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Sample records for n-ethyl-n-nitrosourea enu mutagenesis

  1. Chromoanasynthetic Genomic Rearrangement Identified in a N-Ethyl-N-Nitrosourea (ENU Mutagenesis Screen in Caenorhabditis elegans

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    Omar A. Itani

    2016-02-01

    Full Text Available Chromoanasynthesis is a recently discovered phenomenon in humans with congenital diseases that is characterized by complex genomic rearrangements (CGRs resulting from aberrant repair of catastrophic chromosomal damage. How these CGRs are induced is not known. Here, we describe the structure and function of dpDp667, a causative CGR that emerged from a Caenorhabditis elegans dauer suppressor screen in which animals were treated with the point mutagen N-ethyl-N-nitrosourea (ENU. dpDp667 comprises nearly 3 Mb of sequence on the right arm of the X chromosome, contains three duplications and one triplication, and is devoid of deletions. Sequences from three out of the four breakpoint junctions in dpDp667 reveal microhomologies that are hallmarks of chromoanasynthetic CGRs. Our findings suggest that environmental insults and physiological processes that cause point mutations may give rise to chromoanasynthetic rearrangements associated with congenital disease. The relatively subtle phenotype of animals harboring dpDp667 suggests that the prevalence of CGRs in the genomes of mutant and/or phenotypically unremarkable animals may be grossly underestimated.

  2. A Mutant Mouse with a Highly Specific Contextual Fear-Conditioning Deficit Found in an N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Screen

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    Pletcher, Mathew T.; Wiltshire, Tim; Tarantino, Lisa M.; Mayford, Mark; Reijmers, Leon G.; Coats, Jennifer K.

    2006-01-01

    Targeted mutagenesis in mice has shown that genes from a wide variety of gene families are involved in memory formation. The efficient identification of genes involved in learning and memory could be achieved by random mutagenesis combined with high-throughput phenotyping. Here, we provide the first report of a mutagenesis screen that has…

  3. Spontaneous inflammatory pain model from a mouse line with N-ethyl-N-nitrosourea mutagenesis

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    Chen Tsung-Chieh

    2012-05-01

    Full Text Available Abstract Background N-ethyl-N-nitrosourea mutagenesis was used to induce a point mutation in C57BL/6 J mice. Pain-related phenotype screening was performed in 915 G3 mice. We report the detection of a heritable recessive mutant in meiotic recombinant N1F1 mice that caused an abnormal pain sensitivity phenotype with spontaneous skin inflammation in the paws and ears. Methods We investigated abnormal sensory processing, neuronal peptides, and behavioral responses after the induction of autoinflammatory disease. Single-nucleotide polymorphism (SNP markers and polymerase chain reaction product sequencing were used to identify the mutation site. Results All affected mice developed paw inflammation at 4–8 weeks. Histological examinations revealed hyperplasia of the epidermis in the inflamed paws and increased macrophage expression in the spleen and paw tissues. Mechanical and thermal nociceptive response thresholds were reduced in the affected mice. Locomotor activity was decreased in affected mice with inflamed hindpaws, and this reduction was attributable to the avoidance of contact of the affected paw with the floor. Motor strength and daily activity in the home cage in the affected mice did not show any significant changes. Although Fos immunoreactivity was normal in the dorsal horn of affected mice, calcitonin gene-related peptide immunoreactivity significantly increased in the deep layer of the dorsal horn. The number of microglia increased in the spinal cord, hippocampus, and cerebral cortex in affected mice, and the proliferation of microglia was maintained for a couple of months. Two hundred eighty-five SNP markers were used to reveal the affected gene locus, which was found on the distal part of chromosome 18. A point mutation was detected at A to G in exon 8 of the pstpip2 gene, resulting in a conserved tyrosine residue at amino acid 180 replaced by cysteine (Y180 C. Conclusions The data provide definitive evidence that a mutation

  4. Highly Efficient ENU Mutagenesis in Zebrafish.

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    de Bruijn, E.; Cuppen, E.; Feitsma, H.

    2009-01-01

    ENU (N-ethyl-N-nitrosourea) mutagenesis is a widely accepted and proven method to introduce random point mutations in the genome. Because there are no targeted knockout strategies available for zebrafish so far, random mutagenesis is currently the preferred method in both forward and reverse genetic

  5. Identification of a rat model for usher syndrome type 1B by N-ethyl-N-nitrosourea mutagenesis-driven forward genetics

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    Smits, B.M.; Peters, T.A.; Mul, J.D.; Croes, H.J.; Fransen, J.A.; Beynon, A.J.; Guryev, V.; Plasterk, R.; Cuppen, E.

    2005-01-01

    The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis

  6. ENU mutagenesis to generate genetically modified rat models.

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    van Boxtel, Ruben; Gould, Michael N; Cuppen, Edwin; Smits, Bart M G

    2010-01-01

    The rat is one of the most preferred model organisms in biomedical research and has been extremely useful for linking physiology and pathology to the genome. However, approaches to genetically modify specific genes in the rat germ line remain relatively scarce. To date, the most efficient approach for generating genetically modified rats has been the target-selected N-ethyl-N-nitrosourea (ENU) mutagenesis-based technology. Here, we describe the detailed protocols for ENU mutagenesis and mutant retrieval in the rat model organism.

  7. Longitudinal evaluation of an N-ethyl-N-nitrosourea-created murine model with normal pressure hydrocephalus.

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    Ming-Jen Lee

    Full Text Available BACKGROUND: Normal-pressure hydrocephalus (NPH is a neurodegenerative disorder that usually occurs late in adult life. Clinically, the cardinal features include gait disturbances, urinary incontinence, and cognitive decline. METHODOLOGY/PRINCIPAL FINDINGS: Herein we report the characterization of a novel mouse model of NPH (designated p23-ST1, created by N-ethyl-N-nitrosourea (ENU-induced mutagenesis. The ventricular size in the brain was measured by 3-dimensional micro-magnetic resonance imaging (3D-MRI and was found to be enlarged. Intracranial pressure was measured and was found to fall within a normal range. A histological assessment and tracer flow study revealed that the cerebral spinal fluid (CSF pathway of p23-ST1 mice was normal without obstruction. Motor functions were assessed using a rotarod apparatus and a CatWalk gait automatic analyzer. Mutant mice showed poor rotarod performance and gait disturbances. Cognitive function was evaluated using auditory fear-conditioned responses with the mutant displaying both short- and long-term memory deficits. With an increase in urination frequency and volume, the mutant showed features of incontinence. Nissl substance staining and cell-type-specific markers were used to examine the brain pathology. These studies revealed concurrent glial activation and neuronal loss in the periventricular regions of mutant animals. In particular, chronically activated microglia were found in septal areas at a relatively young age, implying that microglial activation might contribute to the pathogenesis of NPH. These defects were transmitted in an autosomal dominant mode with reduced penetrance. Using a whole-genome scan employing 287 single-nucleotide polymorphic (SNP markers and further refinement using six additional SNP markers and four microsatellite markers, the causative mutation was mapped to a 5.3-cM region on chromosome 4. CONCLUSIONS/SIGNIFICANCE: Our results collectively demonstrate that the p23-ST1

  8. Spectroscopic investigation (FT-IR, FT-Raman), HOMO-LUMO, NBO, and molecular docking analysis of N-ethyl-N-nitrosourea, a potential anticancer agent

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    Singh, Priyanka; Islam, S. S.; Ahmad, Hilal; Prabaharan, A.

    2018-02-01

    Nitrosourea plays an important role in the treatment of cancer. N-ethyl-N-nitrosourea, also known as ENU, (chemical formula C3H7N3O2), is a highly potent mutagen. The chemical is an alkylating agent and acts by transferring the ethyl group of ENU to nucleobases (usually thymine) in nucleic acids. The molecular structure of N-ethyl-N-nitrosourea has been elucidated using experimental (FT-IR and FT-Raman) and theoretical (DFT) techniques. APT charges, Mulliken atomic charges, Natural bond orbital, Electrostatic potential, HOMO-LUMO and AIM analysis were performed to identify the reactive sites and charge transfer interactions. Furthermore, to evaluate the anticancer activity of ENU molecular docking studies were carried out against 2JIU protein.

  9. Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line.

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    Feitsma, Harma; de Bruijn, Ewart; van de Belt, Jose; Nijman, Isaac J; Cuppen, Edwin

    2008-07-01

    S(N)1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches. We applied a standard ENU mutagenesis protocol to adult zebrafish deficient in the MMR gene msh6 and heterozygous controls to study the effect of MMR on ENU-induced DNA damage. Dose-dependent lethality was found to be similar for homozygous and heterozygous mutants, indicating that there is no difference in ENU resistance. Mutation discovery by high-throughput dideoxy resequencing of genomic targets in outcrossed progeny of the mutagenized fish did also not reveal any differences in germ line mutation frequency. These results may indicate that the maximum mutation load for zebrafish has been reached with the currently used, highly optimized ENU mutagenesis protocol. Alternatively, the MMR system in the zebrafish germ line may be saturated very rapidly, thereby having a limited effect on high-dose ENU mutagenesis.

  10. The effect of gamma radiation and N-ethyl-N-nitrosourea on cultured maize callus growth and plant regeneration

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    Moustafa, R.A.K.; Duncan, D.R.; Widholm, J.M.

    1989-01-01

    Regenerable maize calli of two inbred lines were exposed to 0 to 100 Gy of gamma rays or treated with 0 to 30 mM of N-ethyl-N-nitrosourea (ENU) to determine their effect on growth and plant regeneration capability. Both growth and plant regeneration capacity decreased with increasing levels of either gamma radiation or ENU; however, plant regeneration capacity was more sensitive to either agent than growth. The 50% inhibition dose (I 50 ) for callus growth (fresh-weight gain) was approximately 100 Gy of gamma radiation and 30 mM ENU. The I 50 for plant regeneration capacity of treated callus was approximately 25 Gy of gamma radiation and 2.5 mM ENU. The decrease in plant regeneration capacity correlated with a change in tissue composition of the treated callus from a hard, yellow and opaque tissue to a soft, grayish-yellow and translucent tissue. This change was quantified by measuring the reduction of MnO 4 - to MnO 2 (PR assay) by the callus. These results suggest that the effect of gamma radiation or ENU on plant regeneration capacity must be taken into consideration if these potentially mutagenic agents are to be used on maize callus cultures, for the purpose of producing useful mutations at a whole plant level. The data also suggest that the PR assay may be useful for predicting the actual plant regeneration capacity of maize callus. (author)

  11. ENU Mutagenesis in Mice Identifies Candidate Genes For Hypogonadism

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    Weiss, Jeffrey; Hurley, Lisa A.; Harris, Rebecca M.; Finlayson, Courtney; Tong, Minghan; Fisher, Lisa A.; Moran, Jennifer L.; Beier, David R.; Mason, Christopher; Jameson, J. Larry

    2012-01-01

    Genome-wide mutagenesis was performed in mice to identify candidate genes for male infertility, for which the predominant causes remain idiopathic. Mice were mutagenized using N-ethyl-N-nitrosourea (ENU), bred, and screened for phenotypes associated with the male urogenital system. Fifteen heritable lines were isolated and chromosomal loci were assigned using low density genome-wide SNP arrays. Ten of the fifteen lines were pursued further using higher resolution SNP analysis to narrow the candidate gene regions. Exon sequencing of candidate genes identified mutations in mice with cystic kidneys (Bicc1), cryptorchidism (Rxfp2), restricted germ cell deficiency (Plk4), and severe germ cell deficiency (Prdm9). In two other lines with severe hypogonadism candidate sequencing failed to identify mutations, suggesting defects in genes with previously undocumented roles in gonadal function. These genomic intervals were sequenced in their entirety and a candidate mutation was identified in SnrpE in one of the two lines. The line harboring the SnrpE variant retains substantial spermatogenesis despite small testis size, an unusual phenotype. In addition to the reproductive defects, heritable phenotypes were observed in mice with ataxia (Myo5a), tremors (Pmp22), growth retardation (unknown gene), and hydrocephalus (unknown gene). These results demonstrate that the ENU screen is an effective tool for identifying potential causes of male infertility. PMID:22258617

  12. Sequencing analysis of mutations induced by N-ethyl-N-nitrosourea at different sampling times in mouse bone marrow.

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    Wang, Jianyong; Chen, Tao

    2010-03-01

    In our previous study (Wang et al., 2004, Toxicol. Sci. 82: 124-128), we observed that the cII gene mutant frequency (MF) in the bone marrow of Big Blue mice showed significant increase as early as day 1, reached the maximum at day 3 and then decreased to a plateau by day 15 after a single dose of carcinogen N-ethyl-N-nitrosourea (ENU) treatment, which is different from the longer mutation manifestation time and the constancy of MFs after reaching their maximum in some other tissues. To determine the mechanism underlying the quick increase in MF and the peak formation in the mutant manifestation, we examined the mutation frequencies and spectra of the ENU-induced mutants collected from different sampling times in this study. The cII mutants from days 1, 3 and 120 after ENU treatment were randomly selected from different animals. The mutation frequencies were 33, 217, 305 and 144 x 10(-6) for control, days 1, 3, and 120, respectively. The mutation spectra at days 1 and 3 were significantly different from that at day 120. Considering that stem cells are responsible for the ultimate MF plateau (day 120) and transit cells are accountable for the earlier MF induction (days 1 or 3) in mouse bone marrow, we conclude that transit cells are much more sensitive to mutation induction than stem cells in mouse bone marrow, which resulted in the specific mutation manifestation induced by ENU.

  13. Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line.

    NARCIS (Netherlands)

    Feitsma, H.; de Bruijn, E.; van de Belt, J.; Nijman, I.J.; Cuppen, E.

    2008-01-01

    S(N)1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are

  14. Altered IFN-γ-mediated immunity and transcriptional expression patterns in N-Ethyl-N-nitrosourea-induced STAT4 mutants confer susceptibility to acute typhoid-like disease.

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    Eva, Megan M; Yuki, Kyoko E; Dauphinee, Shauna M; Schwartzentruber, Jeremy A; Pyzik, Michal; Paquet, Marilène; Lathrop, Mark; Majewski, Jacek; Vidal, Silvia M; Malo, Danielle

    2014-01-01

    Salmonella enterica is a ubiquitous Gram-negative intracellular bacterium that continues to pose a global challenge to human health. The etiology of Salmonella pathogenesis is complex and controlled by pathogen, environmental, and host genetic factors. In fact, patients immunodeficient in genes in the IL-12, IL-23/IFN-γ pathway are predisposed to invasive nontyphoidal Salmonella infection. Using a forward genomics approach by N-ethyl-N-nitrosourea (ENU) germline mutagenesis in mice, we identified the Ity14 (Immunity to Typhimurium locus 14) pedigree exhibiting increased susceptibility following in vivo Salmonella challenge. A DNA-binding domain mutation (p.G418_E445) in Stat4 (Signal Transducer and Activator of Transcription Factor 4) was the causative mutation. STAT4 signals downstream of IL-12 to mediate transcriptional regulation of inflammatory immune responses. In mutant Ity14 mice, the increased splenic and hepatic bacterial load resulted from an intrinsic defect in innate cell function, IFN-γ-mediated immunity, and disorganized granuloma formation. We further show that NK and NKT cells play an important role in mediating control of Salmonella in Stat4(Ity14/Ity14) mice. Stat4(Ity14/Ity14) mice had increased expression of genes involved in cell-cell interactions and communication, as well as increased CD11b expression on a subset of splenic myeloid dendritic cells, resulting in compromised recruitment of inflammatory cells to the spleen during Salmonella infection. Stat4(Ity14/Ity14) presented upregulated compensatory mechanisms, although inefficient and ultimately Stat4(Ity14/Ity14) mice develop fatal bacteremia. The following study further elucidates the pathophysiological impact of STAT4 during Salmonella infection.

  15. Combined effects of ionizing radiation and N-ethyl-N-nitrosourea in murine T-cell lymphoma-genesis

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    Shizuko, Kakinuma; Yoshiko, Amasaki; Kazumi, Yamauchi; Mayumi, Nishimura; Tatsuhiko, Imaoka; Yoshiya, Shimada

    2006-01-01

    We are living in the environment with numerous natural and man-made radiation and chemicals. Cancer development in human is considered as a result of interaction with these factors. But, the quantitative assessment and mechanistic understanding of combined effects of radiation and chemical carcinogens are still insufficient. The aim of this study is to elucidate the mode and mechanism of the combined effect of X-rays with N-ethyl-N-nitrosourea (ENU) on mouse T-cell leukemogenesis, especially at low or threshold dose range. We previously showed that the dose-response curve for X-ray- or ENU induced thymic lymphoma (TL) had threshold dose. X-ray-induced TL was characteristic for frequent inactivation of Ikaros caused by transcriptional silencing, unusual splicing, point mutation and small insertion, most of which were associated with loss of heterozygosity (LOH). In contrast, ENU-induced tumors had point mutations of Ikaros without LOH. In combination, X-irradiation followed by ENU exposure resulted in the synergistic effect at high dose, while the effect was antagonistic at low or threshold dose. When the order of exposure was reversed, i.e., ENU followed by X-rays, the mode of combined exposure was additive at any doses. Molecular analysis demonstrated that Ikaros mutation in TL after X-irradiation followed by ENLI at high doses was predominantly point mutation without LOH (ENU-type). But, after reverse treatment, mutation spectrum of Ikaros was similar to that observed in TL after X-ray exposure alone. It is concluded that the mode of combined effect is dependent upon the treatment order and the dose of carcinogens. (author)

  16. Mouse ENU Mutagenesis to Understand Immunity to Infection: Methods, Selected Examples, and Perspectives

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    Grégory Caignard

    2014-09-01

    Full Text Available Infectious diseases are responsible for over 25% of deaths globally, but many more individuals are exposed to deadly pathogens. The outcome of infection results from a set of diverse factors including pathogen virulence factors, the environment, and the genetic make-up of the host. The completion of the human reference genome sequence in 2004 along with technological advances have tremendously accelerated and renovated the tools to study the genetic etiology of infectious diseases in humans and its best characterized mammalian model, the mouse. Advancements in mouse genomic resources have accelerated genome-wide functional approaches, such as gene-driven and phenotype-driven mutagenesis, bringing to the fore the use of mouse models that reproduce accurately many aspects of the pathogenesis of human infectious diseases. Treatment with the mutagen N-ethyl-N-nitrosourea (ENU has become the most popular phenotype-driven approach. Our team and others have employed mouse ENU mutagenesis to identify host genes that directly impact susceptibility to pathogens of global significance. In this review, we first describe the strategies and tools used in mouse genetics to understand immunity to infection with special emphasis on chemical mutagenesis of the mouse germ-line together with current strategies to efficiently identify functional mutations using next generation sequencing. Then, we highlight illustrative examples of genes, proteins, and cellular signatures that have been revealed by ENU screens and have been shown to be involved in susceptibility or resistance to infectious diseases caused by parasites, bacteria, and viruses.

  17. Identification of 17 hearing impaired mouse strains in the TMGC ENU-mutagenesis screen

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    Kermany, Mohammad [St. Jude Children' s Research Hospital; Parker, Lisan [St. Jude Children' s Research Hospital; Guo, Yun-Kai [St. Jude Children' s Research Hospital; Miller, Darla R [ORNL; Swanson, Douglas J [ORNL; Yoo, Tai-June [Neuroscience Institute, Memphis, TN; Goldowitz, Daniel [University of Tennessee Health Science Center, Memphis; Zuo, Jian [St. Jude Children' s Research Hospital

    2006-01-01

    The Tennessee Mouse Genome Consortium (TMGC) employed an N-ethyl-N-nitrosourea (ENU)-mutagenesis scheme to identify mouse recessive mutants with hearing phenotypes. We employed auditory brainstem responses (ABR) to click and 8, 16, and 32 kHz stimuli and screened 285 pedigrees (1819 mice of 8-11 weeks old in various mixed genetic backgrounds) each bred to carry a homozygous ENU-induced mutation. To define mutant pedigrees, we measured P12 mice per pedigree in P2 generations and used a criterion where the mean ABR threshold per pedigree was two standard deviations above the mean of all offspring from the same parental strain. We thus identified 17 mutant pedigrees (6%), all exhibiting hearing loss at high frequencies (P16 kHz) with an average threshold elevation of 30-35 dB SPL. Interestingly, four mutants showed sex-biased hearing loss and six mutants displayed wide range frequency hearing loss. Temporal bone histology revealed that six of the first nine mutants displayed cochlear morphological defects: degeneration of spiral ganglia, spiral ligament fibrocytes or inner hair cells (but not outer hair cells) mostly in basal turns. In contrast to other ENU-mutagenesis auditory screens, our screen identified high-frequency, mild and sex-biased hearing defects. Further characterization of these 17 mouse models will advance our understanding of presbycusis and noise-induced hearing loss in humans.

  18. Pilot study of large-scale production of mutant pigs by ENU mutagenesis.

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    Hai, Tang; Cao, Chunwei; Shang, Haitao; Guo, Weiwei; Mu, Yanshuang; Yang, Shulin; Zhang, Ying; Zheng, Qiantao; Zhang, Tao; Wang, Xianlong; Liu, Yu; Kong, Qingran; Li, Kui; Wang, Dayu; Qi, Meng; Hong, Qianlong; Zhang, Rui; Wang, Xiupeng; Jia, Qitao; Wang, Xiao; Qin, Guosong; Li, Yongshun; Luo, Ailing; Jin, Weiwu; Yao, Jing; Huang, Jiaojiao; Zhang, Hongyong; Li, Menghua; Xie, Xiangmo; Zheng, Xuejuan; Guo, Kenan; Wang, Qinghua; Zhang, Shibin; Li, Liang; Xie, Fei; Zhang, Yu; Weng, Xiaogang; Yin, Zhi; Hu, Kui; Cong, Yimei; Zheng, Peng; Zou, Hailong; Xin, Leilei; Xia, Jihan; Ruan, Jinxue; Li, Hegang; Zhao, Weiming; Yuan, Jing; Liu, Zizhan; Gu, Weiwang; Li, Ming; Wang, Yong; Wang, Hongmei; Yang, Shiming; Liu, Zhonghua; Wei, Hong; Zhao, Jianguo; Zhou, Qi; Meng, Anming

    2017-06-22

    N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research.

  19. Induction of 6-thioguanine-resistant lymphocytes in Fischer 344 rats following in vivo exposure to N-ethyl-N-nitrosourea and cyclophosphamide

    International Nuclear Information System (INIS)

    Aidoo, A.; Lyn-Cook, L.E.; Mittelstaedt, R.A.; Heflich, R.H.; Casciano, D.A.

    1991-01-01

    The authors have developed a limiting dilution clonal assay for determining the frequency of 6-thioguanine-resistant (TG r ) lymphocytes produced in rats by in vivo exposure to genotoxic agents. Lymphocyte cloning efficiencies (CEs) were highest in plates containing both irradiated TK6 cells and irradiated autologous feeder cells. To measure the effects of chemical mutagens on the frequency of TG r lymphocytes, rats were given a single i.p. injection of N-ethyl-N-nitrosourea (ENU), a direct-acting alkylating agent, cyclophosphamide (CP), an indirect acting alkylating agent. Lymphocytes were isolated, primed, and cloned at 4 weeks after CP treatment and at 1,2,4 and 6 weeks after ENU treatment. CE in these cultures ranged from 12% to 27%. Cultures were also established for measuring CE in the presence of 6-thioguanine (TG). The dose-dependent responses obtained with both ENU and CP treatments suggest that rat lymphocytes are sensitive to direct- and indirect-acting alkylating agents administered in vivo and that the rat lymphocyte assay is a useful complement to the in vivo/in vitro mouse assay for determining the mutagenicity of environmental toxicants

  20. Differences in micronucleus induction in peripheral blood reticulocytes of mice exposed to N-ethyl-N-nitrosourea at light and dark dosing times.

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    Itoh, Keiichi; Masumori, Shoji; Nakajima, Madoka; Hayashi, Makoto; Sakakibara, Hiroyuki; Shimoi, Kayoko

    2012-01-01

    Mammals, including human beings, have a circadian clock system to regulate behavioral and physiological processes. In this study, we investigated the effect of dosing time on micronucleus induction in the bone marrow by evaluating the frequencies of micronucleated peripheral reticulocytes (MNRETs) in mice exposed to N-ethyl-N-nitrosourea (ENU) to assess any difference in genotoxic sensitivity to chemicals between light and dark periods (inactive phase for rodents and active phase for rodents). Male C3H/He mice were treated intraperitoneally with ENU (12.5 or 25 mg/kg body weight) at zeitgeber time (ZT) 3 in the light period or ZT15 in the dark period, and then the time courses of the frequencies of the MNRETs were determined. The frequencies of the MNRETs induced by ENU increased time-dependently and peaked at 48 hr after treatment for ZT3 and ZT15, and were obviously higher in the ZT15 treatment group than the ZT3 treatment group. The MNRETs were measured at 48 hr after treatment with ENU (25 mg/kg body weight) at various dosing times (ZT0, 3, 6, 12, 15 and 18). The frequencies of the MNRETs in mice treated at ZT0, 15 and 18 were significantly higher than those in mice treated at ZT3, 6 and 12. These results suggest that genotoxic sensitivity to chemicals in mouse bone marrow is different between light and dark periods maybe due to different biological responses (detoxification, cell cycle, DNA repair, etc.) related to circadian rhythms.

  1. Vanillin as a modulator agent in SMART test: inhibition in the steps that precede N-methyl-N-nitrosourea-, N-ethyl-N-nitrosourea-, ethylmethanesulphonate- and bleomycin-genotoxicity.

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    Sinigaglia, Marialva; Lehmann, Maurício; Baumgardt, Paula; do Amaral, Viviane Souza; Dihl, Rafael Rodrigues; Reguly, Maria Luíza; de Andrade, Heloísa Helena Rodrigues

    2006-09-05

    Vanillin (VA), the world's major flavoring compound used in food industry and confectionery products - that has antimutagenic and anticarcinogenic activity against a variety of mutagenic/carcinogenic agents - was tested for the interval between the formation of premutational lesion and it is finalization as a DNA lesion. The overall findings using co-treatment protocols in SMART test suggest that VA can lead to a significant protection against the general genotoxicity of ethylmethanesulphonate (EMS), N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU) and bleomycin sulphate (BLEO). Considering MNU, ENU and EMS the desmutagenic activity observed could result from VA-stimulation of detoxification, via induction of glutathione S-transferase. However, the protector effect related to BLEO could be attributed to its powerful scavenger ability, which has the potential to prevent oxidative damage induced by BLEO.

  2. Genomic analysis of microRNA time-course expression in liver of mice treated with genotoxic carcinogen N-ethyl-N-nitrosourea

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    Guo Lei

    2010-10-01

    Full Text Available Abstract Background Dysregulated expression of microRNAs (miRNAs has been previously observed in human cancer tissues and shown promise in defining tumor status. However, there is little information as to if or when expression changes of miRNAs occur in normal tissues after carcinogen exposure. Results To explore the possible time-course changes of miRNA expression induced by a carcinogen, we treated mice with one dose of 120 mg/kg N-ethyl-N-nitrosourea (ENU, a model genotoxic carcinogen, and vehicle control. The miRNA expression profiles were assessed in the mouse livers in a time-course design. miRNAs were isolated from the livers at days 1, 3, 7, 15, 30 and 120 after the treatment and their expression was determined using a miRNA PCR Array. Principal component analysis of the miRNA expression profiles showed that miRNA expression at post-treatment days (PTDs 7 and 15 were different from those at the other time points and the control. The number of differentially expressed miRNAs (DEMs changed over time (3, 5, 14, 32, 5 and 5 at PTDs 1, 3, 7, 15, 30 and 120, respectively. The magnitude of the expression change varied with time with the highest changes at PTDs 7 or 15 for most of the DEMs. In silico functional analysis of the DEMs at PTDs 7 and 15 indicated that the major functions of these ENU-induced DEMs were associated with DNA damage, DNA repair, apoptosis and other processes related to carcinogenesis. Conclusion Our results showed that many miRNAs changed their expression to respond the exposure of the genotoxic carcinogen ENU and the number and magnitude of the changes were highest at PTDs 7 to 15. Thus, one to two weeks after the exposure is the best time for miRNA expression sampling.

  3. Genome-wide ENU mutagenesis in combination with high density SNP analysis and exome sequencing provides rapid identification of novel mouse models of developmental disease.

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    Georgina Caruana

    Full Text Available Mice harbouring gene mutations that cause phenotypic abnormalities during organogenesis are invaluable tools for linking gene function to normal development and human disorders. To generate mouse models harbouring novel alleles that are involved in organogenesis we conducted a phenotype-driven, genome-wide mutagenesis screen in mice using the mutagen N-ethyl-N-nitrosourea (ENU.ENU was injected into male C57BL/6 mice and the mutations transmitted through the germ-line. ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads. From 52 pedigrees screened 15 were detected with anomalies in one or more of the structures/organs screened. Using single nucleotide polymorphism (SNP-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS we identified novel recessive alleles for Fras1, Ift140 and Lig1.In this study we have generated mouse models in which the anomalies closely mimic those seen in human disorders. The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.

  4. ENU mutagenesis reveals a novel phenotype of reduced limb strength in mice lacking fibrillin 2.

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    Gaynor Miller

    2010-02-01

    Full Text Available Fibrillins 1 (FBN1 and 2 (FBN2 are components of microfibrils, microfilaments that are present in many connective tissues, either alone or in association with elastin. Marfan's syndrome and congenital contractural arachnodactyly (CCA result from dominant mutations in the genes FBN1 and FBN2 respectively. Patients with both conditions often present with specific muscle atrophy or weakness, yet this has not been reported in the mouse models. In the case of Fbn1, this is due to perinatal lethality of the homozygous null mice making measurements of strength difficult. In the case of Fbn2, four different mutant alleles have been described in the mouse and in all cases syndactyly was reported as the defining phenotypic feature of homozygotes.As part of a large-scale N-ethyl-N-nitrosourea (ENU mutagenesis screen, we identified a mouse mutant, Mariusz, which exhibited muscle weakness along with hindlimb syndactyly. We identified an amber nonsense mutation in Fbn2 in this mouse mutant. Examination of a previously characterised Fbn2-null mutant, Fbn2(fp, identified a similar muscle weakness phenotype. The two Fbn2 mutant alleles complement each other confirming that the weakness is the result of a lack of Fbn2 activity. Skeletal muscle from mutants proved to be abnormal with higher than average numbers of fibres with centrally placed nuclei, an indicator that there are some regenerating muscle fibres. Physiological tests indicated that the mutant muscle produces significantly less maximal force, possibly as a result of the muscles being relatively smaller in Mariusz mice.These findings indicate that Fbn2 is involved in integrity of structures required for strength in limb movement. As human patients with mutations in the fibrillin genes FBN1 and FBN2 often present with muscle weakness and atrophy as a symptom, Fbn2-null mice will be a useful model for examining this aspect of the disease process further.

  5. Combined exposure to X-irradiation followed by N-ethyl-N-nitrosourea treatment alters the frequency and spectrum of Ikaros point mutations in murine T-cell lymphoma

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    Kakinuma, Shizuko, E-mail: skakinum@nirs.go.jp [Radiobiology for Children' s Health Research Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Nishimura, Mayumi; Amasaki, Yoshiko; Takada, Mayumi; Yamauchi, Kazumi; Sudo, Satomi; Shang, Yi [Radiobiology for Children' s Health Research Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Doi, Kazutaka; Yoshinaga, Shinji [Regulatory Sciences Research Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Shimada, Yoshiya [Radiobiology for Children' s Health Research Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan)

    2012-09-01

    Ionizing radiation is a well-known carcinogen, but its potency may be influenced by other environmental carcinogens, which is of practical importance in the assessment of risk. Data are scarce, however, on the combined effect of radiation with other environmental carcinogens and the underlying mechanisms involved. We studied the mode and mechanism of the carcinogenic effect of radiation in combination with N-ethyl-N-nitrosourea (ENU) using doses approximately equal to the corresponding thresholds. B6C3F1 mice exposed to fractionated X-irradiation (Kaplan's method) followed by ENU developed T-cell lymphomas in a dose-dependent manner. Radiation doses above an apparent threshold acted synergistically with ENU to promote lymphoma development, whereas radiation doses below that threshold antagonized lymphoma development. Ikaros, which regulates the commitment and differentiation of lymphoid lineage cells, is a critical tumor suppressor gene frequently altered in both human and mouse lymphomas and shows distinct mutation spectra between X-ray- and ENU-induced lymphomas. In the synergistically induced lymphomas, we observed a low frequency of LOH and an inordinate increase of Ikaros base substitutions characteristic of ENU-indcued point mutations, G:C to A:T at non-CpG, A:T to G:C, G:C to T:A and A:T to T:A. This suggests that radiation doses above an apparent threshold activate the ENU mutagenic pathway. This is the first report on the carcinogenic mechanism elicited by combined exposure to carcinogens below and above threshold doses based on the mutation spectrum of the causative gene. These findings constitute a basis for assessing human cancer risk following exposure to multiple carcinogens.

  6. The effects of X-irradiation, N-ethyl-N-nitrosourea or combined treatment on O6-alkylguanine-DNA alkyltransferase activity in fetal rat brain and liver and the induction of CNS tumours

    International Nuclear Information System (INIS)

    Stammberger, I.; Nice, L.; Schmahl, W.

    1990-01-01

    Wistar rats were treated in utero on day 16 of gestation either by X-irradiation, N-ethyl-N-nitrosourea (ENU), or both in combination. The O 6 -alkylguanine-DNA alkyltransferase (AT) activity of the fetal brain and liver was analyzed and long-term observations were made to reveal any relationship between the O 6 -ethylguanine repair capability and tumour incidence in the organs of the offspring. The AT activity in the brain was affected to the same extent in the fetuses as in the dams. There was a 60.9% decrease in AT activity in fetuses 24 h after ENU treatment. This correlates with a significant increase in the incidence of brain tumours in the treated offspring (44.1%) compared to control animals. The inductive effect of X-irradiation on AT activity corresponded in turn with a reduction of the incidence of tumours after the combined treatment. In the liver of the rat fetuses, there was generally no effect of treatment on AT activity in contrast to the results obtained for the dams, where an increased AT activity was observed. There were no tumours of the liver observed in the offspring after either treatment alone or after combined treatment. It is suggested that the combined treatment of rat fetuses could significantly reduce the incidence of brain tumours in adult life. (author)

  7. The effects of X-irradiation, N-ethyl-N-nitrosourea or combined treatment on O sup 6 -alkylguanine-DNA alkyltransferase activity in fetal rat brain and liver and the induction of CNS tumours

    Energy Technology Data Exchange (ETDEWEB)

    Stammberger, I.; Nice, L. (Muenchen Univ. (Germany, F.R.). Walter-Straub-Institut fuer Pharmakologie und Toxikologie); Schmahl, W. (Gesellschaft fuer Strahlen- und Umweltforschung mbH Muenchen, Neuherberg (Germany, F.R.). Inst. fuer Pathologie)

    1990-02-01

    Wistar rats were treated in utero on day 16 of gestation either by X-irradiation, N-ethyl-N-nitrosourea (ENU), or both in combination. The O{sup 6}-alkylguanine-DNA alkyltransferase (AT) activity of the fetal brain and liver was analyzed and long-term observations were made to reveal any relationship between the O{sup 6}-ethylguanine repair capability and tumour incidence in the organs of the offspring. The AT activity in the brain was affected to the same extent in the fetuses as in the dams. There was a 60.9% decrease in AT activity in fetuses 24 h after ENU treatment. This correlates with a significant increase in the incidence of brain tumours in the treated offspring (44.1%) compared to control animals. The inductive effect of X-irradiation on AT activity corresponded in turn with a reduction of the incidence of tumours after the combined treatment. In the liver of the rat fetuses, there was generally no effect of treatment on AT activity in contrast to the results obtained for the dams, where an increased AT activity was observed. There were no tumours of the liver observed in the offspring after either treatment alone or after combined treatment. It is suggested that the combined treatment of rat fetuses could significantly reduce the incidence of brain tumours in adult life. (author).

  8. ENU mutagenesis identifies mice with morbid obesity and severe hyperinsulinemia caused by a novel mutation in leptin.

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    Chen-Jee Hong

    Full Text Available BACKGROUND: Obesity is a multifactorial disease that arises from complex interactions between genetic predisposition and environmental factors. Leptin is central to the regulation of energy metabolism and control of body weight in mammals. METHODOLOGY/PRINCIPAL FINDINGS: To better recapitulate the complexity of human obesity syndrome, we applied N-ethyl-N-nitrosourea (ENU mutagenesis in combination with a set of metabolic assays in screening mice for obesity. Mapping revealed linkage to the chromosome 6 within a region containing mouse Leptin gene. Sequencing on the candidate genes identified a novel T-to-A mutation in the third exon of Leptin gene, which translates to a V145E amino acid exchange in the leptin propeptide. Homozygous Leptin(145E/145E mutant mice exhibited morbid obesity, accompanied by adipose hypertrophy, energy imbalance, and liver steatosis. This was further associated with severe insulin resistance, hyperinsulinemia, dyslipidemia, and hyperleptinemia, characteristics of human obesity syndrome. Hypothalamic leptin actions in inhibition of orexigenic peptides NPY and AgRP and induction of SOCS1 and SOCS3 were attenuated in Leptin(145E/145E mice. Administration of exogenous wild-type leptin attenuated hyperphagia and body weight increase in Leptin(145E/145E mice. However, mutant V145E leptin coimmunoprecipitated with leptin receptor, suggesting that the V145E mutation does not affect the binding of leptin to its receptor. Molecular modeling predicted that the mutated residue would form hydrogen bond with the adjacent residues, potentially affecting the structure and formation of an active complex with leptin receptor within that region. CONCLUSIONS/SIGNIFICANCE: Thus, our evolutionary, structural, and in vivo metabolic information suggests the residue 145 as of special function significance. The mouse model harboring leptin V145E mutation will provide new information on the current understanding of leptin biology and novel mouse

  9. Alkylation of deoxyribonucleic acid by carcinogens dimethyl sulphate, ethyl methanesulphonate, N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea

    International Nuclear Information System (INIS)

    Swenson, D.H.; Lawley, P.D.

    1978-01-01

    The ethyl phosphotriester of thymidylyl(3'-5')thymidine, dTp((Et) dT, was identified as a product from the reaction of DNA with N-ethyl-N-nitrosourea. Enzymic degradation to yield alkyl phosphotriesters from DNA alkylated by this carcinogen, and by N-methyl-N-nitrosourea, dimethyl sulphate and ethyl methanesulphonate was studied quantitatively, and the relative yields of the triesters dTp(Alk)dT were determined. The relative reactivity of the phosphodiester group dTpdT to each of the four carcinogens was thus obtained, and compared with that of DNA overall, or with that of the N-7 atom of guanine in DNA. The results are related to steric factors, and the electrophilic character of each carcinogen. (author)

  10. N-ethyl-N-nitrosourea-induced null mutation at the mouse Car-2 locus: An animal model for human carbonic anhydrase II deficiency syndrome

    International Nuclear Information System (INIS)

    Lewis, S.E.; Barnett, L.B.; Erickson, R.P.; Venta, P.J.; Tashian, R.E.

    1988-01-01

    Electrophoretic screening of (C57BL/6J x DBA/2J)F 1 progeny of male mice treated with N-ethyl-N-nitrosourea revealed a mouse that lacked the paternal carbonic anhydrase II (Ca II). Breeding tests showed that this trait was heritable and due to a null mutation at the Car-2 locus on chromosome 3. Like humans with the same inherited enzyme defect, animals homozygous for the new null allele are runted and have renal tubular acidosis. However, the prominent osteopetrosis found in humans with CA II deficiency could be detected even in very old homozygous null mice. A molecular analysis of the deficient mice shows that the mutant gene is not deleted and is transcribed. The CA II protein, which is normally expressed in most tissues, could not be detected by immunodiffusion analysis in any tissues of the CA II-deficient mice, suggesting a nonsense or a missense mutation at the Car-2 locus

  11. Enzymatic removal of O6-ethylguanine from mitochondrial DNA in rat tissues exposed to N-ethyl-N-nitrosourea in vivo

    International Nuclear Information System (INIS)

    Satoh, M.S.; Huh, N.; Rajewsky, M.F.; Kuroki, T.

    1988-01-01

    DNA repair is essential for maintaining the integrity of the genetic material, and a number of DNA repair mechanisms have been fairly well characterized for the nuclear DNA of eukaryotic cells as well as prokaryotes. However, little is know about DNA repair in mitochondria. Using highly sensitive immunoanalytical methods to detect specific DNA alkylation products, the authors found active removal of O 6 -ethyl-2'-deoxyguanosine (O 6 -EtdGuo) from rat liver mitochondrial DNA after pulse-exposure to N-ethyl-N-nitrosourea in vivo. In the kidney, O 6 -EtdGuo was removed from mitochondrial DNA with moderate efficiency, but nearly no removal was observed from the DNA of brain mitochondria. Among the rat tissues examined, the kinetics of O 6 -EtdGuo elimination from mitochondrial DNA was very similar to the kinetics of removal from nuclear DNA. O 4 -Ethyl-2'-deoxythymidine, another premutagenic DNA ethylation product, was stable in both mitochondrial and nuclear DNA of rat liver

  12. ENU-induced phenovariance in mice: inferences from 587 mutations

    Directory of Open Access Journals (Sweden)

    Arnold Carrie N

    2012-10-01

    Full Text Available Abstract Background We present a compendium of N-ethyl-N-nitrosourea (ENU-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1 to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2 to assess the characteristics of these mutations; and 3 to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype. Findings In the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied. Conclusions Collectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by

  13. Alkylation of deoxyribonucleic acid by carcinogens dimethyl sulphate, ethyl methanesulphonate, N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea. Relative reactivity of the phosphodiester site thymidylyl(3'-5')thymidine.

    Science.gov (United States)

    Swenson, D H; Lawley, P D

    1978-01-01

    1. The ethyl phosphotriester of thymidylyl(3'-5')thymidine, dTp(Et)dT, was identified as a product from reaction of DNA with N-ethyl-N-nitrosourea, by procedures parallel to those reported previously for the methyl homologue produced by N-methyl-N-nitrosourea. 2. Enzymic degradation to yield alkyl phosphotriesters from DNA alkylated by these carcinogens and by dimethyl sulphate and ethyl methanesulphonate was studied quantitatively, and the relative yields of the triesters dTp(Alk)dT were determined. The relative reactivity of the phosphodiester group dTpdT to each of the four carcinogens was thus obtained, and compared with that of DNA overall, or with that of the N-7 atom of guanine in DNA. Relative reactivity of the phosphodiester group was lowest towards dimethyl sulphate, the least electrophilic of the reagents used, and was highest towards N-ethyl-N-nitrosourea, the most electrophilic reagent. 3. The nature of the alkyl group transferred also influenced reactivity of the phosphodiester site, since this site was relatively more reactive towards ethylation than would be predicted simply from the known Swain-Scott s values of the alkylating agents. It was therefore suggested that the steric accessibility of the weakly nucleophilic phosphodiester group on the outside of the DNA macromolecule favours its reaction with ethylating, as opposed to methylating, reagents. 4. Taking a value of the Swain-Scott nucleophilicity (n) of 2.5 for an average DNA nucleotide unit [Walles & Ehrenberg (1969) Acta Chem. Scand. 23, 1080-1084], a value of n of about 1 for the phosphodiester group was deduced, and this value was found to be 2-3 units less than that for the N-7 atom of guanine in DNA. 5. The reactivity of DNA overall was markedly high towards the alkylnitrosoureas, despite their relatively low s values. This was ascribed to an electrostatic factor that favoured reaction of the negatively charged polymer with alkyldiazonium cation intermediates. PMID:208508

  14. A novel ENU-mutation in ankyrin-1 disrupts malaria parasite maturation in red blood cells of mice.

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    Andreas Greth

    Full Text Available The blood stage of the plasmodium parasite life cycle is responsible for the clinical symptoms of malaria. Epidemiological studies have identified coincidental malarial endemicity and multiple red blood cell (RBC disorders. Many RBC disorders result from mutations in genes encoding cytoskeletal proteins and these are associated with increased protection against malarial infections. However the mechanisms underpinning these genetic, host responses remain obscure. We have performed an N-ethyl-N-nitrosourea (ENU mutagenesis screen and have identified a novel dominant (haploinsufficient mutation in the Ank-1 gene (Ank1(MRI23420 of mice displaying hereditary spherocytosis (HS. Female mice, heterozygous for the Ank-1 mutation showed increased survival to infection by Plasmodium chabaudi adami DS with a concomitant 30% decrease in parasitemia compared to wild-type, isogenic mice (wt. A comparative in vivo red cell invasion and parasite growth assay showed a RBC-autonomous effect characterised by decreased proportion of infected heterozygous RBCs. Within approximately 6-8 hours post-invasion, TUNEL staining of intraerythrocytic parasites, showed a significant increase in dead parasites in heterozygotes. This was especially notable at the ring and trophozoite stages in the blood of infected heterozygous mutant mice compared to wt (p<0.05. We conclude that increased malaria resistance due to ankyrin-1 deficiency is caused by the intraerythrocytic death of P. chabaudi parasites.

  15. Workshop on ENU Mutagenesis: Planning for Saturation, July 25-28, 2002

    Energy Technology Data Exchange (ETDEWEB)

    Nadeau, Joseph H

    2002-07-25

    The goal of the conference is to enhance the development of improved technologies and new approaches to the identification of genes underlying chemically-induced mutant phenotypes. The conference brings together ENU mutagenesis experts from the United States and aborad for a small, intensive workshop to consider these issues.

  16. Simulation and estimation of gene number in a biological pathway using almost complete saturation mutagenesis screening of haploid mouse cells.

    Science.gov (United States)

    Tokunaga, Masahiro; Kokubu, Chikara; Maeda, Yusuke; Sese, Jun; Horie, Kyoji; Sugimoto, Nakaba; Kinoshita, Taroh; Yusa, Kosuke; Takeda, Junji

    2014-11-24

    Genome-wide saturation mutagenesis and subsequent phenotype-driven screening has been central to a comprehensive understanding of complex biological processes in classical model organisms such as flies, nematodes, and plants. The degree of "saturation" (i.e., the fraction of possible target genes identified) has been shown to be a critical parameter in determining all relevant genes involved in a biological function, without prior knowledge of their products. In mammalian model systems, however, the relatively large scale and labor intensity of experiments have hampered the achievement of actual saturation mutagenesis, especially for recessive traits that require biallelic mutations to manifest detectable phenotypes. By exploiting the recently established haploid mouse embryonic stem cells (ESCs), we present an implementation of almost complete saturation mutagenesis in a mammalian system. The haploid ESCs were mutagenized with the chemical mutagen N-ethyl-N-nitrosourea (ENU) and processed for the screening of mutants defective in various steps of the glycosylphosphatidylinositol-anchor biosynthetic pathway. The resulting 114 independent mutant clones were characterized by a functional complementation assay, and were shown to be defective in any of 20 genes among all 22 known genes essential for this well-characterized pathway. Ten mutants were further validated by whole-exome sequencing. The predominant generation of single-nucleotide substitutions by ENU resulted in a gene mutation rate proportional to the length of the coding sequence, which facilitated the experimental design of saturation mutagenesis screening with the aid of computational simulation. Our study enables mammalian saturation mutagenesis to become a realistic proposition. Computational simulation, combined with a pilot mutagenesis experiment, could serve as a tool for the estimation of the number of genes essential for biological processes such as drug target pathways when a positive selection of

  17. Phenotype screening for genetically deermined age-onset disorders and increased longevity in ENU-mutagenized mice

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, Dabney K [ORNL; Rinchik, Eugene M [ORNL; Moustaid-Moussa, Naima [University of Tennessee, Knoxville (UTK); Miller, Darla R [ORNL; Williams, Robert [University of Tennessee Health Science Center, Memphis; Michaud III, Edward J [ORNL; Jablonski, Monica M. [University of Tennessee Health Science Center, Memphis; Elberger, Andrea [University of Tennessee Health Science Center, Memphis; Hamre, Kristin M. [University of Tennessee Health Science Center, Memphis; Smeyne, Richard [St. Jude Children' s Research Hospital; Chesler, Elissa J [ORNL; Goldowitz, Daniel [University of Tennessee Health Science Center, Memphis

    2005-01-01

    With the goal of discovering genes that contribute to late-onset neurological and ocular disorders and also genes that extend the healthy life span in mammals, we are phenotyping mice carrying new mutations induced by the chemical N-ethyl-N-nitrosourea (ENU). The phenotyping plan includes basic behavioral, neurohistological, and vision testing in sibling cohorts of mice aged to 18 months, and then evaluation for markers of growth trajectory and stress response in these same cohorts aged up to 28 months. Statistical outliers are identified by comparison to test results of similar aged cohorts, and potential mutants are recovered for re-aging to confirm heritability of the phenotype.

  18. Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci.

    Science.gov (United States)

    Westrick, Randal J; Tomberg, Kärt; Siebert, Amy E; Zhu, Guojing; Winn, Mary E; Dobies, Sarah L; Manning, Sara L; Brake, Marisa A; Cleuren, Audrey C; Hobbs, Linzi M; Mishack, Lena M; Johnston, Alexander J; Kotnik, Emilee; Siemieniak, David R; Xu, Jishu; Li, Jun Z; Saunders, Thomas L; Ginsburg, David

    2017-09-05

    Factor V Leiden ( F5 L ) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized N -ethyl- N -nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5 L ( F5 L/L ) and haploinsufficient for tissue factor pathway inhibitor ( Tfpi +/- ). F8 deficiency enhanced the survival of F5 L/L Tfpi +/- mice, demonstrating that F5 L/L Tfpi +/- lethality is genetically suppressible. ENU-mutagenized F5 L/L males and F5 L/+ Tfpi +/- females were crossed to generate 6,729 progeny, with 98 F5 L/L Tfpi +/- offspring surviving until weaning. Sixteen lines, referred to as "modifier of Factor 5 Leiden ( MF5L1-16 )," exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene ( F3 ). Although no ENU-induced F3 mutation was identified, haploinsufficiency for F3 ( F3 +/- ) suppressed F5 L/L Tfpi +/- lethality. Whole-exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of F5 L/L Tfpi +/- lethality ( P = 1.7 × 10 -6 ), suggesting that Actr2 p.R258G is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent Actr2 knockin/knockout demonstrated that Actr2 haploinsufficiency is lethal, supporting a hypomorphic or gain-of-function mechanism of action for Actr2 p.R258G Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5 L and also suggest a role for Actr2 in this process.

  19. Novel ENU-Induced Mutation in Tbx6 Causes Dominant Spondylocostal Dysostosis-Like Vertebral Malformations in the Rat.

    Science.gov (United States)

    Abe, Koichiro; Takamatsu, Nobuhiko; Ishikawa, Kumiko; Tsurumi, Toshiko; Tanimoto, Sho; Sakurai, Yukina; Lisse, Thomas S; Lisse, Thomas; Imai, Kenji; Serikawa, Tadao; Mashimo, Tomoji

    2015-01-01

    Congenital vertebral malformations caused by embryonic segmentation defects are relatively common in humans and domestic animals. Although reverse genetics approaches in mice have provided information on the molecular mechanisms of embryonic somite segmentation, hypothesis-driven approaches cannot adequately reflect human dysmorphology within the population. In a N-ethyl-N-nitrosourea (ENU) mutagenesis project in Kyoto, the Oune mutant rat strain was isolated due to a short and kinked caudal vertebra phenotype. Skeletal staining of heterozygous rats showed partial loss of the cervical vertebrae as well as hemivertebrae and fused vertebral blocks in lumbar and sacral vertebrae. In homozygous embryos, severe displacement of the whole vertebrae was observed. The Oune locus was genetically mapped to rat chromosome 1 using 202 backcross animals and 50 genome-wide microsatellite markers. Subsequently, a miss-sense mutation in the Tbx6 gene was identified in the critical region. Although the mutation is located within the T-box domain near a predicted dimmer-interface, in vitro experiments revealed that the Tbx6 variant retains normal DNA binding ability and translational efficiency. However, the variant has decreased transcriptional activation potential in response to Notch-mediated signaling. Recently, it was reported that a dominant type of familial spondylocostal dysostosis is caused by a stoploss mutation in TBX6. Thus, we propose that partial dysfunction of Tbx6 leads to similar congenital vertebral malformations in both humans and rats. The Oune strain could be a unique animal model for dominant spondylocostal dysostosis and is useful for molecular dissection of the pathology of congenital vertebral malformations in humans.

  20. Novel ENU-Induced Mutation in Tbx6 Causes Dominant Spondylocostal Dysostosis-Like Vertebral Malformations in the Rat.

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    Koichiro Abe

    Full Text Available Congenital vertebral malformations caused by embryonic segmentation defects are relatively common in humans and domestic animals. Although reverse genetics approaches in mice have provided information on the molecular mechanisms of embryonic somite segmentation, hypothesis-driven approaches cannot adequately reflect human dysmorphology within the population. In a N-ethyl-N-nitrosourea (ENU mutagenesis project in Kyoto, the Oune mutant rat strain was isolated due to a short and kinked caudal vertebra phenotype. Skeletal staining of heterozygous rats showed partial loss of the cervical vertebrae as well as hemivertebrae and fused vertebral blocks in lumbar and sacral vertebrae. In homozygous embryos, severe displacement of the whole vertebrae was observed. The Oune locus was genetically mapped to rat chromosome 1 using 202 backcross animals and 50 genome-wide microsatellite markers. Subsequently, a miss-sense mutation in the Tbx6 gene was identified in the critical region. Although the mutation is located within the T-box domain near a predicted dimmer-interface, in vitro experiments revealed that the Tbx6 variant retains normal DNA binding ability and translational efficiency. However, the variant has decreased transcriptional activation potential in response to Notch-mediated signaling. Recently, it was reported that a dominant type of familial spondylocostal dysostosis is caused by a stoploss mutation in TBX6. Thus, we propose that partial dysfunction of Tbx6 leads to similar congenital vertebral malformations in both humans and rats. The Oune strain could be a unique animal model for dominant spondylocostal dysostosis and is useful for molecular dissection of the pathology of congenital vertebral malformations in humans.

  1. ENU-mutagenesis mice with a non-synonymous mutation in Grin1 exhibit abnormal anxiety-like behaviors, impaired fear memory, and decreased acoustic startle response

    Science.gov (United States)

    2013-01-01

    Background The Grin1 (glutamate receptor, ionotropic, NMDA1) gene expresses a subunit of N-methyl-D-aspartate (NMDA) receptors that is considered to play an important role in excitatory neurotransmission, synaptic plasticity, and brain development. Grin1 is a candidate susceptibility gene for neuropsychiatric disorders, including schizophrenia, bipolar disorder, and attention deficit/hyperactivity disorder (ADHD). In our previous study, we examined an N-ethyl-N-nitrosourea (ENU)-generated mutant mouse strain (Grin1Rgsc174/Grin1+) that has a non-synonymous mutation in Grin1. These mutant mice showed hyperactivity, increased novelty-seeking to objects, and abnormal social interactions. Therefore, Grin1Rgsc174/Grin1+ mice may serve as a potential animal model of neuropsychiatric disorders. However, other behavioral characteristics related to these disorders, such as working memory function and sensorimotor gating, have not been fully explored in these mutant mice. In this study, to further investigate the behavioral phenotypes of Grin1Rgsc174/Grin1+ mice, we subjected them to a comprehensive battery of behavioral tests. Results There was no significant difference in nociception between Grin1Rgsc174/Grin1+ and wild-type mice. The mutants did not display any abnormalities in the Porsolt forced swim and tail suspension tests. We confirmed the previous observations that the locomotor activity of these mutant mice increased in the open field and home cage activity tests. They displayed abnormal anxiety-like behaviors in the light/dark transition and the elevated plus maze tests. Both contextual and cued fear memory were severely deficient in the fear conditioning test. The mutant mice exhibited slightly impaired working memory in the eight-arm radial maze test. The startle amplitude was markedly decreased in Grin1Rgsc174/Grin1+ mice, whereas no significant differences between genotypes were detected in the prepulse inhibition (PPI) test. The mutant mice showed no obvious

  2. CHARACTERIZATION OF ENU-INDUCED MUTATIONS IN RED BLOOD CELL STRUCTURAL PROTEINS

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    Katrina Kildey

    2013-03-01

    Full Text Available Murine models with modified gene function as a result of N-ethyl-N-nitrosourea (ENU mutagenesis have been used to study phenotypes resulting from genetic change. This study investigated genetic factors associated with red blood cell (RBC physiology and structural integrity that may impact on blood component storage and transfusion outcome. Forward and reverse genetic approaches were employed with pedigrees of ENU-treated mice using a homozygous recessive breeding strategy. In a “forward genetic” approach, pedigree selection was based upon identification of an altered phenotype followed by exome sequencing to identify a causative mutation. In a second strategy, a “reverse genetic” approach based on selection of pedigrees with mutations in genes of interest was utilised and, following breeding to homozygosity, phenotype assessed. Thirty-three pedigrees were screened by the forward genetic approach. One pedigree demonstrated reticulocytosis, microcytic anaemia and thrombocytosis. Exome sequencing revealed a novel single nucleotide variation (SNV in Ank1 encoding the RBC structural protein ankyrin-1 and the pedigree was designated Ank1EX34. The reticulocytosis and microcytic anaemia observed in the Ank1EX34 pedigree were similar to clinical features of hereditary spherocytosis in humans. For the reverse genetic approach three pedigrees with different point mutations in Spnb1 encoding RBC protein spectrin-1β, and one pedigree with a mutation in Epb4.1, encoding band 4.1 were selected for study. When bred to homozygosity two of the spectrin-1β pedigrees (a, b demonstrated increased RBC count, haemoglobin (Hb and haematocrit (HCT. The third Spnb1 mutation (spectrin-1β c and mutation in Epb4.1 (band 4.1 did not significantly affect the haematological phenotype, despite these two mutations having a PolyPhen score predicting the mutation may be damaging. Exome sequencing allows rapid identification of causative mutations and development of

  3. Enu mutagenesis identifies a novel platelet phenotype in a loss-of-function Jak2 allele.

    Directory of Open Access Journals (Sweden)

    Nicole M Anderson

    Full Text Available Utilizing ENU mutagenesis, we identified a mutant mouse with elevated platelets. Genetic mapping localized the mutation to an interval on chromosome 19 that encodes the Jak2 tyrosine kinase. We identified a A3056T mutation resulting in a premature stop codon within exon 19 of Jak2 (Jak2(K915X, resulting in a protein truncation and functionally inactive enzyme. This novel platelet phenotype was also observed in mice bearing a hemizygous targeted disruption of the Jak2 locus (Jak2(+/-. Timed pregnancy experiments revealed that Jak2(K915X/K915X and Jak2(-/- displayed embryonic lethality; however, Jak2(K915X/K915X embryos were viable an additional two days compared to Jak2(-/- embryos. Our data suggest that perturbing JAK2 activation may have unexpected consequences in elevation of platelet number and correspondingly, important implications for treatment of hematological disorders with constitutive Jak2 activity.

  4. A mouse model of hereditary coproporphyria identified in an ENU mutagenesis screen

    Directory of Open Access Journals (Sweden)

    Ashlee J. Conway

    2017-08-01

    Full Text Available A genome-wide ethyl-N-nitrosourea (ENU mutagenesis screen in mice was performed to identify novel regulators of erythropoiesis. Here, we describe a mouse line, RBC16, which harbours a dominantly inherited mutation in the Cpox gene, responsible for production of the haem biosynthesis enzyme, coproporphyrinogen III oxidase (CPOX. A premature stop codon in place of a tryptophan at amino acid 373 results in reduced mRNA expression and diminished protein levels, yielding a microcytic red blood cell phenotype in heterozygous mice. Urinary and faecal porphyrins in female RBC16 heterozygotes were significantly elevated compared with that of wild-type littermates, particularly coproporphyrinogen III, whereas males were biochemically normal. Attempts to induce acute porphyric crises were made using fasting and phenobarbital treatment on females. While fasting had no biochemical effect on RBC16 mice, phenobarbital caused significant elevation of faecal coproporphyrinogen III in heterozygous mice. This is the first known investigation of a mutagenesis mouse model with genetic and biochemical parallels to hereditary coproporphyria.

  5. Creation of miniature pig model of human Waardenburg syndrome type 2A by ENU mutagenesis.

    Science.gov (United States)

    Hai, Tang; Guo, Weiwei; Yao, Jing; Cao, Chunwei; Luo, Ailing; Qi, Meng; Wang, Xianlong; Wang, Xiao; Huang, Jiaojiao; Zhang, Ying; Zhang, Hongyong; Wang, Dayu; Shang, Haitao; Hong, Qianlong; Zhang, Rui; Jia, Qitao; Zheng, Qiantao; Qin, Guosong; Li, Yongshun; Zhang, Tao; Jin, Weiwu; Chen, Zheng-Yi; Wang, Hongmei; Zhou, Qi; Meng, Anming; Wei, Hong; Yang, Shiming; Zhao, Jianguo

    2017-11-01

    Human Waardenburg syndrome 2A (WS2A) is a dominant hearing loss (HL) syndrome caused by mutations in the microphthalmia-associated transcription factor (MITF) gene. In mouse models with MITF mutations, WS2A is transmitted in a recessive pattern, which limits the study of hearing loss (HL) pathology. In the current study, we performed ENU (ethylnitrosourea) mutagenesis that resulted in substituting a conserved lysine with a serine (p. L247S) in the DNA-binding domain of the MITF gene to generate a novel miniature pig model of WS2A. The heterozygous mutant pig (MITF +/L247S ) exhibits a dominant form of profound HL and hypopigmentation in skin, hair, and iris, accompanied by degeneration of stria vascularis (SV), fused hair cells, and the absence of endocochlear potential, which indicate the pathology of human WS2A. Besides hypopigmentation and bilateral HL, the homozygous mutant pig (MITF L247S/L247S ) and CRISPR/Cas9-mediated MITF bi-allelic knockout pigs both exhibited anophthalmia. Three WS2 patients carrying MITF mutations adjacent to the corresponding region were also identified. The pig models resemble the clinical symptom and molecular pathology of human WS2A patients perfectly, which will provide new clues for better understanding the etiology and development of novel treatment strategies for human HL.

  6. Defective immuno- and thymoproteasome assembly causes severe immunodeficiency

    DEFF Research Database (Denmark)

    Treise, Irina; Huber, Eva M.; Klein-Rodewald, Tanja

    2018-01-01

    By N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated the mutant mouse line TUB6 that is characterised by severe combined immunodeficiency (SCID) and systemic sterile autoinflammation in homozygotes, and a selective T cell defect in heterozygotes. The causative missense point mutation results...

  7. Circadian Biology and Sleep: Missing Links in Obesity and Metabolism

    Science.gov (United States)

    2009-05-01

    large-scale N-ethyl-N- nitrosourea (ENU) mutagenesis screen using a wide range of nervous system and behavioral phenotypes, we have identified new...physiology. In addition to storing energy in the form of triglycerides, WAT is now known to act as an active endocrine tissue via the synthesis and

  8. The serotonin transporter knockout rat : A review

    NARCIS (Netherlands)

    Olivier, Jocelien; Cools, Alexander; Ellenbroek, Bart A.; Cuppen, E.; Homberg, Judith; Kalueff, Allan V.; LaPorte, Justin L.

    2010-01-01

    This chapter dicusses the most recent data on the serotonin transporter knock-out rat, a unique rat model that has been generated by target-selected N-ethyl-N-nitrosourea (ENU) driven mutagenesis. The knock-out rat is the result of a premature stopcodon in the serotonin transporter gene, and the

  9. Low-dose radiation attenuates chemical mutagenesis in vivo. Cross adaptation

    International Nuclear Information System (INIS)

    Kakinuma, Shizuko; Yamauchi, Kazumi; Amasaki, Yoshiko; Nishimura, Mayumi; Shimada, Yoshiya

    2009-01-01

    The biological effects of low-dose radiation are not only of social concern but also of scientific interest. The radioadaptive response, which is defined as an increased radioresistance by prior exposure to low-dose radiation, has been extensively studied both in vitro and in vivo. Here we briefly review the radioadaptive response with respect to mutagenesis, survival rate, and carcinogenesis in vivo, and introduce our recent findings of cross adaptation in mouse thymic cells, that is, the suppressive effect of repeated low-dose radiation on mutation induction by the alkylating agent N-ethyl-N-nitrosourea. (author)

  10. Discovery of candidate disease genes in ENU-induced mouse mutants by large-scale sequencing, including a splice-site mutation in nucleoredoxin.

    Directory of Open Access Journals (Sweden)

    Melissa K Boles

    2009-12-01

    Full Text Available An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn, inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing.

  11. Mutagenic effect of radionuclides incorporated into DNA of Drosophila melanogaster. Progress report, July 16, 1983-June 30, 1984

    International Nuclear Information System (INIS)

    Lee, W.R.

    1984-01-01

    Progress is reported in the development of methods for economically analyzing mutants at the molecular level so that mutants induced by x-rays, N-ethyl-N-nitrosourea (ENU) and tritium could be classified

  12. Rapid identification and recovery of ENU-induced mutations with next-generation sequencing and Paired-End Low-Error analysis.

    Science.gov (United States)

    Pan, Luyuan; Shah, Arish N; Phelps, Ian G; Doherty, Dan; Johnson, Eric A; Moens, Cecilia B

    2015-02-14

    Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics approach to directly identify point mutations in specific genes of interest in genomic DNA from a large chemically mutagenized population. Classical TILLING processes, based on enzymatic detection of mutations in heteroduplex PCR amplicons, are slow and labor intensive. Here we describe a new TILLING strategy in zebrafish using direct next generation sequencing (NGS) of 250 bp amplicons followed by Paired-End Low-Error (PELE) sequence analysis. By pooling a genomic DNA library made from over 9,000 N-ethyl-N-nitrosourea (ENU) mutagenized F1 fish into 32 equal pools of 288 fish, each with a unique Illumina barcode, we reduce the complexity of the template to a level at which we can detect mutations that occur in a single heterozygous fish in the entire library. MiSeq sequencing generates 250 base-pair overlapping paired-end reads, and PELE analysis aligns the overlapping sequences to each other and filters out any imperfect matches, thereby eliminating variants introduced during the sequencing process. We find that this filtering step reduces the number of false positive calls 50-fold without loss of true variant calls. After PELE we were able to validate 61.5% of the mutant calls that occurred at a frequency between 1 mutant call:100 wildtype calls and 1 mutant call:1000 wildtype calls in a pool of 288 fish. We then use high-resolution melt analysis to identify the single heterozygous mutation carrier in the 288-fish pool in which the mutation was identified. Using this NGS-TILLING protocol we validated 28 nonsense or splice site mutations in 20 genes, at a two-fold higher efficiency than using traditional Cel1 screening. We conclude that this approach significantly increases screening efficiency and accuracy at reduced cost and can be applied in a wide range of organisms.

  13. Modifier action of the chlorophyllin of the mutagenesis induced by the ethyl-nitroso-urea (ENU) in germinal cells of Drosophila melanogaster

    International Nuclear Information System (INIS)

    Morales N, I.

    2006-01-01

    The cupro-sodium chlorophyllin (CCS) it is a soluble porphyrin in water that it includes in its it structures a copper atom instead of the magnesium that has the chlorophyll. Diverse experiments have demonstrated that it possesses a potent activity, reducing or inhibiting, the DNA damage caused by physical and chemical agents of direct action or insinuation. Most of the knowledge about their anti genotoxic activity has been obtained using somatic cells of different organisms, on the other hand it is known very little of their effect in germinal cells. At the moment in the Drosophila laboratory of the ININ it is investigating the protective action of the CCS in germinal cells, with these studies has been observed that its administration to females that were crossed with males irradiated with 20 Gy of gamma radiation, promotes the induction of lethal dominant in the embryonic and post-embryonic states causing a diminution in the viability egg-adult. With the test of lethal recessive bound to the sex one has evidence that it increases the basal frequency of lethal recessive and it doesn't reduce those induced by radiation. In contrast, with the present investigation when the CCS was administered to males that later on were treated with ethyl-nitroso-urea (ENU) caused a reduction of the lethal frequency in all the monitored cellular states, but only it was significant in the post-meiotic cells. On the contrary, when the CCS was administered to the female ones and then they crossed with males treaties with ENU, it was observed a tendency to increase the lethal ones in all the cellular types. With both protocols the CCS caused a diminution of the sterility. The fact that the CCS has antagonistic activities, it deserves special attention to investigate with different protocols and systems, the conditions in that this pigment can work as a true antimutagenic and/or anti carcinogenic before being able to him to propose as a chemopreventor. (Author)

  14. ENU mutagenesis screening for dominant behavioral mutations based on normal control data obtained in home-cage activity, open-field, and passive avoidance tests.

    Science.gov (United States)

    Wada, Yumiko; Furuse, Tamio; Yamada, Ikuko; Masuya, Hiroshi; Kushida, Tomoko; Shibukawa, Yoko; Nakai, Yuji; Kobayashi, Kimio; Kaneda, Hideki; Gondo, Yoichi; Noda, Tetsuo; Shiroishi, Toshihiko; Wakana, Shigeharu

    2010-01-01

    To establish the cutoff values for screening ENU-induced behavioral mutations, normal variations in mouse behavioral data were examined in home-cage activity (HA), open-field (OF), and passive-avoidance (PA) tests. We defined the normal range as one that included more than 95% of the normal control values. The cutoffs were defined to identify outliers yielding values that deviated from the normal by less than 5% for C57BL/6J, DBA/2J, DBF(1), and N(2) (DXDB) progenies. Cutoff values for G1-phenodeviant (DBF(1)) identification were defined based on values over +/- 3.0 SD from the mean of DBF(1) for all parameters assessed in the HA and OF tests. For the PA test, the cutoff values were defined based on whether the mice met the learning criterion during the 2nd (at a shock intensity of 0.3 mA) or the 3rd (at a shock intensity of 0.15 mA) retention test. For several parameters, the lower outliers were undetectable as the calculated cutoffs were negative values. Based on the cutoff criteria, we identified 275 behavioral phenodeviants among 2,646 G1 progeny. Of these, 64 were crossed with wild-type DBA/2J individuals, and the phenotype transmission was examined in the G2 progeny using the cutoffs defined for N(2) mice. In the G2 mice, we identified 15 novel dominant mutants exhibiting behavioral abnormalities, including hyperactivity in the HA or OF tests, hypoactivity in the OF test, and PA deficits. Genetic and detailed behavioral analysis of these ENU-induced mutants will provide novel insights into the molecular mechanisms underlying behavior.

  15. Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

    Science.gov (United States)

    Potter, Paul K; Bowl, Michael R; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E; Simon, Michelle M; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V; Law, Gemma; MacLaren, Robert E; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H; Foster, Russell G; Jackson, Ian J; Peirson, Stuart N; Thakker, Rajesh V; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D M

    2016-08-18

    Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.

  16. Comprehensive behavioral analysis of ENU-induced Disc1-Q31L and -L100P mutant mice

    Directory of Open Access Journals (Sweden)

    Shoji Hirotaka

    2012-02-01

    Full Text Available Abstract Background Disrupted-in-Schizophrenia 1 (DISC1 is considered to be a candidate susceptibility gene for psychiatric disorders, including schizophrenia, bipolar disorder, and major depression. A recent study reported that N-ethyl-N-nitrosourea (ENU-induced mutations in exon 2 of the mouse Disc1 gene, which resulted in the amino acid exchange of Q31L and L100P, caused an increase in depression-like behavior in 31 L mutant mice and schizophrenia-like behavior in 100P mutant mice; thus, these are potential animal models of psychiatric disorders. However, remaining heterozygous mutations that possibly occur in flanking genes other than Disc1 itself might induce behavioral abnormalities in the mutant mice. Here, to confirm the effects of Disc1-Q31L and Disc1-L100P mutations on behavioral phenotypes and to investigate the behaviors of the mutant mice in more detail, the mutant lines were backcrossed to C57BL/6JJcl through an additional two generations and the behaviors were analyzed using a comprehensive behavioral test battery. Results Contrary to expectations, 31 L mutant mice showed no significant behavioral differences when compared with wild-type control mice in any of the behavioral tests, including the Porsolt forced swim and tail suspension tests, commonly used tests for depression-like behavior. Also, 100P mutant mice exhibited no differences in almost all of the behavioral tests, including the prepulse inhibition test for measuring sensorimotor gating, which is known to be impaired in schizophrenia patients; however, 100P mutant mice showed higher locomotor activity compared with wild-type control mice in the light/dark transition test. Conclusions Although these results are partially consistent with the previous study in that there was hyperactivity in 100P mutant mice, the vast majority of the results are inconsistent with those of the previous study; this discrepancy may be explained by differences in the genetic background of the

  17. Modifier action of the chlorophyllin of the mutagenesis induced by the ethyl-nitroso-urea (ENU) in germinal cells of Drosophila melanogaster; Accion modificadora de la clorofilina de la mutagenesis inducida por la etil-nitroso-urea (ENU) en celulas germinales de Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Morales N, I

    2006-07-01

    The cupro-sodium chlorophyllin (CCS) it is a soluble porphyrin in water that it includes in its it structures a copper atom instead of the magnesium that has the chlorophyll. Diverse experiments have demonstrated that it possesses a potent activity, reducing or inhibiting, the DNA damage caused by physical and chemical agents of direct action or insinuation. Most of the knowledge about their anti genotoxic activity has been obtained using somatic cells of different organisms, on the other hand it is known very little of their effect in germinal cells. At the moment in the Drosophila laboratory of the ININ it is investigating the protective action of the CCS in germinal cells, with these studies has been observed that its administration to females that were crossed with males irradiated with 20 Gy of gamma radiation, promotes the induction of lethal dominant in the embryonic and post-embryonic states causing a diminution in the viability egg-adult. With the test of lethal recessive bound to the sex one has evidence that it increases the basal frequency of lethal recessive and it doesn't reduce those induced by radiation. In contrast, with the present investigation when the CCS was administered to males that later on were treated with ethyl-nitroso-urea (ENU) caused a reduction of the lethal frequency in all the monitored cellular states, but only it was significant in the post-meiotic cells. On the contrary, when the CCS was administered to the female ones and then they crossed with males treaties with ENU, it was observed a tendency to increase the lethal ones in all the cellular types. With both protocols the CCS caused a diminution of the sterility. The fact that the CCS has antagonistic activities, it deserves special attention to investigate with different protocols and systems, the conditions in that this pigment can work as a true antimutagenic and/or anti carcinogenic before being able to him to propose as a chemopreventor. (Author)

  18. Mutagenesis

    International Nuclear Information System (INIS)

    Dubinin, N.P.

    1986-01-01

    Problems on radiation mutagenesis, in particular, data on general factors of genetic radiation effects, dependences of mutation frequencies on radiation dose and threshold in genetic radiation effects, problems of low doses, modification of genetic radiation effects, repauir of injuries of genetic material, photoreactivation, causing structure chromosomal mutations under radiation action, on relative genetic efficiency of different types of radiation are considered besides others

  19. Mutagenic effect of radionuclides incorporated into DNA of Drosophila melanogaster. Progress report, December 15, 1982-July 15, 1983

    International Nuclear Information System (INIS)

    Lee, W.R.

    1983-01-01

    The molecular changes in DNA of mutations induced at the well-defined locus alcohol dehydrogenase (Adh) in Drosophila melanogaster were compared between null mutants induced by x-rays, the alkylating agent N-ethyl-N-nitrosourea (ENU) and decay of tritium incorporated into specific sites of DNA

  20. Alkylation damage causes MMR-dependent chromosomal instability in vertebrate embryos.

    NARCIS (Netherlands)

    Feitsma, H.; Akay, A.; Cuppen, E.

    2008-01-01

    S(N)1-type alkylating agents, like N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU), are potent mutagens. Exposure to alkylating agents gives rise to O(6)-alkylguanine, a modified base that is recognized by DNA mismatch repair (MMR) proteins but is not repairable, resulting in

  1. Breeding a PKU-mouse model on Phe-free diet, is it possible?

    DEFF Research Database (Denmark)

    Dagnæs-Hansen, Frederik; Johansen, Karen Singers; Vorup-Jensen, Thomas

    2014-01-01

    The PKU-mouse model mutated in the PAH gene was developed in the 1990s in the laboratory of Dr. Alexandra Shedlovsky at the McArdle Laboratory for Cancer Research, University of Wisconsin. The mutation was generated by ENU (N-ethyl-N-nitrosourea) treatment of BTBR males. Several mutation was found...

  2. Differential genotoxicity of ethyl methanesulphonate, N-ethyl-N-nitrosourea and maleic hydrazide in tobacco seedlings based on data of the Comet assay and two recombination assays

    Czech Academy of Sciences Publication Activity Database

    Gichner, Tomáš

    2003-01-01

    Roč. 538, - (2003), s. 171-179 ISSN 1383-5718 R&D Projects: GA ČR GA521/02/0400; GA MŠk LN00B030 Institutional research plan: CEZ:AV0Z5038910 Keywords : DNA damage * Nicotiana tabacum * Single cell gel electrophoresis (SCGE) Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.748, year: 2003

  3. Comparative studies of dose-response curves for recessive lethal mutations induced by ethylnitrosourea in spermatogonia and in spermatozoa of Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Yoshikawa, I.; Ayaki, T.; Ohshima, K.

    1984-01-01

    Induction of recessive lethal mutation by N-ethyl-N-nitrosourea (ENU) was studied for the second chromosome of spermatogonia and spermatozoa in Drosophila melanogaster. ENU (0.03, 0.3, and 1.0 mM) was given to flies by dissolving it in feeding sucrose solution. When plotted against absorbed doses of ENU, the observed frequencies to recessive lethals showed a linear relationship for induction in spermatozoa but a sigmoidal relationship for induction in spermatogonia. These results suggest that in spermatogonia ENU-induced mutational damage is more repairable in a lower dose range of ENU. Mosaic lethal mutations were induced by ENU but not in spermatogonia.

  4. Identification of Mouse Cytomegalovirus Resistance Loci by ENU Mutagenesis

    Directory of Open Access Journals (Sweden)

    Philippe Georgel

    2009-10-01

    Full Text Available Host resistance to infection depends on the efficiency with which innate immune responses keep the infectious agent in check. Innate immunity encompasses components with sensing, signaling and effector properties. These elements with nonredundant functions are encoded by a set of host genes, the resistome. Here, we review our findings concerning the resistome. We have screened randomly mutagenized mice for susceptibility to a natural opportunistic pathogen, the mouse cytomegalovirus. We found that some genes with initially no obvious functions in innate immunity may be critical for host survival to infections, falling into a newly defined category of genes of the resistome.

  5. ENU mutagenesis to generate genetically modified rat models

    NARCIS (Netherlands)

    van Boxtel, R.; Gould, M.; Cuppen, E.; Smits, B.M.

    2010-01-01

    The rat is one of the most preferred model organisms in biomedical research and has been extremely useful for linking physiology and pathology to the genome. However, approaches to genetically modify specific genes in the rat germ line remain relatively scarce. To date, the most efficient approach

  6. Conserved role of unc-79 in ethanol responses in lightweight mutant mice.

    Directory of Open Access Journals (Sweden)

    David J Speca

    2010-08-01

    Full Text Available The mechanisms by which ethanol and inhaled anesthetics influence the nervous system are poorly understood. Here we describe the positional cloning and characterization of a new mouse mutation isolated in an N-ethyl-N-nitrosourea (ENU forward mutagenesis screen for animals with enhanced locomotor activity. This allele, Lightweight (Lwt, disrupts the homolog of the Caenorhabditis elegans (C. elegans unc-79 gene. While Lwt/Lwt homozygotes are perinatal lethal, Lightweight heterozygotes are dramatically hypersensitive to acute ethanol exposure. Experiments in C. elegans demonstrate a conserved hypersensitivity to ethanol in unc-79 mutants and extend this observation to the related unc-80 mutant and nca-1;nca-2 double mutants. Lightweight heterozygotes also exhibit an altered response to the anesthetic isoflurane, reminiscent of unc-79 invertebrate mutant phenotypes. Consistent with our initial mapping results, Lightweight heterozygotes are mildly hyperactive when exposed to a novel environment and are smaller than wild-type animals. In addition, Lightweight heterozygotes exhibit increased food consumption yet have a leaner body composition. Interestingly, Lightweight heterozygotes voluntarily consume more ethanol than wild-type littermates. The acute hypersensitivity to and increased voluntary consumption of ethanol observed in Lightweight heterozygous mice in combination with the observed hypersensitivity to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants suggests a novel conserved pathway that might influence alcohol-related behaviors in humans.

  7. umuC-mediated misrepair mutagenesis in Escherichia coli: Extent and specificity of SOS mutagenesis

    International Nuclear Information System (INIS)

    Shinoura, Y.; Ise, T.; Kato, T.; Glickman, B.W.

    1983-01-01

    The role of the error-prone misrepair pathway in mutagenesis was examined for a series of mutagens in umuC + and umuC36 strains of Escherichia coli. Mutagenesis by ENU, MNU, MNNG and EMS was independent of the umuC + gene function, while mutagenesis by MMS, 4NQO, γ-rays and UV was largely umuC + -dependent. Residual mutagenesis following UV-treatment of a umuC - strain showed the same mutational specificity seen in the umuC + strain. In contrast, the umuC mutation altered specificity substantially in an excision-repair-defective strain that showed a UV-spectrum strikingly different from that seen in an excision-repair-proficient strain. Only one of nine trpE frameshift mutations examined was reverted by UV-light and its reversion was umuC-dependent. In comparison, the dependence of frameshift mutagenesis following ICR191 treatment was site-specific, suggesting at least two mechanisms of frameshift mutagenesis, one dependent upon misrepair, the other not. (orig./AJ)

  8. Characterization of phospholipase C gamma enzymes with gain-of-function mutations.

    Science.gov (United States)

    Everett, Katy L; Bunney, Tom D; Yoon, Youngdae; Rodrigues-Lima, Fernando; Harris, Richard; Driscoll, Paul C; Abe, Koichiro; Fuchs, Helmut; de Angelis, Martin Hrabé; Yu, Philipp; Cho, Wohnwa; Katan, Matilda

    2009-08-21

    Phospholipase C gamma isozymes (PLC gamma 1 and PLC gamma 2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLC gamma 2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLC gamma 1 and PLC gamma 2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC gamma 2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC gamma 2 and corresponding PLC gamma 1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.

  9. Non-alcoholic fatty liver disease in mice with heterozygous mutation in TMED2.

    Directory of Open Access Journals (Sweden)

    Wenyang Hou

    Full Text Available The transmembrane emp24 domain/p24 (TMED family are essential components of the vesicular transport machinery. Members of the TMED family serve as cargo receptors implicated in selection and packaging of endoplasmic reticulum (ER luminal proteins into coatomer (COP II coated vesicles for anterograde transport to the Golgi. Deletion or mutations of Tmed genes in yeast and Drosophila results in ER-stress and activation of the unfolded protein response (UPR. The UPR leads to expression of genes and proteins important for expanding the folding capacity of the ER, degrading misfolded proteins, and reducing the load of new proteins entering the ER. The UPR is activated in non-alcoholic fatty liver disease (NAFLD in human and mouse and may contribute to the development and the progression of NAFLD. Tmed2, the sole member of the vertebrate Tmed β subfamily, exhibits tissue and temporal specific patterns of expression in embryos and developing placenta but is ubiquitously expressed in all adult organs. We previously identified a single point mutation, the 99J mutation, in the signal sequence of Tmed2 in an N-ethyl-N-nitrosourea (ENU mutagenesis screen. Histological and molecular analysis of livers from heterozygous mice carrying the 99J mutation, Tmed299J/+, revealed a requirement for TMED2 in liver health. We show that Tmed299J/+ mice had decreased levels of TMED2 and TMED10, dilated endoplasmic reticulum membrane, and increased phosphorylation of eIF2α, indicating ER-stress and activation of the UPR. Increased expression of Srebp1a and 2 at the newborn stage and increased incidence of NAFLD were also found in Tmed299J/+ mice. Our data establishes Tmed299J/+ mice as a novel mouse model for NAFLD and supports a role for TMED2 in liver health.

  10. An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse

    Czech Academy of Sciences Publication Activity Database

    Daxinger, L.; Harten, S.K.; Oey, H.; Epp, Trevor; Isbel, L.; Huang, E.; Whitelaw, N.; Apedaile, A.; Sorolla, A.; Yong, J.; Bharti, V.; Sutton, J.; Ashe, A.; Pang, Z.Y.; Wallace, N.; Gerhardt, D.J.; Blewitt, M.E.; Jeddeloh, J.A.; Whitelaw, E.

    2013-01-01

    Roč. 14, č. 9 (2013) ISSN 1465-6906 Institutional support: RVO:68378050 Keywords : epigenetics * variegation * forward genetics Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 10.465, year: 2013

  11. Comparing BMD-derived genotoxic potency estimations across variants of the transgenic rodent gene mutation assay.

    Science.gov (United States)

    Wills, John W; Johnson, George E; Battaion, Hannah L; Slob, Wout; White, Paul A

    2017-12-01

    There is growing interest in quantitative analysis of in vivo genetic toxicity dose-response data, and use of point-of-departure (PoD) metrics such as the benchmark dose (BMD) for human health risk assessment (HHRA). Currently, multiple transgenic rodent (TGR) assay variants, employing different rodent strains and reporter transgenes, are used for the assessment of chemically-induced genotoxic effects in vivo. However, regulatory issues arise when different PoD values (e.g., lower BMD confidence intervals or BMDLs) are obtained for the same compound across different TGR assay variants. This study therefore employed the BMD approach to examine the ability of different TGR variants to yield comparable genotoxic potency estimates. Review of over 2000 dose-response datasets identified suitably-matched dose-response data for three compounds (ethyl methanesulfonate or EMS, N-ethyl-N-nitrosourea or ENU, and dimethylnitrosamine or DMN) across four commonly-used murine TGR variants (Muta™Mouse lacZ, Muta™Mouse cII, gpt delta and BigBlue® lacI). Dose-response analyses provided no conclusive evidence that TGR variant choice significantly influences the derived genotoxic potency estimate. This conclusion was reliant upon taking into account the importance of comparing BMD confidence intervals as opposed to directly comparing PoD values (e.g., comparing BMDLs). Comparisons with earlier works suggested that with respect to potency determination, tissue choice is potentially more important than choice of TGR assay variant. Scoring multiple tissues selected on the basis of supporting toxicokinetic information is therefore recommended. Finally, we used typical within-group variances to estimate preliminary endpoint-specific benchmark response (BMR) values across several TGR variants/tissues. We discuss why such values are required for routine use of genetic toxicity PoDs for HHRA. Environ. Mol. Mutagen. 58:632-643, 2017. © 2017 Her Majesty the Queen in Right of Canada

  12. Measurement of the Absolute Branching Fraction for Lambda(+)(c) -> Lambda e(+)nu(e)

    NARCIS (Netherlands)

    Ablikim, M.; Achasov, M.N.; Ai, X.C.; Albayrak, O.; Albrecht, M.; Ambrose, D.J.; Amorose, A.; Haddadi, Z.; Kalantar-Nayestanaki, Nasser; Kavatsyuk, M.; Messchendorp, J.G; Tiemens, M.

    2015-01-01

    We report the first measurement of the absolute branching fraction for Lambda(+)(c) -> Lambda e(+)nu(e). This measurement is based on 567 pb(-1) of e(+)e(-) annihilation data produced at root s = 4.599 GeV, which is just above the Lambda(+)(c)Lambda(-)(c) threshold. The data were collected with the

  13. Antigenotoxic effects of Citrus aurentium L. fruit peel oil on mutagenicity of two alkylating agents and two metals in the Drosophila wing spot test.

    Science.gov (United States)

    Demir, Eşref; Kocaoğlu, Serap; Cetin, Huseyin; Kaya, Bülent

    2009-07-01

    Antigenotoxic effects of Citrus aurentium L. (Rutaceae) fruit peel oil (CPO) in combination with mutagenic metals and alkylating agents were studied using the wing spot test of D. melanogaster. The four reference mutagens, potassium dichromate (K2Cr2O7), cobalt chloride (CoCl2), ethylmethanesulfonate (EMS), and N-ethyl-N-nitrosourea (ENU) were clearly genotoxic. CPO alone at doses from 0.1 to 0.5% in Tween 80 was not mutagenic and did not enhance the mutagenic effect of the reference mutagens. However, antigenotoxic effects of CPO were clearly demonstrated in chronic cotreatments with mutagens and oil, by a significant decrease in wing spots induced by all four mutagens. The D. melanogaster wing spot test was found to be a suitable assay for detecting antigenotoxic effects in vivo. Copyright 2009 Wiley-Liss, Inc.

  14. Chemical and UV Mutagenesis.

    Science.gov (United States)

    Bose, Jeffrey L

    2016-01-01

    The ability to create mutations is an important step towards understanding bacterial physiology and virulence. While targeted approaches are invaluable, the ability to produce genome-wide random mutations can lead to crucial discoveries. Transposon mutagenesis is a useful approach, but many interesting mutations can be missed by these insertions that interrupt coding and noncoding sequences due to the integration of an entire transposon. Chemical mutagenesis and UV-based random mutagenesis are alternate approaches to isolate mutations of interest with the potential of only single nucleotide changes. Once a standard method, difficulty in identifying mutation sites had decreased the popularity of this technique. However, thanks to the recent emergence of economical whole-genome sequencing, this approach to making mutations can once again become a viable option. Therefore, this chapter provides an overview protocol for random mutagenesis using UV light or DNA-damaging chemicals.

  15. Protracted radiation mutagenesis

    International Nuclear Information System (INIS)

    Dubinina, L.G.; Shanazarova, A.S.; Chernikova, O.P.

    1976-01-01

    The aim of the work is investigation of the dynamics of structural mutations of Cr.capillaris chromosomes induced by irradiation of seeds at different stages of the cell cycle with subsequent storage. The results obtained show that irradiation is followed by mutagenesis wave kinetics under such conditions. The level and the character of this phenomenon depends on the functional state of the nucleus or on the relationship between this state and the amount of water in the seeds. Studies of this phenomenon will bring better understanding to the mechanism of radiation mutagenesis [ru

  16. Computer Simulation of Mutagenesis.

    Science.gov (United States)

    North, J. C.; Dent, M. T.

    1978-01-01

    A FORTRAN program is described which simulates point-substitution mutations in the DNA strands of typical organisms. Its objective is to help students to understand the significance and structure of the genetic code, and the mechanisms and effect of mutagenesis. (Author/BB)

  17. 2004 Mutagenesis Gordon Conference

    Energy Technology Data Exchange (ETDEWEB)

    Dr. Sue Jinks-Robertson

    2005-09-16

    Mutations are genetic alterations that drive biological evolution and cause many, if not all, human diseases. Mutation originates via two distinct mechanisms: ''vertical'' variation is de novo change of one or few bases, whereas ''horizontal'' variation occurs by genetic recombination, which creates new mosaics of pre-existing sequences. The Mutagenesis Conference has traditionally focused on the generation of mutagenic intermediates during normal DNA synthesis or in response to environmental insults, as well as the diverse repair mechanisms that prevent the fixation of such intermediates as permanent mutations. While the 2004 Conference will continue to focus on the molecular mechanisms of mutagenesis, there will be increased emphasis on the biological consequences of mutations, both in terms of evolutionary processes and in terms of human disease. The meeting will open with two historical accounts of mutation research that recapitulate the intellectual framework of this field and thereby place the current research paradigms into perspective. The two introductory keynote lectures will be followed by sessions on: (1) mutagenic systems, (2) hypermutable sequences, (3) mechanisms of mutation, (4) mutation avoidance systems, (5) mutation in human hereditary and infectious diseases, (6) mutation rates in evolution and genotype-phenotype relationships, (7) ecology, mutagenesis and the modeling of evolution and (8) genetic diversity of the human population and models for human mutagenesis. The Conference will end with a synthesis of the meeting as the keynote closing lecture.

  18. Mutagenesis and Teratogenesis Section

    International Nuclear Information System (INIS)

    Anon.

    1976-01-01

    Progress is reported on research with mice in the areas of radioinduced and chemical mutagenesis, cytologic studies, radiation effects on DNA synthesis, radiation effects on germ cells, mutagenicity of coal-conversion products, and others. Research on Drosophila was concerned with mutagenesis and genetics of nucleases. Studies were conducted on hamster cells with regard to cytotoxicity and mutagenicity of alkylating agents, modification of the microtubule system, protein kinase activity, and others. Research on bacteria was concerned with effects of x radiation on bacteriophage of Haemophilus influenzae, x-ray induced DNA polymerase I-directed repair synthesis in Escherichia coli, transformation by DNA polymerase II in Bacillus subtilis, and others. Research on xenopus laevis was conducted in the areas of calcium-induced cleavage of oocytes, yolk degradation in explants, and others

  19. Effects of thermal and fast neutrons and of ENU on generations M3 and M4 of Lens culinaris (medicus)

    International Nuclear Information System (INIS)

    Uhlik, J.; Urban, J.

    1976-01-01

    Plants in which the selection of the most fertile plants had not been made in the preceding generations showed a significantly lower emergence rate in the M3 and M4 generation after an ethyl nitroso urea (ENU) application, in comparison with material treated with neutrons. In the evaluation of the plants obtained after an exposure to the most effective doses in the induction of chlorophyll mutants, significant differences of the average values in relation to the control were found in the M3 generation in the number of seeds per plant after the application of both neutron radiations and ENU. In addition, after the application of thermal neutrons and ENU a significant difference was found in the average values of plant weight. A difference in the overall range of variability in relation to the control was found in plant weight after the application of neutrons and ENU, and in seed weight after the application of ENU and fast neutrons. The differences between the treated plants and controls in the M4 generation plants with fusarium disease were insignificant. The evaluation of the progenies exposed to various doses of the highest mutation effectiveness showed in the M3 generation significant differences (in relation to the control) in the mean values of plant height, seed weight, plant weight, seed proportion in plants, in the bottom-pod insertion level, and in the number of pods set. Despite a considerable attack by fusarium disease, the greatest number of plants having more seeds than 50 was selected in the M4 generation of the material exposed to the dose of 8 fast neutrons (0.95% of plants) while in the control the proportion of highly fertile plants was only 0.05%. The widest range of overall variability in the characteristics under study was found after irradiation with thermal neutrons. From this viewpoint they can be recommended for wide practical utilization. (author)

  20. Sensitivity of Bidens laevis L. to mutagenic compounds. Use of chromosomal aberrations as biomarkers of genotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Perez, D.J. [Laboratorio de Genetica, Estacion Experimental Agropecuaria Balcarce (INTA), Facultad de Ciencias Agrarias, UNMdP, CC 276, 7620 Balcarce (Argentina); Laboratorio de Ecotoxicologia, Departamento de Ciencias Marinas, Facultad de Ciencias Exactas y Naturales, UNMdP, Funes 3350, 7600 Mar del Plata (Argentina); Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Rivadavia 1917, 1033 Buenos Aires (Argentina); Lukaszewicz, G. [Laboratorio de Ecotoxicologia, Departamento de Ciencias Marinas, Facultad de Ciencias Exactas y Naturales, UNMdP, Funes 3350, 7600 Mar del Plata (Argentina); Menone, M.L., E-mail: lujanm@mdp.edu.a [Laboratorio de Ecotoxicologia, Departamento de Ciencias Marinas, Facultad de Ciencias Exactas y Naturales, UNMdP, Funes 3350, 7600 Mar del Plata (Argentina); Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Rivadavia 1917, 1033 Buenos Aires (Argentina); Camadro, E.L. [Laboratorio de Genetica, Estacion Experimental Agropecuaria Balcarce (INTA), Facultad de Ciencias Agrarias, UNMdP, CC 276, 7620 Balcarce (Argentina); Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Rivadavia 1917, 1033 Buenos Aires (Argentina)

    2011-01-15

    The wetland macrophyte Bidens laevis possesses suitable cytological characteristics for genotoxicity testing. To test its sensitivity as compared to terrestrial plants species currently in use in standardized assays, Methyl Methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU) and Maleic Hydrazide (HM) were used. On the other hand, the insecticide Endosulfan (ES) - an environmentally relevant contaminant - was assayed in seeds and two-month old plants. Mitotic Index (MI), frequency of Chromosome Aberrations in Anaphase-Telophase (CAAT) and frequency of Abnormal Metaphases (AM) were analyzed. MH, MMS and ENU caused a significant decrease of the MI. MMS was aneugenic whereas MH and ENU were both aneugenic and clastogenic. ES caused a significant concentration-dependent increase of total- and aneugenic-CAAT in roots and a significant high frequency of AM at high concentrations. Because of its sensitivity to mutagenic substances, B. laevis can be regarded as a reliable and convenient species for genotoxicity assays especially if aquatic contaminants are evaluated. - The wetland macrophyte Bidens laevis is sensitive to genotoxic compounds similarly to terrestrial standardized species.

  1. Sensitivity of Bidens laevis L. to mutagenic compounds. Use of chromosomal aberrations as biomarkers of genotoxicity

    International Nuclear Information System (INIS)

    Perez, D.J.; Lukaszewicz, G.; Menone, M.L.; Camadro, E.L.

    2011-01-01

    The wetland macrophyte Bidens laevis possesses suitable cytological characteristics for genotoxicity testing. To test its sensitivity as compared to terrestrial plants species currently in use in standardized assays, Methyl Methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU) and Maleic Hydrazide (HM) were used. On the other hand, the insecticide Endosulfan (ES) - an environmentally relevant contaminant - was assayed in seeds and two-month old plants. Mitotic Index (MI), frequency of Chromosome Aberrations in Anaphase-Telophase (CAAT) and frequency of Abnormal Metaphases (AM) were analyzed. MH, MMS and ENU caused a significant decrease of the MI. MMS was aneugenic whereas MH and ENU were both aneugenic and clastogenic. ES caused a significant concentration-dependent increase of total- and aneugenic-CAAT in roots and a significant high frequency of AM at high concentrations. Because of its sensitivity to mutagenic substances, B. laevis can be regarded as a reliable and convenient species for genotoxicity assays especially if aquatic contaminants are evaluated. - The wetland macrophyte Bidens laevis is sensitive to genotoxic compounds similarly to terrestrial standardized species.

  2. Theory of misrepair mutagenesis

    International Nuclear Information System (INIS)

    Bresler, S.E.

    1975-01-01

    On the basis of experimental data, a model of induced mutagenesis is proposed that takes into account the repair of DNA damage by the Rec system. The peculiar feature of the Rec system is the cleavage and resynthesis of long sequences near the recognized DNA damage. Up to 1000-2000 nucleotides are replaced in one act. Therefore a definite probability exists of finding a damaged point on the second strand serving as template. It is believed that at this point no requirements of complementarity exist and that a random substitution can take place. This is the origin of a point mutation (transversion or frameshift). From this model, a general formula for the dose-response curve of mutagenesis is deduced which also takes into account the possibility of simultaneously initiated repair on both complementary strands of DNA. The latter leads to a lethal event when the points are situated proximally. This formula fits the observations in different cases studied. Some fundamental observations such as the absence of mutants from predominant single-strand breaks of DNA chains are readily explained

  3. Ethyl methanesulfonate mutagenesis in Schizosaccharomyces pombe

    DEFF Research Database (Denmark)

    Ekwall, Karl; Thon, Genevieve

    2017-01-01

    Here we provide an ethyl methanesulfonate (EMS) mutagenesis protocol for Schizosaccharomyces pombe cells.......Here we provide an ethyl methanesulfonate (EMS) mutagenesis protocol for Schizosaccharomyces pombe cells....

  4. The ENU-3 protein family members function in the Wnt pathway parallel to UNC-6/Netrin to promote motor neuron axon outgrowth in C. elegans.

    Science.gov (United States)

    Florica, Roxana Oriana; Hipolito, Victoria; Bautista, Stephen; Anvari, Homa; Rapp, Chloe; El-Rass, Suzan; Asgharian, Alimohammad; Antonescu, Costin N; Killeen, Marie T

    2017-10-01

    The axons of the DA and DB classes of motor neurons fail to reach the dorsal cord in the absence of the guidance cue UNC-6/Netrin or its receptor UNC-5 in C. elegans. However, the axonal processes usually exit their cell bodies in the ventral cord in the absence of both molecules. Strains lacking functional versions of UNC-6 or UNC-5 have a low level of DA and DB motor neuron axon outgrowth defects. We found that mutations in the genes for all six of the ENU-3 proteins function to enhance the outgrowth defects of the DA and DB axons in strains lacking either UNC-6 or UNC-5. A mutation in the gene for the MIG-14/Wntless protein also enhances defects in a strain lacking either UNC-5 or UNC-6, suggesting that the ENU-3 and Wnt pathways function parallel to the Netrin pathway in directing motor neuron axon outgrowth. Our evidence suggests that the ENU-3 proteins are novel members of the Wnt pathway in nematodes. Five of the six members of the ENU-3 family are predicted to be single-pass trans-membrane proteins. The expression pattern of ENU-3.1 was consistent with plasma membrane localization. One family member, ENU-3.6, lacks the predicted signal peptide and the membrane-spanning domain. In HeLa cells ENU-3.6 had a cytoplasmic localization and caused actin dependent processes to appear. We conclude that the ENU-3 family proteins function in a pathway parallel to the UNC-6/Netrin pathway for motor neuron axon outgrowth, most likely in the Wnt pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Phage transposon mutagenesis.

    Science.gov (United States)

    Siegrist, M Sloan; Rubin, Eric J

    2009-01-01

    Phage transduction is an attractive method of genetic manipulation in mycobacteria. PhiMycoMarT7 is well suited for transposon mutagenesis as it is temperature sensitive for replication and contains T7 promoters that promote transcription, a highly active transposase gene, and an Escherichia coli oriR6 K origin of replication. Mycobacterial transposon mutant libraries produced by PhiMycoMarT7 transduction are amenable to both forward and reverse genetic studies. In this protocol, we detail the preparation of PhiMycoMarT7, including a description of the phage, reconstitution of the phage, purification of plaques, preparation of phage stock, and titering of phage stock. We then describe the transduction procedure and finally outline the isolation of individual transposon mutants.

  6. Forsythia improvement by mutagenesis

    International Nuclear Information System (INIS)

    Cadic, A.; Martin, Denise; Renoux, A.

    1980-01-01

    Mutagenesis is a method used by selectors to modify the genetic heritage of a species. Since about twenty years ago the list of varieties obtained has lengthened steadily. For various reasons, plants which propagate vegetatively, and amongst these a large number of decorative plants, have been especially improved by this method. Of the mutagenic agents known at present a favourite choice has often been the gamma radiations emitted by radioactive cobalt ( 60 Co). Several clones of forsythia, very irregular in decorative value, were exposed to gamma radiation for the purpose of judging the breadth of the easily identifiable mutation range and creating new varieties. From the results it is hoped very soon to release compact varieties with short internodes and varieties better suited to forcing because of their earlier flowering season [fr

  7. Search for the weak decays J/psi -> D-s(()*()-) e(+)nu(e) + c.c.

    NARCIS (Netherlands)

    Ablikim, M.; Achasov, M. N.; Ai, X. C.; Albayrak, O.; Albrecht, M.; Ambrose, D. J.; Amoroso, A.; An, F. F.; An, Q.; Bai, J. Z.; Ferroli, R. Baldini; Ban, Y.; Bennett, D. W.; Bennett, J. V.; Bertani, M.; Bettoni, D.; Bian, J. M.; Bianchi, F.; Boger, E.; Bondarenko, O.; Boyko, I.; Briere, R. A.; Cai, H.; Cai, X.; Cakir, O.; Calcaterra, A.; Cao, G. F.; Cetin, S. A.; Chang, J. F.; Chelkov, G.; Chen, G.; Chen, H. S.; Chen, H. Y.; Chen, J. C.; Chen, M. L.; Chen, S. J.; Chen, X.; Chen, X. R.; Chen, Y. B.; Cheng, H. P.; Chu, X. K.; Chu, Y. P.; Cibinetto, G.; Cronin-Hennessy, D.; Dai, H. L.; Dai, J. P.; Dedovich, D.; Deng, Z. Y.; Denig, A.; Denysenko, I.; Destefanis, M.; De Mori, F.; Ding, Y.; Dong, C.; Dong, J.; Dong, L. Y.; Dong, M. Y.; Du, S. X.; Duan, P. F.; Fan, J. Z.; Fang, J.; Fang, S. S.; Fang, X.; Fang, Y.; Fava, L.; Feldbauer, F.; Felici, G.; Feng, C. Q.; Fioravanti, E.; Fu, C. D.; Gao, Q.; Gao, Y.; Garzia, I.; Goetzen, K.; Gong, W. X.; Gradl, W.; Greco, M.; Gu, M. H.; Gu, Y. T.; Guan, Y. H.; Guo, A. Q.; Guo, L. B.; Guo, T.; Guo, Y.; Guo, Y. P.; Haddadi, Z.; Hafner, A.; Han, S; Han, Y. L.; Harris, F. A.; He, K. L.; He, Z. Y.; Held, T.; Heng, Y. K.; Hou, Z. L.; Hu, C.; Hu, H. M.; Hu, J. F.; Hu, T.; Hu, Y.; Huang, G. M.; Huang, G. S.; Huang, H. P.; Huang, J. S.; Huang, X. T.; Huang, Y.; Hussain, T.; Ji, Q.; Ji, Q. P.; Ji, X. B.; Ji, X. L.; Jiang, L. L.; Jiang, L. W.; Jiang, X. S.; Jiao, J. B.; Jiao, Z.; Jin, D. P.; Jin, S.; Johansson, T.; Julin, A.; Kalantar-Nayestanaki, N.; Kang, X. L.; Kang, X. S.; Kavatsyuk, M.; Ke, B. C.; Kliemt, R.; Kloss, B.; Kolcu, O. B.; Kopf, B.; Kornicer, M.; Kuehn, W.; Kupsc, A.; Lai, W.; Lange, J. S.; Lara, M.; Larin, P.; Li, C. H.; Li, Cheng; Li, D. M.; Li, F.; Li, G.; Li, H. B.; Li, J. C.; Li, Jin; Li, K.; Li, K.; Li, P. R.; Li, T.; Li, W. D.; Li, W. G.; Li, X. L.; Li, X. M.; Li, X. N.; Li, X. Q.; Li, Z. B.; Liang, H.; Liang, Y. F.; Liang, Y. T.; Liao, G. R.; Lin, D. X.; Liu, B. J.; Liu, C. L.; Liu, C. X.; Liu, F. H.; Liu, Fang; Liu, Feng; Liu, H. B.; Liu, H. H.; Liu, H. H.; Liu, H. M.; Liu, J.; Liu, J. P.; Liu, J. Y.; Liu, K.; Liu, K. Y.; Liu, L. D.; Liu, Q.; Liu, S. B.; Liu, X.; Liu, X. X.; Liu, Y. B.; Liu, Z. A.; Liu, Zhiqiang; Liu, Zhiqing; Loehner, H.; Lou, X. C.; Lu, H. J.; Lu, J. G.; Lu, R. Q.; Lu, Y.; Lu, Y. P.; Luo, C. L.; Luo, M. X.; Luo, T.; Luo, X. L.; Lv, M.; Lyu, X. R.; Ma, F. C.; Ma, H. L.; Ma, L. L.; Ma, Q. M.; Ma, S.; Ma, T.; Ma, X. N.; Ma, X. Y.; Maas, F. E.; Maggiora, M.; Malik, Q. A.; Mao, Y. J.; Mao, Z. P.; Marcello, S.; Messchendorp, J. G.; Min, J.; Min, T. J.; Mitchell, R. E.; Mo, X. H.; Mo, Y. J.; Moeini, H.; Morales, C. Morales; Moriya, K.; Muchnoi, N. Yu.; Muramatsu, H.; Nefedov, Y.; Nerling, F.; Nikolaev, I. B.; Ning, Z.; Nisar, S.; Niu, S. L.; Niu, X. Y.; Olsen, S. L.; Ouyang, Q.; Pacetti, S.; Patteri, P.; Pelizaeus, M.; Peng, H. P.; Peters, K.; Ping, J. L.; Ping, R. G.; Poling, R.; Pu, Y. N.; Qi, M.; Qian, S.; Qiao, C. F.; Qin, L. Q.; Qin, N.; Qin, X. S.; Qin, Y.; Qin, Z. H.; Qiu, J. F.; Rashid, K. H.; Redmer, C. F.; Ren, H. L.; Ripka, M.; Rong, G.; Ruan, X. D.; Santoro, V.; Sarantsev, A.; Savrie, M.; Schoenning, K.; Schumann, S.; Shan, W.; Shao, M.; Shen, C. P.; Shen, P. X.; Shen, X. Y.; Sheng, H. Y.; Shepherd, M. R.; Song, W. M.; Song, X. Y.; Sosio, S.; Spataro, S.; Spruck, B.; Sun, G. X.; Sun, J. F.; Sun, S. S.; Sun, Y. J.; Sun, Y. Z.; Sun, Z. J.; Sun, Z. T.; Tang, C. J.; Tang, X.; Tapan, I.; Thorndike, E. H.; Tiemens, M.; Toth, D.; Ullrich, M.; Uman, I.; Varner, G. S.; Wang, B.; Wang, B. L.; Wang, D.; Wang, D. Y.; Wang, K.; Wang, L. L.; Wang, L. S.; Wang, M.; Wang, P.; Wang, P. L.; Wang, Q. J.; Wang, S. G.; Wang, W.; Wang, X. F.; Wang, Y. D.; Wang, Y. F.; Wang, Y. Q.; Wang, Z.; Wang, Z. G.; Wang, Z. H.; Wang, Z. Y.; Wei, D. H.; Wei, J. B.; Weidenkaff, P.; Wen, S. P.; Wiedner, U.; Wolke, M.; Wu, L. H.; Wu, Z.; Xia, L. G.; Xia, Y.; Xiao, D.; Xiao, Z. J.; Xie, Y. G.; Xiu, Q. L.; Xu, G. F.; Xu, L.; Xu, Q. J.; Xu, Q. N.; Xu, X. P.; Yan, L.; Yan, W. B.; Yan, W. C.; Yan, Y. H.; Yang, H. X.; Yang, L.; Yang, Y.; Yang, Y. X.; Ye, H.; Ye, M.; Ye, M. H.; Yin, J. H.; Yu, B. X.; Yu, C. X.; Yu, H. W.; Yu, J. S.; Yuan, C. Z.; Yuan, W. L.; Yuan, Y.; Yuncu, A.; Zafar, A. A.; Zallo, A.; Zeng, Y.; Zhang, B. X.; Zhang, B. Y.; Zhang, C.; Zhang, C. C.; Zhang, D. H.; Zhang, H. H.; Zhang, H. Y.; Zhang, J. J.; Zhang, J. L.; Zhang, J. Q.; Zhang, J. W.; Zhang, J. Y.; Zhang, J. Z.; Zhang, K.; Zhang, L.; Zhang, S. H.; Zhang, X. J.; Zhang, X. Y.; Zhang, Y.; Zhang, Y. H.; Zhang, Z. H.; Zhang, Z. P.; Zhang, Z. Y.; Zhao, G.; Zhao, J. W.; Zhao, J. Y.; Zhao, J. Z.; Zhao, Lei; Zhao, Ling; Zhao, M. G.; Zhao, Q.; Zhao, Q. W.; Zhao, S. J.; Zhao, T. C.; Zhao, Y. B.; Zhao, Z. G.; Zhemchugov, A.; Zheng, B.; Zheng, J. P.; Zheng, W. J.; Zheng, Y. H.; Zhong, B.; Zhou, L.; Zhou, Li; Zhou, X.; Zhou, X. K.; Zhou, X. R.; Zhou, X. Y.; Zhu, K.; Zhu, K. J.; Zhu, S.; Zhu, X. L.; Zhu, Y. C.; Zhu, Y. S.; Zhu, Z. A.; Zhuang, J.; Zou, B. S.; Zou, J. H.

    2014-01-01

    Using a sample of 2.25 x 10(8) J/psi events collected with the BESIII detector at the BEPCII collider, we search for the J/psi semileptonic weak decay J/psi -> D-s(-) e(+)nu(e) +c.c. with a much higher sensitivity than previous searches. We also perform the first search for J/psi -> D-s(*-) e(+)

  8. Optogenetic mutagenesis in Caenorhabditis elegans.

    Science.gov (United States)

    Noma, Kentaro; Jin, Yishi

    2015-12-03

    Reactive oxygen species (ROS) can modify and damage DNA. Here we report an optogenetic mutagenesis approach that is free of toxic chemicals and easy to perform by taking advantage of a genetically encoded ROS generator. This method relies on the potency of ROS generation by His-mSOG, the mini singlet oxygen generator, miniSOG, fused to a histone. Caenorhabditis elegans expressing His-mSOG in the germline behave and reproduce normally, without photoinduction. Following exposure to blue light, the His-mSOG animals produce progeny with a wide range of heritable phenotypes. We show that optogenetic mutagenesis by His-mSOG induces a broad spectrum of mutations including single-nucleotide variants (SNVs), chromosomal deletions, as well as integration of extrachromosomal transgenes, which complements those derived from traditional chemical or radiation mutagenesis. The optogenetic mutagenesis expands the toolbox for forward genetic screening and also provides direct evidence that nuclear ROS can induce heritable and specific genetic mutations.

  9. Antimicrobials, stress and mutagenesis.

    Directory of Open Access Journals (Sweden)

    Alexandro Rodríguez-Rojas

    2014-10-01

    Full Text Available Cationic antimicrobial peptides are ancient and ubiquitous immune effectors that multicellular organisms use to kill and police microbes whereas antibiotics are mostly employed by microorganisms. As antimicrobial peptides (AMPs mostly target the cell wall, a microbial 'Achilles heel', it has been proposed that bacterial resistance evolution is very unlikely and hence AMPs are ancient 'weapons' of multicellular organisms. Here we provide a new hypothesis to explain the widespread distribution of AMPs amongst multicellular organism. Studying five antimicrobial peptides from vertebrates and insects, we show, using a classic Luria-Delbrück fluctuation assay, that cationic antimicrobial peptides (AMPs do not increase bacterial mutation rates. Moreover, using rtPCR and disc diffusion assays we find that AMPs do not elicit SOS or rpoS bacterial stress pathways. This is in contrast to the main classes of antibiotics that elevate mutagenesis via eliciting the SOS and rpoS pathways. The notion of the 'Achilles heel' has been challenged by experimental selection for AMP-resistance, but our findings offer a new perspective on the evolutionary success of AMPs. Employing AMPs seems advantageous for multicellular organisms, as it does not fuel the adaptation of bacteria to their immune defenses. This has important consequences for our understanding of host-microbe interactions, the evolution of innate immune defenses, and also sheds new light on antimicrobial resistance evolution and the use of AMPs as drugs.

  10. Experimental mutagenesis in plants

    International Nuclear Information System (INIS)

    Conger, B.V.

    1979-01-01

    Considerable progress has been made in directed or controlled mutagenesis with bacterial systems, the genetic resolving power of which is much greater than that of higher plants. The mutagen specificity in higher plants has been of great interest, and numerous results and observations have been reported. The advances in the culture of plant cells and tissues have created much interest concerning the possibility of inducing and recovering mutants at the cellular level. There are great problems including the failure to regenerate plants from cells in all but a few species. The genetic and cytogenetic instability in the culture of plant tissues is well known, and the most common nuclear change is polyploidy including aneuploidy. The degree of polyploidy increases with calluses or culture age. In rice, the frequency of aneuploidy is greater in the calluses derived from roots than those derived from stem internodes. Polyploid and/or self-incompatible plant species are not as amenable to conventional mutation breeding techniques as diploid, self-fertilizing species. Inducing mutations in somatic tissues creates the problem of chimeras. However, the new cultivars of highly heterozygous, outcrossing, self-incompatible species are produced by combining several different clones. The performance of the progeny of at least 4 generations removed from the polycross of the parent clones is the important factor, and a high amount of heterozygocity is tolerated within cultivars and even on the same plants. (Yamashita, S.)

  11. Recovery during radiation mutagenesis

    International Nuclear Information System (INIS)

    Deen, D.F.; Shaw, E.I.

    1976-01-01

    Many variables (e.g. cell inoculum size, mutagen dose, expression time, and concentration of the selective agent) are known to affect the induced mutation frequency obtained in cultured mammalian cells. The authors have studied the effects of several parameters on the frequency of radiation-induced resistance to 8-azaguanine in asynchronous V79-171B hamster cells. Inoculation with 10 5 cells was followed by graded doses of radiation, expression times were optimized to maximize mutation frequency, and then the treated cells were challenged with 8-azaguanine for ten days. The optimal expression times which maximized mutation frequency were dose dependent and are in the range of 14-24, 24, and 24-36 hours respectively for doses of 250, 40 and 800 rads. A time interval of 24 hours between two 250-rad fractions resulted in a mutation frequency smaller than that obtained from administration of a single 500-rad dose. With 36 hours between halves of the dose, the induced mutation frequency was an order of magnitude lower than that produced by a single dose and actually below the unirradiated (spontaneous) frequency. Maintenance of cells after irradation first at 18 0 C for 24 hours, and then allowance of expression at 37 0 C for 24 hours, increased both the spontaneous and induced mutation frequency. A one-hour postirradiation balanced salt-solution treatment did not affect the number of spontaneous mutants that arose, but reduced the number of induced mutants. Thus, the balanced salt treatment lowers the induced mutation frequency about a factor of two. The possible significance of these results are discussed with respect to the role of radiation repair mechanisms during mutagenesis, and to recovery at low dose rates. A working hypothesis is advanced to explain the possible mechanism which causes expression time to vary as a function of the dose of mutagen. (author)

  12. DNA methylation in the pathophysiology of hyperphenylalaninemia in the PAH(enu2) mouse model of phenylketonuria.

    Science.gov (United States)

    Dobrowolski, S F; Lyons-Weiler, J; Spridik, K; Vockley, J; Skvorak, K; Biery, A

    2016-09-01

    Phenylalanine hydroxylase deficient phenylketonuria (PKU) is the paradigm for a treatable inborn error of metabolism where maintaining plasma phenylalanine (Phe) in the therapeutic range relates to improved clinical outcomes. While Phe is the presumed intoxicating analyte causal in neurologic damage, the mechanism(s) of Phe toxicity has remained elusive. Altered DNA methylation is a recognized response associated with exposure to numerous small molecule toxic agents. Paralleling this effect, we hypothesized that chronic Phe over-exposure in the brain would lead to aberrant DNA methylation with secondary influence upon gene regulation that would ultimately contribute to PKU neuropathology. The PAH(enu2) mouse models human PKU with intrinsic hyperphenylalaninemia, abnormal response to Phe challenge, and neurologic deficit. To examine this hypothesis, we assessed DNA methylation patterns in brain tissues using methylated DNA immunoprecipitation and paired end sequencing in adult PAH(enu2) animals maintained under either continuous dietary Phe restriction or chronic hyperphenylalaninemia. Heterozygous PAH(enu2/WT) litter mates served as controls for normal Phe exposure. Extensive repatterning of DNA methylation was observed in brain tissue of hyperphenylalaninemic animals while Phe restricted animals displayed an attenuated pattern of aberrant DNA methylation. Affected gene coding regions displayed aberrant hypermethylation and hypomethylation. Gene body methylation of noncoding RNA genes was observed and among these microRNA genes were prominent. Of particular note, observed only in hyperphenylalaninemic animals, was hypomethylation of miRNA genes within the imprinted Dlk1-Dio3 locus on chromosome 12. Aberrant methylation of microRNA genes influenced their expression which has secondary effects upon the expression of targeted protein coding genes. Differential hypermethylation of gene promoters was exclusive to hyperphenylalaninemic PAH(enu2) animals. Genes with

  13. ENU mutagenesis reveals that Notchless homolog 1 (Drosophila affects Cdkn1a and several members of the Wnt pathway during murine pre-implantation development

    Directory of Open Access Journals (Sweden)

    Lossie Amy C

    2012-12-01

    Full Text Available Abstract Background Our interests lie in determining the genes and genetic pathways that are important for establishing and maintaining maternal-fetal interactions during pregnancy. Mutation analysis targeted to a 34 Mb domain flanked by Trp53 and Wnt3 demonstrates that this region of mouse chromosome 11 contains a large number of essential genes. Two mutant alleles (l11Jus1 and l11Jus4, which fall into the same complementation group, survive through implantation but fail prior to gastrulation. Results Through a positional cloning strategy, we discovered that these homozygous mutant alleles contain non-conservative missense mutations in the Notchless homolog 1 (Drosophila (Nle1 gene. NLE1 is a member of the large WD40-repeat protein family, and is thought to signal via the canonical NOTCH pathway in vertebrates. However, the phenotype of the Nle1 mutant mice is much more severe than single Notch receptor mutations or even in animals in which NOTCH signaling is blocked. To test the hypothesis that NLE1 functions in multiple signaling pathways during pre-implantation development, we examined expression of multiple Notch downstream target genes, as well as select members of the Wnt pathway in wild-type and mutant embryos. We did not detect altered expression of any primary members of the Notch pathway or in Notch downstream target genes. However, our data reveal that Cdkn1a, a NOTCH target, was upregulated in Nle1 mutants, while several members of the Wnt pathway are downregulated. In addition, we found that Nle1 mutant embryos undergo caspase-mediated apoptosis as hatched blastocysts, but not as morulae or blastocysts. Conclusions Taken together, these results uncover potential novel functions for NLE1 in the WNT and CDKN1A pathways during embryonic development in mammals.

  14. Forward and reverse mutagenesis in C. elegans

    Science.gov (United States)

    Kutscher, Lena M.; Shaham, Shai

    2014-01-01

    Mutagenesis drives natural selection. In the lab, mutations allow gene function to be deciphered. C. elegans is highly amendable to functional genetics because of its short generation time, ease of use, and wealth of available gene-alteration techniques. Here we provide an overview of historical and contemporary methods for mutagenesis in C. elegans, and discuss principles and strategies for forward (genome-wide mutagenesis) and reverse (target-selected and gene-specific mutagenesis) genetic studies in this animal. PMID:24449699

  15. Comments on mutagenesis risk estimation

    International Nuclear Information System (INIS)

    Russell, W.L.

    1976-01-01

    Several hypotheses and concepts have tended to oversimplify the problem of mutagenesis and can be misleading when used for genetic risk estimation. These include: the hypothesis that radiation-induced mutation frequency depends primarily on the DNA content per haploid genome, the extension of this concept to chemical mutagenesis, the view that, since DNA is DNA, mutational effects can be expected to be qualitatively similar in all organisms, the REC unit, and the view that mutation rates from chronic irradiation can be theoretically and accurately predicted from acute irradiation data. Therefore, direct determination of frequencies of transmitted mutations in mammals continues to be important for risk estimation, and the specific-locus method in mice is shown to be not as expensive as is commonly supposed for many of the chemical testing requirements

  16. Molecular fundamentals of chromosomal mutagenesis

    International Nuclear Information System (INIS)

    Ganassi, E.Eh.; Zaichkina, S.I.; Malakhova, L.V.

    1987-01-01

    Precise quantitative correlation between the yield of chromosome structure damages and the yield of DNA damages is shown when comparing data on molecular and cytogenetic investigations carried out in cultural Mammalia cells. As the chromosome structure damage is to be connected with the damage of its carcass structure, then it is natural that DNA damage in loop regions is not to affect considerably the structure, while DNA damage lying on the loop base and connected with the chromosome carcass is to play a determining role in chromosomal mutagenesis. This DNA constitutes 1-2% from the total quantity of nuclear DNA. If one accepts that damages of these regions of DNA are ''hot'' points of chromosomal mutagenesis, then it becomes clear why 1-2% of preparation damages in a cell are realized in chromosome structural damages

  17. Repairability during G1 of the inductor leisure of exchanges in the sister chromatid induced by alkylating agents in DNA substituted and no substituted with BUDR, in cells of the salivary gland of mouse In vivo; Reparabilidad durante G1 de las lesiones inductoras de intercambios en las cromatidas hermanas inducidos por agentes alquilantes en ADN sustituido y no sustituido con BrdU, en celulas de la glandula salival de raton In vivo

    Energy Technology Data Exchange (ETDEWEB)

    Gonzalez B, F

    2004-07-01

    In this work you determines the repair of the lesions inductoras of Sister chromatid exchange (ICHs) generated in the cells of the salivary gland of mouse, for the treatment with the N-Methyl-N-Nitrosourea (MNU), the N-Ethyl-N-Nitrosourea (ENU), the Methyl methanesulfonate (MMS) and the Ethyl methanesulfonate (EMS) in early and slow G1 of the first one and the second cellular division, that is to say before and after the cells incorporate 5-bromine-2 -Desoxyuridine (BrdU) in the DNA. Groups witness non treaties were included with mutagen. The cells of the salivary gland repaired the generated lesions partially by the MNU, the MMS and the EMS in the 1st division, and only the lesions induced by the ENU and MMS were repaired partially in the 2nd division. The ENU generates injure that they were not repaired in the 1st division and those taken place by the EMS were little repaired in the 2nd division. The methylating agents generated but ICHs that the ethylating. One observes that the BrdU makes to the molecule of the DNA but susceptible to the damage generated by the alkylating agents that induce the formation of the ICHs. This susceptibility was incremented around 150% for the treatment with the MNU, the ENU and the MMS, on the other hand for the EMS it was 3 times minor. It is proposed that the one electronegative atom of this analog of the timine would to work as a nucleophyllic center with which the electrophyllic compounds react. (Author)

  18. Repairability during G1 of the inductor leisure of exchanges in the sister chromatid induced by alkylating agents in DNA substituted and no substituted with BUDR, in cells of the salivary gland of mouse In vivo

    International Nuclear Information System (INIS)

    Gonzalez B, F.

    2004-01-01

    In this work you determines the repair of the lesions inductoras of Sister chromatid exchange (ICHs) generated in the cells of the salivary gland of mouse, for the treatment with the N-Methyl-N-Nitrosourea (MNU), the N-Ethyl-N-Nitrosourea (ENU), the Methyl methanesulfonate (MMS) and the Ethyl methanesulfonate (EMS) in early and slow G1 of the first one and the second cellular division, that is to say before and after the cells incorporate 5-bromine-2 -Desoxyuridine (BrdU) in the DNA. Groups witness non treaties were included with mutagen. The cells of the salivary gland repaired the generated lesions partially by the MNU, the MMS and the EMS in the 1st division, and only the lesions induced by the ENU and MMS were repaired partially in the 2nd division. The ENU generates injure that they were not repaired in the 1st division and those taken place by the EMS were little repaired in the 2nd division. The methylating agents generated but ICHs that the ethylating. One observes that the BrdU makes to the molecule of the DNA but susceptible to the damage generated by the alkylating agents that induce the formation of the ICHs. This susceptibility was incremented around 150% for the treatment with the MNU, the ENU and the MMS, on the other hand for the EMS it was 3 times minor. It is proposed that the one electronegative atom of this analog of the timine would to work as a nucleophyllic center with which the electrophyllic compounds react. (Author)

  19. Mutational specificity of SOS mutagenesis

    International Nuclear Information System (INIS)

    Kato, Takeshi

    1986-01-01

    In an approach to the isolation of mutants of E. coli unable to produce mutations by ultraviolet light, the author has found new umuC-mutants. Their properties could be explained by ''SOS hypothesis of Radman and Witkin'', which has now been justified by many investigators. Analysis of the umuC region of E. coli chromosome cloned in pSK 100 has led to the conclusion that two genes, umuD and umuC, having the capacity of mutation induction express in the same mechanism as that of SOS genes, which is known to be inhibited by LexA protein bonding to ''SOS box'' found at promotor region. Suppressor analysis for mutational specificity has revealed: (i) umuDC-independent mutagens, such as EMS and (oh) 4 Cy, induce selected base substitution alone; and (ii) umuDC-dependent mutagens, such as X-rays and gamma-rays, induce various types of base substitution simultaneously, although they have mutational specificity. In the umuDC-dependent processes of basechange mutagenesis, the spectra of base substitution were a mixture of base substitution reflecting the specific base damages induced by individual mutagens and nonspecific base substitution. In conclusion, base substitution plays the most important role in umuDC-dependent mutagenesis, although mutagenesis of umuDC proteins remains uncertain. (Namekawa, K.)

  20. Mechanisms of umuC-dependent mutagenesis

    International Nuclear Information System (INIS)

    Kato, Takeji; Kitagawa, Yoshinori

    1985-01-01

    Present status of studies on umcDC genes-induced mutagenesis is introduced. Specificity of umuCD-dependent and -independent base substitution and frameshift mutagenesis is presented. Biochemical examinations of U.V.-induced umuCD gene function are described. Previous studies suggest that umuCD genes are induced by SOS inhibitory systems, that gene products are directly responsible for mutagenesis, that base substitution is largely involved in inducible mutagenesis, and that many of frameshifts are induced irrespective of gene function. (Namekawa, K.)

  1. Induced repair and mutagenesis in animal cells

    International Nuclear Information System (INIS)

    Takimoto, Koichi

    1981-01-01

    Induced repair and mutagenesis of animal cells against UV were studied in contrast with SOS repair of E. coli primarily by the use of viruses. Since UV-enhanced reactivation is a phenomenon similar to UV-reactivation (mutagenesis) and the presence of lesion bypass synthsis has been suggested, UV-enhanced reactivation has several common aspects with SOS reactivation of E. coli. However, correlation is not necessarily noted between increase in the viral survival rate and mutagenesis, nor do protease blockers exert any effect. Therefore, SOS repair of E. coli may have different mechansms from induced repair and mutagenesis in animal cells. (Ueda, J.)

  2. Radiation mutagenesis of subtropic plants

    International Nuclear Information System (INIS)

    Kerkadze, I.G.

    1987-01-01

    Possibilities of expansion of subtropic plant changeability and development of new gene bank for future selection-genetic studies are detected. New trends of radiation mutagenesis of subtropic plants are formulated as results of studies during many years. A lot of mutants is subjected to sufficient tests, and concrete results are obtained with the help of these tests for definite species. Summing genetic and selection estimations of the results, it is possible to make the conclusion that mutant selection represents one of the powerful methods of preparation of productive and qualitative species of subtropic plants, which are successfully introduced into practice

  3. Polycomb repressive complex 2 (PRC2 restricts hematopoietic stem cell activity.

    Directory of Open Access Journals (Sweden)

    Ian J Majewski

    2008-04-01

    Full Text Available Polycomb group proteins are transcriptional repressors that play a central role in the establishment and maintenance of gene expression patterns during development. Using mice with an N-ethyl-N-nitrosourea (ENU-induced mutation in Suppressor of Zeste 12 (Suz12, a core component of Polycomb Repressive Complex 2 (PRC2, we show here that loss of Suz12 function enhances hematopoietic stem cell (HSC activity. In addition to these effects on a wild-type genetic background, mutations in Suz12 are sufficient to ameliorate the stem cell defect and thrombocytopenia present in mice that lack the thrombopoietin receptor (c-Mpl. To investigate the molecular targets of the PRC2 complex in the HSC compartment, we examined changes in global patterns of gene expression in cells deficient in Suz12. We identified a distinct set of genes that are regulated by Suz12 in hematopoietic cells, including eight genes that appear to be highly responsive to PRC2 function within this compartment. These data suggest that PRC2 is required to maintain a specific gene expression pattern in hematopoiesis that is indispensable to normal stem cell function.

  4. Direct random insertion mutagenesis of Helicobacter pylori.

    NARCIS (Netherlands)

    Jonge, de R.; Bakker, D.; Vliet, van AH; Kuipers, E.J.; Vandenbroucke-Grauls, C.M.J.E.; Kusters, J.G.

    2003-01-01

    Random insertion mutagenesis is a widely used technique for the identification of bacterial virulence genes. Most strategies for random mutagenesis involve cloning in Escherichia coli for passage of plasmids or for phenotypic selection. This can result in biased selection due to restriction or

  5. Mutagenesis and carcinogenesis resulting from environment pollution

    International Nuclear Information System (INIS)

    Dimitrov, B.

    2001-01-01

    The paper reviews different ways of environmental contamination with natural and artificial harmful substances (chemical and radioactive) and their role in the processes of mutagenesis and carcinogenesis. The recent studies of the mechanism of mutagenesis and carcinogenesis due to environmental pollution are discussed

  6. Direct random insertion mutagenesis of Helicobacter pylori

    NARCIS (Netherlands)

    de Jonge, Ramon; Bakker, Dennis; van Vliet, Arnoud H. M.; Kuipers, Ernst J.; Vandenbroucke-Grauls, Christina M. J. E.; Kusters, Johannes G.

    2003-01-01

    Random insertion mutagenesis is a widely used technique for the identification of bacterial virulence genes. Most strategies for random mutagenesis involve cloning in Escherichia coli for passage of plasmids or for phenotypic selection. This can result in biased selection due to restriction or

  7. Receptor mutagenesis strategies for examination of structure-function relationships

    NARCIS (Netherlands)

    Blomenröhr, Marion; Vischer, Henry F; Bogerd, Jan

    2004-01-01

    This chapter describes three different strategies of receptor mutagenesis with their advantages, disadvantages, and limitations. Oligonucleotide-directed mutagenesis using either the Altered Sites II in vitro mutagenesis system or the GeneTailor site-directed mutagenesis system can generate base

  8. A mutation in synaptojanin 2 causes progressive hearing loss in the ENU-mutagenised mouse strain Mozart.

    Science.gov (United States)

    Manji, Shehnaaz S M; Williams, Louise H; Miller, Kerry A; Ooms, Lisa M; Bahlo, Melanie; Mitchell, Christina A; Dahl, Hans-Henrik M

    2011-03-15

    Hearing impairment is the most common sensory impairment in humans, affecting 1:1,000 births. We have identified an ENU generated mouse mutant, Mozart, with recessively inherited, non-syndromic progressive hearing loss caused by a mutation in the synaptojanin 2 (Synj2), a central regulatory enzyme in the phosphoinositide-signaling cascade. The hearing loss in Mozart is caused by a p.Asn538Lys mutation in the catalytic domain of the inositol polyphosphate 5-phosphatase synaptojanin 2. Within the cochlea, Synj2 mRNA expression was detected in the inner and outer hair cells but not in the spiral ganglion. Synj2(N538K) mutant protein showed loss of lipid phosphatase activity, and was unable to degrade phosphoinositide signaling molecules. Mutant Mozart mice (Synj2(N538K/N538K)) exhibited progressive hearing loss and showed signs of hair cell degeneration as early as two weeks of age, with fusion of stereocilia followed by complete loss of hair bundles and ultimately loss of hair cells. No changes in vestibular or neurological function, or other clinical or behavioral manifestations were apparent. Phosphoinositides are membrane associated signaling molecules that regulate many cellular processes including cell death, proliferation, actin polymerization and ion channel activity. These results reveal Synj2 as a critical regulator of hair cell survival that is essential for hair cell maintenance and hearing function.

  9. Mutagenesis: Interactions with a parallel universe.

    Science.gov (United States)

    Miller, Jeffrey H

    Unexpected observations in mutagenesis research have led to a new perspective in this personal reflection based on years of studying mutagenesis. Many mutagens have been thought to operate via a single principal mechanism, with secondary effects usually resulting in only minor changes in the observed mutation frequencies and spectra. For example, we conceive of base analogs as resulting in direct mispairing as their main mechanism of mutagenesis. Recent studies now show that in fact even these simple mutagens can cause very large and unanticipated effects both in mutation frequencies and in the mutational spectra when used in certain pair-wise combinations. Here we characterize this leap in mutation frequencies as a transport to an alternate universe of mutagenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Cellular components required for mutagenesis

    International Nuclear Information System (INIS)

    Elledge, S.J.; Perry, K.L.; Krueger, J.H.; Mitchell, B.B.; Walker, G.C.

    1983-01-01

    We have cloned the umuD and umuC genes of Escherichia coli and have shown that they code for two proteins of 16,000 and 45,000 daltons respectively; the two genes are organized in an operon that is repressed by the LexA protein. Similarly, we have shown that the mucA and mucB genes of the mutagenesis-enhancing plasmid pKM101 code for proteins of 16,000 and 45,000 daltons respectively and, like umuD/C, the genes are organized in an operon. Preliminary sequencing studies have indicated that the umuD/C and mucA/B loci are approximately 50% homologous at both the nucleic acid and deduced protein sequence levels and that the umuD gene is preceeded by two putative LexA binding sites separated by 4 basepairs. Like umuD/C, the mucA/B genes of pKM101 are induced by DNA damage and are repressed by LexA. In addition to inducing recA + lexA + -regulated din genes, DNA damaging agents such as uv and nalidixic acid also induce the heat shock proteins GroEL and DnaK in an htpR-dependent fashion. 22 references, 1 figure, 1 table

  11. [Stress-induced cellular adaptive mutagenesis].

    Science.gov (United States)

    Zhu, Linjiang; Li, Qi

    2014-04-01

    The adaptive mutations exist widely in the evolution of cells, such as antibiotic resistance mutations of pathogenic bacteria, adaptive evolution of industrial strains, and cancerization of human somatic cells. However, how these adaptive mutations are generated is still controversial. Based on the mutational analysis models under the nonlethal selection conditions, stress-induced cellular adaptive mutagenesis is proposed as a new evolutionary viewpoint. The hypothetic pathway of stress-induced mutagenesis involves several intracellular physiological responses, including DNA damages caused by accumulation of intracellular toxic chemicals, limitation of DNA MMR (mismatch repair) activity, upregulation of general stress response and activation of SOS response. These responses directly affect the accuracy of DNA replication from a high-fidelity manner to an error-prone one. The state changes of cell physiology significantly increase intracellular mutation rate and recombination activity. In addition, gene transcription under stress condition increases the instability of genome in response to DNA damage, resulting in transcription-associated DNA mutagenesis. In this review, we summarize these two molecular mechanisms of stress-induced mutagenesis and transcription-associated DNA mutagenesis to help better understand the mechanisms of adaptive mutagenesis.

  12. Therapeutic brain modulation with targeted large neutral amino acid supplements in the Pah-enu2 phenylketonuria mouse model.

    Science.gov (United States)

    van Vliet, Danique; Bruinenberg, Vibeke M; Mazzola, Priscila N; van Faassen, Martijn Hjr; de Blaauw, Pim; Pascucci, Tiziana; Puglisi-Allegra, Stefano; Kema, Ido P; Heiner-Fokkema, M Rebecca; van der Zee, Eddy A; van Spronsen, Francjan J

    2016-11-01

    Phenylketonuria treatment consists mainly of a Phe-restricted diet, which leads to suboptimal neurocognitive and psychosocial outcomes. Supplementation of large neutral amino acids (LNAAs) has been suggested as an alternative dietary treatment strategy to optimize neurocognitive outcome in phenylketonuria and has been shown to influence 3 brain pathobiochemical mechanisms in phenylketonuria, but its optimal composition has not been established. In order to provide additional pathobiochemical insight and develop optimal LNAA treatment, several targeted LNAA supplements were investigated with respect to all 3 biochemical disturbances underlying brain dysfunction in phenylketonuria. Pah-enu2 (PKU) mice received 1 of 5 different LNAA-supplemented diets beginning at postnatal day 45. Control groups included phenylketonuria mice receiving an isonitrogenic and isocaloric high-protein diet or the AIN-93M diet, and wild-type mice receiving the AIN-93M diet. After 6 wk, brain and plasma amino acid profiles and brain monoaminergic neurotransmitter concentrations were measured. Brain Phe concentrations were most effectively reduced by supplementation of LNAAs, such as Leu and Ile, with a strong affinity for the LNAA transporter type 1. Brain non-Phe LNAAs could be restored on supplementation, but unbalanced LNAA supplementation further reduced brain concentrations of those LNAAs that were not (sufficiently) included in the LNAA supplement. To optimally ameliorate brain monoaminergic neurotransmitter concentrations, LNAA supplementation should include Tyr and Trp together with LNAAs that effectively reduce brain Phe concentrations. The requirement for Tyr supplementation is higher than it is for Trp, and the relative effect of brain Phe reduction is higher for serotonin than it is for dopamine and norepinephrine. The study shows that all 3 biochemical disturbances underlying brain dysfunction in phenylketonuria can be targeted by specific LNAA supplements. The study thus

  13. Evolving artificial metalloenzymes via random mutagenesis

    Science.gov (United States)

    Yang, Hao; Swartz, Alan M.; Park, Hyun June; Srivastava, Poonam; Ellis-Guardiola, Ken; Upp, David M.; Lee, Gihoon; Belsare, Ketaki; Gu, Yifan; Zhang, Chen; Moellering, Raymond E.; Lewis, Jared C.

    2018-03-01

    Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. This improved selectivity carried over to other dirhodium-catalysed transformations, including N-H, S-H and Si-H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.

  14. Mutagenesis and repair of DNA

    International Nuclear Information System (INIS)

    Janion, C.; Grzesiuk, E.; Fabisiewicz, A.; Tudek, B.; Ciesla, J.; Graziewicz, M.; Wojcik, A.; Speina, E.

    1998-01-01

    Full text. The discovery that the mfd gene codes for a transcription-coupling repair factor (TRCF) prompted us to re-investigate the MFD (mutation frequency decline) phenomenon in E.coli K-12 strain when mutations were induced by ultraviolet light, halogen light or MMS-treatment. These studies revealed that: (i) the process of MFD involves the proofreading activity of DNA pol III and the mismatch repair system, as well as, TRCF and the UvrABC-excinuclease (ii) a semi-rich plate test may be replaced by a rich liquid medium, (iii) the T-T pyrimidine dimers are the lesions excised with the highest activity, and (iv) overproduction of UmuD(D'C) proteins leads to a great increase in mutant frequency in irradiated and MMS-treated cells. The role of mismatch repair (MR) in MMS-induced mutagenesis is obscured by the fact that the spectra of mutational specificity are different in bacteria proficient and deficient in MR. It has been found that transposons Tn10 (and Tn5) when inserted into chromosomal DNA of E. coli influence the phenotype lowering the survival and frequency of mutations induced by UV or halogen light irradiation. This is connected with a deficiency of UmuD(D') and UmuC proteins. Transformation of bacteria with plasmids bearing the umuD(D')C genes, suppresses the effects of the transposon insertion, a phenomenon which has not been described before. Single-stranded DNA of M13mp18 phage was oxidized in vitro by a hydroxyl radical generating system including hypoxanthine/xanthine oxidase/Fe3+/EDTA, and it was found that Fapy-Ade, Fapy-Gua, 8-oxyAde and thymine glycol were the main products formed. Replication of the oxidized template by T7 phage DNA polymerase, Klenow fragment of polymerase I, or polymerase beta from bovine thymus has revealed that oxidized pyrimidines are stronger blockers than oxidized purines for T7 phage and Klenow fragment polymerases and the blocking potency depends on the neighboring bases and on the type of polymerase. Studies of

  15. CD48-deficient T-lymphocytes from DMBA-treated rats have de novo mutations in the endogenous Pig-a gene.

    Science.gov (United States)

    Dobrovolsky, Vasily N; Revollo, Javier; Pearce, Mason G; Pacheco-Martinez, M Monserrat; Lin, Haixia

    2015-10-01

    A major question concerning the scientific and regulatory acceptance of the rodent red blood cell-based Pig-a gene mutation assay is the extent to which mutants identified by their phenotype in the assay are caused by mutations in the Pig-a gene. In this study, we identified T-lymphocytes deficient for the glycosylphosphatidylinositol-anchored surface marker, CD48, in control and 7,12-dimethylbenz[a]anthracene (DMBA)-treated rats using a flow cytometric assay and determined the spectra of mutations in the endogenous Pig-a gene in these cells. CD48-deficient T-cells were seeded by sorting at one cell per well into 96-well plates, expanded into clones, and exons of their genomic Pig-a were sequenced. The majority (78%) of CD48-deficient T-cell clones from DMBA-treated rats had mutations in the Pig-a gene. The spectrum of DMBA-induced Pig-a mutations was dominated by mutations at A:T, with the mutated A being on the nontranscribed strand and A → T transversion being the most frequent change. The spectrum of Pig-a mutations in DMBA-treated rats was different from the spectrum of Pig-a mutations in N-ethyl-N-nitrosourea (ENU)-treated rats, but similar to the spectrum of DMBA mutations for another endogenous X-linked gene, Hprt. Only 15% of CD48-deficient mutants from control animals contained Pig-a mutations; T-cell biology may be responsible for a relatively large fraction of false Pig-a mutant lymphocytes in control animals. Among the verified mutants from control rats, the most common were frameshifts and deletions. The differences in the spectra of spontaneous, DMBA-, and ENU-induced Pig-a mutations suggest that the flow cytometric Pig-a assay detects de novo mutation in the endogenous Pig-a gene. © 2015 Wiley Periodicals, Inc.

  16. Diet-induced obesity increases the frequency of Pig-a mutant erythrocytes in male C57BL/6J mice.

    Science.gov (United States)

    Wickliffe, Jeffrey K; Dertinger, Stephen D; Torous, Dorothea K; Avlasevich, Svetlana L; Simon-Friedt, Bridget R; Wilson, Mark J

    2016-12-01

    Obesity increases the risk of a number of chronic diseases in humans including several cancers. Biological mechanisms responsible for such increased risks are not well understood at present. Increases in systemic inflammation and oxidative stress, endogenous production of mutagenic metabolites, altered signaling in proliferative pathways, and increased sensitivity to exogenous mutagens and carcinogens are some of the potential contributing factors. We hypothesize that obesity creates an endogenously mutagenic environment in addition to increasing the sensitivity to environmental mutagens. To test this hypothesis, we examined two in vivo genotoxicity endpoints. Pig-a mutant frequencies and micronucleus frequencies were determined in blood cells in two independent experiments in 30-week old male mice reared on either a high-fat diet (60% calories from fat) that exhibit an obese phenotype or a normal-fat diet (10% calories from fat) that do not exhibit an obese phenotype. Mice were assayed again at 52 weeks of age in one of the experiments. N-ethyl-N-nitrosourea (ENU) was used as a positive mutation control in one experiment. ENU induced a robust Pig-a mutant and micronucleus response in both phenotypes. Obese, otherwise untreated mice, did not differ from non-obese mice with respect to Pig-a mutant frequencies in reticulocytes or micronucleus frequencies. However, such mice, had significantly higher and sustained Pig-a mutant frequencies (increased 2.5-3.7-fold, p obese mice (based on measurements collected at 30 weeks or 30 and 52 weeks of age). This suggests that obesity, in the absence of exposure to an exogenous mutagen, is itself mutagenic. Environ. Mol. Mutagen. 57:668-677, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Modification of Antibody Function by Mutagenesis.

    Science.gov (United States)

    Dasch, James R; Dasch, Amy L

    2017-09-01

    The ability to "fine-tune" recombinant antibodies by mutagenesis separates recombinant antibodies from hybridoma-derived antibodies because the latter are locked with respect to their properties. Recombinant antibodies can be modified to suit the application: Changes in isotype, format (e.g., scFv, Fab, bispecific antibodies), and specificity can be made once the heavy- and light-chain sequences are available. After immunoglobulin heavy and light chains for a particular antibody have been cloned, the binding site-namely, the complementarity determining regions (CDR)-can be manipulated by mutagenesis to obtain antibody variants with improved properties. The method described here is relatively simple, uses commercially available reagents, and is effective. Using the pComb3H vector, a commercial mutagenesis kit, PfuTurbo polymerase (Agilent), and two mutagenic primers, a library of phage with mutagenized heavy and light CDR3 can be obtained. © 2017 Cold Spring Harbor Laboratory Press.

  18. Photodynamic action of the methylene blue: mutagenesis and sinergism

    International Nuclear Information System (INIS)

    Capella, M.A.M.

    1988-01-01

    Two aspects of photodynamic therapy were studied: the associated mutagenesis and the interactions with physical agents, in order to increase its biological effects. The photodynamic action with methylene blue in the mutagenesis and sinergism is studied. (L.M.J.)

  19. Faux Mutagenesis: Teaching Troubleshooting through Controlled Failure

    Science.gov (United States)

    Hartberg, Yasha

    2006-01-01

    By shifting pedagogical goals from obtaining successful mutations to teaching students critical troubleshooting skills, it has been possible to introduce site-directed mutagenesis into an undergraduate teaching laboratory. Described in this study is an inexpensive laboratory exercise in which students follow a slightly modified version of…

  20. Complex epidemiological approach to human mutagenesis

    International Nuclear Information System (INIS)

    Czeizel, A.

    1980-01-01

    The main characteristics of the epidemiological approach are summarised and the criteria discussed for the adoption of this approach for the detection of human mutagenesis. Mutation monitoring systems are described and results of epidemiological studies of higher risk populations are presented. (C.F.)

  1. Target-selected mutagenesis of the rat

    NARCIS (Netherlands)

    Smits, B.M.; Mudde, J.B.; Plasterk, R.; Cuppen, E.

    2004-01-01

    The rat is one of the most extensively studied model organisms, and with its genome being sequenced, tools to manipulate gene function in vivo have become increasingly important. We here report proof of principle for target-selected mutagenesis as a reverse genetic or knockout approach for the rat.

  2. Excision repair and mutagenesis in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Kilbey, Brian

    1987-01-01

    This and succeeding letters discuss the James and Kilbey (1977 and 1978) model for the initiation of u.v. mutagenesis in Saccharomyces cerevisiae and its application to include a number of chemical mutagens. The Baranowska et al (1987) results indicating the role of DNA replication, the differing mechanisms in Escherichia coli, are all discussed. (UK)

  3. Seed mutagenesis in Portulaca grandiflora (Hook)

    International Nuclear Information System (INIS)

    Bennani, F.; Rossi-Hassani, B.D.

    2001-01-01

    Betalain pigments have been used as natural additives. Despite their importance, the biochemistry and genetics of betalain synthesis remain relatively undetermined. Portulaca grandiflora represents an ideal material for genetic analysis. In the present work, seed mutagenesis was examined with a view to enhance the chance of detection of new genetic markers in this species

  4. A mutation in the mitochondrial fission gene Dnm1l leads to cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Houman Ashrafian

    2010-06-01

    Full Text Available Mutations in a number of genes have been linked to inherited dilated cardiomyopathy (DCM. However, such mutations account for only a small proportion of the clinical cases emphasising the need for alternative discovery approaches to uncovering novel pathogenic mutations in hitherto unidentified pathways. Accordingly, as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen, we identified a mouse mutant, Python, which develops DCM. We demonstrate that the Python phenotype is attributable to a dominant fully penetrant mutation in the dynamin-1-like (Dnm1l gene, which has been shown to be critical for mitochondrial fission. The C452F mutation is in a highly conserved region of the M domain of Dnm1l that alters protein interactions in a yeast two-hybrid system, suggesting that the mutation might alter intramolecular interactions within the Dnm1l monomer. Heterozygous Python fibroblasts exhibit abnormal mitochondria and peroxisomes. Homozygosity for the mutation results in the death of embryos midway though gestation. Heterozygous Python hearts show reduced levels of mitochondria enzyme complexes and suffer from cardiac ATP depletion. The resulting energy deficiency may contribute to cardiomyopathy. This is the first demonstration that a defect in a gene involved in mitochondrial remodelling can result in cardiomyopathy, showing that the function of this gene is needed for the maintenance of normal cellular function in a relatively tissue-specific manner. This disease model attests to the importance of mitochondrial remodelling in the heart; similar defects might underlie human heart muscle disease.

  5. Real-time resolution of point mutations that cause phenovariance in mice

    Science.gov (United States)

    Wang, Tao; Zhan, Xiaowei; Bu, Chun-Hui; Lyon, Stephen; Pratt, David; Hildebrand, Sara; Choi, Jin Huk; Zhang, Zhao; Zeng, Ming; Wang, Kuan-wen; Turer, Emre; Chen, Zhe; Zhang, Duanwu; Yue, Tao; Wang, Ying; Shi, Hexin; Wang, Jianhui; Sun, Lei; SoRelle, Jeff; McAlpine, William; Hutchins, Noelle; Zhan, Xiaoming; Fina, Maggy; Gobert, Rochelle; Quan, Jiexia; Kreutzer, McKensie; Arnett, Stephanie; Hawkins, Kimberly; Leach, Ashley; Tate, Christopher; Daniel, Chad; Reyna, Carlos; Prince, Lauren; Davis, Sheila; Purrington, Joel; Bearden, Rick; Weatherly, Jennifer; White, Danielle; Russell, Jamie; Sun, Qihua; Tang, Miao; Li, Xiaohong; Scott, Lindsay; Moresco, Eva Marie Y.; McInerney, Gerald M.; Karlsson Hedestam, Gunilla B.; Xie, Yang; Beutler, Bruce

    2015-01-01

    With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled “superpedigrees” are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening. PMID:25605905

  6. A splice site mutation in laminin-α2 results in a severe muscular dystrophy and growth abnormalities in zebrafish.

    Directory of Open Access Journals (Sweden)

    Vandana A Gupta

    Full Text Available Congenital muscular dystrophy (CMD is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A is a form of CMD caused by a defect in the laminin-α2 gene (LAMA2. Laminin-α2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC complex in membranes providing stability to muscle fibers. In an N-ethyl-N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2(cl501/cl501, exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 8-15 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-α2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-α2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD.

  7. ER stress-mediated apoptosis in a new mouse model of osteogenesis imperfecta.

    Directory of Open Access Journals (Sweden)

    Thomas S Lisse

    2008-02-01

    Full Text Available Osteogenesis imperfecta is an inherited disorder characterized by increased bone fragility, fractures, and osteoporosis, and most cases are caused by mutations affecting the type I collagen genes. Here, we describe a new mouse model for Osteogenesis imperfecta termed Aga2 (abnormal gait 2 that was isolated from the Munich N-ethyl-N-nitrosourea mutagenesis program and exhibited phenotypic variability, including reduced bone mass, multiple fractures, and early lethality. The causal gene was mapped to Chromosome 11 by linkage analysis, and a C-terminal frameshift mutation was identified in the Col1a1 (procollagen type I, alpha 1 gene as the cause of the disorder. Aga2 heterozygous animals had markedly increased bone turnover and a disrupted native collagen network. Further studies showed that abnormal proalpha1(I chains accumulated intracellularly in Aga2/+ dermal fibroblasts and were poorly secreted extracellularly. This was associated with the induction of an endoplasmic reticulum stress-specific unfolded protein response involving upregulation of BiP, Hsp47, and Gadd153 with caspases-12 and -3 activation and apoptosis of osteoblasts both in vitro and in vivo. These studies resulted in the identification of a new model for Osteogenesis imperfecta, and identified a role for intracellular modulation of the endoplasmic reticulum stress-associated unfolded protein response machinery toward osteoblast apoptosis during the pathogenesis of disease.

  8. Histological Characterization of the Dicer1 Mutant Zebrafish Retina

    Directory of Open Access Journals (Sweden)

    Saeed Akhtar

    2015-01-01

    Full Text Available DICER1, a multidomain RNase III endoribonuclease, plays a critical role in microRNA (miRNA and RNA-interference (RNAi functional pathways. Loss of Dicer1 affects different developmental processes. Dicer1 is essential for retinal development and maintenance. DICER1 was recently shown to have another function of silencing the toxicity of Alu RNAs in retinal pigment epithelium (RPE cells, which are involved in the pathogenesis of age related macular degeneration. In this study, we characterized a Dicer1 mutant fish line, which carries a nonsense mutation (W1457Ter induced by N-ethyl-N-nitrosourea mutagenesis. Zebrafish DICER1 protein is highly conserved in the evolution. Zebrafish Dicer1 is expressed at the earliest stages of zebrafish development and persists into late developmental stages; it is widely expressed in adult tissues. Homozygous Dicer1 mutant fish (DICER1W1457Ter/W1457Ter have an arrest in early growth with significantly smaller eyes and are dead at 14–18 dpf. Heterozygous Dicer1 mutant fish have similar retinal structure to that of control fish; the retinal pigment epithelium (RPE cells are normal with no sign of degeneration at the age of 20 months.

  9. Paradoxical effect of aspirin on the growth of C6 rat glioma and on time of development of ENU-induced tumors of the nervous system.

    Science.gov (United States)

    Arrieta, O; Guevara, P; Reyes, S; Palencia, G; Rivera, E; Sotelo, J

    2001-11-01

    Administration of acetylsalicylic acid (ASA), an inhibitor of the synthesis of prostaglandins and thrombzoxanes, decreases the incidence of colorectal cancer and other neoplasms and inhibits in vitro some tumor growth. We studied the effect of various doses of ASA on the growth of C6 glioma implanted in rats as well as the effect of chronic administration of ASA on time of development and incidence of tumors of the central nervous system (CNS) induced by prenatal exposure to ethylnitrosourea (ENU). In a controlled study, various doses of ASA, 12.5, 25, 50, 100, 200, 300, and 400 mg/kg per day, were administered to Wistar rats in whom a subcutaneous C6 glioma had been transplanted. Changes in tumor size, histologic characteristics, mitotic index, cell proliferation, and vascular density were studied. In a parallel experiment, we administered ASA (70 mg/kg per day) to rats who were prenatally exposed to ENU; treatment started on day 50 of age, and continued until the end of the experiment at day 400. The time of tumor development as well as incidence, localization, and histological diagnosis were compared with matched controls. A paradoxical effect of ASA administration was observed on the dynamics of cell proliferation of C6 glioma. When high ASA doses were administered (200 or 400 mg/kg per day), tumor volume, cell proliferation, vascular density, and mitotic index increased. In contrast, when low doses were administered (12.5 or 25 mg/kg per day) the tumor size diminished. In the second experiment, localization and incidence of CNS tumors induced by ENU were similar in animals treated with ASA and in controls; however, in rats treated with ASA the time of tumor development was shortened. The growth-promoting effects of high doses of ASA found in the present study in both transplanted and chemically-induced brain tumors, might be due to the blockage of autocrine inhibitory factors dependent on the cyclooxygenase pathway or by increased vascular permeability and

  10. Novel Random Mutagenesis Method for Directed Evolution.

    Science.gov (United States)

    Feng, Hong; Wang, Hai-Yan; Zhao, Hong-Yan

    2017-01-01

    Directed evolution is a powerful strategy for gene mutagenesis, and has been used for protein engineering both in scientific research and in the biotechnology industry. The routine method for directed evolution was developed by Stemmer in 1994 (Stemmer, Proc Natl Acad Sci USA 91, 10747-10751, 1994; Stemmer, Nature 370, 389-391, 1994). Since then, various methods have been introduced, each of which has advantages and limitations depending upon the targeted genes and procedure. In this chapter, a novel alternative directed evolution method which combines mutagenesis PCR with dITP and fragmentation by endonuclease V is described. The kanamycin resistance gene is used as a reporter gene to verify the novel method for directed evolution. This method for directed evolution has been demonstrated to be efficient, reproducible, and easy to manipulate in practice.

  11. Genetic improvement of soybean through induced mutagenesis

    International Nuclear Information System (INIS)

    Manjaya, J.G.; Nandanwar, R.S.; Thengane, R.J.; Muthiah, A.R.

    2009-01-01

    Soybean (Glycine max (L.) Merril) is one of the important oilseed crops of India. The country produces more than 9.00 million tonnes of soybean per annum and has acquired first place amongst oilseed crops grown in India. Narrow genetic base of cultivated varieties in soybean is of global concern. Efficient mutant production systems, through physical or chemical mutagenesis, have been well established in soybean. A vast amount of genetic variability, of both quantitative and qualitative traits, has been generated through experimental mutagenesis. Two soybean varieties TAMS-38 and TAMS 98-21 have been developed and released for commercial cultivation by Bhabha Atomic Research Centre (BARC). In this paper the role of mutation breeding in soybean improvement has been discussed. (author)

  12. Mechanisms of uv mutagenesis in yeast

    International Nuclear Information System (INIS)

    Lawrence, C.W.; Christensen, R.; Schwartz, A.

    1982-01-01

    The uv mutagenesis in yeast depends on the function of the RAD6 locus, a gene that is also responsible for a substantial fraction of wild-type resistance, suggesting that this eukaryote may possess a misrepair mechanism analogous to that proposed for Escherichia coli. The molecular mechanism responsible for RAD6 repair or recovery is not yet known, but it is different from either excision or recombination-dependent repair, processes carried out by the other two main repair pathways in yeast. RAD6-dependent mutagenesis has been found to have the following characteristics. It is associated at best with only a small fraction of RAD6-dependent repair, the majority of the sensitivity of rad6 mutants being due to their lack of nonmutagenic repair. SRS2 metabolic suppressors restore a substantial fraction of uv resistance to rad6 mutants but do not restore their uv mutability. Strains containing mutations at loci (rev, umr) that are probably more directly involved in mutagenesis are only mildly sensitive, and there is a poor correlation between their sensitivity and mutational deficiency. The uv mutagenesis appears to require a large number of gene functions, perhaps ten or more. Where examined in detail, these genes have been found to be concerned in the production of only a specific range of mutational events, not all of them. Mating experiments have shown that a substantial fraction, probably 40% or more, of uv-induced mutations are untargeted, that is, occur in lesion-free regions of DNA. The uv irradiation, therefore, produces a general reduction in the normally high fidelity with which DNA is replicated on undamaged templates. It does not appear to be necessary for the causal lesion to be present in the same chromosome as the mutation it induces. The reduction in fidelity may be the consequence of the production of a diffusible factor in uv-irradiated cells, but definite evidence supporting this proposal has not yet been obtained

  13. Recovery during radiation and chemical mutagenesis

    International Nuclear Information System (INIS)

    Deen, D.F.

    1975-01-01

    These investigations were directed toward the study of recovery in radiation and chemical mutagenesis in cultured mammalian cells. A mutagenesis system was established in which mutation of V79-17lb Chinese hamster cells to 8-azaguanine resistance was tested. The effects of split dose and postirradiation treatments upon both x-ray and EMS induced mutagenesis were determined. Increasing the cell inoculum by a factor of 5 (from 10 5 to 5 x 10 5 ) decreased both the spontaneous and x-ray induced mutation frequencies by two orders of magnitude. The x-ray induced mutation frequency was found to be higher for those cells allowed to attach for 5 hours before irradiation, in comparison to those allowed to attach for 2 hours. The uv spectrum of 8-azaguanine changes as a function of storage time at low temperature, but not when diluted to either 10 μg/ml or 30 μg/ml and maintained at 37 0 C. The optimal expression time required after irradiation is dose dependent and can be determined from the relationship: E.T. = 1.93(10 -2 )D + 15.5. (E.T. = hours; D = rads). The duration of the optimal expression time can be estimated by summing the cell cycle time and the radiation induced lag time

  14. mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay.

    Directory of Open Access Journals (Sweden)

    Jennifer L Anderson

    2017-11-01

    Full Text Available As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, a low frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes. We analyzed mRNA processing in seven zebrafish lines with mutations expected to disrupt gene function, generated by CRISPR/Cas9 or ENU mutagenesis methods. Five of the seven lines showed evidence of altered mRNA processing: one through a skipped exon that did not lead to a frame shift, one through nonsense-associated splicing that did not lead to a frame shift, and three through the use of cryptic splice sites. These results highlight the need for a methodical analysis of the mRNA produced in mutant lines before making conclusions or embarking on studies that assume loss of function as a result of a given genomic change. Furthermore, recognition of the types of adaptations that can occur may inform the strategies of mutant generation.

  15. Fluorometric method of quantitative cell mutagenesis

    Science.gov (United States)

    Dolbeare, F.A.

    1980-12-12

    A method for assaying a cell culture for mutagenesis is described. A cell culture is stained first with a histochemical stain, and then a fluorescent stain. Normal cells in the culture are stained by both the histochemical and fluorescent stains, while abnormal cells are stained only by the fluorescent stain. The two stains are chosen so that the histochemical stain absorbs the wavelengths that the fluorescent stain emits. After the counterstained culture is subjected to exciting light, the fluorescence from the abnormal cells is detected.

  16. Molecular mechanisms of mutagenesis determined by the recombinant DNA technology

    International Nuclear Information System (INIS)

    Lee, W.R.

    1985-01-01

    A study of the alteration of the DNA in the mutant gene can determine mechanisms of mutation by distinguishing between mutations induced by transition, transversion, frameshifts of a single base and deletions involving many base pairs. The association of a specific pattern of response with a mutagen will permit detecting mutants induced by the mutagen with a reduced background by removing mutations induced by other mechanisms from the pool of potential mutants. From analyses of studies that have been conducted, it is quite apparent that there are substantial differences among mutagens in their modes of action. Of 31 x-ray induced mutants, 20 were large deletions while only 3 showed normal Southern blots. Only one mutant produced a sub-unit polypeptide of normal molecular weight and charge in the in vivo test whereas in vitro synthesis produced a second one. In contrast, nine of thirteen EMS induced mutants produced cross-reacting proteins with sub-unit polypeptide molecular weights equivalent to wild type. Two of three ENU induced mutants recently analyzed in our laboratory produced protein with sub-unit polypeptide molecular weight and electrical charge similar to the wild type stock in which the mutants were induced. One ENU induced mutation is a large deletion. 21 refs., 1 fig

  17. Nitric oxide donors attenuate clongenic potential in rat C6 glioma cells treated with alkylating chemotherapeutic agents.

    Science.gov (United States)

    Yang, Jir-Jei; Yin, Jiu-Haw; Yang, Ding-I

    2007-05-11

    1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) kills tumor cells via multiple actions including alkylation and carbamoylation. Previously, we have reported that formation of S-nitrosoglutathione (GSNO) in glioma cells overexpressing inducible nitric oxide synthase (iNOS) contributed to nitric oxide (NO)-dependent carbamoylating chemoresistance against BCNU. To further characterize the effects of NO on alkylating cytotoxicity, colony formation assay was applied to evaluate the effects of various NO donors on rat C6 glioma cells challenged with alkylating agents. We demonstrate that NO donors including GSNO, diethylamine NONOate (DEA/NO), and sodium nitroprusside (SNP) substantially reduced the extent of colony formation in glioma cells treated with alkylating agents, namely methyl methanesulfonate (MMS), N-methyl-N-nitrosourea (MNU), and N-ethyl-N-nitrosourea (ENU). Without alkylating agents these NO-releasing agents alone had no effects on clongenic potential of rat C6 glioma cells. Among these three NO donors used, the effectiveness in potentiating alkylating cytotoxicity is in the order of "GSNO>DEA/NO>SNP" when applied at the same dosages. GSNO also exerted similar synergistic actions reducing the extents of colony formation when co-administrated with 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-hydrazine (compound #1), another alkylating agent that mimics the chloroethylating action of BCNU. Together with our previous findings, we propose that NO donors may be used as adjunct chemotherapy with alkylating agents for such malignant brain tumors as glioblastoma multiforme (GBM). In contrast, production of NO as a result of iNOS induction, such as that occurring after surgical resection of brain tumors, may compromise the efficacy of carbamoylating chemotherapy.

  18. Expression in mammalian cells of the Escherichia coli O6 alkylguanine-DNA-alkyltransferase gene ogt reduces the toxicity of alkylnitrosoureas.

    Science.gov (United States)

    Harris, L. C.; Margison, G. P.

    1993-01-01

    V79 Chinese hamster cells expressing either the O6-alkylguanine-DNA-alkyltransferase (ATase) encoded by the E. coli ogt gene or a truncated version of the E. coli ada gene have been exposed to various alkylnitrosoureas to investigate the contribution of ATase repairable lesions to the toxicity of these compounds. Both ATases are able to repair O6-alkylguanine (O6-AlkG) and O4-alkylthymine (O4-AlkT) but the ogt ATase is more efficient in the repair of O4-methylthymine (O4-MeT) and higher alkyl derivatives of O6-AlkG than is the ada ATase. Expression of the ogt ATase provided greater protection against the toxic effects of the alkylating agents then the ada ATase particularly with N-ethyl-N-nitrosourea (ENU) and N-butyl-N-nitrosourea (BNU) to which the ada ATase expressing cells were as sensitive as parent vector transfected cells. Although ogt was expressed at slightly higher levels than the truncated ada in the transfected cells, this could not account for the differential protection observed. For-N-methyl-N-nitrosourea (MNU) the increased protection in ogt-transfected cells is consistent with O4-MeT acting as a toxic lesion. For the longer chain alkylating agents and chloroethylating agents, the protection afforded by the ogt protein may be a consequence of the more efficient repair of O6-AlkG, O4-AlkT or both of these lesions in comparison with the ada-encoded ATase. Images Figure 2 Figure 3 PMID:8512805

  19. Evaluation of Tumor Angiogenesis with a Second-Generation US Contrast Medium in a Rat Breast Tumor Model

    International Nuclear Information System (INIS)

    Ko, Eun Young; Lee, Sang Hoon; Kim, Hak Hee; Kim, Sung Moon; Shin, Myung Jin; Kim, Nam Kug; Gong, Gyung Yub

    2008-01-01

    Tumor angiogenesis is an important factor for tumor growth, treatment response and prognosis. Noninvasive imaging methods for the evaluation of tumor angiogenesis have been studied, but a method for the quantification of tumor angiogenesis has not been established. This study was designed to evaluate tumor angiogenesis in a rat breast tumor model by the use of a contrast enhanced ultrasound (US) examination with a second-generation US contrast agent. The alkylating agent 19N-ethyl-N-nitrosourea (ENU) was injected into the intraperitoneal cavity of 30-day-old female Sprague-Dawley rats. Three to four months later, breast tumors were detected along the mammary lines of the rats. A total of 17 breast tumors larger than 1 cm in nine rats were evaluated by gray-scale US, color Doppler US and contrast-enhanced US using SonoVue. The results were recorded as digital video images; time-intensity curves and hemodynamic parameters were analyzed. Pathological breast tumor specimens were obtained just after the US examinations. The tumor specimens were stained with hematoxylin and eosin (H and E) and the expression of CD31, an endothelial cell marker, was determined by immunohistochemical staining. We also evaluated the pathological diagnosis of the tumors and the microvessel density (MVD). Spearman's correlation and the Kruskal-Wallis test were used for the analysis. The pathological diagnoses were 11 invasive ductal carcinomas and six benign intraductal epithelial proliferations. The MVD did not correlate with the pathological diagnosis. However, blood volume (BV) showed a statistically significant correlation with MVD (Spearman's correlation, p < 0.05). Contrast-enhanced US using a second-generation US contrast material was useful for the evaluation of tumor angiogenesis of breast tumors in the rat

  20. Novel allelic mutations in murine Serca2 induce differential development of squamous cell tumors

    Energy Technology Data Exchange (ETDEWEB)

    Toki, Hideaki; Minowa, Osamu; Inoue, Maki; Motegi, Hiromi; Karashima, Yuko; Ikeda, Ami [Team for Advanced Development and Evaluation of Human Disease Models, Riken BioResource Center (BRC), Tsukuba, Ibaraki (Japan); Kaneda, Hideki [Technology and Development Team for Mouse Phenotype Analysis, Riken BRC, Tsukuba, Ibaraki (Japan); Sakuraba, Yoshiyuki [Mutagenesis and Genomics Team, Riken BRC, Tsukuba, Ibaraki (Japan); Saiki, Yuriko [Department of Molecular Pathology, Tohoku University Graduate School of Medicine, Sendai, Miyagi (Japan); Wakana, Shigeharu [Technology and Development Team for Mouse Phenotype Analysis, Riken BRC, Tsukuba, Ibaraki (Japan); Suzuki, Hiroshi [Department of Biochemistry, Asahikawa Medical University, Asahikawa, Hokkaido (Japan); Gondo, Yoichi [Mutagenesis and Genomics Team, Riken BRC, Tsukuba, Ibaraki (Japan); Shiroishi, Toshihiko [Mammalian Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka (Japan); Noda, Tetsuo, E-mail: tnoda@jfcr.or.jp [Team for Advanced Development and Evaluation of Human Disease Models, Riken BioResource Center (BRC), Tsukuba, Ibaraki (Japan); Department of Cell Biology, Cancer Institute, The Japanese Foundation for Cancer Research, Tokyo (Japan)

    2016-08-05

    Dominant mutations in the Serca2 gene, which encodes sarco(endo)plasmic reticulum calcium-ATPase, predispose mice to gastrointestinal epithelial carcinoma [1–4] and humans to Darier disease (DD) [14–17]. In this study, we generated mice harboring N-ethyl-N-nitrosourea (ENU)-induced allelic mutations in Serca2: three missense mutations and one nonsense mutation. Mice harboring these Serca2 mutations developed tumors that were categorized as either early onset squamous cell tumors (SCT), with development similar to null-type knockout mice [2,4] (aggressive form; M682, M814), or late onset tumors (mild form; M1049, M1162). Molecular analysis showed no aberration in Serca2 mRNA or protein expression levels in normal esophageal cells of any of the four mutant heterozygotes. There was no loss of heterozygosity at the Serca2 locus in the squamous cell carcinomas in any of the four lines. The effect of each mutation on Ca{sup 2+}-ATPase activity was predicted using atomic-structure models and accumulated mutated protein studies, suggesting that putative complete loss of Serca2 enzymatic activity may lead to early tumor onset, whereas mutations in which Serca2 retains residual enzymatic activity result in late onset. We propose that impaired Serca2 gene product activity has a long-term effect on squamous cell carcinogenesis from onset to the final carcinoma stage through an as-yet unrecognized but common regulatory pathway. -- Highlights: •Novel mutations in murine Serca2 caused early onset or late onset of tumorigenesis. •They also caused higher or lower incidence of Darier Disease phenotype. •3D structure model suggested the former mutations led to severer defect on ATPase. •Driver gene mutations via long-range effect on Ca2+ distributions are suggested.

  1. B-1a transitional cells are phenotypically distinct and are lacking in mice deficient in IκBNS

    Science.gov (United States)

    Pedersen, Gabriel K.; Àdori, Monika; Khoenkhoen, Sharesta; Dosenovic, Pia; Beutler, Bruce; Karlsson Hedestam, Gunilla B.

    2014-01-01

    B-1 cells mediate early protection against infection by responding to T cell-independent (TI) antigens found on the surface of various pathogens. Mice with impaired expression of the atypical IκB protein IκBNS have markedly reduced frequencies of B-1 cells. We used a mouse strain with dysfunctional IκBNS derived from an N-ethyl-N-nitrosourea (ENU) screen, named bumble, to investigate the point in the development of B-1 cells where IκBNS is required. The presence of wild-type (wt) peritoneal cells in mixed wt/bumble chimeras did not rescue the development of bumble B-1 cells, but wt peritoneal cells transferred to bumble mice restored natural IgM levels and response to TI antigens. The bumble and wt mice displayed similar levels of fetal liver B-1 progenitors and splenic neonatal transitional B (TrB) cells, both of which were previously shown to give rise to B-1 cells. Interestingly, we found that a subset of wt neonatal TrB cells expressed common B-1a markers (TrB-1a) and that this cell population was absent in the bumble neonatal spleen. Sorted TrB-1a (CD93+IgM+CD5+) cells exclusively generated B-1a cells when adoptively transferred, whereas sorted CD93+IgM+CD5− cells gave rise to B-2 cells and, to a lesser extent, B-1b and B-1a cells. This study identifies a phenotypically distinct splenic population of TrB-1a cells and establishes that the development of B-1a cells is blocked before this stage in the absence of IκBNS. PMID:25228759

  2. The role of misrepair in experimental mutagenesis

    International Nuclear Information System (INIS)

    Yamaguchi, Hikoyuki

    1983-01-01

    Mutagenesis is classified as being either mispairing occuring at the time of chromosome replication or as being misrepair occuring when damaged nucleotides are converted to the paired ones. In the cell population of the root meristem of barley which is considered to be steadystate, the possibility of the selective segregation of the newly synthesized and the older template strands of DNA at mitosis was studied by the incorporation of 3 H-thymidine. Stochastic removal of de novo synthesized DNA strand to a zone of non-dividing cell population was unlikely. Thus, it has been concluded that there is special mechanisms for protecting the integrity of the DNA by removing the mispairing lesions. Barly seeds first exposed to a low level γ-radiation before treating with ethylmethane sulfonate. Survival rate of M 1 plants as well as mutation frequency of M 2 were higher for the combined treatment than for single treatment of chemical mutagen. A mutational response of barley cell to DNA damaging agent was much affected by a previous treatment with mutagens. It is suggested that in the experimental mutagenesis misrepair plays rather an important role than mispairing. (author)

  3. New mutations affecting induced mutagenesis in yeast.

    Science.gov (United States)

    Lawrence, C W; Krauss, B R; Christensen, R B

    1985-01-01

    Previously isolated mutations in baker's yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions. To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4-38, and have identified 10 mutants that give much reduced yields of revertants. Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6. The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4+ and REV5+ gene functions are only remotely concerned with this process. The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction. The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.

  4. Laboratory of Mutagenesis and DNA Repair

    International Nuclear Information System (INIS)

    2000-01-01

    Full text: Two main lines of research were continued: the first one concerned the mechanisms controlling the fidelity of DNA replication in Escherichia coli; the second concerned cellular responses of Saccharomyces cerevisiae to DNA damaging agents. We have been investigating the question whether during chromosomal DNA replication in Escherichia coli the two DNA strands may be replicated with differential accuracy. To address this question we set up a new system that allows the examination of mutagenesis either of the leading strand or the lagging strand. Our results suggest that the lagging strand replication of the E. coli chromosome may be more accurate than leading strand replication. More recently, we studied mutagenesis of the two strands in recA730 strains which exhibit constitutive expression of the SOS system. Our results clearly indicate that in recA730 strains there is a significant difference in the fidelity of replication between the two replicating strands. Based on our data we propose a model describing a possible mechanism of SOS mutagenesis. To get more insight into cellular responses to DNA damage we have isolated several novel genes of S. cerevisiae, the transcription of which is induced by DNA lesions. Main effort was concentrated on the characterization of the DIN7 gene. We found that Din7p specifically affects the metabolism of mitochondrial DNA (mtDNA). The elevated level of Din7p results in an increased frequency of mitochondrial petite mutants, as well as in a higher frequency of mitochondrial point mutations. Din7p affects also the stability of microsatellite sequences present in the mitochondrial genome. As expected, Din7p was found to be located in mitochondria. In another project, we found that the DIN8 gene isolated in our laboratory is identical with the UMP1 gene encoding a chaperone-like protein involved in 20S proteasome maturation. Interestingly, induction of UMP1 expression in response to DNA damage is subject to regulation

  5. Radiation mutagenesis in selection of apple trees

    International Nuclear Information System (INIS)

    Kolontaev, V.M.; Kolontaev, Yu.V.

    1977-01-01

    After X-radiation of grafts of antonovka apple trees, three groups of morphological mutants, namely, weak-, average- and violently-growing, have been revealed. Although the mutation spectrum has some indefinite character a dose of 6 kR causes, more frequently and in a greater number, the weak-growing mutants, and a dose of 2 kR, the violently-growing ones. Mutants of each group differ in the precociousness (precocious and latefruiting), type of fruiting (nospur and spur) and yield (high- and low-yielding). Using the method of radiation mutagenesis it is possible to rise the frequency and spectrum of somatic mutability of antonovka apple trees and to induce forms having valuable features

  6. Efficient Mutagenesis Independent of Ligation (EMILI).

    Science.gov (United States)

    Füzik, Tibor; Ulbrich, Pavel; Ruml, Tomáš

    2014-11-01

    Site-directed mutagenesis is one of the most widely used techniques in life sciences. Here we describe an improved and simplified method for introducing mutations at desired sites. It consists of an inverse PCR using a plasmid template and two partially complementary primers. The synthesis step is followed by annealing of the PCR product's sticky ends, which are generated by exonuclease digestion. This method is fast, extremely efficient and cost-effective. It can be used to introduce large insertions and deletions, but also for multiple point mutations in a single step. To show the principle and to prove the efficiency of the method, we present a series of basic mutations (insertions, deletions, point mutations) on pUC19 plasmid DNA. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Scoring function to predict solubility mutagenesis

    Directory of Open Access Journals (Sweden)

    Deutsch Christopher

    2010-10-01

    Full Text Available Abstract Background Mutagenesis is commonly used to engineer proteins with desirable properties not present in the wild type (WT protein, such as increased or decreased stability, reactivity, or solubility. Experimentalists often have to choose a small subset of mutations from a large number of candidates to obtain the desired change, and computational techniques are invaluable to make the choices. While several such methods have been proposed to predict stability and reactivity mutagenesis, solubility has not received much attention. Results We use concepts from computational geometry to define a three body scoring function that predicts the change in protein solubility due to mutations. The scoring function captures both sequence and structure information. By exploring the literature, we have assembled a substantial database of 137 single- and multiple-point solubility mutations. Our database is the largest such collection with structural information known so far. We optimize the scoring function using linear programming (LP methods to derive its weights based on training. Starting with default values of 1, we find weights in the range [0,2] so that predictions of increase or decrease in solubility are optimized. We compare the LP method to the standard machine learning techniques of support vector machines (SVM and the Lasso. Using statistics for leave-one-out (LOO, 10-fold, and 3-fold cross validations (CV for training and prediction, we demonstrate that the LP method performs the best overall. For the LOOCV, the LP method has an overall accuracy of 81%. Availability Executables of programs, tables of weights, and datasets of mutants are available from the following web page: http://www.wsu.edu/~kbala/OptSolMut.html.

  8. Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

    Science.gov (United States)

    Burger, Alexa; Lindsay, Helen; Felker, Anastasia; Hess, Christopher; Anders, Carolin; Chiavacci, Elena; Zaugg, Jonas; Weber, Lukas M; Catena, Raul; Jinek, Martin; Robinson, Mark D; Mosimann, Christian

    2016-06-01

    CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo. © 2016. Published by The Company of Biologists Ltd.

  9. Heat shock and herpes virus: enhanced reactivation without untargeted mutagenesis

    International Nuclear Information System (INIS)

    Lytle, C.D.; Carney, P.G.

    1988-01-01

    Enhanced reactivation of Ultraviolet-irradiated virus has been reported to occur in heat-shocked host cells. Since enhanced virus reactivation is often accompanied by untargeted mutagenesis, we investigated whether such mutagenesis would occur for herpes simplex virus (HSV) in CV-1 monkey kidney cells subjected to heat shock. In addition to expressing enhanced reactivation, the treated cells were transiently more susceptible to infection by unirradiated HSV. No mutagenesis of unirradiated HSV was found whether infection occurred at the time of increased susceptibility to infection or during expression of enhanced viral reactivation

  10. DNA MUTAGENESIS IN PANAX GINSENG CELL CULTURES

    Directory of Open Access Journals (Sweden)

    Kiselev K.V.

    2012-08-01

    Full Text Available At the present time, it is well documented that plant tissue culture induces a number of mutations and chromosome rearrangements termed “somaclonal variations”. However, little is known about the nature and the molecular mechanisms of the tissue culture-induced mutagenesis and the effects of long-term subculturing on the rate and specific features of the mutagenesis. The aim of the present study was to investigate and compare DNA mutagenesis in different genes of Panax ginseng callus cultures of different age. It has previously been shown that the nucleotide sequences of the Agrobacterium rhizogenes rolC locus and the selective marker nptII developed mutations during long-term cultivation of transgenic cell cultures of P. ginseng. In the present work, we analyzed nucleotide sequences of selected plant gene families in a 2-year-old and 20-year-old P. ginseng 1c cell culture and in leaves of cultivated P. ginseng plants. We analysed sequence variability between the Actin genes, which are a family of house-keeping genes; the phenylalanine ammonia-lyase (PAL and dammarenediol synthase (DDS genes, which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinase (SERK genes, which control plant development. The frequency of point mutations in the Actin, PAL, DDS, and SERK genes in the 2-year-old callus culture was markedly higher than that in cultivated plants but lower than that in the 20-year-old callus culture of P. ginseng. Most of the mutations in the 2- and 20-year-old P. ginseng calli were A↔G and T↔C transitions. The number of nonsynonymous mutations was higher in the 2- and 20-year-old callus cultures than the number of nonsynonymous mutations in the cultivated plants of P. ginseng. Interestingly, the total number of N→G or N→C substitutions in the analyzed genes was 1.6 times higher than the total number of N→A or N→T substitutions. Using methylation-sensitive DNA fragmentation

  11. Favipiravir elicits antiviral mutagenesis during virus replication in vivo.

    Science.gov (United States)

    Arias, Armando; Thorne, Lucy; Goodfellow, Ian

    2014-10-21

    Lethal mutagenesis has emerged as a novel potential therapeutic approach to treat viral infections. Several studies have demonstrated that increases in the high mutation rates inherent to RNA viruses lead to viral extinction in cell culture, but evidence during infections in vivo is limited. In this study, we show that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection. Increased mutation frequencies were observed in samples from treated mice and were accompanied with lower or in some cases undetectable levels of infectious virus in faeces and tissues. Viral RNA isolated from treated animals showed reduced infectivity, a feature of populations approaching extinction during antiviral mutagenesis. These results suggest that favipiravir can induce norovirus mutagenesis in vivo, which in some cases leads to virus extinction, providing a proof-of-principle for the use of favipiravir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses.

  12. Effective mutagenesis of Arabidopsis by heavy ion beam-irradiation

    International Nuclear Information System (INIS)

    Yamamoto, Y.Y.; Saito, H.; Ryuto, H.; Fukunishi, N.; Yoshida, S.; Abe, T.

    2005-01-01

    Full text: Arabidopsis researches frequently include the genetic approach, so efficient, convenient, and safe methods for mutagenesis are required. Currently, the most popular method for in house mutagenesis is application of EMS. Although this method is very effective, its base substitution-type mutations often gives leaky mutants with residual gene functions, leading some difficulty in understanding the corresponding gene functions. Heavy ion beam generated by accelerators gives highest energy transfer rates among known radiation-based mutagenesis methods including X ray, gamma ray, fast neutron, electron and proton irradiation. This feature is thought to give high frequency of the double strand break of genomic DNA and resultant short deletions, resulting frame shift-type mutations. At RIKEN Accelerator Research Facility (RARF, http://www.rarf.riken.go.jp/index-e.html), we have optimized conditions for effective mutagenesis of Arabidopsis regarding to ion species and irradiation dose, and achieved comparable mutation rates to the method with EMS. (author)

  13. Symposium on molecular and cellular mechanisms of mutagenesis

    International Nuclear Information System (INIS)

    1981-01-01

    These proceedings contain abstracts only of the 21 papers presented at the Sympsoium. The papers dealt with molecular mechanisms of mutagenesis and cellular responses to chemical and physical mutagenic agents

  14. Symposium on molecular and cellular mechanisms of mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    1981-01-01

    These proceedings contain abstracts only of the 21 papers presented at the Sympsoium. The papers dealt with molecular mechanisms of mutagenesis and cellular responses to chemical and physical mutagenic agents. (ERB)

  15. Mutagenesis and mathematics: The allure of numbers

    International Nuclear Information System (INIS)

    Haynes, R.H.

    1989-01-01

    This paper sets out the formal, empirical, and mechanistic equations that my colleagues and I have developed for the description and analysis of dose-response data on the lethal and genetic effects of mutagens in microorganisms. These three types of equations are interrelated inasmuch as they are all based ultimately on the use of the Poisson distribution in the formal definition of lethal and mutational hit functions. Explicit mathematical expressions for these functions can be written down in either empirical or mechanistic terms. The empirical equations are obtained simply by writing the hit functions as finite polynomials with adjustable coefficients. The mechanistic equations are based on the assumptions of the DNA damage-repair hypothesis. The mathematical formulation of this hypothesis entails an important change in the definition of the word hit from that used in the classical hit/target theory of radiation biology. The theoretical and practical applications of these various equations in mutation research are summarized briefly and their merits are assessed in light of recent advances in our understanding of the biochemical basis of mutagenesis

  16. The Roles of UmuD in Regulating Mutagenesis

    Directory of Open Access Journals (Sweden)

    Jaylene N. Ollivierre

    2010-01-01

    Full Text Available All organisms are subject to DNA damage from both endogenous and environmental sources. DNA damage that is not fully repaired can lead to mutations. Mutagenesis is now understood to be an active process, in part facilitated by lower-fidelity DNA polymerases that replicate DNA in an error-prone manner. Y-family DNA polymerases, found throughout all domains of life, are characterized by their lower fidelity on undamaged DNA and their specialized ability to copy damaged DNA. Two E. coli Y-family DNA polymerases are responsible for copying damaged DNA as well as for mutagenesis. These DNA polymerases interact with different forms of UmuD, a dynamic protein that regulates mutagenesis. The UmuD gene products, regulated by the SOS response, exist in two principal forms: UmuD2, which prevents mutagenesis, and UmuD2′, which facilitates UV-induced mutagenesis. This paper focuses on the multiple conformations of the UmuD gene products and how their protein interactions regulate mutagenesis.

  17. Empirical complexities in the genetic foundations of lethal mutagenesis.

    Science.gov (United States)

    Bull, James J; Joyce, Paul; Gladstone, Eric; Molineux, Ian J

    2013-10-01

    From population genetics theory, elevating the mutation rate of a large population should progressively reduce average fitness. If the fitness decline is large enough, the population will go extinct in a process known as lethal mutagenesis. Lethal mutagenesis has been endorsed in the virology literature as a promising approach to viral treatment, and several in vitro studies have forced viral extinction with high doses of mutagenic drugs. Yet only one empirical study has tested the genetic models underlying lethal mutagenesis, and the theory failed on even a qualitative level. Here we provide a new level of analysis of lethal mutagenesis by developing and evaluating models specifically tailored to empirical systems that may be used to test the theory. We first quantify a bias in the estimation of a critical parameter and consider whether that bias underlies the previously observed lack of concordance between theory and experiment. We then consider a seemingly ideal protocol that avoids this bias-mutagenesis of virions-but find that it is hampered by other problems. Finally, results that reveal difficulties in the mere interpretation of mutations assayed from double-strand genomes are derived. Our analyses expose unanticipated complexities in testing the theory. Nevertheless, the previous failure of the theory to predict experimental outcomes appears to reside in evolutionary mechanisms neglected by the theory (e.g., beneficial mutations) rather than from a mismatch between the empirical setup and model assumptions. This interpretation raises the specter that naive attempts at lethal mutagenesis may augment adaptation rather than retard it.

  18. Environmental stress induces trinucleotide repeat mutagenesis in human cells.

    Science.gov (United States)

    Chatterjee, Nimrat; Lin, Yunfu; Santillan, Beatriz A; Yotnda, Patricia; Wilson, John H

    2015-03-24

    The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)-the cause of multiple human diseases-have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential.

  19. Enamelin (Enam) is essential for amelogenesis: ENU-induced mouse mutants as models for different clinical subtypes of human amelogenesis imperfecta (AI).

    Science.gov (United States)

    Masuya, Hiroshi; Shimizu, Kunihiko; Sezutsu, Hideki; Sakuraba, Yoshiyuki; Nagano, Junko; Shimizu, Aya; Fujimoto, Naomi; Kawai, Akiko; Miura, Ikuo; Kaneda, Hideki; Kobayashi, Kimio; Ishijima, Junko; Maeda, Takahide; Gondo, Yoichi; Noda, Tetsuo; Wakana, Shigeharu; Shiroishi, Toshihiko

    2005-03-01

    Amelogenesis imperfecta (AI) is a group of commonly inherited defects of dental enamel formation, which exhibits marked genetic and clinical heterogeneity. The genetic basis of this heterogeneity is still poorly understood. Enamelin, the affected gene product in one form of AI (AIH2), is an extracellular matrix protein that is one of the components of enamel. We isolated three ENU-induced dominant mouse mutations, M100395, M100514 and M100521, which caused AI-like phenotypes in the incisors and molars of the affected individuals. Linkage analyses mapped each of the three mutations to a region of chromosome 5 that contained the genes encoding enamelin (Enam) and ameloblastin (Ambn). Sequence analysis revealed that each mutation was a single-base substitution in Enam. M100395 (Enam(Rgsc395)) and M100514 (Enam(Rgsc514)) were putative missense mutations that caused S to I and E to G substitutions at positions 55 and 57 of the translated protein, respectively. Enam(Rgsc395) and Enam(Rgsc514) heterozygotes showed severe breakage of the enamel surface, a phenotype that resembled local hypoplastic AI. The M100521 mutation (Enam(Rgsc521)) was a T to A substitution at the splicing donor site in intron 4. This mutation resulted in a frameshift that gave rise to a premature stop codon. The transcript of the Enam(Rgsc521) mutant allele was degraded, indicating that Enam(Rgsc521) is a loss-of-function mutation. Enam(Rgsc521) heterozygotes showed a hypomaturation-type AI phenotype in the incisors, possibly due to haploinsufficiency of Enam. Enam(Rgsc521) homozygotes showed complete loss of enamel on the incisors and the molars. Thus, we report here that the Enam gene is essential for amelogenesis, and that mice with different point mutations at Enam may provide good animal models to study the different clinical subtypes of AI.

  20. Genome-Wide Mutagenesis in Borrelia burgdorferi.

    Science.gov (United States)

    Lin, Tao; Gao, Lihui

    2018-01-01

    Signature-tagged mutagenesis (STM) is a functional genomics approach to identify bacterial virulence determinants and virulence factors by simultaneously screening multiple mutants in a single host animal, and has been utilized extensively for the study of bacterial pathogenesis, host-pathogen interactions, and spirochete and tick biology. The signature-tagged transposon mutagenesis has been developed to investigate virulence determinants and pathogenesis of Borrelia burgdorferi. Mutants in genes important in virulence are identified by negative selection in which the mutants fail to colonize or disseminate in the animal host and tick vector. STM procedure combined with Luminex Flex ® Map™ technology and next-generation sequencing (e.g., Tn-seq) are the powerful high-throughput tools for the determination of Borrelia burgdorferi virulence determinants. The assessment of multiple tissue sites and two DNA resources at two different time points using Luminex Flex ® Map™ technology provides a robust data set. B. burgdorferi transposon mutant screening indicates that a high proportion of genes are the novel virulence determinants that are required for mouse and tick infection. In this protocol, an effective signature-tagged Himar1-based transposon suicide vector was developed and used to generate a sequence-defined library of nearly 4800 mutants in the infectious B. burgdorferi B31 clone. In STM, signature-tagged suicide vectors are constructed by inserting unique DNA sequences (tags) into the transposable elements. The signature-tagged transposon mutants are generated when transposon suicide vectors are transformed into an infectious B. burgdorferi clone, and the transposable element is transposed into the 5'-TA-3' sequence in the B. burgdorferi genome with the signature tag. The transposon library is created and consists of many sub-libraries, each sub-library has several hundreds of mutants with same tags. A group of mice or ticks are infected with a mixed

  1. Improvement of soybean variety 'Bragg' through mutagenesis

    International Nuclear Information System (INIS)

    Bhatnagar, P.S.; Prabhakar; Tiwari, S.P.; Sandhu, J.S.

    1989-01-01

    Full text: Variety 'Bragg' (Jackson x D49-2491) of soybean (Glycine max. (L.) Merrill) was found to be high yielding and widely adaptable throughout India. Its yield stability, however, is unsatisfactory, probably due to low germinability necessitating use of higher seed rate. With the main objective to rectify this defect, mutagenesis involving chemical as well as physical mutagens was used. Dry seeds were treated with EMS or MMS (0.2, 0.4 and 0.6%), or gamma rays (15, 20 and 25 kR) with and without additional exposure to UV (2 hrs at 260 nm) in 1982. In M 2 , a mutation frequency ranging from 2.24 to 22.85% was observed. Screening of M 2 and of subsequent generations yielded a broad spectrum of mutations. Some of the mutants are agronomically useful. Among them, mutant 'T 2 14' resulting from 25 kR gamma rays + UV, was found to possess better germinability (+15%), earliness (5 days) and high yield during both rainy and post-rainy seasons in 1986 and 1987, when compared with the parent variety 'Bragg'. The mutant has smaller seed-size (TGW 125 g) than the parent (145 g). In soybean, large-seeded varieties were reported to have poorer seed germinability. Thus, the better germinability of the mutant might be related to its reduced seed size. Seeds of the mutant show a light brown colour of the hilum in contrast to the black hilum of 'Bragg'. In other characters the mutant is similar to 'Bragg'. The mutant should have potential for commercial cultivation in India. For confirmation of its agronomically superior performance, it is undergoing national evaluation in multilocational trials under 'All India Co-ordinated Research Project on Soybean (ICAR)'. The strain has been named 'NRC-2'. (author)

  2. Improvement of soybean variety 'Bragg' through mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Bhatnagar, P S; Prabhakar,; Tiwari, S P; Sandhu, J S [National Research Centre for Soybean, Indore (India)

    1989-01-01

    Full text: Variety 'Bragg' (Jackson x D49-2491) of soybean (Glycine max. (L.) Merrill) was found to be high yielding and widely adaptable throughout India. Its yield stability, however, is unsatisfactory, probably due to low germinability necessitating use of higher seed rate. With the main objective to rectify this defect, mutagenesis involving chemical as well as physical mutagens was used. Dry seeds were treated with EMS or MMS (0.2, 0.4 and 0.6%), or gamma rays (15, 20 and 25 kR) with and without additional exposure to UV (2 hrs at 260 nm) in 1982. In M{sub 2}, a mutation frequency ranging from 2.24 to 22.85% was observed. Screening of M{sub 2} and of subsequent generations yielded a broad spectrum of mutations. Some of the mutants are agronomically useful. Among them, mutant 'T{sub 2}14' resulting from 25 kR gamma rays + UV, was found to possess better germinability (+15%), earliness (5 days) and high yield during both rainy and post-rainy seasons in 1986 and 1987, when compared with the parent variety 'Bragg'. The mutant has smaller seed-size (TGW 125 g) than the parent (145 g). In soybean, large-seeded varieties were reported to have poorer seed germinability. Thus, the better germinability of the mutant might be related to its reduced seed size. Seeds of the mutant show a light brown colour of the hilum in contrast to the black hilum of 'Bragg'. In other characters the mutant is similar to 'Bragg'. The mutant should have potential for commercial cultivation in India. For confirmation of its agronomically superior performance, it is undergoing national evaluation in multilocational trials under 'All India Co-ordinated Research Project on Soybean (ICAR)'. The strain has been named 'NRC-2'. (author)

  3. Mutagenesis as a breeding method in lentil

    International Nuclear Information System (INIS)

    Mihor, M.; Stoyanova, M.; Mehandjiev, A.

    2001-01-01

    Full text: Mutagenesis was used to develop cultivars with good adaptability to exogenous factors and with increased productivity. By means of this alternative breeding procedure, increases in biological and nutritive value of the seeds were studied. To increase genetic variability in lentil (Lens culinaris Medic.) breeding material, experimental mutagenesis was applied parallel to conventional breeding methods. The aim was to characterize the mutant lines as well as determine whether some of them could be directly registered as cultivars or as gene donors in breeding programme. Within the period 1993-1996, eight mutant lentil lines were studied under field conditions. They were obtained as a result of gamma rays ( 60 Co) and ethyl methanesulfonate (EMS) treatment of the small seeded cultivar 'Tadjikskaya 95'. Air-dried seeds were treated. During the vegetative stage, phenological observation was made. The structural elements of productivity were established by biometrical analysis of 25-30 plants from each of the variants. Phytopathological evaluations were made using the scoring procedure established by ICARDA. Protein content was determined by the Kiejdhal method. The technological qualities of the seeds were determined using the method of Tretyakova and Ustinova. The mutant lines differed considerably in their biological traits from the parent cultivar. The vegetative period ranged from 84 to 89 days. The mutant lines were latermaturing than parent variety Tadjikskaya 95 by 1-5 days. As a result of mutagen treatment, the range in plant height was expanded from 1 to 8.3 cm. Line 96-8, obtained after irradiation with gamma rays, was the tallest (40.3 cm). Lodging of the mutant lines was greater than that of the initial cultivar and ranged from 20.0 to 66.7%. The trait varied to a great extent depending on environmental conditions. Mutagenic treatments also caused changes in seed size and seed coat colour. Development of resistance to important diseases of lentil

  4. Evaluation of the E mu-pim-1 transgenic mouse model for short-term carcinogenicity testing

    DEFF Research Database (Denmark)

    van Kreijl, C. F.; van Oordt, C. W. V.; Kroese, E. D.

    1998-01-01

    of T-cell lymphomas. Because of the low incidence of spontaneous tumors and the increased sensitivity to N-ethyl-N-nitrosourea-induced carcinogenesis, E mu-pim-1 mice were suggested to be one of the first potential and attractive candidates to be used in short-term carcinogenicity testing...

  5. Mutagenesis of metal compounds in bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Nishioka, H

    1974-01-01

    The mutagenic activity of 41 metal compounds was examined by applying the Rec-assay method with Bacillus subtilis H17 (rec/sup +/) and M45 (rec/sup -/) strains. Among these compounds, Na/sub 2/HAsO/sub 4/, CdCl/sub 2/, K/sub 2/CrO/sub 4/, K/sub 2/Cr/sub 2/O/sub 7/, CH/sub 3/HgCl, C/sub 2/H/sub 5/HgCl, CH/sub 3/COOHgC/sub 6/H/sub 5/, MnCl/sub 2/, MnNO/sub 3/, MnSO/sub 4/, Mn(CH/sub 3/COO)/sub 2/, (NH/sub 4/)/sub 2/MoO/sub 4/ and KMoO/sub 4/ showed positive results. The reactions of K/sub 2/Cr/sub 2/O/sub 7/ and (NH/sub 4/)/sub 2/MoO/sub 4/ were especially strong in the assay. Therefore, mutation induction to reversion (try/sup +/) and streptomycin resistance (SM/sup r/) of E. coli B/r WP2 try/sup -/ (hcl/sup +/ and hcr/sup -/) by the two compounds were examined by the following two experimental procedures. Stationary phase bacteria were exposed to the compounds at high concentrations (6.9 x 10/sup -3/ approx. 3.44 x 10/sup -2/M) in M9 buffer for 15 min at 37/sup -/ with shaking. After incubation at 37/sup 0/ for 48 h visible colonies on the plates were scored. Bacteria in M9 buffer were plated in media supplemented with low concentrations (1.7 x 10/sup -5/ approx. 3.4 x 10/sup -5/M) of the compounds. K/sub 2/Cr/sub 2/O/sub 7/ and (NH/sub 4/)/sub 2/MoO/sub 4/ increased the mutation rate of SM/sup r/ and try/sup +/ in both strains treated with either procedure. No marked differences in mutation rate were found between hcr/sup +/ and hcr/sup -/. After treatment with high concentrations of compounds one can imagine that a peroxidation state produced by these peroxides in the media might affect the killing and mutation induction. These results suggest the possibility that the mutagenesis of the metals relate to their atomic values, rather than the peroxidation state as far as these two compounds are concerned.

  6. Genetic analysis of gamma-ray mutagenesis in yeast. II. Allele-specific control of mutagenesis

    International Nuclear Information System (INIS)

    McKee, R.H.; Lawrence, C.W.

    1979-01-01

    We find that partially different sets of gene functions are required for the production of different kinds of mutations induced by 60 Co γ rays in Saccharomyces cerevisiae. This observation is very similar to others made previously with respect to uv mutagenesis and confirms the conclusion that such distinctive patterns of genetic control reflect properties of the test alleles and their genetic locations, rather than the kinds of lesions required to revert them. The data also support the model of mutagenic repair outlined in the first paper of this series in which partially different sets of gene functions are required for the production of different kinds of mutations, the formation of mutations at different genetic sites and the induction of mutations by different mutagens

  7. Use of gamma-radiation and ethyl nitrosourea independently and in combination in experimental mutagenesis in peas. Pt. 1

    International Nuclear Information System (INIS)

    Lejtao, Zh.; Petkova, S.; Dzhondzhurov, L.

    1987-01-01

    Dry seeds from field peas cv. Uladovskij 303 were treated for three years with gamma rays (280 to 250 rad/min) and with the powerful chemical mutagen/carcinogen ethyl nitrosourea - ENU (solution prepared in phosphate buffer, ph=6.0). The seeds were exposed to ENU for 6 h, washed out for 30 min and dried for 30 min. The following qualitative indices were investigated: emergence; survival; sterility; lethality; vegetation period and plant's height one month after sowing. They could be used successfully to determine the injurious effects of the mutagens. A clear-cut dependence on the used dose and concentration was observed independently if the mutagens were applied separately or in combination. Quantitative indices determined at the end of the vegetation: branches per plant, sterile nodes, nodes per plant, number of pods and seeds per plant as well as plant's height did not show any clear-cut tendency when treatments were made in combination. Their determination, in field conditions, seemed to be without significance. As theoretically it was expected, pre-radiation use of ENU leads to a stronger injurious effect in comparison to that of post-radiation treatment. Concerning induced leaf-spots (leaf aberrations) in M 1 -generation, the results were in complete disagreement with the theory: pre-radiation treatment with ENU (at combination 3.12 mM ENU + 12 krad), which provoked a very high injury (lethality - 80-90%), showed a very little percent of plants with a high level of leaf aberrations. On the contrary, post-radiation treatment with ENU (lethality - 40-60%) caused a high level of leaf aberrations in greater number of plants. The presence of 'gamma plantlets' phenomenon is proposed for possible explanation. 3 tabs.; 9 refs

  8. Suppression of radiation mutagenesis by dactinomycin in Chinese hamster cells

    International Nuclear Information System (INIS)

    Tokita, N.; Capenter, S.G.; Chen, D.J.; MacInnes, M.A.; Raju, M.R.

    1985-01-01

    Dactinomycin (AMD) suppression of radiation mutagenesis was investigated using an in vitro mutation assay (6-thioguanine resistance) in Chinese hamster ovary cells. Cells were exposed to acute single doses of x rays followed by 1 hr-treatment with 0.1 or 1 μg/ml AMD. The cell survival curves plotted as a function of x-ray doses were similar for radiation alone and radiation plus AMD. The results suggest that AMD treatment was only slightly mutagenic, however, when given immediately after irradiation, it suppressed radiatiion mutagenesis at higher x-ray dose regions (below 10% survival levels). Higher AMD concentrations appeared more suppressive than lower concentrations. Dose-response data analyzed based on Poisson distribution models suggest the stochastic dependence of x-ray mutagenesis and AMD cytotoxity

  9. A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering.

    Science.gov (United States)

    Belsare, Ketaki D; Andorfer, Mary C; Cardenas, Frida S; Chael, Julia R; Park, Hyun June; Lewis, Jared C

    2017-03-17

    Directed evolution is a powerful tool for optimizing enzymes, and mutagenesis methods that improve enzyme library quality can significantly expedite the evolution process. Here, we report a simple method for targeted combinatorial codon mutagenesis (CCM). To demonstrate the utility of this method for protein engineering, CCM libraries were constructed for cytochrome P450 BM3 , pfu prolyl oligopeptidase, and the flavin-dependent halogenase RebH; 10-26 sites were targeted for codon mutagenesis in each of these enzymes, and libraries with a tunable average of 1-7 codon mutations per gene were generated. Each of these libraries provided improved enzymes for their respective transformations, which highlights the generality, simplicity, and tunability of CCM for targeted protein engineering.

  10. Back to the future: revisiting HIV-1 lethal mutagenesis

    Science.gov (United States)

    Dapp, Michael J.; Patterson, Steven E.; Mansky, Louis M.

    2012-01-01

    The concept of eliminating HIV-1 infectivity by elevating the viral mutation rate was first proposed over a decade ago, even though the general concept had been conceived earlier for RNA viruses. Lethal mutagenesis was originally viewed as a novel chemotherapeutic approach for treating HIV-1 infection in which use of a viral mutagen would over multiple rounds of replication lead to the lethal accumulation of mutations, rendering the virus population non infectious – known as the slow mutation accumulation model. There have been limitations in obtaining good efficacy data with drug leads, leaving some doubt into clinical translation. More recent studies of the APOBEC3 proteins as well as new progress in the use of nucleoside analogs for inducing lethal mutagenesis have helped to refocus attention on rapid induction of HIV-1 lethal mutagenesis in a single or limited number of replication cycles leading to a rapid mutation accumulation model. PMID:23195922

  11. CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

    Science.gov (United States)

    Xue, Zhaoyu; Wu, Menghua; Wen, Kejia; Ren, Menda; Long, Li; Zhang, Xuedi; Gao, Guanjun

    2014-09-05

    Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila. Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency. Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. Furthermore, by including multiple guide RNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. The CRISPR/Cas9-mediated conditional mutagenesis system provides a simple and effective tool for gene function analysis, and complements the existing RNAi approach. Copyright © 2014 Xue et al.

  12. Mechanisms of mutagenesis of E. coli by ultraviolet light

    International Nuclear Information System (INIS)

    Hutchinson, F.; Wood, R.D.

    1986-01-01

    This summary shows that uv mutagenesis involves several processes and several types of mutations. It is important to know, if some step or event affects, say, uv-induced reversion of a his mutant, what kinds of mutation cause the reversion. More, if reversion of the mutant is not affected, it is essential to know what kinds of mutation are involved, because statements can only be made about these mutations, and not about uv mutagenesis in general. It is also clear that the spectrum of mutations will depend on dose. Thus, extrapolation from experimental data at high dose to low dose situations involve considerations both of numbers and of kinds of mutations. Extrapolation of these results to other organisms may be particularly difficult because the SOS functions play such a large role in uv mutagenesis of E. coli. 34 refs., 1 tab

  13. Genetic modifications of established varieties of potato through mutagenesis

    International Nuclear Information System (INIS)

    Brown, C.R.

    1984-01-01

    Owing to the high intercrossability of improved clones with primitive cultivars and many wild species there is little justification for use of induced mutations in potato to increase variability per se. Modification of certain traits while leaving the genotype basically intact is a promising use of mutagenesis in potato. The successful curing of defects in clones will depend on the establishment a priori of three principles. First, the clones undergoing mutagenesis should be well established varieties tolerant or resistant to the major biotic and abiotic stresses in the area of cultivation. The yield and culinary quality should also be considered high. Second, there should exist some indication that the variation desired is induceable, either through reports of natural intra-clone variation or previous mutagenesis studies. Third, initial screening should be done in virus-free materials

  14. Theories of Lethal Mutagenesis: From Error Catastrophe to Lethal Defection.

    Science.gov (United States)

    Tejero, Héctor; Montero, Francisco; Nuño, Juan Carlos

    2016-01-01

    RNA viruses get extinct in a process called lethal mutagenesis when subjected to an increase in their mutation rate, for instance, by the action of mutagenic drugs. Several approaches have been proposed to understand this phenomenon. The extinction of RNA viruses by increased mutational pressure was inspired by the concept of the error threshold. The now classic quasispecies model predicts the existence of a limit to the mutation rate beyond which the genetic information of the wild type could not be efficiently transmitted to the next generation. This limit was called the error threshold, and for mutation rates larger than this threshold, the quasispecies was said to enter into error catastrophe. This transition has been assumed to foster the extinction of the whole population. Alternative explanations of lethal mutagenesis have been proposed recently. In the first place, a distinction is made between the error threshold and the extinction threshold, the mutation rate beyond which a population gets extinct. Extinction is explained from the effect the mutation rate has, throughout the mutational load, on the reproductive ability of the whole population. Secondly, lethal defection takes also into account the effect of interactions within mutant spectra, which have been shown to be determinant for the understanding the extinction of RNA virus due to an augmented mutational pressure. Nonetheless, some relevant issues concerning lethal mutagenesis are not completely understood yet, as so survival of the flattest, i.e. the development of resistance to lethal mutagenesis by evolving towards mutationally more robust regions of sequence space, or sublethal mutagenesis, i.e., the increase of the mutation rate below the extinction threshold which may boost the adaptability of RNA virus, increasing their ability to develop resistance to drugs (including mutagens). A better design of antiviral therapies will still require an improvement of our knowledge about lethal

  15. Genetic and physiological factors affecting repair and mutagenesis in yeast

    Energy Technology Data Exchange (ETDEWEB)

    Lemontt, J F

    1979-01-01

    Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data, and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various pathways of repair are described particularly in relation to their involvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as cell age, DNA replication, and the regulatory state of the mating-type locus, are shown to also play a role in repair and mutagenesis.

  16. Genetic and physiological factors affecting repair and mutagenesis in yeast

    Energy Technology Data Exchange (ETDEWEB)

    Lemontt, J F

    1979-01-01

    Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various pathways of repair are described, particularly in relation to their imvolvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as cell age, DNA replication, and the regulatory state of the mating-type locus are shown to also play a role in repair and mutagenesis.

  17. Effective lethal mutagenesis of influenza virus by three nucleoside analogs.

    Science.gov (United States)

    Pauly, Matthew D; Lauring, Adam S

    2015-04-01

    Lethal mutagenesis is a broad-spectrum antiviral strategy that exploits the high mutation rate and low mutational tolerance of many RNA viruses. This approach uses mutagenic drugs to increase viral mutation rates and burden viral populations with mutations that reduce the number of infectious progeny. We investigated the effectiveness of lethal mutagenesis as a strategy against influenza virus using three nucleoside analogs, ribavirin, 5-azacytidine, and 5-fluorouracil. All three drugs were active against a panel of seasonal H3N2 and laboratory-adapted H1N1 strains. We found that each drug increased the frequency of mutations in influenza virus populations and decreased the virus' specific infectivity, indicating a mutagenic mode of action. We were able to drive viral populations to extinction by passaging influenza virus in the presence of each drug, indicating that complete lethal mutagenesis of influenza virus populations can be achieved when a sufficient mutational burden is applied. Population-wide resistance to these mutagenic agents did not arise after serial passage of influenza virus populations in sublethal concentrations of drug. Sequencing of these drug-passaged viral populations revealed genome-wide accumulation of mutations at low frequency. The replicative capacity of drug-passaged populations was reduced at higher multiplicities of infection, suggesting the presence of defective interfering particles and a possible barrier to the evolution of resistance. Together, our data suggest that lethal mutagenesis may be a particularly effective therapeutic approach with a high genetic barrier to resistance for influenza virus. Influenza virus is an RNA virus that causes significant morbidity and mortality during annual epidemics. Novel therapies for RNA viruses are needed due to the ease with which these viruses evolve resistance to existing therapeutics. Lethal mutagenesis is a broad-spectrum strategy that exploits the high mutation rate and the low

  18. Tissue culture regeneration and radiation induced mutagenesis in banana

    International Nuclear Information System (INIS)

    Kulkarni, V.M.; Ganapathi, T.R.

    2009-01-01

    Radiation induced mutagenesis is an important tool for banana genetic improvement. At BARC, protocols for shoo-tip multiplication of commercial banana varieties have been developed and transferred to user agencies for commercial production. Excellent embryogenic cell suspensions were established in banana cvs. Rasthali and Rajeli, and were maintained at low temperatures for long-term storage. Normal plantlets were successfully regenerated from these cell suspensions. The cell suspensions and shoot-tip cultures were gamma-irradiated for mutagenesis. The mutagenized populations were field screened and a few interesting mutants have been isolated. The existence of genetic variation was confirmed using DNA markers. Further evaluation of these mutants is in progress. (author)

  19. Towards controlled mutagenesis with transposons Ac and Tam3

    Energy Technology Data Exchange (ETDEWEB)

    Haring, M; Veken, J; Windrich, R; Kneppers, T; Rommens, C; Nijkamp, H J.J.; Hille, J [Department of Genetics, Free University, Amsterdam (Netherlands)

    1990-01-01

    Full text: The discovery of mobile genetic elements in plants has permitted the use of these transposons for insertional mutagenesis. This applies so far only to Zea mays and Antirrhinum majus, because other plant transposable elements have not been characterised so thoroughly at the genetic and the molecular level. To establish whether transposons (Ac from maize and Tam3 from Antirrhinum) remain mobile in heterologous hosts, either in somatic tissue or after meiosis, a phenotypic assay system for transposition was developed. The separation of the two transposition functions will allow controlled mutagenesis of plant genes. Our results indicate that both transposable elements remain active in heterologous hosts. (author)

  20. Mutagenesis of Trichoderma Viride by Ultraviolet and Plasma

    International Nuclear Information System (INIS)

    Yao Risheng; Li Manman; Deng Shengsong; Hu Huajia; Wang Huai; Li Fenghe

    2012-01-01

    Considering the importance of a microbial strain capable of increased cellulase production, a mutant strain UP4 of Trichoderma viride was developed by ultraviolet (UV) and plasma mutation. The mutant produced a 21.0 IU/mL FPase which was 98.1% higher than that of the parent strain Trichoderma viride ZY-1. In addition, the effect of ultraviolet and plasma mutagenesis was not merely simple superimposition of single ultraviolet mutation and single plasma mutation. Meanwhile, there appeared a capsule around some of the spores after the ultraviolet and plasma treatment, namely, the spore surface of the strain became fuzzy after ultraviolet or ultraviolet and plasma mutagenesis.

  1. Mutagenesis of Trichoderma Viride by Ultraviolet and Plasma

    Science.gov (United States)

    Yao, Risheng; Li, Manman; Deng, Shengsong; Hu, Huajia; Wang, Huai; Li, Fenghe

    2012-04-01

    Considering the importance of a microbial strain capable of increased cellulase production, a mutant strain UP4 of Trichoderma viride was developed by ultraviolet (UV) and plasma mutation. The mutant produced a 21.0 IU/mL FPase which was 98.1% higher than that of the parent strain Trichoderma viride ZY-1. In addition, the effect of ultraviolet and plasma mutagenesis was not merely simple superimposition of single ultraviolet mutation and single plasma mutation. Meanwhile, there appeared a capsule around some of the spores after the ultraviolet and plasma treatment, namely, the spore surface of the strain became fuzzy after ultraviolet or ultraviolet and plasma mutagenesis.

  2. Genetic and physiological factors affecting repair and mutagenesis in yeast

    International Nuclear Information System (INIS)

    Lemontt, J.F.

    1979-01-01

    Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data, and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various pathways of repair are described particularly in relation to their involvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as cell age, DNA replication, and the regulatory state of the mating-type locus, are shown to also play a role in repair and mutagenesis

  3. Genetic and physiological factors affecting repair and mutagenesis in yeast

    International Nuclear Information System (INIS)

    Lemontt, J.F.

    1979-01-01

    Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various pathways of repair are described, particularly in relation to their imvolvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as cell age, DNA replication, and the regulatory state of the mating-type locus are shown to also play a role in repair and mutagenesis

  4. A Review on Microbial Mutagenesis through Gamma Irradiation for Agricultural Applications

    International Nuclear Information System (INIS)

    Hoe, P.C.K.; Khairuddin Abdul Rahim

    2016-01-01

    Gamma irradiation is widely used in sterilization and mutagenesis, especially for plant breeding and crop protection. Microbial mutagenesis through gamma irradiation is mainly applied in fermentation industry. In agriculture, gamma irradiation is mostly applied in crop improvement. Microbial mutagenesis is mainly applied against fungus and spore-forming bacteria, which are resistant to gamma irradiation. Response of microbes to gamma irradiation varies and depends on various factors. Review of previous works on gamma irradiation for microbial mutagenesis in agriculture may provide some information for the use of this method. The general view on gamma irradiation, its application, and mutagenesis are discussed in this paper. Further investigation on microbial mutagenesis should consider molecular changes, information on which is lacking in previous works. Moreover, studies on microbial mutagenesis are still lacking in Malaysia despite having several gamma irradiation facilities. Therefore, further studies on microbial mutagenesis should be conducted. (author)

  5. Methods for targetted mutagenesis in gram-positive bacteria

    Science.gov (United States)

    Yang, Yunfeng

    2014-05-27

    The present invention provides a method of targeted mutagenesis in Gram-positive bacteria. In particular, the present invention provides a method that effectively integrates a suicide integrative vector into a target gene in the chromosome of a Gram-positive bacterium, resulting in inactivation of the target gene.

  6. Insertional mutagenesis using Tnt1 retrotransposon in potato

    Science.gov (United States)

    Potato is the third most important food crop in the world. However, genetics and genomics research of potato has lagged behind many major crop species due to its autotetraploidy and a highly heterogeneous genome. Insertional mutagenesis using T-DNA or transposable elements, which is available in sev...

  7. CYTOTOXICITY AND MUTAGENESIS METHODS FOR EVALUATING TOXICITY REMOVAL FROM WASTEWATERS

    Science.gov (United States)

    This project was a feasibility study of the effectiveness of a mammalian cell cytotoxicity assay and a mammalian cell mutagenesis assay for monitoring the toxicity and mutagenicity of influent and effluent wastewater at treatment plants. In the cytotoxicity assay, ambient samples...

  8. What Can a Micronucleus Teach? Learning about Environmental Mutagenesis

    Science.gov (United States)

    Linde, Ana R.; Garcia-Vazquez, Eva

    2009-01-01

    The micronucleus test is widely employed in environmental health research. It can also be an excellent tool for learning important concepts in environmental health. In this article we present an inquiry-based laboratory exercise where students explore several theoretical and practical aspects of environmental mutagenesis employing the micronucleus…

  9. p21-ras effector domain mutants constructed by "cassette" mutagenesis

    DEFF Research Database (Denmark)

    Stone, J C; Vass, W C; Willumsen, B M

    1988-01-01

    A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis...

  10. The European dimension for the mouse genome mutagenesis

    Czech Academy of Sciences Publication Activity Database

    Auwerx, J.; Avner, P.; Baldock, R.; Ballabio, A.; Balling, R.; Barbacid, M.; Berns, A.; Bradley, A.; Brown, S.; Carmeliet, P.; Chambon, P.; Cox, R.; Davidson, D.; Davies, K.; Duboule, D.; Forejt, Jiří; Granucci, F.; Hastie, N.; Angelis, M. H. de; Jackson, I.; Kioussis, D.; Kollias, G.; Lathrop, M.; Lendahl, U.; Malumbres, M.; von Melchner, H.; Müller, W.; Partanen, J.; Ricciardi-Castagnoli, P.; Rigby, P.; Rosen, B.; Rosenthal, N.; Skarnes, B.; Stewart, A. F.; Thornton, J.; Tocchini-Valentini, G.; Wagner, E.; Wahli, W.; Wurst, W.

    2004-01-01

    Roč. 16, - (2004), s. 925-927 ISSN 1061-4036 R&D Projects: GA MŠk(CZ) LN00A079 Institutional research plan: CEZ:AV0Z5052915 Keywords : The European Mouse Mutagenesis Consortium Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 24.695, year: 2004

  11. Integrating structural and mutagenesis data to elucidate GPCR ligand binding

    DEFF Research Database (Denmark)

    Munk, Christian; Harpsøe, Kasper; Hauser, Alexander S

    2016-01-01

    is reported that exhibit activity through multiple receptors, binding in allosteric sites, and bias towards different intracellular signalling pathways. Furthermore, a wealth of single point mutants has accumulated in literature and public databases. Integrating these structural and mutagenesis data will help...

  12. Model building of a thermolysin-like protease by mutagenesis

    NARCIS (Netherlands)

    Frigerio, F; Margarit, [No Value; Nogarotto, R; Grandi, G; Vriend, G; Hardy, F; Veltman, OR; Venema, G; Eijsink, VGH

    The present study concerns the use of site-directed mutagenesis experiments to optimize a three-dimensional model of the neutral protease of Bacillus subtilis (NP-sub), An initial model of NP-sub was constructed using the crystal structures of the homologous neutral proteases of Bacillus

  13. Targeted mutagenesis using CRISPR/Cas in inbred potatoes

    Science.gov (United States)

    Targeted mutagenesis using sequence-specific nucleases (SSNs) has been well established in several important crop species, but is in need of improvement in potato (Solanum tuberosum L.). For over a century, potatoes have been bred as autotetraploids (2n = 4x = 48), relying on F1 selections and clona...

  14. A mutation in Nischarin causes otitis media via LIMK1 and NF-κB pathways.

    Directory of Open Access Journals (Sweden)

    Michael Crompton

    2017-08-01

    Full Text Available Otitis media (OM, inflammation of the middle ear (ME, is a common cause of conductive hearing impairment. Despite the importance of the disease, the aetiology of chronic and recurrent forms of middle ear inflammatory disease remains poorly understood. Studies of the human population suggest that there is a significant genetic component predisposing to the development of chronic OM, although the underlying genes are largely unknown. Using N-ethyl-N-nitrosourea mutagenesis we identified a recessive mouse mutant, edison, that spontaneously develops a conductive hearing loss due to chronic OM. The causal mutation was identified as a missense change, L972P, in the Nischarin (NISCH gene. edison mice develop a serous or granulocytic effusion, increasingly macrophage and neutrophil rich with age, along with a thickened, inflamed mucoperiosteum. We also identified a second hypomorphic allele, V33A, with only modest increases in auditory thresholds and reduced incidence of OM. NISCH interacts with several proteins, including ITGA5 that is thought to have a role in modulating VEGF-induced angiogenesis and vascularization. We identified a significant genetic interaction between Nisch and Itga5; mice heterozygous for Itga5-null and homozygous for edison mutations display a significantly increased penetrance and severity of chronic OM. In order to understand the pathological mechanisms underlying the OM phenotype, we studied interacting partners to NISCH along with downstream signalling molecules in the middle ear epithelia of edison mouse. Our analysis implicates PAK1 and RAC1, and downstream signalling in LIMK1 and NF-κB pathways in the development of chronic OM.

  15. Male Hypogonadism and Germ Cell Loss Caused by a Mutation in Polo-Like Kinase 4

    Science.gov (United States)

    Harris, Rebecca M.; Weiss, Jeffrey

    2011-01-01

    The genetic etiologies of male infertility remain largely unknown. To identify genes potentially involved in spermatogenesis and male infertility, we performed genome-wide mutagenesis in mice with N-ethyl-N-nitrosourea and identified a line with dominant hypogonadism and patchy germ cell loss. Genomic mapping and DNA sequence analysis identified a novel heterozygous missense mutation in the kinase domain of Polo-like kinase 4 (Plk4), altering an isoleucine to asparagine at residue 242 (I242N). Genetic complementation studies using a gene trap line with disruption in the Plk4 locus confirmed that the putative Plk4 missense mutation was causative. Plk4 is known to be involved in centriole formation and cell cycle progression. However, a specific role in mammalian spermatogenesis has not been examined. PLK4 was highly expressed in the testes both pre- and postnatally. In the adult, PLK4 expression was first detected in stage VIII pachytene spermatocytes and was present through step 16 elongated spermatids. Because the homozygous Plk4I242N/I242N mutation was embryonic lethal, all analyses were performed using the heterozygous Plk4+/I242N mice. Testis size was reduced by 17%, and histology revealed discrete regions of germ cell loss, leaving only Sertoli cells in these defective tubules. Testis cord formation (embryonic day 13.5) was normal. Testis histology was also normal at postnatal day (P)1, but germ cell loss was detected at P10 and subsequent ages. We conclude that the I242N heterozygous mutation in PLK4 is causative for patchy germ cell loss beginning at P10, suggesting a role for PLK4 during the initiation of spermatogenesis. PMID:21791561

  16. Targeted mutagenesis in sea urchin embryos using TALENs.

    Science.gov (United States)

    Hosoi, Sayaka; Sakuma, Tetsushi; Sakamoto, Naoaki; Yamamoto, Takashi

    2014-01-01

    Genome editing with engineered nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has been reported in various animals. We previously described ZFN-mediated targeted mutagenesis and insertion of reporter genes in sea urchin embryos. In this study, we demonstrate that TALENs can induce mutagenesis at specific genomic loci of sea urchin embryos. Injection of TALEN mRNAs targeting the HpEts transcription factor into fertilized eggs resulted in the impairment of skeletogenesis. Sequence analyses of the mutations showed that deletions and/or insertions occurred at the HpEts target site in the TALEN mRNAs-injected embryos. The results suggest that targeted gene disruption using TALENs is feasible in sea urchin embryos. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  17. Study of UV-induced mutagenesis in Bacillus subtilis

    International Nuclear Information System (INIS)

    Filippov, V.D.; Lotareva, O.V.

    1978-01-01

    The mechanism of UV-induced mutagenesis was studied in Bacillus subtilis departing from the assumption that a lower yield of UV-induced mutations should be found in mutants deficient in the recombination if production of mutations is coupled with the recombination process. Three recombination-deficient strains were used: two (recA and recF) with defects in different recombination pathways and the third (recB) has a block at a stage common for both of them. UV light induced reversions to prototrophy in recB cells and did not in recA and recF strains. Direct mutations, which confer to the cell additional growth requirements, were induced by UV light in recA and recF mutants. It is concluded that UV-induced mutagenesis in B subtilis is independent of the two known recombination mechanisms

  18. Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases.

    Science.gov (United States)

    Ishida, Kentaro; Gee, Peter; Hotta, Akitsu

    2015-10-16

    Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspersed short palindromic repeats associated protein-9 (CRISPR-Cas9), hold tremendous potential for applications in the clinical setting to treat genetic diseases or prevent infectious diseases. However, because the accuracy of DNA recognition by these nucleases is not always perfect, off-target mutagenesis may result in undesirable adverse events in treated patients such as cellular toxicity or tumorigenesis. Therefore, designing nucleases and analyzing their activity must be carefully evaluated to minimize off-target mutagenesis. Furthermore, rigorous genomic testing will be important to ensure the integrity of nuclease modified cells. In this review, we provide an overview of available nuclease designing platforms, nuclease engineering approaches to minimize off-target activity, and methods to evaluate both on- and off-target cleavage of CRISPR-Cas9.

  19. Mutagenesis breeding research of Lactobacillus brevis of nitrite reduction

    Directory of Open Access Journals (Sweden)

    LI Zeli

    2015-10-01

    Full Text Available The pollution of nitrite in food became one of the focus of food safety issues,the use of biotechnology methods degrading nitrite became hotspot.The primitive strain was Lactobacillus brevis C2,preserved in our laboratory,had the ability to degrade nitrite,through composite mutagenesis of 15 W,254 nm,20 cm ultraviolet mutagenesis (UV for 120 s and 0.8% diethyl sulfate(DES in 37℃ mutation for 40 min,after screening,we successfully obtained high efficient strain of nitrite degradation,named UV6-DS2,relative to the starting strain,under the condition of 400 mg/L nitrite,after 12 h degradation,nitrite degradation rate increased from 92.8% to 97.8%,to explore its application in food was able to effectively reduce concentration of nitrite in food.

  20. Molecular techniques as complementary tools in orchid mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Mohd Nazir Basiran; Sakinah Ariffin [Malaysian Institute for Nuclear Technology Research (MINT), Bangi (Malaysia)

    2002-02-01

    Orchid breeders have always been dependent on hybridization technology to produce new orchid hybrids and varieties. The technology has proven very reliable and easy to use and has produced wide range of successful cultivars with attractive combinations of spray length, bud number, flower colour and form, vase life, fragrance, seasonality, and compactness. By introducing mutagenesis however, wide variations of flower colours, form and size can still be obtained in addition to overcoming the problem of sexual incompatibility and sterility. In addition, complementary use of molecular techniques will allow breeders to target more specific characteristic changes and cut short breeding time. PCR-based techniques used to analyse the DNA of mutagenic clones found polymorphic fragments that can be developed as molecular markers. This paper describes how mutagenesis and molecular techniques can be used to enhance orchid breeding efforts. (author)

  1. Transcriptional mutagenesis: causes and involvement in tumor development

    Science.gov (United States)

    Brégeon, Damien; Doetsch, Paul W.

    2013-01-01

    The majority of normal cells in a human do not multiply continuously but are quiescent and devote most of their energy to gene transcription. When DNA damages in the transcribed strand of an active gene are bypassed by an RNA polymerase, they can miscode at the damaged site and produce mutant transcripts. This process known as transcriptional mutagenesis can lead to the production of mutant proteins that could be important in tumor development. PMID:21346784

  2. Photodynamic action of methylene blue: mutagenesis and synergism

    International Nuclear Information System (INIS)

    Capella, M.A.M.

    1988-01-01

    The associated mutagenesis and the interactions with physical agents in order to potencialize its biological effects are studied. The induction of mutation in bacterias due to photodynamic action of methylene blue is presented as well as the induction of single breaks in bacterial DNA and the relationship between the repair systems, especially the SOS one. The interaction of the photodynamic therapy with low intensity electric current is discussed. (M.A.C.) [pt

  3. Himar1 Transposon for Efficient Random Mutagenesis in Aggregatibacter actinomycetemcomitans

    Directory of Open Access Journals (Sweden)

    Qinfeng Ding

    2017-09-01

    Full Text Available Aggregatibacter actinomycetemcomitans is the primary etiological agent of aggressive periodontal disease. Identification of novel virulence factors at the genome-wide level is hindered by lack of efficient genetic tools to perform mutagenesis in this organism. The Himar1 mariner transposon is known to yield a random distribution of insertions in an organism’s genome with requirement for only a TA dinucleotide target and is independent of host-specific factors. However, the utility of this system in A. actinomycetemcomitans is unknown. In this study, we found that Himar1 transposon mutagenesis occurs at a high frequency (×10-4, and can be universally applied to wild-type A. actinomycetemcomitans strains of serotypes a, b, and c. The Himar1 transposon inserts were stably inherited in A. actinomycetemcomitans transconjugants in the absence of antibiotics. A library of 16,000 mutant colonies of A. actinomycetemcomitans was screened for reduced biofilm formation. Mutants with transposon inserts in genes encoding pilus, putative ion transporters, multidrug resistant proteins, transcription regulators and enzymes involved in the synthesis of extracellular polymeric substance, bacterial metabolism and stress response were discovered in this screen. Our results demonstrated the utility of the Himar1 mutagenesis system as a novel genetic tool for functional genomic analysis in A. actinomycetemcomitans.

  4. Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

    Science.gov (United States)

    Huovinen, Tuomas; Brockmann, Eeva-Christine; Akter, Sultana; Perez-Gamarra, Susan; Ylä-Pelto, Jani; Liu, Yuan; Lamminmäki, Urpo

    2012-01-01

    Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material. PMID:22355397

  5. Lethal mutagenesis: targeting the mutator phenotype in cancer.

    Science.gov (United States)

    Fox, Edward J; Loeb, Lawrence A

    2010-10-01

    The evolution of cancer and RNA viruses share many similarities. Both exploit high levels of genotypic diversity to enable extensive phenotypic plasticity and thereby facilitate rapid adaptation. In order to accumulate large numbers of mutations, we have proposed that cancers express a mutator phenotype. Similar to cancer cells, many viral populations, by replicating their genomes with low fidelity, carry a substantial mutational load. As high levels of mutation are potentially deleterious, the viral mutation frequency is thresholded at a level below which viral populations equilibrate in a traditional mutation-selection balance, and above which the population is no longer viable, i.e., the population undergoes an error catastrophe. Because their mutation frequencies are fine-tuned just below this error threshold, viral populations are susceptible to further increases in mutational load and, recently this phenomenon has been exploited therapeutically by a concept that has been termed lethal mutagenesis. Here we review the application of lethal mutagenesis to the treatment of HIV and discuss how lethal mutagenesis may represent a novel therapeutic approach for the treatment of solid cancers. Copyright © 2010 Elsevier Ltd. All rights reserved.

  6. Directed mutagenesis affects recombination in Azospirillum brasilense nif genes

    Directory of Open Access Journals (Sweden)

    C.P. Nunes

    2000-12-01

    Full Text Available In order to improve the gene transfer/mutagenesis system for Azospirillum brasilense, gene-cartridge mutagenesis was used to replace the nifD gene with the Tn5 kanamycin resistance gene. The construct was transferred to A. brasilense by electrotransformation. Of the 12 colonies isolated using the suicide plasmid pSUP202 as vector, only four did not show vector integration into the chromosome. Nevertheless, all 12 colonies were deficient in acetylene reduction, indicating an Nif- phenotype. Four Nif- mutants were analyzed by Southern blot, using six different probes spanning the nif and Km r genes and the plasmid vector. Apparently, several recombination events occurred in the mutant genomes, probably caused mainly by gene disruption owing to the mutagenesis technique used: resistance gene-cartridge mutagenesis combined with electrotransformation.Com o objetivo de melhorar os sistemas de transferência gênica e mutagênese para Azospirillum brasilense, a técnica de mutagênese através do uso de um gene marcador ("gene-cartridge mutagenesis" foi utilizada para substituir a região genômica de A. brasilense correspondente ao gene nifD por um segmento de DNA do transposon Tn5 contendo o gene que confere resistência ao antibiótico canamicina. A construção foi transferida para a linhagem de A. brasilense por eletrotransformação. Doze colônias transformantes foram isoladas com o plasmídeo suicida pSUP202 servindo como vetor. Dessas, somente quatro não possuíam o vetor integrado no cromossomo da bactéria. Independentemente da integração ou não do vetor, as 12 colônias foram deficientes na redução do gás acetileno, evidenciando o fenótipo Nif -. Quatro mutantes Nif - foram analisados através da técnica de Southern blot, utilizando-se seis diferentes fragmentos contendo genes nif, de resistência à canamicina e do vetor como sondas. Os resultados sugerem a ocorrência de eventos recombinacionais variados no genoma dos mutantes. A

  7. Untargeted viral mutagenesis is not found in X-irradiated monkey cells

    International Nuclear Information System (INIS)

    Lytle, C.D.; Carney, P.G.; Lee, W.; Bushar, H.F.

    1988-01-01

    The existence of untargeted viral mutagenesis in X-irradiated cells was investigated in a mammalian virus/cell system, where a low level of such viral mutagenesis can be demonstrated in UV-irradiated cells. In the positive control experiment UV-elicited mutagenesis was shown with cell exposures of 5, 10 and 15 J/m 2 and a delay of 24 h between cell irradiation and infection with unirradiated herpes simplex virus. Although X-ray doses of 1, 3 and 10 Gy elicit enhanced reactivation of UV-irradiated virus, no untargeted mutagenesis for any X-ray dose at post-irradiation infection times of 0, 24 or 72 h was observed in this study. Thus untargeted mutagenesis of herpes simplex virus was not demonstrated in X-irradiated monkey cells, under conditions where X-ray-enhanced reactivation occurs and where untargeted mutagenesis in UV-irradiated cells occurs. (author)

  8. Modification of γ-induced mutagenesis in Ames test-strains

    International Nuclear Information System (INIS)

    Basha, S.G.; Krasavin, E.A.; Kozubek, S.; Amirtaev, K.G.

    1990-01-01

    Glycerine and cysteamine protective effect on mutagenesis was studied in 3 strains of Salmonella typhimurium under γ-radiation. Glycerine modifying effect was shown to be not similar for various test-strains and depended on DNA injury nature. DNA complex injuries were shown to play significant role in mutagenesis of TA100 and TA102 strains. Absence of cysteamine modifying effect on γ-induced mutagenesis testified to cysteamine effect on enzyme balance. 20 refs.; 2 figs.; 1 tab

  9. Revised Mechanism and Improved Efficiency of the QuikChange Site-Directed Mutagenesis Method.

    Science.gov (United States)

    Xia, Yongzhen; Xun, Luying

    2017-01-01

    Site-directed mutagenesis has been widely used for the substitution, addition or deletion of nucleotide residues in a defined DNA sequence. QuikChange™ site-directed mutagenesis and its related protocols have been widely used for this purpose because of convenience and efficiency. We have recently demonstrated that the mechanism of the QuikChange™ site-directed mutagenesis process is different from that being proposed. The new mechanism promotes the use of partially overlapping primers and commercial PCR enzymes for efficient PCR and mutagenesis.

  10. Mechanism of SOS-induced targeted and untargeted mutagenesis in E. coli

    International Nuclear Information System (INIS)

    Maenhaut-Michel, G.

    1985-01-01

    This paper retraces the evolution of hypotheses concerning mechanisms of SOS induced mutagenesis. Moreover, it reports some recent data which support a new model for the mechanism of targeted and untargeted mutagenesis in E. coli. In summary, the SOS mutator effect, which is responsible for untargeted mutagenesis and perhaps for the misincorporation step in targeted mutagenesis, is believed to involve a fidelity function associated with DNA polymerase III and does not require the umuC gene product. umuC and umuD gene products are probably required specifically for elongation of DNA synthesis past blocking lesions, i.e. to allow mutagenic replication of damaged DNA

  11. Beyond the Natural Proteome: Nondegenerate Saturation Mutagenesis-Methodologies and Advantages.

    Science.gov (United States)

    Ferreira Amaral, M M; Frigotto, L; Hine, A V

    2017-01-01

    Beyond the natural proteome, high-throughput mutagenesis offers the protein engineer an opportunity to "tweak" the wild-type activity of a protein to create a recombinant protein with required attributes. Of the various approaches available, saturation mutagenesis is one of the core techniques employed by protein engineers, and in recent times, nondegenerate saturation mutagenesis is emerging as the approach of choice. This review compares the current methodologies available for conducting nondegenerate saturation mutagenesis with traditional, degenerate saturation and briefly outlines the options available for screening the resulting libraries, to discover a novel protein with the required activity and/or specificity. © 2017 Elsevier Inc. All rights reserved.

  12. The relationship between survival and mutagenesis in Escherichia coli after fractionated ultraviolet irradiation

    International Nuclear Information System (INIS)

    Dzidic, S.; Salaj-Smic, E.; Trgovcevic, Z.

    1986-01-01

    The relationship between survival and mutagenesis in Escherichia coli after fractionated ultraviolet (UV) irradiation was studied. The cells were incubated either in buffer or nutrient media. Regardless of incubation conditions, greater survival is observed after fractionated irradiation than after acute irradiation. When the cells are incubated in buffer, UV mutagenesis decreases with an increase in the number of dose fractions. However, when the cells are cultivated in nutrient media, the increased survival is coupled with the enhanced capacity for UV mutagenesis. The authors, therefore, assume that during incubation in nutrient media, fractionated irradiation leads to full and prolonged expression of all UV inducible (SOS) genes, including those required for mutagenesis. (Auth.)

  13. Phenotypic heterogeneity in a bacteriophage population only appears as stress-induced mutagenesis.

    Science.gov (United States)

    Yosef, Ido; Edgar, Rotem; Qimron, Udi

    2016-11-01

    Stress-induced mutagenesis has been studied in cancer cells, yeast, bacteria, and archaea, but not in viruses. In a recent publication, we present a bacteriophage model showing an apparent stress-induced mutagenesis. We show that the stress does not drive the mutagenesis, but only selects the fittest mutants. The mechanism underlying the observed phenomenon is a phenotypic heterogeneity that resembles persistence of the viral population. The new findings, the background for the ongoing debate on stress-induced mutagenesis, and the phenotypic heterogeneity underlying a novel phage infection strategy are discussed in this short manuscript.

  14. [Influence of diethyl sulfate (DES) mutagenesis on growth properties and pigment secondary metabolites of Phellinus igniarius].

    Science.gov (United States)

    Wang, Jing; Wu, Xin-yuan; Ma, Wei; Chen, Jing; Liu, Cheng; Wu, Xiu-li

    2015-06-01

    The diethyl sulfate (DES) mutagenesis was chosen for the mutagenic treatment to Phellinus igniarius, and the relationship of mutagenesis time and death rate was investigated with 0.5% DES. The differences of mycelial growth speed, liquid fermentation mycelia biomass, morphology and pigment classes of secondary metabolites production speed and antioxidant activities of metabolite products were discussed. The study displayed that DES mutagenesis could change mycelial morphology without obvious effect on mycelium growth, and the DES mutagenesis improved antioxidant activities of the active ingredients of P. igniarius and had more antioxidant activity of hypoxia/sugar PC12 nerve cells than that of P. igniarius.

  15. A protocol for chemical mutagenesis in Strongyloides ratti.

    Science.gov (United States)

    Guo, Li; Chang, Zisong; Dieterich, Christoph; Streit, Adrian

    2015-11-01

    Genetic analysis using experimentally induced mutations has been a most valuable tool in the analysis of various organisms. However, genetic analysis of endoparasitic organisms tends to be difficult because of the limited accessibility of the sexually reproducing adults, which are normally located within the host. Nematodes of the genera Strogyloides and Parastrongyloides represent an exception to this because they can form facultative free-living sexually reproducing generations in between parasitic generations. Here we present a protocol for the chemical mutagenesis of Strongyloides ratti. Further we evaluate the feasibility of identifying the induced mutations by whole genome re-sequencing. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. History of the science of mutagenesis from a personal perspective.

    Science.gov (United States)

    Malling, Heinrich V

    2004-01-01

    A career in the study of mutagenesis spanning 50 years is a gift few scientists have been bestowed. My tenure in the field started in 1953, the year the structure of DNA became known (Watson and Crick [1953]: Nature 171:737). Before that time, it was suspected that DNA was the genetic material based on the research of Oswald T. Avery (Avery et al. [1944]: J Exp Med 79:137), but many scientists still believed that proteins or polysaccharides could be the genetic material. The present article describes a lifetime of personal experience in the field of chemical mutagenesis. The methods used to treat viruses with chemical mutagens were well developed in the 1950s. Here I review the early use of nitrous acid and hydroxylamine as mutagens in eukaryotes, the development of methods for the metabolic activation of mutagens by microsomal preparations, and the selection of a mutant tester set for the qualitative characterization of the mutagenic activity of chemicals. These studies provided critical background information that was used by Bruce Ames in the development of his Salmonella/microsome assay, widely known as the Ames test (Ames et al. [1973]: Proc Nat Acad Sci USA 70:2281-2285). This article also describes how a set of diagnostic chemical mutagens was selected and used to identify the molecular nature of gene mutations. Today, DNA sequencing has replaced the use of diagnostic mutagens, but studies of this kind formed the foundation of modern mutation research. They also helped set the stage for the organization of the Environmental Mutagen Society and the Environmental Mutagen Information Center, which are described. The article ends with the development of mammalian single-cell mutation assays, the first system for studying in vivo mutagenesis using recoverable vectors in transgenic animals, other mutation assays in intact mammals, and my thoughts on the critically important area of germ cell mutagenesis. This narrative is not a complete autobiographical account

  17. Radiation mutagenesis in development of genetic fundamentals of cotton selection

    International Nuclear Information System (INIS)

    Musaev, D.A.; Almatov, A.S.

    1987-01-01

    Some results of investigations on preparation and genetic analysis of mutants in inbreeding lines of genetic collections of cotton plants, as well as problems on mutant application in practical selection are covered. The results show that the scientific authenticity and efficiency of fundamental and applied investigations in the field of experimental mutagenesis of cotton plants,being a facultative self-polinator, depend on keeping necessary methodical requirements. Application of inbreeding lines of genetic collection with marker features as the initial material, isolation of plants usinng self-polination of flowers on all stages of investigation are related to these requirements. Several methodical recommendations on genetic-selective investigations are developed

  18. Recent advances of microbial breeding via heavy-ion mutagenesis at IMP.

    Science.gov (United States)

    Hu, W; Li, W; Chen, J

    2017-10-01

    Nowadays, the value of heavy-ion mutagenesis has been accepted as a novel powerful mutagen technique to generate new microbial mutants due to its high linear energy transfer and high relative biological effectiveness. This paper briefly reviews recent progress in developing a more efficient mutagenesis technique for microbial breeding using heavy-ion mutagenesis, and also presents the outline of the beam line for microbial breeding in Heavy Ion Research Facility of Lanzhou. Then, new insights into microbial biotechnology via heavy-ion mutagenesis are also further explored. We hope that our concerns will give deep insight into microbial breeding biotechnology via heavy-ion mutagenesis. We also believe that heavy-ion mutagenesis breeding will greatly contribute to the progress of a comprehensive study industrial strain engineering for bioindustry in the future. There is currently a great interest in developing rapid and diverse microbial mutation tool for strain modification. Heavy-ion mutagenesis has been proved as a powerful technology for microbial breeding due to its broad spectrum of mutation phenotypes with high efficiency. In order to deeply understand heavy-ion mutagenesis technology, this paper briefly reviews recent progress in microbial breeding using heavy-ion mutagenesis at IMP, and also presents the outline of the beam line for microbial breeding in Heavy Ion Research Facility of Lanzhou (HIRFL) as well as new insights into microbial biotechnology via heavy-ion mutagenesis. Thus, this work can provide the guidelines to promote the development of novel microbial biotechnology cross-linking heavy-ion mutagenesis breeding that could make breeding process more efficiently in the future. © 2017 The Society for Applied Microbiology.

  19. The pathologic effect of a novel neomorphic Fgf9(Y162C allele is restricted to decreased vision and retarded lens growth.

    Directory of Open Access Journals (Sweden)

    Oliver Puk

    Full Text Available Fibroblast growth factor (Fgf signalling plays a crucial role in many developmental processes. Among the Fgf pathway ligands, Fgf9 (UniProt: P54130 has been demonstrated to participate in maturation of various organs and tissues including skeleton, testes, lung, heart, and eye. Here we establish a novel Fgf9 allele, discovered in a dominant N-ethyl-N-nitrosourea (ENU screen for eye-size abnormalities using the optical low coherence interferometry technique. The underlying mouse mutant line Aca12 was originally identified because of its significantly reduced lens thickness. Linkage studies located Aca12 to chromosome 14 within a 3.6 Mb spanning interval containing the positional candidate genes Fgf9 (MGI: 104723, Gja3 (MGI: 95714, and Ift88 (MGI: 98715. While no sequence differences were found in Gja3 and Ift88, we identified an A→G missense mutation at cDNA position 770 of the Fgf9 gene leading to an Y162C amino acid exchange. In contrast to previously described Fgf9 mutants, Fgf9(Y162C carriers were fully viable and did not reveal reduced body-size, male-to-female sexual reversal or skeletal malformations. The histological analysis of the retina as well as its basic functional characterization by electroretinography (ERG did not show any abnormality. However, the analysis of head-tracking response of the Fgf9(Y162C mutants in a virtual drum indicated a gene-dosage dependent vision loss of almost 50%. The smaller lenses in Fgf9(Y162C suggested a role of Fgf9 during lens development. Histological investigations showed that lens growth retardation starts during embryogenesis and continues after birth. Young Fgf9(Y162C lenses remained transparent but developed age-related cataracts. Taken together, Fgf9(Y162C is a novel neomorphic allele that initiates microphakia and reduced vision without effects on organs and tissues outside the eye. Our data point to a role of Fgf9 signalling in primary and secondary lens fiber cell growth. The results

  20. The novel mouse mutant, chuzhoi, has disruption of Ptk7 protein and exhibits defects in neural tube, heart and lung development and abnormal planar cell polarity in the ear

    Directory of Open Access Journals (Sweden)

    Paudyal Anju

    2010-08-01

    Full Text Available Abstract Background The planar cell polarity (PCP signalling pathway is fundamental to a number of key developmental events, including initiation of neural tube closure. Disruption of the PCP pathway causes the severe neural tube defect of craniorachischisis, in which almost the entire brain and spinal cord fails to close. Identification of mouse mutants with craniorachischisis has proven a powerful way of identifying molecules that are components or regulators of the PCP pathway. In addition, identification of an allelic series of mutants, including hypomorphs and neomorphs in addition to complete nulls, can provide novel genetic tools to help elucidate the function of the PCP proteins. Results We report the identification of a new N-ethyl-N-nitrosourea (ENU-induced mutant with craniorachischisis, which we have named chuzhoi (chz. We demonstrate that chuzhoi mutant embryos fail to undergo initiation of neural tube closure, and have characteristics consistent with defective convergent extension. These characteristics include a broadened midline and reduced rate of increase of their length-to-width ratio. In addition, we demonstrate disruption in the orientation of outer hair cells in the inner ear, and defects in heart and lung development in chuzhoi mutants. We demonstrate a genetic interaction between chuzhoi mutants and both Vangl2Lp and Celsr1Crsh mutants, strengthening the hypothesis that chuzhoi is involved in regulating the PCP pathway. We demonstrate that chuzhoi maps to Chromosome 17 and carries a splice site mutation in Ptk7. This mutation results in the insertion of three amino acids into the Ptk7 protein and causes disruption of Ptk7 protein expression in chuzhoi mutants. Conclusions The chuzhoi mutant provides an additional genetic resource to help investigate the developmental basis of several congenital abnormalities including neural tube, heart and lung defects and their relationship to disruption of PCP. The chuzhoi mutation

  1. Crowding depression of UV-mutagenesis in E. coli

    International Nuclear Information System (INIS)

    Bockrath, R.; Harper, D.; Kristoff, S.; Stanford Univ., CA

    1980-01-01

    Strains of E. coli Br were exposed to UV radiation and assayed for reversion mutation, using a standard selection medium. If more irradiated bacteria were assayed per petri dish, a proportional increase in the number of indicated reversion mutants was oud only up to a limiting plating density. Beyond a density of about 10 8 viable bacteria per petri dish, the number of indicated revertants per viable bacteriy assayed (the mutation frequency) decreased as the plating density was increased. The crowding depression of mutagenesis was more severe for de novo and converted suppressor mutations, the mutation frequency being reduced 100-fold at a plating density of about 6 x 10 9 viable bacteria per plate. The effect on backmutation was 10 times less. Crowding depression of mutagenesis occured in excision-proficient and -deficient strains, with identical effects in the 2 strains on de novo and converted suppressor mutation, but different effects on backmutations. There were no accompanying effects on viability. Irreversible loss of potential mutants during crowded growth was indicated in wash-off experiments. The kinetics suggested a half-life of approximately 1 h. Kinetics for accumulation by the bacteria of the limiting metabolite (tyrosine) on the assay plate indicated a short period of time for protein synthesis, but direct examination of the proteins synthesized during early growth on a crowded plate demonstrated successful induction of recA protein. (orig.)

  2. Is radiation an appropriate model for chemical mutagenesis and carcinogenesis

    International Nuclear Information System (INIS)

    Bond, V.P.

    1982-01-01

    This chapter attempts to show why the quadratic, or ''linear quadratic,'' relationship holds for organ dose-single cell radiation effects, and to explore the extension of this relationship to chemical exposures in general. Demonstrates that although the ''αD + βD 2 relationship'' may be unexpected for normal pharmacologicalmedical dose-response relationships, a linear, no-threshold curve of this kind is expected for all stochastic-type (accidental or risk) situations with health consequences (e.g. all common accidents) including exposure to ''low-level radiation'' (LLR). Discusses the stochastic or risk approach, relevant radiobiology, and the stochastic for chemicals. Assumes that even though actual mutational rates cannot be expected to apply to the relevance of Tradescantia or any other single cell system as a predictor for mutagenesis and carcinogenesis in animals and man, the cardinal principles of genetics largely transcend species and the particular environment in which the cell is located. Concludes that with regard to LLR, the curve shapes and other relationships developed for Tradescantia would be expected to apply in principle to animal and human mutagenesis and carcinogenesis

  3. In vitro mutagenesis of commercial fern, Asplenium nidus from spores

    International Nuclear Information System (INIS)

    Norazlina Noordin

    2004-01-01

    Asplenium is a largest, most diverse fern genera. One of the common species is Asplenium nidus, well known as Bird's-nest fern, a medium to large fern with erect, stout, unbranched rhizomes. In creating variability of ferns for the benefit of the ornamental plant industry, in vitro mutagenesis is used. In this study, spores of Asplenium nidus were collected from frond bearing mature sporangia. Spores were cultured in modified 1/2 MS basal medium supplemented with various combinations of 6-Benzylaminopurine (BAP) and Naphtalene Acetic Acid (NAA). Spore cultures were incubated in incubation room at 24 degree C with 16 hours photoperiod (3500 lux). It was found that, the most effective combinations were 1 mg/1 BAP + 0. 1 mg/1 NAA and 2mg/1 BAP + 0. 1 mg/1 NAA. Prothallus was formed after 10 days of cultures and gametophytes were formed 1 month later. These gametophytes were irradiated with Gamma ray at doses of 0, 20, 90, 120, 150 and 180 Gy. From the preliminary result obtained from this study, for generating variations and desired phenotypic expression for Asplenium nidus, recommended doses for in vitro mutagenesis using spores are between 90 Gy to 150 Gy. Gametophytes were subcultured at monthly interval to ensure further development and propagation. Frequent monitoring for any changes in the morphology of the irradiated Asplenium nidus plants were carried out. (Author)

  4. Tradescantia bioassays as monitoring systems for environmental mutagenesis: a review

    International Nuclear Information System (INIS)

    Rodrigues, G.S.; Ma, T.H.; Pimentel, D.; Weinstein, L.H.

    1997-01-01

    Since the early studies on the genetic effects of chemical and physical agents, species and clones of Tradescantia have been used as experimental subjects, by virtue of a series of favorable genetic characteristics. Bearing just six pairs (2n = 12) of large, easily observable chromosomes, cells from almost every part of the plant, from the root tips to the developing pollen tube, yield excellent material for cytogenetic studies. As a consequence of the intensive use of Tradescantia in genetic studies, a series of genetic characteristics have been found that offer opportunities for the detection of agents affecting the stability of the genome. At least five such characteristics have been selected as endpoints for the establishment of assays to evaluate mutagenesis. Three of these, root-tip mitosis, pollen-tube, and microspore mitosis are essentially chromosome aberration assays, wherein one observes and evaluates the visible damage in the chromosomes. A fourth, the stamen-hair mutation assay (Trad-SHM), is a point mutation mitotic assay based on the expression of a recessive gene for flower color in heterozygous plants. The fifth assay is a cytogenetic test based on the formation of micronuclei (Trad-MCN) that result from chromosome breakage in the meiotic pollen mother cells. This article examines the characteristics and fundamentals of the Trad-MCN and the Trad-SHM assays and reviews the results obtained to date with these systems in the assessment of environmental mutagenesis. (author)

  5. Tissue culture and mutagenesis of rain lily (zephyranthes)

    International Nuclear Information System (INIS)

    Mohd Nazir Basiran; Zaiton Ahmad; Shakinah Salleh; Shuhaimi Shamsudin; Aiza Shaliha Jamaludin

    2004-01-01

    There are three varieties of Zephyranthes used widely in landscaping due to their robust growth and attractive flowers in pink, yellow and white. Both in vivo and in vitro mutagenesis are an effective approach to increase the flower colour variations of Zephyranthes. In vitro propagation for the three varieties was attempted by using the induction medium developed by Sachar and Kapoor in 1959. The medium contains I ma of each indole 3-acetic acid (IAA), indole 3-butyric acid (IBA) and kinetin. Following surface sterilization of bulb scales, 17.8%, 10.5% and 10.7% of pink, white and yellow varieties respectively, were able to form small bulblets on the induction media. Further development of these bulblets into plantlets was also achieved on the same medium. Work is now being carried out to improve the efficiency of bulblet regeneration. Mutagenesis of Zephyranthes was initiated from bulbs of the pink varieties to develop new varieties with attractive combinations of flower colour and forms, shelf life and growth habits. These bulbs were irradiated using a gamma cell with a 60 Co source. Three variants with different flower colour and morphology have been achieved so far and are now being propagated in the nursery. (Author)

  6. Mechanisms of Base Substitution Mutagenesis in Cancer Genomes

    Directory of Open Access Journals (Sweden)

    Albino Bacolla

    2014-03-01

    Full Text Available Cancer genome sequence data provide an invaluable resource for inferring the key mechanisms by which mutations arise in cancer cells, favoring their survival, proliferation and invasiveness. Here we examine recent advances in understanding the molecular mechanisms responsible for the predominant type of genetic alteration found in cancer cells, somatic single base substitutions (SBSs. Cytosine methylation, demethylation and deamination, charge transfer reactions in DNA, DNA replication timing, chromatin status and altered DNA proofreading activities are all now known to contribute to the mechanisms leading to base substitution mutagenesis. We review current hypotheses as to the major processes that give rise to SBSs and evaluate their relative relevance in the light of knowledge acquired from cancer genome sequencing projects and the study of base modifications, DNA repair and lesion bypass. Although gene expression data on APOBEC3B enzymes provide support for a role in cancer mutagenesis through U:G mismatch intermediates, the enzyme preference for single-stranded DNA may limit its activity genome-wide. For SBSs at both CG:CG and YC:GR sites, we outline evidence for a prominent role of damage by charge transfer reactions that follow interactions of the DNA with reactive oxygen species (ROS and other endogenous or exogenous electron-abstracting molecules.

  7. Mechanisms of base substitution mutagenesis in cancer genomes.

    Science.gov (United States)

    Bacolla, Albino; Cooper, David N; Vasquez, Karen M

    2014-03-05

    Cancer genome sequence data provide an invaluable resource for inferring the key mechanisms by which mutations arise in cancer cells, favoring their survival, proliferation and invasiveness. Here we examine recent advances in understanding the molecular mechanisms responsible for the predominant type of genetic alteration found in cancer cells, somatic single base substitutions (SBSs). Cytosine methylation, demethylation and deamination, charge transfer reactions in DNA, DNA replication timing, chromatin status and altered DNA proofreading activities are all now known to contribute to the mechanisms leading to base substitution mutagenesis. We review current hypotheses as to the major processes that give rise to SBSs and evaluate their relative relevance in the light of knowledge acquired from cancer genome sequencing projects and the study of base modifications, DNA repair and lesion bypass. Although gene expression data on APOBEC3B enzymes provide support for a role in cancer mutagenesis through U:G mismatch intermediates, the enzyme preference for single-stranded DNA may limit its activity genome-wide. For SBSs at both CG:CG and YC:GR sites, we outline evidence for a prominent role of damage by charge transfer reactions that follow interactions of the DNA with reactive oxygen species (ROS) and other endogenous or exogenous electron-abstracting molecules.

  8. Visualization of tandem repeat mutagenesis in Bacillus subtilis.

    Science.gov (United States)

    Dormeyer, Miriam; Lentes, Sabine; Ballin, Patrick; Wilkens, Markus; Klumpp, Stefan; Kohlheyer, Dietrich; Stannek, Lorena; Grünberger, Alexander; Commichau, Fabian M

    2018-03-01

    Mutations are crucial for the emergence and evolution of proteins with novel functions, and thus for the diversity of life. Tandem repeats (TRs) are mutational hot spots that are present in the genomes of all organisms. Understanding the molecular mechanism underlying TR mutagenesis at the level of single cells requires the development of mutation reporter systems. Here, we present a mutation reporter system that is suitable to visualize mutagenesis of TRs occurring in single cells of the Gram-positive model bacterium Bacillus subtilis using microfluidic single-cell cultivation. The system allows measuring the elimination of TR units due to growth rate recovery. The cultivation of bacteria carrying the mutation reporter system in microfluidic chambers allowed us for the first time to visualize the emergence of a specific mutation at the level of single cells. The application of the mutation reporter system in combination with microfluidics might be helpful to elucidate the molecular mechanism underlying TR (in)stability in bacteria. Moreover, the mutation reporter system might be useful to assess whether mutations occur in response to nutrient starvation. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli

    International Nuclear Information System (INIS)

    Wood, R.D.; Hutchinson, F.

    1984-01-01

    Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr + host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one or two mutant phage per mutant burst. From the results of a series of experiments with various mutant host cells, a major pathway of non-targeted mutagenesis by ultraviolet light was proposed which acts in addition to ''SOS induction''. This pathway involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage. (author)

  10. Antimutation effect of an E. coli membrane fraction on UV-mutagenesis

    International Nuclear Information System (INIS)

    Harper, D.; Kristoff, S.; Bockrath, R.; Indiana Univ., Indianapolis; Indiana Univ., Indianapolis

    1980-01-01

    The depression of mutagenesis that occurs when irradiated E. coli are plated at high densities is studied. The number of mutant colonies indicated increases linearly with increasing plate density to about 10 8 bacteria per plate. At higher plate densities, suppressor mutations are very sensitive to crowding depression of mutagenesis and backmutations are somewhat sensitive. (orig./AJ)

  11. Direct Mutagenesis of Thousands of Genomic Targets using Microarray-derived Oligonucleotides

    DEFF Research Database (Denmark)

    Bonde, Mads; Kosuri, Sriram; Genee, Hans Jasper

    2015-01-01

    Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale-up by ...

  12. The relation between repair of DNA and radiation and chemical mutagenesis in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Prakash, L.

    1976-01-01

    The effect of various genes involved in DNA repair functions on radiation and chemical mutagenesis in Escherichia coli is discussed and compared to similar studies done in yeast. Results of the effect of various genes conferring radiation-sensitivty on mutation induction in yeast are presented and related to current ideas of mutagenesis

  13. Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Wood, R.D.; Hutchinson, F. (Yale Univ., New Haven, CT (USA). Dept. of Molecular Biophysics and Biochemistry)

    1984-03-05

    Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr/sup +/ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one or two mutant phage per mutant burst. From the results of a series of experiments with various mutant host cells, a major pathway of non-targeted mutagenesis by ultraviolet light was proposed which acts in addition to ''SOS induction''. This pathway involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage.

  14. Scientific projection paper for mutagenesis, transformation and cell killing

    International Nuclear Information System (INIS)

    Todd, P.

    1980-01-01

    Our knowledge about mutagenesis, transformation, and cell killing by ionizing radiation consists of large bodies of data, which are potentially useful in terms of application to human risk assessment and to the constructive use of radiation, as in cancer treatment. The three end-points discussed above are united by at least five significant concepts in radiation research strategy: (1) The inter-relationships among the important end-points, mutation, carcinogenesis, and cell killing. Research on one is meaningful only in the context of information about the other two. (2) The interaction of radiations with other agents in producing these end-points. (3) The mechanisms of action of other environmental mutagenic, carcinogenic, and cytotoxic agents. (4) The use of repair deficient human mutant cells. (5) The study of radiation damage mechanisms. There is no better way to extrapolate laboratory data to the clinical and public worlds than to understand the underlying biological mechanisms that produced the data

  15. Results and perspectives of mutagenesis applied to durum wheat

    International Nuclear Information System (INIS)

    Bagnara, D.

    1975-01-01

    A review is made of the main aspects and problems of mutagenesis applied to the breeding of durum wheat (Triticum turgidum ssp. durum). Features and type of action of the main physical and chemical mutagens are considered: a comparison is also made between the two classes of mutagens, on the basis of results so far achieved. Mentions is then made of methods of treatment; parts of plant which can be treated; growing of treated material in segregating generations: data to be successively recorded. Methods of estimating mutation frequency and the problem of arising chimerical tissues and its possible overcoming are also discussed. Examination is made of some special effects of mutagens, namely: induction of translocations; diploidization of polyploids; induction of haploids and aneuploids; genetic analysis of specific loci; induction of male sterility. Finally, results are reviewed concerning induction and utilization, either as varieties or in cross breeding programmes, of mutants for characters of agronomic interest. (Bagnara, D.)

  16. Environmental mutagenesis during the end-Permian ecological crisis.

    Science.gov (United States)

    Visscher, Henk; Looy, Cindy V; Collinson, Margaret E; Brinkhuis, Henk; van Konijnenburg-van Cittert, Johanna H A; Kürschner, Wolfram M; Sephton, Mark A

    2004-08-31

    During the end-Permian ecological crisis, terrestrial ecosystems experienced preferential dieback of woody vegetation. Across the world, surviving herbaceous lycopsids played a pioneering role in repopulating deforested terrain. We document that the microspores of these lycopsids were regularly released in unseparated tetrads indicative of failure to complete the normal process of spore development. Although involvement of mutation has long been hinted at or proposed in theory, this finding provides concrete evidence for chronic environmental mutagenesis at the time of global ecological crisis. Prolonged exposure to enhanced UV radiation could account satisfactorily for a worldwide increase in land plant mutation. At the end of the Permian, a period of raised UV stress may have been the consequence of severe disruption of the stratospheric ozone balance by excessive emission of hydrothermal organohalogens in the vast area of Siberian Traps volcanism. Copyright 2004 The National Academy of Sciencs of the USA

  17. Study of UV-mutagenesis in Bacillus subtilis

    International Nuclear Information System (INIS)

    Lotareva, O.V.; Filippov, V.D.

    1974-01-01

    The sensitivity of Bac. subtilis to the inactivating and mutagenic effects of UV-mutants has been determined: uvr, which does not extract pyrimidine dimers from damaged DNA; recsub(x), which exhibits a reduced activity of ATP-dependent DNAase; poll, which is devoid of DNA polymerase, and wild strains (DT). The sensitivity of these strains to the inactivating effects of UV rays increases in the order: DT<= recsub(x) << uvr < poll, and UV mutability in the order: DT = rec(sub(x) < poll<< uvr. A comparison of UV mutagenesis in Bac. subtilis and E. coli suggests the hypothesis that the mechanisms of UV mutation formation are similar in these two organisms. (author)

  18. Mutant fatty acid desaturase and methods for directed mutagenesis

    Science.gov (United States)

    Shanklin, John [Shoreham, NY; Whittle, Edward J [Greenport, NY

    2008-01-29

    The present invention relates to methods for producing fatty acid desaturase mutants having a substantially increased activity towards substrates with fewer than 18 carbon atom chains relative to an unmutagenized precursor desaturase having an 18 carbon chain length specificity, the sequences encoding the desaturases and to the desaturases that are produced by the methods. The present invention further relates to a method for altering a function of a protein, including a fatty acid desaturase, through directed mutagenesis involving identifying candidate amino acid residues, producing a library of mutants of the protein by simultaneously randomizing all amino acid candidates, and selecting for mutants which exhibit the desired alteration of function. Candidate amino acids are identified by a combination of methods. Enzymatic, binding, structural and other functions of proteins can be altered by the method.

  19. Creating Sunflower Mutant Lines (Helianthus Annuus L.) Using Induced Mutagenesis

    International Nuclear Information System (INIS)

    Encheva, J.

    2009-01-01

    Immature sunflower zygotic embryos of sunflower fertility restorer line 374 R were treated with ultrasound and gamma radiation before plating embryos to culture medium. All plants were isolated and self-pollinated for several generations. New sunflower forms with inherited morphological and biochemical changes were obtained. The genetic changes occurring during the mutation procedure included fourteen morphological and biochemical characters. In comparison to the check line 374 R, decreasing of the mean value of the indexes was registered for 33 % of the total number of characters and vise verse, significant increasing was observed for 60 %. Mutation for resistance to the local population of Orobanche cumana race A-E was obtained from the susceptible Bulgarian control line 374 R. Two investigated mutant lines possessed 100 % resistance to Orobanche and stable inheritance in the next generations. Our results showed that induced mutagenesis in sunflower can be successfully used to develop new lines useful for heterosis breeding

  20. Radiation induced DNA damage and repair in mutagenesis

    International Nuclear Information System (INIS)

    Strniste, G.F.; Chen, D.J.; Okinaka, R.T.

    1987-01-01

    The central theme in cellular radiobiological research has been the mechanisms of radiation action and the physiological response of cells to this action. Considerable effort has been directed toward the characterization of radiation-induced DNA damage and the correlation of this damage to cellular genetic change that is expressed as mutation or initiating events leading to cellular transformation and ultimately carcinogenesis. In addition, there has been a significant advancement in their understanding of the role of DNA repair in the process of mutation leading to genetic change in cells. There is extensive literature concerning studies that address radiation action in both procaryotic and eucaryotic systems. This brief report will make no attempt to summarize this voluminous data but will focus on recent results from their laboratory of experiments in which they have examined, at both the cellular and molecular levels, the process of ionizing radiation-induced mutagenesis in cultured human cells

  1. Mechanisms of uv mutagenesis in yeast and E. coli

    International Nuclear Information System (INIS)

    Lawrence, C.; Christensen, R.; Christensen, J.R.; O'Brien, T.

    1983-01-01

    Experiments investigating ultraviolet light mutagenesis in either bakers' yeast, Saccharomyces cerevisiae, or E. coli have led to the following conclusions. First, cyclobutane pyrimidine dimers cause most mutations in both organisms; pyrimidine adducts, such as PyC, can account at best for only a small proportion. 86 percent of forward mutations induced at the E. coli lacI locus can be abolished by photoreactivation under conditions which do not alter the level of recA induction. About 75 percent of the forward mutations induced at the CAN1 locus of yeast could be removed by photoreactivation, a value that lies within the range observed previously for the reversion of CYC1 alleles (60 percent - 97 percent). Second, about 10 percent of the lacI forward mutations are untargeted, a smaller fraction than found previously for cycl-91 reversion in yeast. It is not yet clear whether the two species are really different in this respect, of whether the cycl-91 reversion site is a typical of the yeast genome at large. Third, analysis of reversion frequencies of 20 mutant alleles suggests that about 10 to 25 percent of all replication errors produced by mutagenic mechanisms in uv-irradiated yeast involve additions or deletions of base-pairs, indicating that error-prone repair does not just produce substitutions. Last, the REV1 locus in yeast is concerned with the induction of frameshift mutations at some, but not all, genetic sites, just as found previously for substitution mutations. The function of the REV3 gene is more widely, though not universally, required while the function of the RAD6 gene, like that of the recA locus in E. coli, appears to be necessary for all kinds of uv mutagenesis. E coli genes comparable to REV1 and REV3 have not yet been described; conversely, there does not yet appear to be a yeast equivalent of umuC

  2. Mechanisms of uv mutagenesis in yeast and E. coli

    International Nuclear Information System (INIS)

    Lawrence, C.; Christensen, R.; Christensen, J.R.; O'Brien, T.

    1983-01-01

    Experiments investigating ultraviolet light mutagenesis in either bakers' yeast, Saccharomyces cerevisiae, or E. coli have led to the following conclusions. First, cyclobutane pyrimidine dimers cause most mutations in both organisms; pyrimidine adducts, such as PyC, can account at best for only a small proportion. Eighty-six percent of forward mutations induced at the E. coli lacI locus can be abolished by photoreactivation under conditions which do not alter the level of recA induction. About 75 percent of the forward mutations induced at the CAN1 locus of yeast could be removed by photoreactivation, a value that lies within the range observed previously for the reversion of CYC1 alleles (60 percent - 97 percent). Second, about 10 percent of the lacI forward mutations are untargeted, a smaller fraction than found previously for cycl1-91 reversion in yeast. It is not yet clear whether the two species are really different in this respect, or whether the cyc1-91 reversion site is atypical of the yeast genome at large. Third, analysis of reversion frequencies of 20 mutant alleles suggests that about 10 - 25 percent of all replication errors produced by mutagenic mechanisms in UV-irradiated yeast involve additions or deletions of base-pairs, indicating that error-prone repair does not just produce substitutions. Last, the REV1 locus in yeast is concerned with the induction of frameshift mutations at some, but not all, genetic sites, just as found previously for substitution mutations. The function of the REV3 gene is more widely, though not universally, required while the function of the RAD6 gene, like that of the recA locus in E. coli, appears to be necessary for all kinds of UV mutagenesis. E. coli genes comparable to REV1 and REV3 have not yet been described, conversely, there does not yet appear to be a yeast equivalent of umuC. 13 references, 4 tables

  3. Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli.

    Science.gov (United States)

    Standley, Melissa; Allen, Jennifer; Cervantes, Layla; Lilly, Joshua; Camps, Manel

    2017-01-01

    Mutagenesis in model organisms following exposure to chemicals is used as an indicator of genotoxicity. Mutagenesis assays are also used to study mechanisms of DNA homeostasis. This chapter focuses on detection of mutagenesis in prokaryotes, which boils down to two approaches: reporter inactivation (forward mutation assay) and reversion of an inactivating mutation (reversion mutation assay). Both methods are labor intensive, involving visual screening, quantification of colonies on solid media, or determining a Poisson distribution in liquid culture. Here, we present two reversion reporters for in vivo mutagenesis that produce a quantitative output, and thus have the potential to greatly reduce the amount of test chemical and labor involved in these assays. This output is obtained by coupling a TEM β lactamase-based reversion assay with GFP fluorescence, either by placing the two genes on the same plasmid or by fusing them translationally and interrupting the N-terminus of the chimeric ORF with a stop codon. We also describe a reporter aimed at facilitating the monitoring of continuous mutagenesis in mutator strains. This reporter couples two reversion markers, allowing the temporal separation of mutation events in time, thus providing information about the dynamics of mutagenesis in mutator strains. Here, we describe these reporter systems, provide protocols for use, and demonstrate their key functional features using error-prone Pol I mutagenesis as a source of mutations. © 2017 Elsevier Inc. All rights reserved.

  4. The Yeast Environmental Stress Response Regulates Mutagenesis Induced by Proteotoxic Stress

    Science.gov (United States)

    Shor, Erika; Fox, Catherine A.; Broach, James R.

    2013-01-01

    Conditions of chronic stress are associated with genetic instability in many organisms, but the roles of stress responses in mutagenesis have so far been elucidated only in bacteria. Here, we present data demonstrating that the environmental stress response (ESR) in yeast functions in mutagenesis induced by proteotoxic stress. We show that the drug canavanine causes proteotoxic stress, activates the ESR, and induces mutagenesis at several loci in an ESR-dependent manner. Canavanine-induced mutagenesis also involves translesion DNA polymerases Rev1 and Polζ and non-homologous end joining factor Ku. Furthermore, under conditions of chronic sub-lethal canavanine stress, deletions of Rev1, Polζ, and Ku-encoding genes exhibit genetic interactions with ESR mutants indicative of ESR regulating these mutagenic DNA repair processes. Analyses of mutagenesis induced by several different stresses showed that the ESR specifically modulates mutagenesis induced by proteotoxic stress. Together, these results document the first known example of an involvement of a eukaryotic stress response pathway in mutagenesis and have important implications for mechanisms of evolution, carcinogenesis, and emergence of drug-resistant pathogens and chemotherapy-resistant tumors. PMID:23935537

  5. Random mutagenesis by error-prone pol plasmid replication in Escherichia coli.

    Science.gov (United States)

    Alexander, David L; Lilly, Joshua; Hernandez, Jaime; Romsdahl, Jillian; Troll, Christopher J; Camps, Manel

    2014-01-01

    Directed evolution is an approach that mimics natural evolution in the laboratory with the goal of modifying existing enzymatic activities or of generating new ones. The identification of mutants with desired properties involves the generation of genetic diversity coupled with a functional selection or screen. Genetic diversity can be generated using PCR or using in vivo methods such as chemical mutagenesis or error-prone replication of the desired sequence in a mutator strain. In vivo mutagenesis methods facilitate iterative selection because they do not require cloning, but generally produce a low mutation density with mutations not restricted to specific genes or areas within a gene. For this reason, this approach is typically used to generate new biochemical properties when large numbers of mutants can be screened or selected. Here we describe protocols for an advanced in vivo mutagenesis method that is based on error-prone replication of a ColE1 plasmid bearing the gene of interest. Compared to other in vivo mutagenesis methods, this plasmid-targeted approach allows increased mutation loads and facilitates iterative selection approaches. We also describe the mutation spectrum for this mutagenesis methodology in detail, and, using cycle 3 GFP as a target for mutagenesis, we illustrate the phenotypic diversity that can be generated using our method. In sum, error-prone Pol I replication is a mutagenesis method that is ideally suited for the evolution of new biochemical activities when a functional selection is available.

  6. Role of the RecF gene product in UV mutagenesis of lambda phage

    International Nuclear Information System (INIS)

    Wood, R.D.; Stein, J.

    1986-01-01

    E. coli recF mutants have a greatly reduced capacity for Weigle mutagenesis of ultraviolet light-irradiated lambda phage. A recF 332::Tn3 mutation was introduced into an E. coli recA441 lex A51 strain which constitutively expresses SOS functions. Weigle mutagenesis of phage lambda could occur in the resulting strain in the absence of host cell irradiation, and was increased when the recA441 (tif) allele was activated of recF strains to support Weigle mutagenesis can therefore be ascribed to a defect in expression of SOS functions after irradiation. (orig.)

  7. A function of mutagenesis on rhodotorula RY strain irradiated by heavy ion

    International Nuclear Information System (INIS)

    Li Hongyu; Li Chenghua; Ding Xinchun; Wang Jufang; Zhou Guangming; Xie Hongmei; Li Qiang; Dang bingrong; Wen Xiaoqiong; Li Wenjian; Wei Zengquan

    2004-01-01

    In this paper, red yeast (Rhodotorula RY Strain) that produces carotene is irradiated by 50 MeV/u 12 C 6+ heavy ion from Heavy Ion Accelerator in IMP. Fermentation tests show that 50 MeV/u 12 C 6+ heavy ion has a mutagenesis effect on the red yeast. Some strains of red yeast with changed production of carotene were found by screening. Meanwhile, by RFLP and RAPD analysis, authors have a further evidence that heavy ion can cause mutagenesis in Rhodotorula RY Strain. This presents a new prospect for the mutagenesis breeding by heavy ion in industry

  8. Proceedings of the Annual Conference on Environmental Toxicology (5th) Held at Fairborn, OH on 24-26 September 1974

    Science.gov (United States)

    1974-12-01

    naphthylamine, benzidine and derivatives, 4-biphenylamine, 4-nitrobiphenyl, auramine and magenta) Alkylating Agents (chlornaphthazlne, mustard gas...recently developed carborane-silicone columns (Willeboordse et al., 1968; Yancey and Lynn, 1974). Alkylating Agents Several alkylating agents have emerged...Magee, "Nitrosamine-Induced Carcinogenesis. The Alkylation of N-7 of Guanine of Nucleic Acids of the Rat by Diethylnitrosamine N-Ethyl-N- Nitrosourea

  9. Rapidly Degradable Pyrotechnic System

    Science.gov (United States)

    2009-02-01

    material system (structural polymer and degradation agent ) for producing a high strength, non-corroding, highly inert, environmentally safe, extended...polymer sites in the active enzyme center differs dramatically between alkyl and aromatic polyesters. More specifically, as the degree of backbone...capped and centrifuged at 3,000 g. This procedure was repeated twice. To the remaining biomass pellet 15 mL of 1 mg/mL solution of N-ethyl-N- nitrosourea

  10. Development of potent in vivo mutagenesis plasmids with broad mutational spectra.

    Science.gov (United States)

    Badran, Ahmed H; Liu, David R

    2015-10-07

    Methods to enhance random mutagenesis in cells offer advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency and narrow mutational spectra. Using a mechanism-driven approach, we created a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that enhances mutation 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro methods. We demonstrate that this system can be used to evolve antibiotic resistance in wild-type E. coli in mutagenesis of chromosomes, episomes and viruses in vivo, and are applicable to both bacterial and bacteriophage-mediated laboratory evolution platforms.

  11. Radiation-induced base substitution mutagenesis in single-stranded DNA phage M13

    International Nuclear Information System (INIS)

    Brandenburger, A.; Godson, G.N.; Glickman, B.W.; Sluis, C.A. van

    1981-01-01

    To elucidate the relative contributions of targeted and untargeted mutations to γ and UV radiation mutagenesis, the DNA sequences of 174 M13 revertant phages isolated from stocks of irradiated or unirradiated amber mutants grown in irradiated (SOS-induced) or unirradiated (non-induced) host bacteria, have been determined. Differences in the spectra of base change mutations induced in the various conditions were apparent, but no obvious specificity of mutagenesis was detected. In particular, under the present conditions, pyrimidine dimers did not seem to be the principal sites of UV-induced base substitution mutagenesis, suggesting that such mutagenesis occurs at the sites of lesions other than pyrimidine dimers, or is untargeted. (U.K.)

  12. Multiplex conditional mutagenesis in zebrafish using the CRISPR/Cas system.

    Science.gov (United States)

    Yin, L; Maddison, L A; Chen, W

    2016-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a powerful tool for genome editing in numerous organisms. However, the system is typically used for gene editing throughout the entire organism. Tissue and temporal specific mutagenesis is often desirable to determine gene function in a specific stage or tissue and to bypass undesired consequences of global mutations. We have developed the CRISPR/Cas system for conditional mutagenesis in transgenic zebrafish using tissue-specific and/or inducible expression of Cas9 and U6-driven expression of sgRNA. To allow mutagenesis of multiple targets, we have isolated four distinct U6 promoters and designed Golden Gate vectors to easily assemble transgenes with multiple sgRNAs. We provide experimental details on the reagents and applications for multiplex conditional mutagenesis in zebrafish. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans.

    Science.gov (United States)

    Noma, Kentaro; Jin, Yishi

    2016-11-14

    Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

  14. The use of plant tissue culture system in the mutagenesis of Secale cereale L

    International Nuclear Information System (INIS)

    Rybczynski, J.J.; KozIowska, W.; Turzynski, D.

    1990-01-01

    Full text: Among cereals, Secale cereale L. is the worst species for 'in vitro' mutagenesis. In the case of seed mutagenesis of rye each seed is expected to be a different genotype and only somatic embryogenesis assures propagation towards numerous individuals possessing the same genotype. Therefore, another system of in-vitro mutagenesis is explored. Immature embryos were isolated from spikes of field growing plants. The established cultures were irradiated with 0.5; 1.0 and 1.5 kR gamma rays on the first day of the culture and after 6 weeks in culture. After irradiation all cultures were subcultured. For mutagenesis in general uniformity of the original material is very important. Therefore, in rye, irradiation of regenerated somatic embryos may be a good approach. (author)

  15. Facile Site-Directed Mutagenesis of Large Constructs Using Gibson Isothermal DNA Assembly.

    Science.gov (United States)

    Yonemoto, Isaac T; Weyman, Philip D

    2017-01-01

    Site-directed mutagenesis is a commonly used molecular biology technique to manipulate biological sequences, and is especially useful for studying sequence determinants of enzyme function or designing proteins with improved activity. We describe a strategy using Gibson Isothermal DNA Assembly to perform site-directed mutagenesis on large (>~20 kbp) constructs that are outside the effective range of standard techniques such as QuikChange II (Agilent Technologies), but more reliable than traditional cloning using restriction enzymes and ligation.

  16. Overexpression of metallothioneins, stem cell niches and field cancerization in experimental gliomagenesis Superexpresión de metalotioneínas, nichos de células madre y campos de cancerización en gliomagénesis experimental A superexpressão de metalotioneínas em células-tronco de áreas cancerígenas na gênese de glioma experimental

    Directory of Open Access Journals (Sweden)

    Julio César Fernandes da Silva

    2009-12-01

    Full Text Available INTRODUCTION: stem cells may originate and perpetuate the tumor growth, but they are poorly known in gliomagenesis. Metallothioneins (MTs are proteins involved in oncogenesis and immunopositivity, for MT may be used as a stem cell mutation marker. OBJECTIVE: to study the MT expression in the ENU experimental model and to establish an experimental model to track glioma stem cells in early oncogenesis. METHODS: Thirty-six male Wistar rats were divided into two groups; the experimental group was treated within 24 hours after birth (neonate rats with a single dose of subcutaneously injected N-ethyl N-nitrosourea ENU (40 mg/kg body weight. The control animals were injected with the same volume of saline. These experimental animals were subdivided into three groups according to the euthanize time, as follows: the Group 1 (G1 was euthanized at the age of 30 days; the Group 2 (G2, at the age of 180 days and the Group 3 (G3 was euthanized soon after the appearing of signs of the existence of nervous system tumors, at an average age of 321 days. Immunohistochemical detection of MT protein in cold acetone-fixed paraffin embedded spine cord sections was performed by the streptavidin-avidin-biotin-immuno peroxidase complex method. RESULTS: by using the experimental model of gliomagenesis induced by the N-ethyl N-nitrosourea, it was possible to detect putative tumor stem cells in early oncogenesis, to analyze a field cancerization process and to observe a close morphological relationship between MT positive cells and blood vessels. CONCLUSIONS: this reproducible experimental model allows further studies on the origins, development and regulating factors involved in gliomagenesis.INTRODUCCIÓN: células madre pueden originar y perpetuar el crecimiento tumoral, sin embargo son poco conocidas en la gliomagénesis. Las metalotioneínas (MTs son proteínas involucradas en la oncogénesis, y la inmunopositividad de las MTs puede ser utilizada como marcador de

  17. Retroviral Vectors for Analysis of Viral Mutagenesis and Recombination

    Directory of Open Access Journals (Sweden)

    Jonathan M.O. Rawson

    2014-09-01

    Full Text Available Retrovirus population diversity within infected hosts is commonly high due in part to elevated rates of replication, mutation, and recombination. This high genetic diversity often complicates the development of effective diagnostics, vaccines, and antiviral drugs. This review highlights the diverse vectors and approaches that have been used to examine mutation and recombination in retroviruses. Retroviral vectors for these purposes can broadly be divided into two categories: those that utilize reporter genes as mutation or recombination targets and those that utilize viral genes as targets of mutation or recombination. Reporter gene vectors greatly facilitate the detection, quantification, and characterization of mutants and/or recombinants, but may not fully recapitulate the patterns of mutagenesis or recombination observed in native viral gene sequences. In contrast, the detection of mutations or recombination events directly in viral genes is more biologically relevant but also typically more challenging and inefficient. We will highlight the advantages and disadvantages of the various vectors and approaches used as well as propose ways in which they could be improved.

  18. CRISPR/Cas-mediated targeted mutagenesis in Daphnia magna.

    Directory of Open Access Journals (Sweden)

    Takashi Nakanishi

    Full Text Available The water flea Daphnia magna has been used as an animal model in ecology, evolution, and environmental sciences. Thanks to the recent progress in Daphnia genomics, genetic information such as the draft genome sequence and expressed sequence tags (ESTs is now available. To investigate the relationship between phenotypes and the available genetic information about Daphnia, some gene manipulation methods have been developed. However, a technique to induce targeted mutagenesis into Daphnia genome remains elusive. To overcome this problem, we focused on an emerging genome editing technique mediated by the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas system to introduce genomic mutations. In this study, we targeted a functionally conserved regulator of eye development, the eyeless gene in D. magna. When we injected Cas9 mRNAs and eyeless-targeting guide RNAs into eggs, 18-47% of the survived juveniles exhibited abnormal eye morphology. After maturation, up to 8.2% of the adults produced progenies with deformed eyes, which carried mutations in the eyeless loci. These results showed that CRISPR/Cas system could introduce heritable mutations into the endogenous eyeless gene in D. magna. This is the first report of a targeted gene knockout technique in Daphnia and will be useful in uncovering Daphnia gene functions.

  19. In vitro mutagenesis for the improvement of Josapine pineapple

    International Nuclear Information System (INIS)

    Rusli Ibrahim; Amir Hamzah

    2006-01-01

    Pineapple is the most important fruit in terms of revenue earner in Malaysia. There are about 10,000 ha cultivated with this fruit and half of this is owned by estates and planted for the canning industry. The export of canned pineapple is about 2 million standard cases annually valued at RM 60 million, while the export of fresh pineapple is about 40,000 tonnes worth about RM 10 million. The industry for canning is however, an ailing industry with production on the decline since the 70s. Somaclonal variations and induced mutation using irradiation in breeding are least invasive in changes to genetic make-up of an established variety and will be useful for improving the pineapple varieties. The use of tissue culture to generate somaclones with minute genetic changes that do not damage the overall varietal identity would be the most suitable tool to improve the variety. Protocols for the production of tissue culture plantlets of pineapple using bioreactor technology has been developed and proved to be much more efficient and productive compared to conventional method. In vitro mutagenesis using adventitious buds had produced new plants with smooth leaves, vigorous growth and ornamental-like characters. A total of 30,000 plants derived from tissue culture will be planted and screened in the field for the improvement of Josapine pineapple against bacterial heart rot disease and multiple crown. (Author)

  20. A wheat cold resistance mutant derived from space mutagenesis

    International Nuclear Information System (INIS)

    Li Peng; Sun Mingzhu; Zhang Fengyun; Gao Guoqiang; Qiu Denglin; Li Xinhua

    2012-01-01

    A cold resistance mutant, obtained by spaceflight mutagenesis on the seeds of wheat variety Han6172, and the DNA of cold resistance mutant and contrast Han6172 were compared by SRAP technique. 380 pairs of primers were screened, 6 pairs of them had polymorphisms between mutant and contrast, the rate was 1.58%, and this data indicated that there are no obvious DNA differences between mutant and contrast Six specific fragments were obtained, 3 fragments of them were amplified in mutant. Homology analysis in GenBank showed that Me3-Em7-Mt, Me4-Em11-CK, Me7-Em19-CK and Me6-Em9-Mt all had homologous sequences with wheat chromosome 3B-specific BAC library, and this result indicated that the gene and regulator sequences associated with mutant cold resistance might locate on 3B chromosome. It was speculated that space mutation induced the mutation of 3B chromosome primary structure, and influenced the expressions of cold resistance genes, which resulted in the mutation of cold resistance ability. (authors)

  1. The Fanconi anemia pathway limits the severity of mutagenesis.

    Science.gov (United States)

    Hinz, John M; Nham, Peter B; Salazar, Edmund P; Thompson, Larry H

    2006-08-13

    Fanconi anemia (FA) is a developmental and cancer predisposition disorder in which key, yet unknown, physiological events promoting chromosome stability are compromised. FA cells exhibit excess metaphase chromatid breaks and are universally hypersensitive to DNA interstrand crosslinking agents. Published mutagenesis data from single-gene mutation assays show both increased and decreased mutation frequencies in FA cells. In this review we discuss the data from the literature and from our isogenic fancg knockout hamster CHO cells, and interpret these data within the framework of a molecular model that accommodates these seemingly divergent observations. In FA cells, reduced rates of recovery of viable X-linked hypoxanthine phosphoribosyltransferase (hprt) mutants are characteristically observed for diverse mutagenic agents, but also in untreated cultures, indicating the relevance of the FA pathway for processing assorted DNA lesions. We ascribe these reductions to: (1) impaired mutagenic translesion synthesis within hprt during DNA replication and (2) lethality of mutant cells following replication fork breakage on the X chromosome, caused by unrepaired double-strand breaks or large deletions/translocations encompassing essential genes flanking hprt. These findings, along with studies showing increased spontaneous mutability of FA cells at two autosomal loci, support a model in which FA proteins promote both translesion synthesis at replication-blocking lesions and repair of broken replication forks by homologous recombination and DNA end joining. The essence of this model is that the FANC protein pathway serves to restrict the severity of mutational outcome by favoring base substitutions and small deletions over larger deletions and chromosomal rearrangements.

  2. Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Rongming; Liang, Liya; Garst, Andrew D.; Choudhury, Alaksh; Nogué, Violeta Sànchez i.; Beckham, Gregg T.; Gill, Ryan T.

    2018-05-01

    Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method to engineer rapid glucose and xylose co-consumption and tolerance to hydrolysate inhibitors in E. coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target ~40,000 mutations across 30 genes. These libraries included global and high-level regulators that regulate global gene expression, transcription factors that play important roles in genome-level transcription, enzymes that function in the sugar transport system, NAD(P)H metabolism, and the aldehyde reduction system. Specific mutants that conferred increased growth in mixed sugars and hydrolysate tolerance conditions were isolated, confirmed, and evaluated for changes in genome-wide expression levels. We tested the strain with positive combinatorial mutations for 3-hydroxypropionic acid (3HP) production under high furfural and high acetate hydrolysate fermentation, which demonstrated a 7- and 8-fold increase in 3HP productivity relative to the parent strain, respectively.

  3. A wheat cold resistance mutant derived from space mutagenesis

    International Nuclear Information System (INIS)

    Li Peng; Sun Mingzhu; Zhang Fengyun; Gao Guoqiang; Qiu Denglin; Li Xinhua

    2011-01-01

    A cold resistance mutant, obtained by spaceflight mutagenesis on the seeds of wheat variety Han6172, and the DNA of cold resistance mutant and contrast Han6172 were compared by SRAP technique. 380 pairs of primers were screened, 6 pairs of them had polymorphisms between mutant and contrast, the rate was 1.58%, and this data indicated that there are no obvious DNA differences between mutant and contrast. Six specific fragments were obtained, 3 fragments of them were amplified in mutant. Homology analysis in GenBank showed that Me3-Em7-Mt, Me4-Em11-CK, Me7-Em19-CK and Me6-Em9-Mt all had homologous sequences with wheat chromosome 3B-specific BAC library, and this result indicated that the gene and regulator sequences associated with mutant cold resistance might locate on 3B chromosome. It was speculated that space mutation induced the mutation of 3B chromosome primary structure, and influenced the expressions of cold resistance genes, which resulted in the mutation of cold resistance ability. (authors)

  4. Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs.

    Science.gov (United States)

    Yin, Linlin; Maddison, Lisette A; Li, Mingyu; Kara, Nergis; LaFave, Matthew C; Varshney, Gaurav K; Burgess, Shawn M; Patton, James G; Chen, Wenbiao

    2015-06-01

    Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward. Copyright © 2015 by the Genetics Society of America.

  5. Step-By-Step In Vitro Mutagenesis: Lessons From Fucose-Binding Lectin PA-IIL.

    Science.gov (United States)

    Mrázková, Jana; Malinovská, Lenka; Wimmerová, Michaela

    2017-01-01

    Site-directed mutagenesis is a powerful technique which is used to understand the basis of interactions between proteins and their binding partners, as well as to modify these interactions. Methods of rational design that are based on detailed knowledge of the structure of a protein of interest are often used for preliminary investigations of the possible outcomes which can result from the practical application of site-directed mutagenesis. Also, random mutagenesis can be used in tandem with site-directed mutagenesis for an examination of amino acid "hotspots."Lectins are sugar-binding proteins which, among other functions, mediate the recognition of host cells by a pathogen and its adhesion to the host cell surface. Hence, lectins and their binding properties are studied and engineered using site-directed mutagenesis.In this chapter, we describe a site-directed mutagenesis method used for investigating the sugar binding pattern of the PA-IIL lectin from the pathogenic bacterium Pseudomonas aeruginosa. Moreover, procedures for the production and purification of PA-IIL mutants are described, and several basic methods for characterizing the mutants are discussed.

  6. Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis.

    Directory of Open Access Journals (Sweden)

    Chad K Heazlewood

    2008-03-01

    Full Text Available MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis.By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p < 0.01 and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1beta, TNF-alpha, and IFN-gamma was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-gamma, TNF-alpha, and IL-13. This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar

  7. Mutagenesis and haploid culture for disease resistance in Brassica napus

    Energy Technology Data Exchange (ETDEWEB)

    MacDonald, M V; Ahmad, I; Ingram, D S [Botany School, University of Cambridge, Cambridge (United Kingdom)

    1990-01-01

    Full text: Most winter oilseed rape cultivars share parentage and therefore show little genetic diversity. There is no known resistance to Alternaria spp. in oilseed rape or in any related Brassica species. Experiments with tissue culture yielded only transient, non-genetic resistance. Therefore, mutagenesis may be used to generate heritable resistance to Alternaria spp. Gamma irradiation was applied to seeds of 'Bienvenue', secondary embryoids of cvs 'Primor' and 'Rapora', and buds of cvs 'Primor' and 'Ariana'. Isolated microspores from cv 'Ariana' and rapid cycling B. napus were also treated. The doses used ranged from 0-100 Gy for isolated microspores and buds, up to 600 Gy for seeds and 960 Gy for secondary embryoids. EMS was used to treat seeds of line WRG-42 (supplied by Nickersons RPB) and microspores of cv 'Bienvenue' and rapid cycling B. napus. Seeds were treated with up to 2.0% EMS for 0.2 h. before plating them on the culture medium. Seed irradiation up to 600 Gy did not reduce germination. M{sub 1} and M{sub 2} progenies were tested both in the laboratory and in field trials, and none of these were found to be resistant to Alternaria. However, considerable variation for other characters was observed. Haploid cultures from these plants were extremely difficult to regenerate, and for this reason no regenerant plants have been tested for resistance. For irradiated secondary embryoids the regeneration capacity decreased with increasing dose. Regenerated plants have been tested for resistance to Alternaria, but stable resistance was not observed. Haploid cultures were obtained from irradiated buds, using both anther and microspore culture. Low irradiation treatment was beneficial to developing embryoids. Some regenerants have been obtained from EMS treated microspores and seeds. Four plants have repeatedly given increased levels of resistance to A. brassicicola, and progenies are being tested to determine the genetic nature of the resistance. (author)

  8. Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data.

    Science.gov (United States)

    Tripathi, Arti; Gupta, Kritika; Khare, Shruti; Jain, Pankaj C; Patel, Siddharth; Kumar, Prasanth; Pulianmackal, Ajai J; Aghera, Nilesh; Varadarajan, Raghavan

    2016-11-01

    Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used saturation mutagenesis combined with deep sequencing to determine mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliphatic and charged amino acid substitutions. At nonactive-site positions, the average mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were observed for two other small proteins, PDZ domain (PSD95 pdz3 ) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and solubility. The inability to refold reversibly, as well as a decreased folding rate in vitro, is associated with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones on mutant phenotypes, most deleterious mutants showed an increased in vivo activity and solubility only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  9. Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

    Science.gov (United States)

    Crawford, Lisa; Karr, Laurel J.; Nadarajah, Arunan; Pusey, Marc

    1999-01-01

    A number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 43 axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to (3)500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 (Registered) PHE or ALA and ASN 113 (Registered) ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 43 helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.

  10. Radiation mutagenesis from molecular and genetic points of view

    International Nuclear Information System (INIS)

    Chen, D.J.C.; Park, M.S.; Okinaka, R.T.; Jaberaboansari, A.

    1993-01-01

    An important biological effect of ionizing radiation on living organisms is mutation induction. Mutation is also a primary event in the etiology of cancer. The chain events, from induction of DNA damage by ionizing radiation to processing of these damages by the cellular repair/replication machinery, that lead to mutation are not well understood. The development of quantitative methods for measuring mutation-induction, such as the HPRT system, in cultured mammalian cells has provided an estimate of the mutagenic effects of x- and γ-rays as wen as of high LET radiation in both rodent and human cells. A major conclusion from these mutagenesis data is that high LET radiation induces mutations more efficiently than g-rays. Molecular analysis of mutations induced by sparsely ionizing radiation have detected major structural alterations at the gene level. Our molecular results based on analysis of human HPRT deficient mutants induced by γ-rays, α-particles and high energy charged particles indicate that higher LET radiation induce more total and large deletion mutations than γ-rays. Utilizing molecular techniques including polymerase chain reaction (PCR), Single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and Direct DNA sequencing, mutational spectra induced by ionizing radiation have been compared in different cell systems. Attempts have also been made to determine the mutagenic potential and the nature of mutation induced by low dose rate γ-rays. Defective repair, in the form of either a diminished capability for repair or inaccurate repair, can lead to increased risk of heritable mutations from radiation exposure. Therefore, the effects of DNA repair deficiency on the mutation induction in mammalian cells is reviewed

  11. Expression and site-directed mutagenesis of human dihydrofolate reductase

    Energy Technology Data Exchange (ETDEWEB)

    Prendergast, N.J.; Delcamp, T.J.; Smith, P.L.; Freisheim, J.H.

    1988-05-17

    A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 ..-->.. Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by ..cap alpha..-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.

  12. Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Kai, Mihoko; Wang, Teresa S.-F

    2003-11-27

    Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Pol{kappa}). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks. Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development.

  13. Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis

    International Nuclear Information System (INIS)

    Kai, Mihoko; Wang, Teresa S.-F.

    2003-01-01

    Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Polκ). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks. Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development

  14. Mutagenesis and haploid culture for disease resistance in Brassica napus

    International Nuclear Information System (INIS)

    MacDonald, M.V.; Ahmad, I.; Ingram, D.S.

    1990-01-01

    Full text: Most winter oilseed rape cultivars share parentage and therefore show little genetic diversity. There is no known resistance to Alternaria spp. in oilseed rape or in any related Brassica species. Experiments with tissue culture yielded only transient, non-genetic resistance. Therefore, mutagenesis may be used to generate heritable resistance to Alternaria spp. Gamma irradiation was applied to seeds of 'Bienvenue', secondary embryoids of cvs 'Primor' and 'Rapora', and buds of cvs 'Primor' and 'Ariana'. Isolated microspores from cv 'Ariana' and rapid cycling B. napus were also treated. The doses used ranged from 0-100 Gy for isolated microspores and buds, up to 600 Gy for seeds and 960 Gy for secondary embryoids. EMS was used to treat seeds of line WRG-42 (supplied by Nickersons RPB) and microspores of cv 'Bienvenue' and rapid cycling B. napus. Seeds were treated with up to 2.0% EMS for 0.2 h. before plating them on the culture medium. Seed irradiation up to 600 Gy did not reduce germination. M 1 and M 2 progenies were tested both in the laboratory and in field trials, and none of these were found to be resistant to Alternaria. However, considerable variation for other characters was observed. Haploid cultures from these plants were extremely difficult to regenerate, and for this reason no regenerant plants have been tested for resistance. For irradiated secondary embryoids the regeneration capacity decreased with increasing dose. Regenerated plants have been tested for resistance to Alternaria, but stable resistance was not observed. Haploid cultures were obtained from irradiated buds, using both anther and microspore culture. Low irradiation treatment was beneficial to developing embryoids. Some regenerants have been obtained from EMS treated microspores and seeds. Four plants have repeatedly given increased levels of resistance to A. brassicicola, and progenies are being tested to determine the genetic nature of the resistance. (author)

  15. Expression and site-directed mutagenesis of human dihydrofolate reductase

    International Nuclear Information System (INIS)

    Prendergast, N.J.; Delcamp, T.J.; Smith, P.L.; Freisheim, J.H.

    1988-01-01

    A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 → Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by α-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme

  16. Defect of Fe-S cluster binding by DNA polymerase δ in yeast suppresses UV-induced mutagenesis, but enhances DNA polymerase ζ - dependent spontaneous mutagenesis.

    Science.gov (United States)

    Stepchenkova, E I; Tarakhovskaya, E R; Siebler, H M; Pavlov, Y I

    2017-01-01

    Eukaryotic genomes are duplicated by a complex machinery, utilizing high fidelity replicative B-family DNA polymerases (pols) α, δ and ε. Specialized error-prone pol ζ, the fourth B-family member, is recruited when DNA synthesis by the accurate trio is impeded by replication stress or DNA damage. The damage tolerance mechanism dependent on pol ζ prevents DNA/genome instability and cell death at the expense of increased mutation rates. The pol switches occurring during this specialized replication are not fully understood. The loss of pol ζ results in the absence of induced mutagenesis and suppression of spontaneous mutagenesis. Disruption of the Fe-S cluster motif that abolish the interaction of the C-terminal domain (CTD) of the catalytic subunit of pol ζ with its accessory subunits, which are shared with pol δ, leads to a similar defect in induced mutagenesis. Intriguingly, the pol3-13 mutation that affects the Fe-S cluster in the CTD of the catalytic subunit of pol δ also leads to defective induced mutagenesis, suggesting the possibility that Fe-S clusters are essential for the pol switches during replication of damaged DNA. We confirmed that yeast strains with the pol3-13 mutation are UV-sensitive and defective in UV-induced mutagenesis. However, they have increased spontaneous mutation rates. We found that this increase is dependent on functional pol ζ. In the pol3-13 mutant strain with defective pol δ, there is a sharp increase in transversions and complex mutations, which require functional pol ζ, and an increase in the occurrence of large deletions, whose size is controlled by pol ζ. Therefore, the pol3-13 mutation abrogates pol ζ-dependent induced mutagenesis, but allows for pol ζ recruitment for the generation of spontaneous mutations and prevention of larger deletions. These results reveal differential control of the two major types of pol ζ-dependent mutagenesis by the Fe-S cluster present in replicative pol δ. Copyright © 2016

  17. Site-specific genomic (SSG and random domain-localized (RDL mutagenesis in yeast

    Directory of Open Access Journals (Sweden)

    Honigberg Saul M

    2004-04-01

    Full Text Available Abstract Background A valuable weapon in the arsenal available to yeast geneticists is the ability to introduce specific mutations into yeast genome. In particular, methods have been developed to introduce deletions into the yeast genome using PCR fragments. These methods are highly efficient because they do not require cloning in plasmids. Results We have modified the existing method for introducing deletions in the yeast (S. cerevisiae genome using PCR fragments in order to target point mutations to this genome. We describe two PCR-based methods for directing point mutations into the yeast genome such that the final product contains no other disruptions. In the first method, site-specific genomic (SSG mutagenesis, a specific point mutation is targeted into the genome. In the second method, random domain-localized (RDL mutagenesis, a mutation is introduced at random within a specific domain of a gene. Both methods require two sequential transformations, the first transformation integrates the URA3 marker into the targeted locus, and the second transformation replaces URA3 with a PCR fragment containing one or a few mutations. This PCR fragment is synthesized using a primer containing a mutation (SSG mutagenesis or is synthesized by error-prone PCR (RDL mutagenesis. In SSG mutagenesis, mutations that are proximal to the URA3 site are incorporated at higher frequencies than distal mutations, however mutations can be introduced efficiently at distances of at least 500 bp from the URA3 insertion. In RDL mutagenesis, to ensure that incorporation of mutations occurs at approximately equal frequencies throughout the targeted region, this region is deleted at the same time URA3 is integrated. Conclusion SSG and RDL mutagenesis allow point mutations to be easily and efficiently incorporated into the yeast genome without disrupting the native locus.

  18. Codon cassette mutagenesis: a general method to insert or replace individual codons by using universal mutagenic cassettes.

    OpenAIRE

    Kegler-Ebo, D M; Docktor, C M; DiMaio, D

    1994-01-01

    We describe codon cassette mutagenesis, a simple method of mutagenesis that uses universal mutagenic cassettes to deposit single codons at specific sites in double-stranded DNA. A target molecule is first constructed that contains a blunt, double-strand break at the site targeted for mutagenesis. A double-stranded mutagenic codon cassette is then inserted at the target site. Each mutagenic codon cassette contains a three base pair direct terminal repeat and two head-to-head recognition sequen...

  19. Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice.

    Science.gov (United States)

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2015-08-01

    The CRISPR/Cas9 system is an efficient tool used for genome editing in a variety of organisms. Despite several recent reports of successful targeted mutagenesis using the CRISPR/Cas9 system in plants, in each case the target gene of interest, the Cas9 expression system and guide-RNA (gRNA) used, and the tissues used for transformation and subsequent mutagenesis differed, hence the reported frequencies of targeted mutagenesis cannot be compared directly. Here, we evaluated mutation frequency in rice using different Cas9 and/or gRNA expression cassettes under standardized experimental conditions. We introduced Cas9 and gRNA expression cassettes separately or sequentially into rice calli, and assessed the frequency of mutagenesis at the same endogenous targeted sequences. Mutation frequencies differed significantly depending on the Cas9 expression cassette used. In addition, a gRNA driven by the OsU6 promoter was superior to one driven by the OsU3 promoter. Using an all-in-one expression vector harboring the best combined Cas9/gRNA expression cassette resulted in a much improved frequency of targeted mutagenesis in rice calli, and bi-allelic mutant plants were produced in the T0 generation. The approach presented here could be adapted to optimize the construction of Cas9/gRNA cassettes for genome editing in a variety of plants.

  20. 2012 Gordon Research Conference on Mutagenesis - Formal Schedule and Speaker/Poster Program

    Energy Technology Data Exchange (ETDEWEB)

    Demple, Bruce [Stony Brook Univ., NY (United States). School of Medicine

    2012-08-24

    The delicate balance among cellular pathways that control mutagenic changes in DNA will be the focus of the 2012 Mutagenesis Gordon Research Conference. Mutagenesis is essential for evolution, while genetic stability maintains cellular functions in all organisms from microbes to metazoans. Different systems handle DNA lesions at various times of the cell cycle and in different places within the nucleus, and inappropriate actions can lead to mutations. While mutation in humans is closely linked to disease, notably cancers, mutational systems can also be beneficial. The conference will highlight topics of beneficial mutagenesis, including full establishment of the immune system, cell survival mechanisms, and evolution and adaptation in microbial systems. Equal prominence will be given to detrimental mutation processes, especially those involved in driving cancer, neurological diseases, premature aging, and other threats to human health. Provisional session titles include Branching Pathways in Mutagenesis; Oxidative Stress and Endogenous DNA Damage; DNA Maintenance Pathways; Recombination, Good and Bad; Problematic DNA Structures; Localized Mutagenesis; Hypermutation in the Microbial World; and Mutation and Disease.

  1. Hypoxia induces mitochondrial mutagenesis and dysfunction in inflammatory arthritis.

    LENUS (Irish Health Repository)

    Biniecka, Monika

    2012-02-01

    mitochondrial genome mutagenesis, and antioxidants significantly rescue these events.

  2. Chromosomal damages and mutagenesis in mammalian and human cells induced by ionizing radiations with different LET

    International Nuclear Information System (INIS)

    Govorun, R.D.

    1997-01-01

    On the basis of literature and proper data the inference was made about essential role of structural chromosomal (and gene) damages in spontaneous and radiation-induced mutagenesis of mammalian and human cells on HPRT-loci. The evidences of increasing role of these damages in the mutagenesis after the influence of ionizing radiations with high LET are adduced. The consequences of HPRT-gene damages have been examined hypothetically. The geterogeneity of mutant subclones on their cytogenetical properties were revealed experimentally. The data reflect a phenomenon of the reproductive chromosomal instability in many generations of mutant cell. The mutagenesis of mammalian cells is also accompanied by the impairment of chromosome integrity with high probability as a stage of appropriate genome reorganization because of changed vital conditions

  3. Effect of umuC mutations on targeted and untargeted ultraviolet mutagenesis in bacteriophage lambda

    International Nuclear Information System (INIS)

    Maenhaut-Michel, G.; Caillet-Fauquet, P.

    1984-01-01

    Mutagenesis of phage lambda towards clear-plaque (c + → c) results in two classes of mutants that can be distinguished genetically and morphologically. Indirect mutagenesis, i.e. mutagenesis of unirradiated phage lambdac + stimulated by the ultraviolet irradiation of the Escherichia coli host, results in mixed bursts (c/c + ) of turbid wild-type and clear=plaque mutant phages. Pure bursts of lambdac mutants are induced by irradiation of the phage genome. Irradiation of both phages and host bacteria stimulates the production of the two classes of mutant clones. It is shown that three different mutant alleles of the E. coli umuC gene only prevent the appearance of pure bursts of clear-plaque mutants, while mixed bursts are produced at least as frequently in umuC mutants as in the umuC + parent. (author)

  4. Genetic analysis of gamma-ray mutagenesis in yeast. I. Reversion in radiation-sensitive strains

    International Nuclear Information System (INIS)

    McKee, R.H.; Lawrence, C.W.

    1979-01-01

    The frequency of revertants induced by 60 Co γ rays of the ochre allele, cyc1-9, has been measured in radiation-sensitive strains carrying one of 19 nonallelic mutations and in wild-type strains. The results indicate that ionizing radiation mutagenesis depends on the activity of the RAD6 group of genes and that the gene functions employed are very similar, but probably not identical, to those that mediate uv mutagenesis. Repair activities dependent on the functions of the RAD50 through RAD57 loci, the major pathway for the repair of damage caused by ionizing radiation, do not appear to play any part in mutagenesis. A comparison between the γ-ray data and those obtained previously with uv and chemical mutagens suggests that the RAD6 mutagenic pathway is in fact composed of a set of processes, some of which are concerned with error-prone, and some with error-free, recovery activities

  5. Environmental Stress Induces Trinucleotide Repeat Mutagenesis in Human Cells by Alt-Nonhomologous End Joining Repair.

    Science.gov (United States)

    Chatterjee, Nimrat; Lin, Yunfu; Yotnda, Patricia; Wilson, John H

    2016-07-31

    Multiple pathways modulate the dynamic mutability of trinucleotide repeats (TNRs), which are implicated in neurodegenerative disease and evolution. Recently, we reported that environmental stresses induce TNR mutagenesis via stress responses and rereplication, with more than 50% of mutants carrying deletions or insertions-molecular signatures of DNA double-strand break repair. We now show that knockdown of alt-nonhomologous end joining (alt-NHEJ) components-XRCC1, LIG3, and PARP1-suppresses stress-induced TNR mutagenesis, in contrast to the components of homologous recombination and NHEJ, which have no effect. Thus, alt-NHEJ, which contributes to genetic mutability in cancer cells, also plays a novel role in environmental stress-induced TNR mutagenesis. Published by Elsevier Ltd.

  6. Genome-wide comparison of ultraviolet and ethyl methanesulphonate mutagenesis methods for the brown alga Ectocarpus.

    Science.gov (United States)

    Godfroy, Olivier; Peters, Akira F; Coelho, Susana M; Cock, J Mark

    2015-12-01

    Ectocarpus has emerged as a model organism for the brown algae and a broad range of genetic and genomic resources are being generated for this species. The aim of the work presented here was to evaluate two mutagenesis protocols based on ultraviolet irradiation and ethyl methanesulphonate treatment using genome resequencing to measure the number, type and distribution of mutations generated by the two methods. Ultraviolet irradiation generated a greater number of genetic lesions than ethyl methanesulphonate treatment, with more than 400 mutations being detected in the genome of the mutagenised individual. This study therefore confirms that the ultraviolet mutagenesis protocol is suitable for approaches that require a high density of mutations, such as saturation mutagenesis or Targeting Induced Local Lesions in Genomes (TILLING). Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Mutagenesis and phenotyping resources in zebrafish for studying development and human disease

    Science.gov (United States)

    Varshney, Gaurav Kumar

    2014-01-01

    The zebrafish (Danio rerio) is an important model organism for studying development and human disease. The zebrafish has an excellent reference genome and the functions of hundreds of genes have been tested using both forward and reverse genetic approaches. Recent years have seen an increasing number of large-scale mutagenesis projects and the number of mutants or gene knockouts in zebrafish has increased rapidly, including for the first time conditional knockout technologies. In addition, targeted mutagenesis techniques such as zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short sequences (CRISPR) or CRISPR-associated (Cas), have all been shown to effectively target zebrafish genes as well as the first reported germline homologous recombination, further expanding the utility and power of zebrafish genetics. Given this explosion of mutagenesis resources, it is now possible to perform systematic, high-throughput phenotype analysis of all zebrafish gene knockouts. PMID:24162064

  8. Targeted Mutagenesis in Rice Using TALENs and the CRISPR/Cas9 System.

    Science.gov (United States)

    Endo, Masaki; Nishizawa-Yokoi, Ayako; Toki, Seiichi

    2016-01-01

    Sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system, are powerful tools for understanding gene function and for developing novel traits in plants. In plant species for which transformation and regeneration systems using protoplasts are not yet established, direct delivery to nuclei of SSNs either in the form of RNA or protein is difficult. Thus, Agrobacterium-mediated transformation of SSN expression constructs in cultured cells is a practical means of delivering targeted mutagenesis in some plant species including rice. Because targeted mutagenesis occurs stochastically in transgenic cells and SSN-mediated targeted mutagenesis often leads to no selectable phenotype, identification of highly mutated cell lines is a critical step in obtaining regenerated plants with desired mutations.

  9. The influence of glycerol on γ-induced mutagenesis in Salmonella typhimurium cells

    International Nuclear Information System (INIS)

    Basha, S.G.; Krasavin, E.A.; Kozubek, S.; Amirtaev, K.G.

    1990-01-01

    A study was made of the modifying effect of glycerol on the survival rate and γ-radiation-induced mutagenesis of Salmonella typhimurium cells TA98, TA100 and TA102. The DMF value, with respect to the survival rate, was 2.05-0.20. The dependence of the yield of γ-radiation-induced mutants on radiation dose was described by the curve with a maximum; the mutation frequency M(D) was well described by a gradual function M(D)=kD x . DMF values of the induced mutagenesis amounted to 2 for strains TA100 and TA102, and 1.5 for strain TA98

  10. Ultraviolet mutagenesis and the SOS response in Escherichia coli: A personal perspective

    International Nuclear Information System (INIS)

    Witkin, E.M.

    1989-01-01

    The study of ultraviolet (UV) mutagenesis in Escherichia coli began with the assumption that genes were likely to be changed at the instant of photon absorption. Over many decades, it became clear that postirradiation cellular activities, including enzymatic DNA repair of UV photo products and error-prone modes of tolerating unrepaired DNA lesions can exert profound influences on the mutagenic outcome of irradiation. Current study focusses on the molecular details of radiation-induced translesion DNA replication as the final event in UV mutagenesis

  11. Moderate repetitions (mobile elements) and hot points of chromosomal mutagenesis in Drozophila melanogaster

    International Nuclear Information System (INIS)

    Aleksandrova, M.V.; Sevan'kaev, A.V.; Aleksandrov, I.D.

    1989-01-01

    The results of experimental examination of hypothesis on mobile elements (ME) as target for radiation-induced chromosomal mutagenesis do not confirm it in this direct variant though indicate on the presence of nonincidental connection between sites of ME localization of certain type and points of chromosomal mutagenesis are presented. The presented regularity is explained by assumption that settling of the ME by genome is going along postulated static chromomer-binding-DNA complexes, playing an important role in space organization of interphase chromatin. 11 refs.; 2 figs.; 2 tabs

  12. Autosomal dominant hypercalciuria in a mouse model due to a mutation of the epithelial calcium channel, TRPV5.

    Directory of Open Access Journals (Sweden)

    Nellie Y Loh

    Full Text Available Hypercalciuria is a major cause of nephrolithiasis, and is a common and complex disorder involving genetic and environmental factors. Identification of genetic factors for monogenic forms of hypercalciuria is hampered by the limited availability of large families, and to facilitate such studies, we screened for hypercalciuria in mice from an N-ethyl-N-nitrosourea mutagenesis programme. We identified a mouse with autosomal dominant hypercalciuria (HCALC1. Linkage studies mapped the Hcalc1 locus to a 11.94 Mb region on chromosome 6 containing the transient receptor potential cation channel, subfamily V, members 5 (Trpv5 and 6 (Trpv6 genes. DNA sequence analysis of coding regions, intron-exon boundaries and promoters of Trpv5 and Trpv6 identified a novel T to C transition in codon 682 of TRPV5, mutating a conserved serine to a proline (S682P. Compared to wild-type littermates, heterozygous (Trpv5(682P/+ and homozygous (Trpv5(682P/682P mutant mice had hypercalciuria, polyuria, hyperphosphaturia and a more acidic urine, and ∼10% of males developed tubulointerstitial nephritis. Trpv5(682P/682P mice also had normal plasma parathyroid hormone but increased 1,25-dihydroxyvitamin D(3 concentrations without increased bone resorption, consistent with a renal defect for the hypercalciuria. Expression of the S682P mutation in human embryonic kidney cells revealed that TRPV5-S682P-expressing cells had a lower baseline intracellular calcium concentration than wild-type TRPV5-expressing cells, suggesting an altered calcium permeability. Immunohistological studies revealed a selective decrease in TRPV5-expression from the renal distal convoluted tubules of Trpv5(682P/+ and Trpv5(682P/682P mice consistent with a trafficking defect. In addition, Trpv5(682P/682P mice had a reduction in renal expression of the intracellular calcium-binding protein, calbindin-D(28K, consistent with a specific defect in TRPV5-mediated renal calcium reabsorption. Thus, our findings

  13. Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis.

    Science.gov (United States)

    Zhang, Xue; Zhang, Chong; Zhou, Qian-Qian; Zhang, Xiao-Fei; Wang, Li-Yan; Chang, Hai-Bo; Li, He-Ping; Oda, Yoshimitsu; Xing, Xin-Hui

    2015-07-01

    DNA damage is the dominant source of mutation, which is the driving force of evolution. Therefore, it is important to quantitatively analyze the DNA damage caused by different mutagenesis methods, the subsequent mutation rates, and their relationship. Atmospheric and room temperature plasma (ARTP) mutagenesis has been used for the mutation breeding of more than 40 microorganisms. However, ARTP mutagenesis has not been quantitatively compared with conventional mutation methods. In this study, the umu test using a flow-cytometric analysis was developed to quantify the DNA damage in individual viable cells using Salmonella typhimurium NM2009 as the model strain and to determine the mutation rate. The newly developed method was used to evaluate four different mutagenesis systems: a new ARTP tool, ultraviolet radiation, 4-nitroquinoline-1-oxide (4-NQO), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The mutation rate was proportional to the corresponding SOS response induced by DNA damage. ARTP caused greater DNA damage to individual living cells than the other conventional mutagenesis methods, and the mutation rate was also higher. By quantitatively comparing the DNA damage and consequent mutation rate after different types of mutagenesis, we have shown that ARTP is a potentially powerful mutagenesis tool with which to improve the characteristics of microbial cell factories.

  14. Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis.

    Science.gov (United States)

    Lee, Raymond Teck Ho; Ng, Ashley Shu Mei; Ingham, Philip W

    2016-01-01

    CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter) that was able to induce mutagenesis of its target. Our method provides a streamlined approach to test gRNA efficiency as well as increasing the versatility of conditional gene knock out in zebrafish.

  15. DinB Upregulation Is the Sole Role of the SOS Response in Stress-Induced Mutagenesis in Escherichia coli

    Science.gov (United States)

    Galhardo, Rodrigo S.; Do, Robert; Yamada, Masami; Friedberg, Errol C.; Hastings, P. J.; Nohmi, Takehiko; Rosenberg, Susan M.

    2009-01-01

    Stress-induced mutagenesis is a collection of mechanisms observed in bacterial, yeast, and human cells in which adverse conditions provoke mutagenesis, often under the control of stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e., are stressed. It is therefore important to understand how stress responses increase mutagenesis. In the Escherichia coli Lac assay, stress-induced point mutagenesis requires induction of at least two stress responses: the RpoS-controlled general/starvation stress response and the SOS DNA-damage response, both of which upregulate DinB error-prone DNA polymerase, among other genes required for Lac mutagenesis. We show that upregulation of DinB is the only aspect of the SOS response needed for stress-induced mutagenesis. We constructed two dinB(oc) (operator-constitutive) mutants. Both produce SOS-induced levels of DinB constitutively. We find that both dinB(oc) alleles fully suppress the phenotype of constitutively SOS-“off” lexA(Ind−) mutant cells, restoring normal levels of stress-induced mutagenesis. Thus, dinB is the only SOS gene required at induced levels for stress-induced point mutagenesis. Furthermore, although spontaneous SOS induction has been observed to occur in only a small fraction of cells, upregulation of dinB by the dinB(oc) alleles in all cells does not promote a further increase in mutagenesis, implying that SOS induction of DinB, although necessary, is insufficient to differentiate cells into a hypermutable condition. PMID:19270270

  16. DNA polymerase III of Escherichia coli is required for UV and ethyl methanesulfonate mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Hagensee, M.E.; Timme, T.L.; Bryan, S.K.; Moses, R.E.

    1987-06-01

    Strains of Escherichia coli possessing the pcbA1 mutation, a functional DNA polymerase I, and a temperature-sensitive mutation in DNA polymerase III can survive at the restrictive temperature (43 degrees C) for DNA polymerase III. The mutation rate of the bacterial genome of such strains after exposure to either UV light or ethyl methanesulfonate was measured by its rifampicin resistance or amino acid requirements. In addition, Weigle mutagenesis of preirradiated lambda phage was also measured. In all cases, no increase in mutagenesis was noted at the restrictive temperature for DNA polymerase III. Introduction of a cloned DNA polymerase III gene returned the mutation rate of the bacterial genome as well as the Weigle mutagenesis to normal at 43 degrees C. Using a recA-lacZ fusion, the SOS response after UV irradiation was measured and found to be normal at the restrictive and permissive temperature for DNA polymerase III, as was induction of lambda prophage. Recombination was also normal at either temperature. Our studies demonstrate that a functional DNA polymerase III is strictly required for mutagenesis at a step other than SOS induction.

  17. Deletion mutagenesis identifies a haploinsufficient role for gamma-zein in opaque-2 endosperm modification

    Science.gov (United States)

    Quality Protein Maize (QPM) is a hard kernel variant of the high-lysine mutant, opaque-2. Using gamma irradiation, we created opaque QPM variants to identify opaque-2 modifier genes and to investigate deletion mutagenesis combined with Illumina sequencing as a maize functional genomics tool. A K0326...

  18. Natural selection underlies apparent stress-induced mutagenesis in a bacteriophage infection model.

    Science.gov (United States)

    Yosef, Ido; Edgar, Rotem; Levy, Asaf; Amitai, Gil; Sorek, Rotem; Munitz, Ariel; Qimron, Udi

    2016-04-18

    The emergence of mutations following growth-limiting conditions underlies bacterial drug resistance, viral escape from the immune system and fundamental evolution-driven events. Intriguingly, whether mutations are induced by growth limitation conditions or are randomly generated during growth and then selected by growth limitation conditions remains an open question(1). Here, we show that bacteriophage T7 undergoes apparent stress-induced mutagenesis when selected for improved recognition of its host's receptor. In our unique experimental set-up, the growth limitation condition is physically and temporally separated from mutagenesis: growth limitation occurs while phage DNA is outside the host, and spontaneous mutations occur during phage DNA replication inside the host. We show that the selected beneficial mutations are not pre-existing and that the initial slow phage growth is enabled by the phage particle's low-efficiency DNA injection into the host. Thus, the phage particle allows phage populations to initially extend their host range without mutagenesis by virtue of residual recognition of the host receptor. Mutations appear during non-selective intracellular replication, and the frequency of mutant phages increases by natural selection acting on free phages, which are not capable of mutagenesis.

  19. Alleles conferring improved fiber quality from EMS mutagenesis of elite cotton genotypes

    Science.gov (United States)

    The elite gene pool of cotton (Gossypium spp.) has less diversity than those of most other major crops, making identification of novel alleles important to ongoing crop improvement. A total of 3,164 M5 lines resulting from ethyl methanesulfonate mutagenesis of two G. hirsutum breeding lines, TAM 94L...

  20. Random mutagenesis of the hyperthermophilic archaeon Pyrococcus furiosus using in vitro mariner transposition and natural transformation.

    Science.gov (United States)

    Guschinskaya, Natalia; Brunel, Romain; Tourte, Maxime; Lipscomb, Gina L; Adams, Michael W W; Oger, Philippe; Charpentier, Xavier

    2016-11-08

    Transposition mutagenesis is a powerful tool to identify the function of genes, reveal essential genes and generally to unravel the genetic basis of living organisms. However, transposon-mediated mutagenesis has only been successfully applied to a limited number of archaeal species and has never been reported in Thermococcales. Here, we report random insertion mutagenesis in the hyperthermophilic archaeon Pyrococcus furiosus. The strategy takes advantage of the natural transformability of derivatives of the P. furiosus COM1 strain and of in vitro Mariner-based transposition. A transposon bearing a genetic marker is randomly transposed in vitro in genomic DNA that is then used for natural transformation of P. furiosus. A small-scale transposition reaction routinely generates several hundred and up to two thousands transformants. Southern analysis and sequencing showed that the obtained mutants contain a single and random genomic insertion. Polyploidy has been reported in Thermococcales and P. furiosus is suspected of being polyploid. Yet, about half of the mutants obtained on the first selection are homozygous for the transposon insertion. Two rounds of isolation on selective medium were sufficient to obtain gene conversion in initially heterozygous mutants. This transposition mutagenesis strategy will greatly facilitate functional exploration of the Thermococcales genomes.

  1. Novel Escherichia coli umuD′ Mutants: Structure-Function Insights into SOS Mutagenesis

    Science.gov (United States)

    McLenigan, Mary; Peat, Thomas S.; Frank, Ekaterina G.; McDonald, John P.; Gonzalez, Martín; Levine, Arthur S.; Hendrickson, Wayne A.; Woodgate, Roger

    1998-01-01

    Although it has been 10 years since the discovery that the Escherichia coli UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD′, the function of UmuD′ has yet to be determined. In an attempt to elucidate the role of UmuD′ in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number umuD′ plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent umuD′ mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD′, three were nonsense mutations that resulted in a truncated UmuD′ protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomal umuD1, umuD44, and umuD77 alleles into umuD′. All 17 umuD′ mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD′. PMID:9721309

  2. Functional studies of human intestinal alkaline sphingomyelinase by deglycosylation and mutagenesis

    DEFF Research Database (Denmark)

    Wu, Jun; Hansen, Gert H; Nilsson, Ake

    2005-01-01

    in NPP are conserved, and those of the active core are modified. We examined the functional changes of the enzyme induced by deglycosylation and mutagenesis. Treating alk-SMase cDNA-transfected COS-7 cells with tunicamycin rendered the expressed enzyme completely inactive. Mutations of the five potential...

  3. Building on the Past, Shaping the Future: The Environmental Mutagenesis and Genomics Society

    Science.gov (United States)

    In late 2012 the members of the Environmental Mutagen Society voted to change its name to the Environmental Mutagenesis and Genomics Society. Here we describe the thought process that led to adoption of the new name, which both respects the rich history of a Society founded in 19...

  4. Gene discovery by chemical mutagenesis and whole-genome sequencing in Dictyostelium.

    Science.gov (United States)

    Li, Cheng-Lin Frank; Santhanam, Balaji; Webb, Amanda Nicole; Zupan, Blaž; Shaulsky, Gad

    2016-09-01

    Whole-genome sequencing is a useful approach for identification of chemical-induced lesions, but previous applications involved tedious genetic mapping to pinpoint the causative mutations. We propose that saturation mutagenesis under low mutagenic loads, followed by whole-genome sequencing, should allow direct implication of genes by identifying multiple independent alleles of each relevant gene. We tested the hypothesis by performing three genetic screens with chemical mutagenesis in the social soil amoeba Dictyostelium discoideum Through genome sequencing, we successfully identified mutant genes with multiple alleles in near-saturation screens, including resistance to intense illumination and strong suppressors of defects in an allorecognition pathway. We tested the causality of the mutations by comparison to published data and by direct complementation tests, finding both dominant and recessive causative mutations. Therefore, our strategy provides a cost- and time-efficient approach to gene discovery by integrating chemical mutagenesis and whole-genome sequencing. The method should be applicable to many microbial systems, and it is expected to revolutionize the field of functional genomics in Dictyostelium by greatly expanding the mutation spectrum relative to other common mutagenesis methods. © 2016 Li et al.; Published by Cold Spring Harbor Laboratory Press.

  5. Identification of a halotolerant mutant via in vitro mutagenesis in the cyanobacterium Fremyella diplosiphon

    Science.gov (United States)

    Energy metabolism and photosynthetic pigment accumulation are affected by salt stress in cyanobacteria leading to cessation of growth. The effect of salinity on the fresh water cyanobacteria, Fremyella diplosiphon was investigated and mutagenesis-based efforts were undertaken to enhance salt toleran...

  6. Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus

    DEFF Research Database (Denmark)

    Urbanski, Dorian Fabian; Malolepszy, Anna; Stougaard, Jens

    2012-01-01

    Insertion mutants facilitate functional analysis of genes, but for most plant species it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics. The main challenge is developing efficient high-throughput procedures for both mutagenesis and insert......Insertion mutants facilitate functional analysis of genes, but for most plant species it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics. The main challenge is developing efficient high-throughput procedures for both mutagenesis...... plants. The identified insertions showed that the endogenous LORE1 retrotransposon is well suited for insertion mutagenesis due to its homogenous gene targeting and exonic insertion preference. Since LORE1 transposition occurs in the germline, harvesting seeds from a single founder line and cultivating...... progeny generates a complete mutant population. This ease of LORE1 mutagenesis combined with the efficient FSTpoolit protocol, which exploits 2D pooling, Illumina sequencing, and automated data analysis, allows highly cost-efficient development of a comprehensive reverse genetic resource....

  7. Comparing Different Strategies in Directed Evolution of Enzyme Stereoselectivity: Single- versus Double-Code Saturation Mutagenesis.

    Science.gov (United States)

    Sun, Zhoutong; Lonsdale, Richard; Li, Guangyue; Reetz, Manfred T

    2016-10-04

    Saturation mutagenesis at sites lining the binding pockets of enzymes constitutes a viable protein engineering technique for enhancing or inverting stereoselectivity. Statistical analysis shows that oversampling in the screening step (the bottleneck) increases astronomically as the number of residues in the randomization site increases, which is the reason why reduced amino acid alphabets have been employed, in addition to splitting large sites into smaller ones. Limonene epoxide hydrolase (LEH) has previously served as the experimental platform in these methodological efforts, enabling comparisons between single-code saturation mutagenesis (SCSM) and triple-code saturation mutagenesis (TCSM); these employ either only one or three amino acids, respectively, as building blocks. In this study the comparative platform is extended by exploring the efficacy of double-code saturation mutagenesis (DCSM), in which the reduced amino acid alphabet consists of two members, chosen according to the principles of rational design on the basis of structural information. The hydrolytic desymmetrization of cyclohexene oxide is used as the model reaction, with formation of either (R,R)- or (S,S)-cyclohexane-1,2-diol. DCSM proves to be clearly superior to the likewise tested SCSM, affording both R,R- and S,S-selective mutants. These variants are also good catalysts in reactions of further substrates. Docking computations reveal the basis of enantioselectivity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Random mutagenesis of human serine racemase reveals residues important for the enzymatic activity

    Czech Academy of Sciences Publication Activity Database

    Hoffman, Hillary Elizabeth; Jirásková, Jana; Zvelebil, M.; Konvalinka, Jan

    2010-01-01

    Roč. 75, č. 1 (2010), s. 59-79 ISSN 0010-0765 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : D-serine * serine racemase * random mutagenesis Subject RIV: CE - Biochemistry Impact factor: 0.853, year: 2010

  9. Identification of a novel streptococcal gene cassette mediating SOS mutagenesis in Streptococcus uberis

    NARCIS (Netherlands)

    Varhimo, Emilia; Savijoki, Kirsi; Jalava, Jari; Kuipers, Oscar P.; Varmanen, Pekka

    Streptococci have been considered to lack the classical SOS response, defined by increased mutation after UV exposure and regulation by LexA. Here we report the identification of a potential self-regulated SOS mutagenesis gene cassette in the Streptococcaceae family. Exposure to UV light was found

  10. A high-throughput shotgun mutagenesis approach to mapping B-cell antibody epitopes.

    Science.gov (United States)

    Davidson, Edgar; Doranz, Benjamin J

    2014-09-01

    Characterizing the binding sites of monoclonal antibodies (mAbs) on protein targets, their 'epitopes', can aid in the discovery and development of new therapeutics, diagnostics and vaccines. However, the speed of epitope mapping techniques has not kept pace with the increasingly large numbers of mAbs being isolated. Obtaining detailed epitope maps for functionally relevant antibodies can be challenging, particularly for conformational epitopes on structurally complex proteins. To enable rapid epitope mapping, we developed a high-throughput strategy, shotgun mutagenesis, that enables the identification of both linear and conformational epitopes in a fraction of the time required by conventional approaches. Shotgun mutagenesis epitope mapping is based on large-scale mutagenesis and rapid cellular testing of natively folded proteins. Hundreds of mutant plasmids are individually cloned, arrayed in 384-well microplates, expressed within human cells, and tested for mAb reactivity. Residues are identified as a component of a mAb epitope if their mutation (e.g. to alanine) does not support candidate mAb binding but does support that of other conformational mAbs or allows full protein function. Shotgun mutagenesis is particularly suited for studying structurally complex proteins because targets are expressed in their native form directly within human cells. Shotgun mutagenesis has been used to delineate hundreds of epitopes on a variety of proteins, including G protein-coupled receptor and viral envelope proteins. The epitopes mapped on dengue virus prM/E represent one of the largest collections of epitope information for any viral protein, and results are being used to design better vaccines and drugs. © 2014 John Wiley & Sons Ltd.

  11. Roles for the yeast RAD18 and RAD52 DNA repair genes in UV mutagenesis.

    Science.gov (United States)

    Armstrong, J D; Chadee, D N; Kunz, B A

    1994-11-01

    Experimental evidence indicates that although the Saccharomyces cerevisiae RAD18 and RAD52 genes are not required for nucleotide excision repair, they function in the processing of UV-induced DNA damage in yeast. Conflicting statements regarding the UV mutability of strains deleted for RAD18 prompted us to re-examine the influence of RAD18, and RAD52, on UV mutagenesis. To do so, we characterized mutations induced by UV in SUP4-o, a yeast suppressor tRNA gene. SUP4-o was maintained on a plasmid in isogenic strains that either carried one of two different rad18 deletions (rad18 delta) or had RAD52 disrupted. Both rad18 deletions decreased the frequency of UV-induced SUP4-o mutations to levels close to those for spontaneous mutagenesis in the rad18 delta backgrounds, and prevented a net increase in mutant yield. A detailed analysis of mutations isolated after UV irradiation of one of the rad18 delta strains uncovered little evidence of the specificity features typical for UV mutagenesis in the isogenic repair-proficient (RAD) parent (e.g., predominance of G.C-->A.T transitions). Evidently, UV induction of SUP4-o mutations is highly dependent on the RAD18 gene. Compared to the RAD strain, disruption of RAD52 reduced the frequency and yield of UV mutagenesis by about two-thirds. Closer inspection revealed that 80% of this reduction was due to a decrease in the frequency of G.C-->A.T transitions. In addition, there were differences in the distributions and site specificities of single base-pair substitutions. Thus, RAD52 also participates in UV mutagenesis of a plasmid-borne gene in yeast, but to a lesser extent than RAD18.

  12. One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections.

    Directory of Open Access Journals (Sweden)

    Janire Mingo

    Full Text Available Site-directed mutagenesis (SDM is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis.

  13. Mutagenesis of the somaclones in vitro of cut roses by 60Co γ-rays irradiation

    International Nuclear Information System (INIS)

    Qu Suping; Su Yan; Wang Lihua; Tang Kaixue; Wang Jihua; Zhang Hao

    2009-01-01

    Mutagenesis of cut rose in vitro irradiated by 60 Co γ-rays was studied. The callus of leaves and regenerations adventitious bud was used as the explants for mutagenesis. Effect of 60 Co γ-rays irradiation on the callus's regeneration rate, adventitious bud's multiplication rate and vegetal status were studied. The results showed that the regeneration frequency of callus was decreased by 60 Co γ-rays irradiation. The regenerations adventitious bud was the better experimental materials compared with the callus of leaves. The lethal dose was 122 Gy and the semi-lethal dose was 76 Gy according to the regression equation. The appropriate dose on adventitious bud by irradiation rays was 50-60 Gy. (authors)

  14. Software-Supported USER Cloning Strategies for Site-Directed Mutagenesis and DNA Assembly

    DEFF Research Database (Denmark)

    Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen

    2015-01-01

    USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER...... cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein...... (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at ....

  15. Improvement of DNA transfer frequency and transposon mutagenesis of Erwinia carotovora subsp. betavasculorum.

    Science.gov (United States)

    Rella, M; Axelrood, P E; Weinhold, A R; Schroth, M N

    1989-01-01

    The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled transposon mutagenesis of a wild-type antibiotic-producing strain of E. carotovora subsp. betavasculorum. Twelve antibiotic-negative mutants were isolated, and one of these showed a reduction in antibiotic production in vitro. Many of these mutants also showed a reduction in their ability to macerate potato tissue. The mutants were classified into four genetic groups on the basis of their genetic and phenotypic characteristics, indicating that several genes are involved in antibiotic biosynthesis by E. carotovora subsp. betavasculorum. PMID:2543291

  16. Altered lipid accumulation in Nannochloropsis salina CCAP849/3 following EMS and UV induced mutagenesis

    Directory of Open Access Journals (Sweden)

    T.A. Beacham

    2015-09-01

    Full Text Available Microalgae have potential as a chemical feed stock in a range of industrial applications. Nannochloropsis salina was subject to EMS mutagenesis and the highest lipid containing cells selected using fluorescence-activated cell sorting. Assessment of growth, lipid content and fatty acid composition identified mutant strains displaying a range of altered traits including changes in the PUFA content and a total FAME increase of up to 156% that of the wild type strain. Combined with a reduction in growth this demonstrated a productivity increase of up to 76%. Following UV mutagenesis, lipid accumulation of the mutant cultures was elevated to more than 3 fold that of the wild type strain, however reduced growth rates resulted in a reduction in overall productivity. Changes observed are indicative of alterations to the regulation of the omega 6 Kennedy pathway. The importance of these variations in physiology for industrial applications such as biofuel production is discussed.

  17. Cell-mediated mutagenesis and cell transformation of mammalian cells by chemical carcinogens

    International Nuclear Information System (INIS)

    Huberman, E.; Langenbach, R.

    1977-01-01

    We have developed a cell-mediated mutagenesis assay in which cells with the appropriate markers for mutagenesis are co-cultivated with either lethally irradiated rodent embryonic cells that can metabolize carcinogenic hydrocarbons or with primary rat liver cells that can metabolize chemicals carcinogenic to the liver. During co-cultivation, the reactive metabolites of the procarcinogen appear to be transmitted to the mutable cells and induce mutations in them. Assays of this type make it possible to demonstrate a relationship between carcinogenic potency of the chemicals and their ability to induce mutations in mammalian cells. In addition, by simultaneously comparing the frequencies of transformation and mutation induced in normal diploid hamster cells by benzo(a)pyrene (BP) and one of its metabolites, it is possible to estimate the genetic target size for cell transformation in vitro

  18. Study on mutagenesis of Signal grass (Brachiaria decumbens) by gamma irradiation

    International Nuclear Information System (INIS)

    Abdul Rahim Harun; Shuhami Shamsuddin; Ghazali Hussin

    2000-01-01

    Before starting experiments on induced mutation breeding of crops it is essential to carry out radiosensitivity test when determining the optimum doses of every plant genotype. Factors such as moisture content and oxsigen availability could influence mutagenesis process through ionizing radiation. Many methodologies could be used for determining the effect of radiation on seed of plants such as the 'Sandwich blotter technique' and 'Sand bed technique'. Different species have different sensitivity to mutagenic agents. Even varieties of the same species show variations in sensitivity to irradiation. Brachiaria clecumbens was found to be less sensitive to gamma radiation. At a dose of 800 Gy, the percentage reduction in growth was around 40%. Early result has shown that the optimum doses for mutagenesis was between 700 to 900 Gy

  19. Impact of increased mutagenesis on adaptation to high temperature in bacteriophage Qβ.

    Science.gov (United States)

    Arribas, María; Cabanillas, Laura; Kubota, Kirina; Lázaro, Ester

    2016-10-01

    RNA viruses replicate with very high error rates, which makes them more sensitive to additional increases in this parameter. This fact has inspired an antiviral strategy named lethal mutagenesis, which is based on the artificial increase of the error rate above a threshold incompatible with virus infectivity. A relevant issue concerning lethal mutagenesis is whether incomplete treatments might enhance the adaptive possibilities of viruses. We have addressed this question by subjecting an RNA virus, the bacteriophage Qβ, to different transmission regimes in the presence or the absence of sublethal concentrations of the mutagenic nucleoside analogue 5-azacytidine (AZC). Populations obtained were subsequently exposed to a non-optimal temperature and analyzed to determine their consensus sequences. Our results show that previously mutagenized populations rapidly fixed a specific set of mutations upon propagation at the new temperature, suggesting that the expansion of the mutant spectrum caused by AZC has an influence on later evolutionary behavior. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Pecularities of mutagenesis of T4Br bacteriophage under the direct and indirect radiation effects

    International Nuclear Information System (INIS)

    Yurov, S.S.

    1975-01-01

    Different lethal and mutagenic effects were shown when bacteriophage T4Br + (470 r/min) was irradiated in broth (direct effect) and a buffer solution (direct and indirect action). The survival rate of the bacteriophage in the buffer solution was 0.1 percent for a dose rate of 60 kr; in the broth it was 10 percent. The frequency of mutation of the bacteriophage also showed the greater effect of the irradiation in the buffer solution than in the broth (25 and 5 r-mutants respectively at a dose rate of 10 kr). An analysis of the ratio of the r-groups when the bacteriophage was treated in various ways revealed differences between mutagenesis produced in the broth and the buffer, and spontaneous mutagenesis. (V.A.P.)

  1. The mutagenesis and breeding of high productive strains of streptomyces jingyangensis '5406'

    International Nuclear Information System (INIS)

    Qi Hongyan; Yin Xinyun

    1988-03-01

    The purpose of these experiments is to explore the mutagenesis rhythm and breed high productive strains of actinomycete '5406'. The single colony agar pieces of strain F 358 were treated with fast neutron and 60 Co-γ ray irradiation Two mutants have been selected from 20025 treated single colonies. The output of cytokinins from them is higher than from strain F 358 . The original strain 'Mu-Tan-al' rejuvenated by freezing was treated with several physical and chemical mutagens. The mutagenesis rhythm has been summed up tentatively. Eight mutants obtained from 93014 treated single colonies produced more '5406' antibiotics than that of strain 'Mu-Tan-al,. The effect of mutant 'N2-10-Ra3' was the best

  2. Regulation of mutagenesis by exogenous biological factors in the eukaryotic cell systems

    Directory of Open Access Journals (Sweden)

    Lukash L. L.

    2013-07-01

    Full Text Available The representations of the mutations and the nature of spontaneous mutation process and mutagenesis induced by exogenous oncoviruses, DNAs and proteins-mitogens are analysed. Exogenous biological factors induce DNA damages in regulatory-informational way, acting on the cellular systems for maintenance of genetical stability. Molecular mechanisms are the same as at spontaneous mutagenesis but they are realized with the participation of alien genetical material. Among biological mutagens, the oncoviruses and mobile genetic elements (MGEs are distinguished as the strongest destabilizing factors which direct tumor transformation of somatic mammalian cells. Genetical reprogramming or changing the programs of gene expression at the differentiation of stem and progenitor cells under growth factors and citokines is probably followed by mutations and recombinations as well.

  3. A highly efficient transposon mutagenesis system for the tomato pathogen Clavibacter michiganensis subsp. michiganensis.

    Science.gov (United States)

    Kirchner, O; Gartemann, K H; Zellermann, E M; Eichenlaub, R; Burger, A

    2001-11-01

    A transposon mutagenesis system for Clavibacter michiganensis subsp. michiganensis was developed based on antibiotic resistance transposons that were derived from the insertion element IS1409 from Arthrobacter sp. strain TM1 NCIB12013. As a prerequisite, the electroporation efficiency was optimized by using unmethylated DNA and treatment of the cells with glycine such that about 5 x 10(6) transformants per microg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 x 10(3) transposon mutants per pg of DNA and thus is suitable for saturation mutagenesis. Analysis of Tn1409C insertion sites suggests a random mode of transposition. Transposition of Tn1409C was also demonstrated for other subspecies of C. michiganensis.

  4. Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis

    Science.gov (United States)

    Fedashchin, Andrij; Cernota, William H.; Gonzalez, Melissa C.; Leach, Benjamin I.; Kwan, Noelle; Wesley, Roy K.; Weber, J. Mark

    2015-01-01

    A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ∼3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering. PMID:26468041

  5. Multiple pathways for SOS-induced mutagenesis in Escherichia coli: An overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA

    Science.gov (United States)

    Kim, Su-Ryang; Maenhaut-Michel, Geneviéve; Yamada, Masami; Yamamoto, Yoshihiro; Matsui, Keiko; Sofuni, Toshio; Nohmi, Takehiko; Ohmori, Haruo

    1997-01-01

    dinP is an Escherichia coli gene recently identified at 5.5 min of the genetic map, whose product shows a similarity in amino acid sequence to the E. coli UmuC protein involved in DNA damage-induced mutagenesis. In this paper we show that the gene is identical to dinB, an SOS gene previously localized near the lac locus at 8 min, the function of which was shown to be required for mutagenesis of nonirradiated λ phage infecting UV-preirradiated bacterial cells (termed λUTM for λ untargeted mutagenesis). A newly constructed dinP null mutant exhibited the same defect for λUTM as observed previously with a dinB::Mu mutant, and the defect was complemented by plasmids carrying dinP as the only intact bacterial gene. Furthermore, merely increasing the dinP gene expression, without UV irradiation or any other DNA-damaging treatment, resulted in a strong enhancement of mutagenesis in F′lac plasmids; at most, 800-fold increase in the G6-to-G5 change. The enhanced mutagenesis did not depend on recA, uvrA, or umuDC. Thus, our results establish that E. coli has at least two distinct pathways for SOS-induced mutagenesis: one dependent on umuDC and the other on dinB/P. PMID:9391106

  6. Mutagenesis of Jatropha curcas - Exploring new traits in the breeding of a biofuel plant

    International Nuclear Information System (INIS)

    Azhar Mohamad; Sobri Hussein; Abdul Rahim Harun

    2010-01-01

    Mutagenesis in plant species is considered effective in recovering and producing useful mutants as it leads to a high degree of chimerism and produces high degree of somaclonal variations for further selection in breeding programmes. Jatropha curcas is a species with many attributes and considerable potential, especially as bio diesel. Narrow genetic background of Jatropha spp. gives less selection to growers for better quality plant materials. In this study, a new method through nuclear technology was used to increase the genetic variability of Jatropha towards novel superior potential mutant lines. The objective of the study is to generate new mutant varieties of Jatropha curcas through the mutagenesis approach in getting new sustainable mutants for high oil yield and improved plant characteristics. Seeds of a Jatropha cultivar were from selected materials from Lembaga Kenaf and Tembakau Negara, Kelantan. Radiosensitivity test was done by irradiating a total of each 60 seeds at multiple doses (0 Gy, 20 Gy, 40 Gy, 60 Gy, 80 Gy, 100 Gy, 200 Gy, 300 Gy, 400 Gy, 600 Gy and 700 Gy). After getting the LD 50 , three doses i.e. 250 Gy, 300 Gy and 350 Gy were selected for mutagenesis, where a total of 1000 seeds were exposed to gamma radiation. The seeds were hardened and field planted at close distance of 1 m x 1 m. Pruning was conducted three times at two months interval prior to screening for early flowering, short stature and high branching mutant lines. Radiosensitivity of seeds to acute gamma irradiation revealed that the LD 50 was at 320 Gy. At nursery stage, somatic mutations related to chlorophyll changes were observed on leaves with certain shapes. Screening of Jatropha via seed mutagenesis bore 6 early flowering mutants, 7 dwarf mutants and, 17 high branching plants. In narrowing the mutant lines, cuttings from each selected trait were collected and re-planted for further evaluation. (author)

  7. Improvement of DNA transfer frequency and transposon mutagenesis of Erwinia carotovora subsp. betavasculorum.

    OpenAIRE

    Rella, M; Axelrood, P E; Weinhold, A R; Schroth, M N

    1989-01-01

    The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled tra...

  8. Lack of enhancement of chemical mutagenesis by saccharin in the Salmonella assay

    Energy Technology Data Exchange (ETDEWEB)

    Rao, T.K. (Oak Ridge National Lab., TN); Stoltz, D.R.; Epler, J.L.

    1979-01-01

    A purified batch of the artificial sweetener saccharin (S-1022) was assayed for mutagenicity and comutagenicity by the Ames Salmonella assay system. Saccharin was not mutagenic and failed to enhance the mutagenic activity induced by a wide variety of known mutagens. These results do not argue against the tumor-promoter-like activity of saccharin but only indicate that the Ames Salmonella assay is not capable of detecting saccharin as a promoter of mutagenesis.

  9. The Mechanism of Nucleotide Excision Repair-Mediated UV-Induced Mutagenesis in Nonproliferating Cells

    Science.gov (United States)

    Kozmin, Stanislav G.; Jinks-Robertson, Sue

    2013-01-01

    Following the irradiation of nondividing yeast cells with ultraviolet (UV) light, most induced mutations are inherited by both daughter cells, indicating that complementary changes are introduced into both strands of duplex DNA prior to replication. Early analyses demonstrated that such two-strand mutations depend on functional nucleotide excision repair (NER), but the molecular mechanism of this unique type of mutagenesis has not been further explored. In the experiments reported here, an ade2 adeX colony-color system was used to examine the genetic control of UV-induced mutagenesis in nondividing cultures of Saccharomyces cerevisiae. We confirmed a strong suppression of two-strand mutagenesis in NER-deficient backgrounds and demonstrated that neither mismatch repair nor interstrand crosslink repair affects the production of these mutations. By contrast, proteins involved in the error-prone bypass of DNA damage (Rev3, Rev1, PCNA, Rad18, Pol32, and Rad5) and in the early steps of the DNA-damage checkpoint response (Rad17, Mec3, Ddc1, Mec1, and Rad9) were required for the production of two-strand mutations. There was no involvement, however, for the Pol η translesion synthesis DNA polymerase, the Mms2-Ubc13 postreplication repair complex, downstream DNA-damage checkpoint factors (Rad53, Chk1, and Dun1), or the Exo1 exonuclease. Our data support models in which UV-induced mutagenesis in nondividing cells occurs during the Pol ζ-dependent filling of lesion-containing, NER-generated gaps. The requirement for specific DNA-damage checkpoint proteins suggests roles in recruiting and/or activating factors required to fill such gaps. PMID:23307894

  10. Amino acid substitutions in random mutagenesis libraries: lessons from analyzing 3000 mutations.

    Science.gov (United States)

    Zhao, Jing; Frauenkron-Machedjou, Victorine Josiane; Kardashliev, Tsvetan; Ruff, Anna Joëlle; Zhu, Leilei; Bocola, Marco; Schwaneberg, Ulrich

    2017-04-01

    The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation frequency and SeSaM-Tv P/P) employing lipase A from Bacillus subtilis (bsla). A comparison of the obtained numbers of beneficial variants in the mentioned three random mutagenesis libraries with a site saturation mutagenesis (SSM) (covering the natural diversity at each amino acid position of BSLA) concludes the diversity analysis. Seventy-six percent of the SeSaM-Tv P/P-generated substitutions yield chemically different amino acid substitutions compared to 64% (epPCR-low) and 69% (epPCR-high). Unique substitutions from one amino acid to others are termed distinct amino acid substitutions. In the SeSaM-Tv P/P library, 35% of all theoretical distinct amino acid substitutions were found in the 1000 mutation library compared to 25% (epPCR-low) and 26% (epPCR-high). Thirty-six percent of distinct amino acid substitutions found in SeSaM-Tv P/P were unobtainable by epPCR-low. Comparison with the SSM library showed that epPCR-low covers 15%, epPCR-high 18%, and SeSaM-Tv P/P 21% of obtainable beneficial amino acid positions. In essence, this study provides first insights on the quality of epPCR and SeSaM-Tv P/P libraries in terms of amino acid substitutions, their chemical differences, and the number of obtainable beneficial amino acid positions.

  11. Mutagenesis and cytotoxicity in human epithelial cells by far- and near-ultraviolet radiations: action spectra

    International Nuclear Information System (INIS)

    Jones, C.A.; Huberman, E.; Cunningham, M.L.; Peak, M.J.

    1987-01-01

    Action spectra were determined for cell killing and mutation by monochromatic ultraviolet and visible radiations (254-434 nm) in cultured human epithelial P3 cells. Cell killing was more efficient following radiation at the shorter wavelengths (254-434 nm) than at longer wavelengths (365-434 nm). At 254 nm, for example, a fluence of 11 Jm-2 gave 37% cell survival, while at 365 nm, 17 X 10(5) Jm-2 gave equivalent survival. At 434 nm little killing was observed with fluences up to 3 X 10(6) Jm-2. Mutant induction, determined at the hypoxanthine-guanine phosphoribosyltransferase locus, was caused by radiation at 254, 313, and 365 nm. There was no mutant induction at 334 nm although this wavelength was highly cytotoxic. Mutagenesis was not induced by 434 nm radiation, either. There was a weak response at 405 nm; the mutant frequencies were only slightly increased above background levels. For the mutagenic wavelengths, log-log plots of the mutation frequency against fluence showed linear regressions with positive slopes of 2.5, consistent with data from a previous study using Escherichia coli. The data points of the action spectra for lethality and mutagenesis were similar to the spectrum for DNA damage at wavelengths shorter than 313 nm, whereas at longer wavelengths the lethality spectrum had a shoulder, and the mutagenesis spectrum had a secondary peak at 365 nm. No correlation was observed for the P3 cells between the spectra for cell killing and mutagenesis caused by wavelengths longer than 313 nm and the induction of DNA breakage or the formation of DNA-to-protein covalent bonds in these cells

  12. The contribution of Nth and Nei DNA glycosylases to mutagenesis in Mycobacterium smegmatis.

    Science.gov (United States)

    Moolla, Nabiela; Goosens, Vivianne J; Kana, Bavesh D; Gordhan, Bhavna G

    2014-01-01

    The increased prevalence of drug resistant strains of Mycobacterium tuberculosis (Mtb) indicates that significant mutagenesis occurs during tuberculosis disease in humans. DNA damage by host-derived reactive oxygen/nitrogen species is hypothesized to be critical for the mutagenic process in Mtb thus, highlighting an important role for DNA repair enzymes in maintenance of genome fidelity. Formamidopyrimidine (Fpg/MutM/Fapy) and EndonucleaseVIII (Nei) constitute the Fpg/Nei family of DNA glycosylases and together with EndonucleaseIII (Nth) are central to the base excision repair pathway in bacteria. In this study we assess the contribution of Nei and Nth DNA repair enzymes in Mycobacterium smegmatis (Msm), which retains a single nth homologue and duplications of the Fpg (fpg1 and fpg2) and Nei (nei1 and nei2) homologues. Using an Escherichia coli nth deletion mutant, we confirm the functionality of the mycobacterial nth gene in the base excision repair pathway. Msm mutants lacking nei1, nei2 and nth individually or in combination did not display aberrant growth in broth culture. Deletion of nth individually results in increased UV-induced mutagenesis and combinatorial deletion with the nei homologues results in reduced survival under oxidative stress conditions and an increase in spontaneous mutagenesis to rifampicin. Deletion of nth together with the fpg homolgues did not result in any growth/survival defects or changes in mutation rate. Furthermore, no differential emergence of the common rifampicin resistance conferring genotypes were noted. Collectively, these data confirm a role for Nth in base excision repair in mycobacteria and further highlight a novel interplay between the Nth and Nei homologues in spontaneous mutagenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Modulating effect of dimethylbenzanthracene on gamma-ray mutagenesis in the soybean test system

    Energy Technology Data Exchange (ETDEWEB)

    Fujii, T. [National Inst. of Genetics, Mishima, Shizuoka (Japan); Inoue, T.

    1987-10-15

    Dimethylbenzanthracene (DMBA), a strong tumor initiator, did not show mutagenic activity in the soybean test system either alone or in combination with tumor promoter, TPA. In combination treatments with DMBA and gamma-rays, the mutagenicity of gamma-rays was not affected by post-treatment with DMBA. However, DMBA pre-treatment clearly reduced gammaray-ray induced spotting frequency, suggesting that DMBA affects mutagenesis in gamma-irradiated soybean cells.

  14. Molecular processes as basis for plasmid-mediated bacterial UV-light resistance and mutagenesis

    International Nuclear Information System (INIS)

    Aleshkin, G.I.; Brukhanskij, G.V.; Skavronskaya, A.G.

    1985-01-01

    The increase of UV-resistance and UV-induced mutagenesis by lambda 1 pint intmid as well as molecular-genetic mechanisms of plasmid participation in reparation and DNA replication and its degradation after UV-irradiation in plasmid cells on pKM101 plasmid model have been investigated. Data testifying to the necessity of intmid integration in chromosome as obligatory stage of intmid participation in increasing UV-resistance of bacterial cells are obtained. It has been found that intmid raises UV-resistance of cells and increases respectively the UV-induced reverants efficiency. On the basis of the experiment data the conclusion is drawn that the intmid capacity to raise UV-resistance and, possibly, mutagenesis is bound not only with its integration into chromosome but also with pol A + chromosome replication by dependendent imtmid replication complex. It is shown that pKM101 plasmid ensures functioning in E coli cells of inducible, chloroamphenicol-resistant DNA replication, highly resistant to UV-light harmful effect and that the volume of excision reparation in E. coli cells carrying pKM101 plasmid is increased as compared with the volume of reparation in plasmid legs cells. The combination of the data obtained gives grounds to the authors to assume that inducible replication, inducible reparation of DNA and inducible decrease of DNA degradation determined by pKM101 plasmid may serve as recA + lexA + basis dependent increase of UV-resistance and mutagenesis and that these processes provide the possibility of functioning of integrative replication mechanism of plasmid participation in ensuring UV-resistance and mutagenesis of plants

  15. A plasmid-transposon hybrid mutagenesis system effective in a broad range of Enterobacteria

    Directory of Open Access Journals (Sweden)

    Rita eMonson

    2015-12-01

    Full Text Available Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways.

  16. Site-directed mutagenesis of the foot-and-mouth disease virus RNA-polymerase gene

    International Nuclear Information System (INIS)

    Brindeiro, R.M.; Soares, M.A.; Vianna, A.L.M.; Pontes, O.H.A. de; Pacheco, A.B.F.; Almeida, D.F. de; Tanuri, A.

    1991-01-01

    The foot-and-mouth disease virus RNA-polymerase gene was mutagenised in its active site. Pst I digestion of the polymerase gene (cDNA) generated a 790 bp fragment containing the critical sequence. This fragment was subcloned in M13mp8 for mutagenesis method. The polymerase gene was then reconstructed and subcloned in pUC19. These mutants will be used to study the enzyme structure and activity and to develop intracellular immunization assays in eukaryotic cells. (author)

  17. Random Mutagenesis of the Aspergillus oryzae Genome Results in Fungal Antibacterial Activity

    OpenAIRE

    Leonard, Cory A.; Brown, Stacy D.; Hayman, J. Russell

    2013-01-01

    Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin-res...

  18. Repair and mutagenesis of herpes simplex virus in UV-irradiated monkey cells

    International Nuclear Information System (INIS)

    Lytle, C.D.; Goddard, J.G.; Lin, C.H.

    1980-01-01

    Mutagenic repair in mammalian cells was investigated by determining the mutagenesis of UV-irradiated or unirradiated herpes simplex virus in UV-irradiated CV-1 monkey kidney cells. These results were compared with the results for UV-enhanced virus reactivation (UVER) in the same experimental situation. High and low multiplicities of infection were used to determine the effects of multiplicity reactivation (MR). UVER and MR were readily demonstrable and were approximately equal in amount in an infectious center assay. For this study, a forward-mutation assay was developed to detect virus mutants resistant to iododeoxycytidine (ICdR), probably an indication of the mutant virus being defective at its thymidine kinase locus. ICdR-resistant mutants did not have a growth advantage over wild-type virus in irradiated or unirradiated cells. Thus, higher fractions of mutant virus indicated greater mutagenesis during virus repair and/or replication. The data showed that: (1) unirradiated virus was mutated in unirradiated cells, providing a background level of mutagenesis; (2) unirradiated virus was mutated about 40% more in irradiated cells, indicating that virus replication (DNA synthesis) became more mutagenic as a result of cell irradiation; (3) irradiated virus was mutated much more (about 6-fold) than unirradiated virus, even in unirradiated cells; (4) cell irradiation did not change the mutagenesis of irradiated virus except at high multiplicity of infection. High multiplicity of infection did not demonstrate UVER or MR alone to be either error-free or error-prone. When the two processes were present simultaneously, they were mutagenic. (orig.)

  19. Combined mutagenesis of Rhodosporidium toruloides for improved production of carotenoids and lipids.

    Science.gov (United States)

    Zhang, Chaolei; Shen, Hongwei; Zhang, Xibin; Yu, Xue; Wang, Han; Xiao, Shan; Wang, Jihui; Zhao, Zongbao K

    2016-10-01

    To improve production of lipids and carotenoids by the oleaginous yeast Rhodosporidium toruloides by screening mutant strains. Upon physical mutagenesis of the haploid strain R. toruloides np11 with an atmospheric and room temperature plasma method followed by chemical mutagenesis with nitrosoguanidine, a mutant strain, R. toruloides XR-2, formed dark-red colonies on a screening plate. When cultivated in nitrogen-limited media, XR-2 cells grew slower but accumulated 0.23 g lipids/g cell dry wt and 0.75 mg carotenoids/g CDW. To improve its production capacity, different amino acids and vitamins were supplemented. p-Aminobenzoic acid and tryptophan had beneficial effects on cell growth. When cultivated in nitrogen-limited media in the presence of selected vitamins, XR-2 accumulated 0.41 g lipids/g CDW and 0.69 mg carotenoids/g CDW. A mutant R. toruloides strain with improved production profiles for lipids and carotenoids was obtained, indicating its potential to use combined mutagenesis for a more productive phenotype.

  20. Application of In Vitro Transposon Mutagenesis to Erythromycin Strain Improvement in Saccharopolyspora erythraea.

    Science.gov (United States)

    Weber, J Mark; Reeves, Andrew; Cernota, William H; Wesley, Roy K

    2017-01-01

    Transposon mutagenesis is an invaluable technique in molecular biology for the creation of random mutations that can be easily identified and mapped. However, in the field of microbial strain improvement, transposon mutagenesis has scarcely been used; instead, chemical and physical mutagenic methods have been traditionally favored. Transposons have the advantage of creating single mutations in the genome, making phenotype to genotype assignments less challenging than with traditional mutagens which commonly create multiple mutations in the genome. The site of a transposon mutation can also be readily mapped using DNA sequencing primer sites engineered into the transposon termini. In this chapter an in vitro method for transposon mutagenesis of Saccharopolyspora erythraea is presented. Since in vivo transposon tools are not available for most actinomycetes including S. erythraea, an in vitro method was developed. The in vitro method involves a significant investment in time and effort to create the mutants, but once the mutants are made and screened, a large number of highly relevant mutations of direct interest to erythromycin production can be found.

  1. Transgenic Chinese hamster V79 cell lines which exhibit variable levels of gpt mutagenesis

    International Nuclear Information System (INIS)

    Klein, C.B.; Rossman, T.G.

    1990-01-01

    The Escherichia coli gpt gene coding for xanthine-guanine phosphoribosyl transferase has been stably transfected into HPRT - Chinese hamster V79 cells. Several gpt - cell lines have been established, which retain the sequence(s) even after long-term culture without selection for gpt. While spontaneous mutagenesis to gpt - occurs rather frequently for most cell lines, it cannot be correlated with either the number of plasmid integration sites or deletion of the plasmid sequence(s). One transgenic cell line (g12), which continuously maintains a low spontaneous mutation frequency was used in comparative mutagenesis studies with wild-type V79 cells (gpt vs. hprt). Alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and β-propiolactone (BPL) are shown to be equally toxic and mutagenic in both g12 and V79 cells. UV and X-rays are also equally toxic to both cell lines. The data presented here suggests that g12 cells may be useful to study mammalian mutagenesis by agents which yield limited response at the hprt locus

  2. Ascorbate enhances u.v.-mutagenesis in E. coli but inhibits it in Chinese hamster cells

    International Nuclear Information System (INIS)

    Rossman, T.G.; Klein, C.B.; Naslund, M.

    1986-01-01

    Ascorbic acid (vitamin C) causes an increase in the mutation frequency of u.v.-irradiated Escherichia coli WP2. The enhancement occurs at all u.v. fluences, and is dependent upon the ascorbate concentration in the medium. A maximum effect (approx. 8- to 13-fold) is seen at 100-150 μg/ml, although some enhancement can be seen even at 10 μg/ml. The comutagenic effect of ascorbate with u.v. in E. coli is dependent upon peptone, a constituent of nutrient broth. The enhancement of u.v.-mutagenesis by ascorbate is absent in strains WP2sub(s) (uvrA) amd WP6 (polA), suggesting that ascorbate affects the repair of pyrimidine dimers. The opposite results are observed for u.v.-mutagenesis in Chinese hamster V79 cells. The presence of ascorbate (50 μg/ml) during u.v. irradiation does not enhance the u.v. effect, but rather decreases it approx. 30%. These results are discussed with regard to differences in the mechanism of u.v.-mutagenesis and DNA repair in bacterial and mammalian cells. (author)

  3. Lack of mutational hot spots during decitabine-mediated HIV-1 mutagenesis.

    Science.gov (United States)

    Rawson, Jonathan M O; Landman, Sean R; Reilly, Cavan S; Bonnac, Laurent; Patterson, Steven E; Mansky, Louis M

    2015-11-01

    Decitabine has previously been shown to induce lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1). However, the factors that determine the susceptibilities of individual sequence positions in HIV-1 to decitabine have not yet been defined. To investigate this, we performed Illumina high-throughput sequencing of multiple amplicons prepared from proviral DNA that was recovered from decitabine-treated cells infected with HIV-1. We found that decitabine induced an ≈4.1-fold increase in the total mutation frequency of HIV-1, primarily due to a striking ≈155-fold increase in the G-to-C transversion frequency. Intriguingly, decitabine also led to an ≈29-fold increase in the C-to-G transversion frequency. G-to-C frequencies varied substantially (up to ≈80-fold) depending upon sequence position, but surprisingly, mutational hot spots (defined as upper outliers within the mutation frequency distribution) were not observed. We further found that every single guanine position examined was significantly susceptible to the mutagenic effects of decitabine. Taken together, these observations demonstrate for the first time that decitabine-mediated HIV-1 mutagenesis is promiscuous and occurs in the absence of a clear bias for mutational hot spots. These data imply that decitabine-mediated G-to-C mutagenesis is a highly effective antiviral mechanism for extinguishing HIV-1 infectivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  4. Improvement of lipid production by the oleaginous yeast Rhodosporidium toruloides through UV mutagenesis.

    Science.gov (United States)

    Yamada, Ryosuke; Kashihara, Tomomi; Ogino, Hiroyasu

    2017-05-01

    Oleaginous yeasts are considered a promising alternative lipid source for biodiesel fuel production. In this study, we attempted to improve the lipid productivity of the oleaginous yeast Rhodosporidium toruloides through UV irradiation mutagenesis and selection based on ethanol and H 2 O 2 tolerance or cerulenin, a fatty acid synthetase inhibitor. Glucose consumption, cell growth, and lipid production of mutants were evaluated. The transcription level of genes involved in lipid production was also evaluated in mutants. The ethanol and H 2 O 2 tolerant strain 8766 2-31M and the cerulenin resistant strain 8766 3-11C were generated by UV mutagenesis. The 8766 2-31M mutant showed a higher lipid production rate, and the 8766 3-11C mutant produced a larger amount of lipid and had a higher lipid production rate than the wild type strain. Transcriptional analysis revealed that, similar to the wild type strain, the ACL1 and GND1 genes were expressed at significantly low levels, whereas IDP1 and ME1 were highly expressed. In conclusion, lipid productivity in the oleaginous yeast R. toruloides was successfully improved via UV mutagenesis and selection. The study also identified target genes for improving lipid productivity through gene recombination.

  5. DNA damage and mutagenesis of lambda phage induced by gamma-rays

    International Nuclear Information System (INIS)

    Bertram, Heidi

    1988-01-01

    Lambda phage DNA was gamma irradiated in aqueous solution and strand breakage determined. Twice as much minor structural damage per lethal hit was found in this DNA compared with DNA from irradiated phage suspensions. The in vitro irradiated DNA was repackaged into infectious particles. Induction of mutations in the cI or cII cistron was scored using SOS-induced host cells. In vitro prepared particles were found to have second-order kinetics for mutagenesis induced by gamma rays indicating two pre-mutational events were necessary to produce a mutation, but bacteria-free phage suspensions ('lys-phage') showed single hit kinetics for mutagenesis after irradiation. Increase in the mutation rate in the phage particles was mainly due to minor lesions, i.e. ssb, als and unidentified base damage. In lys-phage, mutagenesis might be enhanced by clustered DNA damage - configuration not existing in pack-phage. Loss of infectivity was analysed in comparison with structural damage. All lesions contributed to biological inactivation. Minor lesions were tolerated by lambda phage to a limited extent. Major lesions (e.g. dsb) contributed most to infectivity loss and were considered lethal events. (U.K.)

  6. Spontaneous mutability and light-induced mutagenesis in Salmonella typhimurium: effects of an R-plasmid

    International Nuclear Information System (INIS)

    Valdivia, L.

    1979-01-01

    The UV-protecting plasmid R46 was transferred by conjugation to a genetically marked mouse-virulent Salmonella typhimurium strain, not derived from LT2; in this host the plasmid conferred UV protection and enhanced UV mutagenesis just as it does in LT2 lines. Tra - derivatives of R46 encountered during transduction retained UV-protecting and mutagenesis-enhancing ability. Stored strains carrying the R46-derived plasmids with strong mutator effect but not UV-protecting had lost most of their original streptomycin resistance but were slightly resistant to spectinomycin; attempts to transfer such plasmids failed. R46 enhanced the weak mutagenic effect of visible light on several his and trp mutants of strain LT2, including some whose frequency of spontaneous reversion was not increased by the plasmid. A mutagenic effect was produced by visible-light irradiation of hisG46(R46), either growing cells or nonmultiplying (histidine-deprived cells at 10 0 C). Presence of catalase or cyanide during irradiation did not prevent mutagenesis, which excludes some hypothetical mechanisms. Visible-light irradiation of hisG46 or hisG46(R46) under strict anaerobiosis had little or no mutagenic effect (controls showed that revertants if produced would have been detected). This is as expected if visible-light irradiation in air causes photodynamic damage to DNA and mutations are produced during error-prone, plasmid-enhanced repair

  7. Ionizing radiation-induced bystander mutagenesis and adaptation: Quantitative and temporal aspects

    International Nuclear Information System (INIS)

    Zhang Ying; Zhou Junqing; Baldwin, Joseph; Held, Kathryn D.; Prise, Kevin M.; Redmond, Robert W.; Liber, Howard L.

    2009-01-01

    This work explores several quantitative aspects of radiation-induced bystander mutagenesis in WTK1 human lymphoblast cells. Gamma-irradiation of cells was used to generate conditioned medium containing bystander signals, and that medium was transferred onto naive recipient cells. Kinetic studies revealed that it required up to 1 h to generate sufficient signal to induce the maximal level of mutations at the thymidine kinase locus in the bystander cells receiving the conditioned medium. Furthermore, it required at least 1 h of exposure to the signal in the bystander cells to induce mutations. Bystander signal was fairly stable in the medium, requiring 12-24 h to diminish. Medium that contained bystander signal was rendered ineffective by a 4-fold dilution; in contrast a greater than 20-fold decrease in the cell number irradiated to generate a bystander signal was needed to eliminate bystander-induced mutagenesis. This suggested some sort of feedback inhibition by bystander signal that prevented the signaling cells from releasing more signal. Finally, an ionizing radiation-induced adaptive response was shown to be effective in reducing bystander mutagenesis; in addition, low levels of exposure to bystander signal in the transferred medium induced adaptation that was effective in reducing mutations induced by subsequent γ-ray exposures.

  8. Yeasts acquire resistance secondary to antifungal drug treatment by adaptive mutagenesis.

    Directory of Open Access Journals (Sweden)

    David Quinto-Alemany

    Full Text Available Acquisition of resistance secondary to treatment both by microorganisms and by tumor cells is a major public health concern. Several species of bacteria acquire resistance to various antibiotics through stress-induced responses that have an adaptive mutagenesis effect. So far, adaptive mutagenesis in yeast has only been described when the stress is nutrient deprivation. Here, we hypothesized that adaptive mutagenesis in yeast (Saccharomyces cerevisiae and Candida albicans as model organisms would also take place in response to antifungal agents (5-fluorocytosine or flucytosine, 5-FC, and caspofungin, CSP, giving rise to resistance secondary to treatment with these agents. We have developed a clinically relevant model where both yeasts acquire resistance when exposed to these agents. Stressful lifestyle associated mutation (SLAM experiments show that the adaptive mutation frequencies are 20 (S. cerevisiae -5-FC, 600 (C. albicans -5-FC or 1000 (S. cerevisiae--CSP fold higher than the spontaneous mutation frequency, the experimental data for C. albicans -5-FC being in agreement with the clinical data of acquisition of resistance secondary to treatment. The spectrum of mutations in the S. cerevisiae -5-FC model differs between spontaneous and acquired, indicating that the molecular mechanisms that generate them are different. Remarkably, in the acquired mutations, an ectopic intrachromosomal recombination with an 87% homologous gene takes place with a high frequency. In conclusion, we present here a clinically relevant adaptive mutation model that fulfils the conditions reported previously.

  9. Quantitative Missense Variant Effect Prediction Using Large-Scale Mutagenesis Data.

    Science.gov (United States)

    Gray, Vanessa E; Hause, Ronald J; Luebeck, Jens; Shendure, Jay; Fowler, Douglas M

    2018-01-24

    Large datasets describing the quantitative effects of mutations on protein function are becoming increasingly available. Here, we leverage these datasets to develop Envision, which predicts the magnitude of a missense variant's molecular effect. Envision combines 21,026 variant effect measurements from nine large-scale experimental mutagenesis datasets, a hitherto untapped training resource, with a supervised, stochastic gradient boosting learning algorithm. Envision outperforms other missense variant effect predictors both on large-scale mutagenesis data and on an independent test dataset comprising 2,312 TP53 variants whose effects were measured using a low-throughput approach. This dataset was never used for hyperparameter tuning or model training and thus serves as an independent validation set. Envision prediction accuracy is also more consistent across amino acids than other predictors. Finally, we demonstrate that Envision's performance improves as more large-scale mutagenesis data are incorporated. We precompute Envision predictions for every possible single amino acid variant in human, mouse, frog, zebrafish, fruit fly, worm, and yeast proteomes (https://envision.gs.washington.edu/). Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Complementation of a pKM101 derivative that decreases resistance to UV killing but increases susceptibility to mutagenesis

    International Nuclear Information System (INIS)

    Langer, P.J.; Perry, K.L.; Walker, G.C.

    1985-01-01

    The drug resistance plasmid pKM101 makes Escherichia coli resistant to the lethal effects of ultraviolet (UV) irradiation and more susceptible to mutagenesis by a variety of agents. The plasmid operon responsible for increasing mutagenesis has been termed mucAB (Mutagenesis, UV and chemical). The authors have isolated a derivative of pKM101 called pGW1975 which makes cells more sensitive to killing by UV but which retains the ability of pKM101 to increase susceptibility to methyl methanesulfonate (MMS) mutagenesis. pGW1975 increases UV mutagenesis less than pKM101 in a uvrA + strain but more than pKM101 in a uvrA - strain. muc - point and insertion mutants of pKM101 and pGW1975 complement to restore the plasmid-mediated: (i) ability to reactivate UV-irradiated phage, (ii) resistance to killing by UV, and (iii) level of susceptibility to UV mutagenesis. They have identified a 2.0 kb region of pKM101 which is responsible for the complementation and which maps counterclockwise of mucAB. (Auth)

  11. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

    Science.gov (United States)

    Gagnon, James A; Valen, Eivind; Thyme, Summer B; Huang, Peng; Akhmetova, Laila; Ahkmetova, Laila; Pauli, Andrea; Montague, Tessa G; Zimmerman, Steven; Richter, Constance; Schier, Alexander F

    2014-01-01

    The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.

  12. A report on 36 years practical work on crop improvement through induced mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Datta, S.K. [CSIR, Madhyamgram Experimental Farm, Bose Institute, Kolkata (India)], E-mail: subodhskdatta@rediffmail.com

    2008-07-01

    Physical and/or chemical mutagens cause random changes in the nuclear DNA or cytoplasmic organelles, resulting in gene, chromosomal or genomic mutations. The author will share his life time experience and achievement on induced mutagenesis. The author initiated induced mutagenesis work in 1971 till July 2007 and used both physical (X-ray and Gamma rays) and chemical (EMS, MMS, Colchicine) mutagens for improvement of vegetables (Trichosanthes anguina L, T. cucumarina , Cucurbita maxima L, Cephalandra indica, Luffa acutangula Roxb., Lagenaria ciceraria), medicinal (Trigonella foenum-graecum L, Mentha citrate Ehrh), pulse (Winged Bean (Psophocarpus tetragonolobus L. D.C.), oil bearing (Jatropha curcas L, Rosa damascene, Cymbopogon flexuosus (Nees) Wats) and ornamental (Bougainvillea, Chrysanthemum, Dahlia, Gladiolus, Hibiscus, Lantana depressa Naud, Rose, Tuberose, Narcissus etc.) crops. All classical and advanced mutagenesis methods have been extensively used for the development of new and novel cultivars of economic importance. Early flowering, late flowering, dwarf, yellow fruit color, crinkled leaf, short thick fruit, increased branching, increased pod and seed number, seed size, seed color (green, brown, chocolate color) high fruit-, seed-, oil- and punicic acidyielding mutants have been developed in T. anguina, T. fornum-graecum, Winged Bean and in J.curcas containing 'curcas oil', an efficient substitute fuel for diesel engines. Induction of flower color and chlorophyll variegated mutants in L. depressa proved the efficiency of mutation technique for domestication of wild relatives. Author was deeply engaged for the last 30 years for improvement of ornamentals and has been most successful to produce quite a large number of new promising mutant varieties in different ornamentals. Colchicine has been successfully used to develop new flower color in chrysanthemum and rose and high yielding strains in T. anguina. A novel direct in vitro regeneration technique has

  13. Origin of Somatic Mutations in β-Catenin versus Adenomatous Polyposis Coli in Colon Cancer: Random Mutagenesis in Animal Models versus Nonrandom Mutagenesis in Humans.

    Science.gov (United States)

    Yang, Da; Zhang, Min; Gold, Barry

    2017-07-17

    Wnt signaling is compromised early in the development of human colorectal cancer (CRC) due to truncating nonsense mutations in adenomatous polyposis coli (APC). CRC induced by chemical carcinogens, such as heterocyclic aromatic amines and azoxymethane, in mice also involves dysregulation of Wnt signaling but via activating missense mutations in the β-catenin oncogene despite the fact that genetically modified mice harboring an inactive APC allele efficiently develop CRC. In contrast, activating mutations in β-catenin are rarely observed in human CRC. Dysregulation of the Wnt signaling pathway by the two distinct mechanisms reveals insights into the etiology of human CRC. On the basis of calculations related to DNA adduct levels produced in mouse CRC models using mutagens, and the number of stem cells in the mouse colon, we show that two nonsense mutations required for biallelic disruption of APC are statistically unlikely to produce CRC in experiments using small numbers of mice. We calculate that an activating mutation in one allele near the critical GSK3β phosphorylation site on β-catenin is >10 5 -times more likely to produce CRC by random mutagenesis due to chemicals than inactivating two alleles in APC, yet it does not occur in humans. Therefore, the mutagenesis mechanism in human CRC cannot be random. We explain that nonsense APC mutations predominate in human CRC because of deamination at 5-methylcytosine at CGA and CAG codons, coupled with the number of human colonic stem cells and lifespan. Our analyses, including a comparison of mutation type and age at CRC diagnosis in U.S. and Chinese patients, also indicate that APC mutations in CRC are not due to environmental mutagens that randomly damage DNA.

  14. High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP receptor expression and function.

    Directory of Open Access Journals (Sweden)

    Anke Bill

    Full Text Available The human prostacyclin receptor (hIP receptor is a seven-transmembrane G protein-coupled receptor (GPCR that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs.

  15. High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor expression and function.

    Science.gov (United States)

    Bill, Anke; Rosethorne, Elizabeth M; Kent, Toby C; Fawcett, Lindsay; Burchell, Lynn; van Diepen, Michiel T; Marelli, Anthony; Batalov, Sergey; Miraglia, Loren; Orth, Anthony P; Renaud, Nicole A; Charlton, Steven J; Gosling, Martin; Gaither, L Alex; Groot-Kormelink, Paul J

    2014-01-01

    The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs.

  16. Misrepair of overlapping daughter strand gaps as a possible mechanism for UV induced mutagenesis in uvr strains of Escherichia coli: a general model for induced mutagenesis by misrepair (SOS repair) of closely spaced DNA lesions

    International Nuclear Information System (INIS)

    Sedgwick, S.G.

    1976-01-01

    It has been previously reported that an inducible form of post-replication repair appeared to be required for UV induced mutagenesis in an uvrA strain of Escherichia coli. It is shown here that the numbers of daughter strand gaps requiring inducible repair were similar to the numbers calculated to be overlapping one another in opposite daughter chromosomes. An estimation of survival with no repair of these gaps resembled the survival predicted with mutagenesis. It is thus proposed that inducible post-replication repair causes mutagenesis by the repair of overlapping daughter strand gaps. A general model for induced mutagenesis is presented. It is proposed that (a) some DNA lesions introduced by any DNA damaging agent may be close enough to interfere with constitutive repair replication of each other, (b) these lesions induce a repair system (SOS repair) which involves the recA + . lexA + and polC + genes (c) repair, and noncomitant mutagenesis occurs during repair replication by the insertion of mismatched bases oppposite the noncoding DNA lesions

  17. Use of Random and Site-Directed Mutagenesis to Probe Protein Structure-Function Relationships: Applied Techniques in the Study of Helicobacter pylori.

    Science.gov (United States)

    Whitmire, Jeannette M; Merrell, D Scott

    2017-01-01

    Mutagenesis is a valuable tool to examine the structure-function relationships of bacterial proteins. As such, a wide variety of mutagenesis techniques and strategies have been developed. This chapter details a selection of random mutagenesis methods and site-directed mutagenesis procedures that can be applied to an array of bacterial species. Additionally, the direct application of the techniques to study the Helicobacter pylori Ferric Uptake Regulator (Fur) protein is described. The varied approaches illustrated herein allow the robust investigation of the structural-functional relationships within a protein of interest.

  18. Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress.

    Science.gov (United States)

    LeBlanc, Chantal; Zhang, Fei; Mendez, Josefina; Lozano, Yamile; Chatpar, Krishna; Irish, Vivian F; Jacob, Yannick

    2018-01-01

    The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off-target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR-induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5-fold in somatic tissues and up to 100-fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double-stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on-target mutagenesis in plants using CRISPR/Cas9. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  19. Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9.

    Science.gov (United States)

    Liu, Yang; Merrick, Paul; Zhang, Zhengzhi; Ji, Chonghui; Yang, Bing; Fei, Shui-Zhang

    2018-02-01

    The CRISPR/Cas9 system has become a powerful tool for targeted mutagenesis. Switchgrass (Panicum virgatum L.) is a high yielding perennial grass species that has been designated as a model biomass crop by the U.S. Department of Energy. The self-infertility and high ploidy level make it difficult to study gene function or improve germplasm. To overcome these constraints, we explored the feasibility of using CRISPR/Cas9 for targeted mutagenesis in a tetraploid cultivar 'Alamo' switchgrass. We first developed a transient assay by which a non-functional green-fluorescent protein gene containing a 1-bp frameshift insertion in its 5' coding region was successfully mutated by a Cas9/sgRNA complex resulting in its restored function. Agrobacterium-mediated stable transformation of embryogenic calli derived from mature caryopses averaged a 3.0% transformation efficiency targeting the genes of teosinte branched 1(tb1)a and b and phosphoglycerate mutase (PGM). With a single construct containing two sgRNAs targeting different regions of tb1a and tb1b genes, primary transformants (T0) containing CRISPR/Cas9-induced mutations were obtained at frequencies of 95.5% (tb1a) and 11% (tb1b), respectively, with T0 mutants exhibiting increased tiller production. Meanwhile, a mutation frequency of 13.7% was obtained for the PGM gene with a CRISPR/Cas9 construct containing a single sgRNA. Among the PGM T0 mutants, six are heterozygous and one is homozygous for a 1-bp deletion in the target region with no apparent phenotypical alterations. We show that CRISPR/Cas9 system can generate targeted mutagenesis effectively and obtain targeted homozygous mutants in T0 generation in switchgrass, circumventing the need of inbreeding. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  20. Modeling insertional mutagenesis using gene length and expression in murine embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Alex S Nord

    2007-07-01

    Full Text Available High-throughput mutagenesis of the mammalian genome is a powerful means to facilitate analysis of gene function. Gene trapping in embryonic stem cells (ESCs is the most widely used form of insertional mutagenesis in mammals. However, the rules governing its efficiency are not fully understood, and the effects of vector design on the likelihood of gene-trapping events have not been tested on a genome-wide scale.In this study, we used public gene-trap data to model gene-trap likelihood. Using the association of gene length and gene expression with gene-trap likelihood, we constructed spline-based regression models that characterize which genes are susceptible and which genes are resistant to gene-trapping techniques. We report results for three classes of gene-trap vectors, showing that both length and expression are significant determinants of trap likelihood for all vectors. Using our models, we also quantitatively identified hotspots of gene-trap activity, which represent loci where the high likelihood of vector insertion is controlled by factors other than length and expression. These formalized statistical models describe a high proportion of the variance in the likelihood of a gene being trapped by expression-dependent vectors and a lower, but still significant, proportion of the variance for vectors that are predicted to be independent of endogenous gene expression.The findings of significant expression and length effects reported here further the understanding of the determinants of vector insertion. Results from this analysis can be applied to help identify other important determinants of this important biological phenomenon and could assist planning of large-scale mutagenesis efforts.

  1. Effect of SOS-induced levels of imuABC on spontaneous and damage-induced mutagenesis in Caulobacter crescentus.

    Science.gov (United States)

    Alves, Ingrid R; Lima-Noronha, Marco A; Silva, Larissa G; Fernández-Silva, Frank S; Freitas, Aline Luiza D; Marques, Marilis V; Galhardo, Rodrigo S

    2017-11-01

    imuABC (imuAB dnaE2) genes are responsible for SOS-mutagenesis in Caulobacter crescentus and other bacterial species devoid of umuDC. In this work, we have constructed operator-constitutive mutants of the imuABC operon. We used this genetic tool to investigate the effect of SOS-induced levels of these genes upon both spontaneous and damage-induced mutagenesis. We showed that constitutive expression of imuABC does not increase spontaneous or damage-induced mutagenesis, nor increases cellular resistance to DNA-damaging agents. Nevertheless, the presence of the operator-constitutive mutation rescues mutagenesis in a recA background, indicating that imuABC are the only genes required at SOS-induced levels for translesion synthesis (TLS) in C. crescentus. Furthermore, these data also show that TLS mediated by ImuABC does not require RecA, unlike umuDC-dependent mutagenesis in E. coli. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Mutational spectrum analysis of umuC-independent and umuC-dependent γ-radiation mutagenesis in Escherichia coli

    International Nuclear Information System (INIS)

    Sargentini, N.J.; Smith, K.C.

    1989-01-01

    γ-radiation mutagenesis Escherichia coli K-12. Mutagenesis (argE3(OC) A rg + ) was blocked in a δ(recA-srlR)306 strain at the same doses that induced mutations in umuC122::Tn5 and wild-type strains, indicating that both umuC-independent and umuC-dependent mechanisms function within recA-dependent misrepair. Analyses of various suppressor and back mutations that result in argE3 and hisG4 ochre reversion and an analysis of trpE9777 reversion were performed on umuC and wild-type cells irradiated in the presence and absence of oxygen. While the umuC strain showed the γ-radiation induction of base substitution and frameshifts when irradiated in the absence of oxygen, the umuC mutation blocked all oxygen-dependent base-substitution mutagenesis, but non all oxygen-dependent frameshift mutagenesis. For anoxically irradiated cells, the yields of GC T and AT GC transitions were essentially umuC independent, while the yields of (AT or GC) TA transversions were heavily umuC dependent. These data suggest new concepts about the nature of the DNA lesions and the mutagenic mechanisms that lead to γ-radiation mutagenesis. (author). 48 refs.; 1 tab.; 6 refs

  3. Use of CRISPR/Cas Genome Editing Technology for Targeted Mutagenesis in Rice.

    Science.gov (United States)

    Xu, Rongfang; Wei, Pengcheng; Yang, Jianbo

    2017-01-01

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system is a newly emerging mutagenesis (gene-editing) tool in genetic engineering. Among the agriculturally important crops, several genes have been successfully mutated by the system, and some agronomic important traits have been rapidly generated, which indicates the potential applications in both scientific research and plant breeding. In this chapter, we describe a standard gene-editing procedure to effectively target rice genes and to make specific rice mutants using the CRISPR/Cas9 system mediated by Agrobacterium transformation.

  4. Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

    Science.gov (United States)

    Taniguchi, Naohiro; Murakami, Hiroshi

    2017-01-01

    Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

  5. Application of radiation induced in vitro mutagenesis for the improvement of sugarcane

    International Nuclear Information System (INIS)

    Penna, Suprasanna; Patade, Vikas Y.; Vaidya, E.R.; Patil, V.D.

    2009-01-01

    Sugarcane varieties with improved tolerance to adverse environmental conditions are highly desirable, as unfavourable environmental factors are the major contributors that can reduce average productivity by 65% to 87%. In this study, we have employed in vitro cultures and radiation induced mutagenesis in three commercially used cultivars. Irradiated callus cultures were also selected for salt tolerance, and radiosensitivity in terms of growth rate and cell viability indicated stress effects. Several mutants with agronomically desirable traits have been isolated that are in field evaluation. (author)

  6. Uvm mutants of Escherichia coli K 12 deficient in UV mutagenesis. Pt. 1

    International Nuclear Information System (INIS)

    Steinborn, G.

    1978-01-01

    Selection for defective reversion induction, after UV treatment of E. coli K 12, yielded uvm mutants. These mutants exhibited highly reduced or no UV mutability for all loci tested although they were moderately and normally mutable by X-rays and EMS, respectively. Uvm mutations confer only a slight sensitivity to killing by UV and X-rays and no clear sensitivity to the lethal effect of HN2, EMS or MMS. Growth and viability of untreated uvm cells were normal. The properties of uvm mutants are discussed in relation to those of other relevant mutant types and to some actual problems of induced mutagenesis. (orig.) 891 AJ [de

  7. Design of Deinococcus radiodurans thioredoxin reductase with altered thioredoxin specificity using computational alanine mutagenesis

    OpenAIRE

    Obiero, Josiah; Sanders, David AR

    2011-01-01

    In this study, the X-ray crystal structure of the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (Trx) was used as a guide to design a Deinococcus radiodurans TrxR (DR TrxR) mutant with altered Trx specificity. Previous studies have shown that TrxRs have higher affinity for cognate Trxs (same species) than that for Trxs from different species. Computational alanine scanning mutagenesis and visual inspection of the EC TrxR–Trx interface suggested...

  8. p53 Mutagenesis by Benzo[a]pyrene derived Radical Cations

    Science.gov (United States)

    Sen, Sushmita; Bhojnagarwala, Pratik; Francey, Lauren; Lu, Ding; Jeffrey Field, Trevor M. Penning

    2013-01-01

    Benzo[a]pyrene (B[a]P), a major human carcinogen in combustion products such as cigarette smoke and diesel exhaust, is metabolically activated into DNA-reactive metabolites via three different enzymatic pathways. The pathways are the anti-(+)-benzo[a]pyrene 7,8-diol 9, 10-epoxide pathway (P450/ epoxide hydrolase catalyzed) (B[a]PDE), the benzo[a]pyrene o-quinone pathway (aldo ketose reductase (AKR) catalyzed) and the B[a]P radical cation pathway (P450 peroxidase catalyzed). We used a yeast p53 mutagenesis system to assess mutagenesis by B[a]P radical cations. Because radical cations are short-lived, they were generated in situ by reacting B[a]P with cumene hydroperoxide (CuOOH) and horse radish peroxidase (HRP) and then monitoring the generation of the more stable downstream products, B[a]P-1,6-dione and B[a]P-3,6-dione. Based on the B[a]P-1,6 and 3,6-dione formation, approximately 4µM of radical cation was generated. In the mutagenesis assays, the radical cations produced in situ showed a dose-dependent increase in mutagenicity from 0.25 µM to 10 µM B[a]P with no significant increase seen with further escalation to 50 µM B[a]P. However, mutagenesis was 200-fold less than with the AKR pathway derived B[a]P, 7–8 dione. Mutant p53 plasmids, which yield red colonies, were recovered from the yeast to study the pattern and spectrum of mutations. The mutation pattern observed was G to T (31%) > G to C (29%) > G to A (14%). The frequency of codons mutated by the B[a]P radical cations was essentially random and not enriched at known cancer hotspots. The quinone products of radical cations, B[a]P-1,6-dione and B[a]P-3,6-dione were more mutagenic than the radical cation reactions, but still less mutagenic than AKR derived B[a]P-7,8-dione. We conclude that B[a]P radical cations and their quinone products are weakly mutagenic in this yeast-based system compared to redox cycling PAH o-quinones. PMID:22768918

  9. The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

    OpenAIRE

    Hoskins, J.T.; Zhou, Z.; Harding, P.A.

    2008-01-01

    A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser108/121, HB-EGF-Cys/Ser116/132, and HB-EGF-Cys/Ser134/143) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with Mr of 6.5, 21 and 24kDa were observed from lys...

  10. Genetic toxicology of metal compounds. II. Enhancement of ultraviolet light-induced mutagenesis in Escherichia coli WP2

    International Nuclear Information System (INIS)

    Rossman, T.G.; Molina, M.

    1986-01-01

    Salts of metals which are carcinogenic, noncarcinogenic, or of unknown carcinogenicity were assayed for their abilities to modulate ultraviolet (UV)-induced mutagenesis in Escherichia coli WP2. In addition to the previously reported comutagenic effect of arsenite, salts of three other compounds were found to enhance UV mutagenesis. CuCl 2 , MnCl 2 (and a small effect by KMnO 4 ), and NaMoO 4 acted as comutagens in E coli WP2, which has wild-type DNA repair capability, but were much less comutagenic in the repair deficient strain WP2/sub s/ (uvrA). The survival of irradiated or unirradiated cells was not affected by these compounds. No effects on UV mutagenesis were seen for 16 other metal compounds. We suggest that the comutagenic effects might occur either via metal-induced decreases in the fidelity of repair replication or via metal-induced depurination

  11. Radioprotective action of glycerol and cysteamine on inactivation and mutagenesis in Salmonella tester strains after gamma and heavy ion irradiation

    International Nuclear Information System (INIS)

    Basha, S.G.; Krasavin, E.A.; Kozubek, S.

    1991-01-01

    Inactivation and mutagenesis were studied in Salmonella tester strains after γ-irradiation and after heavy ion irradiation in the presence of glycerol and cysteamine. Bacterial cells were irradiated at Dubna, JINR. Ions from deuterons to carbon were used with residual energies 2-9 MeV/u. The protective effect of glycerol was found both for γ-radiation and for heavy ions up to 50 keV/μm for both cell inactivation and mutagenesis in Salmonella tester strains with different mutation events. Cell sensitivity slightly increased with LET before falling down. The maximum was shifted in the presence of glycerol to the left and was less pronounced. The radioprotective effect of glycerol diminished gradually with LET from 2.0 for γ-radiation to 1.1 for carbon ions. Mutagenesis increases with LET in TA100 strain; in TA98 strain no marked increase could be detected. 13 refs.; 4 figs.; 5 tabs

  12. The disparity mutagenesis model predicts rescue of living things from catastrophic errors

    Directory of Open Access Journals (Sweden)

    Mitsuru eFurusawa

    2014-12-01

    Full Text Available In animals including humans, mutation rates per generation will exceed a perceived threshold, which should result in an excessive genetic load. Despite this, they have survived without extinction. This is a perplexing problem for human genetics, arising at the end of the last century, and to date still does not have a fully satisfactory explanation. Shortly after we proposed the disparity theory of evolution in 1992, the disparity mutagenesis model was proposed, which forms the basis for an explanation for an acceleration of evolution and species survival. This model predicts a significant increase of the mutation threshold values if there is a high enough fidelity difference in replication between the lagging and leading strands. When applied to biological evolution, the model predicts that living things, including humans, might overcome the lethal effect of accumulated deleterious mutations and be able to survive. Artificially-prepared mutator strains of microorganisms, in which an enhanced lagging-strand-biased mutagenesis was introduced, showed unexpectedly high adaptability to severe environments. The implications of the striking behaviors shown by these disparity mutators will be discussed in relation to how living things with high mutation rates can avoid the self-defeating risk of excess mutations.

  13. Viral Bacterial Artificial Chromosomes: Generation, Mutagenesis, and Removal of Mini-F Sequences

    Directory of Open Access Journals (Sweden)

    B. Karsten Tischer

    2012-01-01

    Full Text Available Maintenance and manipulation of large DNA and RNA virus genomes had presented an obstacle for virological research. BAC vectors provided a solution to both problems as they can harbor large DNA sequences and can efficiently be modified using well-established mutagenesis techniques in Escherichia coli. Numerous DNA virus genomes of herpesvirus and pox virus were cloned into mini-F vectors. In addition, several reverse genetic systems for RNA viruses such as members of Coronaviridae and Flaviviridae could be established based on BAC constructs. Transfection into susceptible eukaryotic cells of virus DNA cloned as a BAC allows reconstitution of recombinant viruses. In this paper, we provide an overview on the strategies that can be used for the generation of virus BAC vectors and also on systems that are currently available for various virus species. Furthermore, we address common mutagenesis techniques that allow modification of BACs from single-nucleotide substitutions to deletion of viral genes or insertion of foreign sequences. Finally, we review the reconstitution of viruses from BAC vectors and the removal of the bacterial sequences from the virus genome during this process.

  14. An inducible tool for random mutagenesis in Aspergillus niger based on the transposon Vader.

    Science.gov (United States)

    Paun, Linda; Nitsche, Benjamin; Homan, Tim; Ram, Arthur F; Kempken, Frank

    2016-07-01

    The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background.

  15. Random mutagenesis of aspergillus niger and process optimization for enhanced production of glucose oxidase

    International Nuclear Information System (INIS)

    Haq, I.; Nawaz, A.; Mukhtar, A.N.H.; Mansoor, H.M.Z.; Ameer, S.M.

    2014-01-01

    The study deals with the improvement of wild strain Aspergillus niger IIB-31 through random mutagenesis using chemical mutagens. The main aim of the work was to enhance the glucose oxidase (GOX) yield of wild strain (24.57+-0.01 U/g of cell mass) through random mutagenesis and process optimization. The wild strain of Aspergillus niger IIB-31 was treated with chemical mutagens such as Ethyl methane sulphonate (EMS) and nitrous acid for this purpose. Mutagen treated 98 variants indicating the positive results were picked and screened for the glucose oxidase production using submerged fermentation. EMS treated E45 mutant strain gave the highest glucose oxidase production (69.47 + 0.01 U/g of cell mass), which was approximately 3-folds greater than the wild strain IIB-31. The preliminary cultural conditions for the production of glucose oxidase using submerged fermentation from strain E45 were also optimized. The highest yield of GOD was obtained using 8% glucose as carbon and 0.3% peptone as nitrogen source at a medium pH of 7.0 after an incubation period of 72 hrs at 30 degree. (author)

  16. Comparative Analysis of Context-Dependent Mutagenesis Using Human and Mouse Models

    Directory of Open Access Journals (Sweden)

    Sofya A. Medvedeva

    2013-01-01

    Full Text Available Substitution rates strongly depend on their nucleotide context. One of the most studied examples is the excess of C > T mutations in the CG context in various groups of organisms, including vertebrates. Studies on the molecular mechanisms underlying this mutation regularity have provided insights into evolution, mutagenesis, and cancer development. Recently several other hypermutable motifs were identified in the human genome. There is an increased frequency of T > C mutations in the second position of the words ATTG and ATAG and an increased frequency of A > C mutations in the first position of the word ACAA. For a better understanding of evolution, it is of interest whether these mutation regularities are human specific or present in other vertebrates, as their presence might affect the validity of currently used substitution models and molecular clocks. A comprehensive analysis of mutagenesis in 4 bp mutation contexts requires a vast amount of mutation data. Such data may be derived from the comparisons of individual genomes or from single nucleotide polymorphism (SNP databases. Using this approach, we performed a systematical comparison of mutation regularities within 2–4 bp contexts in Mus musculus and Homo sapiens and uncovered that even closely related organisms may have notable differences in context-dependent mutation regularities.

  17. Selection of gamma-ray induced salt tolerant rice mutants by in vitro mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dong Sub; Chun, Jae Beom; Lee, Kyung Jun; Kim, Jin Baek; Kim, Sang Hoon; Yun, Song Jong; Kang, Si Yong [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2010-06-15

    The present study had been performed to select the salt tolerant rice mutant lines through an in vivo and in vitro mutagenesis with a gamma-ray. The physiological responses such as MDA and chlorophyll of the selected salt mutant lines were investigated under salt stress. For the selection of the salt tolerant rice mutants by in vitro mutagenesis with gamma-ray, we conducted a second selection procedure with 1,500 mutant lines induced from the original cv. Dongan (wild-type, WT): Ist, selection under a nutrient solution with 171 mM NaCI: 2nd, selection under in vitro conditions. Based on a growth comparison of the entries, out of mutant lines, the putative 2 salt tolerant rice mutant lines, ST-495 and ST-532, were selected. The 2 ST-lines had a lower malonaldehyde (MDA) contents than wild-type (WT) during salt stress. The survival rate of the WT, ST-495 and ST-532 were 36.6%, 70% and 50% in 171 mM NaCI, respectively. The chlorophyll and carotenoid contents were decreased more in a WT plant than the two selected mutant lines. These rice mutant lines will be released for cultivation at the reclaimed land and used as a control plot for genetic research about salt tolerance.

  18. Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.

    Science.gov (United States)

    Iwakuma, Hidekazu; Koyama, Yoshiyuki; Miyachi, Ayako; Nasukawa, Masashi; Matsumoto, Hitoshi; Yano, Shuntaro; Ogihara, Jun; Kasumi, Takafumi

    2016-01-01

    We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

  19. Fragile DNA Motifs Trigger Mutagenesis at Distant Chromosomal Loci in Saccharomyces cerevisiae

    Science.gov (United States)

    Saini, Natalie; Zhang, Yu; Nishida, Yuri; Sheng, Ziwei; Choudhury, Shilpa; Mieczkowski, Piotr; Lobachev, Kirill S.

    2013-01-01

    DNA sequences capable of adopting non-canonical secondary structures have been associated with gross-chromosomal rearrangements in humans and model organisms. Previously, we have shown that long inverted repeats that form hairpin and cruciform structures and triplex-forming GAA/TTC repeats induce the formation of double-strand breaks which trigger genome instability in yeast. In this study, we demonstrate that breakage at both inverted repeats and GAA/TTC repeats is augmented by defects in DNA replication. Increased fragility is associated with increased mutation levels in the reporter genes located as far as 8 kb from both sides of the repeats. The increase in mutations was dependent on the presence of inverted or GAA/TTC repeats and activity of the translesion polymerase Polζ. Mutagenesis induced by inverted repeats also required Sae2 which opens hairpin-capped breaks and initiates end resection. The amount of breakage at the repeats is an important determinant of mutations as a perfect palindromic sequence with inherently increased fragility was also found to elevate mutation rates even in replication-proficient strains. We hypothesize that the underlying mechanism for mutagenesis induced by fragile motifs involves the formation of long single-stranded regions in the broken chromosome, invasion of the undamaged sister chromatid for repair, and faulty DNA synthesis employing Polζ. These data demonstrate that repeat-mediated breaks pose a dual threat to eukaryotic genome integrity by inducing chromosomal aberrations as well as mutations in flanking genes. PMID:23785298

  20. Selection of gamma-ray induced salt tolerant rice mutants by in vitro mutagenesis

    International Nuclear Information System (INIS)

    Kim, Dong Sub; Chun, Jae Beom; Lee, Kyung Jun; Kim, Jin Baek; Kim, Sang Hoon; Yun, Song Jong; Kang, Si Yong

    2010-01-01

    The present study had been performed to select the salt tolerant rice mutant lines through an in vivo and in vitro mutagenesis with a gamma-ray. The physiological responses such as MDA and chlorophyll of the selected salt mutant lines were investigated under salt stress. For the selection of the salt tolerant rice mutants by in vitro mutagenesis with gamma-ray, we conducted a second selection procedure with 1,500 mutant lines induced from the original cv. Dongan (wild-type, WT): Ist, selection under a nutrient solution with 171 mM NaCI: 2nd, selection under in vitro conditions. Based on a growth comparison of the entries, out of mutant lines, the putative 2 salt tolerant rice mutant lines, ST-495 and ST-532, were selected. The 2 ST-lines had a lower malonaldehyde (MDA) contents than wild-type (WT) during salt stress. The survival rate of the WT, ST-495 and ST-532 were 36.6%, 70% and 50% in 171 mM NaCI, respectively. The chlorophyll and carotenoid contents were decreased more in a WT plant than the two selected mutant lines. These rice mutant lines will be released for cultivation at the reclaimed land and used as a control plot for genetic research about salt tolerance

  1. Simple-MSSM: a simple and efficient method for simultaneous multi-site saturation mutagenesis.

    Science.gov (United States)

    Cheng, Feng; Xu, Jian-Miao; Xiang, Chao; Liu, Zhi-Qiang; Zhao, Li-Qing; Zheng, Yu-Guo

    2017-04-01

    To develop a practically simple and robust multi-site saturation mutagenesis (MSSM) method that enables simultaneously recombination of amino acid positions for focused mutant library generation. A general restriction enzyme-free and ligase-free MSSM method (Simple-MSSM) based on prolonged overlap extension PCR (POE-PCR) and Simple Cloning techniques. As a proof of principle of Simple-MSSM, the gene of eGFP (enhanced green fluorescent protein) was used as a template gene for simultaneous mutagenesis of five codons. Forty-eight randomly selected clones were sequenced. Sequencing revealed that all the 48 clones showed at least one mutant codon (mutation efficiency = 100%), and 46 out of the 48 clones had mutations at all the five codons. The obtained diversities at these five codons are 27, 24, 26, 26 and 22, respectively, which correspond to 84, 75, 81, 81, 69% of the theoretical diversity offered by NNK-degeneration (32 codons; NNK, K = T or G). The enzyme-free Simple-MSSM method can simultaneously and efficiently saturate five codons within one day, and therefore avoid missing interactions between residues in interacting amino acid networks.

  2. Mutagenesis in mammalian cells can be modulated by radiation-induced voltage-dependent potassium channels

    International Nuclear Information System (INIS)

    Saad, A.H.; Zhou, L.Y.; Lambe, E.K.; Hahn, G.M.

    1994-01-01

    In mammalian cells, little is known about the initial events whose ultimate consequence is mutagenesis or DNA repair. The role the plasma membrane may play as an initiator of such a pathway is not understood. We show, for the first time, that membrane voltage-dependent potassium (K + ) currents, activated by ionizing radiation play a significant role in radiation mutagenesis. Specifically, we show that the frequency of mutation at the HGPRT locus is increased as expected to 37.6±4.0 mutations per 100,000 survivors by 800 cGy of ionizing radiation from a spontaneous frequency of 1.5±1.5. This increase, however, is abolished if either K + channel blocker, CsCl or BaCl 2 , is present for 2h following irradiation of the cells. RbCl, chemically similar to CsCl but known not to block K + channels, is ineffective in reducing the mutation frequency. Treatment of cells with CsCl or BaCl 2 had no effect on radiation-induced cell killing

  3. Construction of a horseradish peroxidase resistant toward hydrogen peroxide by saturation mutagenesis.

    Science.gov (United States)

    Asad, Sedigheh; Dastgheib, Seyed Mohammad Mehdi; Khajeh, Khosro

    2016-11-01

    Horseradish peroxidase (HRP) with a variety of potential biotechnological applications is still isolated from the horseradish root as a mixture of different isoenzymes with different biochemical properties. There is an increasing demand for preparations of high amounts of pure enzyme but its recombinant production is limited because of the lack of glycosylation in Escherichia coli and different glycosylation patterns in yeasts which affects its stability parameters. The goal of this study was to increase the stability of non-glycosylated enzyme, which is produced in E. coli, toward hydrogen peroxide via mutagenesis. Asparagine 268, one of the N-glycosylation sites of the enzyme, has been mutated via saturation mutagenesis using the megaprimer method. Modification and miniaturization of previously described protocols enabled screening of a library propagated in E. coli XJb (DE3). The library of mutants was screened for stability toward hydrogen peroxide with azinobis (ethylbenzthiazoline sulfonate) as a reducing substrate. Asn268Gly mutant, the top variant from the screening, exhibited 18-fold increased stability toward hydrogen peroxide and twice improved thermal stability compared with the recombinant HRP. Moreover, the substitution led to 2.5-fold improvement in the catalytic efficiency with phenol/4-aminoantipyrine. Constructed mutant represents a stable biocatalyst, which may find use in medical diagnostics, biosensing, and bioprocesses. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

  4. Improving isopropanol tolerance and production of Clostridium beijerinckii DSM 6423 by random mutagenesis and genome shuffling.

    Science.gov (United States)

    Gérando, H Máté de; Fayolle-Guichard, F; Rudant, L; Millah, S K; Monot, F; Ferreira, Nicolas Lopes; López-Contreras, A M

    2016-06-01

    Random mutagenesis and genome shuffling was applied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteria Clostridium beijerinckii DSM 6423. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of putatively improved strains was done by submitting the mutants to toxic levels of inhibitory chemicals or by screening for their tolerance to isopropanol (>35 g/L). Suicide substrates, such as ethyl or methyl bromobutyrate or alcohol dehydrogenase inhibitors like allyl alcohol, were tested and, finally, 36 mutants were isolated. The fermentation profiles of these NTG mutant strains were characterized, and the best performing mutants were used for consecutive rounds of genome shuffling. Screening of strains with further enhancement in isopropanol tolerance at each recursive shuffling step was then used to spot additionally improved strains. Three highly tolerant strains were finally isolated and able to withstand up to 50 g/L isopropanol on plates. Even if increased tolerance to the desired end product was not always accompanied by higher production capabilities, some shuffled strains showed increased solvent titers compared to the parental strains and the original C. beijerinckii DSM 6423. This study confirms the efficiency of genome shuffling to generate improved strains toward a desired phenotype such as alcohol tolerance. This tool also offers the possibility of obtaining improved strains of Clostridium species for which targeted genetic engineering approaches have not been described yet.

  5. Gain-of-function mutagenesis approaches in rice for functional genomics and improvement of crop productivity.

    Science.gov (United States)

    Moin, Mazahar; Bakshi, Achala; Saha, Anusree; Dutta, Mouboni; Kirti, P B

    2017-07-01

    The epitome of any genome research is to identify all the existing genes in a genome and investigate their roles. Various techniques have been applied to unveil the functions either by silencing or over-expressing the genes by targeted expression or random mutagenesis. Rice is the most appropriate model crop for generating a mutant resource for functional genomic studies because of the availability of high-quality genome sequence and relatively smaller genome size. Rice has syntenic relationships with members of other cereals. Hence, characterization of functionally unknown genes in rice will possibly provide key genetic insights and can lead to comparative genomics involving other cereals. The current review attempts to discuss the available gain-of-function mutagenesis techniques for functional genomics, emphasizing the contemporary approach, activation tagging and alterations to this method for the enhancement of yield and productivity of rice. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  6. Development of a Dunaliella tertiolecta Strain with Increased Zeaxanthin Content Using Random Mutagenesis.

    Science.gov (United States)

    Kim, Minjae; Ahn, Junhak; Jeon, Hancheol; Jin, EonSeon

    2017-06-21

    Zeaxanthin is a xanthophyll pigment that is regarded as one of the best carotenoids for the prevention and treatment of degenerative diseases. In the worldwide natural products market, consumers prefer pigments that have been produced from biological sources. In this study, a Dunaliella tertiolecta strain that has 10-15% higher cellular zeaxanthin content than the parent strain ( zea1 ), was obtained by random mutagenesis using ethyl methanesulfonate (EMS) as a mutagen. This mutant, mp3 , was grown under various salinities and light intensities to optimize culture conditions for zeaxanthin production. The highest cellular zeaxanthin content was observed at 1.5 M NaCl and 65-85 μmol photons·m -2 ·s -1 , and the highest daily zeaxanthin productivity was observed at 0.6 M NaCl and 140-160 μmol photons·m -2 ·s -1 . The maximal yield of zeaxanthin from mp3 in fed-batch culture was 8 mg·L -1 , which was obtained at 0.6 M NaCl and 140-160 μmol photons·m -2 ·s -1 . These results suggest that random mutagenesis with EMS is useful for generating D. tertiolecta strains with increased zeaxanthin content, and also suggest optimal culture conditions for the enhancement of biomass and zeaxanthin production by the zeaxanthin accumulating mutant strains.

  7. Single d(ApG)/cis-diamminedichloroplatinum(II) adduct-induced mutagenesis in Escherichia coli

    International Nuclear Information System (INIS)

    Burnouf, D.; Fuchs, R.P.P.; Gauthier, C.; Chottard, J.C.

    1990-01-01

    The mutation spectrum induced by the widely used antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) showed that cisDDP[d(ApG)] adducts, although they account for only 25% of the lesions formed are ∼5 times more mutagenic than the major GG adduct. The authors report the construction of vectors bearing a single cisDDP[d(ApG)] lesion and their use in mutagenesis experiments in Escherichia coli. The mutagenic processing of the lesion is found to depend strictly on induction of the SOS system of the bacterial host cells. In SOS-induced cells, mutation frequencies of 1-2% were detected. All these mutations are targeted to the 5' base of the adduct. Single A → T transversions are mainly observed (80%), whereas A → G transitions account for 10% of the total mutations. Tandem base-pair substitutions involving the adenine residue and the thymine residue immediately 5' to the adduct occur at a comparable frequency (10%). No selective loss of the strand bearing the platinum adduct was seen, suggesting that, in vivo, cisDDP[d(ApG)] adducts are not blocking lesions. The high mutation specificity of cisDDP-[d(ApG)]-induced mutagenesis is discussed in relation to structural data

  8. The role of the bacterial mismatch repair system in SOS-induced mutagenesis: a theoretical background

    International Nuclear Information System (INIS)

    Belov, O.V.; Kapralov, M.I.; Chuluunbaatar, O.; Sweilam, N.H.

    2012-01-01

    A theoretical study is performed of the possible role of the methyl-directed mismatch repair system in the ultraviolet-induced mutagenesis of Escherichia coli bacterial cells. For this purpose, a mathematical model of the bacterial mismatch repair system is developed. Within this model, the key pathways of this type of repair are simulated on the basis of modern experimental data related to its mechanisms. Here we have modelled in detail five main pathways of DNA misincorporation removal with different DNA exonucleases. Using our calculations, we have tested the hypothesis that the bacterial mismatch repair system is responsible for the removal of the nucleotides misincorporated by DNA polymerase V (the UmuD' 2 C complex) during ultraviolet-induced SOS response. For the theoretical analysis of the mutation frequency, we have combined the proposed mathematical approach with the model of SOS-induced mutagenesis in the E.coli bacterial cell developed earlier. Our calculations support the hypothesis that methyl-directed mismatch repair influences the mutagenic effect of ultraviolet radiation

  9. Caffeine enhanced measurement of mutagenesis by low levels of [gamma]-irradiation in human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Puck, T.P.; Johnson, R.; Waldren, C.A. (Eleanor Roosevelt Institute for Cancer Research, Denver, CO (United States)); Morse, H. (Univ. of Colorado Cancer Center, Denver, CO (United States))

    1993-09-01

    The well-known action of caffeine in synergizing mutagenesis (including chromosome aberrations) of agents like ionizing radiation by inhibition of cellular repair processes has been incorporated into a rapid procedure for detection of mutagenicity with high sensitivity. Effects of 5-10 rads of [gamma]-irradiation, which approximate the human lifetime dose accumulation from background radiation, can be detected in a two-day procedure using an immortalized human WBC culture. Chromosomally visible lesions are scored on cells incubated for 2 h after irradiation in the presence and absence of 1.0 mg/ml of caffeine. An eightfold amplification of scorable lesions is achieved over the action of radiation alone. This approach provides a closer approximation to absolute mutagenicity unmitigated by repair processes, which can vary in different situations. It is proposed that mutagenesis testing of this kind, using caffiene or other repair-inhibitory agents, be employed to identify mutagens in their effective concentrations to which human populations may be exposed; to detect agents such as caffeine that may synergize mutagenic actions and pose epidemiologic threats; and to discover effective anti-mutagens. Information derived from the use of such procedures may help prevent cancer and newly acquired genetic disease.

  10. The effect of chronic alcohol consumption on mitochondrial DNA mutagenesis in human blood

    Energy Technology Data Exchange (ETDEWEB)

    Wurmb-Schwark, N. von [Institute of Legal Medicine, Christian Albrecht University of Kiel, Arnold-Heller-Str. 12, 24105 Kiel (Germany)], E-mail: nvonwurmb@rechtsmedizin.uni-kiel.de; Ringleb, A.; Schwark, T. [Institute of Legal Medicine, Christian Albrecht University of Kiel, Arnold-Heller-Str. 12, 24105 Kiel (Germany); Broese, T.; Weirich, S.; Schlaefke, D. [Clinic of Psychiatry and Psychotherapy, University of Rostock, Gehlsheimer Str. 20, Rostock (Germany); Wegener, R. [Institute of Legal Medicine, St-Georg-Str. 108, University of Rostock, 18055 Rostock (Germany); Oehmichen, M. [Institute of Legal Medicine, Christian Albrecht University of Kiel, Arnold-Heller-Str. 12, 24105 Kiel (Germany)

    2008-01-01

    The 4977 bp deletion of mitochondrial DNA (mtDNA) is known to accumulate with increasing age in post mitotic tissues. Recently, studies came out detecting this specific alteration also in fast replicating cells, e.g. in blood or skin tissue, often in correlation to specific diseases or - specifically in skin - external stressors such as UV radiation. In this study, we investigated mitochondrial mutagenesis in 69 patients with a chronic alcoholic disease and 46 age matched controls with a moderate drinking behavior. Two different fragments, specific for total and for deleted mtDNA (dmtDNA) were amplified in a duplex-PCR. A subsequent fragment analysis was performed and for relative quantification, the quotient of the peak areas of amplification products specific for deleted and total mtDNA was determined. Additionally, a real time PCR was performed to quantify mtDNA copy number. The relative amount of 4977 bp deleted mtDNA in alcoholics was significantly increased compared to controls. On the other hand, no difference regarding the mtDNA/nuclear DNA ratio in both investigated groups was detected. Additionally, no age dependence could be found nor in alcoholics, neither in the control group. These findings indicate that mtDNA mutagenesis in blood can be influenced by stressors such as alcohol. Ethanol seems to be a significant factor to alter mitochondrial DNA in blood and might be an additional contributor for the cellular aging process.

  11. Genetic analysis of γ-ray mutagenesis in yeast. Vol. 3

    International Nuclear Information System (INIS)

    McKee, R.H.; Lawrence, C.W.

    1980-01-01

    Comparisons between the 60 Co γ-ray survival curves of diploid strains of the yeast Saccharomyces cerevisiae that are homozygous for two non-allelic radiation-sensitive mutations and the corresponding single-mutant diploids suggest that there are two main types of repair of ionizing radiation damage in this organism. The first, which is defined by the rad52 epistasis group, depends on the activities of the RAD50 through RAD57 genes and is responsible for repairing the larger amount of lethal damage. Previous work [22] shows that this type of repair is essentially error-free. The second, defined by the rad6 epistasis group, depends on the activities of the RAD6, RAD9, RAD18, REV1 and REV3 genes and repairs a smaller, though still substantial, amount of lethal damage. It is also responsible for induced mutagenesis [22,23]. Data for survival and mutation induction after irradiation in air and partial anoxia show that oxygen-dependent damage can be repaired by either of these two pathways. They also show similar oxygen-enhancement ratios for survival and mutagenesis. (orig.)

  12. Chronic low-dose ultraviolet-induced mutagenesis in nucleotide excision repair-deficient cells.

    Science.gov (United States)

    Haruta, Nami; Kubota, Yoshino; Hishida, Takashi

    2012-09-01

    UV radiation induces two major types of DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidine photoproducts, which are both primarily repaired by nucleotide excision repair (NER). Here, we investigated how chronic low-dose UV (CLUV)-induced mutagenesis occurs in rad14Δ NER-deficient yeast cells, which lack the yeast orthologue of human xeroderma pigmentosum A (XPA). The results show that rad14Δ cells have a marked increase in CLUV-induced mutations, most of which are C→T transitions in the template strand for transcription. Unexpectedly, many of the CLUV-induced C→T mutations in rad14Δ cells are dependent on translesion synthesis (TLS) DNA polymerase η, encoded by RAD30, despite its previously established role in error-free TLS. Furthermore, we demonstrate that deamination of cytosine-containing CPDs contributes to CLUV-induced mutagenesis. Taken together, these results uncover a novel role for Polη in the induction of C→T transitions through deamination of cytosine-containing CPDs in CLUV-exposed NER deficient cells. More generally, our data suggest that Polη can act as both an error-free and a mutagenic DNA polymerase, depending on whether the NER pathway is available to efficiently repair damaged templates.

  13. Enhancement of Schizochytrium DHA synthesis by plasma mutagenesis aided with malonic acid and zeocin screening.

    Science.gov (United States)

    Zhao, Ben; Li, Yafei; Li, Changling; Yang, Hailin; Wang, Wu

    2018-03-01

    Schizochytrium sp. accumulates valuable polyunsaturated fatty acid (PUFA), such as docosahexaenoic acid (DHA). In order to increase DHA synthesis in this microorganism, physical or chemical mutagenesis aided with powerful screening methods are still preferable, as its DHA synthetic pathway has not yet been clearly defined for gene manipulation. To breed this agglomerate microorganism of thick cell wall and rather large genome for increasing lipid content and DHA percentage, a novel strategy of atmospheric and room temperature plasma (ARTP) mutagenesis coupled with stepped malonic acid (MA) and zeocin resistance screening was developed. The final resulted mutant strain mz-17 was selected with 1.8-fold increased DHA production. Accompanied with supplementation of Fe 2+ in shake flask cultivation, DHA production of 14.0 g/L on average was achieved. This work suggests that ARTP mutation combined with stepped MA and zeocin resistance screening is an efficient method of breeding Schizochytrium sp. of high DHA production, and might be applied on other microorganisms for obtaining higher desired PUFA products.

  14. Mechanisms of mutagenesis: DNA replication in the presence of DNA damage.

    Science.gov (United States)

    Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F Peter; Zhang, Huidong

    2016-01-01

    Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, Escherichia coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Improvement of Lead Tolerance of Saccharomyces cerevisiae by Random Mutagenesis of Transcription Regulator SPT3.

    Science.gov (United States)

    Zhu, Liying; Gao, Shan; Zhang, Hongman; Huang, He; Jiang, Ling

    2018-01-01

    Bioremediation of heavy metal pollution with biomaterials such as bacteria and fungi usually suffer from limitations because of microbial sensitivity to high concentration of heavy metals. Herein, we adopted a novel random mutagenesis technique called RAISE to manipulate the transcription regulator SPT3 of Saccharomyces cerevisiae to improve cell lead tolerance. The best strain Mutant VI was selected from the random mutagenesis libraries on account of the growth performance, with higher specific growth rate than the control strain (0.068 vs. 0.040 h -1 ) at lead concentration as high as 1.8 g/L. Combined with the transcriptome analysis of S. cerevisiae, expressing the SPT3 protein was performed to make better sense of the global regulatory effects of SPT3. The data analysis revealed that 57 of S. cerevisiae genes were induced and 113 genes were suppressed, ranging from those for trehalose synthesis, carbon metabolism, and nucleotide synthesis to lead resistance. Especially, the accumulation of intracellular trehalose in S. cerevisiae under certain conditions of stress is considered important to lead resistance. The above results represented that SPT3 was acted as global transcription regulator in the exponential phase of strain and accordingly improved heavy metal tolerance in the heterologous host S. cerevisiae. The present study provides a route to complex phenotypes that are not readily accessible by traditional methods.

  16. Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

    Science.gov (United States)

    Weng, Meizhi; Deng, Xiongwei; Bao, Wei; Zhu, Li; Wu, Jieyuan; Cai, Yongjun; Jia, Yan; Zheng, Zhongliang; Zou, Guolin

    2015-09-25

    Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Evidence for a Rad18-independent frameshift mutagenesis pathway in human cell-free extracts.

    Directory of Open Access Journals (Sweden)

    Régine Janel-Bintz

    Full Text Available Bypass of replication blocks by specialized DNA polymerases is crucial for cell survival but may promote mutagenesis and genome instability. To gain insight into mutagenic sub-pathways that coexist in mammalian cells, we examined N-2-acetylaminofluorene (AAF-induced frameshift mutagenesis by means of SV40-based shuttle vectors containing a single adduct. We found that in mammalian cells, as previously observed in E. coli, modification of the third guanine of two target sequences, 5'-GGG-3' (3G and 5'-GGCGCC-3' (NarI site, induces -1 and -2 frameshift mutations, respectively. Using an in vitro assay for translesion synthesis, we investigated the biochemical control of these events. We showed that Pol eta, but neither Pol iota nor Pol zeta, plays a major role in the frameshift bypass of the AAF adduct located in the 3G sequence. By complementing PCNA-depleted extracts with either a wild-type or a non-ubiquitinatable form of PCNA, we found that this Pol eta-mediated pathway requires Rad18 and ubiquitination of PCNA. In contrast, when the AAF adduct is located within the NarI site, TLS is only partially dependent upon Pol eta and Rad18, unravelling the existence of alternative pathways that concurrently bypass this lesion.

  18. Random Mutagenesis of the Aspergillus oryzae Genome Results in Fungal Antibacterial Activity

    Directory of Open Access Journals (Sweden)

    Cory A. Leonard

    2013-01-01

    Full Text Available Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin-resistant Staphylococcus aureus (MRSA in vitro. To randomly generate A. oryzae mutant strains, spores were treated with ethyl methanesulfonate (EMS. Over 3000 EMS-treated A. oryzae cultures were tested in the screen, and one isolate, CAL220, exhibited altered morphology and antibacterial activity. Culture supernatant from this isolate showed antibacterial activity against Methicillin-sensitive Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa, but not Klebsiella pneumonia or Proteus vulgaris. The results of this study support our hypothesis and suggest that the screen used is sufficient and appropriate to detect secreted antibacterial fungal compounds resulting from mutagenesis of A. oryzae. Because the genome of A. oryzae has been sequenced and systems are available for genetic transformation of this organism, targeted as well as random mutations may be introduced to facilitate the discovery of novel antibacterial compounds using this system.

  19. Random Mutagenesis of the Aspergillus oryzae Genome Results in Fungal Antibacterial Activity

    Science.gov (United States)

    Leonard, Cory A.; Brown, Stacy D.; Hayman, J. Russell

    2013-01-01

    Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin-resistant Staphylococcus aureus (MRSA) in vitro. To randomly generate A. oryzae mutant strains, spores were treated with ethyl methanesulfonate (EMS). Over 3000 EMS-treated A. oryzae cultures were tested in the screen, and one isolate, CAL220, exhibited altered morphology and antibacterial activity. Culture supernatant from this isolate showed antibacterial activity against Methicillin-sensitive Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa, but not Klebsiella pneumonia or Proteus vulgaris. The results of this study support our hypothesis and suggest that the screen used is sufficient and appropriate to detect secreted antibacterial fungal compounds resulting from mutagenesis of A. oryzae. Because the genome of A. oryzae has been sequenced and systems are available for genetic transformation of this organism, targeted as well as random mutations may be introduced to facilitate the discovery of novel antibacterial compounds using this system. PMID:23983696

  20. Random Mutagenesis of the Aspergillus oryzae Genome Results in Fungal Antibacterial Activity.

    Science.gov (United States)

    Leonard, Cory A; Brown, Stacy D; Hayman, J Russell

    2013-01-01

    Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin-resistant Staphylococcus aureus (MRSA) in vitro. To randomly generate A. oryzae mutant strains, spores were treated with ethyl methanesulfonate (EMS). Over 3000 EMS-treated A. oryzae cultures were tested in the screen, and one isolate, CAL220, exhibited altered morphology and antibacterial activity. Culture supernatant from this isolate showed antibacterial activity against Methicillin-sensitive Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa, but not Klebsiella pneumonia or Proteus vulgaris. The results of this study support our hypothesis and suggest that the screen used is sufficient and appropriate to detect secreted antibacterial fungal compounds resulting from mutagenesis of A. oryzae. Because the genome of A. oryzae has been sequenced and systems are available for genetic transformation of this organism, targeted as well as random mutations may be introduced to facilitate the discovery of novel antibacterial compounds using this system.

  1. Genetic separation of Escherichia coli recA functions for SOS mutagenesis and repressor cleavage

    International Nuclear Information System (INIS)

    Ennis, D.G.; Ossanna, N.; Mount, D.W.

    1989-01-01

    Evidence is presented that recA functions which promote the SOS functions of mutagenesis, LexA protein proteolysis, and lambda cI repressor proteolysis are each genetically separable from the others. This separation was observed in recombination-proficient recA mutants and rec+ (F' recA56) heterodiploids. recA430, recA433, and recA435 mutants and recA+ (F' recA56) heterodiploids were inducible for only one or two of the three functions and defective for mutagenesis. recA80 and recA432 mutants were constitutively activated for two of the three functions in that these mutants did not have to be induced to express the functions. We propose that binding of RecA protein to damaged DNA and subsequent interaction with small inducer molecules gives rise to conformational changes in RecA protein. These changes promote surface-surface interactions with other target proteins, such as cI and LexA proteins. By this model, the recA mutants are likely to have incorrect amino acids substituted as sites in the RecA protein structure which affect surface regions required for protein-protein interactions. The constitutively activated mutants could likewise insert altered amino acids at sites in RecA which are involved in the activation of RecA protein by binding small molecules or polynucleotides which metabolically regulate RecA protein

  2. Novel patterns of ultraviolet mutagenesis and Weigle reactivation in Staphylococcus aureus and phage phi II

    International Nuclear Information System (INIS)

    Thompson, J.K.; Hart, M.G.R.

    1981-01-01

    The effects of u.v. irradiation on the survival of Staphylococcus aureus and its phage phi11 were studied. The recA and uvr mutations affected their survival like synonymous mutations in Escherichia coli. Weigle reactivation (W-reactivation) of phi11 occurred in wild-type S. aureus and in a uvr mutant. Reactivation was recA-dependent and was accompanied by u.v.-induced mutagenesis in a temperature-sensitive mutant of phi11. Bacterial mutation to streptomycin resistance was induced by u.v. and was also recA-dependent. In S. aureus, as in E. coli, u.v. was a more effective mutagen in the uvr genetic background. However, a dose-squared response for u.v.-induced mutation of wild-type and uvr strains of S. aureus to streptomycin resistance, and of a trp auxotroph to tryptophan independence, was found only with u.v. doses below 1 J m -2 . In relation to the Uvr mechanism of DNA repair, u.v. mutagenesis in S. aureus may involve both repairable and non-repairable lesions. As in E. Coli, the uvr genetic background reduced the u.v. dose required for maximal W-reactivation of u.v.-irradiated phage. However, there was no enhancement of W-reactivation by post-irradiation broth incubation of S. aureus. The results are compatible with a non-inducible mechanism for this phenomenon. (author)

  3. The effect of chronic alcohol consumption on mitochondrial DNA mutagenesis in human blood

    International Nuclear Information System (INIS)

    Wurmb-Schwark, N. von; Ringleb, A.; Schwark, T.; Broese, T.; Weirich, S.; Schlaefke, D.; Wegener, R.; Oehmichen, M.

    2008-01-01

    The 4977 bp deletion of mitochondrial DNA (mtDNA) is known to accumulate with increasing age in post mitotic tissues. Recently, studies came out detecting this specific alteration also in fast replicating cells, e.g. in blood or skin tissue, often in correlation to specific diseases or - specifically in skin - external stressors such as UV radiation. In this study, we investigated mitochondrial mutagenesis in 69 patients with a chronic alcoholic disease and 46 age matched controls with a moderate drinking behavior. Two different fragments, specific for total and for deleted mtDNA (dmtDNA) were amplified in a duplex-PCR. A subsequent fragment analysis was performed and for relative quantification, the quotient of the peak areas of amplification products specific for deleted and total mtDNA was determined. Additionally, a real time PCR was performed to quantify mtDNA copy number. The relative amount of 4977 bp deleted mtDNA in alcoholics was significantly increased compared to controls. On the other hand, no difference regarding the mtDNA/nuclear DNA ratio in both investigated groups was detected. Additionally, no age dependence could be found nor in alcoholics, neither in the control group. These findings indicate that mtDNA mutagenesis in blood can be influenced by stressors such as alcohol. Ethanol seems to be a significant factor to alter mitochondrial DNA in blood and might be an additional contributor for the cellular aging process

  4. Rapid Integration of Multi-copy Transgenes Using Optogenetic Mutagenesis in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Kentaro Noma

    2018-06-01

    Full Text Available Stably transmitted transgenes are indispensable for labeling cellular components and manipulating cellular functions. In Caenorhabditis elegans, transgenes are generally generated as inheritable multi-copy extrachromosomal arrays, which can be stabilized in the genome through a mutagenesis-mediated integration process. Standard methods to integrate extrachromosomal arrays primarily use protocols involving ultraviolet light plus trimethylpsoralen or gamma- or X-ray irradiation, which are laborious and time-consuming. Here, we describe a one-step integration method, following germline-mutagenesis induced by mini Singlet Oxygen Generator (miniSOG. Upon blue light treatment, miniSOG tagged to histone (Histone-miniSOG generates reactive oxygen species (ROS and induces heritable mutations, including DNA double-stranded breaks. We demonstrate that we can bypass the need to first establish extrachromosomal transgenic lines by coupling microinjection of desired plasmids with blue light illumination on Histone-miniSOG worms to obtain integrants in the F3 progeny. We consistently obtained more than one integrant from 12 injected animals in two weeks. This optogenetic approach significantly reduces the amount of time and labor for transgene integration. Moreover, it enables to generate stably expressed transgenes that cause toxicity in animal growth.

  5. DC-Analyzer-facilitated combinatorial strategy for rapid directed evolution of functional enzymes with multiple mutagenesis sites.

    Science.gov (United States)

    Wang, Xiong; Zheng, Kai; Zheng, Huayu; Nie, Hongli; Yang, Zujun; Tang, Lixia

    2014-12-20

    Iterative saturation mutagenesis (ISM) has been shown to be a powerful method for directed evolution. In this study, the approach was modified (termed M-ISM) by combining the single-site saturation mutagenesis method with a DC-Analyzer-facilitated combinatorial strategy, aiming to evolve novel biocatalysts efficiently in the case where multiple sites are targeted simultaneously. Initially, all target sites were explored individually by constructing single-site saturation mutagenesis libraries. Next, the top two to four variants in each library were selected and combined using the DC-Analyzer-facilitated combinatorial strategy. In addition to site-saturation mutagenesis, iterative saturation mutagenesis also needed to be performed. The advantages of M-ISM over ISM were that the screening effort is greatly reduced, and the entire M-ISM procedure was less time-consuming. The M-ISM strategy was successfully applied to the randomization of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) when five interesting sites were targeted simultaneously. After screening 900 clones in total, six positive mutants were obtained. These mutants exhibited 4.0- to 9.3-fold higher k(cat) values than did the wild-type HheC toward 1,3-dichloro-2-propanol. However, with the ISM strategy, the best hit showed a 5.9-fold higher k(cat) value toward 1,3-DCP than the wild-type HheC, which was obtained after screening 4000 clones from four rounds of mutagenesis. Therefore, M-ISM could serve as a simple and efficient version of ISM for the randomization of target genes with multiple positions of interest.

  6. piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART for genetic screens in mice.

    Directory of Open Access Journals (Sweden)

    Sean F Landrette

    Full Text Available Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.

  7. Use of a simian virus 40-based shuttle vector to analyze enhanced mutagenesis in mitomycin C-treated monkey cells

    International Nuclear Information System (INIS)

    Roilides, E.; Munson, P.J.; Levine, A.S.; Dixon, K.

    1988-01-01

    When monkey cells were treated with mitomycin C 24 h before transfection with UV-irradiated pZ189 (a simian virus 40-based shuttle vector), there was a twofold increase in the frequency of mutations in the supF gene of the vector. These results suggest the existence of an enhancible mutagenesis pathway in mammalian cells. However, DNA sequence analysis of the SupF- mutants suggested no dramatic changes in the mechanisms of mutagenesis due to mitomycin C treatment of the cells

  8. Ultra-high frequency ultrasound biomicroscopy and high throughput cardiovascular phenotyping in a large scale mouse mutagenesis screen

    Science.gov (United States)

    Liu, Xiaoqin; Francis, Richard; Tobita, Kimimasa; Kim, Andy; Leatherbury, Linda; Lo, Cecilia W.

    2013-02-01

    Ultrasound biomicroscopy (UBM) is ideally suited for phenotyping fetal mice for congenital heart disease (CHD), as imaging can be carried out noninvasively to provide both hemodynamic and structural information essential for CHD diagnosis. Using the UBM (Vevo 2100; 40Hz) in conjunction with the clinical ultrasound system (Acuson Sequioa C512; 15Hz), we developed a two-step screening protocol to scan thousands fetuses derived from ENU mutagenized pedigrees. A wide spectrum of CHD was detected by the UBM, which were subsequently confirmed with follow-up necropsy and histopathology examination with episcopic fluorescence image capture. CHD observed included outflow anomalies, left/right heart obstructive lesions, septal/valvular defects and cardiac situs anomalies. Meanwhile, various extracardiac defects were found, such as polydactyly, craniofacial defects, exencephaly, omphalocele-cleft palate, most of which were associated with cardiac defects. Our analyses showed the UBM was better at assessing cardiac structure and blood flow profiles, while conventional ultrasound allowed higher throughput low-resolution screening. Our study showed the integration of conventional clinical ultrasound imaging with the UBM for fetal mouse cardiovascular phenotyping can maximize the detection and recovery of CHD mutants.

  9. indCAPS: A tool for designing screening primers for CRISPR/Cas9 mutagenesis events.

    Science.gov (United States)

    Hodgens, Charles; Nimchuk, Zachary L; Kieber, Joseph J

    2017-01-01

    Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be readily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles. However, in the case of most CRISPR-induced alleles, no such restriction sites are present in the target sequences. In this case, a derived CAPS (dCAPS) approach can be used in which mismatches are purposefully introduced in the oligonucleotide primers to create a restriction site in one, but not both, of the amplified templates. Web-based tools exist to aid dCAPS primer design, but when supplied sequences that include indels, the current tools often fail to suggest appropriate primers. Here, we report the development of a Python-based, species-agnostic web tool, called indCAPS, suitable for the design of PCR primers used in dCAPS assays that is compatible with indels. This tool should have wide utility for screening editing events following CRISPR/Cas9 mutagenesis as well as for identifying specific editing events in a pool of CRISPR-mediated mutagenesis events. This tool was field-tested in a CRISPR mutagenesis experiment targeting a cytokinin receptor (AHK3) in Arabidopsis thaliana. The tool suggested primers that successfully distinguished between wild-type and edited alleles of a target locus and facilitated the isolation of two novel ahk3 null alleles. Users can access indCAPS and design PCR primers to employ dCAPS to identify CRISPR/Cas9 alleles at http://indcaps.kieber.cloudapps.unc.edu/.

  10. Characterization of highly efficient heavy-ion mutagenesis in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Kazama Yusuke

    2011-11-01

    Full Text Available Abstract Background Heavy-ion mutagenesis is recognised as a powerful technology to generate new mutants, especially in higher plants. Heavy-ion beams show high linear energy transfer (LET and thus more effectively induce DNA double-strand breaks than other mutagenic techniques. Previously, we determined the most effective heavy-ion LET (LETmax: 30.0 keV μm-1 for Arabidopsis mutagenesis by analysing the effect of LET on mutation induction. However, the molecular structure of mutated DNA induced by heavy ions with LETmax remains unclear. Knowledge of the structure of mutated DNA will contribute to the effective exploitation of heavy-ion beam mutagenesis. Results Dry Arabidopsis thaliana seeds were irradiated with carbon (C ions with LETmax at a dose of 400 Gy and with LET of 22.5 keV μm-1 at doses of 250 Gy or 450 Gy. The effects on mutation frequency and alteration of DNA structure were compared. To characterise the structure of mutated DNA, we screened the well-characterised mutants elongated hypocotyls (hy and glabrous (gl and identified mutated DNA among the resulting mutants by high-resolution melting curve, PCR and sequencing analyses. The mutation frequency induced by C ions with LETmax was two-fold higher than that with 22.5 keV μm-1 and similar to the mutation frequency previously induced by ethyl methane sulfonate. We identified the structure of 22 mutated DNAs. Over 80% of the mutations caused by C ions with both LETs were base substitutions or deletions/insertions of less than 100 bp. The other mutations involved large rearrangements. Conclusions The C ions with LETmax showed high mutation efficiency and predominantly induced base substitutions or small deletions/insertions, most of which were null mutations. These small alterations can be determined by single-nucleotide polymorphism (SNP detection systems. Therefore, C ions with LETmax might be useful as a highly efficient reverse genetic system in conjunction with SNP detection

  11. Tailor-Made Protein Tyrosine Phosphatases: In Vitro Site-Directed Mutagenesis of PTEN and PTPRZ-B

    NARCIS (Netherlands)

    Luna, S.; Mingo, J.; Aurtenetxe, O.; Blanco, L.; Amo, L.; Schepens, J.; Hendriks, W.J.A.J.; Pulido, R.

    2016-01-01

    In vitro site-directed mutagenesis (SDM) of protein tyrosine phosphatases (PTPs) is a commonly used approach to experimentally analyze PTP functions at the molecular and cellular level and to establish functional correlations with PTP alterations found in human disease. Here, using the

  12. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

    Directory of Open Access Journals (Sweden)

    Weir Jerry P

    2007-05-01

    Full Text Available Abstract Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2 BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors.

  13. USP7 Is a Suppressor of PCNA Ubiquitination and Oxidative-Stress-Induced Mutagenesis in Human Cells.

    Science.gov (United States)

    Kashiwaba, Shu-ichiro; Kanao, Rie; Masuda, Yuji; Kusumoto-Matsuo, Rika; Hanaoka, Fumio; Masutani, Chikahide

    2015-12-15

    Mono-ubiquitinated PCNA activates error-prone DNA polymerases; therefore, strict regulation of PCNA mono-ubiquitination is crucial in avoiding undesired mutagenesis. In this study, we used an in vitro assay system to identify USP7 as a deubiquitinating enzyme of mono-ubiquitinated PCNA. Suppression of USP1, a previously identified PCNA deubiquitinase, or USP7 increased UV- and H2O2-induced PCNA mono-ubiquitination in a distinct and additive manner, suggesting that USP1 and USP7 make different contributions to PCNA deubiquitination in human cells. Cell-cycle-synchronization analyses revealed that USP7 suppression increased H2O2-induced PCNA ubiquitination throughout interphase, whereas USP1 suppression specifically increased ubiquitination in S-phase cells. UV-induced mutagenesis was elevated in USP1-suppressed cells, whereas H2O2-induced mutagenesis was elevated in USP7-suppressed cells. These results suggest that USP1 suppresses UV-induced mutations produced in a manner involving DNA replication, whereas USP7 suppresses H2O2-induced mutagenesis involving cell-cycle-independent processes such as DNA repair. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Rational development of an attenuated recombinant cyprinid herpesvirus 3 vaccine using prokaryotic mutagenesis and in vivo bioluminescent imaging

    Science.gov (United States)

    Cyprinid herpesvirus 3 (CyHV-3) is causing severe economic losses worldwide in the carp industry, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open...

  15. β-lactam antibiotics promote bacterial mutagenesis via an RpoS-mediated reduction in replication fidelity

    Science.gov (United States)

    Gutierrez, A.; Laureti, L.; Crussard, S.; Abida, H.; Rodríguez-Rojas, A.; Blázquez, J.; Baharoglu, Z.; Mazel, D.; Darfeuille, F.; Vogel, J.; Matic, I.

    2013-01-01

    Regardless of their targets and modes of action, subinhibitory concentrations of antibiotics can have an impact on cell physiology and trigger a large variety of cellular responses in different bacterial species. Subinhibitory concentrations of β-lactam antibiotics cause reactive oxygen species production and induce PolIV-dependent mutagenesis in Escherichia coli. Here we show that subinhibitory concentrations of β-lactam antibiotics induce the RpoS regulon. RpoS-regulon induction is required for PolIV-dependent mutagenesis because it diminishes the control of DNA-replication fidelity by depleting MutS in E. coli, Vibrio cholerae and Pseudomonas aeruginosa. We also show that in E. coli, the reduction in mismatch-repair activity is mediated by SdsR, the RpoS-controlled small RNA. In summary, we show that mutagenesis induced by subinhibitory concentrations of antibiotics is a genetically controlled process. Because this mutagenesis can generate mutations conferring antibiotic resistance, it should be taken into consideration for the development of more efficient antimicrobial therapeutic strategies. PMID:23511474

  16. RecA-mediated cleavage activates UmuD for mutagenesis: Mechanistic relationship between transcriptional derepression and posttranslational activation

    International Nuclear Information System (INIS)

    Nohmi, Takehiko; Battista, J.R.; Dodson, L.A.; Walker, G.C.

    1988-01-01

    The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. It has been shown that the UmuD protein shares homology with LexA, the repressor of the SOS genes. In this paper the authors describe a series of genetic experiments that indicate that the purpose of RecA-mediated cleavage of UmuD at its bond between Cys-24 and Gly-25 is to activate UmuD for its role in mutagenesis and that the COOH-terminal fragment of UmuD is necessary and sufficient for the role of UmuD in UV mutagenesis. Other genetic experiments are presented that (i) support the hypothesis that the primary role of Ser-60 in UmuD function is to act as a nucleophile in the RecA-mediated cleavage reaction and (ii) raise the possibility that RecA has a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD

  17. Predictive mutagenesis of ligation-independent cloning (LIC) vectors for protein expression and site-specific chemical conjugation

    DEFF Research Database (Denmark)

    Vernet, Erik; Sauer, Jørgen; Andersen, Peter Andreas

    2011-01-01

    Ligation-independent cloning (LIC) allows for cloning of DNA constructs independent of insert restriction sites and ligases. However, any required mutations are typically introduced by additional, time-consuming steps. We present a rapid, inexpensive method for mutagenesis in the 5' LIC site...

  18. Steric hindrance mutagenesis in the conserved extracellular vestibule impedes allosteric binding of antidepressants to the serotonin transporter

    DEFF Research Database (Denmark)

    Plenge, Per; Shi, Lei; Beuming, Thijs

    2012-01-01

    be involved in the allosteric binding in the extracellular vestibule located above the central substrate binding (S1) site. Indeed, mutagenesis of selected residues in the vestibule reduces the allosteric potency of (S)-citalopram and clomipramine. The identified site is further supported by the inhibitory...

  19. From Green to Blue: Site-Directed Mutagenesis of the Green Fluorescent Protein to Teach Protein Structure-Function Relationships

    Science.gov (United States)

    Giron, Maria D.; Salto, Rafael

    2011-01-01

    Structure-function relationship studies in proteins are essential in modern Cell Biology. Laboratory exercises that allow students to familiarize themselves with basic mutagenesis techniques are essential in all Genetic Engineering courses to teach the relevance of protein structure. We have implemented a laboratory course based on the…

  20. Biological effects of DNA repair, including mutagenesis. Progress report, August 15, 1982-August 1, 1983. COO-3571-23

    International Nuclear Information System (INIS)

    Hutchinson, F.

    1983-01-01

    The research supported by this contract for the period covered by this report has concerned mechanisms in mutagenesis. Specifically, the work has been aimed at determining the lesions in DNA formed by particular mutagenic agents which lead to mutations, and to characterization of the pathways by which these lesions lead to changes in the sequence of bases in the genomic DNA

  1. Characterization of the functional epitope on the urokinase receptor. Complete alanine scanning mutagenesis supplemented by chemical cross-linking

    DEFF Research Database (Denmark)

    Gårdsvoll, Henrik; Gilquin, Bernard; Le Du, Marie Hélène

    2006-01-01

    a comprehensive alanine scanning mutagenesis of uPAR combined with low resolution distance constraints defined within the complex using chemical cross-linkers as molecular rulers. The kinetic rate constants for the interaction between pro-uPA and 244 purified uPAR mutants with single-site replacements were...

  2. Mutation of C/EBPalpha predisposes to the development of myeloid leukemia in a retroviral insertional mutagenesis screen

    DEFF Research Database (Denmark)

    Hasemann, Marie S; Damgaard, Inge; Schuster, Mikkel B

    2008-01-01

    and incomplete penetrance, suggesting that accumulation of secondary mutations is necessary for disease progression. Here, we use SRS19-6-driven retroviral insertional mutagenesis to compare the phenotypes of leukemias arising in Cebpa(+/+), Cebpa(+/BRM2), and Cebpa(BRM2/BRM2) mice, with respect to disease type...

  3. [Comparative mutagenesis of human cells in vivo and in vitro]. Progress report, January 1-December 30, 1985

    International Nuclear Information System (INIS)

    1985-01-01

    Annual progress report is made on project focusing on the comparative mutagenesis of human cells in vivo and in vitro. The study employs the HGPRT gene to explore the changes in nucleotide sequence which has occurred in spontaneous mutations or mutations induced by MNNG or ICR191. Reports on the individual projects have been abstracted and indexed for the Energy Data Base. (DT)

  4. Species-scanning mutagenesis of the serotonin transporter reveals residues essential in selective, high-affinity recognition of antidepressants

    DEFF Research Database (Denmark)

    Mortensen, O.V.; Wiborg, O.; Kristensen, A.S.

    2001-01-01

    )tropane, or for 3,4-methylenedioxymethamphetamine (MDMA). Analysis of six hSERT/bSERT chimeras and subsequent species-scanning mutagenesis of each isoform revealed methionine-180, tyrosine-495, and phenylalanine-513 to be responsible for the increase in citalopram and paroxetine potencies at hSERT and methionine...

  5. Codon cassette mutagenesis: a general method to insert or replace individual codons by using universal mutagenic cassettes.

    Science.gov (United States)

    Kegler-Ebo, D M; Docktor, C M; DiMaio, D

    1994-05-11

    We describe codon cassette mutagenesis, a simple method of mutagenesis that uses universal mutagenic cassettes to deposit single codons at specific sites in double-stranded DNA. A target molecule is first constructed that contains a blunt, double-strand break at the site targeted for mutagenesis. A double-stranded mutagenic codon cassette is then inserted at the target site. Each mutagenic codon cassette contains a three base pair direct terminal repeat and two head-to-head recognition sequences for the restriction endonuclease Sapl, an enzyme that cleaves outside of its recognition sequence. The intermediate molecule containing the mutagenic cassette is then digested with Sapl, thereby removing most of the mutagenic cassette, leaving only a three base cohesive overhang that is ligated to generate the final insertion or substitution mutation. A general method for constructing blunt-end target molecules suitable for this approach is also described. Because the mutagenic cassette is excised during this procedure and alters the target only by introducing the desired mutation, the same cassette can be used to introduce a particular codon at all target sites. Each cassette can deposit two different codons, depending on the orientation in which it is inserted into the target molecule. Therefore, a series of eleven cassettes is sufficient to insert all possible amino acids at any constructed target site. Thus codon cassettes are 'off-the-shelf' reagents, and this methodology should be a particularly useful and inexpensive approach for subjecting multiple different positions in a protein sequence to saturation mutagenesis.

  6. One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections

    NARCIS (Netherlands)

    Mingo, J.; Erramuzpe, A.; Luna, S.; Aurtenetxe, O.; Amo, L.; Diez, I.; Schepens, J.T.G.; Hendriks, W.J.A.J.; Cortes, J.M.; Pulido, R.

    2016-01-01

    Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates

  7. R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata

    Energy Technology Data Exchange (ETDEWEB)

    Youvan, D C [Univ. of California, Berkeley; Elder, J T; Sandlin, D E; Zsebo, K; Alder, D P; Panopoulos, N J; Marrs, B L; Hearst, J E

    1982-01-01

    Site-directed mutagenesis of the photosynthetic apparatus (PSA) genes in Rhodopseudomonas capsulata is presented utilizing a transposon Tn7 mutagenized R-prime. The R-prime, pRPS404, bears most of the genes necessary for the differentiation of the photosynthetic apparatus. Mutagenesis of the R-prime with Tn7 in Escherichia coli, conjugation into R. capsulata, and homologous recombination with the wild-type alleles efficiently generates photosynthetic apparatus lesions. Wild-type alleles are lost spontaneously and the Tn7-induced lesions are revealed by subsequent intramolecular recombination between IS21 insertion elements that bracket the prime sequences in direct repeat. The molecular nature of the intermediates involved in the transposition, recombination and deletion have been investigated by Southern hybridization analysis. The spontaneous loss of wild-type alleles after homologous recombination with the chromosome may be of general use to other prokaryotic site-directed transposon mutagenesis schemes. The IS21-mediated deletion of the prime DNA is dependent on the RecA protein in E. coli, generating the parental R-factor bearing one IS21 element. A genetic-physical map exists for a portion of the prime photosynthetic apparatus DNA. When Tn7 is inserted into a bacteriochlorophyll gene in the R-prime and then crossed into R. capsulata, mutants are produced that accumulate a bacteriochlorophyll precursor, which is in excellent agreement with the existing genetic-physical map. This corroborates the mutagenesis scheme.

  8. Effect of radiation-sensitive mutations and mutagens/carcinogens on bacterial recombination and mutagenesis. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Matney, T.S.

    1978-01-01

    Progress is reported on effects of temperature sensitive DNA-initiation mutation in E. coli K-12 mutants; the use of Bacillus subtilis transforming system as an in vitro mutagenesis system; characteristics of the E. coli lysogen used to test the permeability to polycyclic aromatic hydrocarbons; and the genetic toxicology of gentian violet. (PCS)

  9. Inducible pathway is required for mutagenesis in Salmonella typhimurium LT2

    International Nuclear Information System (INIS)

    Orrego, C.; Eisenstadt, E.

    1987-01-01

    UV mutability of Salmonella typhimurium LT2 was eliminated in the presence of a multicopy plasmid carrying the Escherichia coli lexA + gene. This result suggests that inducible, SOS-like functions are required for UV mutagenesis in S. typhimurium. S. typhimurium strains carrying either point or deletion mutations in topA had previously been shown to lose their mutability by UV or methyl methanesulfonate. Mitomycin C induction of the Phi(mucB'-lacZ') fusion (a DNA damage-inducible locus carried on plasmid pSE205) in S. typhimurium topA was normal, suggesting that RecA is activated in topA mutants. These observations lead the authors deduce that S. typhimurium has at least one DNA damage-inducible locus in addition to recA that is required for UV mutability

  10. In vivo elimination of parental clones in general and site-directed mutagenesis.

    Science.gov (United States)

    Holland, Erika G; Acca, Felicity E; Belanger, Kristina M; Bylo, Mary E; Kay, Brian K; Weiner, Michael P; Kiss, Margaret M

    2015-02-01

    The Eco29k I restriction endonuclease is a Sac II isoschizomer that recognizes the sequence 5'-CCGCGG-3' and is encoded, along with the Eco29k I methylase, in the Escherichia coli strain 29k. We have expressed the Eco29k I restriction-methylation system (RM2) in E. coli strain TG1 to produce the strain AXE688. We have developed a directed molecular evolution (DME) mutagenesis method that uses Eco29k I to restrict incoming parental DNA in transformed cells. Using our DME method, we have demonstrated that our AXE688 strain results in mutated directed molecular evolution libraries with diversity greater than 10(7) from a single transformation and with greater than 90% recombinant clones. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Evaluating Risks of Insertional Mutagenesis by DNA Transposons in Gene Therapy

    Science.gov (United States)

    Hackett, Perry B.; Largaespada, David A.; Switzer, Kirsten C.; Cooper, Laurence J.N.

    2013-01-01

    Investigational therapy can be successfully undertaken using viral- and non-viral-mediated ex vivo gene transfer. Indeed, recent clinical trials have established the potential for genetically modified T cells to improve and restore health. Recently the Sleeping Beauty (SB) transposon/transposase system has been applied in clinical trials to stably insert a chimeric antigen receptor (CAR) to redirect T-cell specificity. We discuss the context in which the SB system can be harnessed for gene therapy and describe the human application of SB-modified CAR+ T cells. We have focused on theoretical issues relating to insertional mutagenesis in the context of human genomes that are naturally subjected to remobilization of transposons and the experimental evidence over the last decade of employing SB transposons for defining genes that induce cancer. These findings are put into the context of the use of SB transposons in the treatment of human disease. PMID:23313630

  12. The Glyphosate-Based Herbicide Roundup Does not Elevate Genome-Wide Mutagenesis of Escherichia coli.

    Science.gov (United States)

    Tincher, Clayton; Long, Hongan; Behringer, Megan; Walker, Noah; Lynch, Michael

    2017-10-05

    Mutations induced by pollutants may promote pathogen evolution, for example by accelerating mutations conferring antibiotic resistance. Generally, evaluating the genome-wide mutagenic effects of long-term sublethal pollutant exposure at single-nucleotide resolution is extremely difficult. To overcome this technical barrier, we use the mutation accumulation/whole-genome sequencing (MA/WGS) method as a mutagenicity test, to quantitatively evaluate genome-wide mutagenesis of Escherichia coli after long-term exposure to a wide gradient of the glyphosate-based herbicide (GBH) Roundup Concentrate Plus. The genome-wide mutation rate decreases as GBH concentration increases, suggesting that even long-term GBH exposure does not compromise the genome stability of bacteria. Copyright © 2017 Tincher et al.

  13. Accurate RNA consensus sequencing for high-fidelity detection of transcriptional mutagenesis-induced epimutations.

    Science.gov (United States)

    Reid-Bayliss, Kate S; Loeb, Lawrence A

    2017-08-29

    Transcriptional mutagenesis (TM) due to misincorporation during RNA transcription can result in mutant RNAs, or epimutations, that generate proteins with altered properties. TM has long been hypothesized to play a role in aging, cancer, and viral and bacterial evolution. However, inadequate methodologies have limited progress in elucidating a causal association. We present a high-throughput, highly accurate RNA sequencing method to measure epimutations with single-molecule sensitivity. Accurate RNA consensus sequencing (ARC-seq) uniquely combines RNA barcoding and generation of multiple cDNA copies per RNA molecule to eliminate errors introduced during cDNA synthesis, PCR, and sequencing. The stringency of ARC-seq can be scaled to accommodate the quality of input RNAs. We apply ARC-seq to directly assess transcriptome-wide epimutations resulting from RNA polymerase mutants and oxidative stress.

  14. U.v.-induced and N-methyl-N'-nitrosoguanidine-induced mutagenesis in Bacillus thuringiensis

    International Nuclear Information System (INIS)

    Auffray, Y.; Boutibonnes, P.

    1987-01-01

    The lethal and mutagenic effects of u.v. light and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on Bacillus thuringiensis were investigated. Lethality studies demonstrated that B. thuringiensis was relatively sensitive to these agents. This bacterium was mutated at the rifampicin resistance marker by u.v. light and to a lesser extent by the direct acting alkylating agent MNNG. One mutant selected for its greater sensitivity to u.v. light expressed a higher frequency of mutagenesis after u.v. light treatment and appeared to be defective in an excision repair pathway. However, this mutant was only slightly mutable by MNNG in comparison with the wild-type strain. This unusual phenotype does not yet have a parallel among the radiation sensitive mutants described in other bacterial species. (author)

  15. Genetic recombination as a major cause of mutagenesis in the human globin gene clusters.

    Science.gov (United States)

    Borg, Joseph; Georgitsi, Marianthi; Aleporou-Marinou, Vassiliki; Kollia, Panagoula; Patrinos, George P

    2009-12-01

    Homologous recombination is a frequent phenomenon in multigene families and as such it occurs several times in both the alpha- and beta-like globin gene families. In numerous occasions, genetic recombination has been previously implicated as a major mechanism that drives mutagenesis in the human globin gene clusters, either in the form of unequal crossover or gene conversion. Unequal crossover results in the increase or decrease of the human globin gene copies, accompanied in the majority of cases with minor phenotypic consequences, while gene conversion contributes either to maintaining sequence homogeneity or generating sequence diversity. The role of genetic recombination, particularly gene conversion in the evolution of the human globin gene families has been discussed elsewhere. Here, we summarize our current knowledge and review existing experimental evidence outlining the role of genetic recombination in the mutagenic process in the human globin gene families.

  16. Degeneration and domestication of a selfish gene in yeast: molecular evolution versus site-directed mutagenesis.

    Science.gov (United States)

    Koufopanou, Vassiliki; Burt, Austin

    2005-07-01

    VDE is a homing endonuclease gene in yeasts with an unusual evolutionary history including horizontal transmission, degeneration, and domestication into the mating-type switching locus HO. We investigate here the effects of these features on its molecular evolution. In addition, we correlate rates of evolution with results from site-directed mutagenesis studies. Functional elements have lower rates of evolution than degenerate ones and higher conservation at functionally important sites. However, functionally important and unimportant sites are equally likely to have been involved in the evolution of new function during the domestication of VDE into HO. The domestication event also indicates that VDE has been lost in some species and that VDE has been present in yeasts for more than 50 Myr.

  17. Mutagenesis Strategies for the Enhancement of Glucose Oxidase Production from Corn Steep Liquor

    International Nuclear Information System (INIS)

    Khalaf, M.A.

    2007-01-01

    During screening of the twenty eight Aspergillus and Penicillium species for GOD production, only A. niger (84) was observed to release high extra (3.15 U ml -1 ) and intracellular (9.30 U mg -l ) GOD activity. Conidia of A. niger S4 were subjected to mutagenesis with UV radiation, nitrous acid, and sodium azide, as a single or combined treatments, and GOD activity was detected with the diffusion plate method. Out of 27 over producing mutants tested in shaken flasks, UVNA54 mutant strain showed the highest level of GOD activity (171 %, higher than the wild type). Using CSL at concentration 25 ml rl as the sol nutrient source, the enzyme activity was increased to 5.16 U ml -1 and 22.40 U mg -1 for extra and intracellular, respectively. Viable cell numbers of Pseudomonas and Salmonella spp. decreased as the concentration of the produced enzyme increased from 1 to 3 U ml -1

  18. Ultraviolet mutagenesis studies of [psi], a cytoplasmic determinant of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Tuite, M.F.; Cox, B.S.

    1980-01-01

    uv mutagenesis was used to probe the molecular nature of [psi], a nonmitochondrial cytoplasmic determinant of Saccharomyces cerevisiae involved in the control of nonsense suppression. The uv-induced mutation from [psi + ] to [psi - ] showed characteristics of forward nuclear gene mutation in terms of frequency, induction kinetics, occurrence of whole and sectored mutant clones and the effect of the stage in the growth cycle on mutation frequency. The involvement of pyrimidine dimers in the premutational lesion giving the [psi - ] mutation was demonstrated by photoreactivation. uv-induced damage to the [psi] genetic determinant was shown to be repaired by nuclear-coded repair enzymes that are responsible for the repair of nuclear DNA damage. uv-induced damage to mitochondrial DNA appeared to be, at least partly, under the control of different repair processes. The evidence obtained suggests that the [psi] determinant is DNA

  19. Improved inhibitor tolerance in xylose-fermenting yeast Spathaspora passalidarum by mutagenesis and protoplast fusion

    DEFF Research Database (Denmark)

    Hou, Xiaoru; Yao, Shuo

    2012-01-01

    The xylose-fermenting yeast Spathaspora passalidarum showed excellent fermentation performance utilizing glucose and xylose under anaerobic conditions. But this yeast is highly sensitive to the inhibitors such as furfural present in the pretreated lignocellulosic biomass. In order to improve...... from fusion of the protoplasts of S. passalidarum M7 and a robust yeast, Saccharomyces cerevisiae ATCC 96581, were able to grow in 75% WSLQ and produce around 0.4 g ethanol/g consumed xylose. Among the selected hybrid strains, the hybrid FS22 showed the best fermentation capacity in 75% WSLQ...... the inhibitor tolerance of this yeast, a combination of UV mutagenesis and protoplast fusion was used to construct strains with improved performance. Firstly, UVinduced mutants were screened and selected for improved tolerance towards furfural. The most promised mutant, S. passalidarum M7, produced 50% more...

  20. Identification of NH4+-regulated genes of Herbaspirillum seropedicae by random insertional mutagenesis.

    Science.gov (United States)

    Schwab, Stefan; Ramos, Humberto J; Souza, Emanuel M; Pedrosa, Fábio O; Yates, Marshall G; Chubatsu, Leda S; Rigo, Liu U

    2007-05-01

    Random mutagenesis using transposons with promoterless reporter genes has been widely used to examine differential gene expression patterns in bacteria. Using this approach, we have identified 26 genes of the endophytic nitrogen-fixing bacterium Herbaspirillum seropedicae regulated in response to ammonium content in the growth medium. These include nine genes involved in the transport of nitrogen compounds, such as the high-affinity ammonium transporter AmtB, and uptake systems for alternative nitrogen sources; nine genes coding for proteins responsible for restoring intracellular ammonium levels through enzymatic reactions, such as nitrogenase, amidase, and arginase; and a third group includes metabolic switch genes, coding for sensor kinases or transcription regulation factors, whose role in metabolism was previously unknown. Also, four genes identified were of unknown function. This paper describes their involvement in response to ammonium limitation. The results provide a preliminary profile of the metabolic response of Herbaspirillum seropedicae to ammonium stress.

  1. Mutagenesis for ACMV resistance in a Ghanian cassava cultivar 'Bosom nsia'

    International Nuclear Information System (INIS)

    Ahiabu, R.K.; Lokko, Y.; Danso, K.; Klu, G.Y.P.

    1997-01-01

    Breeding for resistance to the African Cassava Mosaic Virus (ACMV) disease in the Ghanian cassava cultivar 'Bosom nsia' has been on-going for the past four years at the Biotechnology and Nuclear Agricultural Research Institute. Protocols for in vitro culture from shoot meristems and acclimation of plantlets were established. Radiosensitivity tests on the regenerated plantlets indicated LD 50 of 40 Gy, and doses of 25, 30 and 35 Gy were suitable for mutagenesis. These doses were applied to in vivo and in vitro grown plants, and selection was carried out in three propagations. Four variants, selected under field conditions with high viral incidence, were analyzed for virus particles with three virus indexing techniques. Polymerase chain reaction (PCR) product analysis of DNA extracts from Nicotiana benthamiana test plants, inoculated with sap from the leaves of variants, confirmed the presence of virus particles in all variants. Inoculation and ELISA tests suggested ACMV tolerance in selected variants. (author). 24 refs, 3 figs, 1 tab

  2. Laser mutagenesis and producing cellulase condition optimization of Trichoderma virid protoplast

    International Nuclear Information System (INIS)

    Chen Shuli; Zhang Qin; Han Jingjing; Lv Jiangtao; Wang Shilong; Yao Side

    2009-01-01

    The protoplast of Trichoderma virid CICC13038 was mutated using Nd:YAG laser of 266 nm light. And a high-cellulase producing strain JG13 was bred by screening with cellulose microcrystalline. Under the condition of 28 degree C, 180 rpm and 72 h of fermentation time, optimal conditions for the celluase ferment by orthogonal experiment were: 2% bran as the carbon source, 1% (NH 4 ) 2 SO 4 as the nitrogen source, 0.5% Tween-80 as a enzyme-promoting agent,and 25 mL of medium volume in a 250 mL bottle. The cellulase activity of the mutant reached 35.68 U/mL, 25.76% higher than that of the original strain under the same conditions. The mutant JG13 has a great potential in industrial production. And it also can be used as the original strain for further mutagenesis to get the strain of higher cellulase activity. (authors)

  3. Factors influencing transformation and mutagenesis in vitro by high LET radiation

    International Nuclear Information System (INIS)

    Little, J.B.

    1986-01-01

    We have shown that 125 Iododeoxyuridine ( 125 IdUrd) and 3 H-thymidine are more effective than x-rays for the induction of specific gene mutations in TK6 human lymphoblastoid cells. The results of parallel transformation experiments with tritiated water suggest that the enhanced efficiency of 3 H-thymidine as compared with x-rays is due to a higher RBE of the tritium beta particle for mutagenesis and transformation, rather than to the fact that the radioactive decay occurs within the DNA molecule. Studies of the cell cycle specificity for the induction of cell killing and mutagenesis by 125 IdUrd indicate that late S-phase cells are more radiosensitive than early S-phase cells to the localized deposition of energy characteristic of 125 I decay. Cellular localization studies have shown that 125 I decay must occur in close proximity to cellular DNA in order to produce mutations; 125 IdUrd was very mutagenic in cells which incorporated it into DNA, but not in cells in which it remained in the acid soluble pool. A similar result emerged from preliminary experiments with 131 I. These results suggest that effects result largely from a transmutation/fragmentation. Continuous, low dose-rate exposure to fast neutrons (total doses of 1 to 40 rads protracted over periods of 5 to 20 days) was more mutagenic than acute neutron irradiation. No cytotoxicity was observed in cultures continuously irradiated with up to 2 rads/day for a 20 day period (total dose of 40 rads), whereas significant cell killing occurred with acute neutron exposures of 4.6 rads or greater. 11 refs., 6 figs., 1 tab

  4. Specificity determinants for autoproteolysis of LexA, a key regulator of bacterial SOS mutagenesis.

    Science.gov (United States)

    Mo, Charlie Y; Birdwell, L Dillon; Kohli, Rahul M

    2014-05-20

    Bacteria utilize the tightly regulated stress response (SOS) pathway to respond to a variety of genotoxic agents, including antimicrobials. Activation of the SOS response is regulated by a key repressor-protease, LexA, which undergoes autoproteolysis in the setting of stress, resulting in derepression of SOS genes. Remarkably, genetic inactivation of LexA's self-cleavage activity significantly decreases acquired antibiotic resistance in infection models and renders bacteria hypersensitive to traditional antibiotics, suggesting that a mechanistic study of LexA could help inform its viability as a novel target for combating acquired drug resistance. Despite structural insights into LexA, a detailed knowledge of the enzyme's protease specificity is lacking. Here, we employ saturation and positional scanning mutagenesis on LexA's internal cleavage region to analyze >140 mutants and generate a comprehensive specificity profile of LexA from the human pathogen Pseudomonas aeruginosa (LexAPa). We find that the LexAPa active site possesses a unique mode of substrate recognition. Positions P1-P3 prefer small hydrophobic residues that suggest specific contacts with the active site, while positions P5 and P1' show a preference for flexible glycine residues that may facilitate the conformational change that permits autoproteolysis. We further show that stabilizing the β-turn within the cleavage region enhances LexA autoproteolytic activity. Finally, we identify permissive positions flanking the scissile bond (P4 and P2') that are tolerant to extensive mutagenesis. Our studies shed light on the active site architecture of the LexA autoprotease and provide insights that may inform the design of probes of the SOS pathway.

  5. The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation)

    Science.gov (United States)

    Maisnier-Patin, Sophie; Roth, John R.

    2015-01-01

    Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac+) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F′lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F′lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac+ triggers exponential cell growth leading to a stable Lac+ revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac+ colony. Cells with multiple copies of the F′lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac+ revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns–Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions. PMID:26134316

  6. Sleeping Beauty transposon mutagenesis identifies genes that cooperate with mutant Smad4 in gastric cancer development.

    Science.gov (United States)

    Takeda, Haruna; Rust, Alistair G; Ward, Jerrold M; Yew, Christopher Chin Kuan; Jenkins, Nancy A; Copeland, Neal G

    2016-04-05

    Mutations in SMAD4 predispose to the development of gastrointestinal cancer, which is the third leading cause of cancer-related deaths. To identify genes driving gastric cancer (GC) development, we performed a Sleeping Beauty (SB) transposon mutagenesis screen in the stomach of Smad4(+/-) mutant mice. This screen identified 59 candidate GC trunk drivers and a much larger number of candidate GC progression genes. Strikingly, 22 SB-identified trunk drivers are known or candidate cancer genes, whereas four SB-identified trunk drivers, including PTEN, SMAD4, RNF43, and NF1, are known human GC trunk drivers. Similar to human GC, pathway analyses identified WNT, TGF-β, and PI3K-PTEN signaling, ubiquitin-mediated proteolysis, adherens junctions, and RNA degradation in addition to genes involved in chromatin modification and organization as highly deregulated pathways in GC. Comparative oncogenomic filtering of the complete list of SB-identified genes showed that they are highly enriched for genes mutated in human GC and identified many candidate human GC genes. Finally, by comparing our complete list of SB-identified genes against the list of mutated genes identified in five large-scale human GC sequencing studies, we identified LDL receptor-related protein 1B (LRP1B) as a previously unidentified human candidate GC tumor suppressor gene. In LRP1B, 129 mutations were found in 462 human GC samples sequenced, and LRP1B is one of the top 10 most deleted genes identified in a panel of 3,312 human cancers. SB mutagenesis has, thus, helped to catalog the cooperative molecular mechanisms driving SMAD4-induced GC growth and discover genes with potential clinical importance in human GC.

  7. Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice.

    Science.gov (United States)

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2015-10-01

    Frequency of CRISPR/Cas9-mediated targeted mutagenesis varies depending on Cas9 expression level and culture period of rice callus. Recent reports have demonstrated that the CRISPR/Cas9 system can function as a sequence-specific nuclease in various plant species. Induction of mutation in proliferating tissue during embryogenesis or in germline cells is a practical means of generating heritable mutations. In the case of plant species in which cultured cells are used for transformation, non-chimeric plants can be obtained when regeneration occurs from mutated cells. Since plantlets are regenerated from both mutated and non-mutated cells in a random manner, any increment in the proportion of mutated cells in Cas9- and guide RNA (gRNA)-expressing cells will help increase the number of plants containing heritable mutations. In this study, we examined factors affecting mutation frequency in rice calli. Following sequential transformation of rice calli with Cas9- and gRNA- expression constructs, the mutation frequency in independent Cas9 transgenic lines was analyzed. A positive correlation between Cas9 expression level and mutation frequency was found. This positive relationship was observed regardless of whether the transgene or an endogenous gene was used as the target for CRISPR/Cas9-mediated mutagenesis. Furthermore, we found that extending the culture period increased the proportion of mutated cells as well as the variety of mutations obtained. Because mutated and non-mutated cells might proliferate equally, these results suggest that a prolonged tissue culture period increases the chance of inducing de novo mutations in non-mutated cells. This fundamental knowledge will help improve systems for obtaining non-chimeric regenerated plants in many plant species.

  8. Somatic mutagenesis with a Sleeping Beauty transposon system leads to solid tumor formation in zebrafish.

    Directory of Open Access Journals (Sweden)

    Maura McGrail

    2011-04-01

    Full Text Available Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700-6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers.

  9. Mutagenesis in sequence encoding of human factor VII for gene therapy of hemophilia

    Directory of Open Access Journals (Sweden)

    B Kazemi

    2009-12-01

    Full Text Available "nBackground: Current treatment of hemophilia which is one of the most common bleeding disorders, involves replacement therapy using concentrates of FVIII and FIX .However, these concentrates have been associated with viral infections and thromboembolic complications and development of antibodies. "nThe use of recombinant human factor VII (rhFVII is effective  for the treatment of patients with  hemophilia A or B, who develop antibodies ( referred as inhibitors against  replacement therapy , because it induces coagulation independent of FVIII and FIX. However, its short half-life and high cost have limited its use. One potential solution to this problem may be the use of FVIIa gene transfer, which would attain continuing therapeutic levels of expression from a single injection. The aim of this study was to engineer a novel hFVII (human FVII gene containing a cleavage site for the intracellular protease and furin, by PCR mutagenesis "nMethods: The sequence encoding light and heavy chains of hFVII, were amplified by using hFVII/pTZ57R and specific primers, separately. The PCR products were cloned in pTZ57R vector. "nResults and discussion: Cloning was confirmed by restriction analysis or PCR amplification using specific primers and plasmid universal primers. Mutagenesis of sequence encoding light and heavy chain was confirmed by restriction enzyme. "nConclusion: In the present study, it was provided recombinant plasmids based on mutant form of DNA encoding light and heavy chains.  Joining mutant form of DNA encoding light chain with mutant heavy chain led to a new variant of hFVII. This variant can be activated by furin and an increase in the proportion of activated form of FVII. This mutant form of hFVII may be used for gene therapy of hemophilia.

  10. Nutritional value of quinua (chenopodium quinoa) seeds improved by radioinduced mutagenesis

    International Nuclear Information System (INIS)

    De La Cruz, T. E.; Avila R, S.; Garcia R, A.

    2006-01-01

    Quinua (Chenopodium quinoa), a pseudo cereal considered as an alternative crop for areas with marginal agricultural conditions has been subjected since 1990 to a radioinduced mutagenesis programme aiming to obtain lines with low saponin content, good yields and high nutritional value. Seeds obtained from lines grown in M7 generation which exhibited yields averaging 1.5 ton/ha, were analyzed regarding grain quality and nutritive value. Evaluated parameters were diameter and thickness of the seed, weight of 100 seeds and density. Regarding to bromatological analysis, determinations were made of moisture, ash, raw fiber, proteins, oil content and carbohydrates, following the procedures indicated in Official Mexican Norms (NOM). Evaluated genotypes were the varieties Sajama, Barandales and Amarilla de Marangani and the mutant lines 20R110, 94, 20R333, 20R227, 20R342, 20R37 and the advanced line obtained by selection 640304. Mutant genotypes 20R333 and 20R342 exhibited outstanding characteristics regarding to grain quality (diameter 2.0 mm, thickness 1.2mm, weight of 100 seeds 0.42 and 0.22 g respectively and density 710 and 686 grams per liter). In the bromatological analysis the protein content ranged from 11.82 % (genotype 20 R227) to 16.8% in mutant 20R333 while mutant 20R342 exhibited 15.6%. The lipid content was minimum on Barandales and 20R333 both with 3.8%, having the genotype 20R110 the highest value among evaluated genotypes with 4.35%, line 20R342 exhibited 4.2%.. The high percentages of proteins and lipids, found among some analyzed mutants exhibit the feasibility to obtain, through radioinduced mutagenesis, lines with low saponins and high nutritive value

  11. Predicting resistance by mutagenesis: lessons from 45 years of MBC resistance

    Directory of Open Access Journals (Sweden)

    Nichola J. Hawkins

    2016-11-01

    Full Text Available When a new fungicide class is introduced, it is useful to anticipate the resistance risk in advance, attempting to predict both risk level and potential mechanisms. One tool for the prediction of resistance risk is laboratory selection for resistance, with the mutational supply increased through UV or chemical mutagenesis. This enables resistance to emerge more rapidly than in the field, but may produce mutations that would not emerge under field conditions.The methyl-benzimidazole carbamates (MBCs were the first systemic single-site agricultural fungicides, and the first fungicides affected by rapid evolution of target-site resistance. MBC resistance has now been reported in over 90 plant pathogens in the field, and laboratory mutants have been studied in nearly 30 species.The most common field mutations, including β-tubulin E198A/K/G, F200Y and L240F, have all been identified in laboratory mutants. However, of 28 mutations identified in laboratory mutants, only nine have been reported in the field. Therefore, the predictive value of mutagenesis studies would be increased by understanding which mutations are likely to emerge in the field.Our review of the literature indicates that mutations with high resistance factors, and those found in multiple species, are more likely to be reported in the field. However, there are many exceptions, possibly due to fitness penalties. Whether a mutation occurred in the same species appears less relevant, perhaps because β-tubulin is highly conserved so functional constraints are similar across all species. Predictability of mutations in other target sites will depend on the level and conservation of constraints.

  12. BAX and tumor suppressor TRP53 are important in regulating mutagenesis in spermatogenic cells in mice.

    Science.gov (United States)

    Xu, Guogang; Vogel, Kristine S; McMahan, C Alex; Herbert, Damon C; Walter, Christi A

    2010-12-01

    During the first wave of spermatogenesis, and in response to ionizing radiation, elevated mutant frequencies are reduced to a low level by unidentified mechanisms. Apoptosis is occurring in the same time frame that the mutant frequency declines. We examined the role of apoptosis in regulating mutant frequency during spermatogenesis. Apoptosis and mutant frequencies were determined in spermatogenic cells obtained from Bax-null or Trp53-null mice. The results showed that spermatogenic lineage apoptosis was markedly decreased in Bax-null mice and was accompanied by a significantly increased spontaneous mutant frequency in seminiferous tubule cells compared to that of wild-type mice. Apoptosis profiles in the seminiferous tubules for Trp53-null were similar to control mice. Spontaneous mutant frequencies in pachytene spermatocytes and in round spermatids from Trp53-null mice were not significantly different from those of wild-type mice. However, epididymal spermatozoa from Trp53-null mice displayed a greater spontaneous mutant frequency compared to that from wild-type mice. A greater proportion of spontaneous transversions and a greater proportion of insertions/deletions 15 days after ionizing radiation were observed in Trp53-null mice compared to wild-type mice. Base excision repair activity in mixed germ cell nuclear extracts prepared from Trp53-null mice was significantly lower than that for wild-type controls. These data indicate that BAX-mediated apoptosis plays a significant role in regulating spontaneous mutagenesis in seminiferous tubule cells obtained from neonatal mice, whereas tumor suppressor TRP53 plays a significant role in regulating spontaneous mutagenesis between postmeiotic round spermatid and epididymal spermatozoon stages of spermiogenesis.

  13. Role of Ribonucleotide Reductase in Bacillus subtilis Stress-Associated Mutagenesis.

    Science.gov (United States)

    Castro-Cerritos, Karla Viridiana; Yasbin, Ronald E; Robleto, Eduardo A; Pedraza-Reyes, Mario

    2017-02-15

    The Gram-positive microorganism Bacillus subtilis relies on a single class Ib ribonucleotide reductase (RNR) to generate 2'-deoxyribonucleotides (dNDPs) for DNA replication and repair. In this work, we investigated the influence of RNR levels on B. subtilis stationary-phase-associated mutagenesis (SPM). Since RNR is essential in this bacterium, we engineered a conditional mutant of strain B. subtilis YB955 (hisC952 metB5 leu427) in which expression of the nrdEF operon was modulated by isopropyl-β-d-thiogalactopyranoside (IPTG). Moreover, genetic inactivation of ytcG, predicted to encode a repressor (NrdR) of nrdEF in this strain, dramatically increased the expression levels of a transcriptional nrdE-lacZ fusion. The frequencies of mutations conferring amino acid prototrophy in three genes were measured in cultures under conditions that repressed or induced RNR-encoding genes. The results revealed that RNR was necessary for SPM and overexpression of nrdEF promoted growth-dependent mutagenesis and SPM. We also found that nrdEF expression was induced by H 2 O 2 and such induction was dependent on the master regulator PerR. These observations strongly suggest that the metabolic conditions operating in starved B. subtilis cells increase the levels of RNR, which have a direct impact on SPM. Results presented in this study support the concept that the adverse metabolic conditions prevailing in nutritionally stressed bacteria activate an oxidative stress response that disturbs ribonucleotide reductase (RNR) levels. Such an alteration of RNR levels promotes mutagenic events that allow Bacillus subtilis to escape from growth-limited conditions. Copyright © 2017 American Society for Microbiology.

  14. Acute toxicity and mutagenesis of three metabolites mixture of nitrobenzene in mice.

    Science.gov (United States)

    Wang, Guixia; Zhang, Xiuying; Yao, Chunzhu; Tian, Meizhan

    2011-03-01

    Nitrobenzene is a synthetic compound, more than 95% of which is used in the production of aniline. Nitrobenzene has been demonstrated to be substantially metabolized to p-Nitrophenol, p-Aminophenol and p-Nitroaniline in food animals (e.g., bovines, fowls). There have been no studies on the acute toxicity and the mutagenesis of the mixture of the three metabolites mentioned above. The aim of the present study is to testify the acute toxicity and the mutagenesis of the three metabolites mixture. Seventy Kunming mice (half male, half female) received an intragastric administration exposure to metabolites-containing suspension of 750, 638, 542, 461, 392, 333 mg kg(-1) body weight and 0.5% sodium carboxymethyl cellulose (control), followed by a 14-day observation. The medial lethal dose (LD(50)) concentration for nitrobenzene metabolites mixture in this study was 499.92 mg/kg. Their mutagenic toxicology was studied through micronucleus and sperm abnormality test. Kunming mice were twice intragastrically exposed to 1/5 LD(50), 1/10 LD(50), 1/20 LD(50) mg kg(-1) nitrobenzene metabolites-containing suspension spaced 24-h apart. Cyclophosphamide, pure water and sodium carboxymethyl cellulose served as doses of the positive group, the negative group and the solvent control group, respectively. The incidence of micronucleus and sperm abnormality increased significantly in the 1/5 LD(50) and 1/10 LD(50) group compared with the negative and solvent control group. A dose-related increase in the incidence of micronucleus and sperm abnormality was noted. In conclusion, the three metabolites mixture of nitrobenzene was secondary toxicity and mutagenic substances in mice.

  15. Loss of BRCA1 or BRCA2 markedly increases the rate of base substitution mutagenesis and has distinct effects on genomic deletions

    DEFF Research Database (Denmark)

    Zamborszky, J.; Szikriszt, B.; Gervai, J. Z.

    2017-01-01

    -genome sequencing of multiple isogenic chicken DT40 cell clones to precisely determine the consequences of BRCA1/2 loss on all types of genomic mutagenesis. Spontaneous base substitution mutation rates increased sevenfold upon the disruption of either BRCA1 or BRCA2, and the arising mutation spectra showed strong...... of stalled replication forks as the cause of increased mutagenesis. The high rate of base substitution mutagenesis demonstrated by our experiments is likely to significantly contribute to the oncogenic effect of the inactivation of BRCA1 or BRCA2....

  16. Different efficiency of UmuDC and MucAB proteins in UV light induced mutagenesis in Escherichia coli

    International Nuclear Information System (INIS)

    Blanco, M.; Herrera, G.; Aleixandre, V.

    1986-01-01

    Two multicopy plasmids carrying either the umuDC or the mucAB operon were used to compare the efficiency of UmuDC and MucAB proteins in UV mutagenesis of Escherichia coli K12. It was found that in recA + uvr + bacteria, plasmid pIC80, mucAB + mediated UV mutagenesis more efficiently than did plasmid pSE 117, umuDC + . A similar result was obtained in lex A51(Def) cells, excluding the possibility that this was due to a differential regulation by LexA of the umuDC and mucAB operons. We conclude that some structural characteristic of the UmuDC and MucAB proteins determines their different efficiency in UV mutagenesis. This characteristic could be also responsible for the observation that in the recA430 mutant, pIC80 but no pSE117 can mediate UV mutagenesis. In the recAS142 mutant pIC80 also promoted UV mutagenesis more efficiently than pSE117. In this mutant, the recombination proficiency, the protease activity toward LexA and the mutation frequency were increased by the presence of adenine in the medium. In recA + uvrB5 bacteria, plasmid pSE117, umuDC caused both an increase in UV sensitivity as well as a reduction in the mutation frequency. These negative effects resulting from the overproduction of UmuDC proteins were higher in recA142 uvrB5 than in recA + uvrB5 cells. In contrast, overproduction of MucAB proteins in excision-deficient bacteria containing pIC80 led to a large increase in the mutation frequency. We suggest that the functional differences between UmuDC and MucAB proteins might be due to their different dependence on the direct role of RecA protease in UV mutagenesis. (orig.)

  17. Development of a counterselectable seamless mutagenesis system in lactic acid bacteria.

    Science.gov (United States)

    Xin, Yongping; Guo, Tingting; Mu, Yingli; Kong, Jian

    2017-07-05

    Lactic acid bacteria (LAB) are receiving more attention to act as cell factories for the production of high-value metabolites. However, the molecular tools for genetic modifying these strains are mainly vector-based double-crossover strategies, which are laborious and inefficient. To address this problem, several counterselectable markers have been developed, while few of them could be used in the wild-type host cells without pretreatment. The pheS gene encoding phenylalanyl-tRNA synthetase alpha subunit was identified in Lactococcus lactis NZ9000 genome. When mutant pheS gene (pheS*) under the control of the Lc. lactis NZ9000 L-lactate dehydrogenase promoter (P ldh ) was expressed from a plasmid, the resulted PheS* with an A312G substitution rendered cells sensitive to the phenylalanine analog p-chloro-phenylalanine (p-Cl-Phe). This result suggested pheS* was suitable to be used as a counterselectable marker in Lc. lactis. However, the expression level of pheS* from a chromosomal copy was too low to confer p-Cl-Phe sensitivity. Therefore, a strategy of cascading promoters was attempted for strengthening the expression level of pheS*. Expectedly, a cassette 5Pldh-pheS* with five tandem repetitive promoters P ldh resulted in a sensitivity to 15 mM p-Cl-Phe. Subsequently, a counterselectable seamless mutagenesis system PheS*/pG + host9 based on a temperature-sensitive plasmid pG + host9 harboring a 5Pldh-pheS* cassette was developed in Lc. lactis. We also demonstrated the possibility of applying pheS* to be a counterselectable marker in Lactobacillus casei BL23. As reported in E. coli, pheS* as a counterselectable marker has been demonstrated to be functional in targeted gene(s) deletion in Lc. lactis as well as in L. casei. Moreover, the efficiency and timesaving counterselectable seamless mutagenesis system PheS*/pG + host9 could be used in the wild-type host cells without pretreatment.

  18. HTP-OligoDesigner: An Online Primer Design Tool for High-Throughput Gene Cloning and Site-Directed Mutagenesis.

    Science.gov (United States)

    Camilo, Cesar M; Lima, Gustavo M A; Maluf, Fernando V; Guido, Rafael V C; Polikarpov, Igor

    2016-01-01

    Following burgeoning genomic and transcriptomic sequencing data, biochemical and molecular biology groups worldwide are implementing high-throughput cloning and mutagenesis facilities in order to obtain a large number of soluble proteins for structural and functional characterization. Since manual primer design can be a time-consuming and error-generating step, particularly when working with hundreds of targets, the automation of primer design process becomes highly desirable. HTP-OligoDesigner was created to provide the scientific community with a simple and intuitive online primer design tool for both laboratory-scale and high-throughput projects of sequence-independent gene cloning and site-directed mutagenesis and a Tm calculator for quick queries.

  19. Radioprotective action of glycerol and cysteamine on inactivation and mutagenesis in Salmonella tester strains after γ- and heavy ion irradiation

    International Nuclear Information System (INIS)

    Basha, S.G.; Krasavin, E.A.; Kozubek, S.

    1992-01-01

    Inactivation and mutagenesis were studied in Salmonella tester strains after γ-irradiation and after heavy ion irradiation in the presence of glycerol and cysteamine. Ions from deuteron to carbon with residual energies of 2-9 MeV/n were used. Cell sensitivity slightly increased with LET before decreasing. In the presence of glycerol the maximum was shifted to higher values of LET. The radioprotective effect of glycerol for cell killing diminished gradually with increasing LET from 2.0 for γ-radiation to 1.1 for carbon ions. Mutagenic effectiveness increased slightly for deuterium and helium ions. The radioprotective effect of cysteamine on mutagenesis was found to be very small in the case of γ-radiation for the three strains examined. (author). 20 refs.; 4 figs.; 5 tabs

  20. Effects of harman and norharman on spontaneous and ultraviolet light-induced mutagenesis in cultured Chinese hamster cells

    International Nuclear Information System (INIS)

    Chang, C.C.; Castellazzi, M.; Glover, T.W.; Trosko, J.E.

    1978-01-01

    Nontoxic concentrations of harman and norharman were tested in cultured Chinese hamster cells for their effects on DNA repair and mutagenesis. The following effects of harman were observed: (a) the survival of ultraviolet light- or x-ray-damaged cells was reduced; (b) the ultraviolet light-induced unscheduled DNA synthesis was slightly inhibited; and (c) the frequency of spontaneous or ultraviolet light-induced ouabain-resistant (ouar) or 6-thioguanine-resistant (6-TGr) mutations was reduced. Furthermore, the effect of harman on survival and mutagenesis was greater than that of norharman and was detected primarily in treatments in which cells were exposed to harman immediately following ultraviolet light irradiation. Our data clearly indicate that harman decreases the capacity to repair DNA damage and fix mutations in Chinese hamster cells, possibly because of the intercalation properties of this compound

  1. Mutagenesis of lambda phage by tif-expression or host-irradiation functions is largely independent of damage in the phage DNA

    International Nuclear Information System (INIS)

    Von Wright, A.; Bridges, B.A.

    1980-01-01

    The survival and mutagenesis of UV-irradiated phage lambda, as well as bacterial mutagenesis, are enhanced in tif mutants of Escherichia coli when these strains are grown at 43 0 C (Castellazzi et al., 1972). This was interpreted on the basis of a hypothesis (the SOS hypothesis) according to which the UV-inducible phenomena connected with reactivation and mutagenesis of UV-irradiated bacteriophages (Weigle, 1953; Radman, 1975) are constitutively expressed in tif-bacteria at high temperature (Witkin, 1974). In unpublished experiments with phage T3 we found that the survival of UV-irradiated phage is also better at 43 0 C than at 32 0 C in tif + cells and this made us reexamine the significance and nature of tif expression and examine its effects on both unirradiated and UV-irradiated phage lambda. Our results indicate that tif-induced mutagenesis and possibly reactivation of UV-irradiated phage lambda should be reinterpreted. (orig./AJ)

  2. Rice improvement involving altered flower structure more suitable to cross-pollination, using in vitro culture in combination with mutagenesis

    International Nuclear Information System (INIS)

    Min, S.K.

    1998-01-01

    Anther and somatic tissue culture in combination with mutagenesis were carried out to evaluate the efficiency of different mutagenic treatments of various in vitro culture materials, and to obtain some promising variants for rice improvement. Results indicated that in japonica rice radiation treatment of dry seeds and young panicles influenced the percentage of green plantlets regeneration from anther culture. Both treatments increased significantly the percentage of regenerated green plant lets in comparison with the control

  3. Enhancement of antiviral activity of collectin trimers through cross-linking and mutagenesis of the carbohydrate recognition domain

    DEFF Research Database (Denmark)

    White, Mitchell R; Boland, Patrick; Tecle, Tesfaldet

    2010-01-01

    Surfactant protein D (SP-D) plays important roles in innate defense against respiratory viruses [including influenza A viruses (IAVs)]. Truncated trimers composed of its neck and carbohydrate recognition domains (NCRDs) bind various ligands; however, they have minimal inhibitory activity for IAV......., complementary strategies, namely cross-linking of NCRDs through various means and mutagenesis of CRD residues to increase viral binding. These findings may be relevant for antiviral therapy....

  4. Thermostability enhancement of an endo-1,4-β-galactanase from Talaromyces stipitatus by site-directed mutagenesis

    DEFF Research Database (Denmark)

    Larsen, Dorte Møller; Nyffenegger, Christian; Swiniarska, Malgorzata Maria

    2015-01-01

    -life of TSGAL, nine single amino acid residues were selected for site-directed mutagenesis on the basis of semi-rational design. Of these nine mutants, G305A showed half-lives of 114 min at 55 °C and 15 min at 60 °C, respectively. This is 8.6-fold higher than that of the TSGAL at 55 °C, whereas the other...

  5. Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

    Science.gov (United States)

    Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Wang, Wei; Liu, Yuan-Yuan

    2016-01-01

    A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

  6. Combinatorial effect of mutagenesis and medium component optimization on Bacillus amyloliquefaciens antifungal activity and efficacy in eradicating Botrytis cinerea.

    Science.gov (United States)

    Masmoudi, Fatma; Ben Khedher, Saoussen; Kamoun, Amel; Zouari, Nabil; Tounsi, Slim; Trigui, Mohamed

    2017-04-01

    This work is directed towards Bacillus amyloliquefaciens strain BLB371 metabolite production for biocontrol of fungal phytopathogens. In order to maximise antifungal metabolite production by this strain, two approaches were combined: random mutagenesis and medium component optimization. After three rounds of mutagenesis, a hyper active mutant, named M3-7, was obtained. It produces 7 fold more antifungal metabolites (1800AU/mL) than the wild strain in MC medium. A hybrid design was applied to optimise a new medium to enhance antifungal metabolite production by M3-7. The new optimized medium (35g/L of peptone, 32.5g/L of sucrose, 10.5g/L of yeast extract, 2.4g/L of KH 2 PO 4 , 1.3g/L of MgSO 4 and 23mg/L of MnSO 4 ) achieved 1.62 fold enhancement in antifungal compound production (3000AU/mL) by this mutant, compared to that achieved in MC medium. Therefore, combinatory effect of these two approaches (mutagenesis and medium component optimization) allowed 12 fold improvement in antifungal activity (from 250UA/mL to 3000UA/mL). This improvement was confirmed against several phytopathogenic fungi with an increase of MIC and MFC over than 50%. More interestingly, a total eradication of gray mold was obtained on tomato fruits infected by Botrytis cinerea and treated by M3-7, compared to those treated by BLB371. From the practical point of view, combining random mutagenesis and medium optimization could be considered as an excellent tool for obtaining promising biological products useful against phytopathogenic fungi. Copyright © 2017 Elsevier GmbH. All rights reserved.

  7. Cancerous hyper-mutagenesis in p53 genes is possibly associated with transcriptional bypass of DNA lesions

    International Nuclear Information System (INIS)

    Rodin, S.N.; Rodin, A.S.; Juhasz, A.; Holmquist, G.P.

    2002-01-01

    The database of tumor-associated p53 base substitutions includes about 5% of tumors with two or more base substitutions. These multiplet base substitutions in one tumor are evidence for hyper-mutagenesis. Our retrospective analysis of this database indicates that most multiplets arise from a single transient hyper-mutagenic event in one cell that subsequently proliferated into a clonal tumor. The hyper-mutagenesis, 1.8x10 -4 substitutions per base pair, is detected as multiple mutations in p53 genes of tumors. It requires one strongly tumorigenic p53 substitution, usually missense, called the driver mutation. The occurrence frequencies of ancillary base substitutions, those that hitch-hike along with the driver mutation, are independent of their amino acid coding properties. In this respect, they act like neutral mutations. In support of this neutrality, we find that the frequency distribution of hitch-hiking CpG transitions along the p53 exons, their mutational spectrum, approximates the spontaneous pre-selection mutational spectrum of most human tissues and is correlated with the mutational spectrum of p53 pseudogenes in mammalian germ cells. The driver substitutions of multiplets predominantly originate along the transcribed strand while the ancillary substitutions tend to originate along the non-transcribed strand. This data is consistent with a model of time-dependent mutagenesis in non-dividing stem cells for generating multiple strand-asymmetric p53 mutations in tumors. By transcriptional bypass of DNA lesions with concomitant misincorporation, transcriptional mutagenesis generates a transient mutant p53 mRNA. The associated mutant p53 protein could allow the host cell a growth advantage, release from G 1 -arrest. Then, during subsequent DNA replication and misreading of the same lesion, the damaged base along the transcribed DNA strand would serve as the origin of the p53 base substitution that drives the hyper-mutagenic event leading to tumors with

  8. Development of an Asymbiotic Reference Line for Pea cv. Bohatýr by De Novo Mutagenesis

    Czech Academy of Sciences Publication Activity Database

    Novák, Karel; Šlajs, Martin; Biedermannová, Eva; Vondrys, Josef

    2005-01-01

    Roč. 45, - (2005), s. 1837-1843 ISSN 0011-183X R&D Projects: GA ČR GA521/00/0937; GA ČR GA521/03/0192 Institutional research plan: CEZ:AV0Z50200510 Keywords : mutagenesis * bohatýr * nitrogen fixation Subject RIV: EE - Microbiology, Virology Impact factor: 0.925, year: 2005

  9. Mutagenesis-based research aimed at obtaining resistance to Helminthosporium maydis, straint T, in maize with Texas male sterile cytoplasm

    International Nuclear Information System (INIS)

    Cornu, A.; Vuillaume, E.; Cassini, R.; Berville, A.

    1977-01-01

    Mutagenesis was used on the French line F7T, with Texas male sterile cytoplasm, in order to obtain plants resistant to Helminthosporium maydis. The M 3 seedlings were screened for resistance either by using a fungus culture filtrate or through contamination with the fungus itself. In the first tests, carried out after the seeds had been treated with an aqueous solution of EMS, a number of families exhibiting original types of resistance to Helminthosporium maydis, strain T, were isolated. (author)

  10. Agrobacterium-mediated insertional mutagenesis in the mycorrhizal fungus Laccaria bicolor.

    Science.gov (United States)

    Stephan, B I; Alvarez Crespo, M C; Kemppainen, M J; Pardo, A G

    2017-05-01

    Agrobacterium-mediated gene transfer (AMT) is extensively employed as a tool in fungal functional genomics and accordingly, in previous studies we used AMT on a dikaryotic strain of the ectomycorrhizal basidiomycete Laccaria bicolor. The interest in this fungus derives from its capacity to establish a symbiosis with tree roots, thereby playing a major role in nutrient cycling of forest ecosystems. The ectomycorrhizal symbiosis is a highly complex interaction involving many genes from both partners. To advance in the functional characterization of fungal genes, AMT was used on a monokaryotic L. bicolor. A collection of over 1200 transgenic strains was produced, of which 200 randomly selected strains were analyzed for their genomic T-DNA insertion patterns. By means of insertional mutagenesis, a number of transgenic strains were obtained displaying differential growth features. Moreover, mating with a compatible strain resulted in dikaryons that retained altered phenotypic features of the transgenic monokaryon. The analysis of the T-DNA integration pattern revealed mostly similar results to those reported in earlier studies, confirming the usefulness of AMT on different genetic backgrounds of L. bicolor. Taken together, our studies display the great versatility and potentiality of AMT as a tool for the genetic characterization of L. bicolor.

  11. CRISPR-Cas9-mediated saturated mutagenesis screen predicts clinical drug resistance with improved accuracy.

    Science.gov (United States)

    Ma, Leyuan; Boucher, Jeffrey I; Paulsen, Janet; Matuszewski, Sebastian; Eide, Christopher A; Ou, Jianhong; Eickelberg, Garrett; Press, Richard D; Zhu, Lihua Julie; Druker, Brian J; Branford, Susan; Wolfe, Scot A; Jensen, Jeffrey D; Schiffer, Celia A; Green, Michael R; Bolon, Daniel N

    2017-10-31

    Developing tools to accurately predict the clinical prevalence of drug-resistant mutations is a key step toward generating more effective therapeutics. Here we describe a high-throughput CRISPR-Cas9-based saturated mutagenesis approach to generate comprehensive libraries of point mutations at a defined genomic location and systematically study their effect on cell growth. As proof of concept, we mutagenized a selected region within the leukemic oncogene BCR-ABL1 Using bulk competitions with a deep-sequencing readout, we analyzed hundreds of mutations under multiple drug conditions and found that the effects of mutations on growth in the presence or absence of drug were critical for predicting clinically relevant resistant mutations, many of which were cancer adaptive in the absence of drug pressure. Using this approach, we identified all clinically isolated BCR-ABL1 mutations and achieved a prediction score that correlated highly with their clinical prevalence. The strategy described here can be broadly applied to a variety of oncogenes to predict patient mutations and evaluate resistance susceptibility in the development of new therapeutics. Published under the PNAS license.

  12. Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system.

    Science.gov (United States)

    Liang, Zhen; Zhang, Kang; Chen, Kunling; Gao, Caixia

    2014-02-20

    Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have emerged as powerful tools for genome editing in a variety of species. Here, we report, for the first time, targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS, ZmIPK1A, ZmIPK, ZmMRP4, and obtained targeting efficiencies of up to 23.1% in protoplasts, and about 13.3% to 39.1% of the transgenic plants were somatic mutations. Also, we constructed two gRNAs targeting the ZmIPK gene in maize protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition, the CRISPR/Cas system induced targeted mutations in Z. mays protoplasts with efficiencies (13.1%) similar to those obtained with TALENs (9.1%). Our results show that both TALENs and the CRISPR/Cas system can be used for genome modification in maize. Copyright © 2013. Published by Elsevier Ltd.

  13. Enhanced vanillin production from recombinant E. coli using NTG mutagenesis and adsorbent resin.

    Science.gov (United States)

    Yoon, Sang-Hwal; Lee, Eun-Gyeong; Das, Amitabha; Lee, Sook-Hee; Li, Cui; Ryu, Hee-Kyoung; Choi, Myung-Suk; Seo, Weon-Taek; Kim, Seon-Won

    2007-01-01

    Vanillin production was tested with different concentrations of added ferulic acid in E. coli harboring plasmid pTAHEF containing fcs (feruloyl-CoA synthase) and ech (enoyl-CoA hydratase/aldolase) genes cloned from Amycolatopsis sp. strain HR104. The maximum production of vanillin from E. coli DH5alpha harboring pTAHEF was found to be 1.0 g/L at 2.0 g/L of ferulic acid for 48 h of culture. To improve the vanillin production by reducing its toxicity, two approaches were followed: (1) generation of vanillin-resistant mutant of NTG-VR1 through NTG mutagenesis and (2) removal of toxic vanillin from the medium by XAD-2 resin absorption. The vanillin production of NTG-VR1 increased to three times at 5 g/L of ferulic acid when compared with its wild-type strain. When 50% (w/v) of XAD-2 resin was employed in culture with 10 g/L of ferulic acid, the vanillin production of NTG-VR1 was 2.9 g/L, which was 2-fold higher than that obtained with no use of the resin.

  14. AXM mutagenesis: an efficient means for the production of libraries for directed evolution of proteins.

    Science.gov (United States)

    Holland, Erika G; Buhr, Diane L; Acca, Felicity E; Alderman, Dawn; Bovat, Kristin; Busygina, Valeria; Kay, Brian K; Weiner, Michael P; Kiss, Margaret M

    2013-08-30

    Affinity maturation is an important part of the recombinant antibody development process. There are several well-established approaches for generating libraries of mutated antibody genes for affinity maturation, but these approaches are generally too laborious or expensive to allow high-throughput, parallel processing of multiple antibodies. Here, we describe a scalable approach that enables the generation of libraries with greater than 10(8) clones from a single Escherichia coli transformation. In our method, a mutated DNA fragment is produced using PCR conditions that promote nucleotide misincorporation into newly synthesized DNA. In the PCR reaction, one of the primers contains at least three phosphorothioate linkages at its 5' end, and treatment of the PCR product with a 5' to 3' exonuclease is used to preferentially remove the strand synthesized with the non-modified primer, resulting in a single-stranded DNA fragment. This fragment then serves as a megaprimer to prime DNA synthesis on a uracilated, circular, single-stranded template in a Kunkel-like mutagenesis reaction that biases nucleotide base-changes between the megaprimer and uracilated DNA sequence in favor of the in vitro synthesized megaprimer. This method eliminates the inefficient subcloning steps that are normally required for the construction of affinity maturation libraries from randomly mutagenized antibody genes. Copyright © 2013. Published by Elsevier B.V.

  15. Rose (Rosa hybrida L.) tissue culture mutagenesis for new mutants generation

    International Nuclear Information System (INIS)

    Salahbiah Abdul Majid; Rusli Ibrahim

    2004-01-01

    Tissue culture technique can be used to obtain complete regeneration of plant cells from shoots, rots, flowers, axillary buds and other parts of the plant. In this study, axillary buds from stem cuttings of Cutting Red, Christine Dior and Mini Rose varieties were used as the stating explants. Murashige and Skoog (1962) media supplemented with 6-Benzylaminopurine (BAP, at 4.44 - 8.88μM/l), Napthaleneacetic acid (NAA at 0.54μM/l),, nad 3% sucrose were used for plantlet initiation and regeneration. Cultured axillary buds were exposed to gamma ray (0.250 Gy/s) at 0, 15, 25, 35, 45, 55, 65 and 75 Gy for radiosensitivity test. From the dose respond curve, LD 5 0 the value for cutting red variety was 25 Gy, Christion Dior 30 Gy and Mini Rose 38 Gy, yet 22% of Mini Rose samples survived at 65 Gy and another 10% at 70 Gy. Screening of M3 plants of irradiated cultured shoots, 2 colour variations were obtained at 40 Gy for Cutting Red variety, while 3 colour variations for Mini Rose at 20 Gy. When 6 varieties of Fragrance Rose were irradiated at 40 Gy, 1 colour variation was obtained from 99 screened plants. This study suggests that the dose range of 20 to 45 can be considered for rose mutagenesis study to produce mutants. (Author)

  16. Investigation of Trimethyllysine Binding by the HP1 Chromodomain via Unnatural Amino Acid Mutagenesis.

    Science.gov (United States)

    Baril, Stefanie A; Koenig, Amber L; Krone, Mackenzie W; Albanese, Katherine I; He, Cyndi Qixin; Lee, Ga Young; Houk, Kendall N; Waters, Marcey L; Brustad, Eric M

    2017-12-06

    Trimethyllysine (Kme3) reader proteins are targets for inhibition due to their role in mediating gene expression. Although all such reader proteins bind Kme3 in an aromatic cage, the driving force for binding may differ; some readers exhibit evidence for cation-π interactions whereas others do not. We report a general unnatural amino acid mutagenesis approach to quantify the contribution of individual tyrosines to cation binding using the HP1 chromodomain as a model system. We demonstrate that two tyrosines (Y24 and Y48) bind to a Kme3-histone tail peptide via cation-π interactions, but linear free energy trends suggest they do not contribute equally to binding. X-ray structures and computational analysis suggest that the distance and degree of contact between Tyr residues and Kme3 plays an important role in tuning cation-π-mediated Kme3 recognition. Although cation-π interactions have been studied in a number of proteins, this work is the first to utilize direct binding assays, X-ray crystallography, and modeling, to pinpoint factors that influence the magnitude of the individual cation-π interactions.

  17. Mutagenesis of mNeptune Red-Shifts Emission Spectrum to 681-685 nm.

    Science.gov (United States)

    Li, ZhaoYang; Zhang, ZhiPing; Bi, LiJun; Cui, ZongQiang; Deng, JiaoYu; Wang, DianBing; Zhang, Xian-En

    2016-01-01

    GFP-like fluorescent proteins with diverse emission wavelengths have been developed through mutagenesis, offering many possible choices in cellular and tissue imaging, such as multi-targets imaging, deep tissue imaging that require longer emission wavelength. Here, we utilized a combined approach of random mutation and structure-based rational design to develop new NIR fluorescent proteins on the basis of a far-red fluorescent protein, mNeptune (Ex/Em: 600/650 nm). We created a number of new monomeric NIR fluorescent proteins with the emission range of 681-685 nm, which exhibit the largest Stocks shifts (77-80 nm) compared to other fluorescent proteins. Among them, mNeptune681 and mNeptune684 exhibit more than 30 nm redshift in emission relative to mNeptune, owing to the major role of the extensive hydrogen-bond network around the chromophore and contributions of individual mutations to the observed redshift. Furthermore, the two variants still maintain monomeric state in solution, which is a trait crucial for their use as protein tags. In conclusion, our results suggest that there is untapped potential for developing fluorescent proteins with desired properties.

  18. Screening and mutagenesis of lactobacillus brevis for biosynthesis of γ-aminobutyric acid

    International Nuclear Information System (INIS)

    Xia Jiang; Mei Lehe; Huang Jun

    2006-01-01

    γ-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system. In this study, a GABA-producing strain, hjxj-01, was isolated from the milk samples and identified as Lactobacillus brevis. In this GYP medium containing sodium glutamate, the highest GABA concentration accumulated by Lactobacillus brevis hjxj-01 is 7 g/L. The strain was treated with UV and 60 Co γ-rays. Based on high positive mutation rate, the final mutagenesis conditions were UV light 30 W, irradiation distance 45 cm, irradiation time 50 s, and 60 Co γ-rays irradiation of 500 Gy. The mutant strain, hjxj-08119, was bred by GABA resistance selection. Cultured for 12 generations continually, the GABA-producing capacity of hjxj-08119 maintained stably. The fermentation results indicate that compared with the origin strain hjxj-01, the average yield of GABA by hjij-08119 is 17 g/L, which is 142.9% of the origin strain. (authors)

  19. Using heavy-ion mutagenesis technology to select cellulose enzyme vitality of mutants of Aspergillium niger

    International Nuclear Information System (INIS)

    Tang Jiahui; Yang Fumin; Wang Shuyang

    2012-01-01

    In order to improve the cellulose ion beam at 20, 40, 60, 80, 100, 120Gy and 140 enzyme vitality of Aspergillus niger (=AS3.316), heavy Gy doses was used for inducing mutation. Higher cellulose enzyme vitality strains were screened through the primary screening and secondary screening. The result showed that 5 mutants T2-1, T3-1, T5-1, T6-3, T6-4 were selected, and T6-4 had the highest cellulose enzyme activity. The activity of filter paper cellulose enzyme, endo-glucanase, exo-glucanase and 13-glucosidase of T6-4 was 61.3, 116.2, 29.9 U/mL and 35.9 U/mL respectively. Compared with the original A. niger (=AS3.316), the cellulose enzyme activity was increased by 3.5, 3.78, 2.76 and 2.52 times in turn. The activity of cellulose enzyme of the rest mutants sorted from strong to the weak were T6-3T5-1T3-1T2-1. The dose at 120 Gy showed the best mutagenesis effect. Mutants had different degree of changes in the genetic stability, but overall, the performance showed relatively stable

  20. Mutagenesis applied to improve fruit trees. Techniques, methods and evaluation of radiation-induced mutations

    International Nuclear Information System (INIS)

    Donini, B.

    1982-01-01

    Improvement of fruit tree cultivars is an urgent need for a modern and industrialized horticulture on which is based the economic importance of many countries. Both the cross breeding and the mutation breeding are regarded as the methods to be used for creating new varieties. Research carried out at the CNEN Agriculture Laboratory on mutagenesis to improve vegetatively propagated plants, under the FAO-IAEA Co-ordinated Research Programme, has dealt with methods of exposure, types of radiations, conditions during and after the irradiation, mechanisms of mutation induction, methodology of isolation of somatic mutations and evaluation of radiation-induced mutations in fruit trees. Problems associated with these aspects have been evaluated, which is very important for the more efficient use of radiation in the mutation breeding. Mutants of agronomical importance (plant size reduction, early ripening, fruit colour change, nectarine fruit, self-thinning fruit) have been isolated in cherry, grape, apple, olive and peach and they are ready to be released. (author)

  1. Enhancement of Biomass and Lipid Productivities of Water Surface-Floating Microalgae by Chemical Mutagenesis.

    Science.gov (United States)

    Nojima, Daisuke; Ishizuka, Yuki; Muto, Masaki; Ujiro, Asuka; Kodama, Fumito; Yoshino, Tomoko; Maeda, Yoshiaki; Matsunaga, Tadashi; Tanaka, Tsuyoshi

    2017-05-27

    Water surface-floating microalgae have great potential for biofuel applications due to the ease of the harvesting process, which is one of the most problematic steps in conventional microalgal biofuel production. We have collected promising water surface-floating microalgae and characterized their capacity for biomass and lipid production. In this study, we performed chemical mutagenesis of two water surface-floating microalgae to elevate productivity. Floating microalgal strains AVFF007 and FFG039 (tentatively identified as Botryosphaerella sp. and Chlorococcum sp., respectively) were exposed to ethyl methane sulfonate (EMS) or 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), and pale green mutants (PMs) were obtained. The most promising FFG039 PM formed robust biofilms on the surface of the culture medium, similar to those formed by wild type strains, and it exhibited 1.7-fold and 1.9-fold higher biomass and lipid productivities than those of the wild type. This study indicates that the chemical mutation strategy improves the lipid productivity of water surface-floating microalgae without inhibiting biofilm formation and floating ability.

  2. Contribution of increased mutagenesis to the evolution of pollutants-degrading indigenous bacteria

    Science.gov (United States)

    Ilmjärv, Tanel; Naanuri, Eve; Kivisaar, Maia

    2017-01-01

    Bacteria can rapidly evolve mechanisms allowing them to use toxic environmental pollutants as a carbon source. In the current study we examined whether the survival and evolution of indigenous bacteria with the capacity to degrade organic pollutants could be connected with increased mutation frequency. The presence of constitutive and transient mutators was monitored among 53 pollutants-degrading indigenous bacterial strains. Only two strains expressed a moderate mutator phenotype and six were hypomutators, which implies that constitutively increased mutability has not been prevalent in the evolution of pollutants degrading bacteria. At the same time, a large proportion of the studied indigenous strains exhibited UV-irradiation-induced mutagenesis, indicating that these strains possess error-prone DNA polymerases which could elevate mutation frequency transiently under the conditions of DNA damage. A closer inspection of two Pseudomonas fluorescens strains PC20 and PC24 revealed that they harbour genes for ImuC (DnaE2) and more than one copy of genes for Pol V. Our results also revealed that availability of other nutrients in addition to aromatic pollutants in the growth environment of bacteria affects mutagenic effects of aromatic compounds. These results also implied that mutagenicity might be affected by a factor of how long bacteria have evolved to use a particular pollutant as a carbon source. PMID:28777807

  3. Ultraviolet light mutagenesis in bacteria: a result of the failure of normal error-correcting mechanisms

    International Nuclear Information System (INIS)

    Bridges, B.A.

    1980-01-01

    The various mechanisms for maintaining the fidelity of DNA base sequences during replication and repair are described. In excision-deficient bacteria an attractive possibility is that UV-induced mutations may arise as a result of the inhibition of the 3' to 5' exonuclease proof reading function of DNA polymerase III, either by means of the induction of a specific inhibitor or by means of feed-back inhibition by nucleoside 5' monophosphates. There is at present no convincing evidence for this model and little evidence that the process has any significant effect on the viability of UV-irradiated bacteria. In bacteria wild-type for repair functions, a model for UV-mutagenesis is proposed which postulates that after low to moderate UV doses, mutations reflect the normal infidelity of DNA polymerases (mainly III) acting during the filling of excision gaps. At higher doses excision gaps may uncover photoproducts on the complementary DNA strand which may result in 'targeted' metagenesis and be revealed as a quadratic component to the dose response curve. (Auth.)

  4. Improved antimicrobial activity of Pediococcus acidilactici against Salmonella Gallinarum by UV mutagenesis and genome shuffling.

    Science.gov (United States)

    Han, Geon Goo; Song, Ahn Ah; Kim, Eun Bae; Yoon, Seong-Hyun; Bok, Jin-Duck; Cho, Chong-Su; Kil, Dong Yong; Kang, Sang-Kee; Choi, Yun-Jaie

    2017-07-01

    Pediococcus acidilactici is a widely used probiotic, and Salmonella enterica serovar Gallinarum (SG) is a significant pathogen in the poultry industry. In this study, we improved the antimicrobial activity of P. acidilactici against SG using UV mutation and genome shuffling (GS). To improve antimicrobial activity against SG, UV mutagenesis was performed against wild-type P. acidilactici (WT), and five mutants showed improved antimicrobial activity. To further improve antimicrobial activity, GS was performed on five UV mutants. Following GS, four mutants showed improved antimicrobial activity compared with the UV mutants and WT. The antimicrobial activity of GS1 was highest among the mutants; however, the activity was reduced when the culture supernatant was treated with proteinase K, suggesting that the improved antimicrobial activity is due to a proteinous substance such as bacteriocin. To validate the activity of GS1 in vivo, we designed multi-species probiotics and performed broiler feeding experiments. Groups consisted of no treatment (NC), avilamycin-treated (PC), probiotic group 1 containing WT (T1), and probiotic group 2 containing GS1 (T2). In broiler feeding experiments, coliform bacteria were significantly reduced in T2 compared with NC, PC, and T1. The cecal microbiota was modulated and pathogenic bacteria were reduced by GS1 oral administration. In this study, GS1 showed improved antimicrobial activity against SG in vitro and reduced pathogenic bacteria in a broiler feeding experiment. These results suggest that GS1 can serve as an efficient probiotic, as an alternative to antibiotics in the poultry industry.

  5. Enhanced reactivation and mutagenesis of UV-irradiated adenovirus in normal human fibroblasts

    International Nuclear Information System (INIS)

    Bennett, C.B.; Rainbow, A.J.

    1988-01-01

    UV-enhanced reactivation (UVER) and UV-enhanced mutagenesis (UVEM) for two adenovirus temperature-sensitive mutants were examined following the infection of normal human fibroblasts. UV-irradiation of the virus alone resulted in dose-dependent increase in the UV-induced reversion frequency (RF) of viral progeny and a dose-dependent exponential decrease in progeny survival, when infecting non-irradiated cells. Analysis of the slopes of the UV-induced reversion curves suggested that 2.5 ± 0.3 and 2.4 ± 0.5 'hits' were required to produce a targeted reversion event among the viral progeny of Ad5ts36 and Ad5ts125 respectively. UV-irradiation of cells 24 h prior to infection resulted in a significant increase in progeny survival for UV-irradiated virus (UVER factor = 3.4 ± 0.8) concomitant with a significant increase in RF for UV-irradiated virus (targeted increase = 1.9 ± 0.3). The UV-induced RF per lethal hit to the virus was also significantly greater in UV-irradiated compared with non-irradiated cells. These results are consistent with the existence of a UV-inducible error-prone DNA repair mechanism in normal human cells. (author)

  6. Mechanisms of mutagenesis in human cells exposed to 55 MeV protons

    Science.gov (United States)

    Gauny, S.; Wiese, C.; Kronenberg, A.

    2001-01-01

    Protons represent the major type of charged particle radiation in spaceflight environments. The purpose of this study was to assess mutations arising in human lymphoid cells exposed to protons. Mutations were quantitated at the thymidine kinase (TK1) locus in cell lines derived from the same donor: TK6 cells (wt TP53) and WTK1 cells (mutant TP53). WTK1 cells were much more susceptible to mutagenesis following proton exposure than TK6 cells. Intragenic deletions were observed among early-arising TK1 mutants in TK6 cells, but not in WTK1 cells where all of the mutants arose by LOH. Deletion was the predominant mode of LOH in TK6 cells, while allelic recombination was the major mode of LOH in WTK1 cells. Deletions were of variable lengths, from <1 cM to 64 cM, while mutations that arose by allelic recombination often extended to the telomere. In summary, proton exposures elicited many types of mutations at an autosomal locus in human cells. Most involved large scale loss of genetic information, either through deletion or by recombination.

  7. Carcinogen susceptibility is regulated by genome architecture and predicts cancer mutagenesis.

    Science.gov (United States)

    García-Nieto, Pablo E; Schwartz, Erin K; King, Devin A; Paulsen, Jonas; Collas, Philippe; Herrera, Rafael E; Morrison, Ashby J

    2017-10-02

    The development of many sporadic cancers is directly initiated by carcinogen exposure. Carcinogens induce malignancies by creating DNA lesions (i.e., adducts) that can result in mutations if left unrepaired. Despite this knowledge, there has been remarkably little investigation into the regulation of susceptibility to acquire DNA lesions. In this study, we present the first quantitative human genome-wide map of DNA lesions induced by ultraviolet (UV) radiation, the ubiquitous carcinogen in sunlight that causes skin cancer. Remarkably, the pattern of carcinogen susceptibility across the genome of primary cells significantly reflects mutation frequency in malignant melanoma. Surprisingly, DNase-accessible euchromatin is protected from UV, while lamina-associated heterochromatin at the nuclear periphery is vulnerable. Many cancer driver genes have an intrinsic increase in carcinogen susceptibility, including the BRAF oncogene that has the highest mutation frequency in melanoma. These findings provide a genome-wide snapshot of DNA injuries at the earliest stage of carcinogenesis. Furthermore, they identify carcinogen susceptibility as an origin of genome instability that is regulated by nuclear architecture and mirrors mutagenesis in cancer. © 2017 The Authors.

  8. Processing closely spaced lesions during Nucleotide Excision Repair triggers mutagenesis in E. coli

    Science.gov (United States)

    Isogawa, Asako; Fujii, Shingo

    2017-01-01

    It is generally assumed that most point mutations are fixed when damage containing template DNA undergoes replication, either right at the fork or behind the fork during gap filling. Here we provide genetic evidence for a pathway, dependent on Nucleotide Excision Repair, that induces mutations when processing closely spaced lesions. This pathway, referred to as Nucleotide Excision Repair-induced Mutagenesis (NERiM), exhibits several characteristics distinct from mutations that occur within the course of replication: i) following UV irradiation, NER-induced mutations are fixed much more rapidly (t ½ ≈ 30 min) than replication dependent mutations (t ½ ≈ 80–100 min) ii) NERiM specifically requires DNA Pol IV in addition to Pol V iii) NERiM exhibits a two-hit dose-response curve that suggests processing of closely spaced lesions. A mathematical model let us define the geometry (infer the structure) of the toxic intermediate as being formed when NER incises a lesion that resides in close proximity of another lesion in the complementary strand. This critical NER intermediate requires Pol IV / Pol II for repair, it is either lethal if left unrepaired or mutation-prone when repaired. Finally, NERiM is found to operate in stationary phase cells providing an intriguing possibility for ongoing evolution in the absence of replication. PMID:28686598

  9. Towards Understanding the Catalytic Mechanism of Human Paraoxonase 1: Experimental and In Silico Mutagenesis Studies.

    Science.gov (United States)

    Tripathy, Rajan K; Aggarwal, Geetika; Bajaj, Priyanka; Kathuria, Deepika; Bharatam, Prasad V; Pande, Abhay H

    2017-08-01

    Human paraoxonase 1 (h-PON1) is a ~45-kDa serum enzyme that can hydrolyze a variety of substrates, including organophosphate (OP) compounds. It is a potential candidate for the development of antidote against OP poisoning in humans. However, insufficient OP-hydrolyzing activity of native enzyme affirms the urgent need to develop improved variant(s) having enhanced OP-hydrolyzing activity. The crystal structure of h-PON1 remains unsolved, and the molecular details of how the enzyme catalyses hydrolysis of different types of substrates are also not clear. Understanding the molecular details of the catalytic mechanism of h-PON1 is essential to engineer better variant(s) of enzyme. In this study, we have used a random mutagenesis approach to increase the OP-hydrolyzing activity of recombinant h-PON1. The mutants not only showed a 10-340-fold increased OP-hydrolyzing activity against different OP substrates but also exhibited differential lactonase and arylesterase activities. In order to investigate the mechanistic details of the effect of observed mutations on the hydrolytic activities of enzyme, molecular docking studies were performed with selected mutants. The results suggested that the observed mutations permit differential binding of substrate/inhibitor into the enzyme's active site. This may explain differential hydrolytic activities of the enzyme towards different substrates.

  10. Yellow fluorescent protein phiYFPv (Phialidium): structure and structure-based mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Pletneva, Nadya V.; Pletnev, Vladimir Z., E-mail: vzpletnev@gmail.com; Souslova, Ekaterina; Chudakov, Dmitry M. [Russian Academy of Sciences, Moscow (Russian Federation); Lukyanov, Sergey [Russian Academy of Sciences, Moscow (Russian Federation); Nizhny Novgorod State Medical Academy, Nizhny Novgorod (Russian Federation); Martynov, Vladimir I.; Arhipova, Svetlena; Artemyev, Igor [Russian Academy of Sciences, Moscow (Russian Federation); Wlodawer, Alexander [National Cancer Institute, Frederick, MD 21702 (United States); Dauter, Zbigniew [National Cancer Institute, Argonne, IL 60439 (United States); Pletnev, Sergei [National Cancer Institute, Argonne, IL 60439 (United States); SAIC-Frederick, 9700 South Cass Avenue, Argonne, IL 60439 (United States); Russian Academy of Sciences, Moscow (Russian Federation)

    2013-06-01

    The yellow fluorescent protein phiYFPv with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The yellow fluorescent protein phiYFPv (λ{sub em}{sup max} ≃ 537 nm) with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The latter fluorescent protein is one of only two known cases of naturally occurring proteins that exhibit emission spectra in the yellow–orange range (535–555 nm). Here, the crystal structure of phiYFPv has been determined at 2.05 Å resolution. The ‘yellow’ chromophore formed from the sequence triad Thr65-Tyr66-Gly67 adopts the bicyclic structure typical of fluorophores emitting in the green spectral range. It was demonstrated that perfect antiparallel π-stacking of chromophore Tyr66 and the proximal Tyr203, as well as Val205, facing the chromophore phenolic ring are chiefly responsible for the observed yellow emission of phiYFPv at 537 nm. Structure-based site-directed mutagenesis has been used to identify the key functional residues in the chromophore environment. The obtained results have been utilized to improve the properties of phiYFPv and its homologous monomeric biomarker tagYFP.

  11. ADA1 and NET1 genes of yeast mediate both chromosome maintenance and mitochondrial rho- mutagenesis

    International Nuclear Information System (INIS)

    Koltovaya, N.A.; Gerasimova, A.S.; Chekhuta, I.A.; Devin, A.B.

    2002-01-01

    An increase in the mitochondrial (mt) rho - mutagenesis is a well-known response of yeast cells to mutations in the numerous nuclear genes as well as to various kinds of stress. Notwithstanding the extensive studies during several decades the biological significance of this response is not yet fully understood. The genetic approach to solution of this subject includes the study of genes that are required for the high incidence of spontaneous rho - mutants. Previously we found that mutations in certain nuclear genes including CDC28, the central cell-cycle regulation gene, may decrease the spontaneous rho - mutability and simultaneously affect maintenance of the yeast chromosomes and plasmids. The present work provides data on identification of two more genes, resembling CDC28 in this respect. These genes NET1 and ADA1 mediate important regulatory protein-protein interactions in the yeast cell. The effects of net1 and ada1 mutations on the maintenance of yeast mt genome, chromosomes and plasmids as well on cell sensitivity to ionizing radiation are also described. (author)

  12. Mutagenesis and evaluation of cellulase properties and cellulose hydrolysis of Talaromyces piceus.

    Science.gov (United States)

    He, Ronglin; Cai, Pengli; Wu, Gaihong; Zhang, Can; Zhang, Dongyuan; Chen, Shulin

    2015-11-01

    A fungal species with a high yield of β-glucosidase was isolated and identified as Talaromyces piceus 9-3 (anamorph: Penicillium piceum) by morphological and molecular characterization. Through dimethyl sulphate mutagenesis, the cellulase over-producing strain T. piceus H16 was obtained. The FPase activity and β-glucosidase activity of T. piceus H16 were 5.83 and 53.12 IU ml(-1) respectively--a 5.34- and 4.43-times improvement from the parent strain T. piceus 9-3. The optimum pH and temperature for enzyme activity were pH 5.0 and 50 °C for FPase activity and pH 5.0 and 55 °C for β-glucosidase activity, respectively. The cellulase were quite stable at 37 °C, only losing cellulase from T. piceus H16 with that from Trichoderma reesei RUT C30 on hydrolyzing different substrates due to the high β-glucosidase activity of T. piceus H16. These data suggest that T. piceus H16 can be used as a potential cellulase producer with good prospects.

  13. Probing the molecular determinants of aniline dioxygenase substrate specificity by saturation mutagenesis.

    Science.gov (United States)

    Ang, Ee L; Obbard, Jeffrey P; Zhao, Huimin

    2007-02-01

    Aniline dioxygenase is a multicomponent Rieske nonheme-iron dioxygenase enzyme isolated from Acinetobacter sp. strain YAA. Saturation mutagenesis of the substrate-binding pocket residues, which were identified using a homology model of the alpha subunit of the terminal dioxygenase (AtdA3), was used to probe the molecular determinants of AtdA substrate specificity. The V205A mutation widened the substrate specificity of aniline dioxygenase to include 2-isopropylaniline, for which the wild-type enzyme has no activity. The V205A mutation also made 2-isopropylaniline a better substrate for the enzyme than 2,4-dimethylaniline, a native substrate of the wild-type enzyme. The I248L mutation improved the activity of aniline dioxygenase against aniline and 2,4-dimethylaniline approximately 1.7-fold and 2.1-fold, respectively. Thus, it is shown that the alpha subunit of the terminal dioxygenase indeed plays a part in the substrate specificity as well as the activity of aniline dioxygenase. Interestingly, the equivalent residues of V205 and I248 have not been previously reported to influence the substrate specificity of other Rieske dioxygenases. These results should facilitate future engineering of the enzyme for bioremediation and industrial applications.

  14. Enterocin A mutants identified by saturation mutagenesis enhance potency towards vancomycin-resistant Enterococci.

    Science.gov (United States)

    McClintock, Maria K; Kaznessis, Yiannis N; Hackel, Benjamin J

    2016-02-01

    Vancomycin-resistant Enterococci infections are a significant clinical problem. One proposed solution is to use probiotics, such as lactic acid bacteria, to produce antimicrobial peptides at the site of infection. Enterocin A, a class 2a bacteriocin, exhibits inhibitory activity against E. faecium and E. faecalis, which account for 86% of vancomycin-resistant Enterococci infections. In this study, we aimed to engineer enterocin A mutants with enhanced potency within a lactic acid bacterial production system. Peptide mutants resulting from saturation mutagenesis at sites A24 and T27 were efficiently screened in a 96-well plate assay for inhibition of pathogen growth. Several mutants exhibit increased potency relative to wild-type enterocin A in both liquid- and solid-medium growth assays. In particular, A24P and T27G exhibit enhanced inhibition of multiple strains of E. faecium and E. faecalis, including clinically isolated vancomycin-resistant strains. A24P and T27G enhance killing of E. faecium 8 by 13 ± 3- and 18 ± 4-fold, respectively. The engineered enterocin A/lactic acid bacteria systems offer significant potential to combat antibiotic-resistant infections. © 2015 Wiley Periodicals, Inc.

  15. Crizotinib-Resistant Mutants of EML4-ALK Identified Through an Accelerated Mutagenesis Screen

    Science.gov (United States)

    Zhang, Sen; Wang, Frank; Keats, Jeffrey; Zhu, Xiaotian; Ning, Yaoyu; Wardwell, Scott D; Moran, Lauren; Mohemmad, Qurish K; Anjum, Rana; Wang, Yihan; Narasimhan, Narayana I; Dalgarno, David; Shakespeare, William C; Miret, Juan J; Clackson, Tim; Rivera, Victor M

    2011-01-01

    Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance. PMID:22034911

  16. Production of Trichoderma strains with pesticide-polyresistance by mutagenesis and protoplast fusion.

    Science.gov (United States)

    Hatvani, Lóránt; Manczinger, László; Kredics, László; Szekeres, András; Antal, Zsuzsanna; Vágvölgyi, Csaba

    2006-01-01

    The sensitivity of two cold-tolerant Trichoderma strains belonging to the species T. harzianum and T. atroviride was determined to a series of pesticides widely used in agriculture. From the 16 pesticides tested, seven fungicides: copper sulfate, carbendazim, mancozeb, tebuconazole, imazalil, captan and thiram inhibited colony growth of the test strains significantly with minimal inhibitory concentrations of 300, 0.4, 50, 100, 100, 100 and 50 microg/ml, respectively. Mutants resistant to carbendazim and tebuconazole were produced from both wild type strains by means of UV-mutagenesis. The cross-resistance capabilities and in vitro antagonistic properties of the mutants were determined. Carbendazim-resistant mutants showed total cross-resistance to benomyl and thiabendazole at a concentration of 20 microg/ml. Intraspecific protoplast fusion was carried out between carbendazim- and tebuconazole-resistant mutants of both parental strains, and putative haploid recombinants with stable resistance to both pesticides were produced in the case of T. atroviride. These pesticide-polyresistant progenies are potential candidates for application in an integrated pest management system.

  17. Mutagenesis of Xanthomonas campestris and selection of strains with enhanced Xanthan production

    International Nuclear Information System (INIS)

    Kamal, F.; Mehrgan, H.; Mazaheri, M.; Mortazavi, A. R.

    2003-01-01

    Xanthan gum is microbial polysaccharide of great commercial importance as it has been unusual rheological properties in solution and consequent range of applications. In this study, a series of mutants were isolated from Xanthomonas PTSS 1473 by ethyl methanesulfonate mutagenesis. The polysaccharide yield of one mutant, XC1473E 2 , was 30% better than that of the parent strain. It also showed higher xanthan formation of glucose consumption rates compared to the parent strain. xanthan produced by the mutant and enhanced viscosity, higher pseudo plasticity and larger molecular weight. Since mutant XC1473E 2 appeared white on agar plates, it underwent pigment extraction with methanol. Contrary to the parent strain, the mutant showed no absorption at 443 nm, i.e. the wavelength related to yellow pigment. This finding suggested that yellow pigmentation and normal xanthan biosynthesis are not necessarily concurrent. In general, mutant ZC1473e 2 seems to be a strain with interesting characteristics for use in commercial production of Xanthan

  18. Structure-Function Analysis of Chloroplast Proteins via Random Mutagenesis Using Error-Prone PCR.

    Science.gov (United States)

    Dumas, Louis; Zito, Francesca; Auroy, Pascaline; Johnson, Xenie; Peltier, Gilles; Alric, Jean

    2018-06-01

    Site-directed mutagenesis of chloroplast genes was developed three decades ago and has greatly advanced the field of photosynthesis research. Here, we describe a new approach for generating random chloroplast gene mutants that combines error-prone polymerase chain reaction of a gene of interest with chloroplast complementation of the knockout Chlamydomonas reinhardtii mutant. As a proof of concept, we targeted a 300-bp sequence of the petD gene that encodes subunit IV of the thylakoid membrane-bound cytochrome b 6 f complex. By sequencing chloroplast transformants, we revealed 149 mutations in the 300-bp target petD sequence that resulted in 92 amino acid substitutions in the 100-residue target subunit IV sequence. Our results show that this method is suited to the study of highly hydrophobic, multisubunit, and chloroplast-encoded proteins containing cofactors such as hemes, iron-sulfur clusters, and chlorophyll pigments. Moreover, we show that mutant screening and sequencing can be used to study photosynthetic mechanisms or to probe the mutational robustness of chloroplast-encoded proteins, and we propose that this method is a valuable tool for the directed evolution of enzymes in the chloroplast. © 2018 American Society of Plant Biologists. All rights reserved.

  19. groE mutants of Escherichia coli are defective in umuDC-dependent UV mutagenesis

    International Nuclear Information System (INIS)

    Donnelly, C.E.; Walker, G.C.

    1989-01-01

    Overexpression of the SOS-inducible umuDC operon of Escherichia coli results in the inability of these cells to grow at 30 degrees C. Mutations in several heat shock genes suppress this cold sensitivity. Suppression of umuD+C+-dependent cold sensitivity appears to occur by two different mechanisms. We show that mutations in lon and dnaK heat shock genes suppress cold sensitivity in a lexA-dependent manner. In contrast, mutations in groES, groEL, and rpoH heat shock genes suppress cold sensitivity regardless of the transcriptional regulation of the umuDC genes. We have also found that mutations in groES and groEL genes are defective in umuDC-dependent UV mutagenesis. This defect can be suppressed by increased expression of the umuDC operon. The mechanism by which groE mutations affect umuDC gene product function may be related to the stability of the UmuC protein, since the half-life of this protein is shortened because of mutations at the groE locus

  20. Mutagenesis for ACMV resistance in a Ghanian cassava cultivar `Bosom nsia`

    Energy Technology Data Exchange (ETDEWEB)

    Ahiabu, R K; Lokko, Y; Danso, K; Klu, G Y.P. [Biotechnology and Nuclear Agricultural Research Inst., Ghana Atomic Energy Commission, Legon (Ghana)

    1997-07-01

    Breeding for resistance to the African Cassava Mosaic Virus (ACMV) disease in the Ghanian cassava cultivar `Bosom nsia` has been on-going for the past four years at the Biotechnology and Nuclear Agricultural Research Institute. Protocols for in vitro culture from shoot meristems and acclimation of plantlets were established. Radiosensitivity tests on the regenerated plantlets indicated LD{sub 50} of 40 Gy, and doses of 25, 30 and 35 Gy were suitable for mutagenesis. These doses were applied to in vivo and in vitro grown plants, and selection was carried out in three propagations. Four variants, selected under field conditions with high viral incidence, were analyzed for virus particles with three virus indexing techniques. Polymerase chain reaction (PCR) product analysis of DNA extracts from Nicotiana benthamiana test plants, inoculated with sap from the leaves of variants, confirmed the presence of virus particles in all variants. Inoculation and ELISA tests suggested ACMV tolerance in selected variants. (author). 24 refs, 3 figs, 1 tab.