining features of autism: deficits in appropriate reciprocal social interactions, deficits in verbal social communication, and high levels of ritualistic repetitive behaviors. Social approach is tested in an automated three-chambered apparatus that offers the subject a choice between a familiar environment, a novel environment, and a novel environment containing a stranger mouse. Preference for social novelty is tested in the same apparatus, with a choice between the start chamber, the chamber containing a familiar mouse, and the chamber containing a stranger mouse. Social communication is evaluated by measuring the ultrasonic distress vocalizations emitted by infant mouse pups and the parental response of retrieving the pup to the nest. Resistance to change in ritualistic repetitive behaviors is modeled by forcing a change in habit, including reversal of the spatial location of a reinforcer in a T-maze task and in the Morris water maze. Mouse behavioral tasks that may model additional features of autism are discussed, including tasks relevant to anxiety, seizures, sleep disturbances, and sensory hypersensitivity. Applications of these tests include (1) behavioral phenotyping of transgenic and knockout mice with mutations in genes relevant to autism, (2) characterization of mutant mice derived from random chemical mutagenesis, (3) DNA microarray analyses of genes in inbred strains of mice that differ in social interaction, social communication and resistance to change in habit, and (4) evaluation of proposed therapeutics for the treatment of autism. Published 2004 Wiley-Liss, Inc. MRDD Research Reviews 2004;10:248,258. [source] Arrested differentiation and epithelial cell degeneration in zebrafish lens mutantsDEVELOPMENTAL DYNAMICS, Issue 4 2001Thomas S. Vihtelic Abstract In a chemical mutagenesis screen, we identified two zebrafish mutants that possessed small pupils. Genetic complementation revealed these two lines are due to mutations in different genes. The phenotypes of the two mutants were characterized using histologic, immunohistochemical, and tissue transplantation techniques. The arrested lens (arl) mutant exhibits a small eye and pupil phenotype at 48 hr postfertilization (hpf) and lacks any histologically identifiable lens structures by 5 days postfertilization (dpf). In contrast, the disrupted lens (dsl) mutants are phenotypically normal until 5 dpf, and then undergo lens disorganization and cell degeneration that is apparent by 7 dpf. Histology reveals the arl mutant terminates lens cell differentiation by 48 hpf, whereas the dsl lens exhibits a defective lens epithelial cell population at 5 dpf. Lens transplantation experiments demonstrate both mutations are autonomous to the lens tissue. Immunohistochemistry reveals the retinal cells may suffer subtle effects, possibly due to the lens abnormalities. © 2001 Wiley-Liss, Inc. [source] The zebrafish ennui behavioral mutation disrupts acetylcholine receptor localization and motor axon stabilityDEVELOPMENTAL NEUROBIOLOGY, Issue 1 2008Louis Saint-Amant Abstract The zebrafish ennui mutation was identified from a mutagenesis screen for defects in early behavior. Homozygous ennui embryos swam more slowly than wild-type siblings but normal swimming recovered during larval stages and homozygous mutants survived until adulthood. Electrophysiological recordings from motoneurons and muscles suggested that the motor output of the CNS following mechanosensory stimulation was normal in ennui, but the synaptic currents at the neuromuscular junction were significantly reduced. Analysis of acetylcholine receptors (AChRs) in ennui muscles showed a marked reduction in the size of synaptic clusters and their aberrant localization at the myotome segment borders of fast twitch muscle. Prepatterned, nerve-independent AChR clusters appeared normal in mutant embryos and dispersed upon outgrowth of motor axons onto the muscles. Genetic mosaic analysis showed that ennui is required cell autonomously in muscle fibers for normal synaptic localization of AChRs. Furthermore, exogenous agrin failed to induce AChR aggregation, suggesting that ennui is crucial for agrin function. Finally, motor axons branched more extensively in ennui fast twitch muscles especially in the region of the myotome borders. These results suggest that ennui is important for nerve-dependent AChR clustering and the stability of axon growth. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2008 [source] Novel insulin analogues and its mitogenic potentialDIABETES OBESITY & METABOLISM, Issue 6 2006Ivana Zib Abstract:, Insulin analogues were developed to modify the structure of the human insulin molecule in order to more accurately approximate the endogenous secretion of insulin. With the help of recombinant technology and site-directed mutagenesis, the insulin molecule can be modified to either delay or shorten absorption time, providing better insulin treatment options and facilitating the achievement of glycaemic goals. Changing the structure of the insulin molecule, however, may significantly alter both its metabolic and mitogenic activity. Multiple factors such as residence time on the receptor, dissociation rate, rate of receptor internalization and the degree of phosphorylation of signalling proteins can affect the mitogenic potencies of insulin analogues. Changes in the structure of the insulin have raised concern about the safety of the insulin analogues. For example, questions have emerged about the relationship between the use of insulin lispro and insulin glargine and the progression of diabetic retinopathy. Two studies have shown progression of retinopathy with the use of insulin lispro. However, others have not confirmed these results, and causality could not be proven as progression of retinopathy can occur with rapid improvement in glycaemic control, and methods of assessments among studies were not consistent. Therefore, we examine the metabolic and mitogenic characteristics of the three insulin analogues, insulin lispro, insulin aspart and insulin glargine, that are currently on the market, as well as the two insulin analogues, insulin glulisine and insulin detemir, that are soon going to be available for clinical use. [source] Protein-Based Capacitive Biosensors: a New Tool for Structure-Activity Relationship StudiesELECTROANALYSIS, Issue 24 2008Alessia Mortari Abstract The present work reports a new application of a protein-based capacitive biosensor as an in vitro assay for the selectivity study of the bacterial periplasmic protein MerP and four MerP variants. The modified MerP proteins were produced by site-directed mutagenesis of the heavy metal associated motif (HMA). The MerP and modified MerPs selectivity for copper, zinc, cadmium and mercury bivalent ions were investigated and compared. The variations in the proteins affinity were related to the primary structure of the HMA motifs. Key amino acids for copper coordination of metalloproteins that contain the metal binding sequence Gly-Met-Thr-Cys-xxx-xxx-Cys were identified. The results brought insights valid for Menkes and Wilson ATPases. The protein-based capacitive biosensors were a simple and useful tool for studying structure-activity relationships of proteins. [source] Transgenic , medaka as a new model for germ cell mutagenesisENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 3 2008Richard N. Winn Abstract To address the need for improved approaches to study mutations transmitted to progeny from mutagen-exposed parents, we evaluated , transgenic medaka, a small fish that carries the cII mutation target gene, as a new model for germ cell mutagenesis. Mutations in the cII gene in progeny derived from ethyl-nitrosourea (ENU)-exposed males were readily detected. Frequencies of mutant offspring, proportions of mosaic or whole body mutant offspring, and mutational spectra differed according to germ cell stage exposed to ENU. Postmeiotic germ cells (spermatozoa/late spermatids) generated a higher frequency of mutant offspring (11%) compared to premeiotic germ cells (3.5%). Individuals with cII mutant frequencies (MF) elevated more than threefold above the spontaneous MF (3 × 10,5) in the range of 10,4 to 10,3 were mosaic mutant offspring, whereas those with MFs approaching 1 × 10,2 were whole body mutant offspring. Mosaic mutant offspring comprised the majority of mutant offspring derived from postmeiotic germ cells, and unexpectedly, from spermatogonial stem cells. Mutational spectra comprised of two different mutations, but at identical sites were unusual and characteristic of delayed mutations, in which fixation of a second mutation was delayed following fertilization. Delayed mutations and prevalence of mosaic mutant offspring add to growing evidence that implicates germ cells in mediating processes postfertilization that contribute to genomic instability in progeny. This model provides an efficient and sensitive approach to assess germ cell mutations, expands opportunities to increase understanding of fundamental mechanisms of mutagenesis, and provides a means for improved assessment of potential genetic health risks. Environ. Mol. Mutagen., 2008. © 2008 Wiley-Liss, Inc. [source] Assessing human germ-cell mutagenesis in the Postgenome Era: A celebration of the legacy of William Lawson (Bill) Russell,ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2007Andrew J. Wyrobek Abstract Birth defects, de novo genetic diseases, and chromosomal abnormality syndromes occur in ,5% of all live births, and affected children suffer from a broad range of lifelong health consequences. Despite the social and medical impact of these defects, and the 8 decades of research in animal systems that have identified numerous germ-cell mutagens, no human germ-cell mutagen has been confirmed to date. There is now a growing consensus that the inability to detect human germ-cell mutagens is due to technological limitations in the detection of random mutations rather than biological differences between animal and human susceptibility. A multidisciplinary workshop responding to this challenge convened at The Jackson Laboratory in Bar Harbor, Maine. The purpose of the workshop was to assess the applicability of an emerging repertoire of genomic technologies to studies of human germ-cell mutagenesis. Workshop participants recommended large-scale human germ-cell mutation studies be conducted using samples from donors with high-dose exposures, such as cancer survivors. Within this high-risk cohort, parents and children could be evaluated for heritable changes in (a) DNA sequence and chromosomal structure, (b) repeat sequences and minisatellites, and (c) global gene expression profiles and pathways. Participants also advocated the establishment of a bio-bank of human tissue samples from donors with well-characterized exposure, including medical and reproductive histories. This mutational resource could support large-scale, multiple-endpoint studies. Additional studies could involve the examination of transgenerational effects associated with changes in imprinting and methylation patterns, nucleotide repeats, and mitochondrial DNA mutations. The further development of animal models and the integration of these with human studies are necessary to provide molecular insights into the mechanisms of germ-cell mutations and to identify prevention strategies. Furthermore, scientific specialty groups should be convened to review and prioritize the evidence for germ-cell mutagenicity from common environmental, occupational, medical, and lifestyle exposures. Workshop attendees agreed on the need for a full-scale assault to address key fundamental questions in human germ-cell environmental mutagenesis. These include, but are not limited to, the following: Do human germ-cell mutagens exist? What are the risks to future generations? Are some parents at higher risk than others for acquiring and transmitting germ-cell mutations? Obtaining answers to these, and other critical questions, will require strong support from relevant funding agencies, in addition to the engagement of scientists outside the fields of genomics and germ-cell mutagenesis. Environ. Mol. Mutagen., 2007. Published 2007 Wiley-Liss, Inc. [source] Mutation spectrum in UVB-exposed skin epidermis of Xpa -knockout mice: Frequent recovery of triplet mutationsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2007Hironobu Ikehata Abstract Knockout mutations in both alleles of the Xpa gene give rise to a complete deficiency in nucleotide excision repair (NER) in mammalian cells. We used transgenic mice harboring the ,-phage-based lacZ mutational reporter gene to study the effect of Xpa null mutation (Xpa,/,) on damage induction, repair, and mutagenesis in mouse skin epidermis after UVB irradiation. UVB induced equal amounts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (64PPs) in mouse skin epidermis of Xpa,/, and wild-type mice. Neither photolesion was removed in the Xpa,/, epidermis by 12 hr after irradiation whereas removal of 64PPs was observed in the epidermis of wild-type mice. Irradiation with 200 and 300 J/m2 UVB increased the lacZ mutant frequency in the epidermis of Xpa,/, mice, but the induced mutant frequencies were not significantly different from those previously determined for wild-type mice. One-hundred lacZ mutants isolated from the UVB-exposed epidermis of Xpa,/, mice were analyzed and compared with mutant sequences previously determined for irradiated wild-type mice. The distribution of the mutations along the lacZ transgene and the preferred dipyrimidine context of the UV-specific mutations were similar in mutants from the Xpa,/, and wild-type mice. The spectra of the mutations in the two genotypes were both highly UV-specific and similar in a dominance of C , T transitions at dipyrimidine sites; however, Xpa,/, mice had a higher frequency than wild-type mice of two-base tandem substitutions, including CC , TT mutations, three-base tandem mutations and double base substitutions that were separated by one unchanged base in a three-base sequence (alternating mutations). These tandem/alternating mutations included a remarkably large number of triplet mutations, a recently reported, novel type of UV-specific mutation, characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. We conclude that the triplet mutation is a UV-specific mutation that preferably occurs in NER-deficient genetic backgrounds. Environ. Mol. Mutagen., 2007. © 2006 Wiley-Liss, Inc. [source] A model for targeted substitution mutagenesis during SOS replication of double-stranded DNA containing cis-syn cyclobutane thymine dimersENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 9 2006Helen A. Grebneva Abstract A model for ultraviolet mutagenesis is described that is based on the formation of rare tautomeric bases in pyrimidine dimers. It is shown that during SOS synthesis the modified DNA-polymerase inserts canonical bases opposite the dimers; the inserted bases are capable of forming hydrogen bonds with bases in the template DNA. SOS-replication of double-stranded DNA having thymine dimers, with one or both bases in a rare tautomeric conformation, results in targeted transitions, transversions, or one-nucleotide gaps. Structural analysis indicates that one type of dimer containing a single tautomeric base (TT1*, with the "*" indicating a rare tautomeric base and the subscript referring to the particular conformation) can cause A:T , G:C transition or homologous A:T , T:A transversion, while another dimer (TT2*) can cause a one-nucleotide gap. The dimers containing T4* result in A:T , C:G transversion, while TT5* dimers can cause A:T , C:G transversion or homologous A:T , T:A transversion. If both bases in the dimer are in a rare tautomeric form, then tandem mutations or double-nucleotide gaps can be formed. The dimers containing the rare tautomeric forms T1 *,, T2*,, T3*,, T4*,, and T5*, may not result in mutations. The question of whether dimers containing T4*, and T5*, result in mutations requires further investigation. Environ. Mol. Mutagen., 2006. © 2006 Wiley-Liss, Inc. [source] Frameshift mutations induced by four isomeric nitroacridines and their des-nitro counterpart in the lacZ reversion assay in Escherichia coliENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2006George R. Hoffmann Abstract Acridines are well-known as compounds that intercalate noncovalently between DNA base pairs and induce ±1 frameshift mutations at sites of monotonous repeats of a single base. Reactive derivatives of acridines, including acridine mustards and nitroacridines, form covalent adducts in DNA and exhibit mutagenic properties different from the simple intercalators. We compared the frameshift mutagenicity of the cancer chemotherapy drug nitracrine (1-nitro-9-(3,-dimethylaminopropylamino)-acridine), its des-nitro counterpart 9-(3,-dimethylaminopropylamino)-acridine (DAPA), and its 2-, 3-, and 4-nitro isomers (2-, 3-, and 4-nitro-DAPA) in the lacZ reversion assay in Escherichia coli. DAPA is a simple intercalator, much like the widely studied 9-aminoacridine. It most strongly induced ±1 frameshift mutations in runs of guanine residues and more weakly induced ,1 frameshifts in a run of adenine residues. A nitro group in the 1, 3, or 4 position of DAPA reduced the yield of ±1 frameshift mutations. DAPA weakly induced ,2 frameshifts in an alternating CG sequence. In contrast, nitracrine and its 3-nitro isomer resembled the 3-nitroacridine Entozon in effectively inducing ,2 frameshift mutations. The 2- and 4-nitro isomers were less effective than the 1- and 3-nitro compounds in ,2 frameshift mutagenesis. The results are interpreted with respect to intercalation, steric interactions, effects of base strength on DNA binding, enzymatic processing, and a slipped mispairing model of frameshift mutagenesis. Environ. Mol. Mutagen., 2006. © 2005 Wiley-Liss, Inc. [source] Genotoxicity of acrylamide and its metabolite glycidamide administered in drinking water to male and female Big Blue mice,ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2006Mugimane G. Manjanatha Abstract The recent discovery of acrylamide (AA), a probable human carcinogen, in a variety of fried and baked starchy foods has drawn attention to its genotoxicity and carcinogenicity. Evidence suggests that glycidamide (GA), the epoxide metabolite of AA, is responsible for the genotoxic effects of AA. To investigate the in vivo genotoxicity of AA, groups of male and female Big Blue (BB) mice were administered 0, 100, or 500 mg/l of AA or equimolar doses of GA, in drinking water, for 3,4 weeks. Micronucleated reticulocytes (MN-RETs) were assessed in peripheral blood within 24 hr of the last treatment, and lymphocyte Hprt and liver cII mutagenesis assays were conducted 21 days following the last treatment. Further, the types of cII mutations induced by AA and GA in the liver were determined by sequence analysis. The frequency of MN-RETs was increased 1.7,3.3-fold in males treated with the high doses of AA and GA (P , 0.05; control frequency = 0.28%). Both doses of AA and GA produced increased lymphocyte Hprt mutant frequencies (MFs), with the high doses producing responses 16,25-fold higher than that of the respective control (P , 0.01; control MFs = 1.5 ± 0.3 × 10,6 and 2.2 ± 0.5 × 10,6 in females and males, respectively). Also, the high doses of AA and GA produced significant 2,2.5-fold increases in liver cII MFs (P , 0.05; control MFs = 26.5 ± 3.1 × 10,6 and 28.4 ± 4.5 × 10,6). Molecular analysis of the mutants indicated that AA and GA produced similar mutation spectra and that these spectra were significantly different from that of control mutants (P , 0.001). The predominant types of mutations in the liver cII gene from AA- and GA-treated mice were G:C,T:A transversions and ,1/+1 frameshifts in a homopolymeric run of Gs. The results indicate that both AA and GA are genotoxic in mice. The MFs and types of mutations induced by AA and GA in the liver are consistent with AA exerting its genotoxicity in BB mice via metabolism to GA. Environ. Mol. Mutagen., 2006. Published 2005 Wiley-Liss, Inc. [source] Light-dependent mutagenesis by benzo[a]pyrene is mediated via oxidative DNA damageENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 3 2005Su-Ryang Kim Abstract Benzo[a]pyrene (B[a]P) is an environmental carcinogenic polycyclic aromatic hydrocarbon (PAH). Mammalian enzymes such as cytochrome P-450s and epoxide hydrase convert B[a]P to reactive metabolites that can covalently bind to DNA. However, some carcinogenic compounds that normally require metabolic activation can also be directly photoactivated to mutagens. To examine whether B[a]P is directly mutagenic in the presence of light, we exposed Salmonella typhimurium strains with different DNA repair capacities to B[a]P and white fluorescent light at wavelengths of 370,750 nm. B[a]P plus light significantly enhanced the number of His+ revertants. Mutagenesis was completely light-dependent and required no exogenous metabolic activation. The order of mutability of strains with different DNA repair capacities was strain YG3001 (uvrB, mutMST) , strain TA1535 (uvrB) > strain YG3002 (mutMST) > strain TA1975. The uvrB gene product is involved in the excision repair of bulky DNA adducts, and the mutMST gene encodes 8-oxoguanine (8-oxoG) DNA glycosylase, which removes 8-oxoG from DNA. Introduction of a plasmid carrying the mOgg1 gene that is the mouse counterpart of mutMST substantially reduced the light-mediated mutagenicity of B[a]P in strain YG3001. B[a]P plus light induced predominantly G:C , T:A and G:C , C:G transversions. We propose that B[a]P can directly induce bulky DNA adducts if light is present, and that the DNA adducts induce oxidative DNA damage, such as 8-oxoG, when exposed to light. These findings have implications for the photocarcinogenicity of PAHs. Environ. Mol. Mutagen., 2005. © 2005 Wiley-Liss, Inc. [source] Antimutagenicity of green tea polyphenols in the liver of transgenic medakaENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2005Richard N. Winn Abstract We examined the ability of a mixture of the predominant green tea polyphenolic compounds (GTP) to reduce benzo[a]pyrene (B[a]P)-induced mutations in the cII gene of the , transgenic medaka. Fish were treated with 50 ppb B[a]P for 24 hr, followed by exposure to 2 ppm or 10 ppm GTP for 28 days. cII mutations in livers of fish exposed to B[a]P were increased significantly, 2.6-fold above controls. In contrast, the addition of GTP significantly reduced the frequency of cII mutants by 84%, comparable to that of controls. The frequencies of mutations at G:C basepairs, mutations that are highly characteristic of B[a]P exposure, were elevated significantly in treated fish. By comparison, B[a]P-exposed fish also treated with GTP showed reductions in these mutations, demonstrating a protective effect of GTP against B[a]P-induced mutagenesis. The antioxidant mechanism of GTP possibly played an important role in the reduction of B[a]P mutagenicity. These results corroborate findings from rodent models, showing that the protective effects of green tea extend to different species, and suggesting that similar mechanisms of B[a]P mutagenesis and GTP antimutagenesis are shared among the models. These studies illustrate the utility of , transgenic medaka for in vivo mutation analyses and suggest that this fish may be a valuable model in chemoprevention studies. Environ. Mol. Mutagen., 2005. © 2005 Wiley-Liss, Inc. [source] Endogenous estrogen status, but not genistein supplementation, modulates 7,12-dimethylbenz[a]anthracene-induced mutation in the liver cII gene of transgenic big blue rats,ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 5 2005Tao Chen Abstract A growing number of studies suggest that isoflavones found in soybeans have estrogenic activity and may safely alleviate the symptoms of menopause. One of these isoflavones, genistein, is commonly used by postmenopausal women as an alternative to hormone replacement therapy. Although sex hormones have been implicated as an important risk factor for the development of hepatocellular carcinoma, there are limited data on the potential effects of the estrogens, including phytoestrogens, on chemical mutagenesis in liver. Because of the association between mutation induction and the carcinogenesis process, we investigated whether endogenous estrogen and supplemental genistein affect 7,12-dimethylbenz[a]anthracene (DMBA)-induced mutagenesis in rat liver. Intact and ovariectomized female Big Blue rats were treated with 80 mg DMBA/kg body weight. Some of the rats also received a supplement of 1,000 ppm genistein. Sixteen weeks after the carcinogen treatment, the rats were sacrificed, their livers were removed, and mutant frequencies (MFs) and types of mutations were determined in the liver cII gene. DMBA significantly increased the MFs in liver for both the intact and ovariectomized rats. While there was no significant difference in MF between the ovariectomized and intact control animals, the mutation induction by DMBA in the ovariectomized groups was significantly higher than that in the intact groups. Dietary genistein did not alter these responses. Molecular analysis of the mutants showed that DMBA induced chemical-specific types of mutations in the liver cII gene. These results suggest that endogenous ovarian hormones have an inhibitory effect on liver mutagenesis by DMBA, whereas dietary genistein does not modulate spontaneous or DMBA-induced mutagenesis in either intact or ovariectomized rats. Environ. Mol. Mutagen., 2005. Published 2005 Wiley-Liss, Inc. [source] Effect of deletion of SOS-induced polymerases, pol II, IV, and V, on spontaneous mutagenesis in Escherichia coli mutD5ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2004Anetta Nowosielska Abstract The E. coli dnaQ gene encodes the , subunit of DNA polymerase III (pol III) responsible for the proofreading activity of this polymerase. The mutD5 mutant of dnaQ chronically expresses the SOS response and exhibits a mutator phenotype. In this study we have constructed a set of E. coli AB1157 mutD5 derivatives deleted in genes encoding SOS-induced DNA polymerases, pol II, pol IV, and pol V, and estimated the frequency and specificity of spontaneous argE3,Arg+ reversion in exponentially growing and stationary-phase cells of these strains. We found that pol II exerts a profound effect on the specificity of spontaneous mutation in exponentially growing cells. Analysis of growth-dependent Arg+ revertants in mutD5 polB+ strains revealed that Arg+ revertants were due to tRNA suppressor formation, whereas those in mutD5 ,polB strains arose by back mutation at the argE3 ochre site. In stationary-phase bacteria, Arg+revertants arose mainly by back mutation, regardless of whether they were proficient or deficient in pol II. Our results also indicate that in a mutD5 background, the absence of pol II led to increased frequency of Arg+ growth-dependent revertants, whereas the lack of pol V caused its dramatic decrease, especially in mutD5 ,umuDC and mutD5 ,umuDC ,polB strains. In contrast, the rate of stationary-phase Arg+revertants increased in the absence of pol IV in the mutD5 ,dinB strain. We postulate that the proofreading activity of pol II excises DNA lesions in exponentially growing cells, whereas pol V and pol IV are more active in stationary-phase cultures. Environ. Mol. Mutagen. 43:226,234, 2004. © 2004 Wiley-Liss, Inc. [source] Arsenite induces delayed mutagenesis and transformation in human osteosarcoma cells at extremely low concentrationsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 5 2003Kanae Mure Abstract Arsenite is a human multisite carcinogen, but its mechanism of action is not known. We recently found that extremely low concentrations (,0.1 ,M) of arsenite transform human osteosarcoma TE85 (HOS) cells to anchorage-independence. In contrast to other carcinogens which transform these cells within days of exposure, almost 8 weeks of arsenite exposure are required for transformation. We decided to reexamine the question of arsenite mutagenicity using chronic exposure in a spontaneous mutagenesis assay we previously developed. Arsenite was able to cause a delayed increase in mutagenesis at extremely low concentrations (,0.1 ,M) in a dose-dependent manner. The increase in mutant frequency occurred after almost 20 generations of growth in arsenite. Transformation required more than 30 generations of continuous exposure. We also found that arsenite induced gene amplification of the dihydrofolate reductase (DHFR) gene in a dose-dependent manner. Since HOS cells are able to methylate arsenite at a very low rate, it was possible that active metabolites such as monomethylarsonous acid (MMAIII) contributed to the delayed mutagenesis and transformation in these cells. However, when the assay was repeated with MMAIII, we found no significant increase in mutagenesis or transformation, suggesting that arsenite-induced delayed mutagenesis and transformation are not caused by arsenite's metabolites, but by arsenite itself. Our results suggest that long-term exposure to low concentrations of arsenite may affect signaling pathways that result in a progressive genomic instability. Environ. Mol. Mutagen. 41:322,331, 2003. © 2003 Wiley-Liss, Inc. [source] 2-Amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP),induced mutagenesis in cultured Big BlueÔ rat mammary epithelial and fibroblast cellsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2-3 2002Heather M. McDiarmid Abstract Epithelial cells are the primary site of carcinogenesis in most tissues, including the mammary gland. As an alternative to the study of mutation induction in whole tissues in vivo, we have established Big BlueÔ transgenic rat cell lines from the mammary epithelium (BBR/ME) and the mammary stroma (BBR/MFib), to permit a comparison of their mutagenic responses to carcinogens. We previously demonstrated their responsiveness to the alkylating agent N -ethyl- N -nitrosourea (ENU) (McDiarmid H et al. [2001]: Mutat Res 497:39,47). Here, we examined the responses of cultured epithelial and stromal cells to the protein pyrolysis product and mammary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). Rat hepatic S9 was used as a source of bioactivation enzymes. Mutant induction (cII locus) and clonogenic survival were measured as a function of PhIP concentration. PhIP mutagenicity was observed in the fibroblast cells, but the greater toxicity of PhIP to the epithelial cells prevented a definitive evaluation of mutagenicity. Since PhIP may be detoxified by conjugation with glutathione, we measured glutathione levels and glutathione- S -transferase expression and activities in both cell lines. The epithelial cells had higher glutathione- S -transferase enzyme activity and protein expression than did the fibroblast cell line. Because the epithelial cells were more sensitive to toxicity, glutathione conjugation evidently plays only a minor role in PhIP toxicity and mutagenicity in our cell lines. Environ. Mol. Mutagen. 39:245,253, 2002. © 2002 Wiley-Liss, Inc. [source] DNA repair and mutagenesis in Werner syndrome ,ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2-3 2001Vilhelm A. Bohr Abstract Werner syndrome (WS) is the hallmark premature aging syndrome in which the patients appear much older than their actual chronological age. The disorder is associated with significantly increased genome instability and with transcriptional deficiencies. There has been some uncertainty about whether WS cells are defective in DNA repair. We thus examined repair in vitro in nuclear and mitochondrial DNA. Whereas cellular studies so far do not show significant DNA repair deficiencies, biochemical studies with the Werner protein clearly indicate that it plays a role in DNA repair. Environ. Mol. Mutagen. 38:227,234, 2001. Published 2001 Wiley-Liss, Inc. [source] Transplacental mutagenicity of N -ethyl- N -nitrosourea at the hprt locus in T-lymphocytes of exposed B6C3F1 miceENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2001Hillary E. Sussman Abstract Previous studies have compared age-related differences in total mutagenic burden in mice of differing age (preweanling, weanling, or young adult) after single intraperitoneal (i.p.) injections of ethylnitrosourea (ENU). The purpose of the present investigation was to determine the effects of time elapsed since treatment on the frequency of hprt mutant T-cells (Mf) from mice treated transplacentally with single acute vs. multiple split doses of ENU. To this end, pregnant C57BL/6 mice (n = 13,16/group), which had been bred to C3H males, were given i.p. injections of 40 mg ENU/kg bw in a single dose on day 18 of gestation, in a split dose of 6 mg ENU/kg bw on days 12 through 18 of gestation, or DMSO vehicle alone. Groups of pups were necropsied on days 10, 13, 15 (single dose only), 17, 20, 40, and 70 postpartum for T-cell isolations and hprt Mf measurements using the T-cell cloning assay. The time required to reach maximum Mfs in T-cells isolated from thymus of transplacentally treated animals was 2 weeks, the same time span as previously observed after ENU treatment of adult, weanling, and preweanling mice. Mfs in T-cells isolated from spleens of control animals averaged 2.1 ± 0.3 (SE) × 10,6. In spleens of mice treated transplacentally with ENU in a single dose, Mfs reached a maximum at 15 days postpartum [84.7 ± 15.8 (SE) × 10,6] and decreased to lower but still elevated levels at 40 days postpartum. In spleens of mice treated transplacentally with ENU in a split dose, Mfs reached a maximum at 13 days postpartum [74.0 ± 16.3 (SE) × 10,6] and decreased to background levels at 40 days postpartum. The areas under the curves describing the change in hprt Mfs over time for ENU-treated vs. control mice estimate the mutagenic potency for transplacental single- and split-dose exposures to be 1.9 and 0.8 × 103, respectively. Comparison of the mutagenic potency estimates for mice exposed to ENU in utero to 4-week-old mice given a similar dose of the same lot number of ENU indicates that the mouse is more susceptible to ENU-induced mutagenesis during fetal life. Environ. Mol. Mutagen. 38:30,37, 2001 © 2001 Wiley-Liss, Inc. [source] The exopolysaccharide of Rhizobium sp.ENVIRONMENTAL MICROBIOLOGY, Issue 8 2008Brassica napus roots but contributes to root colonization, YAS34 is not necessary for biofilm formation on Arabidopsis thaliana Summary Microbial exopolysaccharides (EPSs) play key roles in plant,microbe interactions, such as biofilm formation on plant roots and legume nodulation by rhizobia. Here, we focused on the function of an EPS produced by Rhizobium sp. YAS34 in the colonization and biofilm formation on non-legume plant roots (Arabidopsis thaliana and Brassica napus). Using random transposon mutagenesis, we isolated an EPS-deficient mutant of strain YAS34 impaired in a glycosyltransferase gene (gta). Wild type and mutant strains were tagged with a plasmid-born GFP and, for the first time, the EPS produced by the wild-type strain was seen in the rhizosphere using selective carbohydrate probing with a fluorescent lectin and confocal laser-scanning microscopy. We show for the fist time that Rhizobium forms biofilms on roots of non-legumes, independently of the EPS synthesis. When produced by strain YAS34 wild type, EPS is targeted at specific parts of the plant root system. Nutrient fluctuations, root exudates and bacterial growth phase can account for such a production pattern. The EPS synthesis in Rhizobium sp. YAS34 is not essential for biofilm formation on roots, but is critical to colonization of the basal part of the root system and increasing the stability of root-adhering soil. Thus, in Rhizobium sp. YAS34 and non-legume interactions, microbial EPS is implicated in root,soil interface, root colonization, but not in biofilm formation. [source] The characterization of functions involved in the establishment and maturation of Klebsiella pneumoniae in vitro biofilm reveals dual roles for surface exopolysaccharidesENVIRONMENTAL MICROBIOLOGY, Issue 3 2008Damien Balestrino Summary The ability to form biofilm is seen as an increasingly important colonization strategy among both pathogenic and environmental Klebsiella pneumoniae strains. The aim of the present study was to identify abiotic surface colonization factors of K. pneumoniae using different models at different phases of biofilm development. A 2200 K. pneumoniae mutant library previously obtained by signature-tagged mutagenesis was screened in static and dynamic culture models to detect clones impaired at early and/or mature stages of biofilm formation. A total of 28 mutants were affected during late phases of biofilm formation, whereas 16 mutants displayed early adhesion defect. These mutants corresponded to genes involved in potential cellular and DNA metabolism pathways and to membrane transport functions. Eight mutants were deficient in capsule or LPS production. Gene disruption and microscopic analyses showed that LPS is involved in initial adhesion on both glass and polyvinyl-chloride and the capsule required for the appropriate initial coverage of substratum and the construction of mature biofilm architecture. These results give new insight into the bacterial factors sequentially associated with the ability to colonize an abiotic surface and reveal the dual roles played by surface exopolysaccharides during K. pneumoniae biofilm formation. [source] Stationary phase mutagenesis: mechanisms that accelerate adaptation of microbial populations under environmental stressENVIRONMENTAL MICROBIOLOGY, Issue 10 2003Maia Kivisaar Summary Microorganisms are exposed to constantly changing environmental conditions. In a growth-restricting environment (e.g. during starvation), mutants arise that are able to take over the population by a process known as stationary phase mutation. Genetic adaptation of a microbial population under environmental stress involves mechanisms that lead to an elevated mutation rate. Under stressful conditions, DNA synthesis may become more erroneous because of the induction of error-prone DNA polymerases, resulting in a situation in which DNA repair systems are unable to cope with increasing amounts of DNA lesions. Transposition may also increase genetic variation. One may ask whether the rate of mutation under stressful conditions is elevated as a result of malfunctioning of systems responsible for accuracy or are there specific mechanisms that regulate the rate of mutations under stress. Evidence for the presence of mutagenic pathways that have probably been evolved to control the mutation rate in a cell will be discussed. [source] Cadmium-regulated gene fusions in Pseudomonas fluorescensENVIRONMENTAL MICROBIOLOGY, Issue 4 2000Silvia Rossbach To study the mechanisms soil bacteria use to cope with elevated concentrations of heavy metals in the environment, a mutagenesis with the lacZ -based reporter gene transposon Tn5 -B20 was performed. Random gene fusions in the genome of the common soil bacterium Pseudomonas fluorescens strain ATCC 13525 were used to create a bank of 5000 P. fluorescens mutants. This mutant bank was screened for differential gene expression in the presence of the toxic metal cadmium. Fourteen mutants were identified that responded with increased or reduced gene expression to the presence of cadmium. The mutants were characterized with respect to their metal-dependent gene expression and their metal tolerance. Half the identified mutants reacted with differential gene expression specifically to the metal cadmium, whereas some of the other mutants also responded to elevated concentrations of copper and zinc ions. One of the mutants, strain C8, also showed increased gene expression in the presence of the solvent ethanol, but otherwise no overlap between cadmium-induced gene expression and general stress response was detected. Molecular analysis of the corresponding genetic loci was performed using arbitrary polymerase chain reaction (PCR), DNA sequencing and comparison of the deduced protein products with sequences deposited in genetic databases. Some of the genetic loci targeted by the transposon did not show any similarities to any known genes; thus, they may represent ,novel' loci. The hypothesis that genes that are differentially expressed in the presence of heavy metals play a role in metal tolerance was verified for one of the mutants. This mutant, strain C11, was hypersensitive to cadmium and zinc ions. In mutant C11, the transposon had inserted into a genetic region displaying similarity to genes encoding the sensor/regulator protein pairs of two-component systems that regulate gene expression in metal-resistant bacteria, including czcRS of Ralstonia eutropha, czrRS of Pseudomonas aeruginosa and copRS of Pseudomonas syringae. Although the P. fluorescens strain used in this study had not been isolated from a metal-rich environment, it nevertheless contained at least one genetic region enabling it to cope with elevated concentrations of heavy metals. [source] Site-directed mutagenesis of the chemokine receptor CXCR6 suggests a novel paradigm for interactions with the ligand CXCL16EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2008Sarah Abstract Chemokine receptor CXCR6 mediates the chemotaxis and adhesion of leukocytes to soluble and membrane-anchored forms of CXCL16, and is an HIV-1 co-receptor. Here, we describe the effects of mutation of acidic extracellular CXCR6 residues on receptor function. Although most CXCR6 mutants examined were expressed at levels similar to wild-type (WT) CXCR6, an N-terminal E3Q mutant was poorly expressed, which may explain previously reported protective effects of a similar single nucleotide polymorphism, with respect to late-stage HIV-1 infection. In contrast to several other chemokine receptors, mutation of the CXCR6 N,terminus and inhibition of post-translational modifications of this region were without effect on receptor function. Likewise, N-terminal extension of CXCL16 resulted in a protein with decent potency and efficacy in chemotaxis and not, as anticipated, a CXCR6 antagonist. D176N and E274Q CXCR6 mutants were unable to interact with soluble CXCL16, suggesting a critical role for D176 and E274 in ligand binding. Intriguingly, although unable to interact with soluble CXCL16, the E274Q mutant could promote robust adhesion to membrane-anchored CXCL16, suggesting that soluble and membrane-bound forms of CXCL16 possess distinct conformations. Collectively, our data suggest a novel paradigm for the CXCR6:CXCL16 interaction, a finding which may impact the discovery of small-molecule antagonists of CXCR6. [source] Pathologic expression of MHC class,II is driven by mitogen-activated protein kinasesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2007Isabelle Martins Abstract The class,II transactivator (CIITA) is the master regulator of MHC class,II molecules (MHC,II). In melanoma, the MHC,II are constitutively expressed due to an abnormal transcription of CIITA from its promoter,III (pIII), and requires the presence of a 1-kb enhancer located upstream from this latter. Since mitogen-activated protein kinases (MAPK) have been shown to be activated in most melanomas, we sought to analyze their possible involvement in CIITA expression. Using chemical inhibitors and dominant-negative constructs of MAPK-ERK kinase (Mek1) and MAPK-JNK, we evidenced the inhibition of MHC,II and CIITA expression in melanoma cell lines displaying activated MAPK. Transcriptional regulation by MAPK is known to involve the AP-1 transcription factor family. Sequence analysis revealed an AP-1-responsive motif in the enhancer of CIITA pIII at ,5954/,5947 from the site of transcription initiation. Its mutagenesis reduced CIITA expression four- to fivefold in melanoma cell lines and alleviated the effect of dominant-negative constructs of the MAPK pathway. Together, our findings demonstrate that MAPK-ERK and MAPK-JNK are regulators of CIITA transcription in melanoma, and pinpoint an AP-1-responsive site in the CIITA gene pIII. This should have considerable impact on our understanding of the physio-pathologic expression of MHC,II. [source] The CD200 and CD200 receptor cell surface proteins interact through their N-terminal immunoglobulin-like domainsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2004Deborah Hatherley Abstract CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a receptor on myeloid cells (CD200R) involved in regulation of macrophage function. Both CD200 and CD200R contain two Ig superfamily domains like many other leukocyte membrane proteins. Site-directed mutagenesis of CD200R showed that, like CD200, it interacted through its N-terminal domain. This indicated that the cell-cell interaction spans four Ig superfamily domains and this distance is similar to many interactions found between T,cells and antigen-presenting cells. This suggests that this topology is also important in interactions of CD200 on a variety of cells with CD200R on myeloid cells, and comparable contact sites may be important mediating regulation in other cell-cell interactions. The mutagenesis showed that the binding involved the predicted GFCC, face of its N-terminal domain, like that of CD200, suggesting that the interaction evolved from a homotypic interaction. [source] Proteolytic cleavage of the voltage-gated Ca2+ channel ,2, subunit: structural and functional featuresEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2007Arturo Andrade Abstract By mediating depolarization-induced Ca2+ influx, high-voltage-activated Ca2+ channels control a variety of cellular events. These heteromultimeric proteins are composed of an ion-conducting (,1) and three auxiliary (,2,, , and ,) subunits. The ,2, subunit enhances the trafficking of the channel complex to the cell surface and increases channel open probability. To exert these effects, ,2, must undergo important post-translational modifications, including a proteolytic cleavage that separates the extracellular ,2 from its transmembrane , domain. After this proteolysis both domains remain linked by disulfide bonds. In spite of its central role in determining the final conformation of the fully mature ,2,, almost nothing is known about the physiological implications of this structural modification. In the current report, by using site-directed mutagenesis, the proteolytic site of ,2, was mapped to amino acid residues Arg-941 and Val-946. Substitution of these residues renders the protein insensitive to proteolytic cleavage as evidenced by the lack of molecular weight shift upon treatment with a disulfide-reducing agent. Interestingly, these mutations significantly decreased whole-cell patch-clamp currents without affecting the voltage dependence or kinetics of the channels, suggesting a reduction in the number of channels targeted to the plasma membrane. [source] | |||||||||||||||||||||||||||||||||||||||||||