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Sample records for insertional mutagenesis identifies

  1. Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer

    PubMed Central

    Ranzani, Marco; Cesana, Daniela; Bartholomae, Cynthia C.; Sanvito, Francesca; Pala, Mauro; Benedicenti, Fabrizio; Gallina, Pierangela; Sergi, Lucia Sergi; Merella, Stefania; Bulfone, Alessandro; Doglioni, Claudio; von Kalle, Christof; Kim, Yoon Jun; Schmidt, Manfred; Tonon, Giovanni; Naldini, Luigi; Montini, Eugenio

    2013-01-01

    Transposons and γ-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells, hampering the identification of early cancer-driving events amongst bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVV) by which we could efficiently induce hepatocellular carcinoma (HCC) in 3 different mouse models. By virtue of LVV’s replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of 4 new liver cancer genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. Our newly identified cancer genes are likely to play a role in human disease, since they are upregulated and/or amplified/deleted in human HCCs and can predict clinical outcome of patients. PMID:23314173

  2. Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis.

    PubMed

    March, H Nikki; Rust, Alistair G; Wright, Nicholas A; ten Hoeve, Jelle; de Ridder, Jeroen; Eldridge, Matthew; van der Weyden, Louise; Berns, Anton; Gadiot, Jules; Uren, Anthony; Kemp, Richard; Arends, Mark J; Wessels, Lodewyk F A; Winton, Douglas J; Adams, David J

    2011-12-01

    The evolution of colorectal cancer suggests the involvement of many genes. To identify new drivers of intestinal cancer, we performed insertional mutagenesis using the Sleeping Beauty transposon system in mice carrying germline or somatic Apc mutations. By analyzing common insertion sites (CISs) isolated from 446 tumors, we identified many hundreds of candidate cancer drivers. Comparison to human data sets suggested that 234 CIS-targeted genes are also dysregulated in human colorectal cancers. In addition, we found 183 CIS-containing genes that are candidate Wnt targets and showed that 20 CISs-containing genes are newly discovered modifiers of canonical Wnt signaling. We also identified mutations associated with a subset of tumors containing an expanded number of Paneth cells, a hallmark of deregulated Wnt signaling, and genes associated with more severe dysplasia included those encoding members of the FGF signaling cascade. Some 70 genes had co-occurrence of CIS pairs, clustering into 38 sub-networks that may regulate tumor development. PMID:22057237

  3. Insertional Mutagenesis Identifies a STAT3/Arid1b/β-catenin Pathway Driving Neurofibroma Initiation.

    PubMed

    Wu, Jianqiang; Keng, Vincent W; Patmore, Deanna M; Kendall, Jed J; Patel, Ami V; Jousma, Edwin; Jessen, Walter J; Choi, Kwangmin; Tschida, Barbara R; Silverstein, Kevin A T; Fan, Danhua; Schwartz, Eric B; Fuchs, James R; Zou, Yuanshu; Kim, Mi-Ok; Dombi, Eva; Levy, David E; Huang, Gang; Cancelas, Jose A; Stemmer-Rachamimov, Anat O; Spinner, Robert J; Largaespada, David A; Ratner, Nancy

    2016-03-01

    To identify genes and signaling pathways that initiate Neurofibromatosis type 1 (NF1) neurofibromas, we used unbiased insertional mutagenesis screening, mouse models, and molecular analyses. We mapped an Nf1-Stat3-Arid1b/β-catenin pathway that becomes active in the context of Nf1 loss. Genetic deletion of Stat3 in Schwann cell progenitors (SCPs) and Schwann cells (SCs) prevents neurofibroma formation, decreasing SCP self-renewal and β-catenin activity. β-catenin expression rescues effects of Stat3 loss in SCPs. Importantly, P-STAT3 and β-catenin expression correlate in human neurofibromas. Mechanistically, P-Stat3 represses Gsk3β and the SWI/SNF gene Arid1b to increase β-catenin. Knockdown of Arid1b or Gsk3β in Stat3(fl/fl);Nf1(fl/fl);DhhCre SCPs rescues neurofibroma formation after in vivo transplantation. Stat3 represses Arid1b through histone modification in a Brg1-dependent manner, indicating that epigenetic modification plays a role in early tumorigenesis. Our data map a neural tumorigenesis pathway and support testing JAK/STAT and Wnt/β-catenin pathway inhibitors in neurofibroma therapeutic trials. PMID:26904939

  4. Retroviral insertional mutagenesis identifies genes that collaborate with NUP98‐HOXD13 during leukemic transformation

    PubMed Central

    Slape, Christopher; Hartung, Helge; Lin, Ying‐Wei; Bies, Juraj; Wolff, Linda; Aplan, Peter D

    2007-01-01

    The t(2;11)(q31;p15) chromosomal translocation results in a fusion between the NUP98 and HOXD13genes and has been observed in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). We previously demonstrated that expression of the NUP98‐HOXD13 (NHD13) fusion gene in transgenic mice results in an invariably fatal MDS; approximately one third of mice die due to complications of severe pancytopenia, and about two thirds progress to a fatal acute leukemia. In the present study, we used retroviral insertional mutagenesis to identify genes that might collaborate with NHD13 as the MDS transformed to an acute leukemia. Newborn NHD13 transgenic mice and littermate controls were infected with the MOL4070LTR retrovirus. The onset of leukemia was accelerated, suggesting a synergistic effect between the NHD13 transgene and the genes neighbouring retroviral insertion events. We identified numerous common insertion sites located near protein‐coding genes, and confirmed dysregulation of a subset of these by expression analyses. Among these genes were Meis1, a known collaborator of HOX and NUP98‐HOX fusion genes, and Mn1, a transcriptional coactivator involved in human leukemia through fusion with the TEL gene. Other putative collaborators included Gata2, Erg and Epor. Of note, we identified a common insertion site that was >100 kb from the nearest coding gene, but within 20 kb of the miR29a/miR29b1 microRNA locus. Both of these miRNA were upregulated, demonstrating that retroviral insertional mutagenesis can target miRNA loci as well as protein‐coding loci. Our data provides new insights into NHD13 mediated leukemogenesis as well as retroviral insertional mutagenesis mechanisms. PMID:17545593

  5. Pigmentation-based insertional mutagenesis is a simple and potent screening approach for identifying neurocristopathy-associated genes in mice.

    PubMed

    Pilon, Nicolas

    2016-01-01

    Neurocristopathies form a specific group of rare genetic diseases in which a defect in neural crest cell development is causal. Because of the large number of neural crest cell derivatives, distinct structures/cell types (isolated or in combination) are affected in each neurocristopathy. The most important issues in this research field is that the underlying genetic cause and associated pathogenic mechanism of most cases of neurocristopathy are poorly understood. This article describes how a relatively simple insertional mutagenesis approach in the mouse has proved useful for identifying new candidate genes and pathogenic mechanisms for diverse neurocristopathies. PMID:27141416

  6. Pigmentation-based insertional mutagenesis is a simple and potent screening approach for identifying neurocristopathy-associated genes in mice

    PubMed Central

    Pilon, Nicolas

    2016-01-01

    ABSTRACT Neurocristopathies form a specific group of rare genetic diseases in which a defect in neural crest cell development is causal. Because of the large number of neural crest cell derivatives, distinct structures/cell types (isolated or in combination) are affected in each neurocristopathy. The most important issues in this research field is that the underlying genetic cause and associated pathogenic mechanism of most cases of neurocristopathy are poorly understood. This article describes how a relatively simple insertional mutagenesis approach in the mouse has proved useful for identifying new candidate genes and pathogenic mechanisms for diverse neurocristopathies. PMID:27141416

  7. Retroviral insertional mutagenesis in telomerase-immortalized hepatocytes identifies RIPK4 as novel tumor suppressor in human hepatocarcinogenesis.

    PubMed

    Heim, D; Cornils, K; Schulze, K; Fehse, B; Lohse, A W; Brümmendorf, T H; Wege, H

    2015-01-15

    Carcinogenesis is a multistep process involving alterations in various cellular pathways. The critical genetic events driving the evolution of primary liver cancer, specifically hepatoblastoma and hepatocellular carcinoma (HCC), are still poorly understood. However, telomere stabilization is acknowledged as prerequisite for cancer progression in humans. In this project, human fetal hepatocytes were utilized as a cell culture model for untransformed, proliferating human liver cells, with telomerase activation as first oncogenic hit. To elucidate critical downstream genetic events driving further transformation of immortalized liver cells, we used retroviral insertional mutagenesis as an unbiased approach to induce genetic alterations. Following isolation of hyperproliferating, provirus-bearing cell clones, we monitored cancer-associated growth properties and characterized changes toward a malignant phenotype. Three transformed clones with the ability to form colonies in soft agar were expanded. As proof-of-principle for our experimental setup, we identified a transforming insertion on chromosome 8 within the pleiomorphic adenoma gene 1 (PLAG1), resulting in a 20-fold increase in PLAG1 expression. Upregulation of PLAG1 has already been described to promote human hepatoblastoma development. In a separate clone, a transforming insertion was detected in close proximity to the receptor-interacting serine-threonine kinase 4 (RIPK4) with an approximately eightfold suppression in RIPK4 expression. As validation for this currently unknown driver in hepatocarcinogenesis, we examined RIPK4 expression in human HCC samples and confirmed a significant suppression of RIPK4 in 80% of the samples. Furthermore, overexpression of RIPK4 in transformed human fetal hepatocytes resulted in an almost complete elimination of anchorage-independent growth. On the basis of these data, we propose RIPK4 as a novel putative tumor suppressor in human hepatocarcinogenesis. PMID:24413083

  8. An insertional mutagenesis screen identifies genes that cooperate with Mll-AF9 in a murine leukemogenesis model

    PubMed Central

    Bergerson, Rachel J.; Collier, Lara S.; Sarver, Aaron L.; Been, Raha A.; Lugthart, Sanne; Diers, Miechaleen D.; Zuber, Johannes; Rappaport, Amy R.; Nixon, Molly J.; Silverstein, Kevin A. T.; Fan, Danhua; Lamblin, Anne-Francoise J.; Wolff, Linda; Kersey, John H.; Delwel, Ruud; Lowe, Scott W.; O'Sullivan, M. Gerard; Kogan, Scott C.; Adams, David J.

    2012-01-01

    Patients with a t(9;11) translocation (MLL-AF9) develop acute myeloid leukemia (AML), and while in mice the expression of this fusion oncogene also results in the development of myeloid leukemia, it is with long latency. To identify mutations that cooperate with Mll-AF9, we infected neonatal wild-type (WT) or Mll-AF9 mice with a murine leukemia virus (MuLV). MuLV-infected Mll-AF9 mice succumbed to disease significantly faster than controls presenting predominantly with myeloid leukemia while infected WT animals developed predominantly lymphoid leukemia. We identified 88 candidate cancer genes near common sites of proviral insertion. Analysis of transcript levels revealed significantly elevated expression of Mn1, and a trend toward increased expression of Bcl11a and Fosb in Mll-AF9 murine leukemia samples with proviral insertions proximal to these genes. Accordingly, FOSB and BCL11A were also overexpressed in human AML harboring MLL gene translocations. FOSB was revealed to be essential for growth in mouse and human myeloid leukemia cells using shRNA lentiviral vectors in vitro. Importantly, MN1 cooperated with Mll-AF9 in leukemogenesis in an in vivo BM viral transduction and transplantation assay. Together, our data identified genes that define transcription factor networks and important genetic pathways acting during progression of leukemia induced by MLL fusion oncogenes. PMID:22427200

  9. Functional mapping of Cre recombinase by pentapeptide insertional mutagenesis.

    PubMed

    Petyuk, Vladislav; McDermott, Jeffrey; Cook, Malcolm; Sauer, Brian

    2004-08-27

    Cre is a site-specific recombinase from bacteriophage P1. It is a member of the tyrosine integrase family and catalyzes reciprocal recombination between specific 34-bp sites called loxP. To analyze the structure-function relationships of this enzyme, we performed large scale pentapeptide insertional mutagenesis to generate insertions of five amino acids at random positions in the protein. The high density of insertion mutations into Cre allowed us to identify an unexpected degree of functional tolerance to insertions into the 4-5 beta-hairpin and into the loop between helices J and K (both of which contact the DNA in the minor groove) and also into helix A. The phenotypes of the majority of inserts allowed us to confirm a variety of predictions made on the basis of sequence conservation, known three-dimensional structure, and proposed catalytic mechanism. In particular, most insertions into conserved regions or secondary structure elements inactivated Cre, and most insertions located in nonconserved, unstructured regions preserved Cre activity. Less expectedly, the non-conserved and poorly structured loops and linkers between helices A-B, E-F, and M-N did not tolerate insertions, thus identifying these as critical regions for recombinase activity. We purified and characterized in vitro several representatives of these "unexpected" Cre insertion mutants. The role of those regions in the recombination process is discussed. PMID:15218019

  10. Misty somites, a maternal effect gene identified by transposon-mediated insertional mutagenesis in zebrafish that is essential for the somite boundary maintenance.

    PubMed

    Kotani, Tomoya; Kawakami, Koichi

    2008-04-15

    Somite boundary formation is crucial for segmentation of vertebrate somites and vertebrae and skeletal muscle morphogenesis. Previously, we developed a Tol2 transposon-mediated gene trap method in zebrafish. In the present study, we aimed to isolate transposon insertions that trap maternally-expressed genes. We found that homozygous female fish carrying a transposon insertion within a maternally-expressed gene misty somites (mys) produced embryos that showed obscure somite boundaries at the early segmentation stage (12-13 hpf). The somite boundaries became clear and distinct after this period and the embryos survived to adulthood. This phenotype was rescued by expression of mys cDNA in the homozygous adults, confirming that it was caused by a decreased mys activity. We analyzed a role of the mys gene by using morpholino oligonucleotides (MOs). The MO-injected embryo exhibited severer phenotypes than the insertional mutant probably because the mys gene was partially active in the insertional mutant. The MO-injected embryo also showed the obscure somite boundary phenotype. Fibronectin and phosphorylated FAK at the intersomitic regions were accumulated at the boundaries at this stage, but, unlike wild type embryos, somitic cells adjacent to the boundaries did not undergo epithelialization, suggesting that Mys is required for epithelialization of the somitic cells. Then in the MO-injected embryos, the boundaries once became clear and distinct, but, in the subsequent stages, disappeared, resulting in abnormal muscle morphogenesis. Accumulation of Fibronectin and phosphorylated FAK observed in the initial stage also disappeared. Thus, Mys is crucial for maintenance of the somite boundaries formed at the initial stage. To analyze the mys defect at the cellular level, we placed cells dissociated from the MO-injected embryo on Fibronectin-coated glasses. By this cell spreading assay, we found that the mys-deficient cells reduced the activity to form lamellipodia on

  11. Insertional mutagenesis using Tnt1 retrotransposon in potato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. Insertional mutagenesis by transposable elements in the mammalian genome.

    PubMed

    Amariglio, N; Rechavi, G

    1993-01-01

    Several mammalian repetitive transposable genetic elements were characterized in recent years, and their role in mutagenesis is delineated in this review. Two main groups have been described: elements with symmetrical termini such as the murine IAP sequences and the human THE 1 elements and elements characterized by a poly-A rich tail at the 3' end such as the SINE and LINE sequences. The characteristic property of such mobile elements to spread and integrate in the host genome leads to insertional mutagenesis. Both germline and somatic mutations have been documented resulting from the insertion of the various types of mammalian repetitive transposable genetic elements. As foreseen by Barbara McClintock, such genetic events can cause either the activation or the inactivation of specific genes, resulting in their identification via an altered phenotype. Several disease states, such as hemophilia and cancer, are the result of this apparent aspect of genome instability. PMID:8385004

  13. Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas.

    PubMed

    Nault, Jean-Charles; Datta, Shalini; Imbeaud, Sandrine; Franconi, Andrea; Mallet, Maxime; Couchy, Gabrielle; Letouzé, Eric; Pilati, Camilla; Verret, Benjamin; Blanc, Jean-Frédéric; Balabaud, Charles; Calderaro, Julien; Laurent, Alexis; Letexier, Mélanie; Bioulac-Sage, Paulette; Calvo, Fabien; Zucman-Rossi, Jessica

    2015-10-01

    Hepatocellular carcinomas (HCCs) are liver tumors related to various etiologies, including alcohol intake and infection with hepatitis B (HBV) or C (HCV) virus. Additional risk factors remain to be identified, particularly in patients who develop HCC without cirrhosis. We found clonal integration of adeno-associated virus type 2 (AAV2) in 11 of 193 HCCs. These AAV2 integrations occurred in known cancer driver genes, namely CCNA2 (cyclin A2; four cases), TERT (telomerase reverse transcriptase; one case), CCNE1 (cyclin E1; three cases), TNFSF10 (tumor necrosis factor superfamily member 10; two cases) and KMT2B (lysine-specific methyltransferase 2B; one case), leading to overexpression of the target genes. Tumors with viral integration mainly developed in non-cirrhotic liver (9 of 11 cases) and without known risk factors (6 of 11 cases), suggesting a pathogenic role for AAV2 in these patients. In conclusion, AAV2 is a DNA virus associated with oncogenic insertional mutagenesis in human HCC. PMID:26301494

  14. Endogenous mutagenesis by an insertion sequence element identifies Aeromonas salmonicida AbcA as an ATP-binding cassette transport protein required for biogenesis of smooth lipopolysaccharide.

    PubMed Central

    Chu, S; Noonan, B; Cavaignac, S; Trust, T J

    1995-01-01

    Analysis of an Aeromonas salmonicida A layer-deficient/O polysaccharide-deficient mutant carrying a Tn5 insertion in the structural gene for A protein (vapA) showed that the abcA gene immediately downstream of vapA had been interrupted by the endogenous insertion sequence element ISAS1. Immunoelectron microscopy showed that O polysaccharides did not accumulate at the inner membrane-cytoplasm interface of this mutant. abcA encodes an unusual protein; it carries both an amino-terminal ATP-binding cassette (ABC) domain showing high sequence similarity to ABC proteins implicated in the transport of certain capsular and O polysaccharides and a carboxyl-terminal potential DNA-binding domain, which distinguishes AbcA from other polysaccharide transport proteins in structural and evolutionary terms. The smooth lipopolysaccharide phenotype was restored by complementation with abcA but not by abcA carrying site-directed mutations in the sequence encoding the ATP-binding site of the protein. The genetic organization of the A. salmonicida ABC polysaccharide system differs from other bacteria. abcA also differs in apparently being required for both O-polysaccharide synthesis and in energizing the transport of O polysaccharides to the cell surface. Images Fig. 2 Fig. 3 Fig. 4 PMID:7777581

  15. Insertional Mutagenesis by a Hybrid PiggyBac and Sleeping Beauty Transposon in the Rat

    PubMed Central

    Furushima, Kenryo; Jang, Chuan-Wei; Chen, Diane W.; Xiao, Ningna; Overbeek, Paul A.; Behringer, Richard R.

    2012-01-01

    A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was ∼10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat. PMID:23023007

  16. Analysis of HIV-2 Vpx by modeling and insertional mutagenesis

    SciTech Connect

    Mahnke, Lisa A. . E-mail: lmahnke@im.wustl.edu; Belshan, Michael; Ratner, Lee . E-mail: lratner@im.wustl.edu

    2006-04-25

    Vpx facilitates HIV-2 nuclear localization by a poorly understood mechanism. We have compared Vpx to an NMR structure HIV-1 Vpr in a central helical domain and probed regions of Vpx by insertional mutagenesis. A predicted loop between helices two and three appears to be unique, overlapping with a known novel nuclear localization signal. Overall, Vpx was found to be surprisingly flexible, tolerating a series of large insertions. We found that insertion within the polyproline-containing C-terminus destabilizes nuclear localization, whereas mutating a second helix in the central domain disrupts viral packaging. Other insertional mutants in the predicted loop and in a linker region between the central domain and the C-terminus may be useful as sites of intramolecular tags as they could be packaged adequately and retained preintegration complex associated integration activity in a serum starvation assay. An unexpected result was found within a previously defined nuclear localization motif near aa 71. This mutant retained robust nuclear localization in a GFP fusion assay and was competent for preintegration complex associated nuclear import. In summary, we have modeled helical content in Vpx and assessed potential sites of intramolecular tags which may prove useful for protein-protein interactions studies.

  17. Functional characterization of the Sindbis virus E2 glycoprotein by transposon linker-insertion mutagenesis

    SciTech Connect

    Navaratnarajah, Chanakha K.; Kuhn, Richard J. . E-mail: kuhnr@purdue.edu

    2007-06-20

    The glycoprotein envelope of alphaviruses consists of two proteins, E1 and E2. E1 is responsible for fusion and E2 is responsible for receptor binding. An atomic structure is available for E1, but one for E2 has not been reported. In this study, transposon linker-insertion mutagenesis was used to probe the function of different domains of E2. A library of mutants, containing 19 amino acid insertions in the E2 glycoprotein sequence of the prototype alphavirus, Sindbis virus (SINV), was generated. Fifty-seven independent E2 insertions were characterized, of which more than half (67%) gave rise to viable virus. The wild-type-like mutants identify regions that accommodate insertions without perturbing virus production and can be used to insert targeting moieties to direct SINV to specific receptors. The defective and lethal mutants give insight into regions of E2 important for protein stability, transport to the cell membrane, E1-E2 contacts, and receptor binding.

  18. Sleeping Beauty transposon insertional mutagenesis based mouse models for cancer gene discovery

    PubMed Central

    Moriarity, Branden S; Largaespada, David A

    2016-01-01

    Large-scale genomic efforts to study human cancer, such as the cancer gene atlas (TCGA), have identified numerous cancer drivers in a wide variety of tumor types. However, there are limitations to this approach, the mutations and expression or copy number changes that are identified are not always clearly functionally relevant, and only annotated genes and genetic elements are thoroughly queried. The use of complimentary, nonbiased, functional approaches to identify drivers of cancer development and progression is ideal to maximize the rate at which cancer discoveries are achieved. One such approach that has been successful is the use of the Sleeping Beauty (SB) transposon-based mutagenesis system in mice. This system uses a conditionally expressed transposase and mutagenic transposon allele to target mutagenesis to somatic cells of a given tissue in mice to cause random mutations leading to tumor development. Analysis of tumors for transposon common insertion sites (CIS) identifies candidate cancer genes specific to that tumor type. While similar screens have been performed in mice with the PiggyBac (PB) transposon and viral approaches, we limit extensive discussion to SB. Here we discuss the basic structure of these screens, screens that have been performed, methods used to identify CIS. PMID:26051241

  19. Mouse Models of Cancer: Sleeping Beauty Transposons for Insertional Mutagenesis Screens and Reverse Genetic Studies

    PubMed Central

    Tschida, Barbara R.; Largaespada, David A.; Keng, Vincent W.

    2014-01-01

    The genetic complexity and heterogeneity of cancer has posed a problem in designing rationally targeted therapies effective in a large proportion of human cancer. Genomic characterization of many cancer types has provided a staggering amount of data that needs to be interpreted to further our understanding of this disease. Forward genetic screening in mice using Sleeping Beauty (SB) based insertional mutagenesis is an effective method for candidate cancer gene discovery that can aid in distinguishing driver from passenger mutations in human cancer. This system has been adapted for unbiased screens to identify drivers of multiple cancer types. These screens have already identified hundreds of candidate cancer-promoting mutations. These can be used to develop new mouse models for further study, which may prove useful for therapeutic testing. SB technology may also hold the key for rapid generation of reverse genetic mouse models of cancer, and has already been used to model glioblastoma and liver cancer. PMID:24468652

  20. Trapping Cardiac Recessive Mutants via Expression-based Insertional Mutagenesis Screening

    PubMed Central

    Ding, Yonghe; Liu, Weibin; Deng, Yun; Jomok, Beninio; Yang, Jingchun; Huang, Wei; Clark, Karl J.; Zhong, Tao P.; Lin, Xueying; Ekker, Stephen C.; Xu, Xiaolei

    2013-01-01

    Rationale Mutagenesis screening is a powerful genetic tool for probing biological mechanisms underlying vertebrate development and human diseases. However, the increased colony management efforts in vertebrates impose a significant challenge for identifying genes affecting a particular organ such as the heart, especially those exhibiting adult phenotypes upon depletion. Objective We aim to develop a facile approach that streamlines colony management efforts via enriching cardiac mutants, which enables us to screen for adult phenotypes. Methods and Results The transparency of the zebrafish embryos enabled us to score 67 stable transgenic lines generated from an insertional mutagenesis screen using a transposon-based protein trapping vector. Fifteen lines with cardiac monomeric red fluorescent protein (mRFP) reporter expression were identified. We defined the molecular nature for 10 lines and bred them to homozygosity, which led to the identification of one embryonic lethal, one larval lethal, and one adult recessive mutant exhibiting cardiac hypertrophy at one year of age. Further characterization of these mutants uncovered an essential function of methionine adenosyltransferase II, alpha a (mat2aa) in cardiogenesis, an essential function of mitochondrial ribosomal protein S18B (mrps18b) in cardiac mitochondrial homeostasis, as well as a function of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b) in adult cardiac hypertrophy. Conclusions We demonstrate that transposon-based gene trapping is an efficient approach for identifying both embryonic and adult recessive mutants with cardiac expression. The generation of a Zebrafish Insertional Cardiac (ZIC) mutant collection shall facilitate the annotation of a vertebrate cardiac genome, as well as enable heart-based adult screens. PMID:23283723

  1. Identification of pathogenicity-related genes in the rice pathogen Ustilaginoidea virens through random insertional mutagenesis.

    PubMed

    Yu, Mina; Yu, Junjie; Hu, Jiankun; Huang, Lei; Wang, Yahui; Yin, Xiaole; Nie, Yafeng; Meng, Xiangkun; Wang, Weiduo; Liu, Yongfeng

    2015-03-01

    Agrobacterium tumefaciens-mediated transformation (ATMT) is becoming a popular effective system as an insertional mutagenesis tool in filamentous fungi. To gain more insight into the cellular and molecular mechanisms in the pathogenesis of Ustilaginoidea virens, the causal agent of rice false smut disease, a T-DNA insertion mutant library of U. virens was established using ATMT. We optimized a range of conditions to improve the transformation efficiency. Transformants were most effectively obtained when the optimal co-cultivation time is 72h, with 50μM AS in medium and 100μl A. tumefaciens for co-cultivation, leading to the production of 160-185 hygromycin B resistant transformants per 1×10(5) conidia. Southern blot analysis indicated that 58.14% of transformants had a single T-DNA copy. Among 5600 transformants tested for virulence, 37 mutants with reproducible pathogenic defects were obtained. The flanking sequences of three avirulent tranformants (B20, B1015 and B1465) and two pathogenicity-reduced transformants (B726 and B785) were amplified by high-efficiency thermal asymmetric interlaced PCR. Sequence analyses revealed that single T-DNA insertion in mutant B20 targeted the coding region of a gene encoding a protein highly similar to SUN family protein, and in mutant B726 targeted upstream of a gene with unknown function. The two T-DNA insertion sites in mutant B785 were found in the coding region of a gene encoding C6 transcription factor, but failed in amplified flanking sequence of another T-DNA. Chromosomal rearrangement occurred in the genome of mutant B1016 and B1465 with single T-DNA insertion. Among avirulent mutants, B20 showed altered colony growth and pigmentation. The T-DNA insert in B20 was detected in the coding region of a gene named UvSUN2. Morphophysiological characterization analysis suggested that UvSUN2 might be a virulence factor, and possibly required for proper fungal growth, cell wall construction, and stress responses in U. virens

  2. Ds insertion mutagenesis as an efficient tool to produce diverse variations for rice breeding.

    PubMed

    Jiang, Shu-Ye; Bachmann, Doris; La, Honggui; Ma, Zhigang; Venkatesh, Prasanna Nori; Ramamoorthy, Rengasamy; Ramachandran, Srinivasan

    2007-11-01

    The availability of diversified germplasm resources is the most important for developing improved rice varieties with higher seed yield or tolerance to various biotic or abiotic stresses. Here we report an efficient tool to create increased variations in rice by maize Ac/Ds transposon (a gene trap system) insertion mutagenesis. We have generated around 20,000 Ds insertion rice lines of which majority are homozygous for Ds element. We subjected these lines to phenotypic and abiotic stress screens and evaluated these lines with respect to their seed yields and other agronomic traits as well as their tolerance to drought, salinity and cold. Based on this evaluation, we observed that random Ds insertions into rice genome have led to diverse variations including a range of morphological and conditional phenotypes. Such differences in phenotype among these lines were accompanied by differential gene expression revealed by GUS histochemical staining of gene trapped lines. Among the various phenotypes identified, some Ds lines showed significantly higher grain yield compared to wild-type plants under normal growth conditions indicating that rice could be improved in grain yield by disrupting certain endogenous genes. In addition, several 1,000s of Ds lines were subjected to abiotic stresses to identify conditional mutants. Subsequent to these screens, over 800 lines responsive to drought, salinity or cold stress were obtained, suggesting that rice has the genetic potential to survive under abiotic stresses when appropriate endogenous genes were suppressed. The mutant lines that have higher seed yielding potential or display higher tolerance to abiotic stresses may be used for rice breeding by conventional backcrossing combining with molecular marker-assisted selection. In addition, by exploiting the behavior of Ds to leave footprints upon remobilization, we have shown an alternative strategy to develop new rice varieties without foreign DNA sequences in their genome. PMID

  3. Insertion mutagenesis of the yeast Candida famata (Debaryomyces hansenii) by random integration of linear DNA fragments.

    PubMed

    Dmytruk, Kostyantyn V; Voronovsky, Andriy Y; Sibirny, Andriy A

    2006-09-01

    The feasibility of using random insertional mutagenesis to isolate mutants of the flavinogenic yeast Candida famata was explored. Mutagenesis was performed by transformation of the yeast with an integrative plasmid containing the Saccharomyces cerevisiae LEU2 gene as a selective marker. The addition of restriction enzyme together with the plasmid (restriction enzyme-mediated integration, REMI) increased the transformation frequency only slightly. Integration of the linearized plasmid occurred randomly in the C. famata genome. To investigate the potential of insertional mutagenesis, it was used for tagging genes involved in positive regulation of riboflavin synthesis in C. famata. Partial DNA sequencing of tagged genes showed that they were homologous to the S. cerevisiae genes RIB1, MET2, and SEF1. Intact orthologs of these genes isolated from Debaryomyces hansenii restored the wild phenotype of the corresponding mutants, i.e., the ability to overproduce riboflavin under iron limitation. The Staphylococcus aureus ble gene conferring resistance to phleomycin was used successfully in the study as a dominant selection marker for C. famata. The results obtained indicate that insertional mutagenesis is a powerful tool for tagging genes in C. famata. PMID:16770625

  4. The use of T-DNA insertional mutagenesis to improve cellulase production by the thermophilic fungus Humicola insolens Y1

    PubMed Central

    Xu, Xinxin; Li, Jinyang; Shi, Pengjun; Ji, Wangli; Liu, Bo; Zhang, Yuhong; Yao, Bin; Fan, Yunliu; Zhang, Wei

    2016-01-01

    Humicola insolens is an excellent producer of pH-neutral active, thermostable cellulases that find many industrial applications. In the present study, we developed an efficient Agrobacterium tumefaciens-mediated transformation system for H. insolens. We transformed plasmids carrying the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of H. insolens driving the transcription of genes encoding neomycin phosphotransferase, hygromycin B phosphotransferase, and enhanced green fluorescent protein. We optimized transformation efficiency to obtain over 300 transformants/106 conidia. T-DNA insertional mutagenesis was employed to generate an H. insolens mutant library, and we isolated a transformant termed T4 with enhanced cellulase and hemicellulase activities. The FPase, endoglucanase, cellobiohydrolase, β-glucosidase, and xylanase activities of T4, measured at the end of fermentation, were 60%, 440%, 320%, 41%, and 81% higher than those of the wild-type strain, respectively. We isolated the sequences flanking the T-DNA insertions and thus identified new genes potentially involved in cellulase and hemicellulase production. Our results show that it is feasible to use T-DNA insertional mutagenesis to identify novel candidate genes involved in cellulase production. This will be valuable when genetic improvement programs seeking to enhance cellulase production are planned, and will also allow us to gain a better understanding of the genetics of the thermophilic fungus H. insolens. PMID:27506519

  5. The use of T-DNA insertional mutagenesis to improve cellulase production by the thermophilic fungus Humicola insolens Y1.

    PubMed

    Xu, Xinxin; Li, Jinyang; Shi, Pengjun; Ji, Wangli; Liu, Bo; Zhang, Yuhong; Yao, Bin; Fan, Yunliu; Zhang, Wei

    2016-01-01

    Humicola insolens is an excellent producer of pH-neutral active, thermostable cellulases that find many industrial applications. In the present study, we developed an efficient Agrobacterium tumefaciens-mediated transformation system for H. insolens. We transformed plasmids carrying the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of H. insolens driving the transcription of genes encoding neomycin phosphotransferase, hygromycin B phosphotransferase, and enhanced green fluorescent protein. We optimized transformation efficiency to obtain over 300 transformants/10(6) conidia. T-DNA insertional mutagenesis was employed to generate an H. insolens mutant library, and we isolated a transformant termed T4 with enhanced cellulase and hemicellulase activities. The FPase, endoglucanase, cellobiohydrolase, β-glucosidase, and xylanase activities of T4, measured at the end of fermentation, were 60%, 440%, 320%, 41%, and 81% higher than those of the wild-type strain, respectively. We isolated the sequences flanking the T-DNA insertions and thus identified new genes potentially involved in cellulase and hemicellulase production. Our results show that it is feasible to use T-DNA insertional mutagenesis to identify novel candidate genes involved in cellulase production. This will be valuable when genetic improvement programs seeking to enhance cellulase production are planned, and will also allow us to gain a better understanding of the genetics of the thermophilic fungus H. insolens. PMID:27506519

  6. piggyBac transposon-based insertional mutagenesis in mouse haploid embryonic stem cells.

    PubMed

    Pettitt, Stephen J; Tan, E-Pien; Yusa, Kosuke

    2015-01-01

    Forward genetic screening is a powerful non-hypothesis-driven approach to unveil the molecular mechanisms and pathways underlying phenotypes of interest. In this approach, a genome-wide mutant library is first generated and then screened for a phenotype of interest. Subsequently, genes responsible for the phenotype are identified. There have been a number of successful screens in yeasts, Caenorhabditis elegans and Drosophila. These model organisms all allow loss-of-function mutants to be generated easily on a genome-wide scale: yeasts have a haploid stage in their reproductive cycles and the latter two organisms have short generation times, allowing mutations to be systematically bred to homozygosity. However, in mammals, the diploid genome and long generation time have always hampered rapid and efficient production of homozygous mutant cells and animals. The recent discovery of several haploid mammalian cell lines promises to revolutionize recessive genetic screens in mammalian cells. In this protocol, we describe an overview of insertional mutagenesis, focusing on DNA transposons, and provide a method for an efficient generation of genome-wide mutant libraries using mouse haploid embryonic stem cells. PMID:25408399

  7. piggyBac-based insertional mutagenesis in the presence of stably integrated P elements in Drosophila.

    PubMed

    Hacker, Udo; Nystedt, Sverker; Barmchi, Mojgan Padash; Horn, Carsten; Wimmer, Ernst A

    2003-06-24

    P element-mediated mutagenesis has been used to disrupt an estimated 25% of genes essential for Drosophila adult viability. Mutation of all genes in the fly genome, however, poses a problem, because P elements show significant hotspots of integration. In addition, advanced screening scenarios often require the use of P element-based tools like the generation of germ-line mosaics using FLP recombinase-mediated recombination or gene misexpression using the UAS/Gal4 system. These techniques are P element-based and can therefore not be combined with the use of P elements as mutagenic agents. To circumvent these limitations, we have developed an insertional mutagenesis system using non-P element transposons. An enhanced yellow fluorescent protein-marked piggyBac-based mutator element was mobilized by a piggyBac specific transposase source expressed from a Hermes-based jump-starter transposon marked with enhanced cyan fluorescent protein. In a pilot screen, we have generated 798 piggyBac insertions on FRT bearing third chromosomes of which 9% have sustained a putatively piggyBac-related lethal hit. The FRTs present on the target chromosome remained stably integrated during the screen and could subsequently be used to generate germ-line clones associated with maternal and zygotic phenotypes. PCR-based analysis of insertion loci shows that 57% of the insertions are in genes for which no P element insertions have been reported. Our data demonstrate the potential of this technique to facilitate the quest for saturation mutagenesis of the Drosophila genome. The system is Drosophila nonspecific and potentially applicable in a broad spectrum of nonmodel organisms. PMID:12802016

  8. Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum

    PubMed Central

    Kemski, Megan M.; Stevens, Bryan; Rappleye, Chad A.

    2012-01-01

    Agrobacterium-mediated transformation is being increasingly used for insertional mutagenesis of fungi. To better evaluate its effectiveness as a mutagen for the fungal pathogen Histoplasma capsulatum, we analyzed a collection of randomly selected T-DNA insertion mutants. Testing of different T-DNA element vectors engineered for transformation of fungi showed that pBHt2 provides the highest transformation efficiency and the lowest rate of vector backbone carryover. Sixty-eight individual T-DNA integrations were characterized by recovery of T-DNA ends and flanking genomic sequences. The right border end of the T-DNA is largely preserved whereas the left border end is frequently truncated. Analysis of T-DNA insertion sites confirms the lack of any integration hotspots in the Histoplasma genome. Relative to genes, T-DNA integrations show significant bias towards promoter regions at the expense of coding sequences. With consideration for potential promoter interruption and the demonstrated efficacy of intronic insertions, 61% of mapped T-DNA insertions should impair gene expression or function. Mapping of T-DNA flanking sequences demonstrates 67% of T-DNA integrations are integrations at a single chromosomal site and 31% of T-DNA integrations are associated with large-scale chromosomal rearrangements. This characterization of T-DNA insertions in mutants selected without regard to phenotype supports application of Agrobacterium-mediated transformation as an insertional mutagen for genome-based screens and functional discovery of genes in Histoplasma. PMID:23332832

  9. Bidirectional promoter trapping T-DNA for insertional mutagenesis in Verticillium dahliae.

    PubMed

    Deng, Sheng; Wang, Cai-yue; Zhang, Xin; Lin, Ling

    2014-07-01

    Transfer DNA (T-DNA)-based random insertional mutagenesis is a universal forward genetic approach for gene identification and cloning in many phytopathogenic fungi. In a large number of randomly selected transformants, screening for mutants with a specific phenotype is laborious, especially for pathogenicity-defective mutants. To accelerate mutant screening and gene identification, a bidirectional promoter-trapping Ti binary vector, 1300-bisGFP-hyg, was constructed and deployed in this study. More than 6000 Verticillium dahliae transformants were obtained by the mediation of Agrobacterium tumefaciens carrying the vector. One thousand randomly selected transformants were cultured on Czapek-Dox and on Czapek-Dox plus cotton root extract media plates. The cultured transformants with green fluorescent protein (GFP) expression or changes in phenotype were selected and used in virulence or promoter-trapping assays. Based on the virulence assay of 60 transformants, the pathogenicity of 17 of these mutants was compromised. Ten pathogenicity-defective mutants were found with GFP expression, and 6 with expression in Czapek-Dox plus cotton root extract media specifically. Using TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction), the T-DNA insertion sites were identified in 8 GFP-expressing transformants, including 5 pathogenicity-defective mutants and 3 unaffected transformants. Promoters of 6 genes were successfully trapped using the T-DNA method in this study. The nonpathogenic transformant 24C9 was the subject of additional investigation. It displayed strong GFP expression on water agar medium supplemented with cotton root extracts and on cotton seedling stems. The results obtained by Southern blot and quantitative real-time PCR confirmed that the transcription level of VdUGPU (encoding UTP-glucose-1-phosphate uridylyltransferase) was significantly reduced owing to T-DNA insertion in the gene promoter region. These results indicate that the bidirectional

  10. High rates of Ac/Ds germinal transposition in Arabidopsis suitable for gene isolation by insertional mutagenesis.

    PubMed Central

    Grevelding, C; Becker, D; Kunze, R; von Menges, A; Fantes, V; Schell, J; Masterson, R

    1992-01-01

    Overexpression of the Activator (Ac) transposase gene in Arabidopsis thaliana resulted in a minimal germinal transposition frequency of 27% in which independent Dissociation (Ds) transposition events were observed. Molecular analysis of 45 F1 generation Ac/Ds plants indicated that high rates of somatic excision had occurred, and independent germinal insertions were identified in F2 generation progeny plants. A tandem cauliflower mosaic virus (CaMV) promoter fused to two different Ac coding sequences significantly increased the rate of Ds transposition. The CaMV-Ac fusions activated single and multiple copies of two different Ds elements, DsDHFR and Ds35S-1, and reciprocal crosses resulted in similar transposition frequencies. The improved rate of independent germinal transposition observed makes Arabidopsis an ideal system for insertional mutagenesis. Images PMID:1321434

  11. Insertion-Duplication Mutagenesis in Streptococcus pneumoniae: Targeting Fragment Length Is a Critical Parameter in Use as a Random Insertion Tool

    PubMed Central

    Lee, Myeong S.; Seok, Chaok; Morrison, Donald A.

    1998-01-01

    To examine whether insertion-duplication mutagenesis with chimeric DNA as a transformation donor could be valuable as a gene knockout tool for genomic analysis in Streptococcus pneumoniae, we studied the transformation efficiency and targeting specificity of the process by using a nonreplicative vector with homologous targeting inserts of various sizes. Insertional recombination was very specific in targeting homologous sites. While the recombination rate did not depend on which site or region was targeted, it did depend strongly on the size of the targeting insert in the donor plasmid, in proportion to the fifth power of its length for inserts of 100 to 500 bp. The dependence of insertion-duplication events on the length of the targeting homology was quite different from that for linear allele replacement and places certain limits on the design of mutagenesis experiments. The number of independent pneumococcal targeting fragments of uniform size required to knock out any desired fraction of the genes in a model genome with a defined probability was calculated from these data by using a combinatorial theory with simplifying assumptions. The results show that efficient and thorough mutagenesis of a large part of the pneumococcal genome should be practical when using insertion-duplication mutagenesis. PMID:9835564

  12. Random insertion and gene disruption via transposon mutagenesis of Ureaplasma parvum using a mini-transposon plasmid.

    PubMed

    Aboklaish, Ali F; Dordet-Frisoni, Emilie; Citti, Christine; Toleman, Mark A; Glass, John I; Spiller, O Brad

    2014-11-01

    While transposon mutagenesis has been successfully used for Mycoplasma spp. to disrupt and determine non-essential genes, previous attempts with Ureaplasma spp. have been unsuccessful. Using a polyethylene glycol-transformation enhancing protocol, we were able to transform three separate serovars of Ureaplasma parvum with a Tn4001-based mini-transposon plasmid containing a gentamicin resistance selection marker. Despite the large degree of homology between Ureaplasma parvum and Ureaplasma urealyticum, all attempts to transform the latter in parallel failed, with the exception of a single clinical U. urealyticum isolate. PCR probing and sequencing were used to confirm transposon insertion into the bacterial genome and identify disrupted genes. Transformation of prototype serovar 3 consistently resulted in transfer only of sequence between the mini-transposon inverted repeats, but some strains showed additional sequence transfer. Transposon insertion occurred randomly in the genome resulting in unique disruption of genes UU047, UU390, UU440, UU450, UU520, UU526, UU582 for single clones from a panel of screened clones. An intergenic insertion between genes UU187 and UU188 was also characterised. Two phenotypic alterations were observed in the mutated strains: Disruption of a DEAD-box RNA helicase (UU582) altered growth kinetics, while the U. urealyticum strain lost resistance to serum attack coincident with disruption of gene UUR10_137 and loss of expression of a 41 kDa protein. Transposon mutagenesis was used successfully to insert single copies of a mini-transposon into the genome and disrupt genes leading to phenotypic changes in Ureaplasma parvum strains. This method can now be used to deliver exogenous genes for expression and determine essential genes for Ureaplasma parvum replication in culture and experimental models. PMID:25444567

  13. Evaluating Risks of Insertional Mutagenesis by DNA Transposons in Gene Therapy

    PubMed Central

    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

  14. Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA.

    PubMed

    Svitashev, Sergei; Young, Joshua K; Schwartz, Christine; Gao, Huirong; Falco, S Carl; Cigan, A Mark

    2015-10-01

    Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology are reported in maize. DNA vectors expressing maize codon-optimized Streptococcus pyogenes Cas9 endonuclease and single guide RNAs were cointroduced with or without DNA repair templates into maize immature embryos by biolistic transformation targeting five different genomic regions: upstream of the liguleless1 (LIG1) gene, male fertility genes (Ms26 and Ms45), and acetolactate synthase (ALS) genes (ALS1 and ALS2). Mutations were subsequently identified at all sites targeted, and plants containing biallelic multiplex mutations at LIG1, Ms26, and Ms45 were recovered. Biolistic delivery of guide RNAs (as RNA molecules) directly into immature embryo cells containing preintegrated Cas9 also resulted in targeted mutations. Editing the ALS2 gene using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded chlorsulfuron-resistant plants. Double-strand breaks generated by RNA-guided Cas9 endonuclease also stimulated insertion of a trait gene at a site near LIG1 by homology-directed repair. Progeny showed expected Mendelian segregation of mutations, edits, and targeted gene insertions. The examples reported in this study demonstrate the utility of Cas9-guide RNA technology as a plant genome editing tool to enhance plant breeding and crop research needed to meet growing agriculture demands of the future. PMID:26269544

  15. Stem cell gene therapy: the risks of insertional mutagenesis and approaches to minimize genotoxicity

    PubMed Central

    Wu, Chuanfeng

    2012-01-01

    Virus-based vectors are widely used in hematopoietic stem cell (HSC) gene therapy, and have the ability to integrate permanently into genomic DNA, thus driving long-term expression of corrective genes in all hematopoietic lineages. To date, HSC gene therapy has been successfully employed in the clinic for improving clinical outcomes in small numbers of patients with X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID), adrenoleukodystrophy (ALD), thalassemia, chronic granulomatous disease (CGD), and Wiskott-Aldrich syndrome (WAS). However, adverse events were observed during some of these HSC gene therapy clinical trials, linked to insertional activation of proto-oncogenes by integrated proviral vectors leading to clonal expansion and eventual development of leukemia. Numerous studies have been performed to understand the molecular basis of vector-mediated genotoxicity, with the aim of developing safer vectors and lower-risk gene therapy protocols. This review will summarize current information on the mechanisms of insertional mutagenesis in hematopoietic stem and progenitor cells due to integrating gene transfer vectors, discuss the available assays for predicting genotoxicity and mapping vector integration sites, and introduce newly-developed approaches for minimizing genotoxicity as a way to further move HSC gene therapy forward into broader clinical application. PMID:22198747

  16. Retrovirus-induced insertional mutagenesis: mechanism of collagen mutation in Mov13 mice.

    PubMed Central

    Barker, D D; Wu, H; Hartung, S; Breindl, M; Jaenisch, R

    1991-01-01

    The Mov13 mouse strain carries a mutation in the alpha 1(I) procollagen gene which is due to the insertion of a Moloney murine leukemia provirus into the first intron. This insertion results in the de novo methylation of the provirus and flanking DNA, the alteration of chromatin structure, and the transcriptional inactivity of the collagen promoter. To address the mechanism of mutagenesis, we reintroduced a cloned and therefore demethylated version of the Mov13 mutant allele into mouse fibroblasts. The transfected gene was not transcribed, indicating that the transcriptional defect was not due to the hypermethylation. Rather, this result strongly suggests that the mutation is due to the displacement or disruption of cis-acting regulatory DNA sequences within the first intron. We also constructed a Mov13 variant allele containing a single long terminal repeat instead of the whole provirus. This construct also failed to express mRNA, indicating that the Mov13 mutation does not revert by provirus excision as has been observed for other retrovirus-induced mutations. Images PMID:1922037

  17. Insertional mutagenesis and deep profiling reveals gene hierarchies and a Myc/p53-dependent bottleneck in lymphomagenesis.

    PubMed

    Huser, Camille A; Gilroy, Kathryn L; de Ridder, Jeroen; Kilbey, Anna; Borland, Gillian; Mackay, Nancy; Jenkins, Alma; Bell, Margaret; Herzyk, Pawel; van der Weyden, Louise; Adams, David J; Rust, Alistair G; Cameron, Ewan; Neil, James C

    2014-02-01

    Retroviral insertional mutagenesis (RIM) is a powerful tool for cancer genomics that was combined in this study with deep sequencing (RIM/DS) to facilitate a comprehensive analysis of lymphoma progression. Transgenic mice expressing two potent collaborating oncogenes in the germ line (CD2-MYC, -Runx2) develop rapid onset tumours that can be accelerated and rendered polyclonal by neonatal Moloney murine leukaemia virus (MoMLV) infection. RIM/DS analysis of 28 polyclonal lymphomas identified 771 common insertion sites (CISs) defining a 'progression network' that encompassed a remarkably large fraction of known MoMLV target genes, with further strong indications of oncogenic selection above the background of MoMLV integration preference. Progression driven by RIM was characterised as a Darwinian process of clonal competition engaging proliferation control networks downstream of cytokine and T-cell receptor signalling. Enhancer mode activation accounted for the most efficiently selected CIS target genes, including Ccr7 as the most prominent of a set of chemokine receptors driving paracrine growth stimulation and lymphoma dissemination. Another large target gene subset including candidate tumour suppressors was disrupted by intragenic insertions. A second RIM/DS screen comparing lymphomas of wild-type and parental transgenics showed that CD2-MYC tumours are virtually dependent on activation of Runx family genes in strong preference to other potent Myc collaborating genes (Gfi1, Notch1). Ikzf1 was identified as a novel collaborating gene for Runx2 and illustrated the interface between integration preference and oncogenic selection. Lymphoma target genes for MoMLV can be classified into (a) a small set of master regulators that confer self-renewal; overcoming p53 and other failsafe pathways and (b) a large group of progression genes that control autonomous proliferation in transformed cells. These findings provide insights into retroviral biology, human cancer genetics

  18. Validation-based insertional mutagenesis for identification of Nup214 as a host factor for EV71 replication in RD cells

    SciTech Connect

    Wang, Bei; Zhang, XiaoYu; Zhao, Zhendong

    2013-08-02

    Highlights: •We introduced a new mutagenesis strategy named VBIM to the viral research. •This method can identify either host factors or host restriction factors. •Using VBIM system, we identified Nup214 as a host factor for EV71 replication in RD cells. -- Abstract: Lentiviral validation-based insertional mutagenesis (VBIM) is a sophisticated, forward genetic approach that is used for the investigation of signal transduction in mammalian cells. Using VBIM, we conducted function-based genetic screening for host genes that affect enterovirus 71 (EV71) viral replication. This included host factors that are required for the life cycle of EV71 and host restriction factors that inhibit EV71 replication. Several cell clones, resistant to EV71, were produced using EV71 infection as a selection pressure and the nuclear pore protein 214 (Nup214) was identified as a host factor required for EV71 replication. In SD2-2, the corresponding VBIM lentivirus transformed clone, the expression of endogenous Nup214 was significantly down-regulated by the reverse inserted VBIM promoter. After Cre recombinase-mediated excision of the VBIM promoter, the expression of Nup214 recovered and the clone regained sensitivity to the EV71 infection. Furthermore, over-expression of Nup214 in the cells suggested that Nup214 was promoting EV71 replication. Results of this study indicate that a successful mutagenesis strategy has been established for screening host genes related to viral replication.

  19. Development of retroviral vectors for insertional mutagenesis in medaka haploid cells.

    PubMed

    Lin, Fan; Liu, Qizhi; Yuan, Yongming; Hong, Yunhan

    2015-12-01

    Insertional mutagenesis (IM) by retrovirus (RV) is a high-throughput approach for interrogating gene functions in model species. Haploid cell provides a unique system for genetic screening by IM and prosperous progress has been achieved in mammal cells. However, little was known in lower vertebrate cells. Here, we report development of retroviral vectors (rvSAchCVgfp, rvSAchCVpf and rvSAchSTpf) and establishment of IM library in medaka haploid cells. Each vector contains a modified gene trapping (GT) cassette, which could extend the mutated cell population including GT insertions not in-frame or in weakly expressed genes. Virus titration determined by flow cytometry showed that rvSAchSTpf possessed the highest supernatant virus titer (1.5×10(5)TU/ml) in medaka haploid cell, while rvSAchCVpf produced the lowest titer (2.8×10(4)TU/ml). However, quantification of proviral DNAs in transduced cells by droplet digital PCR (ddPCR) demonstrated that the "real titer" may be similar among the three vectors. Furthermore, an IM library was established by FACS of haploid cells transduced with rvSAchCVgfp at a MOI of 0.1. A single copy RV integration in the majority of cells was confirmed by ddPCR in the library. Notably, there was a significant decrease of haploid cell percentage after FACS, suggesting potential trapping for survival/growth essential genes. Our results demonstrated successful development of retroviral vectors for IM in medaka haploid cells, serving for haploid genetic screening of host factors for virus infection and genes underlying certain cellular processes in fish model. PMID:26192464

  20. A Chemical Mutagenesis Screen Identifies Mouse Models with ERG Defects.

    PubMed

    Charette, Jeremy R; Samuels, Ivy S; Yu, Minzhong; Stone, Lisa; Hicks, Wanda; Shi, Lan Ying; Krebs, Mark P; Naggert, Jürgen K; Nishina, Patsy M; Peachey, Neal S

    2016-01-01

    Mouse models provide important resources for many areas of vision research, pertaining to retinal development, retinal function and retinal disease. The Translational Vision Research Models (TVRM) program uses chemical mutagenesis to generate new mouse models for vision research. In this chapter, we report the identification of mouse models for Grm1, Grk1 and Lrit3. Each of these is characterized by a primary defect in the electroretinogram. All are available without restriction to the research community. PMID:26427409

  1. Tryptophan Scanning Mutagenesis Identifies the Molecular Determinants of Distinct Barttin Functions.

    PubMed

    Wojciechowski, Daniel; Fischer, Martin; Fahlke, Christoph

    2015-07-24

    CLC-K chloride channels are expressed in the kidney and in the inner ear and require the accessory subunit barttin for proper function and membrane insertion. Barttin exerts multiple functions on CLC-proteins: it modifies protein stability and intracellular trafficking as well as channel activity, ion conduction, and gating. So far, the molecular determinants of these distinct barttin functions have remained elusive. Here we performed serial perturbation mutagenesis to identify the sequence determinants of barttin function. Barttin consists of two transmembrane helices followed by a long intracellular carboxyl terminus, and earlier work demonstrated that the transmembrane core of barttin suffices for most effects on the α-subunit. We individually substituted every amino acid of the predicted transmembrane core (amino acids 9-26 and 35-55) with tryptophan, co-expressed mutant barttin with hClC-Ka or V166E rClC-K1, and characterized CLC-K/barttin channels by patch clamp techniques, biochemistry, and confocal microscopy. The majority of mutations left the chaperone function of barttin, i.e. the effects on endoplasmic reticulum exit and surface membrane insertion, unaffected. In contrast, tryptophan insertion at multiple positions resulted in impaired activity of hClC-Ka/barttin and changes in gating of V166E rClC-K1/barttin. These results demonstrate that mutations in a cluster of hydrophobic residues within transmembrane domain 1 affect barttin-CLC-K interaction and impair gating modification by the accessory subunit. Whereas tight interaction is necessary for functional modification, even impaired association of barttin and CLC-K suffices for normal intracellular trafficking. Our findings allow definition of a likely interaction surface and clarify the mechanisms underlying CLC-K channel modification by barttin. PMID:26063802

  2. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

    PubMed

    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. PMID:24873830

  3. Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae.

    PubMed

    Liu, Ning; Chen, Guo-Qing; Ning, Guo-Ao; Shi, Huan-Bin; Zhang, Chu-Long; Lu, Jian-Ping; Mao, Li-Juan; Feng, Xiao-Xiao; Liu, Xiao-Hong; Su, Zhen-Zhu; Lin, Fu-Cheng

    2016-01-01

    The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate and biomass yield of rice crops, but also induce systemic resistance against the rice blast fungus, Magnaporthe oryzae. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed and optimized to modify the H. oryzae genes by either random DNA fragment integration or targeted gene replacement. Our results showed that co-cultivation of H. oryzae conidia with A. tumefaciens in the presence of acetosyringone for 48 h at 22 °C could lead to a relatively highest frequency of transformation, and 200 μM acetosyringone (AS) pre-cultivation of A. tumefaciens is also suggested. ATMT-mediated knockout mutagenesis was accomplished with the gene-deletion cassettes using a yeast homologous recombination method with a yeast-Escherichia-Agrobacterium shuttle vector pKOHo. Using the ATMT-mediated knockout mutagenesis, we successfully deleted three genes of H. oryzae (HoATG5, HoATG7, and HoATG8), and then got the null mutants ΔHoatg5, ΔHoatg7, and ΔHoatg8. These results suggest that ATMT is an efficient tool for gene modification including randomly insertional mutagenesis and gene deletion mutagenesis in H. oryzae. PMID:26686612

  4. Insertional mutagenesis by random cloning of antibiotic resistance genes into the genome of the cyanobacterium Synechocystis strain PCC 6803.

    PubMed Central

    Labarre, J; Chauvat, F; Thuriaux, P

    1989-01-01

    The facultative heterotrophic cyanobacterium Synechocystis sp. strain PCC 6803 was transformed by HaeII Cmr fragments ligated at random to HaeII DNA fragments of the host genome. A similar transformation was done with an AvaII Kmr marker ligated to AvaII host DNA fragments. Integration of the resistance markers into the host genome led to a high frequency of stable Kmr and Cmr transformants. Physical analysis of individual transformants indicated that this result was due to homologous recombination by conversionlike events leading to insertion of the Cmr (or Kmr) gene between two HaeII (or AvaII) sites of the host genome, with precise deletion of the host DNA between these sites. In contrast, integrative crossover of circular DNA molecules with homology to the host DNA is very rare in this cyanobacterium. Strain PCC 6803 was shown to have about 12 genomic copies per cell in standard growth conditions, which complicates the detection of recessive mutations induced by chemical or UV mutagenesis. Random disruption of the host DNA by insertional transformation provides a convenient alternative to transposon mutagenesis in cyanobacteria and may help to overcome the difficulties encountered in generating recessive mutants by classical mutagenesis. Images PMID:2498291

  5. Isolation and Characterization of FecA- and FeoB-Mediated Iron Acquisition Systems of the Spirochete Leptospira biflexa by Random Insertional Mutagenesis

    PubMed Central

    Louvel, Hélène; Saint Girons, Isabelle; Picardeau, Mathieu

    2005-01-01

    The specific mechanisms by which Leptospira spp. acquire iron from their ecological niches are unknown. A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. In this study, we have developed a system for random transposon mutagenesis of Leptospira biflexa using a mariner transposon, Himar1. To demonstrate the validity of Himar1 in vivo transposon mutagenesis in L. biflexa, a screen of mutants for clones impaired in amino acid biosynthesis was first performed, enabling the identification of tryptophan and glutamate auxotrophs. To investigate iron transporters, 2,000 L. biflexa transposon mutants were screened onto media with and without hemin, thus allowing the identification of five hemin-requiring mutants, and the putative genes responsible for this phenotype were identified. Three mutants had distinct insertions in a gene encoding a protein which shares homology with the TonB-dependent receptor FecA, involved in ferric citrate transport. We also identified two mutants with a Himar1 insertion into a feoB-like gene, the product of which is required for ferrous iron uptake in many bacterial organisms. Interestingly, the growth inhibition exhibited by the fecA and feoB mutants was relieved by deferoxamine, suggesting the presence of a ferric hydroxamate transporter. These results confirm the importance of iron for the growth of Leptospira and its ability to use multiple iron sources. PMID:15838052

  6. Spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene.

    PubMed Central

    Dolja, V V; Herndon, K L; Pirone, T P; Carrington, J C

    1993-01-01

    The RNA genome of tobacco etch potyvirus (TEV) was engineered to express bacterial beta-glucuronidase (GUS) fused to the virus helper component proteinase (HC-Pro). It was shown previously that prolonged periods (approximately 1 month) of TEV-GUS propagation in plants resulted in the appearance of spontaneous deletion variants. Nine deletion mutants were identified by nucleotide sequence analysis of 40 cDNA clones obtained after polymerase chain reaction amplification. The mutants were missing between 1,741 and 2,074 nucleotides from TEV-GUS, including the sequences coding for most of GUS and the N-terminal region of HC-Pro. This region of HC-Pro contains determinants involved in helper component activity during aphid transmission, as well as a highly conserved series of cysteine residues. The deletion variants were shown to replicate and move systemically without the aid of a helper virus. Infectious viruses harboring the two largest HC-Pro deletions (termed TEV-2del and TEV-7del) were reconstructed by subcloning the corresponding mutated regions into full-length DNA copies of the TEV genome. Characterization of these and additional variants derived by site-directed mutagenesis demonstrated that deletion of sequences coding for the HC-Pro N-terminal domain had a negative effect on accumulation of viral RNA and coat protein. The TEV-2del variant possessed an aphid-nontransmissible phenotype that could be rescued partially by prefeeding of aphids on active HC-Pro from another potyvirus. These data suggest that the N-terminal domain of HC-Pro or its coding sequence enhances virus replication or genome expression but does not provide an activity essential for these processes. The function of this domain, as well as a proposed deletion mechanism involving nonhomologous recombination, is discussed. Images PMID:8371351

  7. Retroviral insertions in the VISION database identify molecular pathways in mouse lymphoid leukemia and lymphoma

    PubMed Central

    Weiser, Keith C.; Liu, Bin; Hansen, Gwenn M.; Skapura, Darlene; Hentges, Kathryn E.; Yarlagadda, Sujatha; Morse III, Herbert C.

    2007-01-01

    AKXD recombinant inbred (RI) strains develop a variety of leukemias and lymphomas due to somatically acquired insertions of retroviral DNA into the genome of hematopoetic cells that can mutate cellular proto-oncogenes and tumor suppressor genes. We generated a new set of tumors from nine AKXD RI strains selected for their propensity to develop B-cell tumors, the most common type of human hematopoietic cancers. We employed a PCR technique called viral insertion site amplification (VISA) to rapidly isolate genomic sequence at the site of provirus insertion. Here we describe 550 VISA sequence tags (VSTs) that identify 74 common insertion sites (CISs), of which 21 have not been identified previously. Several suspected proto-oncogenes and tumor suppressor genes lie near CISs, providing supportive evidence for their roles in cancer. Furthermore, numerous previously uncharacterized genes lie near CISs, providing a pool of candidate disease genes for future research. Pathway analysis of candidate genes identified several signaling pathways as common and powerful routes to blood cancer, including Notch, E-protein, NFκB, and Ras signaling. Misregulation of several Notch signaling genes was confirmed by quantitative RT-PCR. Our data suggest that analyses of insertional mutagenesis on a single genetic background are biased toward the identification of cooperating mutations. This tumor collection represents the most comprehensive study of the genetics of B-cell leukemia and lymphoma development in mice. We have deposited the VST sequences, CISs in a genome viewer, histopathology, and molecular tumor typing data in a public web database called VISION (Viral Insertion Sites Identifying Oncogenes), which is located at http://www.mouse-genome.bcm.tmc.edu/vision. PMID:17926094

  8. Transposon mutagenesis identifies genes and cellular processes driving epithelial-mesenchymal transition in hepatocellular carcinoma.

    PubMed

    Kodama, Takahiro; Newberg, Justin Y; Kodama, Michiko; Rangel, Roberto; Yoshihara, Kosuke; Tien, Jean C; Parsons, Pamela H; Wu, Hao; Finegold, Milton J; Copeland, Neal G; Jenkins, Nancy A

    2016-06-14

    Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), leading to their poor prognosis. The genes driving EMT in HCC are not yet fully understood, however. Here, we show that mobilization of Sleeping Beauty (SB) transposons in immortalized mouse hepatoblasts induces mesenchymal liver tumors on transplantation to nude mice. These tumors show significant down-regulation of epithelial markers, along with up-regulation of mesenchymal markers and EMT-related transcription factors (EMT-TFs). Sequencing of transposon insertion sites from tumors identified 233 candidate cancer genes (CCGs) that were enriched for genes and cellular processes driving EMT. Subsequent trunk driver analysis identified 23 CCGs that are predicted to function early in tumorigenesis and whose mutation or alteration in patients with HCC is correlated with poor patient survival. Validation of the top trunk drivers identified in the screen, including MET (MET proto-oncogene, receptor tyrosine kinase), GRB2-associated binding protein 1 (GAB1), HECT, UBA, and WWE domain containing 1 (HUWE1), lysine-specific demethylase 6A (KDM6A), and protein-tyrosine phosphatase, nonreceptor-type 12 (PTPN12), showed that deregulation of these genes activates an EMT program in human HCC cells that enhances tumor cell migration. Finally, deregulation of these genes in human HCC was found to confer sorafenib resistance through apoptotic tolerance and reduced proliferation, consistent with recent studies showing that EMT contributes to the chemoresistance of tumor cells. Our unique cell-based transposon mutagenesis screen appears to be an excellent resource for discovering genes involved in EMT in human HCC and potentially for identifying new drug targets. PMID:27247392

  9. Gene-Trap Mutagenesis Identifies Mammalian Genes Contributing to Intoxication by Clostridium perfringens ε-Toxin

    PubMed Central

    Ivie, Susan E.; Fennessey, Christine M.; Sheng, Jinsong; Rubin, Donald H.; McClain, Mark S.

    2011-01-01

    The Clostridium perfringens ε-toxin is an extremely potent toxin associated with lethal toxemias in domesticated ruminants and may be toxic to humans. Intoxication results in fluid accumulation in various tissues, most notably in the brain and kidneys. Previous studies suggest that the toxin is a pore-forming toxin, leading to dysregulated ion homeostasis and ultimately cell death. However, mammalian host factors that likely contribute to ε-toxin-induced cytotoxicity are poorly understood. A library of insertional mutant Madin Darby canine kidney (MDCK) cells, which are highly susceptible to the lethal affects of ε-toxin, was used to select clones of cells resistant to ε-toxin-induced cytotoxicity. The genes mutated in 9 surviving resistant cell clones were identified. We focused additional experiments on one of the identified genes as a means of validating the experimental approach. Gene expression microarray analysis revealed that one of the identified genes, hepatitis A virus cellular receptor 1 (HAVCR1, KIM-1, TIM1), is more abundantly expressed in human kidney cell lines than it is expressed in human cells known to be resistant to ε-toxin. One human kidney cell line, ACHN, was found to be sensitive to the toxin and expresses a larger isoform of the HAVCR1 protein than the HAVCR1 protein expressed by other, toxin-resistant human kidney cell lines. RNA interference studies in MDCK and in ACHN cells confirmed that HAVCR1 contributes to ε-toxin-induced cytotoxicity. Additionally, ε-toxin was shown to bind to HAVCR1 in vitro. The results of this study indicate that HAVCR1 and the other genes identified through the use of gene-trap mutagenesis and RNA interference strategies represent important targets for investigation of the process by which ε-toxin induces cell death and new targets for potential therapeutic intervention. PMID:21412435

  10. Site-selected insertional mutagenesis of tomato with maize Ac and Ds elements.

    PubMed

    Cooley, M B; Goldsbrough, A P; Still, D W; Yoder, J I

    1996-08-27

    Site-selected insertion (SSI) is a PCR-based technique which uses primers located within the transposon and a target gene for detection of transposon insertions into cloned genes. We screened tomato plants bearing single or multiple copies of maize Ac or Ds transposable elements for somatic insertions at one close-range target and two long-range targets. Eight close-range Ds insertions near the right border of the T-DNA were recovered. Sequence analysis showed a precise junction between the transposon and the target for all insertions. Two insertions in separate plants occurred at the same site, but others appeared dispersed in the region of the right T-DNA border with no target specificity. However, insertions showed a preference for one orientation of the transposon. Use of plants with multiple Ac (HiAc) or Ds (HiDs) elements allowed detection of somatic insertions at two single-copy genes, PG (polygalacturonase) and DFR (dihydroflavonol 4-reductase). Certain HiDs plants showed much higher rates of insertion into PG than others. Insertions in PG and DFR were found throughout the gene regions monitored and, with the exception of one insertion in PG, the junctions between transposon and target were exact. SSI analysis of progeny from the HiDs parents revealed that in some cases the tendency to incur high levels of somatic insertions in PG was inherited. Inheritance of this character is an indication that SSI could be used to direct a search for germinal PG insertions in tomato. PMID:8804392

  11. Insertional Mutagenesis for Genes involved in Otic/Vestibular Development and Function in Xenopus Tropicalis

    NASA Technical Reports Server (NTRS)

    Torrejon, Marcela; Li, Erica; Nguyen, Minh; Winfree, Seth; Wang, Esther; Reinsch, Sigrid; Dalton, Bonnie (Technical Monitor)

    2002-01-01

    Sensitivity to gravity is essential for spatial orientation. Consequently, the gravity receptor system is one of the phylogenetically oldest sensory systems, and the special adaptations that enhance sensitivity to gravity are highly conserved. The main goal of this project is to use Xenopus (frog) to identify genes expressed during vestibular and auditory development. These studies will lead a better understanding of the molecular mechanisms involved in vestibular and auditory development and function. We are using a gene-trap approach in Xenopus tropicalis with the green fluorescent protein (GFP) gene as the transgene reporter. GFP expression occurs only when the GFP gene is correctly integrated in actively transcribed genes. Using the GFP as a tag we can easily identify and clone the mutated gene. In addition, we can study the function of the mutated gene by analyzing the defects generated by insertion of the GFP transgene. To date we have tissue specific GFP expression in X. tropicalis including expression in ear, neural tube, kidney, muscle, eyes and nose. Our transgenic animals will soon reach maturity so that we can outcross them and analyze their progeny. Our next goal is to isolate RNA from our transgenics and clone the tagged genes using RACE-PCR. Currently we are optimizing the RACE-PCR method using transgenics with crystallin GFP expression.

  12. Neurobehavioral Mutants Identified in an ENU Mutagenesis Project

    SciTech Connect

    Cook, Melloni N.; Dunning, Jonathan P; Wiley, Ronald G; Chesler, Elissa J; Johnson, Dabney K; Goldowitz, Daniel

    2007-01-01

    We report on a behavioral screening test battery that successfully identified several neurobehavioral mutants among a large-scale ENU-mutagenized mouse population. Large numbers of ENU mutagenized mice were screened for abnormalities in central nervous system function based on abnormal performance in a series of behavior tasks. We developed and employed a high-throughput screen of behavioral tasks to detect behavioral outliers. Twelve mutant pedigrees, representing a broad range of behavioral phenotypes, have been identified. Specifically, we have identified two open field mutants (one displaying hyper-locomotion, the other hypo-locomotion), four tail suspension mutants (all displaying increased immobility), one nociception mutant (displaying abnormal responsiveness to thermal pain), two prepulse inhibition mutants (displaying poor inhibition of the startle response), one anxiety-related mutant (displaying decreased anxiety in the light/dark test), and one learning and memory mutant (displaying reduced response to the conditioned stimulus) These findings highlight the utility of a set of behavioral tasks used in a high throughput screen to identify neurobehavioral mutants. Further analysis (i.e., behavioral and genetic mapping studies) of mutants is in progress with the ultimate goal of identification of novel genes and mouse models relevant to human disorders as well as the identification of novel therapeutic targets.

  13. Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in medicinal fungus Cordyceps militaris.

    PubMed

    Zheng, Zhuangli; Huang, Chuanhua; Cao, Li; Xie, Cuihong; Han, Richou

    2011-03-01

    Cordyceps militaris is an insect-born fungus with various biological and pharmacological activities. The mutant library of C. militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation (ATMT), for the ultimate identification of genes involved in isolate degeneration during fruiting body production. Successful transformation of C. militaris JM4 by A. tumefaciens AGL-1 carrying vector pATMT1 was performed, with efficiency in the range of 30-600 transformants per 1×10(5) conidia. Acetosyringone (AS) supplement in C. militaris ATMT was not necessary during either precultivation or cocultivation. The transformation procedure was optimised based on the ratios between donor A. tumefaciens and recipient conidia, and pH value of cocultivation media. The integration of the hyg gene into C. militaris genome was determined by PCR and Southern blot analysis, suggesting that 67-88% resulting transformants in cultivation conditions with or without AS were inserted by T-DNA and 55-80% were single-copy. Special mutants with altered phenotypes and growth potentials were characterised. The efficient TAIL-PCR approach was established for identifying T-DNA flanking sequences from C. militaris mutants. The successful construction of the mutant library indicated the usefulness of this approach for functional genetic analysis in this important fungus. PMID:21354533

  14. Identification of the O antigen polymerase (rfc) gene in Escherichia coli O4 by insertional mutagenesis using a nonpolar chloramphenicol resistance cassette.

    PubMed Central

    Lukomski, S; Hull, R A; Hull, S I

    1996-01-01

    Computer analysis of the O4 polysaccharide gene cluster of Escherichia coli revealed the presence of two open reading frames (ORFs) encoding strongly hydrophobic polypeptides. O antigen polymerase, which is encoded by the rfc gene, is a potential membrane protein and therefore should be hydrophobic. To identify the rfc gene, these two ORFs were subjected to insertional mutagenesis. A chloramphenicol resistance cassette was designed which, when properly inserted, does not cause a polar effect in downstream genes. Each of two ORFs, cloned into a plasmid vector, was inactivated with this cassette. Two types of mutants bearing chromosomal insertions of the cassettes in each ORF were constructed by homologous recombination. These mutants were characterized by PCR, Southern blotting, and transverse-alternating-field electrophoresis. Only one class of mutants exhibited the expected O polymerase-deficient phenotype; they produced O4-specific, semirough lipopolysaccharide. Therefore, this ORF was identified as the rfc gene. The chromosomal rfc mutation was complemented in trans by the rfc gene expressed from a plasmid vector. PMID:8550424

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  16. Gene-scrambling mutagenesis: generation and analysis of insertional mutations in the alginate regulatory region of Pseudomonas aeruginosa.

    PubMed

    Mohr, C D; Deretic, V

    1990-11-01

    A novel method for random mutagenesis of targeted chromosomal regions in Pseudomona aeruginosa was developed. This method can be used with a cloned DNA fragment of indefinite size that contains a putative gene of interest. Cloned DNA is digested to produce small fragments that are then randomly reassembled into long DNA inserts by using cosmid vectors and lambda packaging reaction. This DNA is then transferred into P. aeruginosa and forced into the chromosome via homologous recombination, producing in a single step a random set of insertional mutants along a desired region of the chromosome. Application of this method to extend the analysis of the alginate regulatory region, using a cloned 6.2-kb fragment with the algR gene and the previously uncharacterized flanking regions, produced several insertional mutations. One mutation was obtained in algR, a known transcriptional regulatory of mucoidy in P. aeruginosa. The null mutation of algR was generated in a mucoid derivative of the standard genetic strain PAO responsive to different environmental factors. This mutation was used to demonstrate that the algR gene product was not essential for the regulation of its promoters. Additional insertions were obtained in regions downstream and upstream of algR. A mutation that did not affect mucoidy was generated in a gene located 1 kb upstream of algR. This gene was transcribed in the direction opposite that of algR transcription and encoded a polypeptide of 47 kDa. Partial nucleotide sequence analysis revealed strong homology of its predicted gene product with the human and yeast argininosuccinate lyases. An insertion downstream of algR produced a strain showing reduced induction of mucoidy in response to growth on nitrate as the nitrogen source. PMID:2121708

  17. Gene-scrambling mutagenesis: generation and analysis of insertional mutations in the alginate regulatory region of Pseudomonas aeruginosa.

    PubMed Central

    Mohr, C D; Deretic, V

    1990-01-01

    A novel method for random mutagenesis of targeted chromosomal regions in Pseudomona aeruginosa was developed. This method can be used with a cloned DNA fragment of indefinite size that contains a putative gene of interest. Cloned DNA is digested to produce small fragments that are then randomly reassembled into long DNA inserts by using cosmid vectors and lambda packaging reaction. This DNA is then transferred into P. aeruginosa and forced into the chromosome via homologous recombination, producing in a single step a random set of insertional mutants along a desired region of the chromosome. Application of this method to extend the analysis of the alginate regulatory region, using a cloned 6.2-kb fragment with the algR gene and the previously uncharacterized flanking regions, produced several insertional mutations. One mutation was obtained in algR, a known transcriptional regulatory of mucoidy in P. aeruginosa. The null mutation of algR was generated in a mucoid derivative of the standard genetic strain PAO responsive to different environmental factors. This mutation was used to demonstrate that the algR gene product was not essential for the regulation of its promoters. Additional insertions were obtained in regions downstream and upstream of algR. A mutation that did not affect mucoidy was generated in a gene located 1 kb upstream of algR. This gene was transcribed in the direction opposite that of algR transcription and encoded a polypeptide of 47 kDa. Partial nucleotide sequence analysis revealed strong homology of its predicted gene product with the human and yeast argininosuccinate lyases. An insertion downstream of algR produced a strain showing reduced induction of mucoidy in response to growth on nitrate as the nitrogen source. Images PMID:2121708

  18. Microarray analyses reveal that plant mutagenesis may induce more transcriptomic changes than transgene insertion

    PubMed Central

    Batista, Rita; Saibo, Nelson; Lourenço, Tiago; Oliveira, Maria Margarida

    2008-01-01

    Controversy regarding genetically modified (GM) plants and their potential impact on human health contrasts with the tacit acceptance of other plants that were also modified, but not considered as GM products (e.g., varieties raised through conventional breeding such as mutagenesis). What is beyond the phenotype of these improved plants? Should mutagenized plants be treated differently from transgenics? We have evaluated the extent of transcriptome modification occurring during rice improvement through transgenesis versus mutation breeding. We used oligonucleotide microarrays to analyze gene expression in four different pools of four types of rice plants and respective controls: (i) a γ-irradiated stable mutant, (ii) the M1 generation of a 100-Gy γ-irradiated plant, (iii) a stable transgenic plant obtained for production of an anticancer antibody, and (iv) the T1 generation of a transgenic plant produced aiming for abiotic stress improvement, and all of the unmodified original genotypes as controls. We found that the improvement of a plant variety through the acquisition of a new desired trait, using either mutagenesis or transgenesis, may cause stress and thus lead to an altered expression of untargeted genes. In all of the cases studied, the observed alteration was more extensive in mutagenized than in transgenic plants. We propose that the safety assessment of improved plant varieties should be carried out on a case-by-case basis and not simply restricted to foods obtained through genetic engineering. PMID:18303117

  19. Microarray analyses reveal that plant mutagenesis may induce more transcriptomic changes than transgene insertion.

    PubMed

    Batista, Rita; Saibo, Nelson; Lourenço, Tiago; Oliveira, Maria Margarida

    2008-03-01

    Controversy regarding genetically modified (GM) plants and their potential impact on human health contrasts with the tacit acceptance of other plants that were also modified, but not considered as GM products (e.g., varieties raised through conventional breeding such as mutagenesis). What is beyond the phenotype of these improved plants? Should mutagenized plants be treated differently from transgenics? We have evaluated the extent of transcriptome modification occurring during rice improvement through transgenesis versus mutation breeding. We used oligonucleotide microarrays to analyze gene expression in four different pools of four types of rice plants and respective controls: (i) a gamma-irradiated stable mutant, (ii) the M1 generation of a 100-Gy gamma-irradiated plant, (iii) a stable transgenic plant obtained for production of an anticancer antibody, and (iv) the T1 generation of a transgenic plant produced aiming for abiotic stress improvement, and all of the unmodified original genotypes as controls. We found that the improvement of a plant variety through the acquisition of a new desired trait, using either mutagenesis or transgenesis, may cause stress and thus lead to an altered expression of untargeted genes. In all of the cases studied, the observed alteration was more extensive in mutagenized than in transgenic plants. We propose that the safety assessment of improved plant varieties should be carried out on a case-by-case basis and not simply restricted to foods obtained through genetic engineering. PMID:18303117

  20. Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA[OPEN

    PubMed Central

    Schwartz, Christine; Gao, Huirong; Falco, S. Carl; Cigan, A. Mark

    2015-01-01

    Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology are reported in maize. DNA vectors expressing maize codon-optimized Streptococcus pyogenes Cas9 endonuclease and single guide RNAs were cointroduced with or without DNA repair templates into maize immature embryos by biolistic transformation targeting five different genomic regions: upstream of the liguleless1 (LIG1) gene, male fertility genes (Ms26 and Ms45), and acetolactate synthase (ALS) genes (ALS1 and ALS2). Mutations were subsequently identified at all sites targeted, and plants containing biallelic multiplex mutations at LIG1, Ms26, and Ms45 were recovered. Biolistic delivery of guide RNAs (as RNA molecules) directly into immature embryo cells containing preintegrated Cas9 also resulted in targeted mutations. Editing the ALS2 gene using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded chlorsulfuron-resistant plants. Double-strand breaks generated by RNA-guided Cas9 endonuclease also stimulated insertion of a trait gene at a site near LIG1 by homology-directed repair. Progeny showed expected Mendelian segregation of mutations, edits, and targeted gene insertions. The examples reported in this study demonstrate the utility of Cas9-guide RNA technology as a plant genome editing tool to enhance plant breeding and crop research needed to meet growing agriculture demands of the future. PMID:26269544

  1. Insertional Mutagenesis of the Maize P Gene by Intragenic Transposition of Ac

    PubMed Central

    Athma, P.; Grotewold, E.; Peterson, T.

    1992-01-01

    The P-rr allele of the maize P gene regulates the synthesis of pigments derived from flavan-4-ol in the pericarp, cob glumes and other floral organs. We characterized 21 P alleles derived by intragenic transposition of Ac from three known positions. Ac transpositions can occur in either direction in the P gene, and with no apparent minimum distance: in one case Ac transposed just 6 bp from its original insertion site. However, the distribution of transposed Ac elements was markedly nonrandom: of 19 transposed Ac elements derived from a single Ac donor, 15 were inserted in a 1.1-kb region at the 5' end of P, while none had inserted in an adjacent 3.2-kb intronic region. All of the Ac insertions affect both pericarp and cob glume pigmentation, providing further evidence that the P-rr allele contains a single gene required for both pericarp and cob glume pigmentation. The distribution of the inserted Ac elements and the phenotype conditioned by each allele suggests a structure of P-rr which is similar to that previously determined molecularly. Possible explanations for the nonrandom distribution of transposed Ac elements are discussed. PMID:1317315

  2. Novel strategies for gene trapping and insertional mutagenesis mediated by Sleeping Beauty transposon

    PubMed Central

    Song, Guili; Cui, Zongbin

    2013-01-01

    Gene and poly(A) trappings are high-throughput approaches to capture and interrupt the expression of endogenous genes within a target genome. Although a number of trapping vectors have been developed for investigation of gene functions in cells and vertebrate models, there is still room for the improvement of their efficiency and sensitivity. Recently, two novel trapping vectors mediated by Sleeping Beauty (SB) transposon have been generated by the combination of three functional cassettes that are required for finding endogenous genes, disrupting the expression of trapped genes, and inducing the excision of integrated traps from their original insertion sites and then inserting into another gene. In addition, several other strategies are utilized to improve the activities of two trapping vectors. First, activities of all components were examined in vitro before the generation of two vectors. Second, the inducible promoter from the tilapia Hsp70 gene was used to drive the expression of SB gene, which can mediate the excision of integrated transposons upon induction at 37 °C. Third, the Cre/LoxP system was introduced to delete the SB expression cassette for stabilization of gene interruption and bio-safety. Fourth, three stop codons in different reading frames were introduced downstream of a strong splice acceptor (SA) in the gene trapping vector to effectively terminate the translation of trapped endogenous genes. Fifth, the strong splicing donor (SD) and AU-rich RNA-destabilizing element exhibited no obvious insertion bias and markedly reduced SD read-through events, and the combination of an enhanced SA, a poly(A) signal and a transcript terminator in the poly(A) trapping vector efficiently disrupted the transcription of trapped genes. Thus, these two trapping vectors are alternative and effective tools for large-scale identification and disruption of endogenous genes in vertebrate cells and animals. PMID:24251071

  3. Identification of virulence genes in the crucifer anthracnose fungus Colletotrichum higginsianum by insertional mutagenesis.

    PubMed

    Liu, Liping; Zhao, Dian; Zheng, Lu; Hsiang, Tom; Wei, Yangdou; Fu, Yanping; Huang, Junbin

    2013-11-01

    To investigate the molecular and genetic mechanisms underlying virulence of Colletotrichum higginsianum on Arabidopsis thaliana, a T-DNA insertion mutant library of C. higginsianum, the causal agent of crucifer anthracnose, was established using Agrobacterium tumefaciens-mediated transformation. Among 875 transformants tested for virulence on Arabidopsis, six mutants with altered virulence, including an appressorial melanin-deficient mutant T734, two mutants defective in penetration, T45 and B30, and three mutants, T679, T732 and T801, that cause hypersensitive reactions on host Arabidopsis, were obtained. Southern blot analysis indicated that the mutants T732 and T734 harbored single-site T-DNA integrations, while B30 harbored two T-DNA insertions. Border flanking sequences of T-DNAs from these mutants were recovered by inverse polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR. Sequence analyses revealed that single T-DNA insertions in mutant T734 targeted the coding region of a gene with unknown function, and in mutant T732 targeted a gene encoding a copper amine oxidase. The two T-DNA insertion sites in mutant B30 were found in the coding region of a gene encoding an exosome component and in the upstream region of a DUF221-domain gene. None of these genes have previously been implicated in virulence of the phytopathogenic fungi. Among these avirulent mutants, T734 showed altered color in colony growth and produced melanin-deficient, albino appressoria. The T-DNA insert in T734 was detected in the coding region of a gene named C. higginsianum melanin-deficiency gene (Ch-MEL1), which is highly similar to a gene encoding a hypothetical protein in Colletotrichum gloeosporioides (GenBank ELA33048). To validate whether the Ch-MEL1 gene was associated with virulence of the mutant T734, a targeted gene disruption and complementation approach was used. The appressoria of ▵Ch-mel1 null mutants were defective in melanization and failed to penetrate

  4. Infectivity and insertional mutagenesis of endogenous retrovirus in autoimmune NZB and B/W mice.

    PubMed

    Beck-Engeser, Gabriele B; Ahrends, Tomasz; Knittel, Gero; Wabl, Rafael; Metzner, Mirjam; Eilat, Dan; Wabl, Matthias

    2015-11-01

    Murine leukaemia virus has been suggested to contribute to both autoimmune disease and leukaemia in the NZB mouse and in the (NZB × NZW) F1 (abbreviated B/W) mouse. However, with apparently only xenotropic but no ecotropic virus constitutively expressed in these mice, few mechanisms could explain the aetiology of either disease in either mouse strain. Because pseudotyped and/or inducible ecotropic virus may play a role, we surveyed the ability of murine leukaemia virus in NZB, NZW and B/W mice to infect and form a provirus. From the spleen of NZB mice, we isolated circular cDNA of xenotropic and polytropic virus, which indicates ongoing infection by these viruses. From a B/W lymphoma, we isolated and determined the complete sequence of a putative ecotropic NZW virus. From B/W mice, we recovered de novo endogenous retroviral integration sites (tags) from the hyperproliferating cells of the spleen and the peritoneum. The tagged genes seemed to be selected to aid cellular proliferation, as several of them are known cancer genes. The insertions are consistent with the idea that endogenous retrovirus contributes to B-cell hyperproliferation and progression to lymphoma in B/W mice. PMID:26315139

  5. Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

    PubMed Central

    Boretsky, Yuriy R.; Pynyaha, Yuriy V.; Boretsky, Volodymyr Y.; Fedorovych, Dariya V.; Fayura, Lyubov R.; Protchenko, Olha; Philpott, Caroline C.; Sibirny, Andriy A.

    2012-01-01

    Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B2) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondii Δvma1–17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondii Δfra1–45 mutant accumulated 1.8–2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1–17 and Δfes1–77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1–45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1–77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80–22. Complementation analysis revealed that Δvma1–17 and Δfra1–45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast. PMID:21261808

  6. Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis.

    PubMed

    Boretsky, Yuriy R; Pynyaha, Yuriy V; Boretsky, Volodymyr Y; Fedorovych, Dariya V; Fayura, Lyubov R; Protchenko, Olha; Philpott, Caroline C; Sibirny, Andriy A

    2011-05-01

    Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B(2)) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondiiΔvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondiiΔfra1-45 mutant accumulated 1.8-2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1-17 and Δfes1-77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Δvma1-17 and Δfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast. PMID:21261808

  7. Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis

    PubMed Central

    Hingston, Patricia A.; Piercey, Marta J.

    2015-01-01

    Listeria monocytogenes is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm2 relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P < 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses. PMID:26025900

  8. Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis.

    PubMed

    Hingston, Patricia A; Piercey, Marta J; Truelstrup Hansen, Lisbeth

    2015-08-15

    Listeria monocytogenes is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm(2) relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P < 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses. PMID:26025900

  9. Sleeping Beauty transposon mutagenesis identifies genes that cooperate with mutant Smad4 in gastric cancer development.

    PubMed

    Takeda, Haruna; Rust, Alistair G; Ward, Jerrold M; Yew, Christopher Chin Kuan; Jenkins, Nancy A; Copeland, Neal G

    2016-04-01

    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. PMID:27006499

  10. Transposon mutagenesis identifies genes driving hepatocellular carcinoma in a chronic hepatitis B mouse model

    PubMed Central

    Bard-Chapeau, Emilie A.; Nguyen, Anh-Tuan; Rust, Alistair G.; Sayadi, Ahmed; Lee, Philip; Chua, Belinda Q; New, Lee-Sun; de Jong, Johann; Ward, Jerrold M.; Chin, Christopher KY.; Chew, Valerie; Toh, Han Chong; Abastado, Jean-Pierre; Benoukraf, Touati; Soong, Richie; Bard, Frederic A.; Dupuy, Adam J.; Johnson, Randy L.; Radda, George K.; Chan, Eric CY.; Wessels, Lodewyk FA.; Adams, David J.

    2014-01-01

    The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC we performed a near saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers, and a bewildering number (2860) of candidate later stage drivers, that were enriched for genes mutated, deregulated, or that function in signaling pathways important for human HCC, with a striking 1199 genes linked to cellular metabolic processes. Our study provides a comprehensive overview of the genetic landscape of HCC. PMID:24316982

  11. Rational and random mutagenesis of firefly luciferase to identify an efficient emitter of red bioluminescence

    NASA Astrophysics Data System (ADS)

    Branchini, Bruce R.; Southworth, Tara L.; Khattak, Neelum F.; Murtiashaw, Martha H.; Fleet, Sarah E.

    2004-06-01

    Firefly luciferase, which emits yellow-green (557 nm) light, and the corresponding cDNA have been used successfully as a bioluminescence reporter of gene expression. One particularly exciting application is in the area of in vivo bioluminescence imaging. Our interest is in developing improved reagents by identifying Photinus pyralis luciferase mutants that efficiently emit red bioluminescence. In this way, the proven advantages of the P. pyralis protein can be combined with the potential advantages of a red-shifted emitter. Using site-directed mutagenesis techniques, we have identified many mutants emitting red bioluminescence. Unfortunately, these enzymes generally have significantly decreased bioluminescence activity. Interestingly, we discovered a mutation, Ile351Ala, that produced a moderate 16 nm red-shift, while maintaining excellent bioluminescence activity. We then undertook a random mutagenesis approach to identify luciferase mutants that emit further red-shifted bioluminescence with minimal loss of activity. Libraries of mutants were created using an error-prone PCR method and the Ile351Ala luciferase mutant as the template DNA. The libraries were screened by in vivo bacterial assays and the promising mutants were purified to enable accurate determination of bioluminescence emission spectra and total bioluminescence activity. We will report the characterization results, including the identification of the randomly altered amino acids, of several mutants that catalyze bioluminescence with emission maxima of approximately 600 nm.

  12. Transposon mutagenesis identifies genetic drivers of Braf(V600E) melanoma.

    PubMed

    Mann, Michael B; Black, Michael A; Jones, Devin J; Ward, Jerrold M; Yew, Christopher Chin Kuan; Newberg, Justin Y; Dupuy, Adam J; Rust, Alistair G; Bosenberg, Marcus W; McMahon, Martin; Print, Cristin G; Copeland, Neal G; Jenkins, Nancy A

    2015-05-01

    Although nearly half of human melanomas harbor oncogenic BRAF(V600E) mutations, the genetic events that cooperate with these mutations to drive melanogenesis are still largely unknown. Here we show that Sleeping Beauty (SB) transposon-mediated mutagenesis drives melanoma progression in Braf(V600E) mutant mice and identify 1,232 recurrently mutated candidate cancer genes (CCGs) from 70 SB-driven melanomas. CCGs are enriched in Wnt, PI3K, MAPK and netrin signaling pathway components and are more highly connected to one another than predicted by chance, indicating that SB targets cooperative genetic networks in melanoma. Human orthologs of >500 CCGs are enriched for mutations in human melanoma or showed statistically significant clinical associations between RNA abundance and survival of patients with metastatic melanoma. We also functionally validate CEP350 as a new tumor-suppressor gene in human melanoma. SB mutagenesis has thus helped to catalog the cooperative molecular mechanisms driving BRAF(V600E) melanoma and discover new genes with potential clinical importance in human melanoma. PMID:25848750

  13. Transposon mutagenesis identifies genetic drivers of BrafV600E melanoma

    PubMed Central

    Mann, Michael B; Black, Michael A; Jones, Devin J; Ward, Jerrold M; Yew, Christopher Chin Kuan; Newberg, Justin Y; Dupuy, Adam J; Rust, Alistair G; Bosenberg, Marcus W; McMahon, Martin; Print, Cristin G; Copeland, Neal G; Jenkins, Nancy A

    2016-01-01

    Although nearly half of human melanomas harbor oncogenic BRAFV600E mutations, the genetic events that cooperate with these mutations to drive melanogenesis are still largely unknown. Here we show that Sleeping Beauty (SB) transposon-mediated mutagenesis drives melanoma progression in BrafV600E mutant mice and identify 1,232 recurrently mutated candidate cancer genes (CCGs) from 70 SB-driven melanomas. CCGs are enriched in Wnt, PI3K, MAPK and netrin signaling pathway components and are more highly connected to one another than predicted by chance, indicating that SB targets cooperative genetic networks in melanoma. Human orthologs of >500 CCGs are enriched for mutations in human melanoma or showed statistically significant clinical associations between RNA abundance and survival of patients with metastatic melanoma. We also functionally validate CEP350 as a new tumor-suppressor gene in human melanoma. SB mutagenesis has thus helped to catalog the cooperative molecular mechanisms driving BRAFV600E melanoma and discover new genes with potential clinical importance in human melanoma. PMID:25848750

  14. Use of pentapeptide-insertion scanning mutagenesis for functional mapping of the plum pox virus helper component proteinase suppressor of gene silencing.

    PubMed

    Varrelmann, Mark; Maiss, Edgar; Pilot, Ruth; Palkovics, Laszlo

    2007-03-01

    Helper component proteinase (HC-Pro) of Plum pox virus is a multifunctional potyvirus protein that has been examined intensively. In addition to its involvement in aphid transmission, genome amplification and long-distance movement, it is also one of the better-studied plant virus suppressors of RNA silencing. The first systematic analysis using pentapeptide-insertion scanning mutagenesis of the silencing suppression function of a potyvirus HC-Pro is presented here. Sixty-three in-frame insertion mutants, each containing five extra amino acids inserted randomly within the HC-Pro protein, were analysed for their ability to suppress transgene-induced RNA silencing using Agrobacterium infiltration in transgenic Nicotiana benthamiana plants expressing green fluorescent protein. A functional map was obtained, consisting of clearly defined regions with different classes of silencing-suppression activity (wild-type, restricted and disabled). This map confirmed that the N-terminal part of the protein, which is indispensable for aphid transmission, is dispensable for silencing suppression and supports the involvement of the central region in silencing suppression, in addition to its role in maintenance of genome amplification and synergism with other viruses. Moreover, evidence is provided that the C-terminal part of the protein, previously known to be necessary mainly for proteolytic activity, also participates in silencing suppression. Pentapeptide-insertion scanning mutagenesis has been shown to be a fast and powerful tool to functionally characterize plant virus proteins. PMID:17325375

  15. Functional Genomics of Rice Pollen and Seed Development by Genome-wide Transcript Profiling and Ds Insertion Mutagenesis

    PubMed Central

    Jiang, Shu-Ye; Ramachandran, Srinivasan

    2011-01-01

    Rice pollen and seed development are directly related to grain yield. To further improve rice yield, it is important for us to functionally annotate the genes controlling pollen/seed development and to use them for rice breeding. Here we first carried out a genome-wide expression analysis with an emphasis on genes being involved in rice pollen and seed development. Based on the transcript profiling, we have identified and functionally classified 82 highly expressed pollen-specific, 12 developing seed-specific and 19 germinating seed-specific genes. We then presented the utilization of the maize transposon Dissociation (Ds) insertion lines for functional genomics of rice pollen and seed development and as alternative germplasm resources for rice breeding. We have established a two-element Activator/Dissociation (Ac/Ds) gene trap tagging system and generated around 20,000 Ds insertion lines. We have subjected these lines for screens to obtain high and low yield Ds insertion lines. Some interesting lines have been obtained with higher yield or male sterility. Flanking Sequence Tags (FSTs) analyses showed that these Ds-tagged genes encoded various proteins including transcription factors, transport proteins, unknown functional proteins and so on. They exhibited diversified expression patterns. Our results suggested that rice could be improved not only by introducing foreign genes but also by knocking out its endogenous genes. This finding might provide a new way for rice breeder to further improve rice varieties. PMID:21209789

  16. Serotype 1 viruses modified by backpassage or insertional mutagenesis: approaching the threshold of vaccine efficacy in Marek's disease.

    PubMed

    Witter, R L; Kreager, K S

    2004-12-01

    Improved vaccines to control Marek's disease (MD) in chickens are desired by the poultry industry but have been difficult to develop. Studies were conducted to evaluate strategies for deriving MD vaccines of high protective efficacy, irrespective of virulence. Candidate viruses from parent strains representing v and vv+ pathotypes were modified by cell culture passage, backpassage in chickens, or insertional mutagenesis following cocultivation with retroviruses. Ten strains considered most likely to exhibit high protective efficacy were selected for further study. The ability of these modified viruses to protect commercial or maternal antibody-positive (ab+) chickens against virulent MD virus (MDV) challenge was compared with that of strain CVI988, the standard commercial MD vaccine. Modified strains were also evaluated for the ability to induce lymphomas or other pathologic changes in ab+ and antibody-negative (ab-) chickens. Two of the 10 modified viruses, strains RM1 and CVI988/BP5, provided high levels of protection against highly virulent MDV challenge. The magnitude of protection was greater than that of one laboratory and two commercial preparations of CV1988, but was approximately equal to that of two other commercial preparations of CVI988 in laboratory and field tests. Three of the strains, including RMI and CVI988/BP5, induced lymphoid organ atrophy in ab-chicks but not in ab+ commercial chicks, a property designated here as L phenotype. Seven strains, including two L+ strains, were mildly oncogenic for ab- chicks, a property designated here as O phenotype. Five of these strains caused no tumors in ab+ chickens. The two fully attenuated strains induced neither lymphomas nor lymphoid organ atrophy. The L and O phenotypes appeared not to be linked, and both (especially the L phenotype) appeared associated with high levels of protection. These studies also illustrated differences in the protective efficacy of different preparations of CVI988 vaccine

  17. Final technical report for: Insertional Mutagenesis of Brachypodium distachyon DE-AI02-07ER64452

    SciTech Connect

    John, Vogel P.

    2015-10-29

    Several bioenergy grasses are poised to become a major source of energy in the United States. Despite their increasing importance, we know little about the basic biology underlying the traits that control the utility of grasses as energy crops. Better knowledge of grass biology (e.g. identification of the genes that control cell wall composition, plant architecture, cell size, cell division, reproduction, nutrient uptake, carbon flux, etc.) could be used to design rational strategies for crop improvement and shorten the time required to domesticate these species. The use of an appropriate model system is an efficient way to gain this knowledge. Brachypodium distachyon is a small annual grass with all the attributes needed to be a modern model organism including simple growth requirements, fast generation time, small stature, small genome size and self-fertility. These attributes led to the recommendation in the DOE’s “Breaking the Biological Barriers to Cellulosic Ethanol: A Joint Research Agenda” report to propose developing and using B. distachyon as a model for energy crops to accelerate their domestication. Strategic investments (e.g. genome sequencing) in B. distachyon by the DOE are now bearing fruit and B. distachyon is being used as a model grass by hundreds of laboratories worldwide. Sequence indexed insertional mutants are an extremely powerful tool for both forward and reverse genetics. They allow researchers to order mutants in any gene tagged in the collection by simply emailing a request. The goal of this project was to create a collection of sequence indexed insertional mutants (T-DNA lines) for the model grass Brachypodium distachyon in order to facilitate research by the scientific community. During the course of this grant we created a collection of 23,649 B. distachyon T-DNA lines and identified 26,112 unique insertion sites. The collection can be queried through the project website (http://jgi.doe.gov/our-science

  18. Transposon mutagenesis of Campylobacter jejuni identifies a bipartite energy taxis system required for motility.

    PubMed

    Hendrixson, D R; Akerley, B J; DiRita, V J

    2001-04-01

    Campylobacter jejuni constitutes the leading cause of bacterial gastroenteritis in the United States and a major cause of diarrhoea worldwide. Little is known about virulence mechanisms in this organism because of the scarcity of suitable genetic tools. We have developed an efficient system of in vitro transposon mutagenesis using a mariner-based transposon and purified mariner transposase. Through in vitro transposition of C. jejuni chromosomal DNA followed by natural transformation of the transposed DNA, large random transposon mutant libraries consisting of approximately 16 000 individual mutants were generated. The first genetic screen of C. jejuni using a transposon-generated mutant library identified 28 mutants defective for flagellar motility, one of the few known virulence determinants of this pathogen. We developed a second genetic system, which allows for the construction of defined chromosomal deletions in C. jejuni, and demonstrated the requirement of sigma28 and sigma54 for motility. In addition, we show that sigma28 is involved in the transcription of flaA and that sigma54 is required for transcription of three other flagellar genes, flaB and flgDE. We also identified two previously uncharacterized genes required for motility encoding proteins that we call CetA and CetB, which mediate energy taxis responses. Through our analysis of the Cet proteins, we propose a unique mechanism for sensing energy levels and mediating energy taxis in C. jejuni. PMID:11298288

  19. Lymphomagenesis in SCID-X1 Mice Following Lentivirus-mediated Phenotype Correction Independent of Insertional Mutagenesis and γc Overexpression

    PubMed Central

    Ginn, Samantha L; Liao, Sophia HY; Dane, Allison P; Hu, Min; Hyman, Jessica; Finnie, John W; Zheng, Maolin; Cavazzana-Calvo, Marina; Alexander, Stephen I; Thrasher, Adrian J; Alexander, Ian E

    2010-01-01

    The development of leukemia as a consequence of vector-mediated genotoxicity in gene therapy trials for X-linked severe combined immunodeficiency (SCID-X1) has prompted substantial research effort into the design and safety testing of integrating vectors. An important element of vector design is the selection and evaluation of promoter-enhancer elements with sufficient strength to drive reliable immune reconstitution, but minimal propensity for enhancer-mediated insertional mutagenesis. In this study, we set out to explore the effect of promoter-enhancer selection on the efficacy and safety of human immunodeficiency virus-1-derived lentiviral vectors in γc-deficient mice. We observed incomplete or absent T- and B-cell development in mice transplanted with progenitors expressing γc from the phosphoglycerate kinase (PGK) and Wiscott–Aldrich syndrome (WAS) promoters, respectively. In contrast, functional T- and B-cell compartments were restored in mice receiving an equivalent vector containing the elongation factor-1-α (EF1α) promoter; however, 4 of 14 mice reconstituted with this vector subsequently developed lymphoma. Extensive analyses failed to implicate insertional mutagenesis or γc overexpression as the underlying mechanism. These findings highlight the need for detailed mechanistic analysis of tumor readouts in preclinical animal models assessing vector safety, and suggest the existence of other ill-defined risk factors for oncogenesis, including replicative stress, in gene therapy protocols targeting the hematopoietic compartment. PMID:20354504

  20. Development of Safer Gene Delivery Systems to Minimize the Risk of Insertional Mutagenesis-Related Malignancies: A Critical Issue for the Field of Gene Therapy

    PubMed Central

    Romano, Gaetano

    2012-01-01

    Integrating gene delivery systems allow for a more stable transgene expression in mammalian cells than the episomal ones. However, the integration of the shuttle vector within the cellular chromosomal DNA is associated with the risk of insertional mutagenesis, which, in turn, may cause malignant cell transformation. The use of a retroviral-derived vector system was responsible for the development of leukemia in five children, who participated in various clinical trials for the treatment of severe combined immunodeficiency (SCID-X1) in France and in the United Kingdom. Unfortunately, the hematological malignancy claimed the life of one patient in 2004, who was enrolled in the French clinical trial. In addition, adeno-associated-viral-(AAV-) mediated gene transfer induced tumors in animal models, whereas the Sleeping Beauty (SB) DNA transposon system was associated with insertional mutagenesis events in cell culture systems. On these grounds, it is necessary to develop safer gene delivery systems for the genetic manipulation of mammalian cells. This paper discusses the latest achievements that have been reported in the field of vector design. PMID:23209944

  1. Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthesized oxygen-evolving system

    SciTech Connect

    Debus, R.J.; Barry, B.A.; Babcock, G.T.; McIntosh, L.

    1988-01-01

    Photosynthetic oxygen evolution takes place in the thylakoid protein complex known as photosystem II. The reaction center core of this photosystem, where photochemistry occurs, is a heterodimer of homologous polypeptides called D1 and D2. Besides chlorophyll and quinone, photosystem II contains other organic cofactors, including two known as Z and D. Z transfers electrons from the site of water oxidation to the oxidized reaction center primary donor, P/sub 680//sup +/, while D /center dot//sup +/ gives rise to the dark-stable EPR spectrum known as signal II. D/center dot//sup +/ has recently been shown to be a tyrosine radical. Z is probably a second tyrosine located in a similar environment. Indirect evidence indicates that Z and D are associated with the D1 and E2 polypeptides, respectively. To identify the specific tyrosine residue corresponding to D, the authors have changed Tyr-160 of the D2 polypeptide to phenylalanine by site-directed mutagenesis of a psbD gene in the cyanobacterium Synechocystis 6803. The resulting mutant grows photosynthetically, but it lacks the EPR signal of D/center dot//sup +/. The authors conclude that D is Tyr-160 of the D2 polypeptide. They suggest that the C/sub 2/ symmetry in photosystem II extends beyond P/sub 680/ to its immediate electron donor and conclude that Z is Try-161 of the D1 polypeptide.

  2. Coupled Site-Directed Mutagenesis/Transgenesis Identifies Important Functional Domains of the Mouse Agouti Protein

    PubMed Central

    Perry, W. L.; Nakamura, T.; Swing, D. A.; Secrest, L.; Eagleson, B.; Hustad, C. M.; Copeland, N. G.; Jenkins, N. A.

    1996-01-01

    The agouti locus encodes a novel paracrine signaling molecule containing a signal sequence, an N-linked glycosylation site, a central lysine-rich basic domain, and a C-terminal tail containing 10 cysteine (Cys) residues capable of forming five disulfide bonds. When overexpressed, agouti causes a number of pleiotropic effects including yellow coat and adult-onset obesity. Numerous studies suggest that agouti causes yellow coat color by antagonizing the binding of α-melanocyte-stimulating hormone (α-MSH) to the α-MSH-(melanocortin-1) receptor. With the goal of identifying functional domains of agouti important for its diverse biological activities, we have generated 14 agouti mutations by in vitro site-directed mutagenesis and analyzed these mutations in transgenic mice for their effects on coat color and obesity. These studies demonstrate that the signal sequence, the N-linked glycosylation site, and the C-terminal Cys residues are important for full biological activity, while at least a portion of the lysine-rich basic domain is dispensable for normal function. They also show that the same functional domains of agouti important in coat color determination are important for inducing obesity, consistent with the hypothesis that agouti induces obesity by antagonizing melanocortin binding to other melanocortin receptors. PMID:8878691

  3. Induced mutagenesis of plasmid and chromosomal genes inserted into the plasmid DNA. II. Mutagenic action of chemical factors

    SciTech Connect

    Esipova, V.V.; Vedunova, S.L.; Kriviskii, A.S.

    1986-02-01

    Following the study of the mutagenic action of UV and ..gamma..-radiation on plasmid DNA in vitro, they investigated the induction of mutations under the influence of chemical mutagens on the same DNA of plasmid RSF2124, determining the synthesis of colicine E1 and resistance to ampicillin. The inactivating action of the mutagen was assessed from the yield of transformants resistant to the antibiotic and the mutagenic effect from the loss by colonies of transformants that were capable of releasing colicine into the external medium. In these experiments they mainly used chemical compounds whose mutagenic effect if well known in other systems (transforming and transfecting DNA, microbial viruses). As a result all mutagens tested for their activity were divided into four groups: first group, those exceeding the level of mutagenesis by more than 100-fold above the spontaneous background (hydroxylamine, O-methylhydroxylamine); second group, those exceeding it by a factor of 10 (UV radiation (lambda = 254 nm), W-mutagenesis, ionizing radiation, nitrous acid, mitomycin C); third group, those exceeding it by a factor of <10 (indirect UV mutagenesis, nitrous acid, ..beta..-chloroethyldiethylamine hydrochloride, nitrosoguanidine); fourth group, no mutagenic effect (acridine orange, ethyl methane sulfonate, sodium azide, 0-..beta..-diethylaminoethylhydroxylamine).

  4. Construction and characterization of Pseudomonas aeruginosa protein F-deficient mutants after in vitro and in vivo insertion mutagenesis of the cloned gene.

    PubMed Central

    Woodruff, W A; Hancock, R E

    1988-01-01

    Mutants with insertion mutations in the Pseudomonas aeruginosa protein F (oprF) gene were created in vivo by Tn1 mutagenesis of the cloned gene in Escherichia coli and in vitro by insertion of the streptomycin resistance-encoding omega fragment into the cloned gene, followed by transfer of the mutated protein F gene back to P. aeruginosa. Homologous recombination into the P. aeruginosa chromosome was driven by a bacteriophage F116L transduction method in the oprF::Tn1 mutants or Tn5-instability in the oprF::omega mutants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting demonstrated that the resultant oprF insertion mutants had lost protein F, whereas restriction digestion and Southern blotting experiments proved that the mutants contained a single chromosomal oprF gene with either Tn1 or omega inserted into it. It has been proposed that protein F has a role in antibiotic uptake in P. aeruginosa. Measurement of antibiotic resistance levels showed small to marginal increases in resistance, compared with that of the parent P. aeruginosa strain, to a variety of beta-lactam antibiotics. Protein F-deficient mutants had altered barrier properties as revealed by a three- to fivefold increase in the uptake of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine. Images PMID:2836364

  5. Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics

    PubMed Central

    2013-01-01

    Background The development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance. Methods Here we describe a gain-of-function forward genetic approach for identifying mechanisms of resistance. This approach uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activate nearby gene expression. Genes of interest are identified using next-gen high-throughput sequencing and barcode multiplexing is used to reduce experimental cost. Results Using this approach we successfully identify genes involved in paclitaxel resistance in a variety of cancer cell lines, including the multidrug transporter ABCB1, a previously identified major paclitaxel resistance gene. Analysis of co-occurring transposons integration sites in single cell clone allows for the identification of genes that might act cooperatively to produce drug resistance a level of information not accessible using RNAi or ORF expression screening approaches. Conclusion We have developed a powerful pipeline to systematically discover drug resistance in mammalian cells in vitro. This cost-effective approach can be readily applied to different cell lines, to identify canonical or context specific resistance mechanisms. Its ability to probe complex genetic context and non-coding genomic elements as well as cooperative resistance events makes it a good complement to RNAi or ORF expression based screens. PMID:23442791

  6. Identifying novel mycobacterial stress associated genes using a random mutagenesis screen in Mycobacterium smegmatis.

    PubMed

    Viswanathan, Gopinath; Joshi, Shrilaxmi V; Sridhar, Aditi; Dutta, Sayantanee; Raghunand, Tirumalai R

    2015-12-10

    Cell envelope associated components of Mycobacterium tuberculosis (M.tb) have been implicated in stress response, immune modulation and in vivo survival of the pathogen. Although many such factors have been identified, there is a large disparity between the number of genes predicted to be involved in functions linked to the envelope and those described in the literature. To identify and characterise novel stress related factors associated with the mycobacterial cell envelope, we isolated colony morphotype mutants of Mycobacterium smegmatis (M. smegmatis), based on the hypothesis that mutants with unusual colony morphology may have defects in the biosynthesis of cell envelope components. On testing their susceptibility to stress conditions relevant to M.tb physiology, multiple mutants were found to be sensitive to Isoniazid, Diamide and H2O2, indicative of altered permeability due to changes in cell envelope composition. Two mutants showed defects in biofilm formation implying possible roles for the target genes in antibiotic tolerance and/or virulence. These assays identified novel stress associated roles for several mycobacterial genes including sahH, tatB and aceE. Complementation analysis of selected mutants with the M. smegmatis genes and their M.tb homologues showed phenotypic restoration, validating their link to the observed phenotypes. A mutant carrying an insertion in fhaA encoding a forkhead associated domain containing protein, showed reduced survival in THP-1 macrophages, providing in vivo validation to this screen. Taken together, these results suggest that the M.tb homologues of a majority of the identified genes may play significant roles in the pathogenesis of tuberculosis. PMID:26211627

  7. The origin, evolution, and functional impact of short insertion-deletion variants identified in 179 human genomes.

    PubMed

    Montgomery, Stephen B; Goode, David L; Kvikstad, Erika; Albers, Cornelis A; Zhang, Zhengdong D; Mu, Xinmeng Jasmine; Ananda, Guruprasad; Howie, Bryan; Karczewski, Konrad J; Smith, Kevin S; Anaya, Vanessa; Richardson, Rhea; Davis, Joe; MacArthur, Daniel G; Sidow, Arend; Duret, Laurent; Gerstein, Mark; Makova, Kateryna D; Marchini, Jonathan; McVean, Gil; Lunter, Gerton

    2013-05-01

    Short insertions and deletions (indels) are the second most abundant form of human genetic variation, but our understanding of their origins and functional effects lags behind that of other types of variants. Using population-scale sequencing, we have identified a high-quality set of 1.6 million indels from 179 individuals representing three diverse human populations. We show that rates of indel mutagenesis are highly heterogeneous, with 43%-48% of indels occurring in 4.03% of the genome, whereas in the remaining 96% their prevalence is 16 times lower than SNPs. Polymerase slippage can explain upwards of three-fourths of all indels, with the remainder being mostly simple deletions in complex sequence. However, insertions do occur and are significantly associated with pseudo-palindromic sequence features compatible with the fork stalling and template switching (FoSTeS) mechanism more commonly associated with large structural variations. We introduce a quantitative model of polymerase slippage, which enables us to identify indel-hypermutagenic protein-coding genes, some of which are associated with recurrent mutations leading to disease. Accounting for mutational rate heterogeneity due to sequence context, we find that indels across functional sequence are generally subject to stronger purifying selection than SNPs. We find that indel length modulates selection strength, and that indels affecting multiple functionally constrained nucleotides undergo stronger purifying selection. We further find that indels are enriched in associations with gene expression and find evidence for a contribution of nonsense-mediated decay. Finally, we show that indels can be integrated in existing genome-wide association studies (GWAS); although we do not find direct evidence that potentially causal protein-coding indels are enriched with associations to known disease-associated SNPs, our findings suggest that the causal variant underlying some of these associations may be indels. PMID

  8. Insertion Mutagenesis of wca Reduces Acid and Heat Tolerance of Enterohemorrhagic Escherichia coli O157:H7

    PubMed Central

    Mao, Ying; Doyle, Michael P.; Chen, Jinru

    2001-01-01

    Strains of enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 produce under stress copious amounts of exopolysaccharide (EPS) composed of colanic acid (CA). Studies were performed to evaluate the association of production of CA with survival of EHEC under adverse environmental conditions. A CA-deficient mutant, M4020, was obtained from a CA-proficient parental strain, E. coli O157:H7 W6-13, by inserting a kanamycin resistance gene cassette (kan) into wcaD and wcaE, 2 of the 21 genes required for CA biosynthesis. M4020 was defective in CA production as determined from the ratio of uronic acid to protein (UA/P) of cells grown from 1 to 4 days at 25°C on minimal glucose agar (MGA), MacConkey agar, and sorbitol-MacConkey agar, and by colony morphology on MGA. The results of stress treatment revealed that M4020 was substantially less tolerant to acid (pH 4.5 and 5.5) and heat (55 and 60°C) in comparison to W6-13, indicating that CA confers on E. coli O157:H7 a protective effect from the environmental stresses of acid and heat. PMID:11371548

  9. A novel method to identify nucleic acid binding sites in proteins by scanning mutagenesis: application to iron regulatory protein.

    PubMed Central

    Neupert, B; Menotti, E; Kühn, L C

    1995-01-01

    We describe a new procedure to identify RNA or DNA binding sites in proteins, based on a combination of UV cross-linking and single-hit chemical peptide cleavage. Site-directed mutagenesis is used to create a series of mutants with single Asn-Gly sequences in the protein to be analysed. Recombinant mutant proteins are incubated with their radiolabelled target sequence and UV irradiated. Covalently linked RNA- or DNA-protein complexes are digested with hydroxylamine and labelled peptides identified by SDS-PAGE and autoradiography. The analysis requires only small amounts of protein and is achieved within a relatively short time. Using this method we mapped the site at which human iron regulatory protein (IRP) is UV cross-linked to iron responsive element RNA to amino acid residues 116-151. Images PMID:7544459

  10. In Vivo Transcriptional Profiling of Listeria monocytogenes and Mutagenesis Identify New Virulence Factors Involved in Infection

    PubMed Central

    Camejo, Ana; Buchrieser, Carmen; Couvé, Elisabeth; Carvalho, Filipe; Reis, Olga; Ferreira, Pierre; Sousa, Sandra; Cossart, Pascale; Cabanes, Didier

    2009-01-01

    Listeria monocytogenes is a human intracellular pathogen able to colonize host tissues after ingestion of contaminated food, causing severe invasive infections. In order to gain a better understanding of the nature of host–pathogen interactions, we studied the L. monocytogenes genome expression during mouse infection. In the spleen of infected mice, ≈20% of the Listeria genome is differentially expressed, essentially through gene activation, as compared to exponential growth in rich broth medium. Data presented here show that, during infection, Listeria is in an active multiplication phase, as revealed by the high expression of genes involved in replication, cell division and multiplication. In vivo bacterial growth requires increased expression of genes involved in adaptation of the bacterial metabolism and stress responses, in particular to oxidative stress. Listeria interaction with its host induces cell wall metabolism and surface expression of virulence factors. During infection, L. monocytogenes also activates subversion mechanisms of host defenses, including resistance to cationic peptides, peptidoglycan modifications and release of muramyl peptides. We show that the in vivo differential expression of the Listeria genome is coordinated by a complex regulatory network, with a central role for the PrfA-SigB interplay. In particular, L. monocytogenes up regulates in vivo the two major virulence regulators, PrfA and VirR, and their downstream effectors. Mutagenesis of in vivo induced genes allowed the identification of novel L. monocytogenes virulence factors, including an LPXTG surface protein, suggesting a role for S-layer glycoproteins and for cadmium efflux system in Listeria virulence. PMID:19478867

  11. Insertional mutagenesis in transgenic mice

    SciTech Connect

    Woychik, R.P.; Beatty, B.R.; McKinney, W.L.; Andreadis, D.K.; Chang, A.J.; Barker, P.E.

    1989-01-01

    A considerable effort is under way in medicine and biology to characterize the genes associated with development in humans and other species. An important feature of much of this research involves the molecular analysis of mutations because the alteration in gene expression associated with mutations is, in many cases, the basis for establishing the normal function of individual genes. In fact, the remarkable recent success in characterizing genes with specific functions in yeast, C. elegans, and Drosophila followed from earlier genetic analyses, and from the availability of mutations in these organisms that could be analyzed at the molecular level. The mouse, with its well-characterized genetic makeup and accessibility to experimental manipulation, is a good system for studying genes with specific functions in mammals. Many spontaneous, and chemical- or radiation-induced mutations are being studied at the molecular level to learn more about the structure and expression of genes regulating complex developmental processes. However, to be able to use mutations to characterize genes with specific functions, molecular probes must be available to facilitate the cloning and structural characterization of the mutant loci. 18 refs.

  12. New soybean variants in oil composition identified by large scale mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To identify loci involved in the determination of fatty acid content in mature soybean seeds, we subject 3400 seed samples to fatty acid profiling by gas chromatography. In the mutagenized population, a wide range of variation in the content of each of the five major fatty acids was observed. 74 mu...

  13. Transposon Mutagenesis of Salmonella enterica Serovar Enteritidis Identifies Genes That Contribute to Invasiveness in Human and Chicken Cells and Survival in Egg Albumen

    PubMed Central

    Zhou, Xiaohui; Kim, Hye-Young; Call, Douglas R.; Guard, Jean

    2012-01-01

    Salmonella enterica serovar Enteritidis is an important food-borne pathogen, and chickens are a primary reservoir of human infection. While most knowledge about Salmonella pathogenesis is based on research conducted on Salmonella enterica serovar Typhimurium, S. Enteritidis is known to have pathobiology specific to chickens that impacts epidemiology in humans. Therefore, more information is needed about S. Enteritidis pathobiology in comparison to that of S. Typhimurium. We used transposon mutagenesis to identify S. Enteritidis virulence genes by assay of invasiveness in human intestinal epithelial (Caco-2) cells and chicken liver (LMH) cells and survival within chicken (HD-11) macrophages as a surrogate marker for virulence. A total of 4,330 transposon insertion mutants of an invasive G1 Nalr strain were screened using Caco-2 cells. This led to the identification of attenuating mutations in a total of 33 different loci, many of which include genes previously known to contribute to enteric infection (e.g., Salmonella pathogenicity island 1 [SPI-1], SPI-4, SPI-5, CS54, fliH, fljB, csgB, spvR, and rfbMN) in S. Enteritidis and other Salmonella serovars. Several genes or genomic islands that have not been reported previously (e.g., SPI-14, ksgA, SEN0034, SEN2278, and SEN3503) or that are absent in S. Typhimurium or in most other Salmonella serovars (e.g., pegD, SEN1152, SEN1393, and SEN1966) were also identified. Most mutants with reduced Caco-2 cell invasiveness also showed significantly reduced invasiveness in chicken liver cells and impaired survival in chicken macrophages and in egg albumen. Consequently, these genes may play an important role during infection of the chicken host and also contribute to successful egg contamination by S. Enteritidis. PMID:22988017

  14. Transposon mutagenesis with coat color genotyping identifies an essential role for Skor2 in sonic hedgehog signaling and cerebellum development

    PubMed Central

    Wang, Baiping; Harrison, Wilbur; Overbeek, Paul A.; Zheng, Hui

    2011-01-01

    Correct development of the cerebellum requires coordinated sonic hedgehog (Shh) signaling from Purkinje to granule cells. How Shh expression is regulated in Purkinje cells is poorly understood. Using a novel tyrosinase minigene-tagged Sleeping Beauty transposon-mediated mutagenesis, which allows for coat color-based genotyping, we created mice in which the Ski/Sno family transcriptional co-repressor 2 (Skor2) gene is deleted. Loss of Skor2 leads to defective Purkinje cell development, a severe reduction of granule cell proliferation and a malformed cerebellum. Skor2 is specifically expressed in Purkinje cells in the brain, where it is required for proper expression of Shh. Skor2 overexpression suppresses BMP signaling in an HDAC-dependent manner and stimulates Shh promoter activity, suggesting that Skor2 represses BMP signaling to activate Shh expression. Our study identifies an essential function for Skor2 as a novel transcriptional regulator in Purkinje cells that acts upstream of Shh during cerebellum development. PMID:21937600

  15. Alanine Scanning Mutagenesis Identifies an Asparagine-Arginine-Lysine Triad Essential to Assembly of the Shell of the Pdu Microcompartment

    PubMed Central

    Sinha, Sharmistha; Cheng, Shouqiang; Sung, Yea Won; McNamara, Dan E.; Sawaya, Michael R.; Yeates, Todd O.; Bobik, Thomas A.

    2014-01-01

    Bacterial microcompartments (MCPs) are the simplest organelles known. They function to enhance metabolic pathways by confining several related enzymes inside an all protein envelope called the shell. In this study, we investigated the factors that govern MCP assembly by performing scanning mutagenesis on the surface residues of PduA, a major shell protein of the MCP used for 1,2-propanediol degradation. Biochemical, genetic and structural analysis of 20 mutants allowed us to determine that PduA K26, N29 and R79 are crucial residues that stabilize the shell of the 1,2-propanediol MCP. In addition, we identify two PduA mutants (K37A and K55A) that impair MCP function most likely by altering the permeability of its protein shell. These are the first studies to examine the phenotypic effects of shell protein structural mutations in a microcompartment system. The findings reported here may be applicable to engineering protein containers with improved stability for biotechnology applications. PMID:24747050

  16. Transposon mutagenesis with coat color genotyping identifies an essential role for Skor2 in sonic hedgehog signaling and cerebellum development.

    PubMed

    Wang, Baiping; Harrison, Wilbur; Overbeek, Paul A; Zheng, Hui

    2011-10-01

    Correct development of the cerebellum requires coordinated sonic hedgehog (Shh) signaling from Purkinje to granule cells. How Shh expression is regulated in Purkinje cells is poorly understood. Using a novel tyrosinase minigene-tagged Sleeping Beauty transposon-mediated mutagenesis, which allows for coat color-based genotyping, we created mice in which the Ski/Sno family transcriptional co-repressor 2 (Skor2) gene is deleted. Loss of Skor2 leads to defective Purkinje cell development, a severe reduction of granule cell proliferation and a malformed cerebellum. Skor2 is specifically expressed in Purkinje cells in the brain, where it is required for proper expression of Shh. Skor2 overexpression suppresses BMP signaling in an HDAC-dependent manner and stimulates Shh promoter activity, suggesting that Skor2 represses BMP signaling to activate Shh expression. Our study identifies an essential function for Skor2 as a novel transcriptional regulator in Purkinje cells that acts upstream of Shh during cerebellum development. PMID:21937600

  17. ENU mutagenesis identifies mice modeling Warburg Micro Syndrome with sensory axon degeneration caused by a deletion in Rab18.

    PubMed

    Cheng, Chih-Ya; Wu, Jaw-Ching; Tsai, Jin-Wu; Nian, Fang-Shin; Wu, Pei-Chun; Kao, Lung-Sen; Fann, Ming-Ji; Tsai, Shih-Jen; Liou, Ying-Jay; Tai, Chin-Yin; Hong, Chen-Jee

    2015-05-01

    Mutations in the gene of RAB18, a member of Ras superfamily of small G-proteins, cause Warburg Micro Syndrome (WARBM) which is characterized by defective neurodevelopmental and ophthalmological phenotypes. Despite loss of Rab18 had been reported to induce disruption of the endoplasmic reticulum structure and neuronal cytoskeleton organization, parts of the pathogenic mechanism caused by RAB18 mutation remain unclear. From the N-ethyl-N-nitrosourea (ENU)-induced mutagenesis library, we identified a mouse line whose Rab18 was knocked out. This Rab18(-/-) mouse exhibited stomping gait, smaller testis and eyes, mimicking several features of WARBM. Rab18(-/-) mice were obviously less sensitive to pain and touch than WT mice. Histological examinations on Rab18(-/-) mice revealed progressive axonal degeneration in the optic nerves, dorsal column of the spinal cord and sensory roots of the spinal nerves while the motor roots were spared. All the behavioral and pathological changes that resulted from abnormalities in the sensory axons were prevented by introducing an extra copy of Rab18 transgene in Rab18(-/-) mice. Our results reveal that sensory axonal degeneration is the primary cause of stomping gait and progressive weakness of the hind limbs in Rab18(-/-) mice, and optic nerve degeneration should be the major pathology of progressive optic atrophy in children with WARBM. Our results indicate that the sensory nervous system is more vulnerable to Rab18 deficiency and WARBM is not only a neurodevelopmental but also neurodegenerative disease. PMID:25779931

  18. Alanine Scanning Mutagenesis Identifies an Asparagine–Arginine–Lysine Triad Essential to Assembly of the Shell of the Pdu Microcompartment

    SciTech Connect

    Sinha, Sharmistha; Cheng, Shouqiang; Sung, Yea Won; McNamara, Dan E.; Sawaya, Michael R.; Yeates, Todd O.; Bobik, Thomas A.

    2014-06-01

    Bacterial microcompartments (MCPs) are the simplest organelles known. They function to enhance metabolic pathways by confining several related enzymes inside an all-protein envelope called the shell. In this study, we investigated the factors that govern MCP assembly by performing scanning mutagenesis on the surface residues of PduA, a major shell protein of the MCP used for 1,2-propanediol degradation. Biochemical, genetic, and structural analysis of 20 mutants allowed us to determine that PduA K26, N29, and R79 are crucial residues that stabilize the shell of the 1,2-propanediol MCP. In addition, we identify two PduA mutants (K37A and K55A) that impair MCP function most likely by altering the permeability of its protein shell. These are the first studies to examine the phenotypic effects of shell protein structural mutations in an MCP system. The findings reported here may be applicable to engineering protein containers with improved stability for biotechnology applications.

  19. Transposon Mutagenesis Identified Chromosomal and Plasmid Genes Essential for Adaptation of the Marine Bacterium Dinoroseobacter shibae to Anaerobic Conditions

    PubMed Central

    Ebert, Matthias; Laaß, Sebastian; Burghartz, Melanie; Petersen, Jörn; Koßmehl, Sebastian; Wöhlbrand, Lars; Rabus, Ralf; Wittmann, Christoph; Jahn, Dieter

    2013-01-01

    Anaerobic growth and survival are integral parts of the life cycle of many marine bacteria. To identify genes essential for the anoxic life of Dinoroseobacter shibae, a transposon library was screened for strains impaired in anaerobic denitrifying growth. Transposon insertions in 35 chromosomal and 18 plasmid genes were detected. The essential contribution of plasmid genes to anaerobic growth was confirmed with plasmid-cured D. shibae strains. A combined transcriptome and proteome approach identified oxygen tension-regulated genes. Transposon insertion sites of a total of 1,527 mutants without an anaerobic growth phenotype were determined to identify anaerobically induced but not essential genes. A surprisingly small overlap of only three genes (napA, phaA, and the Na+/Pi antiporter gene Dshi_0543) between anaerobically essential and induced genes was found. Interestingly, transposon mutations in genes involved in dissimilatory and assimilatory nitrate reduction (napA, nasA) and corresponding cofactor biosynthesis (genomic moaB, moeB, and dsbC and plasmid-carried dsbD and ccmH) were found to cause anaerobic growth defects. In contrast, mutation of anaerobically induced genes encoding proteins required for the later denitrification steps (nirS, nirJ, nosD), dimethyl sulfoxide reduction (dmsA1), and fermentation (pdhB1, arcA, aceE, pta, acs) did not result in decreased anaerobic growth under the conditions tested. Additional essential components (ferredoxin, cccA) of the anaerobic electron transfer chain and central metabolism (pdhB) were identified. Another surprise was the importance of sodium gradient-dependent membrane processes and genomic rearrangements via viruses, transposons, and insertion sequence elements for anaerobic growth. These processes and the observed contributions of cell envelope restructuring (lysM, mipA, fadK), C4-dicarboxylate transport (dctM1, dctM3), and protease functions to anaerobic growth require further investigation to unravel the

  20. Genome-Wide Transposon Mutagenesis in Saccharomyces cerevisiae and Candida albicans

    PubMed Central

    Xu, Tao; Bharucha, Nikë; Kumar, Anuj

    2016-01-01

    Transposon mutagenesis is an effective method for generating large sets of random mutations in target DNA, with applicability toward numerous types of genetic screens in prokaryotes, single-celled eukaryotes, and metazoans alike. Relative to methods of random mutagenesis by chemical/UV treatment, transposon insertions can be easily identified in mutants with phenotypes of interest. The construction of transposon insertion mutants is also less labor-intensive on a genome-wide scale than methods for targeted gene replacement, although transposon insertions are not precisely targeted to a specific residue, and thus coverage of the target DNA can be problematic. The collective advantages of transposon mutagenesis have been well demonstrated in studies of the budding yeast Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans, as transposon mutagenesis has been used extensively for phenotypic screens in both yeasts. Consequently, we present here protocols for the generation and utilization of transposon-insertion DNA libraries in S. cerevisiae and C. albicans. Specifically, we present methods for the large-scale introduction of transposon insertion alleles in a desired strain of S. cerevisiae. Methods are also presented for transposon mutagenesis of C. albicans, encompassing both the construction of the plasmid-based transposon-mutagenized DNA library and its introduction into a desired strain of Candida. In total, these methods provide the necessary information to implement transposon mutagenesis in yeast, enabling the construction of large sets of identifiable gene disruption mutations, with particular utility for phenotypic screening in nonstandard genetic backgrounds. PMID:21815095

  1. Directed optimization of a newly identified squalene synthase from Mortierella alpine based on sequence truncation and site-directed mutagenesis.

    PubMed

    Huang, Di; Yao, Yongpeng; Zhang, Hang; Mei, Zhu; Wang, Ru; Feng, Lu; Liu, Bin

    2015-10-01

    Terpenoids, a class of isoprenoids usually isolated from plants, are always used as commercial flavor and anticancer drugs. As a key precursor for triterpenes and sterols, biosynthesis of squalene (SQ) can be catalyzed by squalene synthase (SQS) from two farnesyl diphosphate molecules. In this work, the key SQS gene involved in sterols synthesis by Mortierella alpine, an industrial strain often used to produce unsaturated fatty acid such as γ-linolenic acid and arachidonic acid, was identified and characterized. Bioinformatic analysis indicated that MaSQS contained 416 amino acid residues involved in four highly conserved regions. Phylogenetic analysis revealed the closest relationship of MaSQS with Ganoderma lucidum and Aspergillus, which also belonged to the member of the fungus. Subsequently, the recombinant protein was expressed in Escherichia coli BL21(DE3) and detected by SDS-PAGE. To improve the expression and solubility of protein, 17 or 27 amino acids in the C-terminal were deleted. In vitro activity investigation based on gas chromatography-mass spectrometry revealed that both the truncated enzymes could functionally catalyze the reaction from FPP to SQ and the enzymatic activity was optimal at 37 °C, pH 7.2. Moreover, based on the site-directed mutagenesis, the mutant enzyme mMaSQSΔC17 (E186K) displayed a 3.4-fold improvement in catalytic efficiency (k(cat)/K(m)) compared to the control. It was the first report of characterization and modification of SQS from M. alpine, which facilitated the investigation of isoprenoid biosynthesis in the fungus. The engineered mMaSQSΔC17 (E186K) can be a potential candidate of the terpenes and steroids synthesis employed for synthetic biology. PMID:26275528

  2. eQTL mapping identifies insertion- and deletion-specific eQTLs in multiple tissues.

    PubMed

    Huang, Jinyan; Chen, Jun; Esparza, Jorge; Ding, Jun; Elder, James T; Abecasis, Goncalo R; Lee, Young-Ae; Mark Lathrop, G; Moffatt, Miriam F; Cookson, William O C; Liang, Liming

    2015-01-01

    Genome-wide gene expression quantitative trait loci (eQTL) mapping have been focused on single-nucleotide polymorphisms and have helped interpret findings from diseases mapping studies. The functional effect of structure variants, especially short insertions and deletions (indel) has not been well investigated. Here we impute 1,380,133 indels based on the latest 1,000 Genomes Project panel into three eQTL data sets from multiple tissues. Imputation of indels increased 9.9% power and identifies indel-specific eQTLs for 325 genes. We find introns and vicinities of UTRs are more enriched of indel eQTLs and 3.6 (single-tissue)-9.2%(multi-tissue) of previous identified eSNPs were taggers of eindels. Functional analyses identifies epigenetics marks, gene ontology categories and disease GWAS loci affected by SNPs and indels eQTLs showing tissue-consistent or tissue-specific effects. This study provides new insights into the underlying genetic architecture of gene expression across tissues and new resource to interpret function of diseases and traits associated structure variants. PMID:25951796

  3. eQTL mapping identify insertion and deletion specific eQTLs in multiple tissues

    PubMed Central

    Huang, Jinyan; Chen, Jun; Esparza, Jorge; Ding, Jun; Elder, James; Abecasis, Goncalo R; Lee, Young-Ae; Lathrop, G. Mark; Moffatt, Miriam F; Cookson, William O C; Liang, Liming

    2016-01-01

    GenomeC wide gene expression quantitative trait loci (eQTL) mapping have been focused on single nucleotide polymorphisms and have helped interpret findings from diseases mapping studies. The functional effect of structure variants, especially short insertions and deletions (indel) has not been well investigated. Here we imputed 1,380,133 indels based on the latest 1000 Genomes Project panel into 3 eQTL datasets from multiple tissues. Imputation of indels increased 9.9% power and identified indel specific eQTLs for 325 genes. We found introns and vicinities of UTRs were more enriched of indel eQTLs and 3.6 (singleC tissue)C 9.2%(multiC tissue) of previous identified eSNPs were taggers of eindels. Functional analyses identified epigenetics marks, gene ontology categories and disease GWAS loci affected by SNPs and indels eQTLs showing tissueC consistent or tissueC specific effects. This study provides new insights into the underlying genetic architecture of gene expression across tissues and new resource to interpret function of diseases and traits associated structure variants. PMID:25951796

  4. Random T-DNA mutagenesis identifies a Cu-Zn-superoxide dismutase gene as a virulence factor of Sclerotinia sclerotiorum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Ascomycetous fungus Sclerotinia sclerotiorum is a devastating pathogen capable of infecting more than 400 plant species including many economically important crops. In order to gain a better mechanistic understanding of its non-specific host-pathogen interactions, random mutagenesis through Agro...

  5. Transcriptome Analysis of Neisseria meningitidis in Human Whole Blood and Mutagenesis Studies Identify Virulence Factors Involved in Blood Survival

    PubMed Central

    Del Tordello, Elena; Seib, Kate L.; Francois, Patrice; Rappuoli, Rino; Pizza, Mariagrazia; Serruto, Davide

    2011-01-01

    During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by

  6. Multipurpose Transposon-Insertion Libraries in Yeast.

    PubMed

    Kumar, Anuj

    2016-01-01

    Libraries of transposon-insertion alleles constitute powerful and versatile tools for large-scale analysis of yeast gene function. Transposon-insertion libraries are constructed most simply through mutagenesis of a plasmid-based genomic DNA library; modification of the mutagenizing transposon by incorporation of yeast selectable markers, recombination sites, and an epitope tag enables the application of insertion alleles for phenotypic screening and protein localization. In particular, yeast genomic DNA libraries have been mutagenized with modified bacterial transposons carrying the URA3 marker, lox recombination sites, and sequence encoding multiple copies of the hemagglutinin (HA) epitope. Mutagenesis with these transposons has yielded a large resource of insertion alleles affecting nearly 4000 yeast genes in total. Through well-established protocols, these insertion libraries can be introduced into the desired strain backgrounds and the resulting insertional mutants can be screened or systematically analyzed. Relative to alternative methods of UV irradiation or chemical mutagenesis, transposon-insertion alleles can be easily identified by PCR-based approaches or high-throughput sequencing. Transposon-insertion libraries also provide a cost-effective alternative to targeted deletion approaches, although, in contrast to start-codon to stop-codon deletions, insertion alleles might not represent true null-mutants. For protein-localization studies, transposon-insertion alleles can provide encoded epitope tags in-frame with internal codons; in many cases, these transposon-encoded epitope tags can provide a more accurate localization for proteins in which terminal sequences are crucial for intracellular targeting. Thus, overall, transposon-insertion libraries can be used quickly and economically and have a particular utility in screening for desired phenotypes and localization patterns in nonstandard genetic backgrounds. PMID:27250950

  7. Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti

    PubMed Central

    Basu, Sanjay; Aryan, Azadeh; Overcash, Justin M.; Samuel, Glady Hazitha; Anderson, Michelle A. E.; Dahlem, Timothy J.; Myles, Kevin M.; Adelman, Zach N.

    2015-01-01

    Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24–90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2–3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems. PMID:25775608

  8. Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti.

    PubMed

    Basu, Sanjay; Aryan, Azadeh; Overcash, Justin M; Samuel, Glady Hazitha; Anderson, Michelle A E; Dahlem, Timothy J; Myles, Kevin M; Adelman, Zach N

    2015-03-31

    Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24-90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2-3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems. PMID:25775608

  9. Use of Illumina sequencing to identify transposon insertions underlying mutant phenotypes in high-copy Mutator lines of maize.

    PubMed

    Williams-Carrier, Rosalind; Stiffler, Nicholas; Belcher, Susan; Kroeger, Tiffany; Stern, David B; Monde, Rita-Ann; Coalter, Robert; Barkan, Alice

    2010-07-01

    High-copy transposons have been effectively exploited as mutagens in a variety of organisms. However, their utility for phenotype-driven forward genetics has been hampered by the difficulty of identifying the specific insertions responsible for phenotypes of interest. We describe a new method that can substantially increase the throughput of linking a disrupted gene to a known phenotype in high-copy Mutator (Mu) transposon lines in maize. The approach uses the Illumina platform to obtain sequences flanking Mu elements in pooled, bar-coded DNA samples. Insertion sites are compared among individuals of suitable genotype to identify those that are linked to the mutation of interest. DNA is prepared for sequencing by mechanical shearing, adapter ligation, and selection of DNA fragments harboring Mu flanking sequences by hybridization to a biotinylated oligonucleotide corresponding to the Mu terminal inverted repeat. This method yields dense clusters of sequence reads that tile approximately 400 bp flanking each side of each heritable insertion. The utility of the approach is demonstrated by identifying the causal insertions in four genes whose disruption blocks chloroplast biogenesis at various steps: thylakoid protein targeting (cpSecE), chloroplast gene expression (polynucleotide phosphorylase and PTAC12), and prosthetic group attachment (HCF208/CCB2). This method adds to the tools available for phenotype-driven Mu tagging in maize, and could be adapted for use with other high-copy transposons. A by-product of the approach is the identification of numerous heritable insertions that are unrelated to the targeted phenotype, which can contribute to community insertion resources. PMID:20409008

  10. Isolation of the transposable element hupfer from the entomopathogenic fungus Beauveria bassiana by insertion mutagenesis of the nitrate reductase structural gene.

    PubMed

    Maurer, P; Réjasse, A; Capy, P; Langin, T; Riba, G

    1997-09-01

    A transposable element has been isolated from the entomopathogenic fungus Beauveria bassiana by trapping it in the nitrate reductase structural gene, which has been cloned from this species. The element had inserted in the first exon of the nia gene and appeared to have duplicated the sequence TA at the site of insertion. It was 3336 bp long with 30-bp imperfect, inverted, terminal repeats. The element, called hupfer, contained an open reading frame encoding a 321-amino acid protein similar to the IS630- or mariner-Tcl-like transposases, and a residual sequence of about 2 kb which was not significantly similar to any published sequence. There are fewer than five copies of this transposable element present per genome in the fungus. PMID:9349711

  11. Identifying microbial fitness determinants by Insertion Sequencing (INSeq) using genome-wide transposon mutant libraries

    PubMed Central

    Goodman, Andrew L.; Wu, Meng; Gordon, Jeffrey I.

    2012-01-01

    Insertion Sequencing (INSeq) is a method for determining the insertion site and relative abundance of large numbers of transposon mutants in a mixed population of isogenic mutants of a sequenced microbial species. INSeq is based on a modified mariner transposon containing MmeI sites at its ends, allowing cleavage at chromosomal sites 16–17bp from the inserted transposon. Genomic regions adjacent to the transposons are amplified by linear PCR with a biotinylated primer. Products are bound to magnetic beads, digested with MmeI, and barcoded with sample-specific linkers appended to each restriction fragment. After limited PCR amplification, fragments are sequenced using a high-throughput instrument. The sequence of each read can be used to map the location of a transposon in the genome. Read count measures the relative abundance of that mutant in the population. Solid-phase library preparation makes this protocol rapid (18h), easy to scale-up, amenable to automation, and useful for a variety of samples. A protocol for characterizing libraries of transposon mutant strains clonally arrayed in multi-well format is provided. PMID:22094732

  12. Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders

    PubMed Central

    da Silva, Luciana Madeira; Vandepas, Lauren; Bianco, Suzy D.C.

    2011-01-01

    The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in adulthood 1,2,3. Inactivating mutations in KISS1R identified in patients have been associated with iodiopathic hypogonadotropic hypogonadism (IHH) 1,2 and precocious puberty 4. Functional studies of these mutants are crucial for our understanding of the mechanisms underlying the regulation of reproduction by this receptor as well as those shaping the disease outcomes, which result from abnormal KISS1R signaling and function. However, the highly GC-rich sequence of the KISS1R gene makes it rather difficult to introduce mutations or amplify the gene encoding this receptor by PCR. Here we describe a method to introduce mutations of interest into this highly GC-rich sequence that has been used successfully to generate over a dozen KISS1R mutants in our laboratory. We have optimized the PCR conditions to facilitate the amplification of a range of KISS1R mutants that include substitutions, deletions or insertions in the KISS1R sequence. The addition of a PCR enhancer solution, as well as of a small percentage of DMSO were especially helpful to improve amplification. This optimized procedure may be useful for other GC-rich templates as well. The expression vector encoding the KISS1R is been used to characterize signaling and function of this receptor in order to understand how mutations may change KISS1R function and lead to the associated reproductive phenotypes. Accordingly, potential applications of KISS1R mutants generated by site-directed mutagenesis can be illustrated by many studies 1,4,5,6,7,8. As an example, the gain-of-function mutation in the KISS1R (Arg386Pro), which is associated with precocious puberty, has been shown to prolong responsiveness of the receptor to ligand stimulation 4 as well as to alter the rate of degradation of KISS1R 9. Interestingly, our studies indicate that KISS1R is degraded by the

  13. Insertional Mutagenesis in Neurospora Crassa: Cloning and Molecular Analysis of the Preg(+) Gene Controlling the Activity of the Transcriptional Activator Nuc-1

    PubMed Central

    Kang, S.; Metzenberg, R. L.

    1993-01-01

    The transcriptional activator NUC-1 controls the transcription of the genes for phosphorus acquisition enzymes, and its activity is regulated by the negative regulatory factors, PREG and PGOV. In this report, we describe the cloning and molecular analysis of the preg(+) gene. In Neurospora crassa, as in higher eukaryotes, transformation frequently results in nonhomologous integration of transforming DNA. Insertion of transforming DNA into host genes mutates the gene and provides a molecular tag for cloning it. We obtained two mutants that have an insertion in the preg(+) and pgov(+) genes, respectively, among 2 X 10(5) transformants. The preg(+) gene was cloned by screening a Neurospora genomic DNA library with DNA sequences flanking the transforming DNA of the rescued plasmid. Northern analysis showed that the transcription of the preg(+) gene is not regulated by phosphate. The carboxy-terminal half of PREG shows strong homology with Saccharomyces cerevisiae PHO80 whose function is analogous to that of PREG. The preg(c) mutations are located in the well conserved residues which may directly interact with the residues in the regulatory domain of NUC-1. PMID:8436269

  14. Site-directed mutagenesis of the heterotrimeric killer toxin zymocin identifies residues required for early steps in toxin action.

    PubMed

    Wemhoff, Sabrina; Klassen, Roland; Meinhardt, Friedhelm

    2014-10-01

    Zymocin is a Kluyveromyces lactis protein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activity in vitro that might be important during γ import. Saccharomyces cerevisiae strains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase from Pichia acaciae (P. acaciae Orf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells. PMID:25128337

  15. The Hermes Transposon of Musca domestica Is an Efficient Tool for the Mutagenesis of Schizosaccharomyces pombe

    PubMed Central

    Evertts, Adam G.; Plymire, Christopher; Craig, Nancy L.; Levin, Henry L.

    2007-01-01

    Currently, no transposon-based method for the mutagenesis of Schizosaccharomyces pombe exists. We have developed such a system based on the introduction of the hermes transposon from the housefly into S. pombe. This system efficiently disrupts open reading frames and allows the insertion sites to be readily identified. PMID:17947404

  16. The hermes transposon of Musca domestica is an efficient tool for the mutagenesis of Schizosaccharomyces pombe.

    PubMed

    Evertts, Adam G; Plymire, Christopher; Craig, Nancy L; Levin, Henry L

    2007-12-01

    Currently, no transposon-based method for the mutagenesis of Schizosaccharomyces pombe exists. We have developed such a system based on the introduction of the hermes transposon from the housefly into S. pombe. This system efficiently disrupts open reading frames and allows the insertion sites to be readily identified. PMID:17947404

  17. A two-component enhancer-inhibitor transposon mutagenesis system for functional analysis of the Arabidopsis genome.

    PubMed Central

    Speulman, E; Metz, P L; van Arkel, G; te Lintel Hekkert, B; Stiekema, W J; Pereira, A

    1999-01-01

    A modified Enhancer-Inhibitor transposon system was used to generate a series of mutant lines by single-seed descent such that multiple I insertions occurred per plant. The distribution of original insertions in the population was assessed by isolating transposon-flanking DNA, and a database of insertion sites was created. Approximately three-quarters of the identified insertion sites show similarity to sequences stored in public databases, which demonstrates the power of this regimen of insertional mutagenesis. To isolate insertions in specific genes, we developed three-dimensional pooling and polymerase chain reaction strategies that we then validated by identifying mutants for the regulator genes APETALA1 and SHOOT MERISTEMLESS. The system then was used to identify inserts in a class of uncharacterized genes involved in lipid biosynthesis; one such insertion conferred a fiddlehead mutant phenotype. PMID:10521517

  18. Using Yeast Transposon-Insertion Libraries for Phenotypic Screening and Protein Localization.

    PubMed

    Kumar, Anuj

    2016-01-01

    This protocol details how to use a transposon-insertion library for phenotypic screening and protein localization. The insertion library was generated by mutagenesis of a plasmid-based yeast genomic DNA library by using a multipurpose transposon; the transposon produces gene disruptions, and, by Cre-mediated recombination at lox sites incorporated within the transposon, alleles with an in-frame insertion can be truncated to a residual transposon encoding multiple copies of the hemagglutinin epitope. Insertions are generated in yeast by shuttle mutagenesis. Yeast genomic DNA containing a transposon insertion is released from the library, and the mutagenized DNA sequences are introduced into a desired strain of yeast, where the insertion alleles replace native loci by homologous recombination. The insertion mutants can be screened for phenotypes, and the site of transposon insertion can subsequently be identified in selected mutants by inverse polymerase chain reaction (PCR). In-frame insertions within genes of interest can be truncated to an epitope-tagged allele by Cre-lox recombination, and the subcellular localization of the encoded protein product can be identified by standard methods of indirect immunofluorescence. In summary, the transposon-insertion libraries represent an informative resource for large-scale mutagenesis, presenting a straightforward alternative to labor-intensive targeted approaches for the construction of deletion alleles and fluorescent protein fusions. PMID:27250939

  19. Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

  20. Characterization of Insertions of IS476 and Two Newly Identified Insertion Sequences, IS1478 and IS1479, in Xanthomonas campestris pv. campestris

    PubMed Central

    Chen, Jiann-Hwa; Hsieh, Yu-Ying; Hsiau, Su-Lian; Lo, Ta-Chun; Shau, Chen-Chun

    1999-01-01

    Thirty-two plasmid insertion mutants were independently isolated from two strains of Xanthomonas campestris pv. campestris in Taiwan. Of the 32 mutants, 14 (44%), 8 (25%), and 4 (12%) mutants resulted from separate insertions of an IS3 family member, IS476, and two new insertion sequences (IS), IS1478 and IS1479. While IS1478 does not have significant sequence homology with any IS elements in the EMBL/GenBank/DDBJ database, IS1479 demonstrated 73% sequence homology with IS1051 in X. campestris pv. dieffenbachiae, 62% homology with IS52 in Pseudomonas syringae pv. glycinea, and 60% homology with IS5 in Escherichia coli. Based on the predicted transposase sequences as well as the terminal nucleotide sequences, IS1478 by itself constitutes a new subfamily of the widespread IS5 family, whereas IS1479, along with IS1051, IS52, and IS5, belongs to the IS5 subfamily of the IS5 family. All but one of the IS476 insertions had duplications of 4 bp at the target sites without sequence preference and were randomly distributed. An IS476 insertion carried a duplication of 952 bp at the target site. A model for generating these long direct repeats is proposed. Insertions of IS1478 and IS1479, on the other hand, were not random, and IS1478 and IS1479 each showed conservation of PyPuNTTA and PyTAPu sequences (Py is a pyrimidine, Pu is a purine, and N is any nucleotide) for duplications at the target sites. The results of Southern blot hybridization analysis indicated that multiple copies of IS476, IS1478, and IS1479 are present in the genomes of all seven X. campestris pv. campestris strains tested and several X. campestris pathovars. PMID:9973349

  1. Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity

    PubMed Central

    Wu, Shih-Lu; Li, Chia-Cheng; Chen, Jaw-Chyun; Chen, Yi-Jin; Lin, Ching-Ting; Ho, Tin-Yun; Hsiang, Chien-Yun

    2009-01-01

    Background Endonuclease G (EndoG), a member of DNA/RNA nonspecific ββα-Me-finger nucleases, is involved in apoptosis and normal cellular proliferation. In this study, we analyzed the critical amino acid residues of EndoG and proposed the catalytic mechanism of EndoG. Methods To identify the critical amino acid residues of human EndoG, we replaced the conserved histidine, asparagine, and arginine residues with alanine. The catalytic efficacies of Escherichia coli-expressed EndoG variants were further analyzed by kinetic studies. Results Diethyl pyrocarbonate modification assay revealed that histidine residues were involved in EndoG activity. His-141, Asn-163, and Asn-172 in the H-N-H motif of EndoG were critical for catalysis and substrate specificity. H141A mutant required a higher magnesium concentration to achieve its activity, suggesting the unique role of His-141 in both catalysis and magnesium coordination. Furthermore, an additional catalytic residue (Asn-251) and an additional metal ion binding site (Glu-271) of human EndoG were identified. Conclusion Based on the mutational analysis and homology modeling, we proposed that human EndoG shared a similar catalytic mechanism with nuclease A from Anabaena. PMID:19272175

  2. Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: sequence analysis and mutagenesis identify receptor binding epitopes.

    PubMed Central

    Jerva, L. F.; Sullivan, G.; Lolis, E.

    1997-01-01

    Murine macrophage inflammatory protein-2 (MIP-2), a member of the alpha-chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the alpha-chemokines, such as interleukin-8, gro-alpha/MGSA, and neutrophil activating peptide-2 (NAP-2), are associated with neutrophil activation and chemotaxis. We describe the expression, purification, and characterization of murine MIP-2 from Pichia pastoris. Circular dichroism spectroscopy reveals that MIP-2 exhibits a highly ordered secondary structure consistent with the alpha/beta structures of other chemokines. Recombinant MIP-2 is chemotactic for human and murine neutrophils and up-regulates cell surface expression of Mac-1. MIP-2 binds to human and murine neutrophils with dissociation constants of 6.4 nM and 2.9 nM, respectively. We further characterize the binding of MIP-2 to the human types A and B IL-8 receptors and the murine homologue of the IL-8 receptor. MIP-2 displays low-affinity binding to the type A IL-8 receptor (Kd > 120 nM) and high-affinity binding to the type B IL-8 receptor (Kd 5.7 nM) and the murine receptor (Kd 6.8 nM). The three-dimensional structure of IL-8 and sequence analysis of six chemokines (IL-8, gro-alpha, NAP-2, ENA-78, KC, and MIP-2) that display high-affinity binding to the IL-8 type B receptor are used to identify an extended N-terminal surface that interacts with this receptor. Two mutants of MIP-2 establish that this region is also involved in binding and activating the murine homologue of the IL-8 receptor. Differences in the sequence between IL-8 and related chemokines identify a unique hydrophobic/aromatic region surrounded by charged residues that is likely to impart specificity to IL-8 for binding to the type A receptor. PMID:9260277

  3. Toward the Analysis of the Petunia MADS Box Gene Family by Reverse and Forward Transposon Insertion Mutagenesis Approaches: B, C, and D Floral Organ Identity Functions Require SEPALLATA-Like MADS Box Genes in Petunia

    PubMed Central

    Vandenbussche, Michiel; Zethof, Jan; Souer, Erik; Koes, Ronald; Tornielli, Giovanni B.; Pezzotti, Mario; Ferrario, Silvia; Angenent, Gerco C.; Gerats, Tom

    2003-01-01

    We have initiated a systematic functional analysis of the MADS box, intervening region, K domain, C domain-type MADS box gene family in petunia. The starting point for this has been a reverse-genetics approach, aiming to select for transposon insertions into any MADS box gene. We have developed and applied a family signature insertion screening protocol that is highly suited for this purpose, resulting in the isolation of 32 insertion mutants in 20 different MADS box genes. In addition, we identified three more MADS box gene insertion mutants using a candidate-gene approach. The defined insertion lines provide a sound foundation for a systematic functional analysis of the MADS box gene family in petunia. Here, we focus on the analysis of Floral Binding Protein2 (FBP2) and FBP5 genes that encode the E-function, which in Arabidopsis has been shown to be required for B and C floral organ identity functions. fbp2 mutants display sepaloid petals and ectopic inflorescences originating from the third floral whorl, whereas fbp5 mutants appear as wild type. In fbp2 fbp5 double mutants, reversion of floral organs to leaf-like organs is increased further. Strikingly, ovules are replaced by leaf-like structures in the carpel, indicating that in addition to the B- and C-functions, the D-function, which specifies ovule development, requires E-function activity. Finally, we compare our data with results obtained using cosuppression approaches and conclude that the latter might be less suited for assigning functions to individual members of the MADS box gene family. PMID:14576291

  4. Virulence determinants of Salmonella Gallinarum biovar Pullorum identified by PCR signature-tagged mutagenesis and the spiC mutant as a candidate live attenuated vaccine.

    PubMed

    Geng, Shizhong; Jiao, Xinan; Barrow, Paul; Pan, Zhiming; Chen, Xiang

    2014-01-31

    Salmonella Gallinarum biovar Pullorum (S. Gallinarum biovar Pullorum) is the causative agent of pullorum disease (PD) in chickens which results in considerable economic losses to the poultry industries in developing countries. PCR-Signature Tagged Mutagenesis was used to identify virulence determinants of S. Gallinarum biovar Pullorum and novel attenuated live vaccine candidates for use against this disease. A library of 1800 signature-tagged S. Gallinarum biovar Pullorum mutants was constructed and screened for virulence-associated genes in chickens. The attenuation of 10 mutants was confirmed by in vivo and in vitro competitive index (CI) studies. The transposons were found to be located in SPI-1 (2/10 mutants), SPI-2 (3/10), the virulence plasmid (1/10) and non-SPI genes (4/10). One highly attenuated spiC mutant persisted in spleen and liver for less than 10 days and induced high levels of circulating antibody and protective immunity against oral challenge in young broiler chickens. The spiC mutant is a potential new vaccine candidate for use with chickens against this disease. PMID:24355532

  5. Transposon Mutagenesis of Probiotic Lactobacillus casei Identifies asnH, an Asparagine Synthetase Gene Involved in Its Immune-Activating Capacity

    PubMed Central

    Ito, Masahiro; Kim, Yun-Gi; Tsuji, Hirokazu; Takahashi, Takuya; Kiwaki, Mayumi; Nomoto, Koji; Danbara, Hirofumi; Okada, Nobuhiko

    2014-01-01

    Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139. PMID:24416179

  6. Genome Sequencing and Transposon Mutagenesis of Burkholderia seminalis TC3.4.2R3 Identify Genes Contributing to Suppression of Orchid Necrosis Caused by B. gladioli.

    PubMed

    Araújo, Welington L; Creason, Allison L; Mano, Emy T; Camargo-Neves, Aline A; Minami, Sonia N; Chang, Jeff H; Loper, Joyce E

    2016-06-01

    From a screen of 36 plant-associated strains of Burkholderia spp., we identified 24 strains that suppressed leaf and pseudobulb necrosis of orchid caused by B. gladioli. To gain insights into the mechanisms of disease suppression, we generated a draft genome sequence from one suppressive strain, TC3.4.2R3. The genome is an estimated 7.67 megabases in size, with three replicons, two chromosomes, and the plasmid pC3. Using a combination of multilocus sequence analysis and phylogenomics, we identified TC3.4.2R3 as B. seminalis, a species within the Burkholderia cepacia complex that includes opportunistic human pathogens and environmental strains. We generated and screened a library of 3,840 transposon mutants of strain TC3.4.2R3 on orchid leaves to identify genes contributing to plant disease suppression. Twelve mutants deficient in suppression of leaf necrosis were selected and the transposon insertions were mapped to eight loci. One gene is in a wcb cluster that is related to synthesis of extracellular polysaccharide, a key determinant in bacterial-host interactions in other systems, and the other seven are highly conserved among Burkholderia spp. The fundamental information developed in this study will serve as a resource for future research aiming to identify mechanisms contributing to biological control. PMID:26959838

  7. Large-Scale Insertional Mutagenesis of the Coleopteran Stored Grain Pest, the Red Flour Beetle Tribolium castaneum, Identifies Embryonic Lethal Mutations and Enhancer Traps

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Given its sequenced genome and efficient systemic RNA interference response, Tribolium castaneum is a model organism well suited for the application of “reverse genetics” approaches to elucidate the function of candidate genes. However, there is still a continuing need for “forward genetic” analysi...

  8. Signature-Tagged Mutagenesis of Edwardsiella ictaluri Identifies Virulence Related Genes, 3 Including a Salmonella Pathogenicity Island-2 Class of Type III Secretion System

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Edwardsiella ictaluri is the leading cause of mortality in channel catfish culture, but little is known about its pathogenesis. The use of signature-tagged mutagenesis in a waterborne infection model resulted in the identification of 50 mutants that were unable to infect/survive in catfish. Nineteen...

  9. Sleeping Beauty-mediated somatic mutagenesis implicates CSF1 in the formation of high grade astrocytomas

    PubMed Central

    Bender, Aaron M.; Collier, Lara S.; Rodriguez, Fausto J.; Tieu, Christina; Larson, Jon D.; Halder, Chandralekha; Mahlum, Eric; Kollmeyer, Thomas M.; Akagi, Keiko; Sarkar, Gobinda; Largaespada, David A.; Jenkins, Robert B.

    2010-01-01

    The Sleeping Beauty (SB) transposon system has been used as an insertional mutagenesis tool to identify novel cancer genes. To identify glioma-associated genes, we evaluated tumor formation in brain tissue from 117 transgenic mice that had undergone constitutive SB-mediated transposition. Upon analysis, 21 samples (18%) contained neoplastic tissue with features of high grade astrocytomas. These tumors expressed glial markers and were histologically similar to human glioma. Genomic DNA from SB-induced astrocytoma tissue was extracted and transposon insertion sites were identified. Insertions in the growth factor gene Csf1 were found in 13 of the 21 tumors (62%), clustered in introns 5 and 8. Using RT-PCR, we documented increased Csf1 RNAs in tumor versus adjacent normal tissue, with identification of transposon-terminated Csf1 mRNAs in astrocytomas with SB insertions in intron 8. Analysis of human glioblastomas revealed increased levels of Csf1 RNA and protein. Together, these results indicate that SB-insertional mutagenesis can identify high-grade astrocytoma-associated genes, and they imply an important role for CSF1 in the development of these tumors. PMID:20388773

  10. Optimization of Combinatorial Mutagenesis

    NASA Astrophysics Data System (ADS)

    Parker, Andrew S.; Griswold, Karl E.; Bailey-Kellogg, Chris

    Protein engineering by combinatorial site-directed mutagenesis evaluates a portion of the sequence space near a target protein, seeking variants with improved properties (stability, activity, immunogenicity, etc.). In order to improve the hit-rate of beneficial variants in such mutagenesis libraries, we develop methods to select optimal positions and corresponding sets of the mutations that will be used, in all combinations, in constructing a library for experimental evaluation. Our approach, OCoM (Optimization of Combinatorial Mutagenesis), encompasses both degenerate oligonucleotides and specified point mutations, and can be directed accordingly by requirements of experimental cost and library size. It evaluates the quality of the resulting library by one- and two-body sequence potentials, averaged over the variants. To ensure that it is not simply recapitulating extant sequences, it balances the quality of a library with an explicit evaluation of the novelty of its members. We show that, despite dealing with a combinatorial set of variants, in our approach the resulting library optimization problem is actually isomorphic to single-variant optimization. By the same token, this means that the two-body sequence potential results in an NP-hard optimization problem. We present an efficient dynamic programming algorithm for the one-body case and a practically-efficient integer programming approach for the general two-body case. We demonstrate the effectiveness of our approach in designing libraries for three different case study proteins targeted by previous combinatorial libraries - a green fluorescent protein, a cytochrome P450, and a beta lactamase. We found that OCoM worked quite efficiently in practice, requiring only 1 hour even for the massive design problem of selecting 18 mutations to generate 107 variants of a 443-residue P450. We demonstrate the general ability of OCoM in enabling the protein engineer to explore and evaluate trade-offs between quality and

  11. Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

    PubMed

    Lazar Adler, Natalie R; Stevens, Mark P; Dean, Rachel E; Saint, Richard J; Pankhania, Depesh; Prior, Joann L; Atkins, Timothy P; Kessler, Bianca; Nithichanon, Arnone; Lertmemongkolchai, Ganjana; Galyov, Edouard E

    2015-01-01

    Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were

  12. Systematic Mutagenesis of Genes Encoding Predicted Autotransported Proteins of Burkholderia pseudomallei Identifies Factors Mediating Virulence in Mice, Net Intracellular Replication and a Novel Protein Conferring Serum Resistance

    PubMed Central

    Adler, Natalie R. Lazar; Stevens, Mark P.; Dean, Rachel E.; Saint, Richard J.; Pankhania, Depesh; Prior, Joann L.; Atkins, Timothy P.; Kessler, Bianca; Nithichanon, Arnone; Lertmemongkolchai, Ganjana; Galyov, Edouard E.

    2015-01-01

    Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were

  13. Mutational analysis identifies leucine-rich repeat insertions crucial for pigeon toll-like receptor 7 recognition and signaling.

    PubMed

    Xiong, Dan; Song, Li; Jiao, Yang; Kang, Xilong; Chen, Xiang; Geng, Shizhong; Pan, Zhiming; Jiao, Xinan

    2015-11-15

    Toll-like receptor 7 (TLR7) is responsible for recognizing viral single-stranded RNA and antiviral imidazoquinoline compounds, leading to the activation of the innate immune response. In this study, mutated pigeon TLR7 fragments, in which the insertion at position 10 of leucine-rich repeat 10 (LRR10) or at position 15 of LRR2/11/13/14 was deleted, were amplified with an overlap-PCR method, and inserted into the expression vector pCMV. The immune functions of the TLR7 mutants were determined with an NF-κB luciferase assay of transfected cells. The deletion of the insertions absolutely abolished TLR7-NF-κB signaling. With quantitative real-time PCR and sandwich enzyme-linked immunosorbent assay, we observed that stimulation with R848 failed to induce the expression of interleukin 8 (IL-8) in any of the mutant-TLR7-transfected cells, consistent with their lack of NF-κB activity. However, the expression of interferon α (IFN-α) and tumor necrosis factor α (TNF-α) was significantly upregulated in the Del10IN10 and Del14IN15 groups. Remarkably, the levels of pigeon TLR7 expression were significantly increased in all the TLR7-mutated groups. Therefore, we speculate that another part of the deficient TLR7 mediates the induction of IFN-α and TNF-α by increasing the expression of TLR7 as compensation. However, the increased expression of TLR7 in the Del11IN15 group failed to induce the production of IFN-α, IL-8, or TNF-α, indicating that a false compensation occurred when the crucial LRR insertion was deleted. PMID:26553562

  14. Genome-wide transposon mutagenesis in pathogenic Leptospira species.

    PubMed

    Murray, Gerald L; Morel, Viviane; Cerqueira, Gustavo M; Croda, Julio; Srikram, Amporn; Henry, Rebekah; Ko, Albert I; Dellagostin, Odir A; Bulach, Dieter M; Sermswan, Rasana W; Adler, Ben; Picardeau, Mathieu

    2009-02-01

    Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans. PMID:19047402

  15. Signature-tagged mutagenesis of Vibrio vulnificus

    PubMed Central

    YAMAMOTO, Mai; KASHIMOTO, Takashige; TONG, Ping; XIAO, Jianbo; SUGIYAMA, Michiko; INOUE, Miyuki; MATSUNAGA, Rie; HOSOHARA, Kohei; NAKATA, Kazue; YOKOTA, Kenji; OGUMA, Keiji; YAMAMOTO, Koichiro

    2015-01-01

    Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus. PMID:25755021

  16. A facile and efficient transposon mutagenesis method for generation of multi-codon deletions in protein sequences.

    PubMed

    Liu, Shu-Su; Wei, Xuan; Ji, Qun; Xin, Xiu; Jiang, Biao; Liu, Jia

    2016-06-10

    Substitutions, insertions and deletions are all important mutation events in natural and laboratory protein evolution. However, protein engineering using insertions and deletions (indels) is hindered by the lack of a convenient mutagenesis method. Here, we describe a general transposon mutagenesis method that allows for removal of up to five consecutive in-frame codons from a random position of a target protein. This method, referred to as codon deletion mutagenesis (CDM), relies on an engineered Mu transposon that carries asymmetric terminal sequences flanking the MuA transposase recognition sites. CDM requires minimal DNA manipulations, and can generate multi-codon deletions with high efficiency (>90%). As a proof of principle, we constructed five libraries of green fluorescent protein (GFP) containing one to five random codon deletions, respectively. Several variants with multi-codon deletions remained fluorescent, none of which could be easily identified using traditional mutagenesis method. CDM provides a facile and efficient approach to sampling a protein sequence with multi-codon deletions. It will not only facilitate our understanding of the effects of amino acid deletions on protein function but also expedite protein engineering using deletion mutagenesis. PMID:27071724

  17. Protein-Trap Insertional Mutagenesis Uncovers New Genes Involved in Zebrafish Skin Development, Including a Neuregulin 2a-Based ErbB Signaling Pathway Required during Median Fin Fold Morphogenesis

    PubMed Central

    Urban, Mark D.; Richetti, Stefânia K.; Skuster, Kimberly J.; Harm, Rhianna M.; Lopez Cervera, Roberto; Umemoto, Noriko; McNulty, Melissa S.; Clark, Karl J.; Hammerschmidt, Matthias; Ekker, Stephen C.

    2015-01-01

    Skin disorders are widespread, but available treatments are limited. A more comprehensive understanding of skin development mechanisms will drive identification of new treatment targets and modalities. Here we report the Zebrafish Integument Project (ZIP), an expression-driven platform for identifying new skin genes and phenotypes in the vertebrate model Danio rerio (zebrafish). In vivo selection for skin-specific expression of gene-break transposon (GBT) mutant lines identified eleven new, revertible GBT alleles of genes involved in skin development. Eight genes—fras1, grip1, hmcn1, msxc, col4a4, ahnak, capn12, and nrg2a—had been described in an integumentary context to varying degrees, while arhgef25b, fkbp10b, and megf6a emerged as novel skin genes. Embryos homozygous for a GBT insertion within neuregulin 2a (nrg2a) revealed a novel requirement for a Neuregulin 2a (Nrg2a) – ErbB2/3 – AKT signaling pathway governing the apicobasal organization of a subset of epidermal cells during median fin fold (MFF) morphogenesis. In nrg2a mutant larvae, the basal keratinocytes within the apical MFF, known as ridge cells, displayed reduced pAKT levels as well as reduced apical domains and exaggerated basolateral domains. Those defects compromised proper ridge cell elongation into a flattened epithelial morphology, resulting in thickened MFF edges. Pharmacological inhibition verified that Nrg2a signals through the ErbB receptor tyrosine kinase network. Moreover, knockdown of the epithelial polarity regulator and tumor suppressor lgl2 ameliorated the nrg2a mutant phenotype. Identifying Lgl2 as an antagonist of Nrg2a – ErbB signaling revealed a significantly earlier role for Lgl2 during epidermal morphogenesis than has been described to date. Furthermore, our findings demonstrated that successive, coordinated ridge cell shape changes drive apical MFF development, making MFF ridge cells a valuable model for investigating how the coordinated regulation of cell polarity

  18. Sleeping Beauty mutagenesis in a mouse medulloblastoma model defines networks that discriminate between human molecular subgroups.

    PubMed

    Genovesi, Laura A; Ng, Ching Ging; Davis, Melissa J; Remke, Marc; Taylor, Michael D; Adams, David J; Rust, Alistair G; Ward, Jerrold M; Ban, Kenneth H; Jenkins, Nancy A; Copeland, Neal G; Wainwright, Brandon J

    2013-11-12

    The Sleeping Beauty (SB) transposon mutagenesis screen is a powerful tool to facilitate the discovery of cancer genes that drive tumorigenesis in mouse models. In this study, we sought to identify genes that functionally cooperate with sonic hedgehog signaling to initiate medulloblastoma (MB), a tumor of the cerebellum. By combining SB mutagenesis with Patched1 heterozygous mice (Ptch1(lacZ/+)), we observed an increased frequency of MB and decreased tumor-free survival compared with Ptch1(lacZ/+) controls. From an analysis of 85 tumors, we identified 77 common insertion sites that map to 56 genes potentially driving increased tumorigenesis. The common insertion site genes identified in the mutagenesis screen were mapped to human orthologs, which were used to select probes and corresponding expression data from an independent set of previously described human MB samples, and surprisingly were capable of accurately clustering known molecular subgroups of MB, thereby defining common regulatory networks underlying all forms of MB irrespective of subgroup. We performed a network analysis to discover the likely mechanisms of action of subnetworks and used an in vivo model to confirm a role for a highly ranked candidate gene, Nfia, in promoting MB formation. Our analysis implicates candidate cancer genes in the deregulation of apoptosis and translational elongation, and reveals a strong signature of transcriptional regulation that will have broad impact on expression programs in MB. These networks provide functional insights into the complex biology of human MB and identify potential avenues for intervention common to all clinical subgroups. PMID:24167280

  19. Sleeping Beauty mutagenesis in a mouse medulloblastoma model defines networks that discriminate between human molecular subgroups

    PubMed Central

    Genovesi, Laura A.; Ng, Ching Ging; Davis, Melissa J.; Remke, Marc; Taylor, Michael D.; Adams, David J.; Rust, Alistair G.; Ward, Jerrold M.; Ban, Kenneth H.; Jenkins, Nancy A.; Copeland, Neal G.; Wainwright, Brandon J.

    2013-01-01

    The Sleeping Beauty (SB) transposon mutagenesis screen is a powerful tool to facilitate the discovery of cancer genes that drive tumorigenesis in mouse models. In this study, we sought to identify genes that functionally cooperate with sonic hedgehog signaling to initiate medulloblastoma (MB), a tumor of the cerebellum. By combining SB mutagenesis with Patched1 heterozygous mice (Ptch1lacZ/+), we observed an increased frequency of MB and decreased tumor-free survival compared with Ptch1lacZ/+ controls. From an analysis of 85 tumors, we identified 77 common insertion sites that map to 56 genes potentially driving increased tumorigenesis. The common insertion site genes identified in the mutagenesis screen were mapped to human orthologs, which were used to select probes and corresponding expression data from an independent set of previously described human MB samples, and surprisingly were capable of accurately clustering known molecular subgroups of MB, thereby defining common regulatory networks underlying all forms of MB irrespective of subgroup. We performed a network analysis to discover the likely mechanisms of action of subnetworks and used an in vivo model to confirm a role for a highly ranked candidate gene, Nfia, in promoting MB formation. Our analysis implicates candidate cancer genes in the deregulation of apoptosis and translational elongation, and reveals a strong signature of transcriptional regulation that will have broad impact on expression programs in MB. These networks provide functional insights into the complex biology of human MB and identify potential avenues for intervention common to all clinical subgroups. PMID:24167280

  20. Optimization of combinatorial mutagenesis.

    PubMed

    Parker, Andrew S; Griswold, Karl E; Bailey-Kellogg, Chris

    2011-11-01

    Protein engineering by combinatorial site-directed mutagenesis evaluates a portion of the sequence space near a target protein, seeking variants with improved properties (e.g., stability, activity, immunogenicity). In order to improve the hit-rate of beneficial variants in such mutagenesis libraries, we develop methods to select optimal positions and corresponding sets of the mutations that will be used, in all combinations, in constructing a library for experimental evaluation. Our approach, OCoM (Optimization of Combinatorial Mutagenesis), encompasses both degenerate oligonucleotides and specified point mutations, and can be directed accordingly by requirements of experimental cost and library size. It evaluates the quality of the resulting library by one- and two-body sequence potentials, averaged over the variants. To ensure that it is not simply recapitulating extant sequences, it balances the quality of a library with an explicit evaluation of the novelty of its members. We show that, despite dealing with a combinatorial set of variants, in our approach the resulting library optimization problem is actually isomorphic to single-variant optimization. By the same token, this means that the two-body sequence potential results in an NP-hard optimization problem. We present an efficient dynamic programming algorithm for the one-body case and a practically-efficient integer programming approach for the general two-body case. We demonstrate the effectiveness of our approach in designing libraries for three different case study proteins targeted by previous combinatorial libraries--a green fluorescent protein, a cytochrome P450, and a beta lactamase. We found that OCoM worked quite efficiently in practice, requiring only 1 hour even for the massive design problem of selecting 18 mutations to generate 10⁷ variants of a 443-residue P450. We demonstrate the general ability of OCoM in enabling the protein engineer to explore and evaluate trade-offs between quality and

  1. Sleeping beauty-mediated somatic mutagenesis implicates CSF1 in the formation of high-grade astrocytomas.

    PubMed

    Bender, Aaron M; Collier, Lara S; Rodriguez, Fausto J; Tieu, Christina; Larson, Jon D; Halder, Chandralekha; Mahlum, Eric; Kollmeyer, Thomas M; Akagi, Keiko; Sarkar, Gobinda; Largaespada, David A; Jenkins, Robert B

    2010-05-01

    The Sleeping Beauty (SB) transposon system has been used as an insertional mutagenesis tool to identify novel cancer genes. To identify glioma-associated genes, we evaluated tumor formation in the brain tissue from 117 transgenic mice that had undergone constitutive SB-mediated transposition. Upon analysis, 21 samples (18%) contained neoplastic tissue with features of high-grade astrocytomas. These tumors expressed glial markers and were histologically similar to human glioma. Genomic DNA from SB-induced astrocytoma tissue was extracted and transposon insertion sites were identified. Insertions in the growth factor gene Csf1 were found in 13 of the 21 tumors (62%), clustered in introns 5 and 8. Using reverse transcription-PCR, we documented increased Csf1 RNAs in tumor versus adjacent normal tissue, with the identification of transposon-terminated Csf1 mRNAs in astrocytomas with SB insertions in intron 8. Analysis of human glioblastomas revealed increased levels of Csf1 RNA and protein. Together, these results indicate that SB-insertional mutagenesis can identify high-grade astrocytoma-associated genes and they imply an important role for CSF1 in the development of these tumors. PMID:20388773

  2. Structure-Based Mutagenesis of the Substrate-Recognition Domain of Nrdp1/FLRF Identifies the Binding Site for the Receptor Tyrosine Kinase ErbB3

    SciTech Connect

    Bouyain,S.; Leahy, D.

    2007-01-01

    The E3 ubiquitin ligase neuregulin receptor degrading protein 1 (Nrdp1) mediates the ligand-independent degradation of the epidermal growth factor receptor family member ErbB3/HER3. By regulating cellular levels of ErbB3, Nrdp1 influences ErbB3-mediated signaling, which is essential for normal vertebrate development. Nrdp1 belongs to the tripartite or RBCC (RING, B-box, coiled-coil) family of ubiquitin ligases in which the RING domain is responsible for ubiquitin ligation and a variable C-terminal region mediates substrate recognition. We report here the 1.95 A crystal structure of the C-terminal domain of Nrdp1 and show that this domain is sufficient to mediate ErbB3 binding. Furthermore, we have used site-directed mutagenesis to map regions of the Nrdp1 surface that are important for interacting with ErbB3 and mediating its degradation in transfected cells. The ErbB3-binding site localizes to a region of Nrdp1 that is conserved from invertebrates to vertebrates, in contrast to ErbB3, which is only found in vertebrates. This observation suggests that Nrdp1 uses a common binding site to recognize its targets in different species.

  3. Generation of Single-Copy Transposon Insertions in Clostridium perfringens by Electroporation of Phage Mu DNA Transposition Complexes▿

    PubMed Central

    Lanckriet, A.; Timbermont, L.; Happonen, L. J.; Pajunen, M. I.; Pasmans, F.; Haesebrouck, F.; Ducatelle, R.; Savilahti, H.; Van Immerseel, F.

    2009-01-01

    Transposon mutagenesis is a tool that is widely used for the identification of genes involved in the virulence of bacteria. Until now, transposon mutagenesis in Clostridium perfringens has been restricted to the use of Tn916-based methods with laboratory reference strains. This system yields primarily multiple transposon insertions in a single genome, thus compromising its use for the identification of virulence genes. The current study describes a new protocol for transposon mutagenesis in C. perfringens, which is based on the bacteriophage Mu transposition system. The protocol was successfully used to generate a single-insertion mutant library both for a laboratory strain and for a field isolate. Thus, it can be used as a tool in large-scale screening to identify virulence genes of C. perfringens. PMID:19270116

  4. Feasibility studies on newly identified LiCrP2O7 compound for lithium insertion behavior

    NASA Astrophysics Data System (ADS)

    Gangulibabu; Bhuvaneswari, D.; Kalaiselvi, N.

    2009-08-01

    A new category of lithium intercalating cathode candidates, namely LiCrP2O7, was synthesized at 800°C using a citric acid assisted modified (CAM) sol-gel method and examined for possible lithium insertion behavior. The formation of a phase pure and monoclinic LiCrP2O7 compound with finer crystallite size was confirmed from the X-ray diffraction patterns. The presence of nano-sized particles as observed from a transmittance electron microscope image of LiCrP2O7 and the presence of a preferred local cation environment, evidenced from Fourier transform infra-red and 7Li nuclear magnetic resonance studies, are the added advantages of the present study. Further, cyclic voltametry study performed on 2016 coin cells consisting of the synthesized LiCrP2O7 cathode revealed an excellent cycling reversibility and structural stability. Hence, CAM sol-gel synthesized LiCrP2O7 is found to possess desirable physical as well as electrochemical properties, leading one to consider the same as a possible lithium intercalating cathode material.

  5. Second-Site Mutagenesis of a Hypomorphic argonaute1 Allele Identifies SUPERKILLER3 as an Endogenous Suppressor of Transgene Posttranscriptional Gene Silencing.

    PubMed

    Yu, Agnès; Saudemont, Baptiste; Bouteiller, Nathalie; Elvira-Matelot, Emilie; Lepère, Gersende; Parent, Jean-Sébastien; Morel, Jean-Benoit; Cao, Jun; Elmayan, Taline; Vaucheret, Hervé

    2015-10-01

    Second-site mutagenesis was performed on the argonaute1-33 (ago1-33) hypomorphic mutant, which exhibits reduced sense transgene posttranscriptional gene silencing (S-PTGS). Mutations in FIERY1, a positive regulator of the cytoplasmic 5'-to-3' EXORIBONUCLEASE4 (XRN4), and in SUPERKILLER3 (SKI3), a member of the SKI complex that threads RNAs directly to the 3'-to-5' exoribonuclease of the cytoplasmic exosome, compensated AGO1 partial deficiency and restored S-PTGS with 100% efficiency. Moreover, xrn4 and ski3 single mutations provoked the entry of nonsilenced transgenes into S-PTGS and enhanced S-PTGS on partially silenced transgenes, indicating that cytoplasmic 5'-to-3' and 3'-to-5' RNA degradation generally counteract S-PTGS, likely by reducing the amount of transgene aberrant RNAs that are used by the S-PTGS pathway to build up small interfering RNAs that guide transgene RNA cleavage by AGO1. Constructs generating improperly terminated transgene messenger RNAs (mRNAs) were not more sensitive to ski3 or xrn4 than regular constructs, suggesting that improperly terminated transgene mRNAs not only are degraded from both the 3' end but also from the 5' end, likely after decapping. The facts that impairment of either 5'-to-3' or 3'-to-5' RNA degradation is sufficient to provoke the entry of transgene RNA into the S-PTGS pathway, whereas simultaneous impairment of both pathways is necessary to provoke the entry of endogenous mRNA into the S-PTGS pathway, suggest poor RNA quality upon the transcription of transgenes integrated at random genomic locations. PMID:26286717

  6. Targeted Mutagenesis in Zebrafish Using Customized Zinc Finger Nucleases

    PubMed Central

    Foley, Jonathan E.; Maeder, Morgan L.; Pearlberg, Joseph; Joung, J. Keith; Peterson, Randall T.; Yeh, Jing-Ruey J.

    2009-01-01

    Zebrafish mutants have traditionally been obtained using random mutagenesis or retroviral insertions, methods that cannot be targeted to a specific gene and require laborious gene mapping and sequencing. Recently, we and others have shown that customized zinc finger nucleases (ZFNs) can introduce targeted frame-shift mutations with high efficiency, thereby enabling directed creation of zebrafish gene mutations. Here we describe a detailed protocol for constructing ZFN expression vectors, for generating and introducing ZFN-encoding RNAs into zebrafish embryos, and for identifying ZFN-generated mutations in targeted genomic sites. All of our vectors and methods are compatible with previously described Zinc Finger Consortium reagents for constructing engineered zinc finger arrays. Using these methods, zebrafish founders carrying targeted mutations can be identified within four months. PMID:20010934

  7. PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice.

    PubMed

    Rad, Roland; Rad, Lena; Wang, Wei; Cadinanos, Juan; Vassiliou, George; Rice, Stephen; Campos, Lia S; Yusa, Kosuke; Banerjee, Ruby; Li, Meng Amy; de la Rosa, Jorge; Strong, Alexander; Lu, Dong; Ellis, Peter; Conte, Nathalie; Yang, Fang Tang; Liu, Pentao; Bradley, Allan

    2010-11-19

    Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here, we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon-transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed that PiggyBac is capable of genome-wide mutagenesis. The PiggyBac screen uncovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 21 mouse lines to be compatible with both transposon systems in constitutive, tissue- or temporal-specific mutagenesis. Mice with different transposon types, copy numbers, and chromosomal locations support wide applicability. PMID:20947725

  8. A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria

    PubMed Central

    Monson, Rita; Smith, Debra S.; Matilla, Miguel A.; Roberts, Kevin; Richardson, Elizabeth; Drew, Alison; Williamson, Neil; Ramsay, Josh; Welch, Martin; Salmond, George P. C.

    2015-01-01

    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. PMID:26733980

  9. An ENU Mutagenesis Screen in Zebrafish for Visual System Mutants Identifies a Novel Splice-Acceptor Site Mutation in patched2 that Results in Colobomas

    PubMed Central

    Lee, Jiwoon; Cox, Ben D.; Daly, Christina M. S.; Lee, Chanjae; Nuckels, Richard J.; Tittle, Rachel K.; Uribe, Rosa A.; Gross, Jeffrey M.

    2012-01-01

    Purpose. To identify recessive mutations affecting development and/or maintenance of the zebrafish visual system. Methods. A three-generation ENU (N-Nitroso-N-ethylurea)-based forward genetic screen was performed. F3 embryos were screened visually from 1 to 5 days postfertilization (dpf) for ocular abnormalities, and 5 dpf embryos were fixed and processed for cryosectioning, after which eye sections were screened for defects in cellular organization within the retina, lens, and cornea. A combination of PCR and DNA sequencing, in situ hybridization, and pharmacological treatments were used to clone and characterize a coloboma mutant. Results. A total of 126 F2 families were screened, and, from these, 18 recessive mutations were identified that affected eye development. Phenotypes included lens malformations and cataracts, photoreceptor defects, oculocutaneous albinism, microphthalmia, and colobomas. Analysis of one such coloboma mutant, uta1, identified a splice-acceptor mutation in the patched2 gene that resulted in an in-frame deletion of 19 amino acids that are predicted to contribute to the first extracellular loop of Patched2. ptch2uta1 mutants possessed elevated Hedgehog (Hh) pathway activity, and blocking the Hh pathway with cyclopamine prevented colobomas in ptch2uta1 mutant embryos. Conclusions. We have identified 18 recessive mutations affecting development of the zebrafish visual system and we have characterized a novel splice-acceptor site mutation in patched2 that results in enhanced Hh pathway activity and colobomas. PMID:23150614

  10. Structure-function relationship of a plant NCS1 member--homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis.

    PubMed

    Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M Joanne; Möhlmann, Torsten

    2014-01-01

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. PMID:24621654

  11. Structure-Function Relationship of a Plant NCS1 Member – Homology Modeling and Mutagenesis Identified Residues Critical for Substrate Specificity of PLUTO, a Nucleobase Transporter from Arabidopsis

    PubMed Central

    Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M. Joanne; Möhlmann, Torsten

    2014-01-01

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. PMID:24621654

  12. Protein engineering: single or multiple site-directed mutagenesis.

    PubMed

    Hsieh, Pei-Chung; Vaisvila, Romualdas

    2013-01-01

    Site-directed mutagenesis techniques are invaluable tools in molecular biology to study the structural and functional properties of a protein. To expedite the time required and simplify methods for mutagenesis, we recommend two protocols in this chapter. The first method for single site-directed mutagenesis, which includes point mutations, insertions, or deletions, can be achieved by an inverse PCR strategy with mutagenic primers and the high-fidelity Phusion(®) DNA Polymerase to introduce a site-directed mutation with exceptional efficiency. The second method is for engineering multiple mutations into a gene of interest. This can be completed in one step by PCR with mutagenic primers and by assembling all mutagenized PCR products using the Gibson Assembly™ Master Mix. This method allows multiple nucleotides to be changed simultaneously, which not only saves time but also reagents compared to traditional methods of mutagenesis. PMID:23423897

  13. Computer Simulation of Mutagenesis.

    ERIC Educational Resources Information Center

    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)

  14. 2004 Mutagenesis Gordon Conference

    SciTech Connect

    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.

  15. A Sleeping Beauty mutagenesis screen reveals a tumor suppressor role for Ncoa2/Src-2 in liver cancer.

    PubMed

    O'Donnell, Kathryn A; Keng, Vincent W; York, Brian; Reineke, Erin L; Seo, Daekwan; Fan, Danhua; Silverstein, Kevin A T; Schrum, Christina T; Xie, Wei Rose; Mularoni, Loris; Wheelan, Sarah J; Torbenson, Michael S; O'Malley, Bert W; Largaespada, David A; Boeke, Jef D

    2012-05-22

    The Sleeping Beauty (SB) transposon mutagenesis system is a powerful tool that facilitates the discovery of mutations that accelerate tumorigenesis. In this study, we sought to identify mutations that cooperate with MYC, one of the most commonly dysregulated genes in human malignancy. We performed a forward genetic screen with a mouse model of MYC-induced liver cancer using SB-mediated mutagenesis. We sequenced insertions in 63 liver tumor nodules and identified at least 16 genes/loci that contribute to accelerated tumor development. RNAi-mediated knockdown in a liver progenitor cell line further validate three of these genes, Ncoa2/Src-2, Zfx, and Dtnb, as tumor suppressors in liver cancer. Moreover, deletion of Ncoa2/Src-2 in mice predisposes to diethylnitrosamine-induced liver tumorigenesis. These findings reveal genes and pathways that functionally restrain MYC-mediated liver tumorigenesis and therefore may provide targets for cancer therapy. PMID:22556267

  16. piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART) for genetic screens in mice.

    PubMed

    Landrette, Sean F; Cornett, Jonathan C; Ni, Thomas K; Bosenberg, Marcus W; Xu, Tian

    2011-01-01

    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. PMID:22039523

  17. Insertion derivatives containing segments of up to 16 amino acids identify surface- and periplasm-exposed regions of the FhuA outer membrane receptor of Escherichia coli K-12.

    PubMed Central

    Koebnik, R; Braun, V

    1993-01-01

    The FhuA receptor in the outer membrane of Escherichia coli K-12 is involved in the uptake of ferrichrome, colicin M, and the antibiotic albomycin and in infection by phages T1, T5, and phi 80. Fragments of up to 16 amino acid residues were inserted into FhuA and used to determine FhuA active sites and FhuA topology in the outer membrane. For this purpose antibiotic resistance boxes flanked by symmetric polylinkers were inserted into fhuA and subsequently partially deleted. Additional in-frame insertions were generated by mutagenesis with transposon Tn1725. The 68 FhuA protein derivatives examined contained segments of 4, 8, 12, 16, and 22 additional amino acid residues at 34 different locations from residues 5 to 646 of the mature protein. Most of the FhuA derivatives were found in normal amounts in the outer membrane fraction. Half of these were fully active toward all ligands, demonstrating proper insertion into the outer membrane. Seven of the 12- and 16-amino-acid-insertion derivatives (at residues 378, 402, 405, 415, 417, 456, and 646) were active toward all of the ligands and could be cleaved by subtilisin in whole cells, suggesting a surface location of the extra loops at sites which did not affect FhuA function. Two mutants were sensitive to subtilisin (insertions at residues 511 and 321) but displayed a strongly reduced sensitivity to colicin M and to phages phi 80 and T1. Four of the insertion derivatives (at residues 162, 223, 369, and 531) were cleaved only in spheroplasts and probably form loops at the periplasmic side of the outer membrane. The number and size of the proteolytic fragments indicate cleavage at or close to the sites of insertion, which has been proved for five insertions by amino acid sequencing. Most mutants with functional defects were affected in their sensitivity to all ligands, yet frequently to different degrees. Some mutants showed a specifically altered sensitivity to a few ligands; for example, mutant 511-04 was partially

  18. Isolation of Insertion Sequence ISRLdTAL1145-1 from a Rhizobium sp. (Leucaena diversifolia) and Distribution of Homologous Sequences Identifying Cross-Inoculation Group Relationships †

    PubMed Central

    Rice, Douglas J.; Somasegaran, Padma; MacGlashan, Kathryn; Bohlool, B. Ben

    1994-01-01

    Insertion sequence (IS) element ISRLdTAL1145-1 from Rhizobium sp. (Leucaena diversifolia) strain TAL 1145 was entrapped in the sacB gene of the positive selection vector pUCD800 by insertional inactivation. A hybridization probe prepared from the whole 2.5-kb element was used to determine the distribution of homologous sequences in a diverse collection of 135 Rhizobium and Bradyrhizobium strains. The IS probe hybridized strongly to Southern blots of genomic DNAs from 10 rhizobial strains that nodulate both Phaseolus vulgaris (beans) and Leucaena leucocephala (leguminous trees), 1 Rhizobium sp. that nodulates Leucaena spp., 9 R. meliloti (alfalfa) strains, 4 Rhizobium spp. that nodulate Sophora chrysophylla (leguminous trees), and 1 nonnodulating bacterium associated with the nodules of Pithecellobium dulce from the Leucaena cross-inoculation group, producing distinguishing IS patterns for each strain. Hybridization analysis revealed that ISRLdTAL1145-1 was strongly homologous with and closely related to a previously isolated element, ISRm USDA1024-1 from R. meliloti, while restriction enzyme analysis found structural similarities and differences between the two IS homologs. Two internal segments of these IS elements were used to construct hybridization probes of 1.2 kb and 380 bp that delineate a structural similarity and a difference, respectively, of the two IS homologs. The internal segment probes were used to analyze the structures of homologous IS elements in other strains. Five types of structural variation in homolog IS elements were found. The predominate IS structural type naturally occurring in a strain can reasonably identify the strain's cross-inoculation group relationships. Three IS structural types were found in Rhizobium species that nodulate beans and Leucaena species, one of which included the designated type IIB strain of R. tropici (CIAT 899). Weak homology to the whole IS probe, but not with the internal segments, was found with two

  19. X-ray microtomographic confirmation of the reliability of CBCT in identifying the scalar location of cochlear implant electrode after round window insertion.

    PubMed

    Zou, Jing; Hannula, Markus; Lehto, Kalle; Feng, Hao; Lähelmä, Jaakko; Aula, Antti S; Hyttinen, Jari; Pyykkö, Ilmari

    2015-08-01

    Cone-beam computed tomography (CBCT) plays a key role in cochlear implantation in both planning implantation before surgery and quality control during surgery due to the high spatial resolution and convenience of application in the operation theater. We recently designed a novel, highresolution cone-beam acquisition system that has been tested in temporal bones with cochlear implantation to identify the scalar localization of the electrode arrays. The current study aimed to verify the reliability of the experimental CBCT set-up using high-resolution in vitro X-ray microtomography (μCT) imaging as a reference. Nine human temporal bones were studied by inserting a straight electrode of a cochlear implant using the round window approach followed by sequential imaging using experimental CBCT and μCT with and without 1% iodine as the contrast agent. In the CBCT images, the electrodes were located in the scala tympani and near the lateral wall in all temporal bones. In the μCT images, the cochlear fine structures, including Reissner's membrane, stria vascularis, spiral ligament, basilar membrane, spiral limbus, osseous spiral lamina, and Rosenthal's canal that hosts the spiral ganglion cells, were clearly delineated; the electrode array avoided the lateral wall of the scala tympani in the hook region and then ran along the lateral wall of the scala tympani without any exception, a feature that was also detected in a temporal bone with ruptures in the basilar and Reissner's membranes. In conclusion, the current in vitro μCT imaging system produced high-quality images that could demonstrate the fine cochlear structures faithfully and verify the reliability of a novel experimental CBCT set-up aimed for clinical application in identifying the scalar localization of the electrode array. The straight electrode is safe for cochlear structures with low risk of translocation and is suitable for atraumatic implantation, although a large gap between the contacts and the

  20. Design, synthesis and evaluation of a potent substrate analog inhibitor identified by scanning Ala/Phe mutagenesis, mimicking substrate co-evolution, against multidrug-resistant HIV-1 protease

    SciTech Connect

    Yedidi, Ravikiran S.; Muhuhi, Joseck M.; Liu, Zhigang; Bencze, Krisztina Z.; Koupparis, Kyriacos; O’Connor, Carrie E.; Kovari, Iulia A.; Spaller, Mark R.; Kovari, Ladislau C.

    2013-09-06

    Highlights: •Inhibitors against MDR HIV-1 protease were designed, synthesized and evaluated. •Lead peptide (6a) showed potent inhibition (IC{sub 50}: 4.4 nM) of MDR HIV-1 protease. •(6a) Showed favorable binding isotherms against NL4-3 and MDR proteases. •(6a) Induced perturbations in the {sup 15}N-HSQC spectrum of MDR HIV-1 protease. •Molecular modeling suggested that (6a) may induce total flap closure inMDR protease. -- Abstract: Multidrug-resistant (MDR) clinical isolate-769, human immunodeficiency virus type-1 (HIV-1) protease (PDB ID: (1TW7)), was shown to exhibit wide-open flaps and an expanded active site cavity, causing loss of contacts with protease inhibitors. In the current study, the expanded active site cavity of MDR769 HIV-1 protease was screened with a series of peptide-inhibitors that were designed to mimic the natural substrate cleavage site, capsid/p2. Scanning Ala/Phe chemical mutagenesis approach was incorporated into the design of the peptide series to mimic the substrate co-evolution. Among the peptides synthesized and evaluated, a lead peptide (6a) with potent activity (IC{sub 50}: 4.4 nM) was identified against the MDR769 HIV-1 protease. Isothermal titration calorimetry data showed favorable binding profile for 6aagainst both wild type and MDR769 HIV-1 protease variants. Nuclear magnetic resonance spectrum of {sup 15}N-labeled MDR769 HIV-1 protease in complex with 6a showed some major perturbations in chemical shift, supporting the peptide induced conformational changes in protease. Modeling analysis revealed multiple contacts between 6a and MDR769 HIV-1 protease. The lead peptide-inhibitor, 6a, with high potency and good binding profile can be used as the basis for developing potent small molecule inhibitors against MDR variants of HIV.

  1. Function search in a large transcription factor gene family in Arabidopsis: assessing the potential of reverse genetics to identify insertional mutations in R2R3 MYB genes.

    PubMed Central

    Meissner, R C; Jin, H; Cominelli, E; Denekamp, M; Fuertes, A; Greco, R; Kranz, H D; Penfield, S; Petroni, K; Urzainqui, A; Martin, C; Paz-Ares, J; Smeekens, S; Tonelli, C; Weisshaar, B; Baumann, E; Klimyuk, V; Marillonnet, S; Patel, K; Speulman, E; Tissier, A F; Bouchez, D; Jones, J J; Pereira, A; Wisman, E

    1999-01-01

    More than 92 genes encoding MYB transcription factors of the R2R3 class have been described in Arabidopsis. The functions of a few members of this large gene family have been described, indicating important roles for R2R3 MYB transcription factors in the regulation of secondary metabolism, cell shape, and disease resistance, and in responses to growth regulators and stresses. For the majority of the genes in this family, however, little functional information is available. As the first step to characterizing these genes functionally, the sequences of >90 family members, and the map positions and expression profiles of >60 members, have been determined previously. An important second step in the functional analysis of the MYB family, through a process of reverse genetics that entails the isolation of insertion mutants, is described here. For this purpose, a variety of gene disruption resources has been used, including T-DNA-insertion populations and three distinct populations that harbor transposon insertions. We report the isolation of 47 insertions into 36 distinct MYB genes by screening a total of 73 genes. These defined insertion lines will provide the foundation for subsequent detailed functional analyses for the assignment of specific functions to individual members of the R2R3 MYB gene family. PMID:10521515

  2. Whole Genome Sequencing Identifies a 78 kb Insertion from Chromosome 8 as the Cause of Charcot-Marie-Tooth Neuropathy CMTX3.

    PubMed

    Brewer, Megan H; Chaudhry, Rabia; Qi, Jessica; Kidambi, Aditi; Drew, Alexander P; Menezes, Manoj P; Ryan, Monique M; Farrar, Michelle A; Mowat, David; Subramanian, Gopinath M; Young, Helen K; Zuchner, Stephan; Reddel, Stephen W; Nicholson, Garth A; Kennerson, Marina L

    2016-07-01

    With the advent of whole exome sequencing, cases where no pathogenic coding mutations can be found are increasingly being observed in many diseases. In two large, distantly-related families that mapped to the Charcot-Marie-Tooth neuropathy CMTX3 locus at chromosome Xq26.3-q27.3, all coding mutations were excluded. Using whole genome sequencing we found a large DNA interchromosomal insertion within the CMTX3 locus. The 78 kb insertion originates from chromosome 8q24.3, segregates fully with the disease in the two families, and is absent from the general population as well as 627 neurologically normal chromosomes from in-house controls. Large insertions into chromosome Xq27.1 are known to cause a range of diseases and this is the first neuropathy phenotype caused by an interchromosomal insertion at this locus. The CMTX3 insertion represents an understudied pathogenic structural variation mechanism for inherited peripheral neuropathies. Our finding highlights the importance of considering all structural variation types when studying unsolved inherited peripheral neuropathy cases with no pathogenic coding mutations. PMID:27438001

  3. Whole Genome Sequencing Identifies a 78 kb Insertion from Chromosome 8 as the Cause of Charcot-Marie-Tooth Neuropathy CMTX3

    PubMed Central

    Brewer, Megan H.; Chaudhry, Rabia; Qi, Jessica; Kidambi, Aditi; Drew, Alexander P.; Ryan, Monique M.; Subramanian, Gopinath M.; Young, Helen K.; Zuchner, Stephan; Reddel, Stephen W.; Nicholson, Garth A.; Kennerson, Marina L.

    2016-01-01

    With the advent of whole exome sequencing, cases where no pathogenic coding mutations can be found are increasingly being observed in many diseases. In two large, distantly-related families that mapped to the Charcot-Marie-Tooth neuropathy CMTX3 locus at chromosome Xq26.3-q27.3, all coding mutations were excluded. Using whole genome sequencing we found a large DNA interchromosomal insertion within the CMTX3 locus. The 78 kb insertion originates from chromosome 8q24.3, segregates fully with the disease in the two families, and is absent from the general population as well as 627 neurologically normal chromosomes from in-house controls. Large insertions into chromosome Xq27.1 are known to cause a range of diseases and this is the first neuropathy phenotype caused by an interchromosomal insertion at this locus. The CMTX3 insertion represents an understudied pathogenic structural variation mechanism for inherited peripheral neuropathies. Our finding highlights the importance of considering all structural variation types when studying unsolved inherited peripheral neuropathy cases with no pathogenic coding mutations. PMID:27438001

  4. Feeding tube insertion - gastrostomy

    MedlinePlus

    ... tube insertion; G-tube insertion; PEG tube insertion; Stomach tube insertion; Percutaneous endoscopic gastrostomy tube insertion ... and down the esophagus, which leads to the stomach. After the endoscopy tube is inserted, the skin ...

  5. Two novel PRPF31 premessenger ribonucleic acid processing factor 31 homolog mutations including a complex insertion-deletion identified in Chinese families with retinitis pigmentosa

    PubMed Central

    Dong, Bing; Chen, Jieqiong; Zhang, Xiaohui; Pan, Zhe; Bai, Fengge

    2013-01-01

    Objective To identify the causative mutations in two Chinese families with retinitis pigmentosa (RP), and to describe the associated phenotype. Methods Individuals from two unrelated families underwent full ophthalmic examinations. After informed consent was obtained, genomic DNA was extracted from the venous blood of all participants. Linkage analysis was performed on the known genetic loci for autosomal dominant retinitis pigmentosa with a panel of polymorphic markers in the two families, and then all coding exons of the PRP31 premessenger ribonucleic acid processing factor 31 homolog (PRPF31) gene were screened for mutations with direct sequencing of PCR-amplified DNA fragments. Allele-specific PCR was used to validate a substitution in all available family members and 100 normal controls. A large deletion was detected with real-time quantitative PCR (RQ-PCR) using a panel of primers from regions around the PRPF31 gene. Long-range PCR, followed by DNA sequencing, was used to define the breakpoints. Results Clinical examination and pedigree analysis revealed two four-generation families (RP24 and RP106) with autosomal dominant retinitis pigmentosa. A significant two-point linkage odd disequilibrium score was generated at marker D19S926 (Zmax=3.55, θ=0) for family RP24 and D19S571 (Zmax=3.21, θ=0) for family RP106, and further linkage and haplotype studies confined the disease locus to chromosome 19q13.42 where the PRPF31 gene is located. Mutation screening of the PRPF31 gene revealed a novel deletion c.1215delG (p.G405fs+7X) in family RP106. The deletion cosegregated with the family’s disease phenotype, but was not found in 100 normal controls. No disease-causing mutation was detected in family RP24 with PCR-based sequencing analysis. RQ-PCR and long-range PCR analysis revealed a complex insertion-deletion (indel) in the patients of family RP24. The deletion is more than 19 kb and encompasses part of the PRPF31 gene (exons 1–3), together with three adjacent

  6. Oligonucleotide-directed site-specific mutagenesis in Drosophila melanogaster.

    PubMed Central

    Banga, S S; Boyd, J B

    1992-01-01

    An efficient technique has been developed for performing in vivo site-directed mutagenesis in Drosophila melanogaster. This procedure involves directed repair of P-element-induced DNA lesions after injection of a modified DNA sequence into early embryos. An oligonucleotide of 50 base pairs, whose sequence spans the P-element insertion site, mediates base replacement in the endogenous gene. Restriction mapping, DNA sequencing, and polymerase chain reaction analysis demonstrate that base substitutions present in an injected oligonucleotide are incorporated into genomic sequences flanking a P insertion site in the white gene. This analysis suggests that progeny bearing directed mutations are recovered with a frequency of about 0.5 x 10(-3). Because Drosophila remains a premier organism for the analysis of eukaryotic gene regulation, this system should find strong application in that analysis as well as in the analysis of DNA recombination, conversion, repair, and mutagenesis. Images PMID:1311850

  7. Homemade Site Directed Mutagenesis of Whole Plasmids

    PubMed Central

    Laible, Mark; Boonrod, Kajohn

    2009-01-01

    Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers. PMID:19488024

  8. Insertion Sequences

    PubMed Central

    Mahillon, Jacques; Chandler, Michael

    1998-01-01

    Insertion sequences (ISs) constitute an important component of most bacterial genomes. Over 500 individual ISs have been described in the literature to date, and many more are being discovered in the ongoing prokaryotic and eukaryotic genome-sequencing projects. The last 10 years have also seen some striking advances in our understanding of the transposition process itself. Not least of these has been the development of various in vitro transposition systems for both prokaryotic and eukaryotic elements and, for several of these, a detailed understanding of the transposition process at the chemical level. This review presents a general overview of the organization and function of insertion sequences of eubacterial, archaebacterial, and eukaryotic origins with particular emphasis on bacterial elements and on different aspects of the transposition mechanism. It also attempts to provide a framework for classification of these elements by assigning them to various families or groups. A total of 443 members of the collection have been grouped in 17 families based on combinations of the following criteria: (i) similarities in genetic organization (arrangement of open reading frames); (ii) marked identities or similarities in the enzymes which mediate the transposition reactions, the recombinases/transposases (Tpases); (iii) similar features of their ends (terminal IRs); and (iv) fate of the nucleotide sequence of their target sites (generation of a direct target duplication of determined length). A brief description of the mechanism(s) involved in the mobility of individual ISs in each family and of the structure-function relationships of the individual Tpases is included where available. PMID:9729608

  9. Intraoperative Management of an Incidentally Identified Ectopic Ureter Inserting Into the Prostate of a Patient Undergoing Radical Prostatectomy for Prostate Cancer: A Case Report.

    PubMed

    Singhal, Udit; Dauw, Casey A; Li, Amy Y; Miller, David C; Wolf, J Stuart; Morgan, Todd M

    2015-08-01

    Congenital variations in urinary tract anatomy present unique surgical challenges when they present without prior knowledge. Ectopic ureters occur as a rare anatomic variation of the urinary tract and are often associated with duplicated renal collecting systems. While the condition is uncommon, even more atypical is its discovery and subsequent diagnosis during surgical intervention for treatment of localized prostate cancer.We describe the intraoperative management of a unique case of bilateral ectopic ureters, with a right-sided ureter inserting into the prostate of a 54-year-old male undergoing robotic-assisted radical prostatectomy. While unknown at the time of surgery, this right-sided ureter was associated with a nonfunctioning right upper renal moiety of a duplex renal collecting system. This aberration was discovered intraoperatively and confirmed with imaging, and a robotic-assisted radical prostatectomy with right distal ureterectomy was performed. PMID:26266359

  10. Inserting IUDs safely.

    PubMed

    Burnhill, M S

    1989-01-01

    A comprehensive discussion of safe insertion of IUDs in the contemporary U.S. setting, when any IUD complication may provoke litigation, includes explanations of complications listed on package inserts, what to look for in the pelvic exam, now to handle the inserter, whether to give prophylactic antibiotics or a cervical block, follow-up management, and advice on safe sex and hygiene. The similarities and differences in listed contraindications for the ParaGard and Progestasert IUDs are analyzed. It is important to know these listed contraindications to avoid being the sole defendant in a court case. Neither explicitly rules out nulliparas, and some women who have completed childbearing may be willing to risk ectopic pregnancy. The physician must be sure to avoid any possible risks of pelvic infection, however. It is important to postpone IUD insertion if there is any suggestion of lower genital tract infection. Similarly, IUD insertion is intended to last for years, so a paracervical block is recommended if access is difficult. Tips for ensuring scrupulous asepsis are suggested. Women for whom prophylactic antibiotics are advised include diabetics, those with heart valve disease or transplants. IUD patients should be clearly identified when they call in with complaints, and seen urgently. Finally, a sexual history should be taken to avoid candidates who engage in anal sex practices. PMID:12284992

  11. Systematic Mutagenesis of the Escherichia coli Genome†

    PubMed Central

    Kang, Yisheng; Durfee, Tim; Glasner, Jeremy D.; Qiu, Yu; Frisch, David; Winterberg, Kelly M.; Blattner, Frederick R.

    2004-01-01

    A high-throughput method has been developed for the systematic mutagenesis of the Escherichia coli genome. The system is based on in vitro transposition of a modified Tn5 element, the Sce-poson, into linear fragments of each open reading frame. The transposon introduces both positive (kanamycin resistance) and negative (I-SceI recognition site) selectable markers for isolation of mutants and subsequent allele replacement, respectively. Reaction products are then introduced into the genome by homologous recombination via the λRed proteins. The method has yielded insertion alleles for 1976 genes during a first pass through the genome including, unexpectedly, a number of known and putative essential genes. Sce-poson insertions can be easily replaced by markerless mutations by using the I-SceI homing endonuclease to select against retention of the transposon as demonstrated by the substitution of amber and/or in-frame deletions in six different genes. This allows a Sce-poson-containing gene to be specifically targeted for either designed or random modifications, as well as permitting the stepwise engineering of strains with multiple mutations. The promiscuous nature of Tn5 transposition also enables a targeted gene to be dissected by using randomly inserted Sce-posons as shown by a lacZ allelic series. Finally, assessment of the insertion sites by an iterative weighted matrix algorithm reveals that these hyperactive Tn5 complexes generally recognize a highly degenerate asymmetric motif on one end of the target site helping to explain the randomness of Tn5 transposition. PMID:15262929

  12. A piggyBac insertion disrupts Foxl2 expression that mimics BPES syndrome in mice.

    PubMed

    Shi, Fubiao; Ding, Sheng; Zhao, Shimin; Han, Min; Zhuang, Yuan; Xu, Tian; Wu, Xiaohui

    2014-07-15

    Blepharophimosis, ptosis, epicanthus inversus syndrome (BPES) is an autosomal dominant genetic disorder characterized by small palpebral fissures and other craniofacial malformations, often with (type I) but could also without (type II) premature ovarian failure. While mutations of the forkhead transcription factor FOXL2 are associated with and likely be responsible for many BPES cases, how FOXL2 affects craniofacial development remain to be understood. Through a large-scale piggyBac (PB) insertion mutagenesis, we have identified a mouse mutant carrying a PB insertion ∼160 kb upstream of the transcription start site (TSS) of Foxl2. The insertion reduces, but not eliminates, the expression of Foxl2. This mutant, but not its revertant, displays BPES-like conditions such as midface hypoplasia, eyelid abnormalities and female subfertility. Further analysis indicates that the mutation does not affect mandible, but causes premature fusion of the premaxilla-maxilla suture, smaller premaxilla and malformed maxilla during midface development. We further identified an evolutionarily conserved fragment near the insertion site and observed enhancer activity of this element in tissue culture cells. Analyses using DNase I hypersensitivity assay and chromosome conformation capture assay in developing maxillary and periocular tissues suggest that the DNA region near the insertion site likely interacts with Foxl2 TSS. Therefore, this mutant presents an excellent animal model for mechanistic study of BPES and regulation of Foxl2. PMID:24565867

  13. PAX5 is a tumor suppressor in mouse mutagenesis models of acute lymphoblastic leukemia

    PubMed Central

    Dang, Jinjun; Wei, Lei; de Ridder, Jeroen; Su, Xiaoping; Rust, Alistair G.; Roberts, Kathryn G.; Payne-Turner, Debbie; Cheng, Jinjun; Ma, Jing; Qu, Chunxu; Wu, Gang; Song, Guangchun; Huether, Robert G.; Schulman, Brenda; Janke, Laura; Zhang, Jinghui; Downing, James R.; van der Weyden, Louise; Adams, David J.

    2015-01-01

    Alterations of genes encoding transcriptional regulators of lymphoid development are a hallmark of B-progenitor acute lymphoblastic leukemia (B-ALL) and most commonly involve PAX5, encoding the DNA-binding transcription factor paired-box 5. The majority of PAX5 alterations in ALL are heterozygous, and key PAX5 target genes are expressed in leukemic cells, suggesting that PAX5 may be a haploinsufficient tumor suppressor. To examine the role of PAX5 alterations in leukemogenesis, we performed mutagenesis screens of mice heterozygous for a loss-of-function Pax5 allele. Both chemical and retroviral mutagenesis resulted in a significantly increased penetrance and reduced latency of leukemia, with a shift to B-lymphoid lineage. Genomic profiling identified a high frequency of secondary genomic mutations, deletions, and retroviral insertions targeting B-lymphoid development, including Pax5, and additional genes and pathways mutated in ALL, including tumor suppressors, Ras, and Janus kinase-signal transducer and activator of transcription signaling. These results show that in contrast to simple Pax5 haploinsufficiency, multiple sequential alterations targeting lymphoid development are central to leukemogenesis and contribute to the arrest in lymphoid maturation characteristic of ALL. This cross-species analysis also validates the importance of concomitant alterations of multiple cellular growth, signaling, and tumor suppression pathways in the pathogenesis of B-ALL. PMID:25855603

  14. P-Element Insertion Alleles of Essential Genes on the Third Chromosome of Drosophila Melanogaster: Mutations Affecting Embryonic Pns Development

    PubMed Central

    Salzberg, A.; Prokopenko, S. N.; He, Y.; Tsai, P.; Pal, M.; Maroy, P.; Glover, D. M.; Deak, P.; Bellen, H. J.

    1997-01-01

    To identify novel genes and to isolate tagged mutations in known genes that are required for the development of the peripheral nervous system (PNS), we have screened a novel collection of 2460 strains carrying lethal or semilethal P-element insertions on the third chromosome. Monoclonal antibody 22C10 was used as a marker to visualize the embryonic PNS. We identified 109 mutant strains that exhibited reproducible phenotypes in the PNS. Cytological and genetic analyses of these strains indicated that 87 mutations affect previously identified genes: tramtrack (n = 18 alleles), string (n = 15), cyclin A (n = 13), single-minded (n = 13), Delta (n = 9), neuralized (n = 4), pointed (n = 4), extra macrochaetae (n = 4), prospero (n = 3), tartan (n = 2), and pebble (n = 2). In addition, 13 mutations affect genes that we identified recently in a chemical mutagenesis screen designed to isolate similar mutants: hearty (n = 3), dorsotonals (n = 2), pavarotti (n = 2), sanpodo (n = 2), dalmatian (n = 1), missensed (n = 1), senseless (n = 1), and sticky ch1 (n = 1). The remaining nine mutations define seven novel complementation groups. The data presented here demonstrate that this collection of P elements will be useful for the identification and cloning of novel genes on the third chromosome, since >70% of mutations identified in the screen are caused by the insertion of a P element. A comparison between this screen and a chemical mutagenesis screen undertaken earlier highlights the complementarity of the two types of genetic screens. PMID:9409832

  15. Forward and reverse mutagenesis in C. elegans

    PubMed Central

    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

  16. Optogenetic mutagenesis in Caenorhabditis elegans

    PubMed Central

    Noma, Kentaro; Jin, Yishi

    2015-01-01

    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. PMID:26632265

  17. A mouse chromosome 4 balancer ENU-mutagenesis screen isolates eleven lethal lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ENU-mutagenesis is a powerful technique to identify genes regulating mammalian development. To functionally annotate the distal region of mouse chromosome 4, we performed an ENU-mutagenesis screen using a balancer chromosome targeted to this region of the genome. We isolated 11 lethal lines that map...

  18. Structure-function study of MalF protein by random mutagenesis.

    PubMed

    Tapia, M I; Mourez, M; Hofnung, M; Dassa, E

    1999-04-01

    MalF is one of the two integral inner membrane proteins of the maltose-maltodextrin transport system. To identify functional regions in this protein, we characterized a collection of malF mutants obtained by random mutagenesis. We analyzed their growth on maltose and maltodextrins, the steady-state levels and subcellular localization of the mutant proteins, and the subcellular localization of MalK. Only 2 of the 21 MalF mutant proteins allowed growth on maltose and maltodextrins. Most mutations resulting in immunodetectable proteins mapped to hydrophilic domains, indicating that insertions affecting transmembrane segments gave rise to unstable or lethal proteins. All MalF mutant proteins, even those C-terminally truncated or with large N-terminal deletions, were inserted into the cytoplasmic membrane. Having identified mutations leading to reduced steady-state level, to partial mislocation, and/or to misfolding, we were able to assign to some regions of MalF a role in the assembly of the MalFGK2 complex and/or in the transport mechanism. PMID:10094708

  19. Structure-Function Study of MalF Protein by Random Mutagenesis

    PubMed Central

    Tapia, María Isabel; Mourez, Michaël; Hofnung, Maurice; Dassa, Elie

    1999-01-01

    MalF is one of the two integral inner membrane proteins of the maltose-maltodextrin transport system. To identify functional regions in this protein, we characterized a collection of malF mutants obtained by random mutagenesis. We analyzed their growth on maltose and maltodextrins, the steady-state levels and subcellular localization of the mutant proteins, and the subcellular localization of MalK. Only 2 of the 21 MalF mutant proteins allowed growth on maltose and maltodextrins. Most mutations resulting in immunodetectable proteins mapped to hydrophilic domains, indicating that insertions affecting transmembrane segments gave rise to unstable or lethal proteins. All MalF mutant proteins, even those C-terminally truncated or with large N-terminal deletions, were inserted into the cytoplasmic membrane. Having identified mutations leading to reduced steady-state level, to partial mislocation, and/or to misfolding, we were able to assign to some regions of MalF a role in the assembly of the MalFGK2 complex and/or in the transport mechanism. PMID:10094708

  20. Chest tube insertion

    MedlinePlus

    Chest drainage tube insertion; Insertion of tube into chest; Tube thoracostomy; Pericardial drain ... When your chest tube is inserted, you will lie on your side or sit partly upright, with one arm over your ...

  1. Dihalocarbene Insertion Experiment

    ERIC Educational Resources Information Center

    Goh, S. H.

    1975-01-01

    Describes the insertion reaction using the insertion of carbenes into carbon-hydrogen bonds as an example. Outlines an experiment that will illustrate dihalocarbene insertions into diisopropyl ether. (GS)

  2. A simple and efficient method to identify replacements of P-lacZ by P-Gal4 lines allows obtaining Gal4 insertions in the bithorax complex of Drosophila.

    PubMed

    de Navas, Luis; Foronda, David; Suzanne, Magali; Sánchez-Herrero, Ernesto

    2006-11-01

    The functional replacement of one gene product by another one is a powerful method to study specificity in development and evolution. In Drosophila, the Gal4/UAS method has been used to analyze in vivo such functional substitutions. To this aim, Gal4 lines that inactivate a gene and reproduce its expression pattern are required, and they can be frequently obtained by replacing pre-existing P-lacZ lines with such characteristics. We have devised a new method to quickly identify replacements of P-lacZ lines by P-Gal4 lines, and applied it successfully to obtain Gal4 insertions in the Ultrabithorax and Abdominal-B Hox genes. We have used these lines to study the functional replacement of a Hox gene by another one. Our experiments confirm that the abdominal-A gene can replace Ultrabithorax in haltere development but that it cannot substitute for Abdominal-B in the formation of the genitalia. PMID:16971094

  3. Exploiting the power of LINE-1 retrotransposon mutagenesis for identification of genes involved in embryonic stem cell differentiation.

    PubMed

    Lenka, Nibedita; Krishnan, Shruthi; Board, Philip; Rangasamy, Danny

    2014-06-01

    Identifying the genes or epigenetic factors that control the self-renewal and differentiation of stem cells is critical to understanding the molecular basis of cell commitment. Although a number of insertional mutagenesis vectors have been developed for identifying gene functions in animal models, the L1 retrotransposition system offers additional advantages as a tool to disrupt genes in embryonic stem cells in order to identify their functions and the phenotypes associated with them. Recent advances in producing synthetic versions of L1 retrotransposon vector system and the optimization of techniques to accurately identify retrotransposon integration sites have increased their utility for gene discovery applications. We have developed a novel episomal, nonviral L1 retrotransposon vector using scaffold/matrix attachment regions that provides stable, sustained levels of retrotransposition in cell cultures without being affected by epigenetic silencing or from some of the common problems of vector integration. This modified vector contains a GFP marker whose expression occurs only after successful gene disruption events and thus the cells with disrupted genes can be easily picked for functional analysis. Here we present a method to disrupt gene function in embryonic stem cells that aid in the identification of genes involved in stem cell differentiation processes. The methods presented here can be easily adapted to the study of other types of cancer stem cells or induced pluripotent stem cells using the L1 retrotransposon as an insertional mutagen. PMID:24610122

  4. Coupled mutagenesis screens and genetic mapping in zebrafish.

    PubMed Central

    Rawls, John F; Frieda, Matthew R; McAdow, Anthony R; Gross, Jason P; Clayton, Chad M; Heyen, Candy K; Johnson, Stephen L

    2003-01-01

    Forward genetic analysis is one of the principal advantages of the zebrafish model system. However, managing zebrafish mutant lines derived from mutagenesis screens and mapping the corresponding mutations and integrating them into the larger collection of mutations remain arduous tasks. To simplify and focus these endeavors, we developed an approach that facilitates the rapid mapping of new zebrafish mutations as they are generated through mutagenesis screens. We selected a minimal panel of 149 simple sequence length polymorphism markers for a first-pass genome scan in crosses involving C32 and SJD inbred lines. We also conducted a small chemical mutagenesis screen that identified several new mutations affecting zebrafish embryonic melanocyte development. Using our first-pass marker panel in bulked-segregant analysis, we were able to identify the genetic map positions of these mutations as they were isolated in our screen. Rapid mapping of the mutations facilitated stock management, helped direct allelism tests, and should accelerate identification of the affected genes. These results demonstrate the efficacy of coupling mutagenesis screens with genetic mapping. PMID:12663538

  5. Tailoring of global transcription sigma D factor by random mutagenesis to improve Escherichia coli tolerance towards low-pHs.

    PubMed

    Gao, Xi; Jiang, Ling; Zhu, Liying; Xu, Qing; Xu, Xian; Huang, He

    2016-04-20

    Bioconversion processes of organic acid or acid hydrolysis of raw material for microbial metabolism often suffer limitations as a result of microbial sensitivity in low-pH conditions. We adopted a three-step method called RAndom Insertional-deletional Strand Exchange mutagenesis (RAISE) to engineer the components of global regulator Sigma D factor (RpoD) of Escherichia coli to improve its acid tolerance. The best strain Mutant VII was identified from random mutagenesis libraries based on the growth performance, which exhibited much higher growth rate than the control (0.22h(-1) vs. 0.15h(-1)) at pH as low as 3.17. Combined transcriptome and phenome analysis of E. coli was carried out to better understand the global effects of RpoD on the regulatory networks. Our analysis showed that 95 (2.1%) of all E. coli genes were induced and 178 (4.0%) genes were repressed, including those for trehalose biosynthesis, nucleotides biosynthesis, carbon metabolism, amino acid utilization, except for acid resistance. Also regulated were the master regulators (ArcA, EvgA, H-NS and RpoS) and gene/operon-specific transcription factors (GadX, GadW, AppY, YdeO, KdgR). These results demonstrated that RpoD acts as global regulator in the growth phase of E. coli and consequently improves acid tolerances. PMID:26971973

  6. Arabidopsis genes essential for seedling viability: isolation of insertional mutants and molecular cloning.

    PubMed Central

    Budziszewski, G J; Lewis, S P; Glover, L W; Reineke, J; Jones, G; Ziemnik, L S; Lonowski, J; Nyfeler, B; Aux, G; Zhou, Q; McElver, J; Patton, D A; Martienssen, R; Grossniklaus, U; Ma, H; Law, M; Levin, J Z

    2001-01-01

    We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening approximately 38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype. PMID:11779813

  7. Construction of targeted insertion mutations in Francisella tularensis subsp. novicida.

    PubMed

    Liu, Jirong; Zogaj, Xhavit; Barker, Jeffrey R; Klose, Karl E

    2007-10-01

    Francisella tularensis is one of the most deadly bacterial agents, yet most of the genetic determinants of pathogenesis are still unknown. We have developed an efficient targeted mutagenesis strategy in the model organism F. tularensis subsp. novicida by utilizing universal priming of optimized antibiotic resistance cassettes and splicing by overlap extension (SOE). This process enables fast and efficient construction of targeted insertion mutations in F. tularensis subsp. novicida that have characteristics of nonpolar mutations; optimized targeted mutagenesis strategies will promote the study of this mysterious bacterium and facilitate vaccine development against tularemia. Moreover the general strategy of gene disruption by PCR-based antibiotic resistance cassette insertion is broadly applicable to many bacterial species. PMID:18019340

  8. Insertional hypermutation in mineral oil-induced plasmacytomas

    PubMed Central

    Knittel, Gero; Metzner, Mirjam; Beck-Engeser, Gabriele; Kan, Ada; Ahrends, Tomasz; Eilat, Dan; Huppi, Konrad; Wabl, Matthias

    2014-01-01

    Unless stimulated by a chronic inflammatory agent such as mineral oil, plasma cell tumors are rare in young BALB/c mice. This raises the questions, What do inflammatory tissues provide to promote mutagenesis? and What is the nature of mutagenesis? We determined that mineral oil-induced plasmacytomas produce large amounts of endogenous retroelements—ecotropic and polytropic murine leukemia virus and intracisternal A-particles. Therefore, plasmacytoma formation might occur, in part, by de novo insertion of these retroelements, induced or helped by the inflammation. We recovered up to 10 de novo insertions in a single plasmacytoma, mostly in genes with common retroviral integration sites. Additional integrations accompany tumor evolution from a solid tumor through several generations in cell culture. The high frequency of de novo integrations into cancer genes suggests that endogenous retroelements are coresponsible for plasmacytoma formation and progression in BALB/c mice. PMID:24975032

  9. Identification of Legionella pneumophila Genes Important for Infection of Amoebas by Signature-Tagged Mutagenesis

    PubMed Central

    Polesky, Andrea H.; Ross, Julianna T. D.; Falkow, Stanley; Tompkins, Lucy S.

    2001-01-01

    Legionella pneumophila is a facultative intracellular gram-negative rod that causes pneumonia in humans. Free-living amoebas are thought to serve as a reservoir for Legionella infections. Signature-tagged mutagenesis was employed to identify Legionella pneumophila genes necessary for survival in the amoeba Acanthamoeba castellanii. Six mutant strains were defective in assays of invasion and intracellular growth. Four mutants also exhibited invasion and replication defects in Hartmannella vermiformis, an amoeba linked to hospital outbreaks of Legionella pneumonia. The six mutants also were tested in macrophages derived from peripheral blood mononuclear cells. Two mutants had intracellular replication defects, and two different strains entered cells less efficiently. Two transposon insertions were in known L. pneumophila genes, lspK and aroB. The other four were in novel genes. One gene has similarity to a cytochrome c-type biogenesis protein of Pseudomonas fluorescens. Another has similarity to a transcriptional activator regulating flagellar biosynthesis in Vibrio cholera. The third is similar to traA of Rhizobium sp. strain NGR234, which is involved in conjugal transfer of DNA. The fourth has no homology. By using survival in amoeba as a selection, we have isolated mutant strains with a range of phenotypes; and we have potentially identified new L. pneumophila virulence genes. PMID:11159993

  10. Genome-Wide Synthetic Genetic Screening by Transposon Mutagenesis in Candida albicans

    PubMed Central

    Horton, Brooke N.; Kumar, Anuj

    2016-01-01

    Transposon-based mutagenesis is an effective method for genetic screening on a genome-wide scale, with particular applicability in organisms possessing compact genomes where transforming DNA tends to integrate by homologous recombination. Methods for transposon mutagenesis have been applied with great success in the budding yeast Saccharomyces cerevisiae and in the related pathogenic yeast Candida albicans. In C. albicans, we have implemented transposon mutagenesis to generate heterozygous mutations for the analysis of complex haploinsufficiency, a type of synthetic genetic interaction wherein a pair of non-complementing heterozygous mutations results in a stronger phenotype then either individual mutation in isolation. Genes exhibiting complex haploinsufficiency typically function within a regulatory pathway, in parallel pathways, or in parallel branches within a single pathway. Here, we present protocols to implement transposon mutagenesis for complex haploinsufficiency screening in C. albicans, indicating methods for transposon construction, mutagenesis, phenotypic screening, and identification of insertion sites in strains of interest. In total, the approach is a useful means to implement large-scale synthetic genetic screening in the diploid C. albicans. PMID:25636616

  11. Application of a novel multi-screening signature-tagged mutagenesis assay for identification of Klebsiella pneumoniae genes essential in colonization and infection.

    PubMed

    Struve, Carsten; Forestier, Christiane; Krogfelt, Karen A

    2003-01-01

    Klebsiella pneumoniae is a common cause of urinary tract infections (UTIs) and pneumonia, especially in immunocompromised individuals. Epidemiological studies have revealed that K. pneumoniae infections are frequently preceded by gastrointestinal colonization and the gastrointestinal tract is believed to be the most important reservoir for transmission of the bacteria. To identify genes involved in the ability of K. pneumoniae to colonize the intestine and infect the urinary tract, a novel multi-screening signature-tagged mutagenesis (MS-STM) assay was implemented. In the MS-STM assay, PCR-amplified tags present in the inoculum as well as recovered pools from each infection model are simultaneously subjected to hybridization using each specific tag as a probe. Therefore, screenings of a mutant library in more than one infection model is significantly eased compared to the traditional signature-tagged mutagenesis methodology. From a total of 1,440 K. pneumoniae transposon mutants screened, 13 mutants were identified as attenuated in intestinal colonization as well as the UTI model. In addition, six mutants attenuated only in the UTI model were identified. Transposon insertion sites in attenuated mutants were, among others, in genes encoding well-known K. pneumoniae virulence factors such as lipopolysaccharide and capsule, as well as in genes of unknown function. PMID:12576590

  12. Functional genomics identifies drivers of medulloblastoma dissemination.

    PubMed

    Mumert, Michael; Dubuc, Adrian; Wu, Xiaochong; Northcott, Paul A; Chin, Steven S; Pedone, Carolyn A; Taylor, Michael D; Fults, Daniel W

    2012-10-01

    Medulloblastomas are malignant brain tumors that arise in the cerebellum in children and disseminate via the cerebrospinal fluid to the leptomeningeal spaces of the brain and spinal cord. Challenged by the poor prognosis for patients with metastatic dissemination, pediatric oncologists have developed aggressive treatment protocols, combining surgery, craniospinal radiation, and high-dose chemotherapy, that often cause disabling neurotoxic effects in long-term survivors. Insights into the genetic control of medulloblastoma dissemination have come from transposon insertion mutagenesis studies. Mobilizing the Sleeping Beauty transposon in cerebellar neural progenitor cells caused widespread dissemination of typically nonmetastatic medulloblastomas in Patched(+/-) mice, in which Shh signaling is hyperactive. Candidate metastasis genes were identified by sequencing the insertion sites and then mapping these sequences back to the mouse genome. To determine whether genes located at transposon insertion sites directly caused medulloblastomas to disseminate, we overexpressed candidate genes in Nestin(+) neural progenitors in the cerebella of mice by retroviral transfer in combination with Shh. We show here that ectopic expression of Eras, Lhx1, Ccrk, and Akt shifted the in vivo growth characteristics of Shh-induced medulloblastomas from a localized pattern to a disseminated pattern in which tumor cells seeded the leptomeningeal spaces of the brain and spinal cord. PMID:22875024

  13. Marker reconstitution mutagenesis: a simple and efficient reverse genetic approach.

    PubMed

    Tang, Xie; Huang, Junqi; Padmanabhan, Anup; Bakka, Kavya; Bao, Yun; Tan, Brenda Yuelin; Cande, W Zacheus; Balasubramanian, Mohan K

    2011-03-01

    A novel reverse genetic approach termed 'marker reconstitution mutagenesis' was designed to generate mutational allelic series in genes of interest. This approach consists of two simple steps which utilize two selective markers. First, using one selective marker, a partial fragment of another selective marker gene is inserted adjacently to a gene of interest by homologous recombination. Second, random mutations are introduced precisely into the gene of interest, together with the reconstitution of the latter selective marker by homologous recombination. This approach was successfully tested for several genes in the fission yeast Schizosaccharomyces pombe. It circumvents the problems encountered with other methods and should be adaptable to any organism that incorporates exogenous DNA by homologous recombination. PMID:21360732

  14. [Suprapubic catheter insertion].

    PubMed

    Neumann, Eva; Schwentner, Christian

    2016-01-01

    The suprapubic catheter enables a percutaneous drainage of urine. The insertion is made superior of the pubic bone through the abdominal wall into the bladder. It allows a permanent drainage of urine bypassing the urethra. The insertion of a suprapubic catheter requires knowledge and expertise. This paper summarizes the basic background and allows to follow the practical application step by step. PMID:26800072

  15. MECHANISM OF CHEMICAL MUTAGENESIS IV.

    PubMed Central

    Lorkiewicz, Z.; Szybalski, Waclaw

    1961-01-01

    Lorkiewicz, Z. (University of Wisconsin, Madison), and Waclaw Szybalski. Mechanism of chemical mutagenesis. IV. Reaction between triethylene melamine and nucleic acid components. J. Bacteriol. 82: 195–201. 1961.—Triethylene melamine interacts primarily with phosphorylated intracellular deoxyribonucleic acid (DNA) precursors and not with DNA. It was found by direct chemical and chromatographic analysis that only pyrimidine precursors of nucleic acids are attacked by triethylene melamine. In the course of the triethylene melamine-deoxycytidine reaction the mutagenicity of the reaction mixture is lost, but the mutagenicity of the triethylene melamine-thymidine reaction products significantly increases above that of the reaction substrates. Several steps are postulated to explain the mechanism of the triethylene melamine-initiated mutagenic reaction: (i) Reaction I, semireversible uptake of triethylene melamine; (ii) reaction II, chemical interaction between triethylene melamine and intracellular thymidine mono- or triphosphate with the production of a functional analogue of the latter; (iii) incorporation of this fraudulent analogue into the newly formed DNA strand; (iv) occurrence of self-perpetuating errors in the sequence of natural bases during subsequent rounds of replication of the analogue-containing DNA strand. It is postulated that the mechanism of mutagenic responses to different types of mutagens can fit either a simplified (mutagenic base analogues) or extended version (radiation) of this schema. PMID:16561917

  16. Mutagenesis and functional analysis of the pore-forming toxin HALT-1 from Hydra magnipapillata.

    PubMed

    Liew, Yvonne Jing Mei; Soh, Wai Tuck; Jiemy, William Febry; Hwang, Jung Shan

    2015-02-01

    Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1-4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding. PMID:25654788

  17. Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata

    PubMed Central

    Liew, Yvonne Jing Mei; Soh, Wai Tuck; Jiemy, William Febry; Hwang, Jung Shan

    2015-01-01

    Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1–4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding. PMID:25654788

  18. High-resolution definition of the Vibrio cholerae essential gene set with hidden Markov model–based analyses of transposon-insertion sequencing data

    PubMed Central

    Chao, Michael C.; Pritchard, Justin R.; Zhang, Yanjia J.; Rubin, Eric J.; Livny, Jonathan; Davis, Brigid M.; Waldor, Matthew K.

    2013-01-01

    The coupling of high-density transposon mutagenesis to high-throughput DNA sequencing (transposon-insertion sequencing) enables simultaneous and genome-wide assessment of the contributions of individual loci to bacterial growth and survival. We have refined analysis of transposon-insertion sequencing data by normalizing for the effect of DNA replication on sequencing output and using a hidden Markov model (HMM)-based filter to exploit heretofore unappreciated information inherent in all transposon-insertion sequencing data sets. The HMM can smooth variations in read abundance and thereby reduce the effects of read noise, as well as permit fine scale mapping that is independent of genomic annotation and enable classification of loci into several functional categories (e.g. essential, domain essential or ‘sick’). We generated a high-resolution map of genomic loci (encompassing both intra- and intergenic sequences) that are required or beneficial for in vitro growth of the cholera pathogen, Vibrio cholerae. This work uncovered new metabolic and physiologic requirements for V. cholerae survival, and by combining transposon-insertion sequencing and transcriptomic data sets, we also identified several novel noncoding RNA species that contribute to V. cholerae growth. Our findings suggest that HMM-based approaches will enhance extraction of biological meaning from transposon-insertion sequencing genomic data. PMID:23901011

  19. Identification of virulence determinants in uropathogenic Proteus mirabilis using signature-tagged mutagenesis.

    PubMed

    Himpsl, Stephanie D; Lockatell, C Virginia; Hebel, J Richard; Johnson, David E; Mobley, Harry L T

    2008-09-01

    The Gram-negative bacterium Proteus mirabilis causes urinary tract infections (UTIs) in individuals with long-term indwelling catheters or those with functional or structural abnormalities of the urinary tract. Known virulence factors include urease, haemolysin, fimbriae, flagella, DsbA, a phosphate transporter and genes involved in cell-wall synthesis and metabolism, many of which have been identified using the technique of signature-tagged mutagenesis (STM). To identify additional virulence determinants and to increase the theoretical coverage of the genome, this study generated and assessed 1880 P. mirabilis strain HI4320 mutants using this method. Mutants with disruptions in genes vital for colonization of the CBA mouse model of ascending UTI were identified after performing primary and secondary in vivo screens in approximately 315 CBA mice, primary and secondary in vitro screens in both Luria broth and minimal A medium to eliminate mutants with minor growth deficiencies, and co-challenge competition experiments in approximately 500 CBA mice. After completion of in vivo screening, a total of 217 transposon mutants were attenuated in the CBA mouse model of ascending UTI. Following in vitro screening, this number was reduced to 196 transposon mutants with a probable role in virulence. Co-challenge competition experiments confirmed significant attenuation for 37 of the 93 transposon mutants tested, being outcompeted by wild-type HI4320. Following sequence analysis of the 37 mutants, transposon insertions were identified in genes including the peptidyl-prolyl isomerases surA and ppiA, glycosyltransferase cpsF, biopolymer transport protein exbD, transcriptional regulator nhaR, one putative fimbrial protein, flagellar M-ring protein fliF and hook protein flgE, and multiple metabolic genes. PMID:18719175

  20. Ear tube insertion - slideshow

    MedlinePlus

    ... this page: //medlineplus.gov/ency/presentations/100045.htm Ear tube insertion - series—Normal anatomy To use the ... 4 Overview The eardrum (tympanic membrane) separates the ear canal from the middle ear. Update Date 8/ ...

  1. PEG tube insertion -- discharge

    MedlinePlus

    ... Search Search MedlinePlus GO GO About MedlinePlus Site ... To use the sharing features on this page, please enable JavaScript. A PEG (percutaneous endoscopic gastrostomy) feeding tube insertion is the placement of ...

  2. Chest tube insertion

    MedlinePlus

    ... leaks from inside the lung into the chest ( pneumothorax ) Fluid buildup in the chest (called a pleural ... on the reason a chest tube is inserted. Pneumothorax usually improves, but sometimes needs minimally invasive surgery. ...

  3. Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch.

    PubMed

    Oakes, Benjamin L; Nadler, Dana C; Flamholz, Avi; Fellmann, Christof; Staahl, Brett T; Doudna, Jennifer A; Savage, David F

    2016-06-01

    The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disruption of the enzyme's binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the estrogen receptor-α ligand-binding domain. This protein showed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9. PMID:27136077

  4. Transposon Mutagenesis of the Extremely Thermophilic Bacterium Thermus thermophilus HB27

    PubMed Central

    Carr, Jennifer F.; Gregory, Steven T.; Dahlberg, Albert E.

    2014-01-01

    Thermus thermophilus is an extremely thermophilic bacterium that grows between 50 °C and 80 °C and is an excellent model organism not only for understanding life at high temperature but also for its biotechnological and industrial applications. Multiple molecular capabilities are available including targeted gene inactivation and the use of shuttle plasmids that replicate in T. thermophilus and Escherichia coli; however the ability to disrupt gene function randomly by transposon insertion has not been developed. Here we report a detailed method of transposon mutagenesis of T. thermophilus HB27 based on the EZ-Tn5 system from Epicentre Biotechnologies. We were able to generate insertion mutations throughout the chromosome by in vitro transposition and transformation with mutagenized genomic DNA. We also report that an additional step, one that fills in single stranded gaps in donor DNA generated by the transposition reaction, was essential for successful mutagenesis. We anticipate that our method of transposon mutagenesis will enable further genetic development of T. thermophilus and may also be valuable for similar endeavors with other under-developed organisms. PMID:24948436

  5. Economical analysis of saturation mutagenesis experiments.

    PubMed

    Acevedo-Rocha, Carlos G; Reetz, Manfred T; Nov, Yuval

    2015-01-01

    Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced. PMID:26190439

  6. Economical analysis of saturation mutagenesis experiments

    PubMed Central

    Acevedo-Rocha, Carlos G.; Reetz, Manfred T.; Nov, Yuval

    2015-01-01

    Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced. PMID:26190439

  7. Phenotypes Associated with Second Chromosome P Element Insertions in Drosophila melanogaster

    PubMed Central

    Kahsai, Lily; Millburn, Gillian H.; Cook, Kevin R.

    2016-01-01

    In Drosophila melanogaster, P element transposition has been a productive means of insertional mutagenesis. Thousands of genes have been tagged with natural and engineered P element constructs. Nevertheless, chromosomes carrying P element insertions tend to have high levels of background mutations from P elements inserting and excising during transposition. Consequently, the phenotypes seen when P element-bearing chromosomes are homozygous are often not attributable to the P insertions themselves. In this study, 178 strains in the Bloomington Drosophila Stock Center collection with P insertions on the second chromosome were complementation tested against molecularly defined chromosomal deletions and previously characterized single-gene mutations to determine if recessive lethality or sterility is associated with the P insertions rather than background mutations. This information should prove valuable to geneticists using these strains for experimental studies of gene function. PMID:27317776

  8. Phenotypes Associated with Second Chromosome P Element Insertions in Drosophila melanogaster.

    PubMed

    Kahsai, Lily; Millburn, Gillian H; Cook, Kevin R

    2016-01-01

    In Drosophila melanogaster, P element transposition has been a productive means of insertional mutagenesis. Thousands of genes have been tagged with natural and engineered P element constructs. Nevertheless, chromosomes carrying P element insertions tend to have high levels of background mutations from P elements inserting and excising during transposition. Consequently, the phenotypes seen when P element-bearing chromosomes are homozygous are often not attributable to the P insertions themselves. In this study, 178 strains in the Bloomington Drosophila Stock Center collection with P insertions on the second chromosome were complementation tested against molecularly defined chromosomal deletions and previously characterized single-gene mutations to determine if recessive lethality or sterility is associated with the P insertions rather than background mutations. This information should prove valuable to geneticists using these strains for experimental studies of gene function. PMID:27317776

  9. ALS insertion devices

    SciTech Connect

    Hoyer, E.; Chin, J.; Halbach, K.; Hassenzahl, W.V.; Humphries, D.; Kincaid, B.; Lancaster, H.; Plate, D.

    1990-11-01

    The Advanced Light Source (ALS), the first US third generation synchrotron radiation source, is currently under construction at the Lawrence Berkeley Laboratory. The low-emittance, 1.5 GeV electron storage ring and the insertion devices are specifically designed to produce high brightness beams in the UV to soft X-Ray range. The planned initial complement of insertion devices includes four 4.6 m long undulators, with period lengths of 3.9 cm, 5.0 cm (2) and 8.0 cm, and a 2.9 m long wiggler of 16 cm period length. Undulator design is well advanced and fabrication has begun on the 5.0 cm and 8.0 cm period length undulators. This paper discusses ALS insertion device requirements; general design philosophy; and design of the magnetic structure, support structure/drive systems, control system and vacuum system. 18 refs., 9 figs., 5 tabs.

  10. Transposon mutagenesis as an approach to improved understanding of Borrelia pathogenesis and biology

    PubMed Central

    Lin, Tao; Troy, Erin B.; Hu, Linden T.; Gao, Lihui; Norris, Steven J.

    2014-01-01

    Transposon insertion provides a method for near-random mutation of bacterial genomes, and has been utilized extensively for the study of bacterial pathogenesis and biology. This approach is particularly useful for organisms that are relatively refractory to genetic manipulation, including Lyme disease Borrelia. In this review, progress to date in the application of transposon mutagenesis to the study of Borrelia burgdorferi is reported. An effective Himar1-based transposon vector has been developed and used to acquire a sequence-defined library of nearly 4500 mutants in the infectious, moderately transformable B. burgdorferi B31 derivative 5A18NP1. Analysis of these transposon mutants using signature-tagged mutagenesis (STM) and Tn-seq approaches has begun to yield valuable information regarding the genes important in the pathogenesis and biology of this organism. PMID:24904839

  11. Software-supported USER cloning strategies for site-directed mutagenesis and DNA assembly.

    PubMed

    Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen; Jespersen, Jakob Berg; Sommer, Morten O A; Wernersson, Rasmus; Olsen, Lars Rønn

    2015-03-20

    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 http://www.cbs.dtu.dk/services/AMUSER/. PMID:24847672

  12. CHALLENGES FOR THE FUTURE IN ENVIRONMENTAL MUTAGENESIS

    EPA Science Inventory

    CHALLENGES FOR THE FUTURE IN ENVIRONMENTAL MUTAGENESIS
    Michael D. Waters
    US Environmental Protection Agency, MD-51A, Research Triangle Park, NC 27711 USA

    Our rapidly growing understanding of the structure of the human genome is forming the basis for numerous new...

  13. Faux Mutagenesis: Teaching Troubleshooting through Controlled Failure

    ERIC Educational Resources Information Center

    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…

  14. MAMMALIAN CELL MUTAGENESIS, BANBURY CONFERENCE (JOURNAL VERSION)

    EPA Science Inventory

    A conference on mammalian cell mutagenesis was held at the Banbury Center, Cold Spring Harbor, NY, USA, March 22-25, 1987. The objective of the conference was to provide a forum for discussions concerning the genetic, biochemical, and molecular basis of induced mutations in stand...

  15. Interfacial insert for electrical connectors

    NASA Technical Reports Server (NTRS)

    Macavay, D.

    1975-01-01

    The development of interfacial inserts for improved electric connectors is discussed. The inserts were manufactured from epoxy resins. The design features of the inserts and the manufacturing equipment are described. The reliability test program is reported. Drawings of the interfacial inserts are provided.

  16. Insertion in Persian

    ERIC Educational Resources Information Center

    Kambuziya, Aliyeh Kord-e Zafaranlu; Dehghan, Masoud

    2011-01-01

    This paper investigates epenthesis process in Persian to catch some results in relating to vowel and consonant insertion in Persian lexicon. This survey has a close relationship to the description of epenthetic consonants and the conditions in which these consonants are used. Since no word in Persian may begin with a vowel, so that hiatus can't be…

  17. EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining TILLING mutant populations

    PubMed Central

    2014-01-01

    Background TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that combines chemical mutagenesis with high-throughput genome-wide screening for point mutation detection in genes of interest. However, this mutation discovery approach faces a particular problem which is how to obtain a mutant population with a sufficiently high mutation density. Furthermore, plant mutagenesis protocols require two successive generations (M1, M2) for mutation fixation to occur before the analysis of the genotype can begin. Results Here, we describe a new TILLING approach for rice based on ethyl methanesulfonate (EMS) mutagenesis of mature seed-derived calli and direct screening of in vitro regenerated plants. A high mutagenesis rate was obtained (i.e. one mutation in every 451 Kb) when plants were screened for two senescence-related genes. Screening was carried out in 2400 individuals from a mutant population of 6912. Seven sense change mutations out of 15 point mutations were identified. Conclusions This new strategy represents a significant advantage in terms of time-savings (i.e. more than eight months), greenhouse space and work during the generation of mutant plant populations. Furthermore, this effective chemical mutagenesis protocol ensures high mutagenesis rates thereby saving in waste removal costs and the total amount of mutagen needed thanks to the mutagenesis volume reduction. PMID:24475756

  18. Identifying assembly-inhibiting and assembly-tolerant sites in the SbsB S-layer protein from Geobacillus stearothermophilus.

    PubMed

    Kinns, Helen; Badelt-Lichtblau, Helga; Egelseer, Eva Maria; Sleytr, Uwe B; Howorka, Stefan

    2010-01-29

    Surface layer (S-layer) proteins self-assemble into two-dimensional crystalline lattices that cover the cell wall of all archaea and many bacteria. We have generated assembly-negative protein variants of high solubility that will facilitate high-resolution structure determination. Assembly-negative versions of the S-layer protein SbsB from Geobacillus stearothermophilus PV72/p2 were obtained using an insertion mutagenesis screen. The haemagglutinin epitope tag was inserted at 23 amino acid positions known to be located on the monomer protein surface from a previous cysteine accessibility screen. Limited proteolysis, circular dichroism, and fluorescence were used to probe whether the epitope insertion affected the secondary and tertiary structures of the monomer, while electron microscopy and size-exclusion chromatography were employed to examine proteins' ability to self-assemble. The screen not only identified assembly-compromised mutants with native fold but also yielded correctly folded, self-assembling mutants suitable for displaying epitopes for biomedical and biophysical applications, as well as cryo-electron microscopy imaging. Our study marks an important step in the analysis of the S-layer structure. In addition, the approach of concerted insertion and cysteine mutagenesis can likely be applied for other supramolecular assemblies. PMID:19836402

  19. Quantitative analysis of bristle number in Drosophila mutants identifies genes involved in neural development

    NASA Technical Reports Server (NTRS)

    Norga, Koenraad K.; Gurganus, Marjorie C.; Dilda, Christy L.; Yamamoto, Akihiko; Lyman, Richard F.; Patel, Prajal H.; Rubin, Gerald M.; Hoskins, Roger A.; Mackay, Trudy F.; Bellen, Hugo J.

    2003-01-01

    BACKGROUND: The identification of the function of all genes that contribute to specific biological processes and complex traits is one of the major challenges in the postgenomic era. One approach is to employ forward genetic screens in genetically tractable model organisms. In Drosophila melanogaster, P element-mediated insertional mutagenesis is a versatile tool for the dissection of molecular pathways, and there is an ongoing effort to tag every gene with a P element insertion. However, the vast majority of P element insertion lines are viable and fertile as homozygotes and do not exhibit obvious phenotypic defects, perhaps because of the tendency for P elements to insert 5' of transcription units. Quantitative genetic analysis of subtle effects of P element mutations that have been induced in an isogenic background may be a highly efficient method for functional genome annotation. RESULTS: Here, we have tested the efficacy of this strategy by assessing the extent to which screening for quantitative effects of P elements on sensory bristle number can identify genes affecting neural development. We find that such quantitative screens uncover an unusually large number of genes that are known to function in neural development, as well as genes with yet uncharacterized effects on neural development, and novel loci. CONCLUSIONS: Our findings establish the use of quantitative trait analysis for functional genome annotation through forward genetics. Similar analyses of quantitative effects of P element insertions will facilitate our understanding of the genes affecting many other complex traits in Drosophila.

  20. Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells

    PubMed Central

    Tsakiridis, Anestis; Tzouanacou, Elena; Rahman, Afifah; Colby, Douglas; Axton, Richard; Chambers, Ian; Wilson, Valerie; Forrester, Lesley; Brickman, Joshua M.

    2009-01-01

    Promoterless gene trap vectors have been widely used for high-efficiency gene targeting and random mutagenesis in embryonic stem (ES) cells. Unfortunately, such vectors are only effective for genes expressed in ES cells and this has prompted the development of expression-independent vectors. These polyadenylation (poly A) trap vectors employ a splice donor to capture an endogenous gene's polyadenylation sequence and provide transcript stability. However, the spectrum of mutations generated by these vectors appears largely restricted to the last intron of target loci due to nonsense-mediated mRNA decay (NMD) making them unsuitable for gene targeting applications. Here, we present novel poly A trap vectors that overcome the effect of NMD and also employ RNA instability sequences to improve splicing efficiency. The set of random insertions generated with these vectors show a significantly reduced insertional bias and the vectors can be targeted directly to a 5′ intron. We also show that this relative positional independence is linked to the human β-actin promoter and is most likely a result of its transcriptional activity in ES cells. Taken together our data indicate that these vectors are an effective tool for insertional mutagenesis that can be used for either gene trapping or gene targeting. PMID:19692586

  1. REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering

    PubMed Central

    Trehan, Ashutosh; Kiełbus, Michał; Czapinski, Jakub; Stepulak, Andrzej; Huhtaniemi, Ilpo; Rivero-Müller, Adolfo

    2016-01-01

    Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins. Recently, the use of in vitro seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these in vitro isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes in vitro is not only expensive but also more inaccurate than the high-fidelity recombination inside bacteria. Here we present a single-step method, named REPLACR-mutagenesis (Recombineering of Ends of linearised PLAsmids after PCR), for creating mutations (deletions, substitutions and additions) in plasmids by in vivo recombineering. REPLACR-mutagenesis only involves transformation of PCR products in bacteria expressing Red/ET recombineering proteins. Modifications in a variety of plasmids up to bacterial artificial chromosomes (BACs; 144 kb deletion) have been achieved by this method. The presented method is more robust, involves fewer steps and is cost-efficient. PMID:26750263

  2. REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering.

    PubMed

    Trehan, Ashutosh; Kiełbus, Michał; Czapinski, Jakub; Stepulak, Andrzej; Huhtaniemi, Ilpo; Rivero-Müller, Adolfo

    2016-01-01

    Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins. Recently, the use of in vitro seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these in vitro isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes in vitro is not only expensive but also more inaccurate than the high-fidelity recombination inside bacteria. Here we present a single-step method, named REPLACR-mutagenesis (Recombineering of Ends of linearised PLAsmids after PCR), for creating mutations (deletions, substitutions and additions) in plasmids by in vivo recombineering. REPLACR-mutagenesis only involves transformation of PCR products in bacteria expressing Red/ET recombineering proteins. Modifications in a variety of plasmids up to bacterial artificial chromosomes (BACs; 144 kb deletion) have been achieved by this method. The presented method is more robust, involves fewer steps and is cost-efficient. PMID:26750263

  3. Random mutagenesis strategies for construction of large and diverse clone libraries of mutated DNA fragments.

    PubMed

    Chusacultanachai, Sudsanguan; Yuthavong, Yongyuth

    2004-01-01

    The first important step toward a successful preparation of large and diverse DNA libraries with desired complexity is to select a suitable mutagenesis strategy. This chapter describes three different methods for random mutagenesis, the use of XL1-red cells, error-prone polymerase chain reaction (PCR), and degenerate oligonucleotides-Pfu (DOP). These mutagenesis strategies possess different benefits and pitfalls; thus, they are differentially useful for production of DNA libraries with different density and complexity. The use of XL1-red, an engineered Escherichia coli with DNA repair deficiency, is one of the simplest mutagenesis and requires no subcloning step. After plasmid encoding DNA of inter-est is transformed into the cells, the mutations are simply generated during each round of DNA replication. The mutation frequency of this method is reported to be 1 base change per 2000 nucleotides; however, it can be slightly increased by extending the culture period to allow the accumulation of more mutations. This strategy is suitable for generation of random mutations with low frequency in a large target DNA. Error-prone PCR is one of the most widely used random mutagenesis. During DNA amplification, misincorporation of nucleotides can be promoted by altering the nucleotide ratio and the concentration of divalent cations in the reaction. We discovered that, by adjusting template concentration, frequency of mutation could be controlled easily and a library with desired mutation rate could be obtained. Additionally, efficiency of subsequent cloning steps to insert the PCR product into plasmid DNA is also a key factor determining size and complexity of the libraries. DOP mutagenesis is a rapid and effective method for random mutagenesis of small DNA and peptides. This strategy uses two chemically synthesized degenerate oligonucleotides as primers. By controlling the positions and ratios of degenerate nucleotides used during oligonucleotide synthesis, it is possible to

  4. Agrobacterium tumefaciens-Mediated Transformation of Valsa mali: An Efficient Tool for Random Insertion Mutagenesis

    PubMed Central

    Wang, Caixia; Guan, Xiangnan; Wang, Hanyan; Li, Guifang; Dong, Xiangli; Wang, Guoping

    2013-01-01

    Valsa mali is a causal agent of apple and pear trees canker disease, which is a destructive disease that causes serious economic losses in eastern Asia, especially in China. The lack of an efficient transformation system for Valsa mali retards its investigation, which poses difficulties to control the disease. In this research, a transformation system for this pathogen was established for the first time using A. tumefaciens-mediated transformation (ATMT), with the optimal transformation conditions as follows: 106/mL conidia suspension, cocultivation temperature 22°C, cocultivation time 72 hours, and 200 μM acetosyringone (AS) in the inductive medium. The average transformation efficiency was 1015.00 ± 37.35 transformants per 106 recipient conidia. Thirty transformants were randomly selected for further confirmation and the results showed the presence of T-DNA in all hygromycin B resistant transformants and also revealed random and single gene integration with genetic stability. Compared with wild-type strain, those transformants exhibited various differences in morphology, conidia production, and conidia germination ability. In addition, pathogenicity assays revealed that 14 transformants had mitigated pathogenicity, while one had enhanced infection ability. The results suggest that ATMT of V. mali is a useful tool to gain novel insight into this economically important pathogen at molecular levels. PMID:24381526

  5. [Orthopedic shoe treatment : Inserts].

    PubMed

    Schuh, R; Windhager, R

    2016-03-01

    The use of inserts and orthopedic shoe adjustment represents an essential component of the conservative therapy of degenerative diseases and deformities of the musculoskeletal system. Inserts can have supportive, bedding and corrective effects and are used in particular for complaints of the feet and ankles. The combination of diverse materials allows a high level of cushioning and supporting features and corresponding longevity to be accomplished. The production is carried out on an individual basis and if necessary computer-assisted in order to achieve an optimal fit. For severe and rigid deformities the formation of pressure ulcers can be prevented by orthopedic shoe adjustment and by the use of orthopedic tailor-made shoes. PMID:26861757

  6. Insertion Profiles of 4 Headless Compression Screws

    PubMed Central

    Hart, Adam; Harvey, Edward J.; Lefebvre, Louis-Philippe; Barthelat, Francois; Rabiei, Reza; Martineau, Paul A.

    2013-01-01

    Purpose In practice, the surgeon must rely on screw position (insertion depth) and tactile feedback from the screwdriver (insertion torque) to gauge compression. In this study, we identified the relationship between interfragmentary compression and these 2 factors. Methods The Acutrak Standard, Acutrak Mini, Synthes 3.0, and Herbert-Whipple implants were tested using a polyurethane foam scaphoid model. A specialized testing jig simultaneously measured compression force, insertion torque, and insertion depth at half-screw-turn intervals until failure occurred. Results The peak compression occurs at an insertion depth of −3.1 mm, −2.8 mm, 0.9 mm, and 1.5 mm for the Acutrak Mini, Acutrak Standard, Herbert-Whipple, and Synthes screws respectively (insertion depth is positive when the screw is proud above the bone and negative when buried). The compression and insertion torque at a depth of −2 mm were found to be 113 ± 18 N and 0.348 ± 0.052 Nm for the Acutrak Standard, 104 ± 15 N and 0.175 ± 0.008 Nm for the Acutrak Mini, 78 ± 9 N and 0.245 ± 0.006 Nm for the Herbert-Whipple, and 67 ± 2N, 0.233 ± 0.010 Nm for the Synthes headless compression screws. Conclusions All 4 screws generated a sizable amount of compression (> 60 N) over a wide range of insertion depths. The compression at the commonly recommended insertion depth of −2 mm was not significantly different between screws; thus, implant selection should not be based on compression profile alone. Conically shaped screws (Acutrak) generated their peak compression when they were fully buried in the foam whereas the shanked screws (Synthes and Herbert-Whipple) reached peak compression before they were fully inserted. Because insertion torque correlated poorly with compression, surgeons should avoid using tactile judgment of torque as a proxy for compression. Clinical relevance Knowledge of the insertion profile may improve our understanding of the implants, provide a better basis for comparing screws

  7. Identification of Zebrafish Insertional Mutants With Defects in Visual System Development and Function

    PubMed Central

    Gross, Jeffrey M.; Perkins, Brian D.; Amsterdam, Adam; Egaña, Ana; Darland, Tristan; Matsui, Jonathan I.; Sciascia, Salvatore; Hopkins, Nancy; Dowling, John E.

    2005-01-01

    Genetic analysis in zebrafish has been instrumental in identifying genes necessary for visual system development and function. Recently, a large-scale retroviral insertional mutagenesis screen, in which 315 different genes were mutated, that resulted in obvious phenotypic defects by 5 days postfertilization was completed. That the disrupted gene has been identified in each of these mutants provides unique resource through which the formation, function, or physiology of individual organ systems can be studied. To that end, a screen for visual system mutants was performed on 250 of the mutants in this collection, examining each of them histologically for morphological defects in the eye and behaviorally for overall visual system function. Forty loci whose disruption resulted in defects in eye development and/or visual function were identified. The mutants have been divided into the following phenotypic classes that show defects in: (1) morphogenesis, (2) growth and central retinal development, (3) the peripheral marginal zone, (4) retinal lamination, (5) the photoreceptor cell layer, (6) the retinal pigment epithelium, (7) the lens, (8) retinal containment, and (9) behavior. The affected genes in these mutants highlight a diverse set of proteins necessary for the development, maintenance, and function of the vertebrate visual system. PMID:15716491

  8. Final report [DNA Repair and Mutagenesis - 1999

    SciTech Connect

    Walker, Graham C.

    2001-05-30

    The meeting, titled ''DNA Repair and Mutagenesis: Mechanism, Control, and Biological Consequences'', was designed to bring together the various sub-disciplines that collectively comprise the field of DNA Repair and Mutagenesis. The keynote address was titled ''Mutability Doth Play Her Cruel Sports to Many Men's Decay: Variations on the Theme of Translesion Synthesis.'' Sessions were held on the following themes: Excision repair of DNA damage; Transcription and DNA excision repair; UmuC/DinB/Rev1/Rad30 superfamily of DNA polymerases; Cellular responses to DNA damage, checkpoints, and damage tolerance; Repair of mismatched bases, mutation; Genome-instability, and hypermutation; Repair of strand breaks; Replicational fidelity, and Late-breaking developments; Repair and mutation in challenging environments; and Defects in DNA repair: consequences for human disease and aging.

  9. Z-2 Threaded Insert Design and Testing

    NASA Technical Reports Server (NTRS)

    Ross, Amy; Rhodes, Richard; Jones, Robert J.; Graziosi, David; Ferl, Jinny; Sweeny, Mitch; Scarborough, Stephen

    2016-01-01

    NASA's Z-2 prototype space suit contains several components fabricated from an advanced hybrid composite laminate consisting of IM10 carbon fiber and fiber glass. One requirement was to have removable, replaceable helicoil inserts to which other suit components would be fastened. An approach utilizing bonded in inserts with helicoils inside of them was implemented. During initial assembly, cracking sounds were heard followed by the lifting of one of the blind inserts out of its hole when the screws were torqued. A failure investigation was initiated to understand the mechanism of the failure. Ultimately, it was determined that the pre-tension caused by torqueing the fasteners is a much larger force than induced from the pressure loads of the suit which was not considered in the insert design. Bolt tension is determined by dividing the torque on the screw by a k value multiplied by the thread diameter of the bolt. The k value is a factor that accounts for friction in the system. A common value used for k for a non-lubricated screw is 0.2. The k value can go down by as much as 0.1 if the screw is lubricated which means for the same torque, a much larger tension could be placed on the bolt and insert. This paper summarizes the failure investigation that was performed to identify the root cause of the suit failure and details how the insert design was modified to resist a higher pull out tension.

  10. Mutant fatty acid desaturase and methods for directed mutagenesis

    DOEpatents

    Shanklin, John; Whittle, Edward J.

    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.

  11. SDM-Assist software to design site-directed mutagenesis primers introducing “silent” restriction sites

    PubMed Central

    2013-01-01

    Background Over the past decades site-directed mutagenesis (SDM) has become an indispensable tool for biological structure-function studies. In principle, SDM uses modified primer pairs in a PCR reaction to introduce a mutation in a cDNA insert. DpnI digestion of the reaction mixture is used to eliminate template copies before amplification in E. coli; however, this process is inefficient resulting in un-mutated clones which can only be distinguished from mutant clones by sequencing. Results We have developed a program – ‘SDM-Assist’ which creates SDM primers adding a specific identifier: through additional silent mutations a restriction site is included or a previous one removed which allows for highly efficient identification of ‘mutated clones’ by a simple restriction digest. Conclusions The direct identification of SDM clones will save time and money for researchers. SDM-Assist also scores the primers based on factors such as Tm, GC content and secondary structure allowing for simplified selection of optimal primer pairs. PMID:23522286

  12. Genome-wide mining of potential virulence-associated genes in Riemerella anatipestifer using random transposon mutagenesis.

    PubMed

    Ni, Xintao; Jiang, Pan; Xing, Linlin; Ou, Changcan; Yu, Hui; Qi, Jingjing; Sun, Bingqing; Cui, Junsheng; Wang, Guijun; Hu, Qinghai

    2016-06-30

    Riemerella anatipestifer infection is a severe disease confronting the duck industry worldwide. However, little is known about the molecular basis of R. anatipestifer pathogenesis. In this study, we screened 3580 transposon Tn4351 insertion mutagenesis mutants of the highly virulent strain YZb1 in a duckling infection experiment and found 29 of them to be attenuated and 28 potential virulence-associated genes were identified. Molecular characterization of transposon insertion sites showed that of the 28 screened genes, two were predicted to encode TonB-dependent outer membrane receptor (plugs), sixteen encoded enzymes, and seven encoded hypothetical proteins. In addition, of the 28 affected genes, 19 were only found in bacteria belonging to the phylum Bacteroidetes and 10 were only found in the family Flavobacteriaceae. The median lethal dose of the mutants M11 and M29, which was affected in Riean_0060 and Riean_1537 respectively, were about 1700-fold and 210-fold higher than that of the wild-type strain YZb1, and those of the complemented strains M11(pRES-Riean_0060) and M29(pRES-Riean_1537) were decreased by 25- and 3-fold respectively compared to those of the mutants M11 and M29. Additional analysis indicated that the blood bacterial loading of ducklings infected with M11 or M29 was decreased significantly, as compared with that in ducklings infected with the wild-type strain YZb1. Thus, our results indicate that Riean_0060 and Riean_1537 were involved in R. anatipestifer pathogenesis. PMID:27259827

  13. A Mariner Transposon-Based Signature-Tagged Mutagenesis System for the Analysis of Oral Infection by Listeria monocytogenes

    PubMed Central

    Cummins, Joanne; Casey, Pat G.; Joyce, Susan A.; Gahan, Cormac G. M.

    2013-01-01

    Listeria monocytogenes is a Gram-positive foodborne pathogen and the causative agent of listerosis a disease that manifests predominately as meningitis in the non-pregnant individual or infection of the fetus and spontaneous abortion in pregnant women. Common-source outbreaks of foodborne listeriosis are associated with significant morbidity and mortality. However, relatively little is known concerning the mechanisms that govern infection via the oral route. In order to aid functional genetic analysis of the gastrointestinal phase of infection we designed a novel signature-tagged mutagenesis (STM) system based upon the invasive L. monocytogenes 4b serotype H7858 strain. To overcome the limitations of gastrointestinal infection by L. monocytogenes in the mouse model we created a H7858 strain that is genetically optimised for oral infection in mice. Furthermore our STM system was based upon a mariner transposon to favour numerous and random transposition events throughout the L. monocytogenes genome. Use of the STM bank to investigate oral infection by L. monocytogenes identified 21 insertion mutants that demonstrated significantly reduced potential for infection in our model. The sites of transposon insertion included lmOh7858_0671 (encoding an internalin homologous to Lmo0610), lmOh7858_0898 (encoding a putative surface-expressed LPXTG protein homologous to Lmo0842), lmOh7858_2579 (encoding the HupDGC hemin transport system) and lmOh7858_0399 (encoding a putative fructose specific phosphotransferase system). We propose that this represents an optimised STM system for functional genetic analysis of foodborne/oral infection by L. monocytogenes. PMID:24069416

  14. CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening.

    PubMed

    Ito, Yasuhiro; Nishizawa-Yokoi, Ayako; Endo, Masaki; Mikami, Masafumi; Toki, Seiichi

    2015-11-01

    Site-directed mutagenesis using genetic approaches can provide a wealth of resources for crop breeding as well as for biological research. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 endonuclease (CRISPR/Cas9) system is a novel strategy used to induce mutations in a specific genome region; the system functions in a variety of organisms, including plants. Here, we report application of the CRISPR/Cas9 system to efficient mutagenesis of the tomato genome. In this study, we targeted the tomato RIN gene, which encodes a MADS-box transcription factor regulating fruit ripening. Three regions within the gene were targeted and mutations consisting either of a single base insertion or deletion of more than three bases were found at the Cas9 cleavage sites in T0 regenerated plants. The RIN-protein-defective mutants produced incomplete-ripening fruits in which red color pigmentation was significantly lower than that of wild type, while heterologous mutants expressing the remaining wild-type gene reached full-ripening red color, confirming the important role of RIN in ripening. Several mutations that were generated at three independent target sites were inherited in the T1 progeny, confirming the applicability of this mutagenesis system in tomato. PMID:26408904

  15. Inserts Automatically Lubricate Ball Bearings

    NASA Technical Reports Server (NTRS)

    Hager, J. A.

    1983-01-01

    Inserts on ball-separator ring of ball bearings provide continuous film of lubricant on ball surfaces. Inserts are machined or molded. Small inserts in ball pockets provide steady supply of lubricant. Technique is utilized on equipment for which maintenance is often poor and lubrication interval is uncertain, such as household appliances, automobiles, and marine engines.

  16. Transposon mutagenesis of Pseudomonas aeruginosa exoprotease genes.

    PubMed Central

    Stapleton, M J; Jagger, K S; Warren, R L

    1984-01-01

    Transposon Tn5 was used to generate protease-deficient insertion mutants of Pseudomonas aeruginosa. The presence of Tn5 in the chromosome of P. aeruginosa was demonstrated by transduction and DNA-DNA hybridization. The altered protease production and kanamycin resistance were cotransduced into a wild-type P. aeruginosa strain. A radiolabeled probe of Tn5 DNA hybridized to specific BamHI fragments isolated from the insertion mutants. Two independently isolated Tn5 insertion mutants had reduced protease production, partially impaired elastase activity, and no immunologically reactive alkaline protease. Images PMID:6317657

  17. The Parasol Protocol for computational mutagenesis.

    PubMed

    Aronica, P G A; Verma, C; Popovic, B; Leatherbarrow, R J; Gould, I R

    2016-07-01

    To aid in the discovery and development of peptides and proteins as therapeutic agents, a virtual screen can be used to predict trends and direct workflow. We have developed the Parasol Protocol, a dynamic method implemented using the AMBER MD package, for computational site-directed mutagenesis. This tool can mutate between any pair of amino acids in a computationally expedient, automated manner. To demonstrate the potential of this methodology, we have employed the protocol to investigate a test case involving stapled peptides, and have demonstrated good agreement with experiment. PMID:27255759

  18. Fluorometric method of quantitative cell mutagenesis

    DOEpatents

    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.

  19. Fluorometric method of quantitative cell mutagenesis

    DOEpatents

    Dolbeare, Frank A.

    1982-01-01

    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.

  20. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

    PubMed Central

    Schmeisser, Falko; Weir, Jerry P

    2007-01-01

    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. PMID:17501993

  1. Identification of three core regions essential for protein splicing of the yeast Vma1 protozyme. A random mutagenesis study of the entire Vma1-derived endonuclease sequence.

    PubMed

    Kawasaki, M; Nogami, S; Satow, Y; Ohya, Y; Anraku, Y

    1997-06-20

    The translation product of the VMA1 gene of Saccharomyces cerevisiae undergoes protein splicing, in which the intervening region is autocatalytically excised and the franking regions are ligated. The splicing reaction is catalyzed essentially by the in-frame insert, VMA1-derived endonuclease (VDE), which is a site-specific endonuclease to mediate gene homing. Previous mutational analysis of the splicing reaction has been concentrated extensively upon the splice junctions. However, it still remains unknown which amino acid residues are crucial for the splicing reaction within the entire region of VDE and its neighboring elements. In this work, a polymerase chain reaction-based random mutagenesis strategy was used to identify such residues throughout the overall intervening sequence of the VMA1 gene. Splicing-defective mutant proteins were initially screened using a bacterial expression system and then analyzed further in yeast cells. Mutations were mapped at the N- and C-terminal splice junctions and around the N-terminal one-third of VDE. We identified four potent mutants that yielded aberrant products with molecular masses of 200, 90, and 80 kDa. We suggest that the conserved His362, newly identified as the essential residue for the splicing reaction, contributes to the first cleavage at the N-terminal junction, whereas His736 assists the second cleavage by Asn cyclization at the C-terminal junction. Mutations in these regions did not appear to destroy the endonuclease activity of VDE. PMID:9188457

  2. Facility target insert shielding assessment

    SciTech Connect

    Mocko, Michal

    2015-10-06

    Main objective of this report is to assess the basic shielding requirements for the vertical target insert and retrieval port. We used the baseline design for the vertical target insert in our calculations. The insert sits in the 12”-diameter cylindrical shaft extending from the service alley in the top floor of the facility all the way down to the target location. The target retrieval mechanism is a long rod with the target assembly attached and running the entire length of the vertical shaft. The insert also houses the helium cooling supply and return lines each with 2” diameter. In the present study we focused on calculating the neutron and photon dose rate fields on top of the target insert/retrieval mechanism in the service alley. Additionally, we studied a few prototypical configurations of the shielding layers in the vertical insert as well as on the top.

  3. High Throughput Mutagenesis for Identification of Residues Regulating Human Prostacyclin (hIP) Receptor Expression and Function

    PubMed Central

    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. PMID:24886841

  4. Impedance calculation for ferrite inserts

    SciTech Connect

    Breitzmann, S.C.; Lee, S.Y.; Ng, K.Y.; /Fermilab

    2005-01-01

    Passive ferrite inserts were used to compensate the space charge impedance in high intensity space charge dominated accelerators. They study the narrowband longitudinal impedance of these ferrite inserts. they find that the shunt impedance and the quality factor for ferrite inserts are inversely proportional to the imaginary part of the permeability of ferrite materials. They also provide a recipe for attaining a truly passive space charge impedance compensation and avoiding narrowband microwave instabilities.

  5. Nucleotide sequence of an insertion sequence (IS) element identified in the T-DNA region of a spontaneous variant of the Ti-plasmid pTiT37.

    PubMed Central

    Vanderleyden, J; Desair, J; De Meirsman, C; Michiels, K; Van Gool, A P; Chilton, M D; Jen, G C

    1986-01-01

    We have identified and determined the nucleotide sequence of an IS element (IS136) of Agrobacterium tumefaciens. This is the first IS element isolated and sequenced from a nopaline type Ti-plasmid. Our IS element has 32/30 bp inverted repeats with 6 mismatches, is 1,313 bp long and generates 9 bp direct repeats upon integration. IS136 has 3 main open reading frames (ORF's). Only ORF1 (159 codons) is preceded by sequences that are proposed to serve functional roles in transcriptional and translational initiation. No DNA sequence homology was found between IS136 and IS66, an IS element isolated from an octopine type Ti-plasmid. PMID:3018677

  6. A modifier screen identifies DNAJB6 as a cardiomyopathy susceptibility gene

    PubMed Central

    Ding, Yonghe; Long, Pamela A.; Bos, J. Martijn; Shih, Yu-Huan; Ma, Xiao; Sundsbak, Rhianna S.; Chen, Jianhua; Jiang, Yiwen; Zhao, Liqun; Hu, Xinyang; Wang, Jianan; Shi, Yongyong; Ackerman, Michael J.; Lin, Xueying; Ekker, Stephen C.; Redfield, Margaret M.; Olson, Timothy M.; Xu, Xiaolei

    2016-01-01

    Mutagenesis screening is a powerful forward genetic approach that has been successfully applied in lower-model organisms to discover genetic factors for biological processes. This phenotype-based approach has yet to be established in vertebrates for probing major human diseases, largely because of the complexity of colony management. Herein, we report a rapid strategy for identifying genetic modifiers of cardiomyopathy (CM). Based on the application of doxorubicin stress to zebrafish insertional cardiac (ZIC) mutants, we identified 4 candidate CM-modifying genes, of which 3 have been linked previously to CM. The long isoform of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b(L)) was identified as a CM susceptibility gene, supported by identification of rare variants in its human ortholog DNAJB6 from CM patients. Mechanistic studies indicated that the deleterious, loss-of-function modifying effects of dnajb6b(L) can be ameliorated by inhibition of ER stress. In contrast, overexpression of dnajb6(L) exerts cardioprotective effects on both fish and mouse CM models. Together, our findings establish a mutagenesis screening strategy that is scalable for systematic identification of genetic modifiers of CM, feasible to suggest therapeutic targets, and expandable to other major human diseases.

  7. The ClosTron: Mutagenesis in Clostridium refined and streamlined.

    PubMed

    Heap, John T; Kuehne, Sarah A; Ehsaan, Muhammad; Cartman, Stephen T; Cooksley, Clare M; Scott, Jamie C; Minton, Nigel P

    2010-01-01

    The recent development of the ClosTron Group II intron directed mutagenesis tool for Clostridium has advanced genetics in this genus, and here we present several significant improvements. We have shown how marker re-cycling can be used to construct strains with multiple mutations, demonstrated using FLP/FRT in Clostridium acetobutylicum; tested the capacity of the system for the delivery of transgenes to the chromosome of Clostridium sporogenes, which proved feasible for 1.0kbp transgenes in addition to a marker; and extended the host range of the system, constructing mutants in Clostridium beijerinckii and, for the first time, in a B1/NAP1/027 'epidemic' strain of Clostridium difficile. Automated intron design bioinformatics are now available free-of-charge at our website http://clostron.com; the out-sourced construction of re-targeted intron plasmids has become cost-effective as well as rapid; and the combination of constitutive intron expression with direct selection for intron insertions has made mutant isolation trivial. These developments mean mutants can now be constructed with very little time and effort for the researcher. Those who prefer to construct plasmids in-house are no longer reliant on a commercial kit, as a mixture of two new plasmids provides unlimited template for intron re-targeting by Splicing by Overlap Extension (SOE) PCR. The new ClosTron plasmids also offer blue-white screening and other options for identification of recombinant plasmids. The improved ClosTron system supersedes the prototype plasmid pMTL007 and the original method, and exploits the potential of Group II introns more fully. PMID:19891996

  8. Synthetic approach to stop-codon scanning mutagenesis.

    PubMed

    Nie, Lihua; Lavinder, Jason J; Sarkar, Mohosin; Stephany, Kimberly; Magliery, Thomas J

    2011-04-27

    A general combinatorial mutagenesis strategy using common dimethoxytrityl-protected mononucleotide phosphoramidites and a single orthogonally protected trinucleotide phosphoramidite (Fmoc-TAG; Fmoc = 9-fluorenylmethoxycarbonyl) was developed to scan a gene with the TAG amber stop codon with complete synthetic control. In combination with stop-codon suppressors that insert natural (e.g., alanine) or unnatural (e.g., p-benzoylphenylalanine, Bpa) amino acids, a single DNA library can be used to incorporate different amino acids for diverse purposes. Here, we scanned TAG codons through part of the gene for a model four-helix bundle protein, Rop, which regulates the copy number of ColE1 plasmids. Alanine was incorporated into Rop for mapping its binding site using an in vivo activity screen, and subtle but important differences from in vitro gel-shift studies of Rop function are evident. As a test, Bpa was incorporated using a Phe14 amber mutant isolated from the scanning library. Surprisingly, Phe14Bpa-Rop is weakly active, despite the critical role of Phe14 in Rop activity. Bpa is a photoaffinity label unnatural amino acid that can form covalent bonds with adjacent molecules upon UV irradiation. Irradiation of Phe14Bpa-Rop, which is a dimer in solution like wild-type Rop, results in covalent dimers, trimers, and tetramers. This suggests that Phe14Bpa-Rop weakly associates as a tetramer in solution and highlights the use of Bpa cross-linking as a means of trapping weak and transient interactions. PMID:21452871

  9. Insertion device vacuum system designs

    SciTech Connect

    Hoyer, E.

    1988-05-01

    Synchrotron light source insertion device vacuum systems now in operation and systems proposed for the future are reviewed. An overview of insertion devices is given and four generic vacuum chamber designs, transition section design and pumping considerations are discussed. Examples of vacuum chamber systems are presented.

  10. Mutagenesis as a Genetic Research Strategy

    PubMed Central

    Falk, Raphael

    2010-01-01

    Morgan's three students (Muller, Sturtevant, and Bridges) introduced reductionist empirical methods to the study of the chromosomal theory of heredity. Herman J. Muller concentrated on mutations, namely changes in the heterocatalytic properties of genes, without losing their autocatalytic (self-replication) properties. Experimental induction of mutations allowed quantitative analyses of genes' parameters, but hopes to deduce their chemicophysical character were never fulfilled. Once the model for DNA structure was proposed, the reductionist notions of mutation analysis were successfully applied to the molecular genes. However, it was soon realized that the concept of the particulate gene was inadequate. The more the molecular analysis of the genome advanced, the clearer it became that the entities of heredity must be conceived within systems' perspectives, for which special tools for handling large number of variables were developed. Analytic mutagenesis, however, continues to be a major strategy for the study of the cellular and chromosomal mechanisms that control mutation inductions. PMID:20713742

  11. Mutagenesis assays of human amniotic fluid

    SciTech Connect

    Everson, R.B.; Milne, K.L.; Warbuton, D.; McClamrock, H.D.; Buchanan, P.D.

    1985-01-01

    Extracts of amniocentesis samples from 144 women were tested for the presence of mutagenic substances using tester strain TA1538 in the Ames Salmonella/mammalian-microsome mutagenicity test. Because the volume of amniotic fluid in these samples was limited (generally less than 10 ml), the authors investigated modifications of this mutagenesis assay that could increase its ability to detect effects from small quantities of test material. Using mutagenicity in samples of urine from smokers as a model, it appeared that improved ability to detect small amounts of mutagen could be obtained by reducing volumes of media and reagents while keeping the amount of test sample constant. Tests of amniotic fluid extracts by this modified procedure showed small increases in revertants, about 50% above dimethylsulfoxide solvent control values. The increases suggest the presence of small amounts of mutagenic material in many of the amniotic fluid samples. At the doses employed, mutagenic activity in these samples was not associated with maternal smoking.

  12. PiggyBac transposon-based polyadenylation-signal trap for genome-wide mutagenesis in mice

    PubMed Central

    Li, Limei; Liu, Peng; Sun, Liangliang; Bin Zhou; Fei, Jian

    2016-01-01

    We designed a new type of polyadenylation-signal (PAS) trap vector system in living mice, the piggyBac (PB) (PAS-trapping (EGFP)) gene trapping vector, which takes advantage of the efficient transposition ability of PB and efficient gene trap and insertional mutagenesis of PAS-trapping. The reporter gene of PB(PAS-trapping (EGFP)) is an EGFP gene with its own promoter, but lacking a poly(A) signal. Transgenic mouse lines carrying PB(PAS-trapping (EGFP)) and protamine 1 (Prm1) promoter-driven PB transposase transgenes (Prm1-PBase) were generated by microinjection. Male mice doubly positive for PB(PAS-trapping (EGFP)) and Prm1-PBase were crossed with WT females, generating offspring with various insertion mutations. We found that 44.8% (26/58) of pups were transposon-positive progenies. New transposon integrations comprised 26.9% (7/26) of the transposon-positive progenies. We found that 100% (5/5) of the EGFP fluorescence-positive mice had new trap insertions mediated by a PB transposon in transcriptional units. The direction of the EGFP gene in the vector was consistent with the direction of the endogenous gene reading frame. Furthermore, mice that were EGFP-PCR positive, but EGFP fluorescent negative, did not show successful gene trapping. Thus, the novel PB(PAS-trapping (EGFP)) system is an efficient genome-wide gene-trap mutagenesis in mice. PMID:27292714

  13. A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice.

    PubMed

    Nishizawa-Yokoi, Ayako; Cermak, Tomas; Hoshino, Tomoki; Sugimoto, Kazuhiko; Saika, Hiroaki; Mori, Akiko; Osakabe, Keishi; Hamada, Masao; Katayose, Yuichi; Starker, Colby; Voytas, Daniel F; Toki, Seiichi

    2016-02-01

    We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing. PMID:26668331

  14. A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice1[OPEN

    PubMed Central

    Cermak, Tomas; Sugimoto, Kazuhiko; Saika, Hiroaki; Mori, Akiko; Osakabe, Keishi; Hamada, Masao; Katayose, Yuichi; Voytas, Daniel F.

    2016-01-01

    We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing. PMID:26668331

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

    PubMed

    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. PMID:27130213

  16. Construction of small-insert and large-insert metagenomic libraries.

    PubMed

    Simon, Carola; Daniel, Rolf

    2010-01-01

    The vast majority of the Earth's biological diversity is hidden in uncultured and yet uncharacterized microbial genomes. The construction of metagenomic libraries is a cultivation-independent molecular approach to assess this unexplored genetic reservoir. In the last few years, a high number of novel biocatalysts have been identified by function-based or sequence-based screening of metagenomic libraries. Here, we describe detailed protocols for the construction of metagenomic small-insert and large-insert libraries in plasmids and fosmids, respectively, from environmental DNA. PMID:20830554

  17. Bidirectional-genetics platform, a dual-purpose mutagenesis strategy for filamentous fungi.

    PubMed

    Park, Jaejin; Lee, Yong-Hwan

    2013-11-01

    Rapidly increasing fungal genome sequences call for efficient ways of generating mutants to translate quickly gene sequences into their functions. A reverse genetic strategy via targeted gene replacement (TGR) has been inefficient for many filamentous fungi due to dominant production of undesirable ectopic transformants. Although large-scale random insertional mutagenesis via transformation (i.e., forward genetics) facilitates high-throughput uncovering of novel genes of interest, generating a huge number of transformants, which is necessary to ensure the likelihood of mutagenizing most genes, is time-consuming. We propose a new strategy, entitled the Bidirectional-Genetics (BiG) platform, which combines both forward and reverse genetic strategies by recycling ectopic transformants derived from TGR as a source for random insertional mutants. The BiG platform was evaluated using the rice blast fungus Magnaporthe oryzae as a model. Over 10% of >1,000 M. oryzae ectopic transformants, generated during disruption of specific genes, displayed abnormality in vegetative growth, pigmentation, and/or asexual reproduction. In this pool of putative mutants, we isolated insertional mutants with mutations in three genes involved in histidine biosynthesis (MoHIS5), vegetative growth (MoVPS74), or conidiophore formation (MoFRQ) (where "Mo" indicates "M. oryzae"), supporting the utility of this platform for systematic gene function studies. PMID:24058171

  18. Molecular Characterization of a Foot-and-Mouth Disease Virus (FMDV) Containing a 57-Nucleotide Insertion in the 3' Untranslated Region (3'UTR)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A foot-and-mouth disease virus (FMDV) virus containing a 57 nt insertion in the 3’ untranslated region (3’UTR) was generated by a transposon (tn) mediated mutagenesis. Characterization of the mutant virus (A24-3’UTR8110) revealed no significant differences in virus growth, translation efficiency and...

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

    SciTech Connect

    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.

  20. Percutaneously inserted central catheter - infants

    MedlinePlus

    PICC - infants; PQC - infants; Pic line - infants; Per-Q cath - infants ... A percutaneously inserted central catheter (PICC) is a long, very thin, soft plastic tube that is put into a small blood vessel. This article addresses PICCs in ...

  1. Immediate postabortal insertion of intrauterine devices.

    PubMed

    Okusanya, Babasola O; Oduwole, Olabisi; Effa, Emmanuel E

    2014-01-01

    Background The use of an effective contraceptive may be necessary after an abortion. Insertion of an intrauterine device (IUD) may be done the same day or later. Immediate IUD insertion is an option since the woman is not pregnant, pain of insertion is less because the cervical os is open, and her motivation to use contraception may be high. However, insertion of an IUD immediately after a pregnancy ends carries risks, such as spontaneous expulsion.Objectives To assess the safety and efficacy of IUD insertion immediately after spontaneous or induced abortion.Search methods We searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, POPLINE, ClinicalTrials.gov,and ICTRP in January 27, 2014. We also contacted investigators to identify other trials.Selection criteria We sought all randomised controlled trials (RCTs) with at least one treatment arm that involved IUD insertion immediately after an induced abortion or after curettage for spontaneous abortion.Data collection and analysis We evaluated the methodological quality of each report and abstracted the data. We focused on discontinuation rates for accidental pregnancy, perforation, expulsion, and pelvic inflammatory disease.We computed the weighted average of the rate ratios.We compute drisk ratios (RRs) with 95% Confidence Intervals (CIs).We performed an intention-to-treat (ITT) analysis by including all randomised participants in the analysis according to the Cochrane Handbook for Systematic Reviews of Interventions.Main results We identified 12 trials most of which are of moderate risk of bias involving 7,119 participants which described random assignment.Five trials randomised to either immediate or delayed insertion of IUD. One of them randomised to immediate versus delayed insertion of Copper 7 showed immediate insertion of the Copper 7 was associated with a higher risk of expulsion than was delayed insertion(RR 11.98, 95% CI 1.61 to 89.35,1 study, 259 participants); the

  2. Tool Removes Coil-Spring Thread Inserts

    NASA Technical Reports Server (NTRS)

    Collins, Gerald J., Jr.; Swenson, Gary J.; Mcclellan, J. Scott

    1991-01-01

    Tool removes coil-spring thread inserts from threaded holes. Threads into hole, pries insert loose, grips insert, then pulls insert to thread it out of hole. Effects essentially reverse of insertion process to ease removal and avoid further damage to threaded inner surface of hole.

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

    PubMed Central

    Beacham, T.A.; Macia, V. Mora; Rooks, P.; White, D.A.; Ali, S.T.

    2015-01-01

    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. PMID:26753128

  4. A mouse forward genetics screen identifies LISTERIN as an E3 ubiquitin ligase involved in neurodegeneration

    PubMed Central

    Chu, Jessie; Hong, Nancy A.; Masuda, Claudio A.; Jenkins, Brian V.; Nelms, Keats A.; Goodnow, Christopher C.; Glynne, Richard J.; Wu, Hua; Masliah, Eliezer; Joazeiro, Claudio A. P.; Kay, Steve A.

    2009-01-01

    A mouse neurological mutant, lister, was identified through a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Homozygous lister mice exhibit profound early-onset and progressive neurological and motor dysfunction. lister encodes a RING finger protein, LISTERIN, which functions as an E3 ubiquitin ligase in vitro. Although lister is widely expressed in all tissues, motor and sensory neurons and neuronal processes in the brainstem and spinal cord are primarily affected in the mutant. Pathological signs include gliosis, dystrophic neurites, vacuolated mitochondria, and accumulation of soluble hyperphosphorylated tau. Analysis with a different lister allele generated through targeted gene trap insertion reveals LISTERIN is required for embryonic development and confirms that direct perturbation of a LISTERIN-regulated process causes neurodegeneration. The lister mouse uncovers a pathway involved in neurodegeneration and may serves as a model for understanding the molecular mechanisms underlying human neurodegenerative disorders. PMID:19196968

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

    PubMed Central

    Caignard, Grégory; Eva, Megan M.; van Bruggen, Rebekah; Eveleigh, Robert; Bourque, Guillaume; Malo, Danielle; Gros, Philippe; Vidal, Silvia M.

    2014-01-01

    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. PMID:25268389

  6. Herpesvirus mutagenesis facilitated by infectious bacterial artificial chromosomes (iBACs).

    PubMed

    Robinson, Karl E; Mahony, Timothy J

    2015-01-01

    A critical factor in the study of herpesviruses, their genes and gene functions is the capacity to derive mutants that harbor deletions, truncations, or insertions within the genetic elements of interest. Once constructed the impact of the introduced mutation on the phenotypic properties of the rescued virus can be determined in either in vitro or in vivo systems. However, the construction of such mutants by traditional virological mutagenesis techniques can be a difficult and laborious undertaking. The maintenance of a viral genome as an infectious bacterial artificial chromosome (iBAC), however, endows the capacity to manipulate the viral genome for mutagenesis studies with relative ease. Here, the construction and characterization of two gene deletion mutants of an alphaherpesvirus maintained as iBAC in combination with an inducible homologous recombination system in Escherichia coli is detailed. The methodology is generally applicable to any iBAC and is demonstrated to be a highly efficient and informative approach for mutant virus construction. PMID:25239746

  7. Development of an inducible transposon system for efficient random mutagenesis in Clostridium acetobutylicum

    PubMed Central

    Zhang, Ying; Xu, Shu; Chai, Changsheng; Yang, Sheng; Jiang, Weihong; Minton, Nigel P.; Gu, Yang

    2016-01-01

    Clostridium acetobutylicum is an industrially important Gram-positive organism, which is capable of producing economically important chemicals in the ABE (Acetone, Butanol and Ethanol) fermentation process. Renewed interests in the ABE process necessitate the availability of additional genetics tools to facilitate the derivation of a greater understanding of the underlying metabolic and regulatory control processes in operation through forward genetic strategies. In this study, a xylose inducible, mariner-based, transposon system was developed and shown to allow high-efficient random mutagenesis in the model strain ATCC 824. Of the thiamphenicol resistant colonies obtained, 91.9% were shown to be due to successful transposition of the catP-based mini-transposon element. Phenotypic screening of 200 transposon clones revealed a sporulation-defective clone with an insertion in spo0A, thereby demonstrating that this inducible transposon system can be used for forward genetic studies in C. acetobutylicum. PMID:27001972

  8. Molecular basis of transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis

    PubMed Central

    Xu, Liang; Da, Lintai; Plouffe, Steven W.; Chong, Jenny; Kool, Eric; Wang, Dong

    2014-01-01

    Maintaining high transcriptional fidelity is essential for life. Some DNA lesions lead to significant changes in transcriptional fidelity. In this review, we will summarize recent progress towards understanding the molecular basis of RNA polymerase II (Pol II) transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis. In particular, we will focus on the three key checkpoint steps of controlling Pol II transcriptional fidelity: insertion (specific nucleotide selection and incorporation), extension (differentiation of RNA transcript extension of a matched over mismatched 3'-RNA terminus), and proofreading (preferential removal of misincorporated nucleotides from the 3'-RNA end). We will also discuss some novel insights into the molecular basis and chemical perspectives of controlling Pol II transcriptional fidelity through structural, computational, and chemical biology approaches. PMID:24767259

  9. Development of an inducible transposon system for efficient random mutagenesis in Clostridium acetobutylicum.

    PubMed

    Zhang, Ying; Xu, Shu; Chai, Changsheng; Yang, Sheng; Jiang, Weihong; Minton, Nigel P; Gu, Yang

    2016-04-01

    Clostridium acetobutylicum is an industrially important Gram-positive organism, which is capable of producing economically important chemicals in the ABE (Acetone, Butanol and Ethanol) fermentation process. Renewed interests in the ABE process necessitate the availability of additional genetics tools to facilitate the derivation of a greater understanding of the underlying metabolic and regulatory control processes in operation through forward genetic strategies. In this study, a xylose inducible, mariner-based, transposon system was developed and shown to allow high-efficient random mutagenesis in the model strain ATCC 824. Of the thiamphenicol resistant colonies obtained, 91.9% were shown to be due to successful transposition of the catP-based mini-transposon element. Phenotypic screening of 200 transposon clones revealed a sporulation-defective clone with an insertion in spo0A, thereby demonstrating that this inducible transposon system can be used for forward genetic studies in C. acetobutylicum. PMID:27001972

  10. Identification and Characterization of Non-Cellulose-Producing Mutants of Gluconacetobacter hansenii Generated by Tn5 Transposon Mutagenesis

    PubMed Central

    Deng, Ying; Nagachar, Nivedita; Xiao, Chaowen; Tien, Ming

    2013-01-01

    The acs operon of Gluconacetobacter is thought to encode AcsA, AcsB, AcsC, and AcsD proteins that constitute the cellulose synthase complex, required for the synthesis and secretion of crystalline cellulose microfibrils. A few other genes have been shown to be involved in this process, but their precise role is unclear. We report here the use of Tn5 transposon insertion mutagenesis to identify and characterize six non-cellulose-producing (Cel−) mutants of Gluconacetobacter hansenii ATCC 23769. The genes disrupted were acsA, acsC, ccpAx (encoding cellulose-complementing protein [the subscript “Ax” indicates genes from organisms formerly classified as Acetobacter xylinum]), dgc1 (encoding guanylate dicyclase), and crp-fnr (encoding a cyclic AMP receptor protein/fumarate nitrate reductase transcriptional regulator). Protein blot analysis revealed that (i) AcsB and AcsC were absent in the acsA mutant, (ii) the levels of AcsB and AcsC were significantly reduced in the ccpAx mutant, and (iii) the level of AcsD was not affected in any of the Cel− mutants. Promoter analysis showed that the acs operon does not include acsD, unlike the organization of the acs operon of several strains of closely related Gluconacetobacter xylinus. Complementation experiments confirmed that the gene disrupted in each Cel− mutant was responsible for the phenotype. Quantitative real-time PCR and protein blotting results suggest that the transcription of bglAx (encoding β-glucosidase and located immediately downstream from acsD) was strongly dependent on Crp/Fnr. A bglAx knockout mutant, generated via homologous recombination, produced only ∼16% of the wild-type cellulose level. Since the crp-fnr mutant did not produce any cellulose, Crp/Fnr may regulate the expression of other gene(s) involved in cellulose biosynthesis. PMID:24013627

  11. Human Diallelic Insertion/Deletion Polymorphisms

    PubMed Central

    Weber, James L.; David, Donna; Heil, Jeremy; Fan, Ying; Zhao, Chengfeng; Marth, Gabor

    2002-01-01

    We report the identification and characterization of 2,000 human diallelic insertion/deletion polymorphisms (indels) distributed throughout the human genome. Candidate indels were identified by comparison of overlapping genomic or cDNA sequences. Average confirmation rate for indels with a ⩾2-nt allele-length difference was 58%, but the confirmation rate for indels with a 1-nt length difference was only 14%. The vast majority of the human diallelic indels were monomorphic in chimpanzees and gorillas. The ratio of deletion:insertion mutations was 4.1. Allele frequencies for the indels were measured in Europeans, Africans, Japanese, and Native Americans. New alleles were generally lower in frequency than old alleles. This tendency was most pronounced for the Africans, who are likely to be closest among the four groups to the original modern human population. Diallelic indels comprise ∼8% of all human polymorphisms. Their abundance and ease of analysis make them useful for many applications. PMID:12205564

  12. Symposium on molecular and cellular mechanisms of mutagenesis

    SciTech Connect

    Not Available

    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)

  13. Mutagenesis during plant responses to UVB radiation.

    PubMed

    Holá, M; Vágnerová, R; Angelis, K J

    2015-08-01

    We tested an idea that induced mutagenesis due to unrepaired DNA lesions, here the UV photoproducts, underlies the impact of UVB irradiation on plant phenotype. For this purpose we used protonemal culture of the moss Physcomitrella patens with 50% of apical cells, which mimics actively growing tissue, the most vulnerable stage for the induction of mutations. We measured the UVB mutation rate of various moss lines with defects in DNA repair (pplig4, ppku70, pprad50, ppmre11), and in selected clones resistant to 2-Fluoroadenine, which were mutated in the adenosine phosphotrasferase gene (APT), we analysed induced mutations by sequencing. In parallel we followed DNA break repair and removal of cyclobutane pyrimidine dimers with a half-life τ = 4 h 14 min determined by comet assay combined with UV dimer specific T4 endonuclease V. We show that UVB induces massive, sequence specific, error-prone bypass repair that is responsible for a high mutation rate owing to relatively slow, though error-free, removal of photoproducts by nucleotide excision repair (NER). PMID:25542779

  14. Codon compression algorithms for saturation mutagenesis.

    PubMed

    Pines, Gur; Pines, Assaf; Garst, Andrew D; Zeitoun, Ramsey I; Lynch, Sean A; Gill, Ryan T

    2015-05-15

    Saturation mutagenesis is employed in protein engineering and genome-editing efforts to generate libraries that span amino acid design space. Traditionally, this is accomplished by using degenerate/compressed codons such as NNK (N = A/C/G/T, K = G/T), which covers all amino acids and one stop codon. These solutions suffer from two types of redundancy: (a) different codons for the same amino acid lead to bias, and (b) wild type amino acid is included within the library. These redundancies increase library size and downstream screening efforts. Here, we present a dynamic approach to compress codons for any desired list of amino acids, taking into account codon usage. This results in a unique codon collection for every amino acid to be mutated, with the desired redundancy level. Finally, we demonstrate that this approach can be used to design precise oligo libraries amendable to recombineering and CRISPR-based genome editing to obtain a diverse population with high efficiency. PMID:25303315

  15. In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis.

    PubMed Central

    McAdam, R A; Weisbrod, T R; Martin, J; Scuderi, J D; Brown, A M; Cirillo, J D; Bloom, B R; Jacobs, W R

    1995-01-01

    Insertional mutagenesis in Mycobacterium bovis BCG, a member of the slow-growing M. tuberculosis complex, was accomplished with transposons engineered from the Mycobacterium smegmatis insertion element IS1096. Transposons were created by placing a kanamycin resistance gene in several different positions in IS1096, and the resulting transposons were electroporated into BCG on nonreplicating plasmids. These analyses demonstrated that only one of the two open reading frames was necessary for transposition. A library of insertions was generated. Southern analysis of 23 kanamycin-resistant clones revealed that the transposons had inserted directly, with no evidence of cointegrate formation, into different restriction fragments in each clone. Sequence analysis of nine of the clones revealed junctional direct 8-bp repeats with only a slight similarity in target sites. These results suggest that IS1096-derived transposons transposed into the BCG genome in a relatively random fashion. Three auxotrophs, two for leucine and one for methionine, were isolated from the library of transposon insertions in BCG. They were characterized by sequencing and found to be homologous to the leuD gene of Escherichia coli and a sulfate-binding protein of cyanobacteria, respectively. When inoculated intravenously into C57BL/6 mice, the leucine auxotrophs, in contrast to the parent BCG strain or the methionine auxotroph, showed an inability to grow in vivo and were cleared within 7 weeks from the lungs and spleen. PMID:7868221

  16. Distal Insertions of the Biceps Femoris

    PubMed Central

    Branch, Eric A.; Anz, Adam W.

    2015-01-01

    Background: Avulsion of the biceps femoris from the fibula and proximal tibia is encountered in clinical practice. While the anatomy of the primary posterolateral corner structures has been qualitatively and quantitatively described, a quantitative analysis regarding the insertions of the biceps femoris on the fibula and proximal tibia is lacking. Purpose: To quantitatively assess the insertions of the biceps femoris, fibular collateral ligament (FCL), and anterolateral ligament (ALL) on the fibula and proximal tibia as well as establish relationships among these structures and to pertinent surgical anatomy. Study Design: Descriptive laboratory study. Methods: Dissections were performed on 12 nonpaired, fresh-frozen cadaveric specimens identifying the biceps femoris, FCL, and ALL, and their insertions on the proximal tibia and fibula. The footprint areas, orientations, and distances from relevant osseous landmarks were measured using a 3-dimensional coordinate measurement device. Results: Dissection produced 6 easily identifiable and reproducible anatomic footprints. Tibial footprints included the insertion of the ALL and an insertion of the biceps femoris (TBF). Fibular footprints included the insertion of the FCL, a distal insertion of the biceps femoris (DBF), a medial footprint of the biceps femoris (MBF), and a proximal footprint of the biceps femoris (PBF). The mean area of these footprints (95% CI) was as follows: ALL, 53.0 mm2 (38.4-67.6); TBF, 93.9 mm2 (72.0-115.8); FCL, 86.8 mm2 (72.3-101.2); DBF, 119 mm2 (91.1-146.9); MBF, 46.8 mm2 (29.0-64.5); and PBF, 215 mm2 (192.4-237.5). The mean distance (95% CI) from the Gerdy tubercle to the center of the ALL footprint was 24.3 mm (21.6-27.0) and to the center of the TBF was 22.5 mm (21.0-24.0). The center of the DBF was 8.68 mm (7.0-10.3) from the anterior border of the fibula, the center of the FCL was 14.6 mm (12.5-16.7) from the anterior border of the fibula and 20.7 mm (19.0-22.4) from the tip of the fibular

  17. Insertion Sequence Diversity in Archaea

    PubMed Central

    Filée, J.; Siguier, P.; Chandler, M.

    2007-01-01

    Insertion sequences (ISs) can constitute an important component of prokaryotic (bacterial and archaeal) genomes. Over 1,500 individual ISs are included at present in the ISfinder database (www-is.biotoul.fr), and these represent only a small portion of those in the available prokaryotic genome sequences and those that are being discovered in ongoing sequencing projects. In spite of this diversity, the transposition mechanisms of only a few of these ubiquitous mobile genetic elements are known, and these are all restricted to those present in bacteria. This review presents an overview of ISs within the archaeal kingdom. We first provide a general historical summary of the known properties and behaviors of archaeal ISs. We then consider how transposition might be regulated in some cases by small antisense RNAs and by termination codon readthrough. This is followed by an extensive analysis of the IS content in the sequenced archaeal genomes present in the public databases as of June 2006, which provides an overview of their distribution among the major archaeal classes and species. We show that the diversity of archaeal ISs is very great and comparable to that of bacteria. We compare archaeal ISs to known bacterial ISs and find that most are clearly members of families first described for bacteria. Several cases of lateral gene transfer between bacteria and archaea are clearly documented, notably for methanogenic archaea. However, several archaeal ISs do not have bacterial equivalents but can be grouped into Archaea-specific groups or families. In addition to ISs, we identify and list nonautonomous IS-derived elements, such as miniature inverted-repeat transposable elements. Finally, we present a possible scenario for the evolutionary history of ISs in the Archaea. PMID:17347521

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

    PubMed

    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. PMID:25775519

  19. Environmental stress induces trinucleotide repeat mutagenesis in human cells

    PubMed Central

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

    2015-01-01

    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. PMID:25775519

  20. Genomic Loci of the Porphyromonas gingivalis Insertion Element IS1126

    PubMed Central

    Dong, Hong; Chen, Tsute; Dewhirst, Floyd E.; Fleischmann, Robert D.; Fraser, Claire M.; Duncan, Margaret J.

    1999-01-01

    The Porphyromonas gingivalis genome contains multiple copies of insertion element IS1126. When chromosomal DNA digests of different strains were probed with IS1126, between 25 and 35 hybridizing fragments per genome were detected, depending on the strain. Unrelated strains had very different restriction fragment length polymorphism (RFLP) patterns. When different laboratory copies of a specific strain were examined, the IS1126 RFLP patterns were very similar but small differences were observed, indicating that element-associated changes had occurred during laboratory passage. Within the next year, genome sequencing, assembly, and annotation for P. gingivalis W83 will be completed. Because repetitive elements complicate the assembly of randomly sequenced DNA fragments, we isolated and sequenced the flanking regions of IS1126 copies in strain W83. We also isolated and sequenced the flanking regions of IS1126 copies in strain ATCC 33277 in order to compare insertion sites in phylogenetically divergent strains. We identified 37 new sequences flanking IS1126 from strain ATCC 33277 and 30 from strain W83. The insertion element was found between genes except where it transposed into another insertion element. Examination of identifiable flanking genes or open reading frames indicated that the insertion sites were different in the two strains, except that both strains possess an insertion adjacent to the Lys-gingipain gene (J. P. Lewis and F. L. Macrina, Infect. Immun. 66:3035–3042, 1998). Most of the genes or sequences flanking IS1126 in ATCC 33277 were present in W83 but were contiguous and not insertion element associated. Thus, where genes were identified in both strains, their order was maintained, indicating that the two genomes are organized similarly, but the loci of IS1126 are different. In both strains, insertion element-associated duplicated target sites were lost from several copies of IS1126, providing evidence of homologous recombination between elements

  1. The genomic landscape of polymorphic human nuclear mitochondrial insertions

    PubMed Central

    Dayama, Gargi; Emery, Sarah B.; Kidd, Jeffrey M.; Mills, Ryan E.

    2014-01-01

    The transfer of mitochondrial genetic material into the nuclear genomes of eukaryotes is a well-established phenomenon that has been previously limited to the study of static reference genomes. The recent advancement of high throughput sequencing has enabled an expanded exploration into the diversity of polymorphic nuclear mitochondrial insertions (NumtS) within human populations. We have developed an approach to discover and genotype novel Numt insertions using whole genome, paired-end sequencing data. We have applied this method to a thousand individuals in 20 populations from the 1000 Genomes Project and other datasets and identified 141 new sites of Numt insertions, extending our current knowledge of existing NumtS by almost 20%. We find that recent Numt insertions are derived from throughout the mitochondrial genome, including the D-loop, and have integration biases that differ in some respects from previous studies on older, fixed NumtS in the reference genome. We determined the complete inserted sequence for a subset of these events and have identified a number of nearly full-length mitochondrial genome insertions into nuclear chromosomes. We further define their age and origin of insertion and present an analysis of their potential impact to ongoing studies of mitochondrial heteroplasmy and disease. PMID:25348406

  2. Efficient Gene Transfer and Targeted Mutagenesis in Fusobacterium nucleatum

    PubMed Central

    Haake, Susan Kinder; Yoder, Sean; Gerardo, Sharon Hunt

    2006-01-01

    Fusobacterium nucleatum is a Gram-negative anaerobe important in dental biofilm ecology and infectious diseases with significant societal impact. The lack of efficient genetic systems has hampered molecular analyses in this microorganism. We previously reported construction of a shuttle plasmid, pHS17, using the native fusobacterial plasmid pFN1 and an erythromycin resistance cassette. However, the host range of pHS17 was restricted to F. nucleatum, ATCC 10953 and the transformation efficiency was limited. This study was undertaken to improve genetic systems for molecular analysis in F. nucleatum. We identified a second F. nucleatum strain, ATCC 23726, which is transformed with improved efficiency compared to ATCC 10953. Two novel second generation pFN1-based shuttle plasmids, pHS23 and pHS30, were developed and enable transformation of ATCC 23726 at 6.2 x 104 and 1.5 x 106 transformants/microgram of plasmid DNA, respectively. The transformation efficiency of pHS30, which harbors a catP gene conferring resistance to chloramphenicol, was more than 1,000-fold greater than that of pHS17. The improved transformation efficiency facilitated disruption of the chromosomal rnr gene using a suicide plasmid pHS19, the first demonstration of targeted mutagenesis in F. nucleatum. These results provide significant advances in the development of systems for molecular analysis in F. nucleatum. PMID:16115683

  3. Site-directed mutagenesis and gene deletion using reverse genetics.

    PubMed

    Muhl, Daniela; Filloux, Alain

    2014-01-01

    Understanding gene function is far easier when tools are available to engineer a bacterial strain lacking a specific gene and phenotypically compare its behavior with the corresponding parental strain. Such mutants could be selected randomly, either by natural selection under particular stress conditions or by random mutagenesis using transposon delivery as described elsewhere in this book. However, with the advent of the genomic era there are now hundreds of bacterial genomes whose sequence is available, and thus, genes can be identified, chosen, and strategies designed to specifically inactivate them. This can be done by using suicide plasmids and is most convenient when the bacterium of interest is easily amenable to genetic manipulation. The method presented here will describe the use of a suicide vector, pKNG101, which allows the selection of a double-recombination event. The first event results in the integration of the pKNG101 derivative carrying the "mutator" fragment onto the chromosome, and could be selected on plates containing appropriate antibiotics. The pKNG101 carries the sacB gene, which induces death when cells are grown on sucrose. Growth on sucrose plates will thus select the second homologous recombination event, which results in removing the plasmid backbone and leaving behind the mutated target gene. This method has been widely used over the last 20 years to inactivate genes in a wide range of gram-negative bacteria and in particular in Pseudomonas aeruginosa. PMID:24818930

  4. Induction of Pectinase Hyper Production by Multistep Mutagenesis Using a Fungal Isolate--Aspergillus flavipes.

    PubMed

    Akbar, Sabika; Prasuna, R Gyana; Khanam, Rasheeda

    2014-04-01

    Aspergillus flavipes, a slow growing pectinase producing ascomycete, was isolated from soil identified and characterised in the previously done preliminary studies. Optimisation studies revealed that Citrus peel--groundnut oil cake [CG] production media is the best media for production of high levels of pectinase up to 39 U/ml using wild strain of A. flavipes. Strain improvement of this isolated strain for enhancement of pectinase production using multistep mutagenesis procedure is the endeavour of this project. For this, the wild strain of A. flavipes was treated with both physical (UV irradiation) and chemical [Colchicine, Ethidium bromide, H2O2] mutagens to obtain Ist generation mutants. The obtained mutants were assayed and differentiated basing on pectinase productivity. The better pectinase producing strains were further subjected to multistep mutagenesis to attain stability in mutants. The goal of this project was achieved by obtaining the best pectinase secreting mutant, UV80 of 45 U/ml compared to wild strain and sister mutants. This fact was confirmed by quantitatively analysing 3rd generation mutants obtained after multistep mutagenesis. PMID:26563068

  5. Genetic Regulation of Charged Particle Mutagenesis in Human Cells

    NASA Technical Reports Server (NTRS)

    Kronenberg, Amy; Gauny, S.; Cherbonnel-Lasserre, C.; Liu, W.; Wiese, C.

    1999-01-01

    Our studies use a series of syngeneic, and where possible, isogenic human B-lymphoblastoid cell lines to assess the genetic factors that modulate susceptibility apoptosis and their impact on the mutagenic risks of low fluence exposures to 1 GeV Fe ions and 55 MeV protons. These ions are representative of the types of charged particle radiation that are of particular significance for human health in the space radiation environment. The model system employs cell lines derived from the male donor WIL-2. These cells have a single X chromosome and they are hemizygous for one mutation marker, hypoxanthine phosphoribosyltransferase (HPRT). TK6 and WTK1 cells were each derived from descendants of WIL-2 and were each selected as heterozygotes for a second mutation marker, the thymidine kinase (TK) gene located on chromosome 17q. The HPRT and TK loci can detect many different types of mutations, from single basepair substitutions up to large scale loss of heterozygosity (LOH). The single expressing copy of TK in the TK6 and WTKI cell lines is found on the same copy of chromosome 17, and this allele can be identified by a restriction fragment length polymorphism (RFLP) identified when high molecular weight DNA is digested by the SacI restriction endonuclease and hybridized against the cDNA probe for TK. A large series of polymorphic linked markers has been identified that span more than 60 cM of DNA (approx. 60 megabasepairs) and distinguish the copy of chromosome 17 bearing the initially active TK allele from the copy of chromosome 17 bearing the silent TK allele in both TK6 and WTKI cells. TK6 cells express normal p53 protein while WTKI cells express homozygous mutant p53. Expression of mutant p53 can increase susceptibility to x-ray-induced mutations. It's been suggested that the increased mutagenesis in p53 mutant cells might be due to reduced apoptosis.

  6. [Rapid site-directed mutagenesis on full-length plasmid DNA by using designed restriction enzyme assisted mutagenesis].

    PubMed

    Zhang, Baozhong; Ran, Duoliang; Zhang, Xin; An, Xiaoping; Shan, Yunzhu; Zhou, Yusen; Tong, Yigang

    2009-02-01

    To use the designed restriction enzyme assisted mutagenesis technique to perform rapid site-directed mutagenesis on double-stranded plasmid DNA. The target amino acid sequence was reversely translated into DNA sequences with degenerate codons, resulting in large amount of silently mutated sequences containing various restriction endonucleases (REs). Certain mutated sequence with an appropriate RE was selected as the target DNA sequence for designing mutation primers. The full-length plasmid DNA was amplified with high-fidelity Phusion DNA polymerase and the amplified product was 5' phosphorylated by T4 polynucleotide kinase and then self-ligated. After transformation into an E. coli host the transformants were rapidly screened by cutting with the designed RE. With this strategy we successfully performed the site-directed mutagenesis on an 8 kb plasmid pcDNA3.1-pIgR and recovered the wild-type amino acid sequence of human polymeric immunoglobulin receptor (pIgR). A novel site-directed mutagenesis strategy based on DREAM was developed which exploited RE as a rapid screening measure. The highly efficient, high-fidelity Phusion DNA polymerase was applied to ensure the efficient and faithful amplification of the full-length sequence of a plasmid of up to 8 kb. This rapid mutagenesis strategy avoids using any commercial site-directed mutagenesis kits, special host strains or isotopes. PMID:19459340

  7. Mutational effects of retrovirus insertion on the genome of V79 cells by an attenuated retrovirus vector: implications for gene therapy.

    PubMed

    Themis, M; May, D; Coutelle, C; Newbold, R F

    2003-09-01

    Attenuated retroviruses are currently the most widely used vectors in clinical gene therapy because of their potential to effect stable and permanent gene transfer. Since gene delivery is accompanied by random insertion of foreign genetic material into the recipient chromosomal DNA, the potential for insertional mutagenesis exists. In this study, we used a defective retrovirus vector containing a selectable marker, the hygromycin phosphotransferase gene, to investigate the mutagenic effects of vector integration on the mammalian genome. V79 Chinese hamster cells were infected with virus supernatants or by coculture with virus producer cells, and provirus insertion events occurred at low and high frequencies, respectively. The frequency of hprt mutagenesis was increased by a factor of 2.3 over the spontaneous hprt mutation frequency only following multiple provirus insertions/cell genome. Multiple provirus insertions (>3/genome) resulted in instability at the hprt locus in 63% of the virally induced hprt mutants, as indicated by rearrangements at the molecular level, whereas no rearrangements were found when the provirus copy number was 1-2/genome. To demonstrate direct proviral involvement in mutagenesis, the defective MLV vector was retrieved along with flanking genomic hprt sequences from one mutant, and localized within intron 5 of the hprt gene. These data suggest that provirus copy number is a key factor when considering the potential hazards of using retrovirus vectors for gene therapy. PMID:12923569

  8. Gene Insertion Patterns and Sites

    NASA Astrophysics Data System (ADS)

    Vain, Philippe; Thole, Vera

    During the past 25 years, the molecular analysis of transgene insertion patterns and sites in plants has greatly contributed to our understanding of the mechanisms underlying transgene integration, expression, and stability in the nuclear genome. Molecular characterization is also an essential step in the safety assessment of genetically modified crops. This chapter describes the standard experimental procedures used to analyze transgene insertion patterns and loci in cereals and grasses transformed using Agrobacterium tumefaciens or direct transfer of DNA. Methods and protocols enabling the determination of the number and configuration of transgenic loci via a combination of inheritance studies, polymerase chain reaction, and Southern analyses are presented. The complete characterization of transgenic inserts in plants is, however, a holistic process relying on a wide variety of experimental approaches. In this chapter, these additional approaches are not detailed but references to relevant bibliographic records are provided.

  9. Applications of CRISPR/Cas9 technology for targeted mutagenesis, gene replacement and stacking of genes in higher plants.

    PubMed

    Luo, Ming; Gilbert, Brian; Ayliffe, Michael

    2016-07-01

    Mutagenesis continues to play an essential role for understanding plant gene function and, in some instances, provides an opportunity for plant improvement. The development of gene editing technologies such as TALENs and zinc fingers has revolutionised the targeted mutation specificity that can now be achieved. The CRISPR/Cas9 system is the most recent addition to gene editing technologies and arguably the simplest requiring only two components; a small guide RNA molecule (sgRNA) and Cas9 endonuclease protein which complex to recognise and cleave a specific 20 bp target site present in a genome. Target specificity is determined by complementary base pairing between the sgRNA and target site sequence enabling highly specific, targeted mutation to be readily engineered. Upon target site cleavage, error-prone endogenous repair mechanisms produce small insertion/deletions at the target site usually resulting in loss of gene function. CRISPR/Cas9 gene editing has been rapidly adopted in plants and successfully undertaken in numerous species including major crop species. Its applications are not restricted to mutagenesis and target site cleavage can be exploited to promote sequence insertion or replacement by recombination. The multiple applications of this technology in plants are described. PMID:27146973

  10. In Vivo Site-Specific Mutagenesis and Gene Collage Using the Delitto Perfetto System in Yeast Saccharomyces cerevisiae

    PubMed Central

    Stuckey, Samantha; Mukherjee, Kuntal; Storici, Francesca

    2016-01-01

    Delitto perfetto is a site-specific in vivo mutagenesis system that has been developed to generate changes at will in the genome of the yeast Saccharomyces cerevisiae. Using this technique, it is possible to rapidly and efficiently engineer yeast strains without requiring several intermediate steps as it functions in only two steps, both of which rely on homologous recombination to drive the changes to the target DNA region. The first step involves the insertion of a cassette containing two markers at or near the locus to be altered. The second step involves complete removal of this cassette with oligonucleotides and/or other genetic material and transfer of the expected genetic modification(s) to the chosen DNA locus. Here we provide a detailed protocol of the delitto perfetto approach and present examples of the most common and useful applications for in vivo mutagenesis to generate base substitutions, deletions, insertions, as well as for precise in vivo assembly and integration of multiple genetic elements, or gene collage. PMID:21660695

  11. Insertion device for pressure testing

    NASA Technical Reports Server (NTRS)

    Howland, B. T.; Maurin, A. L.

    1969-01-01

    Test device which introduces either pressure or vacuum into a test pipe or tube, is insertable into the tested item where it secures itself into position and requires no external support. The unit has an operating range from zero to 25,000 psig and to any vacuum level that available equipment can reach.

  12. Precise-Conductance Valve Insert

    NASA Technical Reports Server (NTRS)

    Outlaw, R. A.; Hoyt, R. F.

    1986-01-01

    Valve modification provides two operating modes fully open and small, precise leak. Copper insert with radially oriented holes allows small, controllable, precise effusion rate when valve closed or nearly unobstructed flow when valve open. Numerous applications in surface physics, vacuum physics, materials science, gas kinetics, thin films, and other areas of research requiring measured flows of gas into or out of system.

  13. Targeted Mutagenesis in Bombyx mori Using TALENs.

    PubMed

    Takasu, Yoko; Tamura, Toshiki; Goldsmith, Marian; Zurovec, Michal

    2016-01-01

    Bombyx mori is a valuable model organism of high economic importance. Its genome sequence is available, as well as basic genetic and molecular genetic tools and markers. The introduction of genome editing methods based on engineered nucleases enables precise manipulations with genomic DNA, including targeted DNA deletions, insertions, or replacements in the genome allowing gene analysis and various applications. We describe here the use of TALENs which have a simple modular design of their DNA-binding domains, are easy to prepare and proved to be efficient in targeting of a wide range of cleavage sites. Our procedure often allows the production of individuals carrying homozygous mutations as early as in the G1 generation. PMID:26443219

  14. Identifying Francisella tularensis Genes Required for Growth in Host Cells

    PubMed Central

    Brunton, J.; Steele, S.; Miller, C.; Lovullo, E.; Taft-Benz, S.

    2015-01-01

    Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence. PMID:25987704

  15. 2012 MUTAGENESIS GORDON RESEARCH CONFERENCE, AUGUST 19-23, 2012

    SciTech Connect

    Demple, Bruce

    2012-08-23

    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.

  16. Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

    PubMed Central

    Janik, Callie L.; Starr, Timothy K.

    2013-01-01

    Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM

  17. Identification of Sleeping Beauty transposon insertions in solid tumors using linker-mediated PCR.

    PubMed

    Janik, Callie L; Starr, Timothy K

    2013-01-01

    Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer(1). Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation(2). Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method(3). The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker

  18. A Genetic Screen for Pathogenicity Genes in the Hemibiotrophic Fungus Colletotrichum higginsianum Identifies the Plasma Membrane Proton Pump Pma2 Required for Host Penetration.

    PubMed

    Korn, Martin; Schmidpeter, Johannes; Dahl, Marlis; Müller, Susanne; Voll, Lars M; Koch, Christian

    2015-01-01

    We used insertional mutagenesis by Agrobacterium tumefaciens mediated transformation (ATMT) to isolate pathogenicity mutants of Colletotrichum higginsianum. From a collection of 7200 insertion mutants we isolated 75 mutants with reduced symptoms. 19 of these were affected in host penetration, while 17 were affected in later stages of infection, like switching to necrotrophic growth. For 16 mutants the location of T-DNA insertions could be identified by PCR. A potential plasma membrane H(+)-ATPase Pma2 was targeted in five independent insertion mutants. We genetically inactivated the Ku80 component of the non-homologous end-joining pathway in C. higginsianum to establish an efficient gene knockout protocol. Chpma2 deletion mutants generated by homologous recombination in the ΔChku80 background form fully melanized appressoria but entirely fail to penetrate the host tissue and are non-pathogenic. The ChPMA2 gene is induced upon appressoria formation and infection of A. thaliana. Pma2 activity is not important for vegetative growth of saprophytically growing mycelium, since the mutant shows no growth penalty under these conditions. Colletotrichum higginsianum codes for a closely related gene (ChPMA1), which is highly expressed under most growth conditions. ChPMA1 is more similar to the homologous yeast genes for plasma membrane pumps. We propose that expression of a specific proton pump early during infection may be common to many appressoria forming fungal pathogens as we found ChPMA2 orthologs in several plant pathogenic fungi. PMID:25992547

  19. A Genetic Screen for Pathogenicity Genes in the Hemibiotrophic Fungus Colletotrichum higginsianum Identifies the Plasma Membrane Proton Pump Pma2 Required for Host Penetration

    PubMed Central

    Dahl, Marlis; Müller, Susanne; Voll, Lars M.; Koch, Christian

    2015-01-01

    We used insertional mutagenesis by Agrobacterium tumefaciens mediated transformation (ATMT) to isolate pathogenicity mutants of Colletotrichum higginsianum. From a collection of 7200 insertion mutants we isolated 75 mutants with reduced symptoms. 19 of these were affected in host penetration, while 17 were affected in later stages of infection, like switching to necrotrophic growth. For 16 mutants the location of T-DNA insertions could be identified by PCR. A potential plasma membrane H+-ATPase Pma2 was targeted in five independent insertion mutants. We genetically inactivated the Ku80 component of the non-homologous end-joining pathway in C. higginsianum to establish an efficient gene knockout protocol. Chpma2 deletion mutants generated by homologous recombination in the ΔChku80 background form fully melanized appressoria but entirely fail to penetrate the host tissue and are non-pathogenic. The ChPMA2 gene is induced upon appressoria formation and infection of A. thaliana. Pma2 activity is not important for vegetative growth of saprophytically growing mycelium, since the mutant shows no growth penalty under these conditions. Colletotrichum higginsianum codes for a closely related gene (ChPMA1), which is highly expressed under most growth conditions. ChPMA1 is more similar to the homologous yeast genes for plasma membrane pumps. We propose that expression of a specific proton pump early during infection may be common to many appressoria forming fungal pathogens as we found ChPMA2 orthologs in several plant pathogenic fungi. PMID:25992547

  20. A novel mouse model identifies cooperating mutations and therapeutic targets critical for chronic myeloid leukemia progression

    PubMed Central

    Giotopoulos, George; van der Weyden, Louise; Osaki, Hikari; Rust, Alistair G.; Gallipoli, Paolo; Meduri, Eshwar; Horton, Sarah J.; Chan, Wai-In; Foster, Donna; Prinjha, Rab K.; Pimanda, John E.; Tenen, Daniel G.; Vassiliou, George S.; Koschmieder, Steffen; Adams, David J.

    2015-01-01

    The introduction of highly selective ABL-tyrosine kinase inhibitors (TKIs) has revolutionized therapy for chronic myeloid leukemia (CML). However, TKIs are only efficacious in the chronic phase of the disease and effective therapies for TKI-refractory CML, or after progression to blast crisis (BC), are lacking. Whereas the chronic phase of CML is dependent on BCR-ABL, additional mutations are required for progression to BC. However, the identity of these mutations and the pathways they affect are poorly understood, hampering our ability to identify therapeutic targets and improve outcomes. Here, we describe a novel mouse model that allows identification of mechanisms of BC progression in an unbiased and tractable manner, using transposon-based insertional mutagenesis on the background of chronic phase CML. Our BC model is the first to faithfully recapitulate the phenotype, cellular and molecular biology of human CML progression. We report a heterogeneous and unique pattern of insertions identifying known and novel candidate genes and demonstrate that these pathways drive disease progression and provide potential targets for novel therapeutic strategies. Our model greatly informs the biology of CML progression and provides a potent resource for the development of candidate therapies to improve the dismal outcomes in this highly aggressive disease. PMID:26304963

  1. Landscape of insertion polymorphisms in the human genome.

    PubMed

    Onozawa, Masahiro; Goldberg, Liat; Aplan, Peter D

    2015-04-01

    Nucleotide substitutions, small (<50 bp) insertions or deletions (indels), and large (>50 bp) deletions are well-known causes of genetic variation within the human genome. We recently reported a previously unrecognized form of polymorphic insertions, termed templated sequence insertion polymorphism (TSIP), in which the inserted sequence was templated from a distant genomic region, and was inserted in the genome through reverse transcription of an RNA intermediate. TSIPs can be grouped into two classes based on nucleotide sequence features at the insertion junctions; class 1 TSIPs show target site duplication, polyadenylation, and preference for insertion at a 5'-TTTT/A-3' sequence, suggesting a LINE-1 based insertion mechanism, whereas class 2 TSIPs show features consistent with repair of a DNA double strand break by nonhomologous end joining. To gain a more complete picture of TSIPs throughout the human population, we evaluated whole-genome sequence from 52 individuals, and identified 171 TSIPs. Most individuals had 25-30 TSIPs, and common (present in >20% of individuals) TSIPs were found in individuals throughout the world, whereas rare TSIPs tended to cluster in specific geographic regions. The number of rare TSIPs was greater than the number of common TSIPs, suggesting that TSIP generation is an ongoing process. Intriguingly, mitochondrial sequences were a frequent template for class 2 insertions, used more commonly than any nuclear chromosome. Similar to single nucleotide polymorphisms and indels, we suspect that these TSIPs may be important for the generation of human diversity and genetic diseases, and can be useful in tracking historical migration of populations. PMID:25745018

  2. Mutagenesis of Burkholderia pseudomallei with Tn5-OT182: isolation of motility mutants and molecular characterization of the flagellin structural gene.

    PubMed Central

    DeShazer, D; Brett, P J; Carlyon, R; Woods, D E

    1997-01-01

    Burkholderia pseudomallei is a human and animal pathogen in tropical regions, especially Southeast Asia and northern Australia. Currently little is known about the genetics and molecular biology of this organism. In this report, we describe the mutagenesis of B. pseudomallei with the transposon Tn5-OT182. B. pseudomallei 1026b transposon mutants were obtained at a frequency of 4.6 x 10(-4) per initial donor cell, and the transposon inserted randomly into the chromosome. We used Tn5-OT182 to identify the flagellin structural gene, fliC. We screened 3,500 transposon mutants and identified 28 motility mutants. Tn5-OT182 integrated into 19 unique genetic loci encoding proteins with homology to Escherichia coli and Salmonella typhimurium flagellar and chemotaxis proteins. Two mutants, MM35 and MM36, contained Tn5-OT182 integrations in fliC. We cloned and sequenced fliC and used it to complement MM35 and MM36 in trans. The fliC transcriptional start site and a sigmaF-like promoter were identified by primer extension analysis. We observed a significant difference in the expression of two distinct fliC-lacZ transcriptional fusions during bacterial growth, suggesting the presence of a latent intragenic transcriptional terminator in fliC. There was no significant difference in the virulence of 1026b compared to that of MM36 in diabetic rats or Syrian hamsters, suggesting that flagella and/or motility are probably not virulence determinants in these animal models of B. pseudomallei infection. A phylogenetic analysis based on the flagellins from a variety of bacterial species supported the recent transfer of B. pseudomallei from the genus Pseudomonas to Burkholderia. PMID:9079894

  3. Graphene oxide can induce in vitro and in vivo mutagenesis

    NASA Astrophysics Data System (ADS)

    Liu, Yuanyuan; Luo, Yi; Wu, Jing; Wang, Yinsong; Yang, Xiaoying; Yang, Rui; Wang, Baiqi; Yang, Jinrong; Zhang, Ning

    2013-12-01

    Graphene oxide (GO) has attracted enormous interests due to its extraordinary properties. Recent studies have confirmed the cytotoxicity of GO, we further investigate its mutagenic potential in this study. The results showed that GO interfered with DNA replication and induced mutagenesis at molecular level. GO treatments at concentrations of 10 and 100 μg/mL altered gene expression patterns at cellular level, and 101 differentially expressed genes mediated DNA-damage control, cell apoptosis, cell cycle, and metabolism. Intravenous injection of GO at 4 mg/kg for 5 consecutive days clearly induced formation of micronucleated polychromic erythrocytes in mice, and its mutagenesis potential appeared to be comparable to cyclophosphamide, a classic mutagen. In conclusion, GO can induce mutagenesis both in vitro and in vivo, thus extra consideration is required for its biomedical applications.

  4. An algorithm for protein engineering: simulations of recursive ensemble mutagenesis.

    PubMed Central

    Arkin, A P; Youvan, D C

    1992-01-01

    An algorithm for protein engineering, termed recursive ensemble mutagenesis, has been developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Starting from partially randomized "wild-type" DNA sequences, a highly parallel search of sequence space for peptides fitting an experimenter's criteria is performed. Each iteration uses information gained from the previous rounds to search the space more efficiently. Simulations of the technique indicate that, under a variety of conditions, the algorithm can rapidly produce a diverse population of proteins fitting specific criteria. In the experimental analog, genetic selection or screening applied during recursive ensemble mutagenesis should force the evolution of an ensemble of mutants to a targeted cluster of related phenotypes. Images PMID:1502200

  5. Sleeping Beauty Transposon Mutagenesis as a Tool for Gene Discovery in the NOD Mouse Model of Type 1 Diabetes

    PubMed Central

    Elso, Colleen M.; Chu, Edward P. F.; Alsayb, May A.; Mackin, Leanne; Ivory, Sean T.; Ashton, Michelle P.; Bröer, Stefan; Silveira, Pablo A.; Brodnicki, Thomas C.

    2015-01-01

    A number of different strategies have been used to identify genes for which genetic variation contributes to type 1 diabetes (T1D) pathogenesis. Genetic studies in humans have identified >40 loci that affect the risk for developing T1D, but the underlying causative alleles are often difficult to pinpoint or have subtle biological effects. A complementary strategy to identifying “natural” alleles in the human population is to engineer “artificial” alleles within inbred mouse strains and determine their effect on T1D incidence. We describe the use of the Sleeping Beauty (SB) transposon mutagenesis system in the nonobese diabetic (NOD) mouse strain, which harbors a genetic background predisposed to developing T1D. Mutagenesis in this system is random, but a green fluorescent protein (GFP)-polyA gene trap within the SB transposon enables early detection of mice harboring transposon-disrupted genes. The SB transposon also acts as a molecular tag to, without additional breeding, efficiently identify mutated genes and prioritize mutant mice for further characterization. We show here that the SB transposon is functional in NOD mice and can produce a null allele in a novel candidate gene that increases diabetes incidence. We propose that SB transposon mutagenesis could be used as a complementary strategy to traditional methods to help identify genes that, when disrupted, affect T1D pathogenesis. PMID:26438296

  6. Sleeping Beauty Transposon Mutagenesis as a Tool for Gene Discovery in the NOD Mouse Model of Type 1 Diabetes.

    PubMed

    Elso, Colleen M; Chu, Edward P F; Alsayb, May A; Mackin, Leanne; Ivory, Sean T; Ashton, Michelle P; Bröer, Stefan; Silveira, Pablo A; Brodnicki, Thomas C

    2015-12-01

    A number of different strategies have been used to identify genes for which genetic variation contributes to type 1 diabetes (T1D) pathogenesis. Genetic studies in humans have identified >40 loci that affect the risk for developing T1D, but the underlying causative alleles are often difficult to pinpoint or have subtle biological effects. A complementary strategy to identifying "natural" alleles in the human population is to engineer "artificial" alleles within inbred mouse strains and determine their effect on T1D incidence. We describe the use of the Sleeping Beauty (SB) transposon mutagenesis system in the nonobese diabetic (NOD) mouse strain, which harbors a genetic background predisposed to developing T1D. Mutagenesis in this system is random, but a green fluorescent protein (GFP)-polyA gene trap within the SB transposon enables early detection of mice harboring transposon-disrupted genes. The SB transposon also acts as a molecular tag to, without additional breeding, efficiently identify mutated genes and prioritize mutant mice for further characterization. We show here that the SB transposon is functional in NOD mice and can produce a null allele in a novel candidate gene that increases diabetes incidence. We propose that SB transposon mutagenesis could be used as a complementary strategy to traditional methods to help identify genes that, when disrupted, affect T1D pathogenesis. PMID:26438296

  7. Ultrasound guided spine needle insertion

    NASA Astrophysics Data System (ADS)

    Chen, Elvis C. S.; Mousavi, Parvin; Gill, Sean; Fichtinger, Gabor; Abolmaesumi, Purang

    2010-02-01

    An ultrasound (US) guided, CT augmented, spine needle insertion navigational system is introduced. The system consists of an electromagnetic (EM) sensor, an US machine, and a preoperative CT volume of the patient anatomy. Three-dimensional (3D) US volume is reconstructed intraoperatively from a set of two-dimensional (2D) freehand US slices, and is coregistered with the preoperative CT. This allows the preoperative CT volume to be used in the intraoperative clinical coordinate. The spatial relationship between the patient anatomy, surgical tools, and the US transducer are tracked using the EM sensor, and are displayed with respect to the CT volume. The pose of the US transducer is used to interpolate the CT volume, providing the physician with a 2D "x-ray vision" to guide the needle insertion. Many of the system software components are GPU-accelerated, allowing real-time performance of the guidance system in a clinical setting.

  8. Insertion device calculations with mathematica

    SciTech Connect

    Carr, R.; Lidia, S.

    1995-02-01

    The design of accelerator insertion devices such as wigglers and undulators has usually been aided by numerical modeling on digital computers, using code in high level languages like Fortran. In the present era, there are higher level programming environments like IDL{reg_sign}, MatLab{reg_sign}, and Mathematica{reg_sign} in which these calculations may be performed by writing much less code, and in which standard mathematical techniques are very easily used. The authors present a suite of standard insertion device modeling routines in Mathematica to illustrate the new techniques. These routines include a simple way to generate magnetic fields using blocks of CSEM materials, trajectory solutions from the Lorentz force equations for given magnetic fields, Bessel function calculations of radiation for wigglers and undulators and general radiation calculations for undulators.

  9. A computer program to display codon changes caused by mutagenesis.

    PubMed

    Sirotkin, K

    1988-04-01

    A FORTRAN program for displaying the correspondence between codon changes and different possible base changes is presented. Changes of both single bases and dimers are considered. The user can specify the mutagenesis spectrum. Additionally, the user can choose whether or not to consider single or double events in a codon and whether or not to consider the possibility that the change of two bases (a dimer) can overlap a codon boundary. Furthermore, a variety of ways may be chosen to display and summarize the codon changes that can result from the specified mutagenesis. A user-supplied sequence or the genetic code table can be analyzed. PMID:3167596

  10. Mutagenesis protocols in Saccharomyces cerevisiae by in vivo overlap extension.

    PubMed

    Alcalde, Miguel

    2010-01-01

    A high recombination frequency and its ease of manipulation has made Saccharomyces cerevisiae a unique model eukaryotic organism to study homologous recombination. Indeed, the well-developed recombination machinery in S. cerevisiae facilitates the construction of mutant libraries for directed evolution experiments. In this context, in vivo overlap extension (IVOE) is a particularly attractive protocol that takes advantage of the eukaryotic apparatus to carry out combinatorial saturation mutagenesis, site-directed recombination or site-directed mutagenesis, avoiding ligation steps and additional PCR reactions that are common to standard in vitro protocols. PMID:20676972

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

    SciTech Connect

    Kermany, Mohammad; Parker, Lisan; Guo, Yun-Kai; Miller, Darla R; Swanson, Douglas J; Yoo, Tai-June; Goldowitz, Daniel; Zuo, Jian

    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.

  12. Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis

    PubMed Central

    Mueller, Konrad E.; Wolf, Katerina

    2016-01-01

    ABSTRACT Although progress in Chlamydia genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Herein, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM). We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis serovar L2. Furthermore, this approach permits the monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms. As proof of principle, trpA was successfully deleted and replaced with a sequence encoding both green fluorescent protein (GFP) and β-lactamase. The trpA-deficient strain was unable to grow in indole-containing medium, and this phenotype was reversed by complementation with trpA expressed in trans. To assess reproducibility at alternate sites, FRAEM was repeated for genes encoding type III secretion effectors CTL0063, CTL0064, and CTL0065. In all four cases, stable mutants were recovered one passage after the observation of transformants, and allelic exchange was limited to the specific target gene, as confirmed by whole-genome sequencing. Deleted sequences were not detected by quantitative real-time PCR (qPCR) from isogenic mutant populations. We demonstrate that utilization of the chlamydial suicide vector with FRAEM renders C. trachomatis highly amenable to versatile and efficient genetic manipulation. PMID:26787828

  13. Therapeutic genome mutagenesis using synthetic donor DNA and triplex-forming molecules.

    PubMed

    Reza, Faisal; Glazer, Peter M

    2015-01-01

    Genome mutagenesis can be achieved in a variety of ways, though a select few are suitable for therapeutic settings. Among them, the harnessing of intracellular homologous recombination affords the safety and efficacy profile suitable for such settings. Recombinagenic donor DNA and mutagenic triplex-forming molecules co-opt this natural recombination phenomenon to enable the specific, heritable editing and targeting of the genome. Editing the genome is achieved by designing the sequence-specific recombinagenic donor DNA to have base mismatches, insertions, and deletions that will be incorporated into the genome when it is used as a template for recombination. Targeting the genome is similarly achieved by designing the sequence-specific mutagenic triplex-forming molecules to further recruit the recombination machinery thereby upregulating its activity with the recombinagenic donor DNA. This combination of extracellularly introduced, designed synthetic molecules and intercellularly ubiquitous, evolved natural machinery enables the mutagenesis of chromosomes and engineering of whole genomes with great fidelity while limiting nonspecific interactions. Herein, we demonstrate the harnessing of recombinagenic donor DNA and mutagenic triplex-forming molecular technology for potential therapeutic applications. These demonstrations involve, among others, utilizing this technology to correct genes so that they become physiologically functional, to induce dormant yet functional genes in place of non-functional counterparts, to place induced genes under regulatory elements, and to disrupt genes to abrogate a cellular vulnerability. Ancillary demonstrations of the design and synthesis of this recombinagenic and mutagenic molecular technology as well as their delivery and assayed interaction with duplex DNA reveal a potent technological platform for engineering specific changes into the living genome. PMID:25408401

  14. BAX and tumor suppressor TRP53 are important in regulating mutagenesis in spermatogenic cells in mice.

    PubMed

    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. PMID:20739667

  15. Targeted Mutagenesis in Zebrafish Using CRISPR RNA-Guided Nucleases.

    PubMed

    Hwang, Woong Y; Fu, Yanfang; Reyon, Deepak; Gonzales, Andrew P W; Joung, J Keith; Yeh, Jing-Ruey Joanna

    2015-01-01

    In recent years, the zebrafish has become a critical contributor to various areas of biomedical research, advancing our fundamental understanding of biomedicine and helping discover candidate therapeutics for human diseases. Nevertheless, to further extend the power of this important model organism requires a robust and simple-to-use genome editing platform that will enable targeted gene knockouts and introduction of specific mutations identified in human diseases into the zebrafish genome. We describe here protocols for creating insertion or deletion (indel) mutations or precise sequence modifications in zebrafish genes using customizable CRISPR-Cas9 RNA-guided nucleases (RGNs). These methods can be easily implemented in any lab and may also potentially be extended for use in other organisms. PMID:25981483

  16. Insertional mutagenesis and characterization of a polyketide synthase gene (PKS1) required for melanin biosynthesis in Bipolaris oryzae.

    PubMed

    Moriwaki, Akihiro; Kihara, Junichi; Kobayashi, Tsutomu; Tokunaga, Toshiko; Arase, Sakae; Honda, Yuichi

    2004-09-01

    A polyketide synthase gene named PKS1, involved in the melanin biosynthesis pathway of the phytopathogenic fungus Bipolaris oryzae, was isolated using restriction enzyme-mediated integration. Sequence analysis showed that the PKS1 encodes a putative protein that has 2155 amino acids and significant similarity to other fungal polyketide synthases. Targeted disruption of the PKS1 gene showed that it is necessary for melanin biosynthesis in B. oryzae. Northern blot analysis showed that PKS1 transcripts were specifically enhanced by near-ultraviolet radiation (300-400 nm) and that its temporal transcriptional patterns were similar to those of THR1 and SCD1 genes involved in the melanin biosynthesis pathway of B. oryzae. PMID:15336395

  17. Methods for targetted mutagenesis in gram-positive bacteria

    SciTech Connect

    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.

  18. 2002 Gordon Research Conference on Mutagenesis. Final Progress Report

    SciTech Connect

    2002-08-02

    The Gordon Research Conference (GRC) on MUTAGENESIS was held at Bates College from 7/28/02 thru 8/2/02. The Conference was well-attended with 157 participants. The attendees represented the spectrum of endeavor in this field coming from academia, industry, and government laboratories, both U.S. and foreign scientists, senior researchers, young investigators, and students.

  19. Transposon mutagenesis of Bacteroides fragilis using a mariner transposon vector.

    PubMed

    Veeranagouda, Yaligara; Husain, Fasahath; Wexler, Hannah M

    2013-08-01

    The mariner transposon vector pYV07 was tested for use in the mutagenesis of Bacteroides fragilis 638R. The transposon vector efficiently generated mutants in B. fragilis 638R. The transposon disrupted genes were scattered throughout the genome of B. fragilis 638R. This method serves as a powerful tool to study B. fragilis. PMID:23664906

  20. CRISPR/Cas9-targeted mutagenesis in Caenorhabditis elegans.

    PubMed

    Waaijers, Selma; Portegijs, Vincent; Kerver, Jana; Lemmens, Bennie B L G; Tijsterman, Marcel; van den Heuvel, Sander; Boxem, Mike

    2013-11-01

    The generation of genetic mutants in Caenorhabditis elegans has long relied on the selection of mutations in large-scale screens. Directed mutagenesis of specific loci in the genome would greatly speed up analysis of gene function. Here, we adapt the CRISPR/Cas9 system to generate mutations at specific sites in the C. elegans genome. PMID:23979586

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

    ERIC Educational Resources Information Center

    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…

  2. Favipiravir elicits antiviral mutagenesis during virus replication in vivo

    PubMed Central

    Arias, Armando; Thorne, Lucy; Goodfellow, Ian

    2014-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.03679.001 PMID:25333492

  3. GERM-LINE SPECIFIC FACTORS IN CHEMICAL MUTAGENESIS

    EPA Science Inventory

    Chemical mutagenesis test results ave not revealed evidence of germ-line specific mutagens. owever, conventional assays have indicated that there are male-female differences in mutagenic response, as well as quantitative/qualitative differences in induced mutations which depend u...

  4. Shoe Inserts and Prescription Custom Orthotics

    MedlinePlus

    ... Feet » Foot Health Information Shoe Inserts and Prescription Custom Orthotics What are Shoe Inserts? You've seen ... hold on to your receipt.) What are Prescription Custom Orthotics? Custom orthotics are specially-made devices designed ...

  5. Field errors in hybrid insertion devices

    SciTech Connect

    Schlueter, R.D.

    1995-02-01

    Hybrid magnet theory as applied to the error analyses used in the design of Advanced Light Source (ALS) insertion devices is reviewed. Sources of field errors in hybrid insertion devices are discussed.

  6. Transposon assisted gene insertion technology (TAGIT): a tool for generating fluorescent fusion proteins.

    PubMed

    Gregory, James A; Becker, Eric C; Jung, James; Tuwatananurak, Ida; Pogliano, Kit

    2010-01-01

    We constructed a transposon (transposon assisted gene insertion technology, or TAGIT) that allows the random insertion of gfp (or other genes) into chromosomal loci without disrupting operon structure or regulation. TAGIT is a modified Tn5 transposon that uses Kan(R) to select for insertions on the chromosome or plasmid, beta-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the kan and lacZ genes in vivo. The resulting gfp insertions maintain target gene reading frame (to the 5' and 3' of gfp) and are integrated at the native chromosomal locus, thereby maintaining native expression signals. Libraries can be screened to identify GFP insertions that maintain target protein function at native expression levels, allowing more trustworthy localization studies. We here use TAGIT to generate a library of GFP insertions in the Escherichia coli lactose repressor (LacI). We identified fully functional GFP insertions and partially functional insertions that bind DNA but fail to repress the lacZ operon. Several of these latter GFP insertions localize to lacO arrays integrated in the E. coli chromosome without producing the elongated cells frequently observed when functional LacI-GFP fusions are used in chromosome tagging experiments. TAGIT thereby faciliates the isolation of fully functional insertions of fluorescent proteins into target proteins expressed from the native chromosomal locus as well as potentially useful partially functional proteins. PMID:20090956

  7. Insert metering plates for gas turbine nozzles

    DOEpatents

    Burdgick, Steven S.; Itzel, Gary; Chopra, Sanjay; Abuaf, Nesim; Correia, Victor H.

    2004-05-11

    The invention comprises a metering plate which is assembled to an impingement insert for use in the nozzle of a gas turbine. The metering plate can have one or more metering holes and is used to balance the cooling flow within the nozzle. A metering plate with multiple holes reduces static pressure variations which result from the cooling airflow through the metering plate. The metering plate can be assembled to the insert before or after the insert is inserted into the nozzle.

  8. Porous pseudoalloy electrode gap inserts

    SciTech Connect

    Pustogarov, A.V.; Chernyshev, L.I.; Kostornov, A.G.; Mel'nikov, G.N.

    1985-06-01

    The authors make a comparative assessment of the service characteristics of porous electrode gap inserts (EGI) made of tungsten, molybdenum, and W-Cu and Mo-Cu pseudoalloys, tested using a high pressure apparatus. Charts present the data gathered on maximum thermal flux entering the EGI wall with specific nitrogen flow rates; variation of temperature of the inner surface of the EGI wall with thermal conductivity of the material; and variation of viscous and inertia coefficients of hydraulic resistance of pseudoalloys with duration of EGI operation. The authors conclude that porous EGIs made of W-Cu and Mo-Cu pseudoalloys are characterized by the same operating performance.

  9. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

    PubMed Central

    Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

    2016-01-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

  10. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.

    PubMed

    Hehle, Verena K; Paul, Matthew J; Roberts, Victoria A; van Dolleweerd, Craig J; Ma, Julian K-C

    2016-04-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformedNicotiana tabacum Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy and light chain mutant combinations were expressed transiently inN. tabacumand demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.-Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

  11. Illegitimate recombination: An efficient method for random mutagenesis in Mycobacterium avium subsp. hominissuis

    PubMed Central

    2012-01-01

    Background The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed. Results We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS). Conclusions We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence. PMID:22966811

  12. Quench Module Insert (QMI) and the Diffusion Module Insert (DMI) Furnace Development

    NASA Technical Reports Server (NTRS)

    Crouch, Myscha; Carswell, William; Farmer, Jeff; Rose, Fred; Tidwell, Paul

    2000-01-01

    This paper presents, in viewgraph form, QMI (Quench Module Insert) and DMI (Diffusion Module Insert) furnace development. The topics include: 1) Furnace Module in Rack; 2) Quench Module Insert; 3) QMI in MSL Core; 4) Diffusion Module Insert; 5) QMI; and 6) QMI Development and Testing.

  13. Insertional Inactivation of Genes Responsible for the d-Alanylation of Lipoteichoic Acid in Streptococcus gordonii DL1 (Challis) Affects Intrageneric Coaggregations

    PubMed Central

    Clemans, Daniel L.; Kolenbrander, Paul E.; Debabov, Dmitri V.; Zhang, Qunying; Lunsford, R. Dwayne; Sakone, Holly; Whittaker, Catherine J.; Heaton, Michael P.; Neuhaus, Francis C.

    1999-01-01

    Most human oral viridans streptococci participate in intrageneric coaggregations, the cell-to-cell adherence among genetically distinct streptococci. Two genes relevant to these intrageneric coaggregations were identified by transposon Tn916 mutagenesis of Streptococcus gordonii DL1 (Challis). A 626-bp sequence flanking the left end of the transposon was homologous to dltA and dltB of Lactobacillus rhamnosus ATCC 7469 (formerly called Lactobacillus casei). A 60-kb probe based on this flanking sequence was used to identify the homologous DNA in a fosmid library of S. gordonii DL1. This DNA encoded d-alanine-d-alanyl carrier protein ligase that was expressed in Escherichia coli from the fosmid clone. The cloned streptococcal dltA was disrupted by inserting an ermAM cassette, and then it was linearized and transformed into S. gordonii DL1 for allelic replacement. Erythromycin-resistant transformants containing a single insertion in dltA exhibited a loss of d-alanyl esters in lipoteichoic acid (LTA) and a loss of intrageneric coaggregation. This phenotype was correlated with the loss of a 100-kDa surface protein reported previously to be involved in mediating intrageneric coaggregation (C. J. Whittaker, D. L. Clemans, and P. E. Kolenbrander, Infect. Immun. 64:4137–4142, 1996). The mutants retained the parental ability to participate in intergeneric coaggregation with human oral actinomyces, indicating the specificity of the mutation in altering intrageneric coaggregations. The mutants were altered morphologically and exhibited aberrant cell septa in a variety of pleomorphs. The natural DNA transformation frequency was reduced 10-fold in these mutants. Southern analysis of chromosomal DNAs from various streptococcal species with the dltA probe revealed the presence of this gene in most viridans streptococci. Thus, it is hypothesized that d-alanyl LTA may provide binding sites for the putative 100-kDa adhesin and scaffolding for the proper presentation of this adhesin

  14. Insertional inactivation of genes responsible for the D-alanylation of lipoteichoic acid in Streptococcus gordonii DL1 (Challis) affects intrageneric coaggregations.

    PubMed

    Clemans, D L; Kolenbrander, P E; Debabov, D V; Zhang, Q; Lunsford, R D; Sakone, H; Whittaker, C J; Heaton, M P; Neuhaus, F C

    1999-05-01

    Most human oral viridans streptococci participate in intrageneric coaggregations, the cell-to-cell adherence among genetically distinct streptococci. Two genes relevant to these intrageneric coaggregations were identified by transposon Tn916 mutagenesis of Streptococcus gordonii DL1 (Challis). A 626-bp sequence flanking the left end of the transposon was homologous to dltA and dltB of Lactobacillus rhamnosus ATCC 7469 (formerly called Lactobacillus casei). A 60-kb probe based on this flanking sequence was used to identify the homologous DNA in a fosmid library of S. gordonii DL1. This DNA encoded D-alanine-D-alanyl carrier protein ligase that was expressed in Escherichia coli from the fosmid clone. The cloned streptococcal dltA was disrupted by inserting an ermAM cassette, and then it was linearized and transformed into S. gordonii DL1 for allelic replacement. Erythromycin-resistant transformants containing a single insertion in dltA exhibited a loss of D-alanyl esters in lipoteichoic acid (LTA) and a loss of intrageneric coaggregation. This phenotype was correlated with the loss of a 100-kDa surface protein reported previously to be involved in mediating intrageneric coaggregation (C. J. Whittaker, D. L. Clemans, and P. E. Kolenbrander, Infect. Immun. 64:4137-4142, 1996). The mutants retained the parental ability to participate in intergeneric coaggregation with human oral actinomyces, indicating the specificity of the mutation in altering intrageneric coaggregations. The mutants were altered morphologically and exhibited aberrant cell septa in a variety of pleomorphs. The natural DNA transformation frequency was reduced 10-fold in these mutants. Southern analysis of chromosomal DNAs from various streptococcal species with the dltA probe revealed the presence of this gene in most viridans streptococci. Thus, it is hypothesized that D-alanyl LTA may provide binding sites for the putative 100-kDa adhesin and scaffolding for the proper presentation of this adhesin to

  15. The BDGP gene disruption project: Single transposon insertions associated with 40 percent of Drosophila genes

    SciTech Connect

    Bellen, Hugo J.; Levis, Robert W.; Liao, Guochun; He, Yuchun; Carlson, Joseph W.; Tsang, Garson; Evans-Holm, Martha; Hiesinger, P. Robin; Schulze, Karen L.; Rubin, Gerald M.; Hoskins, Roger A.; Spradling, Allan C.

    2004-01-13

    The Berkeley Drosophila Genome Project (BDGP) strives to disrupt each Drosophila gene by the insertion of a single transposable element. As part of this effort, transposons in more than 30,000 fly strains were localized and analyzed relative to predicted Drosophila gene structures. Approximately 6,300 lines that maximize genomic coverage were selected to be sent to the Bloomington Stock Center for public distribution, bringing the size of the BDGP gene disruption collection to 7,140 lines. It now includes individual lines predicted to disrupt 5,362 of the 13,666 currently annotated Drosophila genes (39 percent). Other lines contain an insertion at least 2 kb from others in the collection and likely mutate additional incompletely annotated or uncharacterized genes and chromosomal regulatory elements. The remaining strains contain insertions likely to disrupt alternative gene promoters or to allow gene mis-expression. The expanded BDGP gene disruption collection provides a public resource that will facilitate the application of Drosophila genetics to diverse biological problems. Finally, the project reveals new insight into how transposons interact with a eukaryotic genome and helps define optimal strategies for using insertional mutagenesis as a genomic tool.

  16. The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes.

    PubMed Central

    Bellen, Hugo J; Levis, Robert W; Liao, Guochun; He, Yuchun; Carlson, Joseph W; Tsang, Garson; Evans-Holm, Martha; Hiesinger, P Robin; Schulze, Karen L; Rubin, Gerald M; Hoskins, Roger A; Spradling, Allan C

    2004-01-01

    The Berkeley Drosophila Genome Project (BDGP) strives to disrupt each Drosophila gene by the insertion of a single transposable element. As part of this effort, transposons in >30,000 fly strains were localized and analyzed relative to predicted Drosophila gene structures. Approximately 6300 lines that maximize genomic coverage were selected to be sent to the Bloomington Stock Center for public distribution, bringing the size of the BDGP gene disruption collection to 7140 lines. It now includes individual lines predicted to disrupt 5362 of the 13,666 currently annotated Drosophila genes (39%). Other lines contain an insertion at least 2 kb from others in the collection and likely mutate additional incompletely annotated or uncharacterized genes and chromosomal regulatory elements. The remaining strains contain insertions likely to disrupt alternative gene promoters or to allow gene misexpression. The expanded BDGP gene disruption collection provides a public resource that will facilitate the application of Drosophila genetics to diverse biological problems. Finally, the project reveals new insight into how transposons interact with a eukaryotic genome and helps define optimal strategies for using insertional mutagenesis as a genomic tool. PMID:15238527

  17. Cotranslational insertion of selenocysteine into formate dehydrogenase from Escherichia coli directed by a UGA codon

    SciTech Connect

    Zinoni, F.; Birkmann, A.; Leinfelder, W.; Boeck, A.

    1987-05-01

    The structural gene (fdhF) for the 80-kDa selenopolypeptide of formate dehydrogenase from Escherichia coli contains an in-frame UGA codon at amino acid position 140 that is translated. Translation of gene fusions between N-terminal parts of fdhF with lacZ depends on the availability of selenium in the medium when the hybrid gene contains the UGA codon; it is independent of the presence of selenium when an fdhF portion upstream of the UGA position is fused to lacZ. Transcription does not require the presence of selenium in either case. By localized mutagenesis, the UGA codon was converted into serine (UCA) and cysteine (UGC and UGU) codons. Each mutagion relieved the selenium dependency of fdhF mRNA translation. Selenium incorporation was completely abolished in the case of the UCA insertion and was reduced to about 10% when the UGA was replaced by a cysteine codon. Insertion of UCA yielded an inactive fdhF gene product, while insertion of UGC and UGU resulted in polypeptides with lowered activities as components in the system formerly known as formate hydrogenlyase. Altogether the results indicate that the UGA codon at position 140 directs the cotranslational insertion of selenocysteine into the fdhF polypeptide chain.

  18. Space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b and screening of higher yielding strains.

    PubMed

    Wang, Junfeng; Liu, Changting; Liu, Jinyi; Fang, Xiangqun; Xu, Chen; Guo, Yinghua; Chang, De; Su, Longxiang

    2014-03-01

    The aim of this study was to investigate the space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b. The genetically engineered bacteria expressing the recombinant interferon α1b were sent into outer space on the Chinese Shenzhou VIII spacecraft. After the 17 day space flight, mutant strains that highly expressed the target gene were identified. After a series of screening of spaceflight-treated bacteria and the quantitative comparison of the mutant strains and original strain, we found five strains that showed a significantly higher production of target proteins, compared with the original strain. Our results support the notion that the outer space environment has unique effects on the mutation breeding of microorganisms, including genetically engineered strains. Mutant strains that highly express the target protein could be obtained through spaceflight-induced mutagenesis. PMID:24096450

  19. Optimizing glycosyltransferase specificity via ‘hot spot’ saturation mutagenesis presents a new catalyst for novobiocin glycorandomization

    PubMed Central

    Williams, Gavin J.; Goff, Randal D.; Zhang, Changsheng; Thorson, Jon S.

    2010-01-01

    Summary A comprehensive two phase ‘hot spot’ saturation mutagenesis strategy to rapidly evolve glycosyltransferase specificity for non-natural acceptors is described. Specifically, the application of a high throughput screen (based upon the fluorescent acceptor umbelliferone) was used to identify key amino acid ‘hot spots’ that contribute to GT proficiency and/or promiscuity. Saturation mutagenesis of the corresponding hot spots facilitated the utilization of a lower throughput screen to provide OleD prodigy capable of efficiently glycosylating the non-natural acceptor novobiocic acid with an array of unique sugars. Incredibly, even in the absence of a high-throughput screen for novobiocic acid glycosylation, this approach rapidly led to improvements in the desired catalytic activity of several hundred-fold. PMID:18420146

  20. Improving the solubility of anti-LINGO-1 monoclonal antibody Li33 by isotype switching and targeted mutagenesis

    SciTech Connect

    Pepinsky, R. Blake; Silvian, Laura; Berkowitz, Steven A.; Farrington, Graham; Lugovskoy, Alexey; Walus, Lee; Eldredge, John; Capili, Allan; Mi, Sha; Graff, Christilyn; Garber, Ellen

    2010-11-15

    Monoclonal antibodies (Mabs) are a favorite drug platform of the biopharmaceutical industry. Currently, over 20 Mabs have been approved and several hundred others are in clinical trials. The anti-LINGO-1 Mab Li33 was selected from a large panel of antibodies by Fab phage display technology based on its extraordinary biological activity in promoting oligodendrocyte differentiation and myelination in vitro and in animal models of remyelination. However, the Li33 Fab had poor solubility when converted into a full antibody in an immunoglobulin G1 framework. A detailed analysis of the biochemical and structural features of the antibody revealed several possible reasons for its propensity to aggregate. Here, we successfully applied three molecular approaches (isotype switching, targeted mutagenesis of complementarity determining region residues, and glycosylation site insertion mutagenesis) to address the solubility problem. Through these efforts we were able to improve the solubility of the Li33 Mab from 0.3 mg/mL to >50 mg/mL and reduce aggregation to an acceptable level. These strategies can be readily applied to other proteins with solubility issues.

  1. Predictive mutagenesis of ligation-independent cloning (LIC) vectors for protein expression and site-specific chemical conjugation.

    PubMed

    Vernet, Erik; Sauer, Jørgen; Andersen, Andreas; Jensen, Knud J; Voldborg, Bjørn

    2011-07-15

    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 of expression constructs and report on the construction of expression vectors with N-terminal serine, cysteine, threonine, or tyrosine residues after tobacco etch virus (TEV) protease cleavage. In a practical application, the N-terminal serine was oxidized to an aldehyde, subsequently reacted with an amino-oxy functionalized polyethylene glycol (PEG) ligand under aniline catalysis to provide a protein selectively modified at the N-terminus. PMID:21414287

  2. Construction of a mutagenesis cartridge for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants

    SciTech Connect

    Kuhn, R.J.; Tada, H.; Ypma-Wong, M.F.; Dunn, J.J.; Semler, B.L.; Wimmer, E.

    1988-01-01

    By following a strategy of genetic analysis of poliovirus, the authors have constructed a synthetic mutagenesis cartridge spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type I (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed them to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alteration of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. They suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s).

  3. European Community research on environmental mutagenesis and carcinogenesis.

    PubMed Central

    Sors, A I

    1993-01-01

    Within the 12 Member States of the European Community (EC), environmental policy is now formulated primarily at Community level. As a result, the EC has important regulatory responsibilities for the protection of workers, consumers, and the general public from risks that may arise from environmental chemicals, foremost among them potential carcinogens and mutagens. An important part of EC environmental research and development is intended to provide a scientific basis for these regulations as well as increasing understanding of the basic mechanisms involved in environmental carcinogenesis and mutagenesis. This paper contains a brief introduction to EC environment policy and research, followed by an overview of EC chemicals control activities that are of particular relevance to the research and development program. Community-level research on environmental mutagenesis and carcinogenesis is then reviewed in some detail, including the achievements of recent projects, the scientific content of the current program, and perspectives for the future. PMID:8143645

  4. Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis

    SciTech Connect

    Wang, Weina; Hellinga, Homme W.; Beese, Lorena S.

    2012-05-10

    Even though high-fidelity polymerases copy DNA with remarkable accuracy, some base-pair mismatches are incorporated at low frequency, leading to spontaneous mutagenesis. Using high-resolution X-ray crystallographic analysis of a DNA polymerase that catalyzes replication in crystals, we observe that a C {center_dot} A mismatch can mimic the shape of cognate base pairs at the site of incorporation. This shape mimicry enables the mismatch to evade the error detection mechanisms of the polymerase, which would normally either prevent mismatch incorporation or promote its nucleolytic excision. Movement of a single proton on one of the mismatched bases alters the hydrogen-bonding pattern such that a base pair forms with an overall shape that is virtually indistinguishable from a canonical, Watson-Crick base pair in double-stranded DNA. These observations provide structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis, a long-standing concept that has been difficult to demonstrate directly.

  5. Antiviral Strategies Based on Lethal Mutagenesis and Error Threshold.

    PubMed

    Perales, Celia; Domingo, Esteban

    2016-01-01

    The concept of error threshold derived from quasispecies theory is at the basis of lethal mutagenesis, a new antiviral strategy based on the increase of virus mutation rate above an extinction threshold. Research on this strategy is justified by several inhibitor-escape routes that viruses utilize to ensure their survival. Successive steps in the transition from an organized viral quasispecies into loss of biologically meaningful genomic sequences are dissected. The possible connections between theoretical models and experimental observations on lethal mutagenesis are reviewed. The possibility of using combination of virus-specific mutagenic nucleotide analogues and broad-spectrum, non-mutagenic inhibitors is evaluated. We emphasize the power that quasispecies theory has had to stimulate exploration of new means to combat pathogenic viruses. PMID:26294225

  6. Efficient site-directed saturation mutagenesis using degenerate oligonucleotides.

    PubMed

    Steffens, David L; Williams, John G K

    2007-07-01

    We describe a reliable protocol for constructing single-site saturation mutagenesis libraries consisting of all 20 naturally occurring amino acids at a specific site within a protein. Such libraries are useful for structure-function studies and directed evolution. This protocol extends the utility of Stratagene's QuikChange Site-Directed Mutagenesis Kit, which is primarily recommended for single amino acid substitutions. Two complementary primers are synthesized, containing a degenerate mixture of the four bases at the three positions of the selected codon. These primers are added to starting plasmid template and thermal cycled to produce mutant DNA molecules, which are subsequently transformed into competent bacteria. The protocol does not require purification of mutagenic oligonucleotides or PCR products. This reduces both the cost and turnaround time in high-throughput directed evolution applications. We have utilized this protocol to generate over 200 site-saturation libraries in a DNA polymerase, with a success rate of greater than 95%. PMID:17595310

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

    PubMed Central

    Ishida, Kentaro; Gee, Peter; Hotta, Akitsu

    2015-01-01

    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. PMID:26501275

  8. Specific mutagenesis of a chlorophyll-binding protein. Progress report.

    SciTech Connect

    Eaton-Rye, Dr., Julian; Shen, Gaozhong

    1990-01-01

    During the first phase of the project regarding specific mutagenesis of the chlorophyll-binding protein CP47 in photosystem II (PS II) most of the time has been devoted to (1) establishment of an optimal procedure for the reintroduction of psbB (the gene encoding CP47) carrying a site-directed mutation into the experimental organism, the cyanobacterium Synechocystis sp. PCC 6803, (2) preparations for site-directed mutagenesis, and (3) creation and analysis of chimaeric spinach/cyanobacterial CP47 mutants of Synechocystis. In the coming year, psbB constructs with site-directed mutations in potential chlorophyll-binding regions of CP47 will be introduced into the Synechocystis genome, and site-directed mutants will be characterized according to procedures described in the original project description. In addition, analysis of chimaeric CP47 mutants will be continued.

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

    PubMed

    Ishida, Kentaro; Gee, Peter; Hotta, Akitsu

    2015-01-01

    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. PMID:26501275

  10. Defining essential genes and identifying virulence factors of Porphyromonas gingivalis by massively-parallel sequencing of transposon libraries (Tn-seq)

    PubMed Central

    Klein, Brian A.; Duncan, Margaret J.; Hu, Linden T.

    2016-01-01

    Summary Porphyromonas gingivalis is a keystone pathogen in the development and progression of periodontal disease. Obstacles to the development of saturated transposon libraries have previously limited transposon mutant-based screens as well as essential gene studies. We have developed a system for efficient transposon mutagenesis of P. gingivalis using a modified mariner transposon. Tn-seq is a technique that allows for quantitative assessment of individual mutants within a transposon mutant library by sequencing the transposon-genome junctions and then compiling mutant presence by mapping to a base genome. Using Tn-seq, it is possible to quickly define all the insertional mutants in a library and thus identify non-essential genes under the conditions in which the library was produced. Identification of fitness of individual mutants under specific conditions can be performed by exposing the library to selective pressures. PMID:25636611

  11. Transcriptional mutagenesis: causes and involvement in tumor development

    PubMed Central

    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

  12. Nitrosoguanidine and ultraviolet light mutagenesis in Eudorina elegans (chlorophyceae)

    SciTech Connect

    Toby, A.L.; Kemp, C.L.

    1980-06-01

    Reversion of an acetate requiring strain and the induction of sectored colonies are used to establish optimal conditions for nitrosoguanidine and ultraviolet light mutagenesis in Eudorina elegans Ehrenberg. Nitrosoguanidine is more effective in causing reversion of the acetate requiring strain and inducing auxotrophs. Morphogenetic mutants are more readily induced by ultraviolet light. The effectiveness of ultraviolet light as a mutagen is cell cycle dependent whereas the mutagenic action of nitrosoguanidine is not.

  13. Metal mutagenesis in transgenic Chinese hamster cell lines.

    PubMed Central

    Klein, C B; Kargacin, B; Su, L; Cosentino, S; Snow, E T; Costa, M

    1994-01-01

    Metals are toxic agents for which genotoxic effects are often difficult to demonstrate. To study metal mutagenesis, we have used two stable hprt/gpt+ transgenic cell lines that were derived from Chinese hamster V79 cells. Both the G12 and G10 cell lines are known to be very sensitive to clastogens such as X-rays and bleomycin, with the mutagenic response of the integrated xanthine guanine phosphoribosyl transferase (gpt) gene in G10 usually exceeding that of the same gene in the transgenic G12 cells. In studies with carcinogenic insoluble nickel compounds, a high level of mutagenesis was found at the gpt locus of G12 cells but not at the endogenous hypoxanthine phosphoribosyl transferase (hprt) locus of V79 cells. We have since demonstrated the similar recovery of a high frequency of viable G12 mutants with other insoluble nickel salts including nickel oxides (black and green). The relative mutant yield for the insoluble nickel compounds (G12 > G10) is the opposite of that obtained with nonmetal clastogens (G10 > G12). In the G12 cells, nickel mutagenesis may be related to the integration of the gpt sequence into a heterochromatic region of the genome. For some of the insoluble nickel compounds, significant inhibition of both cytotoxicity and mutant yield resulted when the G12 cells were pretreated with vitamin E. In comparison with the nickel studies, the mutagenic responses to chromium compounds in these cell lines were not as dramatic. Mutagenesis of the gpt target could not be demonstrated with other metals such as mercury or vanadium. PMID:7843139

  14. Inducibility of a gene product required for UV and chemical mutagenesis in Escherichia coli.

    PubMed Central

    Bagg, A; Kenyon, C J; Walker, G C

    1981-01-01

    The product of the umuC gene is required for UV and chemical mutagenesis in Escherichia coli. By the use of the Mud(Ap, lac) bacteriophage, we have obtained an operon fusion of the lac structural genes to the promoter/regulatory region of the umuC gene. The strain containing the umuC::Mud(Ap, lac) fusion was identified on the basis of its UV nonmutability. Strains containing this putative null allele of umuC were (i) nonmutable by UV and other agents, (ii) slightly UV sensitive, and (iii) deficient in their ability to carry out Weigle reactivation of UV-irradiation bacteriophage lambda. The UV nonmutability of the strain could be suppressed by a derivative of the mutagenesis-enhancing plasmid pKM101. beta-Galactosidase synthesis in umuC::Mud(Ap, lac) fusion strains was inducible by UV and other DNA-damaging agents. Genetic analysis of the regulation of beta-galactosidase in umuC::Mud(Ap, lac) strains suggests that the lexA protein is the direct repressor of the umuC gene and that a function of the recA protein, probably its protease activity, is required for the removal of the lexA repressor at the time of umuC induction. PMID:7029544

  15. Caffeine enhanced measurement of mutagenesis by low levels of [gamma]-irradiation in human lymphocytes

    SciTech Connect

    Puck, T.P.; Johnson, R.; Waldren, C.A. ); Morse, H. )

    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.

  16. Involvement of a joker mutation in a polymerase-independent lethal mutagenesis escape mechanism.

    PubMed

    Agudo, Rubén; de la Higuera, Ignacio; Arias, Armando; Grande-Pérez, Ana; Domingo, Esteban

    2016-07-01

    We previously characterized a foot-and-mouth disease virus (FMDV) with three amino acid replacements in its polymerase (3D) that conferred resistance to the mutagenic nucleoside analogue ribavirin. Here we show that passage of this mutant in the presence of high ribavirin concentrations resulted in selection of viruses with the additional replacement I248T in 2C. This 2C substitution alone (even in the absence of replacements in 3D) increased FMDV fitness mainly in the presence of ribavirin, prevented an incorporation bias in favor of A and U associated with ribavirin mutagenesis, and conferred the ATPase activity of 2C decreased sensitivity to ribavirin-triphosphate. Since in previous studies we described that 2C with I248T was selected under different selective pressures, this replacement qualifies as a joker substitution in FMDV evolution. The results have identified a role of 2C in nucleotide incorporation, and have unveiled a new polymerase-independent mechanism of virus escape to lethal mutagenesis. PMID:27136067

  17. A Multifunctional Mutagenesis System for Analysis of Gene Function in Zebrafish

    PubMed Central

    Quach, Helen Ngoc Bao; Tao, Shijie; Vrljicak, Pavle; Joshi, Adita; Ruan, Hua; Sukumaran, Rashmi; Varshney, Gaurav K.; LaFave, Matthew C.; Burgess, Shawn M.; Winkler, Christoph; Emelyanov, Alexander; Parinov, Sergey; Sampath, Karuna

    2015-01-01

    Since the sequencing of the human reference genome, many human disease-related genes have been discovered. However, understanding the functions of all the genes in the genome remains a challenge. The biological activities of these genes are usually investigated in model organisms such as mice and zebrafish. Large-scale mutagenesis screens to generate disruptive mutations are useful for identifying and understanding the activities of genes. Here, we report a multifunctional mutagenesis system in zebrafish using the maize Ds transposon. Integration of the Ds transposable element containing an mCherry reporter for protein trap events and an EGFP reporter for enhancer trap events produced a collection of transgenic lines marking distinct cell and tissue types, and mutagenized genes in the zebrafish genome by trapping and prematurely terminating endogenous protein coding sequences. We obtained 642 zebrafish lines with dynamic reporter gene expression. The characterized fish lines with specific expression patterns will be made available through the European Zebrafish Resource Center (EZRC), and a database of reporter expression is available online (http://fishtrap.warwick.ac.uk/). Our approach complements other efforts using zebrafish to facilitate functional genomic studies in this model of human development and disease. PMID:25840430

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

    PubMed

    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

  19. Transformation and transposon mutagenesis of Leifsonia xyli subsp. xyli, causal organism of ratoon stunting disease of sugarcane.

    PubMed

    Brumbley, Stevens M; Petrasovits, Lars A; Birch, Robert G; Taylor, Paul W J

    2002-03-01

    Conditions have been developed for genetic transformation and insertional mutagenesis in Leifsonia xyli subsp. xyli (Lxx), the causal organism of ratoon stunting disease (RSD), one of the most damaging and intractable diseases of sugarcane internationally. Transformation frequencies ranged from 1 to 10 colony forming units (CFU)/microg of plasmid DNA using Clavibacter/Escherichia coli shuttle vectors pCG188, pDM302, and pDM306 and ranged from 50 to 500 CFU/microg using cosmid cloning vectors pLAFR3 and pLAFR5-km. The transformation/transposition frequency was 0 to 70 CFU/microg of DNA, using suicide vectors pUCD623 and pSUP2021 containing transposable elements Tn4431 and Tn5, respectively. It was necessary to grow Lxx in media containing 0.1% glycine for electroporation and to amplify large plasmids in a dam-/dcm- E. coli strain and purify the DNA by anion exchange. To keep selection pressure at an optimum, the transformants were grown on nitrocellulose filters (0.2-microm pore size) on media containing the appropriate antibiotics. Transposon Tn4431 containing a promoterless lux operon from Vibrio fischeri and a tetracycline-resistance gene was introduced on the suicide vector pUCD623. All but 1% of the putative transposon mutants produce light, indicating transposition into functional Lxx genes. Southern blot analysis of these transformants indicates predominantly single transposon insertions at unique sites. The cosmid cloning vector pLAFR5-km was stably maintained in Lxx. The development of a transformation and transposon mutagenesis system opens the way for molecular analysis of pathogenicity determinants in Lxx. PMID:11952129

  20. Highly Efficient Targeted Mutagenesis in Mice Using TALENs

    PubMed Central

    Panda, Sudeepta Kumar; Wefers, Benedikt; Ortiz, Oskar; Floss, Thomas; Schmid, Bettina; Haass, Christian; Wurst, Wolfgang; Kühn, Ralf

    2013-01-01

    Targeted mouse mutants are instrumental for the analysis of gene function in health and disease. We recently provided proof-of-principle for the fast-track mutagenesis of the mouse genome, using transcription activator-like effector nucleases (TALENs) in one-cell embryos. Here we report a routine procedure for the efficient production of disease-related knockin and knockout mutants, using improved TALEN mRNAs that include a plasmid-coded poly(A) tail (TALEN-95A), circumventing the problematic in vitro polyadenylation step. To knock out the C9orf72 gene as a model of frontotemporal lobar degeneration, TALEN-95A mutagenesis induced sequence deletions in 41% of pups derived from microinjected embryos. Using TALENs together with mutagenic oligodeoxynucleotides, we introduced amyotrophic lateral sclerosis patient-derived missense mutations in the fused in sarcoma (Fus) gene at a rate of 6.8%. For the simple identification of TALEN-induced mutants and their progeny we validate high-resolution melt analysis (HRMA) of PCR products as a sensitive and universal genotyping tool. Furthermore, HRMA of off-target sites in mutant founder mice revealed no evidence for undesired TALEN-mediated processing of related genomic sequences. The combination of TALEN-95A mRNAs for enhanced mutagenesis and of HRMA for simplified genotyping enables the accelerated, routine production of new mouse models for the study of genetic disease mechanisms. PMID:23979585

  1. Targeted mutagenesis of Arabidopsis thaliana using engineered TAL effector nucleases.

    PubMed

    Christian, Michelle; Qi, Yiping; Zhang, Yong; Voytas, Daniel F

    2013-10-01

    Custom TAL effector nucleases (TALENs) are increasingly used as reagents to manipulate genomes in vivo. Here, we used TALENs to modify the genome of the model plant, Arabidopsis thaliana. We engineered seven TALENs targeting five Arabidopsis genes, namely ADH1, TT4, MAPKKK1, DSK2B, and NATA2. In pooled seedlings expressing the TALENs, we observed somatic mutagenesis frequencies ranging from 2-15% at the intended targets for all seven TALENs. Somatic mutagenesis frequencies as high as 41-73% were observed in individual transgenic plant lines expressing the TALENs. Additionally, a TALEN pair targeting a tandemly duplicated gene induced a 4.4-kb deletion in somatic cells. For the most active TALEN pairs, namely those targeting ADH1 and NATA2, we found that TALEN-induced mutations were transmitted to the next generation at frequencies of 1.5-12%. Our work demonstrates that TALENs are useful reagents for achieving targeted mutagenesis in this important plant model. PMID:23979944

  2. Mutational spectrum at GATA1 provides insights into mutagenesis and leukemogenesis in Down syndrome

    PubMed Central

    Cabelof, Diane C.; Patel, Hiral V.; Chen, Qing; van Remmen, Holly; Matherly, Larry H.; Ge, Yubin

    2009-01-01

    Down syndrome (DS) children have a unique genetic susceptibility to develop leukemia, in particular, acute megakaryocytic leukemia (AMkL) associated with somatic GATA1 mutations. The study of this genetic susceptibility with the use of DS as a model of leukemogenesis has broad applicability to the understanding of leukemia in children overall. On the basis of the role of GATA1 mutations in DS AMkL, we analyzed the mutational spectrum of GATA1 mutations to begin elucidating possible mechanisms by which these sequence alterations arise. Mutational analysis revealed a predominance of small insertion/deletion, duplication, and base substitution mutations, including G:C>T:A, G:C>A:T, and A:T>G:C. This mutational spectrum points to potential oxidative stress and aberrant folate metabolism secondary to genes on chromosome 21 (eg, cystathionine-β-synthase, superoxide dismutase) as potential causes of GATA1 mutations. Furthermore, DNA repair capacity evaluated in DS and non-DS patient samples provided evidence that the base excision repair pathway is compromised in DS tissues, suggesting that inability to repair DNA damage also may play a critical role in the unique susceptibility of DS children to develop leukemia. A model of leukemogenesis in DS is proposed in which mutagenesis is driven by cystathionine-β-synthase overexpression and altered folate homeostasis that becomes fixed as the ability to repair DNA damage is compromised. PMID:19633202

  3. Facilitating Structure-Function Studies of CFTR Modulator Sites with Efficiencies in Mutagenesis and Functional Screening.

    PubMed

    Molinski, Steven V; Ahmadi, Saumel; Hung, Maurita; Bear, Christine E

    2015-12-01

    There are nearly 2000 mutations in the CFTR gene associated with cystic fibrosis disease, and to date, the only approved drug, Kalydeco, has been effective in rescuing the functional expression of a small subset of these mutant proteins with defects in channel activation. However, there is currently an urgent need to assess other mutations for possible rescue by Kalydeco, and further, definition of the binding site of such modulators on CFTR would enhance our understanding of the mechanism of action of such therapeutics. Here, we describe a simple and rapid one-step PCR-based site-directed mutagenesis method to generate mutations in the CFTR gene. This method was used to generate CFTR mutants bearing deletions (p.Gln2_Trp846del, p.Ser700_Asp835del, p.Ile1234_Arg1239del) and truncation with polyhistidine tag insertion (p.Glu1172-3Gly-6-His*), which either recapitulate a disease phenotype or render tools for modulator binding site identification, with subsequent evaluation of drug responses using a high-throughput (384-well) membrane potential-sensitive fluorescence assay of CFTR channel activity within a 1 wk time frame. This proof-of-concept study shows that these methods enable rapid and quantitative comparison of multiple CFTR mutants to emerging drugs, facilitating future large-scale efforts to stratify mutants according to their "theratype" or most promising targeted therapy. PMID:26385858

  4. Marinomonas mediterranea MMB-1 Transposon Mutagenesis: Isolation of a Multipotent Polyphenol Oxidase Mutant

    PubMed Central

    Solano, Francisco; Lucas-Elío, Patricia; Fernández, Eva; Sanchez-Amat, Antonio

    2000-01-01

    Marinomonas mediterranea is a melanogenic marine bacterium expressing a multifunctional polyphenol oxidase (PPO) able to oxidize substrates characteristic for laccases and tyrosinases, as well as produce a classical tyrosinase. A new and quick method has been developed for screening laccase activity in culture plates to detect mutants differentially affected in this PPO activity. Transposon mutagenesis has been applied for the first time to M. mediterranea by using different minitransposons loaded in R6K-based suicide delivery vectors mobilizable by conjugation. Higher frequencies of insertions were obtained by using mini-Tn10 derivatives encoding kanamycin or gentamycin resistance. After applying this protocol, a multifunctional PPO-negative mutant was obtained. By using the antibiotic resistance cassette as a marker, flanking regions were cloned. Then the wild-type gene was amplified by PCR and was cloned and sequenced. This is the first report on cloning and sequencing of a gene encoding a prokaryotic enzyme with laccase activity. The deduced amino acid sequence shows the characteristic copper-binding sites of other blue copper proteins, including fungal laccases. In addition, it shows some extra copper-binding sites that might be related to its multipotent enzymatic capability. PMID:10850991

  5. Two distinct promoter elements in the human rRNA gene identified by linker scanning mutagenesis.

    PubMed Central

    Haltiner, M M; Smale, S T; Tjian, R

    1986-01-01

    A cell-free RNA polymerase I transcription system was used to evaluate the transcription efficiency of 21 linker scanning mutations that span the human rRNA gene promoter. Our analysis revealed the presence of two major control elements, designated the core and upstream elements, that affect the level of transcription initiation. The core element extends from -45 to +18 relative to the RNA start site, and transcription is severely affected (up to 100-fold) by linker scanning mutations in this region. Linker scanning and deletion mutations in the upstream element, located between nucleotides -156 and -107, cause a three- to fivefold reduction in transcription. Under certain reaction conditions, such as the presence of a high ratio of protein to template or supplementation of the reaction with partially purified protein fractions, sequences upstream of the core element can have an even greater effect (20- to 50-fold) on RNA polymerase I transcription. Primer extension analysis showed that RNA synthesized from all of these mutant templates is initiated at the correct in vivo start site. To examine the functional relationship between the core and the upstream region, mutant promoters were constructed that alter the orientation, distance, or multiplicity of these control elements relative to each other. The upstream control element appears to function in only one orientation, and its position relative to the core is constrained within a fairly narrow region. Moreover, multiple core elements in close proximity to each other have an inhibitory effect on transcription. Images PMID:3785147

  6. Transposon Mutagenesis of the Plant-Associated Bacillus amyloliquefaciens ssp. plantarum FZB42 Revealed That the nfrA and RBAM17410 Genes Are Involved in Plant-Microbe-Interactions

    PubMed Central

    Dietel, Kristin; Beator, Barbara; Dolgova, Olga; Fan, Ben; Bleiss, Wilfrid; Ziegler, Jörg; Schmid, Michael; Hartmann, Anton; Borriss, Rainer

    2014-01-01

    Bacillus amyloliquefaciens ssp. plantarum FZB42 represents the prototype of Gram-positive plant growth promoting and biocontrol bacteria. In this study, we applied transposon mutagenesis to generate a transposon library, which was screened for genes involved in multicellular behavior and biofilm formation on roots as a prerequisite of plant growth promoting activity. Transposon insertion sites were determined by rescue-cloning followed by DNA sequencing. As in B. subtilis, the global transcriptional regulator DegU was identified as an activator of genes necessary for swarming and biofilm formation, and the DegU-mutant of FZB42 was found impaired in efficient root colonization. Direct screening of 3,000 transposon insertion mutants for plant-growth-promotion revealed the gene products of nfrA and RBAM_017140 to be essential for beneficial effects exerted by FZB42 on plants. We analyzed the performance of GFP-labeled wild-type and transposon mutants in the colonization of lettuce roots using confocal laser scanning microscopy. While the wild-type strain heavily colonized root surfaces, the nfrA mutant did not colonize lettuce roots, although it was not impaired in growth in laboratory cultures, biofilm formation and swarming motility on agar plates. The RBAM17410 gene, occurring in only a few members of the B. subtilis species complex, was directly involved in plant growth promotion. None of the mutant strains were affected in producing the plant growth hormone auxin. We hypothesize that the nfrA gene product is essential for overcoming the stress caused by plant response towards bacterial root colonization. PMID:24847778

  7. Nozzle insert for mixed mode fuel injector

    DOEpatents

    Lawrence, Keith E.

    2006-11-21

    A fuel injector includes a homogenous charge nozzle outlet set and a conventional nozzle outlet set controlled respectively, by first and second needle valve members. The homogeneous charged nozzle outlet set is defined by a nozzle insert that is attached to an injector body, which defines the conventional nozzle outlet set. The nozzle insert is a one piece metallic component with a large diameter segment separated from a small diameter segment by an annular engagement surface. One of the needle valve members is guided on an outer surface of the nozzle insert, and the nozzle insert has an interference fit attachment to the injector body.

  8. Mars Observer Orbit Insertion Briefing

    NASA Technical Reports Server (NTRS)

    1993-01-01

    For the first part of this briefing, see NONP-NASA-VT-2000081556. Marvin Traxler continues his discussion on signal tracking from the Mars Observer. Julie Webster, Lead Engineer, Telecommunications Subsystem, is introduced. She explains how signals coming back from Mars are detected. Dr. Pasquale Esposito talks about flyby orbits and capture orbits. He says that frequencies coming from the spacecraft can determine if the spacecraft has flown by Mars, or if a capture orbit has occurred. Charles Whetsel, System Engineer Spacecraft Team, presents a computer program. He shows where the signal will appear on the computer from the Spacecraft. Suzanne Dodd presents orbit insertion geometry. Dr. Arden Albee, Project Scientist Mars Observer Project, Cal Tech tech, says that Mars is studied to get more data to confirm their hypotheses derived from previous Mars Missions such as the Viking Mars Program and the Mariner Program. Dr. Albee also describes instrumentation on the Mars Observer such as the Ultra Stable Oscillator, Mars Orbiter Laser Altimeter, and Magnetometer. The camera on the spacecraft is similar to a fax machine because it scans one line at a time as the spacecraft orbits Mars. Dr. Michael Malin, Principle Investigator Mars Observer Camera, Malin Space Science Systems, Inc., describe this process.

  9. Normal Activation of Discoidin Domain Receptor 1 Mutants with Disulfide Cross-links, Insertions, or Deletions in the Extracellular Juxtamembrane Region

    PubMed Central

    Xu, Huifang; Abe, Takemoto; Liu, Justin K. H.; Zalivina, Irina; Hohenester, Erhard; Leitinger, Birgit

    2014-01-01

    The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by collagen. DDR activation does not appear to occur by the common mechanism of ligand-induced receptor dimerization: the DDRs form stable noncovalent dimers in the absence of ligand, and ligand-induced autophosphorylation of cytoplasmic tyrosines is unusually slow and sustained. Here we sought to identify functionally important dimer contacts within the extracellular region of DDR1 by using cysteine-scanning mutagenesis. Cysteine substitutions close to the transmembrane domain resulted in receptors that formed covalent dimers with high efficiency, both in the absence and presence of collagen. Enforced covalent dimerization did not result in constitutive activation and did not affect the ability of collagen to induce receptor autophosphorylation. Cysteines farther away from the transmembrane domain were also cross-linked with high efficiency, but some of these mutants could no longer be activated. Furthermore, the extracellular juxtamembrane region of DDR1 tolerated large deletions as well as insertions of flexible segments, with no adverse effect on activation. These findings indicate that the extracellular juxtamembrane region of DDR1 is exceptionally flexible and does not constrain the basal or ligand-activated state of the receptor. DDR1 transmembrane signaling thus appears to occur without conformational coupling through the juxtamembrane region, but requires specific receptor interactions farther away from the cell membrane. A plausible mechanism to explain these findings is signaling by DDR1 clusters. PMID:24671415

  10. Loss of function of OsMADS3 via the insertion of a novel retrotransposon leads to recessive male sterility in rice (Oryza sativa).

    PubMed

    Zhang, Li; Mao, Donghai; Xing, Feng; Bai, Xufeng; Zhao, Hu; Yao, Wen; Li, Guangwei; Xie, Weibo; Xing, Yongzhong

    2015-09-01

    Natural mutation is the source of natural variation, which is the fundamental basis for the genetic improvement of crops. During the process of developing a recombinant inbred line (RI), a spontaneous mutagenesis in RI127 led to the production of the recessive male-sterile line RI127S. Via a map-based cloning approach, the gene controlling the male sterility was identified as OsMADS3, which was previously reported to be associated with floral organ development and male sterility. Thermal asymmetric interlaced PCR isolated one 1633-bp insertion in OsMADS3 in RI127S, which damaged its function due to failed transcription. The 1633-bp insertion was derived from a fragment flanked by retrotransposon genes on chromosome 5. Seven haplotypes of OsMADS3 were observed among 529 cultivars and 107 wild rice accessions, and 98% of the investigated genotypes carried the same H2 haplotype, indicating that OsMADS3 is highly conserved. RI127S has the combined genome constitution of its parents, indica rice Teqing and japonica 02428, and carries the widely compatible S5 gene donated by 02428. RI127 exhibits good performance in regard to its agronomic traits and has a wide compatibility. Therefore, RI127S would be an elite mediator for recurrent breeding in cases requiring a tedious hand-crossing-based inter-crossing phase. RI127S can be crossed not only with indica rice but also with japonica rice, thus providing breeders with flexible arrangements in recurrent breeding programs. PMID:26259187

  11. Turbine vane segment and impingement insert configuration for fail-safe impingement insert retention

    SciTech Connect

    Burdgick, Steven Sebastian; Kellock, Iain Robertson

    2003-05-13

    An impingement insert sleeve is provided that is adapted to be disposed in a coolant cavity defined through a stator vane. The insert has a generally open inlet end and first and second pairs of diametrically opposed side walls, and at least one fail-safe tab defined at a longitudinal end of the insert for limiting radial displacement of the insert with respect to the stator vane.

  12. Site-directed mutagenesis studies of acetylglutamate synthase delineate the site for the arginine inhibitor.

    PubMed

    Sancho-Vaello, Enea; Fernández-Murga, M Leonor; Rubio, Vicente

    2008-04-01

    N-acetyl-L-glutamate synthase (NAGS), the first enzyme of bacterial/plant arginine biosynthesis and an essential activator of the urea cycle in animals, is, respectively, arginine-inhibited and activated. Site-directed mutagenesis of recombinant Pseudomonas aeruginosa NAGS (PaNAGS) delineates the arginine site in the PaNAGS acetylglutamate kinase-like domain, and, by extension, in human NAGS. Key residues for glutamate binding are identified in the acetyltransferase domain. However, the acetylglutamate kinase-like domain may modulate glutamate binding, since one mutation affecting this domain increases the K(m) for glutamate. The effects on PaNAGS of two mutations found in human NAGS deficiency support the similarity of bacterial and human NAGSs despite their low sequence identity. PMID:18319063

  13. Mutagenesis as a Tool in Plant Genetics, Functional Genomics, and Breeding

    PubMed Central

    Sikora, Per; Chawade, Aakash; Larsson, Mikael; Olsson, Johanna; Olsson, Olof

    2011-01-01

    Plant mutagenesis is rapidly coming of age in the aftermath of recent developments in high-resolution molecular and biochemical techniques. By combining the high variation of mutagenised populations with novel screening methods, traits that are almost impossible to identify by conventional breeding are now being developed and characterised at the molecular level. This paper provides a comprehensive overview of the various techniques and workflows available to researchers today in the field of molecular breeding, and how these tools complement the ones already used in traditional breeding. Both genetic (Targeting Induced Local Lesions in Genomes; TILLING) and phenotypic screens are evaluated. Finally, different ways of bridging the gap between genotype and phenotype are discussed. PMID:22315587

  14. Features of MotA proton channel structure revealed by tryptophan-scanning mutagenesis.

    PubMed Central

    Sharp, L L; Zhou, J; Blair, D F

    1995-01-01

    The MotA protein of Escherichia coli is a component of the flagellar motors that functions in transmembrane proton conduction. Here, we report several features of MotA structure revealed by use of a mutagenesis-based approach. Single tryptophan residues were introduced at many positions within the four hydrophobic segments of MotA, and the effects on function were measured. Function was disrupted according to a periodic pattern that implies that the membrane-spanning segments are alpha-helices and that identifies the lipid-facing parts of each helix. The results support a hypothesis for MotA structure and mechanism in which water molecules form most of the proton-conducting pathway. The success of this approach in studying MotA suggests that it could be useful in structure-function studies of other integral membrane proteins. Images Fig. 1 PMID:7644518

  15. Random Transposon Mutagenesis for Cell-Envelope Resistant to Phage Infection.

    PubMed

    Reyes-Cortés, Ruth; Arguijo-Hernández, Emma S; Carballo-Ontiveros, Marco A; Martínez-Peñafiel, Eva; Kameyama, Luis

    2016-01-01

    In order to identify host components involved in the infective process of bacteriophages, we developed a wide-range strategy to obtain cell envelope mutants, using Escherichia coli W3110 and its specific phage mEp213. The strategy consisted in four steps: (1) random mutagenesis using transposon miniTn10Km(r); (2) selection of phage-resistant mutants by replica-plating; (3) electroporation of the phage-resistant mutants with mEp213 genome, followed by selection of those allowing phage development; and (4) sequencing of the transposon-disrupted genes. This strategy allowed us to distinguish the host factors related to phage development or multiplication within the cell, from those involved in phage infection at the level of the cell envelope. PMID:27311665

  16. Excavating the Genome: Large Scale Mutagenesis Screening for the Discovery of New Mouse Models

    PubMed Central

    Sundberg, John P.; Dadras, Soheil S.; Silva, Kathleen A.; Kennedy, Victoria E.; Murray, Stephen A.; Denegre, James; Schofield, Paul N.; King, Lloyd E.; Wiles, Michael; Pratt, C. Herbert

    2016-01-01

    Technology now exists for rapid screening of mutated laboratory mice to identify phenotypes associated with specific genetic mutations. Large repositories exist for spontaneous mutants and those induced by chemical mutagenesis, many of which have never been studied or comprehensively evaluated. To supplement these resources, a variety of techniques have been consolidated in an international effort to create mutations in all known protein coding genes in the mouse. With targeted embryonic stem cell lines now available for almost all protein coding genes and more recently CRISPR/Cas9 technology, large-scale efforts are underway to create novel mutant mouse strains and to characterize their phenotypes. However, accurate diagnosis of skin, hair, and nail diseases still relies on careful gross and histological analysis. While not automated to the level of the physiological phenotyping, histopathology provides the most direct and accurate diagnosis and correlation with human diseases. As a result of these efforts, many new mouse dermatological disease models are being developed. PMID:26551941

  17. Rapid fine conformational epitope mapping using comprehensive mutagenesis and deep sequencing.

    PubMed

    Kowalsky, Caitlin A; Faber, Matthew S; Nath, Aritro; Dann, Hailey E; Kelly, Vince W; Liu, Li; Shanker, Purva; Wagner, Ellen K; Maynard, Jennifer A; Chan, Christina; Whitehead, Timothy A

    2015-10-30

    Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions and show the method effectiveness by mapping the fine epitope for different antibodies targeting TNF, pertussis toxin, and the cancer target TROP2. In all three cases, the experimentally determined conformational epitope was consistent with previous experimental datasets, confirming the reliability of the experimental pipeline. Once the comprehensive library is generated, fine conformational epitope maps can be prepared at a rate of four per day. PMID:26296891

  18. Mechanism of alamethicin insertion into lipid bilayers.

    PubMed Central

    He, K; Ludtke, S J; Heller, W T; Huang, H W

    1996-01-01

    Alamethicin adsorbs on the membrane surface at low peptide concentrations. However, above a critical peptide-to-lipid ratio (P/L), a fraction of the peptide molecules insert in the membrane. This critical ratio is lipid dependent. For diphytanoyl phosphatidylcholine it is about 1/40. At even higher concentrations P/L > or = 1/15, all of the alamethicin inserts into the membrane and forms well-defined pores as detected by neutron in-plane scattering. A previous x-ray diffraction measurement showed that alamethicin adsorbed on the surface has the effect of thinning the bilayer in proportion to the peptide concentration. A theoretical study showed that the energy cost of membrane thinning can indeed lead to peptide insertion. This paper extends the previous studies to the high-concentration region P/L > 1/40. X-ray diffraction shows that the bilayer thickness increases with the peptide concentration for P/L > 1/23 as the insertion approaches 100%. The thickness change with the percentage of insertion is consistent with the assumption that the hydrocarbon region of the bilayer matches the hydrophobic region of the inserted peptide. The elastic energy of a lipid bilayer including both adsorption and insertion of peptide is discussed. The Gibbs free energy is calculated as a function of P/L and the percentage of insertion phi in a simplified one-dimensional model. The model exhibits an insertion phase transition in qualitative agreement with the data. We conclude that the membrane deformation energy is the major driving force for the alamethicin insertion transition. Images FIGURE 1 PMID:8913604

  19. Analysis of PBase Binding Profile Indicates an Insertion Target Selection Mechanism Dependent on TTAA, But Not Transcriptional Activity

    PubMed Central

    Yang, Dong; Liao, Ruiqi; Zheng, Yun; Sun, Ling; Xu, Tian

    2016-01-01

    Transposons and retroviruses are important pathogenic agents and tools for mutagenesis and transgenesis. Insertion target selection is a key feature for a given transposon or retrovirus. The piggyBac (PB) transposon is highly active in mice and human cells, which has a much better genome-wide distribution compared to the retrovirus and P-element. However, the underlying reason is not clear. Utilizing a tagged functional PB transposase (PBase), we were able to conduct genome-wide profiling for PBase binding sites in the mouse genome. We have shown that PBase binding mainly depends on the distribution of the tetranucleotide TTAA, which is not affected by the presence of PB DNA. Furthermore, PBase binding is negatively influenced by the methylation of CG sites in the genome. Analysis of a large collection of PB insertions in mice has revealed an insertion profile similar to the PBase binding profile. Interestingly, this profile is not correlated with transcriptional active genes in the genome or transcriptionally active regions within a transcriptional unit. This differs from what has been previously shown for P-element and retroviruses insertions. Our study provides an explanation for PB's genome-wide insertion distribution and also suggests that PB target selection relies on a new mechanism independent of active transcription and open chromatin structure. PMID:27570481

  20. Analysis of PBase Binding Profile Indicates an Insertion Target Selection Mechanism Dependent on TTAA, But Not Transcriptional Activity.

    PubMed

    Yang, Dong; Liao, Ruiqi; Zheng, Yun; Sun, Ling; Xu, Tian

    2016-01-01

    Transposons and retroviruses are important pathogenic agents and tools for mutagenesis and transgenesis. Insertion target selection is a key feature for a given transposon or retrovirus. The piggyBac (PB) transposon is highly active in mice and human cells, which has a much better genome-wide distribution compared to the retrovirus and P-element. However, the underlying reason is not clear. Utilizing a tagged functional PB transposase (PBase), we were able to conduct genome-wide profiling for PBase binding sites in the mouse genome. We have shown that PBase binding mainly depends on the distribution of the tetranucleotide TTAA, which is not affected by the presence of PB DNA. Furthermore, PBase binding is negatively influenced by the methylation of CG sites in the genome. Analysis of a large collection of PB insertions in mice has revealed an insertion profile similar to the PBase binding profile. Interestingly, this profile is not correlated with transcriptional active genes in the genome or transcriptionally active regions within a transcriptional unit. This differs from what has been previously shown for P-element and retroviruses insertions. Our study provides an explanation for PB's genome-wide insertion distribution and also suggests that PB target selection relies on a new mechanism independent of active transcription and open chromatin structure. PMID:27570481

  1. Central Solenoid Insert Technical Specification

    SciTech Connect

    Martovetsky, Nicolai N; Smirnov, Alexandre

    2011-09-01

    The US ITER Project Office (USIPO) is responsible for the ITER central solenoid (CS) contribution to the ITER project. The Central Solenoid Insert (CSI) project will allow ITER validation the appropriate lengths of the conductors to be used in the full-scale CS coils under relevant conditions. The ITER Program plans to build and test a CSI to verify the performance of the CS conductor. The CSI is a one-layer solenoid with an inner diameter of 1.48 m and a height of 4.45 m between electric terminal ends. The coil weight with the terminals is approximately 820 kg without insulation. The major goal of the CSI is to measure the temperature margin of the CS under the ITER direct current (DC) operating conditions, including determining sensitivity to load cycles. Performance of the joints, ramp rate sensitivity, and stability against thermal or electromagnetic disturbances, electrical insulation, losses, and instrumentation are addressed separately and therefore are not major goals in this project. However, losses and joint performance will be tested during the CSI testing campaign. The USIPO will build the CSI that will be tested at the Central Solenoid Model Coil (CSMC) Test Facility at the Japan Atomic Energy Agency (JAEA), Naka, Japan. The industrial vendors (the Suppliers) will report to the USIPO (the Company). All approvals to proceed will be issued by the Company, which in some cases, as specified in this document, will also require the approval of the ITER Organization. Responsibilities and obligations will be covered by respective contracts between the USIPO, called Company interchangeably, and the industrial Prime Contractors, called Suppliers. Different stages of work may be performed by more than one Prime Contractor, as described in this specification. Technical requirements of the contract between the Company and the Prime Contractor will be covered by the Fabrication Specifications developed by the Prime Contractor based on this document and approved by

  2. Mutagenesis of the borage Delta(6) fatty acid desaturase.

    PubMed

    Sayanova, O; Beaudoin, F; Libisch, B; Shewry, P; Napier, J

    2000-12-01

    The consensus sequence of the third histidine box of a range of Delta(5), Delta(6), Delta(8) and sphingolipid desaturases differs from that of the membrane-bound non-fusion Delta(12) and Delta(15) desaturases in the presence of glutamine instead of histidine. We have used site-directed mutagenesis to determine the importance of glutamine and other residues of the third histidine box and created a chimaeric enzyme to determine the ability of the Cyt b(5) fusion domain from the plant sphingolipid desaturase to substitute for the endogenous domain of the Delta(6) desaturase. PMID:11171152

  3. Pollen tetrads in the detection of environmental mutagenesis

    SciTech Connect

    Mulcahy, D.L.

    1981-01-01

    Although pollen is a very sensitive indicator of environmental mutagenesis, it is also sensitive to nonmutagenic environmental stress. By analyzing pollen tetrads, rather than individual pollen grains, it is possible to distinguish between mutagenic and nonmutagenic influences. Another advantage of using pollen tetrads in mutagenicity studies is that it is possible to discriminate between pre- and post-pachytene mutations. This eliminates the mutant sector problem of a single mutational event giving rise to a large number of mutant cells. Methods of analyzing pollen tetrads are described.

  4. Chemical mutagenesis: an emerging issue for public health.

    PubMed Central

    Claxton, L D; Barry, P Z

    1977-01-01

    Chemical mutagens are recognized as prevalent in the environment and a potential threat to the health of future generations. This paper presents an overview of chemical mutagenesis as an issue for public health. Several problems in the determination of risk to human populations are discussed, including difficulties of extrapolating scientific data to humans, the latency period between exposure and recognizable genetic damage, and the large number of chemicals which must be tested. Test systems are described. Possibilities of control through federal regulation are discussed. PMID:911015

  5. Mutagenesis in Newts: Protocol for Iberian Ribbed Newts.

    PubMed

    Hayashi, Toshinori; Takeuchi, Takashi

    2016-01-01

    Newts have the remarkable capability of organ/tissue regeneration, and have been used as a unique experimental model for regenerative biology. The Iberian ribbed newt (Pleurodeles waltl) is suitable as a model animal. We have established methods for artificial insemination and efficient transgenesis using P. waltl newts. In addition to the transgenic technique, development of TALENs enables targeting mutagenesis in the newts. We have reported that TALENs efficiently disrupted targeted genes in newt embryos. In this chapter, we introduce a protocol for TALEN-mediated gene targeting in Iberian ribbed newts. PMID:26443218

  6. Development and applications of an orbital insertion surface for the space shuttle orbiter/tug

    NASA Technical Reports Server (NTRS)

    Deaton, A. W.; Brandon, P. D.

    1973-01-01

    The concept of a space shuttle orbit/tug orbital insertion surface is developed. Practical applications in the area of ascent targeting, on-orbit rendezvous targeting, payloads/OMS propellant off-loading, and mission analysis are identified.

  7. Large-scale discovery of insertion hotspots and preferential integration sites of human transposed elements

    PubMed Central

    Levy, Asaf; Schwartz, Schraga; Ast, Gil

    2010-01-01

    Throughout evolution, eukaryotic genomes have been invaded by transposable elements (TEs). Little is known about the factors leading to genomic proliferation of TEs, their preferred integration sites and the molecular mechanisms underlying their insertion. We analyzed hundreds of thousands nested TEs in the human genome, i.e. insertions of TEs into existing ones. We first discovered that most TEs insert within specific ‘hotspots’ along the targeted TE. In particular, retrotransposed Alu elements contain a non-canonical single nucleotide hotspot for insertion of other Alu sequences. We next devised a method for identification of integration sequence motifs of inserted TEs that are conserved within the targeted TEs. This method revealed novel sequences motifs characterizing insertions of various important TE families: Alu, hAT, ERV1 and MaLR. Finally, we performed a global assessment to determine the extent to which young TEs tend to nest within older transposed elements and identified a 4-fold higher tendency of TEs to insert into existing TEs than to insert within non-TE intergenic regions. Our analysis demonstrates that TEs are highly biased to insert within certain TEs, in specific orientations and within specific targeted TE positions. TE nesting events also reveal new characteristics of the molecular mechanisms underlying transposition. PMID:20008508

  8. Force modeling for needle insertion into soft tissue.

    PubMed

    Okamura, Allison M; Simone, Christina; O'Leary, Mark D

    2004-10-01

    The modeling of forces during needle insertion into soft tissue is important for accurate surgical simulation, preoperative planning, and intelligent robotic assistance for percutaneous therapies. We present a force model for needle insertion and experimental procedures for acquiring data from ex vivo tissue to populate that model. Data were collected from bovine livers using a one-degree-of-freedom robot equipped with a load cell and needle attachment. computed tomography imaging was used to segment the needle insertion process into phases identifying different relative velocities between the needle and tissue. The data were measured and modeled in three parts: 1) capsule stiffness, a nonlinear spring model; 2) friction, a modified Karnopp model; and 3) cutting, a constant for a given tissue. In addition, we characterized the effects of needle diameter and tip type on insertion force using a silicone rubber phantom. In comparison to triangular and diamond tips, a bevel tip causes more needle bending and is more easily affected by tissue density variations. Forces for larger diameter needles are higher due to increased cutting and friction forces. PMID:15490818

  9. Contrast-free endoscopic stent insertion in malignant biliary obstruction

    PubMed Central

    De Palma, Giovanni D; Lombardi, Giovanni; Rega, Maria; Simeoli, Immacolata; Masone, Stefania; Siciliano, Saverio; Maione, Francesco; Salvatori, Francesca; Balzano, Antonio; Persico, Giovanni

    2007-01-01

    AIM: To present a case series of MRCP-guided endoscopic biliary stent placement, performed entirely without contrast injection. METHODS: Contrast-free endoscopic biliary drainage was attempted in 20 patients with malignant obstruction, unsuitable for resection on the basis of tumor extent or medical illness. MRCP images were used to confirm the diagnosis of tumor, to exclude other biliary diseases and to demonstrate the stenoses as well as dilation of proximal liver segments. The procedure was carried out under conscious sedation. Patients were placed in the left lateral decubitus position. The endoscope was inserted, the papilla identified and cannulated by a papillotome. A guide wire was inserted and guided deeply into the biliary tree, above the stenosis, by fluoroscopy. A papillotomy approximately 1 cm. long was performed and the papillotome was exchanged with a guiding-catheter. A 10 Fr, Amsterdam-type plastic stent, 7 to 15 cm long, was finally inserted over the guide wire/guiding catheter by a pusher tube system. RESULTS: Successful stent insertion was achieved in all patients. There were no major complications. Successful drainage, with substantial reduction in bilirubin levels, was achieved in all patients. CONCLUSION: This new method of contrast-free endoscopic stenting in malignant biliary obstruction is a safe and effective method of palliation. However, a larger, randomized study comparing this new approach with the standard procedure is needed to confirm the findings of the present study. PMID:17663512

  10. Discovery of unfixed endogenous retrovirus insertions in diverse human populations.

    PubMed

    Wildschutte, Julia Halo; Williams, Zachary H; Montesion, Meagan; Subramanian, Ravi P; Kidd, Jeffrey M; Coffin, John M

    2016-04-19

    Endogenous retroviruses (ERVs) have contributed to more than 8% of the human genome. The majority of these elements lack function due to accumulated mutations or internal recombination resulting in a solitary (solo) LTR, although members of one group of human ERVs (HERVs), HERV-K, were recently active with members that remain nearly intact, a subset of which is present as insertionally polymorphic loci that include approximately full-length (2-LTR) and solo-LTR alleles in addition to the unoccupied site. Several 2-LTR insertions have intact reading frames in some or all genes that are expressed as functional proteins. These properties reflect the activity of HERV-K and suggest the existence of additional unique loci within humans. We sought to determine the extent to which other polymorphic insertions are present in humans, using sequenced genomes from the 1000 Genomes Project and a subset of the Human Genome Diversity Project panel. We report analysis of a total of 36 nonreference polymorphic HERV-K proviruses, including 19 newly reported loci, with insertion frequencies ranging from <0.0005 to >0.75 that varied by population. Targeted screening of individual loci identified three new unfixed 2-LTR proviruses within our set, including an intact provirus present at Xq21.33 in some individuals, with the potential for retained infectivity. PMID:27001843

  11. Small bowel injury after suprapubic catheter insertion presenting 3 years after initial insertion

    PubMed Central

    Gallagher, Kevin M; Good, Daniel W; Brush, John P; Al-hasso, Ammar; Stewart, Grant D

    2013-01-01

    A 77-year-old woman was referred to urology with blockages of her suprapubic catheter (SPC). The catheter was replaced easily in the emergency department, however, no urine was draining, only a cloudy green fluid was visible. On cystoscopy bilious material was identified in the bladder. There was no catheter visible. There seemed to be a fistulous tract entering the bladder at the left dome. The urethra was dilated, a urethral catheter was placed and the SPC was removed. A CT demonstrated that the SPC tract transfixed a loop of pelvic small bowel and entered the bladder with no intraperitoneal contrast leak. The patient recovered well and did not require laparotomy. This case emphasises that bowel perforation, although rare, must be considered as a complication of SPC placement even years after initial insertion when catheter problems arise. Unusually, we learn that this complication may not present with abdominal pain or peritonism. PMID:24326435

  12. Mapping of cystic fibrosis transmembrane conductance regulator membrane topology by glycosylation site insertion.

    PubMed

    Chang, X B; Hou, Y X; Jensen, T J; Riordan, J R

    1994-07-15

    Technical difficulties in obtaining three-dimensional structures of intrinsic membrane proteins continues to limit understanding of their function. However, considerable insight can be gained from their two-dimensional topological arrangement in the lipid bilayer. Efficient molecular genetic approaches are available to discern the topology of prokaryotic but not of eukaryotic membrane proteins. The absolute asymmetry of the sidedness of their N-glycosylation was employed here to develop such a method using the cystic fibrosis transmembrane conductance regulator (CFTR). Insertion by in vitro mutagenesis of N-glycosylation consensus sequences (NXS/T) in predicted cytoplasmic and extracytoplasmic loops between hydrophobic sequences capable of traversing the membrane established the membrane topology of CFTR. This provides the first experimental evaluation of the original topological model of CFTR based solely on hydropathy algorithms and a method which may be generally applicable for the in vivo evaluation of the topology of other mammalian membrane proteins. PMID:7518437

  13. Elliptically polarizing adjustable phase insertion device

    DOEpatents

    Carr, Roger

    1995-01-01

    An insertion device for extracting polarized electromagnetic energy from a beam of particles is disclosed. The insertion device includes four linear arrays of magnets which are aligned with the particle beam. The magnetic field strength to which the particles are subjected is adjusted by altering the relative alignment of the arrays in a direction parallel to that of the particle beam. Both the energy and polarization of the extracted energy may be varied by moving the relevant arrays parallel to the beam direction. The present invention requires a substantially simpler and more economical superstructure than insertion devices in which the magnetic field strength is altered by changing the gap between arrays of magnets.

  14. Sequential cooling insert for turbine stator vane

    SciTech Connect

    Jones, Russel B; Krueger, Judson J; Plank, William L

    2014-11-04

    A sequential impingement cooling insert for a turbine stator vane that forms a double impingement for the pressure and suction sides of the vane or a triple impingement. The insert is formed from a sheet metal formed in a zigzag shape that forms a series of alternating impingement cooling channels with return air channels, where pressure side and suction side impingement cooling plates are secured over the zigzag shaped main piece. Another embodiment includes the insert formed from one or two blocks of material in which the impingement channels and return air channels are machined into each block.

  15. Sequential cooling insert for turbine stator vane

    SciTech Connect

    Jones, Russell B; Krueger, Judson J; Plank, William L

    2014-04-01

    A sequential impingement cooling insert for a turbine stator vane that forms a double impingement for the pressure and suction sides of the vane or a triple impingement. The insert is formed from a sheet metal formed in a zigzag shape that forms a series of alternating impingement cooling channels with return air channels, where pressure side and suction side impingement cooling plates are secured over the zigzag shaped main piece. Another embodiment includes the insert formed from one or two blocks of material in which the impingement channels and return air channels are machined into each block.

  16. Breeding of New Strains of Mushroom by Basidiospore Chemical Mutagenesis

    PubMed Central

    Lee, Jia; Kang, Hyeon-Woo; Kim, Sang-Woo; Lee, Chang-Yun

    2011-01-01

    Chemical mutagenesis of basidiospores of Hypsizygus marmoreus generated new mushroom strains. The basidospores were treated with methanesulfonate methylester, an alkylating agent, to yield 400 mutant monokaryotic mycelia. Twenty fast-growing mycelia were selected and mated each other by hyphal fusion. Fifty out of the 190 matings were successful (mating rate of 26.3%), judged by the formation of clamp connections. The mutant dikaryons were cultivated to investigate their morphological and cultivation characteristics. Mutant strains No. 3 and No. 5 showed 10% and 6% increase in fruiting body production, respectively. Eight mutant strains showed delayed and reduced primordia formation, resulting in the reduced production yield with prolonged cultivation period. The number of the fruiting bodies of mutant No. 31, which displayed reduced primordial formation, was only 15, compared to the parental number of 65. Another interesting phenotype was a fruiting body with a flattened stipe and pileus. Dikaryons generated by mating with the mutant spore No. 14 produced flat fruiting bodies. Further molecular biological studies will provide details of the mechanism. This work shows that the chemical mutagenesis approach is highly utilizable in the development of mushroom strains as well as in the generation of resources for molecular genetic studies. PMID:22783115

  17. Mechanisms of Base Substitution Mutagenesis in Cancer Genomes

    PubMed Central

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

    2014-01-01

    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. PMID:24705290

  18. Fitness Loss and Library Size Determination in Saturation Mutagenesis

    PubMed Central

    Nov, Yuval

    2013-01-01

    Saturation mutagenesis is a widely used directed evolution technique, in which a large number of protein variants, each having random amino acids in certain predetermined positions, are screened in order to discover high-fitness variants among them. Several metrics for determining the library size (the number of variants screened) have been suggested in the literature, but none of them incorporates the actual fitness of the variants discovered in the experiment. We present the results of an extensive simulation study, which is based on probabilistic models for protein fitness landscape, and which investigates how the result of a saturation mutagenesis experiment – the fitness of the best variant discovered – varies as a function of the library size. In particular, we study the loss of fitness in the experiment: the difference between the fitness of the best variant discovered, and the fitness of the best variant in variant space. Our results are that the existing criteria for determining the library size are conservative, so smaller libraries are often satisfactory. Reducing the library size can save labor, time, and expenses in the laboratory. PMID:23844158

  19. Minor displacements in the insertion site provoke major differences in the induction of antibody responses by chimeric parvovirus-like particles.

    PubMed

    Rueda, P; Hurtado, A; del Barrio, M; Martínez-Torrecuadrada, J L; Kamstrup, S; Leclerc, C; Casal, J I

    1999-10-10

    An antigen-delivery system based on hybrid virus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of canine parvovirus (CPV) and expressing foreign peptides was investigated. In this report, we have studied the effects of inserting the poliovirus C3:B epitope in the four loops and the C terminus of the CPV VP2 on the particle structure and immunogenicity. Epitope insertions in the four loops allowed the recovery of capsids in all of the mutants. However, only insertions of the C3:B epitope in VP2 residue 225 of the loop 2 were able to elicit a significant anti-peptide antibody response, but not poliovirus-neutralizing antibodies, probably because residue 225 is located in an small depression of the surface. To fine modulate the insertion site in loop 2, a cassette-mutagenesis was carried out to insert the epitope in adjacent positions 226, 227, and 228. The epitope C3:B inserted into these positions was well recognized by the specific monoclonal antibody C3 by immunoelectron microscopy. BALB/c mice immunized with these chimeric C3:B CPV:VLPs were able to elicit an strong neutralizing antibody response (>3 log(10) units) against poliovirus type 1 (Mahoney strain). Therefore, minor displacements in the insertion place cause dramatic changes in the accessibility of the epitope and the induction of antibody responses. PMID:10544085

  20. Mapping of neutralizing epitopes on Renibacterium salmoninarum p57 by use of transposon mutagenesis and synthetic peptides.

    PubMed

    Wiens, Gregory D; Owen, Jennifer

    2005-06-01

    Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish. PMID:15932983

  1. Mapping of Neutralizing Epitopes on Renibacterium salmoninarum p57 by Use of Transposon Mutagenesis and Synthetic Peptides

    PubMed Central

    Wiens, Gregory D.; Owen, Jennifer

    2005-01-01

    Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish. PMID:15932983

  2. Insertable fluid flow passage bridgepiece and method

    DOEpatents

    Jones, Daniel O.

    2000-01-01

    A fluid flow passage bridgepiece for insertion into an open-face fluid flow channel of a fluid flow plate is provided. The bridgepiece provides a sealed passage from a columnar fluid flow manifold to the flow channel, thereby preventing undesirable leakage into and out of the columnar fluid flow manifold. When deployed in the various fluid flow plates that are used in a Proton Exchange Membrane (PEM) fuel cell, bridgepieces of this invention prevent mixing of reactant gases, leakage of coolant or humidification water, and occlusion of the fluid flow channel by gasket material. The invention also provides a fluid flow plate assembly including an insertable bridgepiece, a fluid flow plate adapted for use with an insertable bridgepiece, and a method of manufacturing a fluid flow plate with an insertable fluid flow passage bridgepiece.

  3. Enhancing the efficiency and regioselectivity of P450 oxidation catalysts by unnatural amino acid mutagenesis.

    PubMed

    Kolev, Joshua N; Zaengle, Jacqueline M; Ravikumar, Rajesh; Fasan, Rudi

    2014-05-01

    The development of effective strategies for modulating the reactivity and selectivity of cytochrome P450 enzymes represents a key step toward expediting the use of these biocatalysts for synthetic applications. We have investigated the potential of unnatural amino acid mutagenesis to aid efforts in this direction. Four unnatural amino acids with diverse aromatic side chains were incorporated at 11 active-site positions of a substrate-promiscuous CYP102A1 variant. The resulting "uP450s" were then tested for their catalytic activity and regioselectivity in the oxidation of two representative substrates: a small-molecule drug and a natural product. Large shifts in regioselectivity resulted from these single mutations, and in particular, for para-acetyl-Phe substitutions at positions close to the heme cofactor. Screening this mini library of uP450s enabled us to identify P450 catalysts for the selective hydroxylation of four aliphatic positions in the target substrates, including a C(sp(3))-H site not oxidized by the parent enzyme. Furthermore, we discovered a general activity-enhancing effect of active-site substitutions involving the unnatural amino acid para-amino-Phe, which resulted in P450 catalysts capable of supporting the highest total turnover number reported to date on a complex molecule (34,650). The functional changes induced by the unnatural amino acids could not be reproduced by any of the 20 natural amino acids. This study thus demonstrates that unnatural amino acid mutagenesis constitutes a promising new strategy for improving the catalytic activity and regioselectivity of P450 oxidation catalysts. PMID:24692265

  4. Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis

    PubMed Central

    GORA, M; GARDAS, A; WIKTOROWICZ, W; HOBBY, P; WATSON, P F; WEETMAN, A P; SUTTON, B J; BANGA, J P

    2004-01-01

    Autoantibodies to thyroid peroxidase (TPO) recognize predominantly conformational epitopes, which are restricted to two distinct determinants, termed immunodominant domain region (IDR) A and B. These dominant determinants reside in the region with structural homology to myeloperoxidase (MPO)-like domain and may extend into the adjacent complement control protein (CCP) domain. We have explored the location of these determinants on the MPO-like domain of the structural model of TPO, by identifying exposed hydrophilic loops that are potential candidates for the autoantigenic sites, generating rabbit antipeptide antisera, and competing with well characterized murine monoclonal antibodies (mabs) specific for these two IDRs. We recently defined the location of IDR-B, and here report our findings on the location of IDR-A and its relationship to IDR-B, defined with a new panel of 15 antipeptide antisera. Moreover, in combination with single amino acid replacements by in vitro mutagenesis, we have defined the limits of the IDR-B region on the TPO model. The combination of antisera to peptides P12 (aa 549–563), P14 (aa 599–617) and P18 (aa 210–225) inhibited the binding of the mab specific for IDR-A (mab 2) by 75. The same combination inhibited the binding of autoantibodies to native TPO from 67 to 94% (mean 81·5%) at autoantibody levels of 5 IU. Fabs prepared from the antipeptide IgG and pooled in this combination were also effective in competition assays, thus defining the epitopes more precisely. IDR-A was found to lie immediately adjacent to IDR-B and thus the two immunodominant epitopes form an extended patch on the surface of TPO. Finally, by single amino acid mutagenesis, we show that IDR-B extends to residue N642, thus further localizing the boundary of this autoantigenic region on the structural model. PMID:15030525

  5. Efficient instruction sequencing with Inline Target Insertion

    NASA Technical Reports Server (NTRS)

    Hwu, Wen-Mei W.; Chang, Pohua P.

    1992-01-01

    Inline target insertion, a specific compiler and pipeline implementation method for delayed branches with squashing, is defined. The method is shown to offer two important features not discovered in previous studies. First, branches inserted into branch slots are correctly executed. Second, the execution returns correctly from interrupts or exceptions with only one program counter. These two features result in better performance and less software/hardware complexity than conventional delayed branching mechanisms.

  6. Sleeping Beauty mutagenesis reveals cooperating mutations and pathways in pancreatic adenocarcinoma

    PubMed Central

    Mann, Karen M.; Ward, Jerrold M.; Yew, Christopher Chin Kuan; Kovochich, Anne; Dawson, David W.; Black, Michael A.; Brett, Benjamin T.; Sheetz, Todd E.; Dupuy, Adam J.; Chang, David K.; Biankin, Andrew V.; Waddell, Nicola; Kassahn, Karin S.; Grimmond, Sean M.; Rust, Alistair G.; Adams, David J.; Jenkins, Nancy A.; Copeland, Neal G.

    2012-01-01

    Pancreatic cancer is one of the most deadly cancers affecting the Western world. Because the disease is highly metastatic and difficult to diagnosis until late stages, the 5-y survival rate is around 5%. The identification of molecular cancer drivers is critical for furthering our understanding of the disease and development of improved diagnostic tools and therapeutics. We have conducted a mutagenic screen using Sleeping Beauty (SB) in mice to identify new candidate cancer genes in pancreatic cancer. By combining SB with an oncogenic Kras allele, we observed highly metastatic pancreatic adenocarcinomas. Using two independent statistical methods to identify loci commonly mutated by SB in these tumors, we identified 681 loci that comprise 543 candidate cancer genes (CCGs); 75 of these CCGs, including Mll3 and Ptk2, have known mutations in human pancreatic cancer. We identified point mutations in human pancreatic patient samples for another 11 CCGs, including Acvr2a and Map2k4. Importantly, 10% of the CCGs are involved in chromatin remodeling, including Arid4b, Kdm6a, and Nsd3, and all SB tumors have at least one mutated gene involved in this process; 20 CCGs, including Ctnnd1, Fbxo11, and Vgll4, are also significantly associated with poor patient survival. SB mutagenesis provides a rich resource of mutations in potential cancer drivers for cross-comparative analyses with ongoing sequencing efforts in human pancreatic adenocarcinoma. PMID:22421440

  7. Sleeping Beauty mutagenesis reveals cooperating mutations and pathways in pancreatic adenocarcinoma.

    PubMed

    Mann, Karen M; Ward, Jerrold M; Yew, Christopher Chin Kuan; Kovochich, Anne; Dawson, David W; Black, Michael A; Brett, Benjamin T; Sheetz, Todd E; Dupuy, Adam J; Chang, David K; Biankin, Andrew V; Waddell, Nicola; Kassahn, Karin S; Grimmond, Sean M; Rust, Alistair G; Adams, David J; Jenkins, Nancy A; Copeland, Neal G

    2012-04-17

    Pancreatic cancer is one of the most deadly cancers affecting the Western world. Because the disease is highly metastatic and difficult to diagnosis until late stages, the 5-y survival rate is around 5%. The identification of molecular cancer drivers is critical for furthering our understanding of the disease and development of improved diagnostic tools and therapeutics. We have conducted a mutagenic screen using Sleeping Beauty (SB) in mice to identify new candidate cancer genes in pancreatic cancer. By combining SB with an oncogenic Kras allele, we observed highly metastatic pancreatic adenocarcinomas. Using two independent statistical methods to identify loci commonly mutated by SB in these tumors, we identified 681 loci that comprise 543 candidate cancer genes (CCGs); 75 of these CCGs, including Mll3 and Ptk2, have known mutations in human pancreatic cancer. We identified point mutations in human pancreatic patient samples for another 11 CCGs, including Acvr2a and Map2k4. Importantly, 10% of the CCGs are involved in chromatin remodeling, including Arid4b, Kdm6a, and Nsd3, and all SB tumors have at least one mutated gene involved in this process; 20 CCGs, including Ctnnd1, Fbxo11, and Vgll4, are also significantly associated with poor patient survival. SB mutagenesis provides a rich resource of mutations in potential cancer drivers for cross-comparative analyses with ongoing sequencing efforts in human pancreatic adenocarcinoma. PMID:22421440

  8. Establishment of a counter-selectable markerless mutagenesis system in Veillonella atypica.

    PubMed

    Zhou, Peng; Li, Xiaoli; Qi, Fengxia

    2015-05-01

    Using an alternative sigma factor ecf3 as target, we successfully established the first markerless mutagenesis system in the Veillonella genus. This system will be a valuable tool for mutagenesis of multiple genes for gene function analysis as well as for gene regulation studies in Veillonella. PMID:25771833

  9. Earth boring tool with improved inserts

    SciTech Connect

    Dill, H.; Scales, S.

    1980-07-08

    A description is given of an improved earth boring tool of the type having at least one sintered tungsten carbide insert with a substantially cylindrical retaining surface interferringly secured to the wall of a retaining hole, the improvement comprising said retaining surface roughened such that in any lineal increment of at least 010 inch, there are at least three asperities at least 0.001 in ch long and at least 0.00005 inch deep when measured with a stylus point with a 0.00005 inch radius. An improved method of retaining tungsten carbide inserts in an earth boring tool, said method comprising the steps of: drilling and reaming a plurality of holes in selected locations on the earth boring tool; sintering a plurality of tungsten carbide inserts with a binder selected from the group consisting of cobalt, nickel or iron; grinding a substantially cylindrical wall surface on the inserts that are nominally larger in diameter than the drilled and reamed holes; roughening the wall surface of the inserts such that any lineal increment of at least 0.010 inch has at least three asperities which are at least 0.001 inch long and at least 00005 inch deep when measured with a stylus point with a 0.00005 inch radius; and forcing the inserts interferringly into the mating drilled and reamed holes.

  10. The Basques according to polymorphic Alu insertions.

    PubMed

    de Pancorbo, M M; López-Martínez, M; Martínez-Bouzas, C; Castro, A; Fernández-Fernández, I; de Mayolo, G A; de Mayolo, A A; de Mayolo, P A; Rowold, D J; Herrera, R J

    2001-08-01

    Polymorphic Alu insertions provide a set of DNA markers of interest in human population genetics. Approximately 1000-2000 of these insertions have not reached fixation within the human genome. Each one of these polymorphic loci most probably resulted from a unique insertional event, and therefore all individuals possessing the insertion are related by descent not just state. In addition, the direction of mutational change is toward the gain of the Alu element at a particular locus. Therefore, the improved knowledge of both the ancestral state and the direction of mutational change greatly facilitates the analysis of population relationships. As a result, Alu insertion polymorphisms represent a significant tool for population genetic studies. In this study, polymorphic Alu insertions have been employed to ascertain phylogenetic relationships among Basque groups and worldwide populations. The Basques are considered to be a geographic isolate with a unique language and customs. They may be direct descendants of Cro-Magnon enclaves from the upper Paleolithic (38,000 to 10,000 years). The Basques are distributed among narrow valleys in northeastern Spain with little migration between them until recently. This characteristic may have had an effect on allelic frequency distributions. With the aim of studying this possible effect, we have analyzed six autosomal polymorphic Alu loci from four different sites within the Spanish Basque region in order to ascertain any genetic heterogeneity among the Basques. The results are consistent with a lack of homogeneity among these four autochthonous Basque groups. PMID:11511929

  11. Rectal suppository: commonsense and mode of insertion.

    PubMed

    Abd-el-Maeboud, K H; el-Naggar, T; el-Hawi, E M; Mahmoud, S A; Abd-el-Hay, S

    1991-09-28

    Rectal suppository is a well-known form of medication and its use is increasing. The commonest shape is one with an apex (pointed end) tapering to a base (blunt end). Because of a general lack of information about mode of insertion, we asked 360 lay subjects (Egyptians and non-Egyptians) and 260 medical personnel (physicians, pharmacists, and nurses) by questionnaire which end they inserted foremost. Apart from 2 individuals, all subjects suggested insertion with the apex foremost. Commonsense was the most frequent basis for this practice (86.9% of lay subjects and 84.6% of medical personnel) followed by information from a relative, a friend, or medical personnel, or from study at medical school. Suppository insertion with the base or apex foremost was compared in 100 subjects (60 adults, 40 infants and children). Retention with the former method was more easily achieved in 98% of the cases, with no need to introduce a finger in the anal canal (1% vs 83%), and lower expulsion rate (0% vs 3%). The designer of the "torpedo-shaped" suppository suggested its insertion with apex foremost. Our data suggest that a suppository is better inserted with the base foremost. Reversed vermicular contractions or pressure gradient of the anal canal might press it inwards. PMID:1681170

  12. Strength of inserts in titanium alloy machining

    NASA Astrophysics Data System (ADS)

    Kozlov, V.; Huang, Z.; Zhang, J.

    2016-04-01

    In this paper, a stressed state of a non-worn cutting wedge in a machined titanium alloy (Ti6Al2Mo2Cr) is analyzed. The distribution of contact loads on the face of a cutting tool was obtained experimentally with the use of a ‘split cutting tool’. Calculation of internal stresses in the indexable insert made from cemented carbide (WC8Co) was carried out with the help of ANSYS 14.0 software. Investigations showed that a small thickness of the cutting insert leads to extremely high compressive stresses near the cutting edge, stresses that exceed the ultimate compressive strength of cemented carbide. The face and the base of the insert experience high tensile stresses, which approach the ultimate tensile strength of cemented carbide and increase a probability of cutting insert destruction. If the thickness of the cutting insert is bigger than 5 mm, compressive stresses near the cutting edge decrease, and tensile stresses on the face and base decrease to zero. The dependences of the greatest normal and tangential stresses on thickness of the cutting insert were found. Abbreviation and symbols: m/s - meter per second (cutting speed v); mm/r - millimeter per revolution (feed rate f); MPa - mega Pascal (dimension of specific contact loads and stresses); γ - rake angle of the cutting tool [°] α - clearance angle of the sharp cutting tool [°].

  13. Construction and Characterization of a recA Mutant of Thiobacillus ferrooxidans by Marker Exchange Mutagenesis

    PubMed Central

    Liu, Zhenying; Guiliani, Nicolas; Appia-Ayme, Corinne; Borne, Françoise; Ratouchniak, Jeanine; Bonnefoy, Violaine

    2000-01-01

    To construct Thiobacillus ferrooxidans mutants by marker exchange mutagenesis, a genetic transfer system is required. The transfer of broad-host-range plasmids belonging to the incompatibility groups IncQ (pKT240 and pJRD215), IncP (pJB3Km1), and IncW (pUFR034) from Escherichia coli to two private T. ferrooxidans strains (BRGM1 and Tf-49) and to two collection strains (ATCC 33020 and ATCC 19859) by conjugation was analyzed. To knock out the T. ferrooxidans recA gene, a mobilizable suicide plasmid carrying the ATCC 33020 recA gene disrupted by a kanamycin resistance gene was transferred from E. coli to T. ferrooxidans ATCC 33020 by conjugation under the best conditions determined. The two kanamycin-resistant clones, which have retained the kanamycin-resistant phenotype after growth for several generations in nonselective medium, were shown to have the kanamycin resistance gene inserted within the recA gene, indicating that the recA::Ω-Km mutated allele was transferred from the suicide plasmid to the chromosome by homologous recombination. These mutants exhibited a slightly reduced growth rate and an increased sensitivity to UV and γ irradiation compared to the wild-type strain. However, the T. ferrooxidans recA mutants are less sensitive to these physical DNA-damaging agents than the recA mutants described in other bacterial species, suggesting that RecA plays a minor role in DNA repair in T. ferrooxidans. PMID:10735871

  14. Improved Somatic Mutagenesis in Zebrafish Using Transcription Activator-Like Effector Nucleases (TALENs)

    PubMed Central

    Moore, Finola E.; Reyon, Deepak; Sander, Jeffry D.; Martinez, Sarah A.; Blackburn, Jessica S.; Khayter, Cyd; Ramirez, Cherie L.; Joung, J. Keith; Langenau, David M.

    2012-01-01

    Zinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%–76.8% compared to 1.1%–3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish. PMID:22655075

  15. A molecular characterization of spontaneous frameshift mutagenesis within the trpA gene of Escherichia coli

    PubMed Central

    Hardin, Aaron; Villalta, Christopher F.; Doan, Michael; Jabri, Mouna; Chockalingham, Valliammal; White, Steven J.; Fowler, Robert G.

    2007-01-01

    Spontaneous frameshift mutations are an important source of genetic variation in all species and cause a large number of genetic disorders in humans. To enhance our understanding of the molecular mechanisms of frameshift mutagenesis, 583 spontaneous Trp+ revertants of two trpA frameshift alleles in Escherichia coli were isolated and DNA sequenced. In order to measure the contribution of methyl-directed mismatch repair to frameshift production, mutational spectra were constructed for both mismatch repair-proficient and repair-defective strains. The molecular origins of practically all of the frameshifts analyzed could be explained by one of six simple models based upon misalignment of the template or nascent DNA strands with or without misincoroporation of primer nucleotides during DNA replication. Most frameshifts occurred within mononucleotide runs as has been shown often in previous studies but the location of the 76 frameshift sites was usually outside of runs. Mismatch repair generally was most effective in preventing the occurrence of frameshifts within runs but there was much variation from site to site. Most frameshift sites outside of runs appear to be refractory to mismatch repair although the small number of occurrences at most of these sites make firm conclusions impossible. There was a dense pattern of reversion sites within the trpA DNA region where reversion events could occur, suggesting that, in general, most DNA sequences are capable of undergoing spontaneous mutational events during replication that can lead to small deletions and insertions. Many of these errors are likely to occur at low frequencies and be tolerated as events too costly to prevent or repair. These studies also revealed an unpredicted flexibility in the primary amino acid sequence of the trpA product, the α subunit of tryptophan synthase. PMID:17084112

  16. Mutagenesis of Bordetella pertussis with transposon Tn5tac1: conditional expression of virulence-associated genes.

    PubMed Central

    Cookson, B T; Berg, D E; Goldman, W E

    1990-01-01

    The Tn5tac1 transposon contains a strong outward-facing promoter, Ptac, a lacI repressor gene, and a selectable Kanr gene. Transcription from Ptac is repressed by the lacI protein unless an inducer (isopropyl-beta-D-thiogalactopyranoside [IPTG]) is present. Thus, Tn5tac1 generates insertion mutations in Escherichia coli with conditional phenotypes because it is polar on distal gene expression when IPTG is absent and directs transcription of these genes when the inducer is present. To test the usefulness of Tn5tac1 in Bordetella pertussis, a nonenteric gram-negative bacterial pathogen, we chose the bifunctional adenylate cyclase-hemolysin determinant as an easily scored marker to monitor insertional mutagenesis. Tn5tac1 delivered to B. pertussis on conjugal suicide plasmids resulted in Kanr exconjugants at a frequency of 10(-3) per donor cell, and nonhemolytic (Hly-) mutants were found among the Kanr colonies at a frequency of about 1%. Of eight independent Kanr Hly- mutants, two were conditional and exhibited an Hly+ phenotype only in the presence of IPTG. Using a new quantitative assay for adenylate cyclase based on high-pressure liquid chromatography, we found that enzymatic activity in these two strains was specifically induced at least 500-fold in a dose-dependent fashion over the range of 0 to 125 microM IPTG. These data show that Ptac serves as a promoter, lacI is expressed and is functional, and IPTG can induce Ptac transcription in B. pertussis. Adenylate cyclase expression in whole cells, culture supernatants, and cell extracts from these strains depended upon IPTG, suggesting that the insertions do not merely alter secretion of adenylate cyclase-hemolysin. Other virulence determinants under control of the vir locus are expressed normally, implying that these Tn5tac1 insertions specifically regulate adenylate cyclase-hemolysin expression. We conclude that Tn5tac1 insertion mutations permit sensitive, exogenous control over the expression of genes of

  17. The Ziegler—Natta olefin insertion reaction for cationic metals

    NASA Astrophysics Data System (ADS)

    Jensen, Vidar R.; Siegban, Per E. M.

    1993-09-01

    The catalytic Ziegler—Natta polymerization reaction has been studied for a set of metal cations, in order to identify the role of the positive charge on this process. Geometry optimizations have been performed for the reactant metal—methyl systems, the π-coordinated olefin systems, the transition states for the olefin insertion and finally for the product metal—propyl systems. All valence electrons are correlated. The cations selected for this study are the transition metals Zr + and Ti +, the non-transition metals Be +, Mg +, Al + and finally also Si +. The transition metal cations are found to have very low barriers for the insertion, but the lowest barrier is actually found for Be +. The results are discussed in terms of the ionization energies and the accessibility to valence p and d orbitals. Comparisons are made to previous theoretical work on cationic model systems.

  18. When can a woman have an intrauterine device inserted? A systematic review☆

    PubMed Central

    Whiteman, Maura K.; Tyler, Crystal P.; Folger, Suzanne G.; Gaffield, Mary E.; Curtis, Kathryn M.

    2015-01-01

    Background Intrauterine device (IUD) insertion during menses may be viewed as preferable by some providers, as it provides reassurance that the woman is not pregnant. However, this practice may result in unnecessary inconvenience and cost to women. The objective of this systematic review is to evaluate the evidence for the effect of inserting IUDs on different days of the menstrual cycle on contraceptive continuation, effectiveness and safety. Study Design We searched the MEDLINE database for peer-reviewed articles published in any language from database inception through March 2012 concerning the effect of inserting copper IUDs (Cu-IUD) or levonorgestrel-releasing IUDs (LNG-IUDs) on different days of the menstrual cycle on contraceptive continuation, effectiveness, and safety. The quality of each individual piece of evidence was assessed using the United States Preventive Services Task Force grading system. Results We identified eight articles that met the criteria for review. Each study examined the Cu-IUD; no studies were identified that examined the LNG-IUD. Overall, these studies suggest that timing of Cu-IUD insertion has little effect on longer term outcomes (rates of continuation, removal, expulsion, or pregnancy) or on shorter term outcomes (pain at insertion, bleeding at insertion, immediate expulsion). Specifically, there was no evidence to suggest that outcomes were better when Cu-IUD insertions were performed during menses. Limitations of the studies include small sample sizes for insertions performed during later days of the menstrual cycle and non-randomized assignment to timing of insertion. Conclusions There is fair evidence (body of evidence grading: II-2, fair) indicating that timing of Cu-IUD insertion has little effect on contraceptive continuation, effectiveness or safety. PMID:22995537

  19. Mutagenesis and differentiation induction in mammalian cells by environmental chemicals

    SciTech Connect

    Friedman, J.; Huberman, E.

    1980-01-01

    These studies indicate that in agreement with the somatic mutation hypothesis, chemical carcinogens: (1) are mutagenic for mammalian cells as tested in the cell-mediated assay; (2) the degree of mutagenicity is correlated with their degree of carcinogenicity; (3) that at least in cases when analyzed carefully the metabolites responsible for mutagenesis are also responsible for initiating the carcinogenic event; and (4) that a cell organ type specificity can be established using the cell-mediated assay. Studies with HL-60 cells and HO melanoma cells and those of others suggest that tumor-promoting phorbol diesters can alter cell differentiation in various cell types and that the degree of the observed alteration in the differentiation properties may be related to the potency of the phorbol esters. Thus these and similar systems may serve as models for both studies and identification of certain types of tumor promoting agents. (ERB)

  20. CRISPR-Cas9 enables conditional mutagenesis of challenging loci

    PubMed Central

    Schick, Joel A.; Seisenberger, Claudia; Beig, Joachim; Bürger, Antje; Iyer, Vivek; Maier, Viola; Perera, Sajith; Rosen, Barry; Skarnes, William C.; Wurst, Wolfgang

    2016-01-01

    The International Knockout Mouse Consortium (IKMC) has produced a genome-wide collection of 15,000 isogenic targeting vectors for conditional mutagenesis in C57BL/6N mice. Although most of the vectors have been used successfully in murine embryonic stem (ES) cells, there remain a set of nearly two thousand genes that have failed to target even after several attempts. Recent attention has turned to the use of new genome editing technology for the generation of mutant alleles in mice. Here, we demonstrate how Cas9-assisted targeting can be combined with the IKMC targeting vector resource to generate conditional alleles in genes that have previously eluded targeting using conventional methods. PMID:27580957

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

    PubMed

    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. PMID:15282373

  2. Radiosensitivity Parameters For Lethal Mutagenesis In Caenorhabditis Elegans

    SciTech Connect

    Cucinotta, F.A.; Wilson, J.W.; Katz, R.

    1994-01-01

    For the first time track structure theory has been applied to radiobiological effects in a living organism. Data for lethal mutagenesis in Caenorhabditis elegans, obtained after irradiation with nine different types of ions of atomic number 1-57 and gamma rays have yielded radiosensitivity parameters (E{sub 0}, sigma{sub 0}, Kappa, m = 68 Gy, 2.5 x 10(exp {minus}9) cm (exp 2), 750, 2) comparable with those found for the transformation of C3HT10 1/2 cells (180 Gy, 1.15 x 10(exp {minus}10) cm(exp 2), 750, 2) but remote from those (E{sub 0} and sigma{sub 0} = approx. 2 Gy, approx. 5 x 10(exp {minus}7) cm(exp 2)) for mammalian cell survival.

  3. CRISPR-Cas9 enables conditional mutagenesis of challenging loci.

    PubMed

    Schick, Joel A; Seisenberger, Claudia; Beig, Joachim; Bürger, Antje; Iyer, Vivek; Maier, Viola; Perera, Sajith; Rosen, Barry; Skarnes, William C; Wurst, Wolfgang

    2016-01-01

    The International Knockout Mouse Consortium (IKMC) has produced a genome-wide collection of 15,000 isogenic targeting vectors for conditional mutagenesis in C57BL/6N mice. Although most of the vectors have been used successfully in murine embryonic stem (ES) cells, there remain a set of nearly two thousand genes that have failed to target even after several attempts. Recent attention has turned to the use of new genome editing technology for the generation of mutant alleles in mice. Here, we demonstrate how Cas9-assisted targeting can be combined with the IKMC targeting vector resource to generate conditional alleles in genes that have previously eluded targeting using conventional methods. PMID:27580957

  4. Environmental mutagenesis during the end-Permian ecological crisis

    PubMed Central

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

    2004-01-01

    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. PMID:15282373

  5. Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria

    PubMed Central

    Rodríguez-Rojas, Alexandro; Makarova, Olga; Müller, Uta; Rolff, Jens

    2015-01-01

    Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection. PMID:26430769

  6. Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria.

    PubMed

    Rodríguez-Rojas, Alexandro; Makarova, Olga; Müller, Uta; Rolff, Jens

    2015-10-01

    Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection. PMID:26430769

  7. Precision Targeted Mutagenesis via Cas9 Paired Nickases in Rice

    PubMed Central

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2016-01-01

    Recent reports of CRISPR- (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) mediated heritable mutagenesis in plants highlight the need for accuracy of the mutagenesis directed by this system. Off-target mutations are an important issue when considering functional gene analysis, as well as the molecular breeding of crop plants with large genome size, i.e. with many duplicated genes, and where the whole-genome sequence is still lacking. In mammals, off-target mutations can be suppressed by using Cas9 paired nickases together with paired guide RNAs (gRNAs). However, the performance of Cas9 paired nickases has not yet been fully assessed in plants. Here, we analyzed on- and off-target mutation frequency in rice calli and regenerated plants using Cas9 nuclease or Cas9 nickase with paired gRNAs. When Cas9 paired nickases were used, off-target mutations were fully suppressed in rice calli and regenerated plants. However, on-target mutation frequency also decreased compared with that induced by the Cas9 paired nucleases system. Since the gRNA sequence determines specific binding of Cas9 protein–gRNA ribonucleoproteins at the targeted sequence, the on-target mutation frequency of Cas9 paired nickases depends on the design of paired gRNAs. Our results suggest that a combination of gRNAs that can induce mutations at high efficiency with Cas9 nuclease should be used together with Cas9 nickase. Furthermore, we confirmed that a combination of gRNAs containing a one nucleotide (1 nt) mismatch toward the target sequence could not induce mutations when expressed with Cas9 nickase. Our results clearly show the effectiveness of Cas9 paired nickases in delivering on-target specific mutations. PMID:26936792

  8. Steady-state transposon mutagenesis in inbred maize.

    PubMed

    McCarty, Donald R; Settles, Andrew Mark; Suzuki, Masaharu; Tan, Bao Cai; Latshaw, Susan; Porch, Tim; Robin, Kevin; Baier, John; Avigne, Wayne; Lai, Jinsheng; Messing, Joachim; Koch, Karen E; Hannah, L Curtis

    2005-10-01

    We implement a novel strategy for harnessing the power of high-copy transposons for functional analysis of the maize genome, and report behavioral features of the Mutator system in a uniform inbred background. The unique UniformMu population and database facilitate high-throughput molecular analysis of Mu-tagged mutants and gene knockouts. Key features of the population include: (i) high mutation frequencies (7% independent seed mutations) and moderation of copy number (approximately 57 total Mu elements; 1-2 MuDR copies per plant) were maintained by continuous back-crossing into a phenotypically uniform inbred background; (ii) a bz1-mum9 marker enabled selection of stable lines (loss of MuDR), inhibiting further transpositions in lines selected for molecular analysis; (iii) build-up of mutation load was prevented by screening Mu-active parents to exclude plants carrying pre-existing seed mutations. To create a database of genomic sequences flanking Mu insertions, selected mutant lines were analyzed by sequencing of MuTAIL PCR clone libraries. These sequences were annotated and clustered to facilitate bioinformatic subtraction of ancestral elements and identification of insertions unique to mutant lines. New insertions targeted low-copy, gene-rich sequences, and in silico mapping revealed a random distribution of insertions over the genome. Our results indicate that Mu populations differ markedly in the occurrence of Mu insertion hotspots and the frequency of suppressible mutations. We suggest that controlled MuDR copy number in UniformMu lines is a key determinant of these differences. The public database (http://uniformmu.org; http://endosperm.info) includes pedigree and phenotypic data for over 2000 independent seed mutants selected from a population of 31 548 F2 lines and integrated with analyses of 34 255 MuTAIL sequences. PMID:16167895

  9. [Mechanisms of targeted frameshift mutations--insertion formation under error-prone or SOS synthesis of DNA containing CIS-SYN cyncyclobutane thymine dimers].

    PubMed

    Grebneva, E A

    2014-01-01

    Up to now the mechanism of formation of frameshift mutations caused by cyclobutane pyrimidine dimers has not been yet explained satisfactorily. Mechanisms of different mutations are usually considered in polymerase model. Here, the alternative polymerase-tautomer model of ultraviolet mutagenesis is developed. The mechanism of targeted insertion formation caused by cis-syn cyclobutane thymine dimers is proposed. Insertions are mutations when one or several DNA bases are inserted.Targeted insertions are mutations of a frameshift type--when one or severalnucleotides are inserted opposite damageswhich may stop synthesis of DNA. Targeted insertions are induced bycyclobutane pyrimidine dimmers. Ultraviolet irradiation may result in a change of tautomer state of DNA bases. A thymine base may form 5 rare tautomer forms that are stable if the base is a part of cyclobutane dimer. As it was shown by structural analysis, one rare tautomeric form of thymine forms hydrogen bonds with no one canonical DNA base. Therefore, under SOS or error-prone synthesis of DNA containing cis-syn cyclobutane thymine dimers with such rare tautomeric_form a specialize or modified DNA polymerase leaves a single nucleotide gap opposite the cis-syn cyclobutane thymine dimer. According to Streisinger model, if the DNA composition within this region is homogeneous, the end of the growing DNA strand can slip and form complementary pairs with a template nucleotide neighboring to the dimer of such type a loop is formed. Further elongation of the daughter strand leads to the appearance of targeted insertion in the daughter strand. Here, it is first shown that cis-syn cyclobutane thymine dimers with one or both bases in the specific tautomer conformation--opposite which it is impossible to insert a canonical base with a hydrogen bond formation--results in targeted insertions. Moreover, the model of forming targeted single--and several-base insertions is developed. The polymerase-tautomer model of

  10. Mos1 mutagenesis reveals a diversity of mechanisms affecting response of Caenorhabditis elegans to the bacterial pathogen Microbacterium nematophilum.

    PubMed

    Yook, Karen; Hodgkin, Jonathan

    2007-02-01

    A specific host-pathogen interaction exists between Caenorhabditis elegans and the gram-positive bacterium Microbacterium nematophilum. This bacterium is able to colonize the rectum of susceptible worms and induces a defensive tail-swelling response in the host. Previous mutant screens have identified multiple loci that affect this interaction. Some of these loci correspond to known genes, but many bus genes [those with a bacterially unswollen (Bus) mutant phenotype] have yet to be cloned. We employed Mos1 transposon mutagenesis as a means of more rapidly cloning bus genes and identifying new mutants with altered pathogen response. This approach revealed new infection-related roles for two well-characterized and much-studied genes, egl-8 and tax-4. It also allowed the cloning of a known bus gene, bus-17, which encodes a predicted galactosyltransferase, and of a new bus gene, bus-19, which encodes a novel, albeit ancient, protein. The results illustrate advantages and disadvantages of Mos1 transposon mutagenesis in this system. PMID:17151260

  11. Lesion insertion in the projection domain: Methods and initial results

    SciTech Connect

    Chen, Baiyu; Leng, Shuai; Yu, Lifeng; Yu, Zhicong; Ma, Chi; McCollough, Cynthia

    2015-12-15

    acquired for the ACR phantom in terms of Hounsfield unit and high-contrast resolution. For the validation of the lesion realism, lesions of various types were successfully inserted, including well circumscribed and invasive lesions, homogeneous and heterogeneous lesions, high-contrast and low-contrast lesions, isolated and vessel-attached lesions, and small and large lesions. The two experienced radiologists who reviewed the original and inserted lesions could not identify the lesions that were inserted. The same lesion, when inserted into the projection domain and reconstructed with different parameters, demonstrated a parameter-dependent appearance. Conclusions: A framework has been developed for projection-domain insertion of lesions into commercial CT images, which can be potentially expanded to all geometries of CT scanners. Compared to conventional image-domain methods, the authors’ method reflected the impact of scan and reconstruction parameters on lesion appearance. Compared to prior projection-domain methods, the authors’ method has the potential to achieve higher anatomical complexity by employing clinical patient projections and real patient lesions.

  12. Elliptically polarizing adjustable phase insertion device

    DOEpatents

    Carr, R.

    1995-01-17

    An insertion device for extracting polarized electromagnetic energy from a beam of particles is disclosed. The insertion device includes four linear arrays of magnets which are aligned with the particle beam. The magnetic field strength to which the particles are subjected is adjusted by altering the relative alignment of the arrays in a direction parallel to that of the particle beam. Both the energy and polarization of the extracted energy may be varied by moving the relevant arrays parallel to the beam direction. The present invention requires a substantially simpler and more economical superstructure than insertion devices in which the magnetic field strength is altered by changing the gap between arrays of magnets. 3 figures.

  13. Membrane Protein Insertion at the Endoplasmic Reticulum

    PubMed Central

    Shao, Sichen; Hegde, Ramanujan S.

    2014-01-01

    Integral membrane proteins of the cell surface and most intracellular compartments of eukaryotic cells are assembled at the endoplasmic reticulum. Two highly conserved and parallel pathways mediate membrane protein targeting to and insertion into this organelle. The classical cotranslational pathway, utilized by most membrane proteins, involves targeting by the signal recognition particle followed by insertion via the Sec61 translocon. A more specialized posttranslational pathway, employed by many tail-anchored membrane proteins, is composed of entirely different factors centered around a cytosolic ATPase termed TRC40 or Get3. Both of these pathways overcome the same biophysical challenges of ferrying hydrophobic cargo through an aqueous milieu, selectively delivering it to one among several intracellular membranes and asymmetrically integrating its transmembrane domain(s) into the lipid bilayer. Here, we review the conceptual and mechanistic themes underlying these core membrane protein insertion pathways, the complexities that challenge our understanding, and future directions to over-come these obstacles. PMID:21801011

  14. Rack Insertion End Effector (RIEE) automation

    NASA Technical Reports Server (NTRS)

    Malladi, Narasimha

    1993-01-01

    NASA is developing a mechanism to manipulate and insert Racks into the Space Station Logistic modules. The mechanism consists of the following: a base with three motorized degrees of freedom, a 3 section motorized boom that goes from 15 to 44 feet in length, and a Rack Insertion End Effector (RIEE) with 5 hand wheels for precise alignment. The robotics section was tasked with the automation of the RIEE unit. In this report, for the automation of the RIEE unit, application of the Perceptics Vision System was conceptually developed to determine the position and orientation of the RIEE relative to the logistic module, and a MathCad program is written to display the needed displacements for precise alignment and final insertion of the Rack. The uniqueness of this report is that the whole report is in fact a MathCad program including text, derivations, and executable equations with example inputs and outputs.

  15. Key Sites for P2X Receptor Function and Multimerization: Overview of Mutagenesis Studies on a Structural Basis

    PubMed Central

    Hausmann, Ralf; Kless, Achim; Schmalzing, Günther

    2015-01-01

    P2X receptors constitute a seven-member family (P2X1-7) of extracellular ATP-gated cation channels of widespread expression. Because P2X receptors have been implicated in neurological, inflammatory and cardiovascular diseases, they constitute promising drug targets. Since the first P2X cDNA sequences became available in 1994, numerous site-directed mutagenesis studies have been conducted to disclose key sites of P2X receptor function and oligomerization. The publication of the 3-Å crystal structures of the zebrafish P2X4 (zfP2X4) receptor in the homotrimeric apo-closed and ATP-bound open states in 2009 and 2012, respectively, has ushered a new era by allowing for the interpretation of the wealth of molecular data in terms of specific three-dimensional models and by paving the way for designing more-decisive experiments. Thanks to these structures, the last five years have provided invaluable insight into our understanding of the structure and function of the P2X receptor class of ligandgated ion channels. In this review, we provide an overview of mutagenesis studies of the pre- and post-crystal structure eras that identified amino acid residues of key importance for ligand binding, channel gating, ion flow, formation of the pore and the channel gate, and desensitization. In addition, the sites that are involved in the trimerization of P2X receptors are reviewed based on mutagenesis studies and interface contacts that were predicted by the zfP2X4 crystal structures. PMID:25439586

  16. A Mycobacterium tuberculosis IS6110 preferential locus (ipl) for insertion into the genome.

    PubMed Central

    Fang, Z; Forbes, K J

    1997-01-01

    A 267-nucleotide Mycobacterium tuberculosis genomic sequence (ipl, the IS6110 preferential locus) which can harbor the insertion sequence IS6110 at six alternative locations has been identified in some three-quarters of the isolates tested. Only one IS6110 copy was observed at this locus in the ipl::IS6110(+)-containing isolates tested, and all insertions had the same orientation. The implications of this finding for IS6110 fingerprint typing methods is discussed in this work. PMID:9003621

  17. Rapid construction of metabolite biosensors using domain-insertion profiling

    PubMed Central

    Nadler, Dana C.; Morgan, Stacy-Anne; Flamholz, Avi; Kortright, Kaitlyn E.; Savage, David F.

    2016-01-01

    Single-fluorescent protein biosensors (SFPBs) are an important class of probes that enable the single-cell quantification of analytes in vivo. Despite advantages over other detection technologies, their use has been limited by the inherent challenges of their construction. Specifically, the rational design of green fluorescent protein (GFP) insertion into a ligand-binding domain, generating the requisite allosteric coupling, remains a rate-limiting step. Here, we describe an unbiased approach, termed domain-insertion profiling with DNA sequencing (DIP-seq), that combines the rapid creation of diverse libraries of potential SFPBs and high-throughput activity assays to identify functional biosensors. As a proof of concept, we construct an SFPB for the important regulatory sugar trehalose. DIP-seq analysis of a trehalose-binding-protein reveals allosteric hotspots for GFP insertion and results in high-dynamic range biosensors that function robustly in vivo. Taken together, DIP-seq simultaneously accelerates metabolite biosensor construction and provides a novel tool for interrogating protein allostery. PMID:27470466

  18. Rapid construction of metabolite biosensors using domain-insertion profiling.

    PubMed

    Nadler, Dana C; Morgan, Stacy-Anne; Flamholz, Avi; Kortright, Kaitlyn E; Savage, David F

    2016-01-01

    Single-fluorescent protein biosensors (SFPBs) are an important class of probes that enable the single-cell quantification of analytes in vivo. Despite advantages over other detection technologies, their use has been limited by the inherent challenges of their construction. Specifically, the rational design of green fluorescent protein (GFP) insertion into a ligand-binding domain, generating the requisite allosteric coupling, remains a rate-limiting step. Here, we describe an unbiased approach, termed domain-insertion profiling with DNA sequencing (DIP-seq), that combines the rapid creation of diverse libraries of potential SFPBs and high-throughput activity assays to identify functional biosensors. As a proof of concept, we construct an SFPB for the important regulatory sugar trehalose. DIP-seq analysis of a trehalose-binding-protein reveals allosteric hotspots for GFP insertion and results in high-dynamic range biosensors that function robustly in vivo. Taken together, DIP-seq simultaneously accelerates metabolite biosensor construction and provides a novel tool for interrogating protein allostery. PMID:27470466

  19. True anteroposterior view pedicle screw insertion technique

    PubMed Central

    Bai, Jia-yue; Zhang, Wei; An, Ji-long; Sun, Ya-peng; Ding, Wen-yuan; Shen, Yong

    2016-01-01

    Background The wide use of minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) surgery in the treatment of degenerative disc disease of lumbar spine in spinal surgery highlights the gradual decrease in the use of traditional pedicle screw insertion technology. This study aims to analyze the accuracy of the true anteroposterior view pedicle screw insertion technique in MIS-TLIF surgery, compare it with conventional pedicle screw insertion technology, and discuss its clinical application value. Methods Fifty-two patients undergoing true anteroposterior view (group A) and 87 patients undergoing conventional pedicle screw insertion (group B) were diagnosed with lumbar disc herniation or lumbar spinal stenosis. Time for screw placement, intraoperative irradiation exposure, accuracy rate of pedicle screw insertion, and incidence of neurovascular injury were compared between the two groups. Results The time for screw placement and intraoperative irradiation exposure was significantly less in group A. Penetration rates of the paries lateralis of vertebral pedicle, medial wall of vertebral pedicle, and anterior vertebral wall were 1.44%, 0%, and 2.40%, respectively, all of which were significantly lower than that in group B. No additional serious complications caused by the placement of screw were observed during the follow-up period in patients in group A, but two patients with medial penetration underwent revision for unbearable radicular pain. Conclusion The application of true anteroposterior view pedicle screw insertion technique in MIS-TLIF surgery shortens time for screw placement and reduces the intraoperative irradiation exposure along with a higher accuracy rate of screw placement, which makes it a safe, accurate, and efficient technique. PMID:27418828

  20. Mosaic retroposon insertion patterns in placental mammals

    PubMed Central

    Churakov, Gennady; Kriegs, Jan Ole; Baertsch, Robert; Zemann, Anja; Brosius, Jürgen; Schmitz, Jürgen

    2009-01-01

    One and a half centuries after Charles Darwin and Alfred Russel Wallace outlined our current understanding of evolution, a new scientific era is dawning that enables direct observations of genetic variation. However, pure sequence-based molecular attempts to resolve the basal origin of placental mammals have so far resulted only in apparently conflicting hypotheses. By contrast, in the mammalian genomes where they were highly active, the insertion of retroelements and their comparative insertion patterns constitute a neutral, virtually homoplasy-free archive of evolutionary histories. The “presence” of a retroelement at an orthologous genomic position in two species indicates their common ancestry in contrast to its “absence” in more distant species. To resolve the placental origin controversy we extracted ∼2 million potentially phylogenetically informative, retroposon-containing loci from representatives of the major placental mammalian lineages and found highly significant evidence challenging all current single hypotheses of their basal origin. The Exafroplacentalia hypothesis (Afrotheria as the sister group to all remaining placentals) is significantly supported by five retroposon insertions, the Epitheria hypothesis (Xenarthra as the sister group to all remaining placentals) by nine insertion patterns, and the Atlantogenata hypothesis (a monophyletic clade comprising Xenarthra and Afrotheria as the sister group to Boreotheria comprising all remaining placentals) by eight insertion patterns. These findings provide significant support for a “soft” polytomy of the major mammalian clades. Ancestral successive hybridization events and/or incomplete lineage sorting associated with short speciation intervals are viable explanations for the mosaic retroposon insertion patterns of recent placental mammals and for the futile search for a clear root dichotomy. PMID:19261842

  1. Mosaic retroposon insertion patterns in placental mammals.

    PubMed

    Churakov, Gennady; Kriegs, Jan Ole; Baertsch, Robert; Zemann, Anja; Brosius, Jürgen; Schmitz, Jürgen

    2009-05-01

    One and a half centuries after Charles Darwin and Alfred Russel Wallace outlined our current understanding of evolution, a new scientific era is dawning that enables direct observations of genetic variation. However, pure sequence-based molecular attempts to resolve the basal origin of placental mammals have so far resulted only in apparently conflicting hypotheses. By contrast, in the mammalian genomes where they were highly active, the insertion of retroelements and their comparative insertion patterns constitute a neutral, virtually homoplasy-free archive of evolutionary histories. The "presence" of a retroelement at an orthologous genomic position in two species indicates their common ancestry in contrast to its "absence" in more distant species. To resolve the placental origin controversy we extracted approximately 2 million potentially phylogenetically informative, retroposon-containing loci from representatives of the major placental mammalian lineages and found highly significant evidence challenging all current single hypotheses of their basal origin. The Exafroplacentalia hypothesis (Afrotheria as the sister group to all remaining placentals) is significantly supported by five retroposon insertions, the Epitheria hypothesis (Xenarthra as the sister group to all remaining placentals) by nine insertion patterns, and the Atlantogenata hypothesis (a monophyletic clade comprising Xenarthra and Afrotheria as the sister group to Boreotheria comprising all remaining placentals) by eight insertion patterns. These findings provide significant support for a "soft" polytomy of the major mammalian clades. Ancestral successive hybridization events and/or incomplete lineage sorting associated with short speciation intervals are viable explanations for the mosaic retroposon insertion patterns of recent placental mammals and for the futile search for a clear root dichotomy. PMID:19261842

  2. Aspartate Embedding Depth Affects pHLIP’s Insertion pKa

    PubMed Central

    Fendos, Justin; Barrera, Francisco N.; Engelman, Donald M.

    2014-01-01

    We have used the pHLIP® (pH Low Insertion Peptide) peptide family to study the role of aspartate embedding depth in pH-dependent transmembrane peptide insertion. pHLIP binds to the surface of a lipid bilayer as a largely unstructured monomer at neutral pH. When pH is lowered, pHLIP inserts spontaneously across the membrane as a spanning α-helix. pHLIP insertion is reversible when pH is adjusted back to a neutral value. One of the critical events facilitating pHLIP insertion is the protonation of aspartates in the spanning domain of the peptide: the negative side chains of these residues convert to uncharged, polar forms, facilitating insertion by altering the hydrophobicity of the spanning domain. To further examine this protonation mechanism, we created pHLIP sequence variants in which the position of the two spanning aspartates (D14, D25) was moved up or down in the sequence. We hypothesized that aspartate depth in the inserted state would directly affect the proton affinity of the acidic side chains, altering the pKa of pH-dependent insertion. To this end, we also mutated the arginine at position 11 to see if arginine snorkeling modulates the insertion pKa by affecting aspartate depth. Our results indicate both types of mutations change the insertion pKa, supporting the idea that aspartate depth is a participating parameter in determining pH dependence. We also show that pHLIP’s resistance to aggregation can be altered with our mutations, identifying a new criterion for improving pHLIP performance in vivo when targeting acidic disease tissues such as cancer and inflammation. PMID:23721379

  3. Genomic stability of murine leukemia viruses containing insertions at the Env-3' untranslated region boundary.

    PubMed

    Logg, C R; Logg, A; Tai, C K; Cannon, P M; Kasahara, N

    2001-08-01

    Retroviruses containing inserts of exogenous sequences frequently eliminate the inserted sequences upon spread in susceptible cells. We have constructed replication-competent murine leukemia virus (MLV) vectors containing internal ribosome entry site (IRES)-transgene cassettes at the env-3' untranslated region boundary in order to examine the effects of insert sequence and size on the loss of inserts during viral replication. A virus containing an insertion of 1.6 kb replicated with greatly attenuated kinetics relative to wild-type virus and lost the inserted sequences in a single infection cycle. In contrast, MLVs containing inserts of 1.15 to 1.30 kb replicated with kinetics only slightly attenuated compared to wild-type MLV and exhibited much greater stability, maintaining their genomic integrity over multiple serial infection cycles. Eventually, multiple species of deletion mutants were detected simultaneously in later infection cycles; once detected, these variants rapidly dominated the population and thereafter appeared to be maintained at a relative equilibrium. Sequence analysis of these variants identified preferred sites of recombination in the parental viruses, including both short direct repeats and inverted repeats. One instance of insert deletion through recombination with an endogenous retrovirus was also observed. When specific sequences involved in these recombination events were eliminated, deletion variants still arose with the same kinetics upon virus passage and by apparently similar mechanisms, although at different locations in the vectors. Our results suggest that while lengthened, insert-containing genomes can be maintained over multiple replication cycles, preferential deletions resulting in loss of the inserted sequences confer a strong selective advantage. PMID:11435579

  4. RERTR-12 Insertion 2 Irradiation Summary Report

    SciTech Connect

    D. M. Perez; G. S. Chang; D. M. Wachs; G. A. Roth; N. E. Woolstenhulme

    2012-09-01

    The Reduced Enrichment for Research and Test Reactor (RERTR) experiment RERTR-12 was designed to provide comprehensive information on the performance of uranium-molybdenum (U-Mo) based monolithic fuels for research reactor applications.1 RERTR-12 insertion 2 includes the capsules irradiated during the last three irradiation cycles. These capsules include Z, Y1, Y2 and Y3 type capsules. The following report summarizes the life of the RERTR-12 insertion 2 experiment through end of irradiation, including as-run neutronic analysis results, thermal analysis results and hydraulic testing results.

  5. Circular permutant GFP insertion folding reporters

    SciTech Connect

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2013-04-16

    Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

  6. Circular permutant GFP insertion folding reporters

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2008-06-24

    Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

  7. Circular permutant GFP insertion folding reporters

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2011-06-14

    Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

  8. Circular permutant GFP insertion folding reporters

    DOEpatents

    Waldo, Geoffrey S; Cabantous, Stephanie

    2013-02-12

    Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

  9. Insertion element analysis and mapping of the Pseudomonas plasmid alk regulon.

    PubMed Central

    Fennewald, M; Benson, S; Oppici, M; Shapiro, J

    1979-01-01

    We characterized and mapped new mutations of the alk (alkane utilization) genes found on Pseudomonas plasmids of the Inc P-2 group. These mutations were isolated after (i) nitrosoguanidine mutagenesis, (ii) transposition of the Tn7 trimethoprim and streptomycin resistance determinant, and (iii) reversion of polarity effects of alk::Tn7 insertion mutations. Our results indicate the existence of two alk loci not previously described--alkD, whose product is required for synthesis of membrane alkane-oxidizing activities, and alkE, whose product is required for synthesis of inducible membrane alcohol dehydrogenase activity. Polarity of alk::Tn7 insertion mutations indicates the existence of an alkBAE operon. Mapping of alk loci by transduction in P. aeruginosa shows that there are at least three alk clusters in the CAM-OCT plasmid--alkRD, containing regulatory genes; alkBAE, containing genes for specific biochemical activities; and alkC, containing one or more genes needed for normal synthesis of membrane alcohol dehydrogenase. The alkRD and alkBAE clusters are linked but separated by about 42 kilobases. The alkC cluster is not linked to either of the other two alk regions. Altogether, these results indicate a complex genetic control of the alkane utilization phenotype in P. putida and P. aeruginosa involving at least six separate genes. Images PMID:479111

  10. DNA transposon Hermes inserts into DNA in nucleosome-free regions in vivo.

    PubMed

    Gangadharan, Sunil; Mularoni, Loris; Fain-Thornton, Jennifer; Wheelan, Sarah J; Craig, Nancy L

    2010-12-21

    Transposons are mobile genetic elements that are an important source of genetic variation and are useful tools for genome engineering, mutagenesis screens, and vectors for transgenesis including gene therapy. We have used second-generation sequencing to analyze ≈2 × 10(5) unique de novo transposon insertion sites of the transposon Hermes in the Saccharomyces cerevisiae genome from both in vitro transposition reactions by using purified yeast genomic DNA, to better characterize intrinsic sequence specificity, and sites recovered from in vivo transposition events, to characterize the effect of intracellular factors such as chromatin on target site selection. We find that Hermes transposon targeting in vivo is profoundly affected by chromatin structure: The subset of genome-wide target sites used in vivo is strongly associated (P < 2e-16 by Fisher's exact test) with nucleosome-free chromatin. Our characterization of the insertion site preferences of Hermes not only assists in the future use of this transposon as a molecular biology tool but also establishes methods to more fully determine targeting mechanisms of other transposons. We have also discovered a long-range sequence motif that defines S. cerevisiae nucleosome-free regions. PMID:21131571

  11. DNA transposon Hermes inserts into DNA in nucleosome-free regions in vivo

    PubMed Central

    Gangadharan, Sunil; Mularoni, Loris; Fain-Thornton, Jennifer; Wheelan, Sarah J.; Craig, Nancy L.

    2010-01-01

    Transposons are mobile genetic elements that are an important source of genetic variation and are useful tools for genome engineering, mutagenesis screens, and vectors for transgenesis including gene therapy. We have used second-generation sequencing to analyze ≈2 × 105 unique de novo transposon insertion sites of the transposon Hermes in the Saccharomyces cerevisiae genome from both in vitro transposition reactions by using purified yeast genomic DNA, to better characterize intrinsic sequence specificity, and sites recovered from in vivo transposition events, to characterize the effect of intracellular factors such as chromatin on target site selection. We find that Hermes transposon targeting in vivo is profoundly affected by chromatin structure: The subset of genome-wide target sites used in vivo is strongly associated (P < 2e-16 by Fisher's exact test) with nucleosome-free chromatin. Our characterization of the insertion site preferences of Hermes not only assists in the future use of this transposon as a molecular biology tool but also establishes methods to more fully determine targeting mechanisms of other transposons. We have also discovered a long-range sequence motif that defines S. cerevisiae nucleosome-free regions. PMID:21131571

  12. Salt-Sensitive Mutants of Chlamydomonas reinhardtii Isolated after Insertional Tagging.

    PubMed Central

    Prieto, R.; Pardo, J. M.; Niu, X.; Bressan, R. A.; Hasegawa, P. M.

    1996-01-01

    We describe the isolation of salt-sensitive Chlamydomonas reinhardtii mutants by insertional mutagenesis using the nitrate reductase (Nit1) gene. The plasmid pMN24, containing Nit1, was used for transformation of 305CW15 (nit1 cw15 mt+), and transformants were selected for complementation of the nit- phenotype. From 6875 nit+ colonies, four transformants (S4, S18, S46, and S66) were isolated that exhibited both Na+ and Li+ sensitivity (sod-), and another transformant (S33) was selected that exhibited sensitivity to Li+ but not Na+ (lit-) based on relative growth comparisons with the wild-type strain. S33, S46, and S66 were no more growth inhibited by sorbitol than was 305CW15. In comparison, S4 and S18 exhibited substantial growth inhibition in medium supplemented with sorbitol. Genetic analyses indicated that the salt-sensitive mutants were each defective in a single recessive gene. The mutant genes in S4 (sod1), S33 (lit1), and S66 (sod3) are linked to a functional copy of Nit1 and are presumably tagged with a pMN24 insertion. PMID:12226377

  13. Isolation and transposition properties of ISBlo11, an active insertion sequence belonging to the IS3 family, from Bifidobacterium longum 105-A.

    PubMed

    Sakanaka, Mikiyasu; Fukiya, Satoru; Kobayashi, Ryoko; Abe, Arisa; Hirayama, Yosuke; Kano, Yasunobu; Yokota, Atsushi

    2015-04-01

    Transposon mutagenesis systems are still under development in bifidobacteria, partly because intrinsic active insertion sequences are not well characterized in bifidobacteria. Here, we isolated an active insertion sequence, ISBlo11, from Bifidobacterium longum 105-A using a sacB-based counterselection system, which is generally used to screen for active insertion sequences from bacterial genomes. ISBlo11 is 1432 bp long and belongs to the IS3 family. It has a single ORF encoding a transposase and 25-bp inverted repeats at its termini. Full-length copies of ISBlo11 are specifically conserved among certain B. longum genomes and exist in different sites. Transposition analysis of an artificial ISBlo11 transposon using an Escherichia coli conjugation system revealed that ISBlo11 has adequate transposition activity, comparable to the reported activity of IS629, another IS3 family element initially isolated from Shigella sonnei. ISBlo11 also showed low transposition selectivity for non-conserved 3- or 4-bp target sequences. These characteristics of ISBlo11 seem suitable for the development of a new transposon mutagenesis system in bifidobacteria. PMID:25724534

  14. Bone metabolism induced by denture insertion in positron emission tomography.

    PubMed

    Suenaga, H; Chen, J; Yamaguchi, K; Sugazaki, M; Li, W; Swain, M; Li, Q; Sasaki, K

    2016-03-01

    18F-fluoride positron emission tomogra-phy (PET) can identify subtle functional variation prior to the major structural change detectable by X-ray. This study aims to investigate the mechanobiological bone reaction around the abutment tooth and in the residual ridge, induced by insertion of removable partial denture (RPD) within two different groups of patients: patients without denture experience (Group 1) and patients with denture experience before (Group 2), using 18F-fluoride PET imaging technique. 18F-fluoride PET/computerised tomography (CT) scan was performed to examine the bone metabolic change in mandible before and after the RPD treatment. Region of interests (ROIs) were placed in alveolar bone around abutment tooth and in residual bone beneath the RPD. Standardised uptake value (SUV), reflecting the accumulation of 18F-fluoride, was measured for each ROI. In all subjects of Group 1, SUVs after insertion were higher than before in both alveolar bone and residual bone, while there was less significant change in SUV in subjects of Group 2. This study demonstrated using longitudinal 18F-fluoride PET scans to effectively examine the bone metabolic change in mandible induced by occlusal loading after RPD insertion. Using this technique, within the six subjects in this study, it was shown that bone metabolism around abutment tooth and residual ridge increased after RPD insertion in case of first-time denture user, while there was no big change in the patient with experience of denture before. This study revealed the effectiveness of applying PET to evaluate bone metabolic activity as mechanobiolo-gical reaction. PMID:26431672

  15. Association of elevated mutagenesis in the spleen with genetic susceptibility to induced plasmacytoma development in mice.

    PubMed

    Felix, K; Kelliher, K; Bornkamm, G W; Janz, S

    1998-04-15

    Using the phage lambdaLIZ-based transgenic in vivo mutagenesis assay, mean mutant rates were determined in the spleen of mice exposed to sustained oxidative stress and were found to be increased approximately 3-fold in plasmacytoma-susceptible BALB/c and C.D2-Idh1-Pep3 mice, but not in plasmacytoma-resistant DBA/2N mice. This finding suggests a correlation between the genetic susceptibility to inflammation-induced peritoneal plasmacytomagenesis and the phenotype of increased mutagenesis in lymphoid tissues, raising the possibility that plasmacytoma resistance genes may inhibit tumor development by minimizing oxidative mutagenesis in B cells. PMID:9563470

  16. Neutron absorber inserts for 55-gal drums

    SciTech Connect

    Wilson, R.E.; Kim, Y.S.; Toffer, H.

    2000-07-01

    Transport and temporary storage of more than 200 g of fissile material in 55-gal drums at the Rocky Flats Environmental Technology Site (RFETS) have received significant attention during the cleanup mission. This paper discusses successful applications and results of extensive computer studies. Interim storage and movement of fissile material in excess of standard drum limits (200 g) in a safe configuration have been accomplished using special drum inserts. Such inserts have constrained the contents of a drum to two 4-{ell} bottles. The content of the bottles was limited to 600 g Pu or U in solution or a total of 1200 g for the entire drum. The inserts were a simple design constructed of stainless steel, forming a vertical cylindrical pipe into which two bottles, one on top of the other, could be centered in the drum. The remaining drum volume was configured to preclude any additional bottle placement external to the vertical cylinder. Such inserts in drums were successfully used in moving high-concentration solution from one building to another for chemical processing. Concern about the knowledge of fissile material concentration in bottles prompted another study for drum inserts. The past practice had been to load up to fourteen 4-{ell} bottles into 55-gal drums, provided the fissile material concentration was <6 g fissile/{ell}, and the total drum contents of 200 g fissile was not exceeded. Only one determination of the solution concentration was needed. An extensive safety analysis concluded that a single measurement of bottle content could not ensure compliance with double-contingency-criterion requirements. A second determination of the bottle contents was required before bottles could be placed in a 55-gal drum. Al alternative to a dual-measurement protocol, which is for bolstering administrative control, was to develop an engineered safety feature that would eliminate expensive tests and administrative decisions. A drum insert design was evaluated that

  17. Molecular Mechanisms for High Hydrostatic Pressure-Induced Wing Mutagenesis in Drosophila melanogaster.

    PubMed

    Wang, Hua; Wang, Kai; Xiao, Guanjun; Ma, Junfeng; Wang, Bingying; Shen, Sile; Fu, Xueqi; Zou, Guangtian; Zou, Bo

    2015-01-01

    Although High hydrostatic pressure (HHP) as an important physical and chemical tool has been increasingly applied to research of organism, the response mechanisms of organism to HHP have not been elucidated clearly thus far. To identify mutagenic mechanisms of HHP on organisms, here, we treated Drosophila melanogaster (D. melanogaster) eggs with HHP. Approximately 75% of the surviving flies showed significant morphological abnormalities from the egg to the adult stages compared with control flies (p < 0.05). Some eggs displayed abnormal chorionic appendages, some larvae were large and red, and some adult flies showed wing abnormalities. Abnormal wing phenotypes of D. melanogaster induced by HHP were used to investigate the mutagenic mechanisms of HHP on organism. Thus 285 differentially expressed genes associated with wing mutations were identified using Affymetrix Drosophila Genome Array 2.0 and verified with RT-PCR. We also compared wing development-related central genes in the mutant flies with control flies using DNA sequencing to show two point mutations in the vestigial (vg) gene. This study revealed the mutagenic mechanisms of HHP-induced mutagenesis in D. melanogaster and provided a new model for the study of evolution on organisms. PMID:26446369

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

    PubMed Central

    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.

    2016-01-01

    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. PMID:27534441

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

    PubMed

    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

    2016-01-01

    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. PMID:27534441

  20. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis

    PubMed Central

    Canver, Matthew C.; Smith, Elenoe C.; Sher, Falak; Pinello, Luca; Sanjana, Neville E.; Shalem, Ophir; Chen, Diane D.; Schupp, Patrick G.; Vinjamur, Divya S.; Garcia, Sara P.; Luc, Sidinh; Kurita, Ryo; Nakamura, Yukio; Fujiwara, Yuko; Maeda, Takahiro; Yuan, Guo-Cheng; Feng, Zhang; Orkin, Stuart H.; Bauer, Daniel E.

    2015-01-01

    Summary Enhancers, critical determinants of cellular identity, are commonly identified by correlative chromatin marks and gain-of-function potential, though only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously we identified an erythroid enhancer of BCL11A, subject to common genetic variation associated with fetal hemoglobin (HbF) level, whose mouse ortholog is necessary for erythroid BCL11A expression. Here we develop pooled CRISPR-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for HbF reinduction. The detailed enhancer map will inform therapeutic genome editing. The screening approach described here is generally applicable to functional interrogation of noncoding genomic elements. PMID:26375006

  1. Reliability Technology to Achieve Insertion of Advanced Packaging (RELTECH) program

    NASA Technical Reports Server (NTRS)

    Fayette, Daniel F.; Speicher, Patricia; Stoklosa, Mark J.; Evans, Jillian V.; Evans, John W.; Gentile, Mike; Pagel, Chuck A.; Hakim, Edward

    1993-01-01

    A joint military-commercial effort to evaluate multichip module (MCM) structures is discussed. The program, Reliability Technology to Achieve Insertion of Advanced Packaging (RELTECH), has been designed to identify the failure mechanisms that are possible in MCM structures. The RELTECH test vehicles, technical assessment task, product evaluation plan, reliability modeling task, accelerated and environmental testing, and post-test physical analysis and failure analysis are described. The information obtained through RELTECH can be used to address standardization issues, through development of cost effective qualification and appropriate screening criteria, for inclusion into a commercial specification and the MIL-H-38534 general specification for hybrid microcircuits.

  2. Systemic Air Embolism Associated with Pleural Pigtail Chest Tube Insertion

    PubMed Central

    Alkhankan, Emad; Nusair, Ahmad; Mazagri, Rida

    2016-01-01

    Pleural pigtail catheter placement is associated with many complications including pneumothorax, hemorrhage, and chest pain. Air embolism is a known but rare complication of pleural pigtail catheter insertion and has a high risk of occurrence with positive pressure ventilation. In this case report, we present a 50-year-old male with bilateral pneumonia who developed a pneumothorax while on mechanical ventilation with continuous positive airway pressure mode. During the placement of the pleural pigtail catheter to correct the pneumothorax, the patient developed a sudden left sided body weakness and became unresponsive. An air embolism was identified in the right main cerebral artery, which was fatal.

  3. CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme.

    PubMed

    Michno, Jean-Michel; Wang, Xiaobo; Liu, Junqi; Curtin, Shaun J; Kono, Thomas Jy; Stupar, Robert M

    2015-10-01

    The CRISPR/Cas9 system is rapidly becoming the reagent of choice for targeted mutagenesis and gene editing in crop species. There are currently intense research efforts in the crop sciences to identify efficient CRISPR/Cas9 platforms to carry out targeted mutagenesis and gene editing projects. These efforts typically result in the incremental tweaking of various platform components including the identification of crop-specific promoters and terminators for optimal expression of the Cas9 enzyme and identification of promoters for expression of the CRISPR guide RNA. In this report, we demonstrate the development of an online web tool for fast identification of CRISPR/Cas9 target loci within soybean gene models, and generic DNA sequences. The web-tool described in this work can quickly identify a high number of potential CRISPR/Cas9 target sites, including restriction enzyme sites that can facilitate the detection of new mutations. In conjunction with the web tool, a soybean codon-optimized CRISPR/Cas9 platform was designed to direct double-stranded breaks to the targeted loci in hairy root transformed cells. The modified Cas9 enzyme was shown to successfully mutate target genes in somatic cells of 2 legume species, soybean and Medicago truncatula. These new tools may help facilitate targeted mutagenesis in legume and other plant species. PMID:26479970

  4. Use of random systematic mutagenesis to generate viable human rhinovirus 14 chimeras displaying human immunodeficiency virus type 1 V3 loop sequences.

    PubMed Central

    Smith, A D; Resnick, D A; Zhang, A; Geisler, S C; Arnold, E; Arnold, G F

    1994-01-01

    Random systematic mutagenesis was used to generate a library of human rhinovirus 14 chimeras that each display a segment from the V3 loop of human immunodeficiency virus type 1. The sequence XXIGPGRAXX, where X could be any of the 20 amino acids, was inserted at the neutralizing immunogenic site II of human rhinovirus 14 between VP2 residues 159 and 160. Twenty-five unique chimeric viruses were isolated, and the identity of their randomized residues was determined. A nonrandom amino acid distribution that may reflect structural requirements for viability was observed at the randomized positions. Fifteen of 25 chimeras were neutralized by one or more of a panel of four anti-human immunodeficiency virus type 1 V3 loop antibody preparations, indicating that antigenicity had been successfully transplanted. Libraries of chimeric viruses produced by using the techniques described may be a source of vaccines and other immunotherapeutic reagents. The random systematic mutagenesis methodology described should be generally useful for the rapid transplantation of foreign sequences into viral coat and other proteins to produce libraries containing members with the desired properties. PMID:8254775

  5. International Consortium of Rice Mutagenesis: resources and beyond

    PubMed Central

    2013-01-01

    Rice is one of the most important crops in the world. The rice community needs to cooperate and share efforts and resources so that we can understand the functions of rice genes, especially those with a role in important agronomical traits, for application in agricultural production. Mutation is a major source of genetic variation that can be used for studying gene function. We will present here the status of mutant collections affected in a random manner by physical/chemical and insertion mutageneses. As of early September 2013, a total of 447, 919 flanking sequence tags from rice mutant libraries with T-DNA, Ac/Ds, En/Spm, Tos17, nDART/aDART insertions have been collected and publicly available. From these, 336,262 sequences are precisely positioned on the japonica rice chromosomes, and 67.5% are in gene interval. We discuss the genome coverage and preference of the insertion, issues limiting the exchange and use of the current collections, as well as new and improved resources. We propose a call to renew all mutant populations as soon as possible. We also suggest that a common web portal should be established for ordering seeds. PMID:24341871

  6. 21 CFR 886.5420 - Contact lens inserter/remover.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Contact lens inserter/remover. 886.5420 Section... (CONTINUED) MEDICAL DEVICES OPHTHALMIC DEVICES Therapeutic Devices § 886.5420 Contact lens inserter/remover. (a) Identification. A contact lens inserter/remover is a handheld device intended to insert or...

  7. 21 CFR 886.5420 - Contact lens inserter/remover.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Contact lens inserter/remover. 886.5420 Section... (CONTINUED) MEDICAL DEVICES OPHTHALMIC DEVICES Therapeutic Devices § 886.5420 Contact lens inserter/remover. (a) Identification. A contact lens inserter/remover is a handheld device intended to insert or...

  8. 21 CFR 886.5420 - Contact lens inserter/remover.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Contact lens inserter/remover. 886.5420 Section... (CONTINUED) MEDICAL DEVICES OPHTHALMIC DEVICES Therapeutic Devices § 886.5420 Contact lens inserter/remover. (a) Identification. A contact lens inserter/remover is a handheld device intended to insert or...

  9. 21 CFR 886.5420 - Contact lens inserter/remover.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Contact lens inserter/remover. 886.5420 Section... (CONTINUED) MEDICAL DEVICES OPHTHALMIC DEVICES Therapeutic Devices § 886.5420 Contact lens inserter/remover. (a) Identification. A contact lens inserter/remover is a handheld device intended to insert or...

  10. 21 CFR 886.5420 - Contact lens inserter/remover.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Contact lens inserter/remover. 886.5420 Section... (CONTINUED) MEDICAL DEVICES OPHTHALMIC DEVICES Therapeutic Devices § 886.5420 Contact lens inserter/remover. (a) Identification. A contact lens inserter/remover is a handheld device intended to insert or...

  11. Embedded Multiprocessor Technology for VHSIC Insertion

    NASA Technical Reports Server (NTRS)

    Hayes, Paul J.

    1990-01-01

    Viewgraphs on embedded multiprocessor technology for VHSIC insertion are presented. The objective was to develop multiprocessor system technology providing user-selectable fault tolerance, increased throughput, and ease of application representation for concurrent operation. The approach was to develop graph management mapping theory for proper performance, model multiprocessor performance, and demonstrate performance in selected hardware systems.

  12. Insertion Loss of Personal Protective Clothing

    SciTech Connect

    Shull D.J.; Biesel, V.B.; Cunefare, K.A.

    1999-05-13

    'The use of personal protective clothing that covers the head is a common practice in many industries. Such personal protective clothing will impact the sound pressure level and the frequency content of sounds to which the wearer will be exposed. The use of such clothing, then, may impact speech and alarm audibility. A measure of the impact of such clothing is its insertion loss. Insertion loss measurements were performed on four types of personal protective clothing in use by Westinghouse Savannah River Company personnel which utilize cloth and plastic hood configurations to protect the head. All clothing configurations tested at least partially cover the ears. The measurements revealed that insertion loss of the items tested was notable at frequencies above 1000 Hz only and was a function of material stiffness and acoustic flanking paths to the ear. Further, an estimate of the clothing''s noise reduction rating reveals poor performance in that regard, even though the insertion loss of the test articles was significant at frequencies at and above 1000 Hz.'

  13. Lymphatic Leak Complicating Central Venous Catheter Insertion

    SciTech Connect

    Barnacle, Alex M. Kleidon, Tricia M.

    2005-12-15

    Many of the risks associated with central venous access are well recognized. We report a case of inadvertent lymphatic disruption during the insertion of a tunneled central venous catheter in a patient with raised left and right atrial pressures and severe pulmonary hypertension, which led to significant hemodynamic instability. To our knowledge, this rare complication is previously unreported.

  14. A design for vertical crossing insertions

    SciTech Connect

    Garren, A.

    1985-10-01

    A crossing insertion designed for an SSC with vertically separated 1-in-1 beam lines is presented in this note. The author supposes that the beam lines consist of separate magnets in separate cryostats separated by about 70 cm. He then describes the design, where vertical separation is done with four vertical dipoles producing a steplike beam line.

  15. Frequency, Gradience, and Variation in Consonant Insertion

    ERIC Educational Resources Information Center

    An, Young-ran

    2010-01-01

    This dissertation addresses the extent to which linguistic behavior can be described in terms of the projection of patterns from existing lexical items, through an investigation of Korean reduplication. Korean has a productive pattern of reduplication in which a consonant is inserted in a vowel-initial base, illustrated by forms such as "alok"--"t…

  16. Magnetic measurements on insertion devices at NSLS

    SciTech Connect

    Decker, G.; Galayda, J.; Solomon, L.; Kitamura, M.

    1989-07-01

    Magnetic measurements on three insertion devices for use in the National Synchrotron Light Source (NSLS) x-ray ring are described. Hall probes were used for field mapping, and rotating long coils were used to determine integrated multipole coefficients. Results are presented for an iron pole-REC hybrid indulator, a vanadium permendur pole-REC hybrid wiggler, and a 5 T superconducting wiggler.

  17. Thermal Performance of the XRS Helium Insert

    NASA Technical Reports Server (NTRS)

    Breon, Susan R.; DiPirro, Michael J.; Tuttle, James G.; Shirron, Peter J.; Warner, Brent A.; Boyle, Robert F.; Canavan, Edgar R.

    1999-01-01

    The X-Ray Spectrometer (XRS) is an instrument on the Japanese Astro-E satellite, scheduled for launch early in the year 2000. The XRS Helium Insert comprises a superfluid helium cryostat, an Adiabatic Demagnetization Refrigerator (ADR), and the XRS calorimeters with their cold electronics. The calorimeters are capable of detecting X-rays over the energy range 0.1 to 10 keV with a resolution of 12 eV. The Helium Insert completed its performance and verification testing at Goddard in January 1999. It was shipped to Japan, where it has been integrated with the neon dewar built by Sumitomo Heavy Industries. The Helium Insert was given a challenging lifetime requirement of 2.0 years with a goal of 2.5 years. Based on the results of the thermal performance tests, the predicted on-orbit lifetime is 2.6 years with a margin of 30%. This is the result of both higher efficiency in the ADR cycle and the low temperature top-off, more than compensating for an increase in the parasitic heat load. This paper presents a summary of the key design features and the results of the thermal testing of the XRS Helium Insert.

  18. Mars Observer Lecture: Mars Orbit Insertion

    NASA Technical Reports Server (NTRS)

    Dodd, Suzanne R. (Personal Name)

    1993-01-01

    The Mars Observer mission spacecraft was primarily designed for exploring Mars and the Martian environment. The Mars Observer was launched on September 25, 1992. The spacecraft was lost in the vicinity of Mars on August 21, 1993 when the spacecraft began its maneuvering sequence for Martian orbital insertion. This videotape shows a lecture by Suzanne R. Dodd, the Mission Planning Team Chief for the Mars Observer Project. Ms Dodd begins with a brief overview of the mission and the timeline from the launch to orbital insertion. Ms Dodd then reviews slides showing the trajectory of the spacecraft on its trip to Mars. Slides of the spacecraft being constructed are also shown. She then discusses the Mars orbit insertion and the events that will occur to move the spacecraft from the capture orbit into a mapping orbit. During the trip to Mars, scientists at JPL had devised a new strategy, called Power In that would allow for an earlier insertion into the mapping orbit. The talk summarizes this strategy, showing on a slide the planned transition orbits. There are shots of the Martian moon, Phobos, taken from the Viking spacecraft, as Ms Dodd explains that the trajectory will allow the orbiter to make new observations of that moon. She also explains the required steps to prepare for mapping after the spacecraft has achieved the mapping orbit around Mars. The lecture ends with a picture of Mars from the Observer on its approach to the planet.

  19. Inserting new technology into small missions

    NASA Technical Reports Server (NTRS)

    Deutsch, L. J.

    2001-01-01

    Part of what makes small missions small is that they have less money. Executing missions at low cost implies extensive use of cost sharing with other missions or use of existing solutions. Luckily, there are methods for creating new technology and inserting it into faster-better-cheaper missions.

  20. The interface between bone and tendon at an insertion site: a study of the quadriceps tendon insertion

    PubMed Central

    CLARK, JOHN; STECHSCHULTE, DANIEL J.

    1998-01-01

    Traumatic avulsions of ligament or tendon insertions rarely occur at the actual interface with bone, which suggests that this attachment is strong or otherwise protected from injury by the structure of the insertion complex. In this study we describe the terminal extent of quadriceps tendon fibres where they insert into the patellae of adult rabbits, humans, dogs and sheep. Specimens were examined by scanning electron microscopy (SEM) and light microscopy (LM). To facilitate tracing of tendon fibres the specimens were decalcified for SEM, and polarised light microscopy (PLM) was used in the LM segment of the study. By SEM it was possible to identify mature bone by the presence of osteocytes and a lamellar organisation. PLM and SEM showed that, unlike tendon fibres elsewhere, those in the calcified fibrocartilage were not crimped. No specific cement line was identified by SEM. Tendon fibres interdigitated among separate bone lamellar systems, (osteons or marrow spaces), but did not merge with the collagen systems of individual lamellae. The interdigitation was more extensive and the margin between tendon and bone was less distinct in the anterior third of the insertion. The segment of calcified tendon which interdigitated with bone stained less intensely blue and was less cellular than the more proximal calcified fibrocartilage zone adjacent to the tidemark. Lamellar collagen fibres of the bony trabeculae in the anterior patella were unusually parallel and longitudinal in orientation, making distinction of interposed tendon fibres difficult on LM and PLM sections. LM, SEM and transmission electron microscopy of rabbit patellae at birth revealed that anterior quadriceps tendon fibres extended over the patella in a fibrous cellular layer. By 2 wk of age, this layer had acquired chondroid features (i.e. cell lacunae and metachromasia) and contained vessels extending from patellar marrow. At 6 wk of age, part of this fibrocartilaginous layer was replaced by mature bone

  1. Toward a Molecular Genetic Analysis of Spermatogenesis in Drosophila Melanogaster: Characterization of Male-Sterile Mutants Generated by Single P Element Mutagenesis

    PubMed Central

    Castrillon, D. H.; Gonczy, P.; Alexander, S.; Rawson, R.; Eberhart, C. G.; Viswanathan, S.; DiNardo, S.; Wasserman, S. A.

    1993-01-01

    We describe 83 recessive autosomal male-sterile mutations, generated by single P element mutagenesis in Drosophila melanogaster. Each mutation has been localized to a lettered subdivision of the polytene map. Reversion analyses, as well as complementation tests using available chromosomal deficiencies, indicate that the insertions are responsible for the mutant phenotypes. These mutations represent 63 complementation groups, 58 of which are required for spermatogenesis. Phenotypes of the spermatogenesis mutants were analyzed by light microscopy. Mutations in 12 loci affect germline proliferation, spermatocyte growth, or meiosis. Mutations in 46 other loci disrupt differentiation and maturation of spermatids into motile sperm. This collection of male-sterile mutants provides the basis for a molecular genetic analysis of spermatogenesis. PMID:8244010

  2. High-quality mutant libraries of Xanthomonas oryzae pv. oryzae and X. campestris pv. campestris generated by an efficient transposon mutagenesis system.

    PubMed

    Sun, Qihong; Wu, Wei; Qian, Wei; Hu, Jun; Fang, Rongxiang; He, Chaozu

    2003-09-12

    A novel transposon mutagenesis system for the phytopathogenic bacteria Xanthomonas oryzae pv. oryzae (Xoo) and X. campestris pv. campestris (Xcc) was developed using a Tn5-based transposome. A highly efficient transformation up to 10(6) transformants per microg transposon DNA was obtained. Southern blot and thermal asymmetric interlaced polymerase chain reaction analyses of Tn5 insertion sites suggested a random mode of transposition. The transposition was stable in the transformants for 20 subcultures. Eighteen thousand and 17000 transformants for Xoo and Xcc, respectively, were generated, corresponding to 4X ORF coverage of the genomes. The libraries will facilitate the identification of pathogenicity-related genes as well as functional genomic analysis in Xoo and Xcc. PMID:13129620

  3. Association between Peripherally Inserted Central Venous Catheter Insertion Site and Complication Rates in Preterm Infants.

    PubMed

    Bashir, Rani A; Swarnam, Kamala; Vayalthrikkovil, Sakeer; Yee, Wendy; Soraisham, Amuchou S

    2016-08-01

    Objective To examine whether there is an association between peripherally inserted central venous catheter (PICC) insertion site and complication rates among preterm infants. Design We performed a retrospective analysis of the first PICCs placed in preterm infants in a tertiary neonatal intensive care unit between January 2006 and December 2010. The PICC-related complications resulting in catheter removal were compared based on site of insertion. Results Of the 827 PICCs, 593 (72%) were inserted in upper extremity. Lower extremity PICC group infants had higher illness severity (SNAP-II) score and more likely to be inserted later as compared with the upper extremity group. There was no significant difference in the total PICC-related complications between upper and lower extremity PICCs (31.3 vs. 26%; p > 0.05). Logistic regression analysis after adjusting for gestational age, day of line insertion, and SNAP-II score revealed that upper extremity PICCs were associated with increased risk of line infiltration (adjusted odds ratio [aOR], 2.41; 95% confidence interval [CI], 1.36-4.29) but not the total PICC complication (aOR, 1.29; 95% CI, 0.91-1.83). Conclusion There is no difference in total PICC-related complication between upper and lower extremity PICCs; however, the PICC-related mechanical complications vary depending on the site of insertion in preterm infants. PMID:27057766

  4. Consolidation and disposal of PWR fuel inserts

    SciTech Connect

    Wakeman, B.H. )

    1992-08-01

    Design and licensing of the Surry Power Station Independent Spent Fuel Storage Installation was initiated in 1982 by Virginia Power as part of a comprehensive strategy to increase spent fuel storage capacity at the Station. Designed to use large, metal dry storage casks, the Surry Installation will accommodate 84 such casks with a total storage capacity of 811 MTU of spent pressurized water reactor fuel assemblies. Virginia Power provided three storage casks for testing at the Idaho National Engineerinq Laboratory's Test Area North and the testing results have been published by the Electric Power Research Institute. Sixty-nine spent fuel assemblies were transported in truck casks from the Surry Power Station to Test Area North for testing in the three casks. Because of restrictions imposed by the cask testing equipment at Test Area North, the irradiated insert components stored in these fuel assemblies at Surry were removed prior to transport of the fuel assemblies. Retaining these insert components proved to be a problem because of a shortage of spent fuel assemblies in the spent fuel storage pool that did not already contain insert components. In 1987 Virginia Power contracted with Chem-Nuclear Systems, Inc. to process and dispose of 136 irradiated insert components consisting of 125 burnable poison rod assemblies, 10 thimble plugging devices and 1 part-length rod cluster control assembly. This work was completed in August and September 1987, culminating in the disposal at the Barnwell, SC low-level radioactive waste facility of two CNS 3-55 liners containing the consolidated insert components.

  5. Screw-Thread Inserts As Temporary Flow Restrictors

    NASA Technical Reports Server (NTRS)

    Trimarchi, Paul

    1992-01-01

    Coil-spring screw-thread inserts found useful as temporary flow restrictors. Inserts placed in holes through which flow restricted, effectively reducing cross sections available for flow. Friction alone holds inserts against moderate upstream pressures. Use of coil-spring thread inserts as flow restrictors conceived as inexpensive solution to problem of adjusting flow of oxygen through orifices in faceplate into hydrogen/oxygen combustion chamber. Installation and removal of threaded inserts gentle enough not to deform orifice tubes.

  6. Retroviral Vectors for Analysis of Viral Mutagenesis and Recombination

    PubMed Central

    Rawson, Jonathan M.O.; Mansky, Louis M.

    2014-01-01

    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. PMID:25254386

  7. Oligonucleotide-directed mutagenesis for precision gene editing.

    PubMed

    Sauer, Noel J; Mozoruk, Jerry; Miller, Ryan B; Warburg, Zachary J; Walker, Keith A; Beetham, Peter R; Schöpke, Christian R; Gocal, Greg F W

    2016-02-01

    Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide-directed mutagenesis (ODM), one of the many tools of Cibus' Rapid Trait Development System (RTDS(™) ) technology, offers a rapid, precise and non-transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide-mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS, is reviewed. PMID:26503400

  8. Targeted mutagenesis using CRISPR/Cas system in medaka

    PubMed Central

    Ansai, Satoshi; Kinoshita, Masato

    2014-01-01

    ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-based RNA-guided endonuclease (RGEN) has recently emerged as a simple and efficient tool for targeted genome editing. In this study, we showed successful targeted mutagenesis using RGENs in medaka, Oryzias latipes. Somatic and heritable mutations were induced with high efficiency at the targeted genomic sequence on the DJ-1 gene in embryos that had been injected with the single guide RNA (sgRNA) transcribed by a T7 promoter and capped RNA encoding a Cas9 nuclease. The sgRNAs that were designed for the target genomic sequences without the 5′ end of GG required by the T7 promoter induced the targeted mutations. This suggests that the RGEN can target any sequence adjacent to an NGG protospacer adjacent motif (PAM) sequence, which occurs once every 8 bp. The off-target alterations at 2 genomic loci harboring double mismatches in the 18-bp targeting sequences were induced in the RGEN-injected embryos. However, we also found that the off-target effects could be reduced by lower dosages of sgRNA. Taken together, our results suggest that CRISPR/Cas-mediated RGENs may be an efficient and flexible tool for genome editing in medaka. PMID:24728957

  9. Lethal Mutagenesis of HIV with Mutagenic Nucleoside Analogs

    NASA Astrophysics Data System (ADS)

    Loeb, Lawrence A.; Essigmann, John M.; Kazazi, Farhad; Zhang, Jue; Rose, Karl D.; Mullins, James I.

    1999-02-01

    The human immunodeficiency virus (HIV) replicates its genome and mutates at exceptionally high rates. As a result, the virus is able to evade immunological and chemical antiviral agents. We tested the hypothesis that a further increase in the mutation rate by promutagenic nucleoside analogs would abolish viral replication. We evaluated deoxynucleoside analogs for lack of toxicity to human cells, incorporation by HIV reverse transcriptase, resistance to repair when incorporated into the DNA strand of an RNA\\cdot DNA hybrid, and mispairing at high frequency. Among the candidates tested, 5-hydroxydeoxycytidine (5-OH-dC) fulfilled these criteria. In seven of nine experiments, the presence of this analog resulted in the loss of viral replicative potential after 9-24 sequential passages of HIV in human CEM cells. In contrast, loss of viral replication was not observed in 28 control cultures passaged in the absence of the nucleoside analog, nor with other analogs tested. Sequence analysis of a portion of the HIV reverse transcriptase gene demonstrated a disproportionate increase in G -> A substitutions, mutations predicted to result from misincorporation of 5-OH-dC into the cDNA during reverse transcription. Thus, "lethal mutagenesis" driven by the class of deoxynucleoside analogs represented by 5-OH-dC could provide a new approach to treating HIV infections and, potentially, other viral infections.

  10. Particulate matter inhibits DNA repair and enhances mutagenesis.

    PubMed

    Mehta, Manju; Chen, Lung-Chi; Gordon, Terry; Rom, William; Tang, Moon-Shong

    2008-12-01

    Exposure to ambient air pollution has been associated with adverse health effects including lung cancer. A recent epidemiology study has established that each 10 microg/m3 elevation in long-term exposure to average PM2.5 ambient concentration was associated with approximately 8% of lung cancer mortality. The underlying mechanisms of how PM contributes to lung carcinogenesis, however, remain to be elucidated. We have recently found that transition metals such as nickel and chromium and oxidative stress induced lipid peroxidation metabolites such as aldehydes can greatly inhibit nucleotide excision repair (NER) and enhance carcinogen-induced mutations. Because PM is rich in metal and aldehyde content and can induce oxidative stress, we tested the effect of PM on DNA repair capacity in cultured human lung cells using in vitro DNA repair synthesis and host cell reactivation assays. We found that PM greatly inhibits NER for ultraviolet (UV) light and benzo(a)pyrene diol epoxide (BPDE) induced DNA damage in human lung cells. We further demonstrated that PM exposure can significantly increase both spontaneous and UV-induced mutagenesis. These results together suggest that the carcinogenicity of PM may act through its combined effect on suppression of DNA repair and enhancement of DNA replication errors. PMID:18804180

  11. N-ethyl-N-nitrosourea mutagenesis produced a small number of mice with altered plasma electrolyte levels

    PubMed Central

    Aigner, Bernhard; Rathkolb, Birgit; Klempt, Martina; Wagner, Sibylle; Michel, Dian; Hrabé de Angelis, Martin; Wolf, Eckhard

    2009-01-01

    Background Clinical chemical blood analysis including plasma electrolytes is routinely carried out for the diagnosis of various organ diseases. Phenotype-driven N-ethyl-N-nitrosourea (ENU) mouse mutagenesis projects used plasma electrolytes as parameters for the generation of novel animal models for human diseases. Methods Here, we retrospectively evaluated the use of the plasma electrolytes calcium, chloride, inorganic phosphorus, potassium and sodium in the Munich ENU mouse mutagenesis project where clinical chemical blood analysis was carried out on more than 20,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in various plasma parameter levels. Results We identified a small number of animals consistently exhibiting altered plasma electrolyte values. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for the parameters calcium and potassium. Published data from other phenotype-driven ENU projects also included only a small number of mutant lines which were generated according to altered plasma electrolyte levels. Conclusion Thus, use of plasma electrolytes detected few mouse mutants in ENU projects compared to other clinical chemical blood parameters. PMID:19505327

  12. A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

    PubMed Central

    Heijink, Anne Margriet; Blomen, Vincent A.; Bisteau, Xavier; Degener, Fabian; Matsushita, Felipe Yu; Foijer, Floris; van Vugt, Marcel A. T. M.

    2015-01-01

    The Wee1 cell cycle checkpoint kinase prevents premature mitotic entry by inhibiting cyclin-dependent kinases. Chemical inhibitors of Wee1 are currently being tested clinically as targeted anticancer drugs. Wee1 inhibition is thought to be preferentially cytotoxic in p53-defective cancer cells. However, TP53 mutant cancers do not respond consistently to Wee1 inhibitor treatment, indicating the existence of genetic determinants of Wee1 inhibitor sensitivity other than TP53 status. To optimally facilitate patient selection for Wee1 inhibition and uncover potential resistance mechanisms, identification of these currently unknown genes is necessary. The aim of this study was therefore to identify gene mutations that determine Wee1 inhibitor sensitivity. We performed a genome-wide unbiased functional genetic screen in TP53 mutant near-haploid KBM-7 cells using gene-trap insertional mutagenesis. Insertion site mapping of cells that survived long-term Wee1 inhibition revealed enrichment of G1/S regulatory genes, including SKP2, CUL1, and CDK2. Stable depletion of SKP2, CUL1, or CDK2 or chemical Cdk2 inhibition rescued the γ-H2AX induction and abrogation of G2 phase as induced by Wee1 inhibition in breast and ovarian cancer cell lines. Remarkably, live cell imaging showed that depletion of SKP2, CUL1, or CDK2 did not rescue the Wee1 inhibition-induced karyokinesis and cytokinesis defects. These data indicate that the activity of the DNA replication machinery, beyond TP53 mutation status, determines Wee1 inhibitor sensitivity, and could serve as a selection criterion for Wee1-inhibitor eligible patients. Conversely, loss of the identified S-phase genes could serve as a mechanism of acquired resistance, which goes along with development of severe genomic instability. PMID:26598692

  13. Engineering a Chemical Switch into the Light-driven Proton Pump Proteorhodopsin by Cysteine Mutagenesis and Thiol Modification.

    PubMed

    Harder, Daniel; Hirschi, Stephan; Ucurum, Zöhre; Goers, Roland; Meier, Wolfgang; Müller, Daniel J; Fotiadis, Dimitrios

    2016-07-25

    For applications in synthetic biology, for example, the bottom-up assembly of biomolecular nanofactories, modules of specific and controllable functionalities are essential. Of fundamental importance in such systems are energizing modules, which are able to establish an electrochemical gradient across a vesicular membrane as an energy source for powering other modules. Light-driven proton pumps like proteorhodopsin (PR) are excellent candidates for efficient energy conversion. We have extended the versatility of PR by implementing an on/off switch based on reversible chemical modification of a site-specifically introduced cysteine residue. The position of this cysteine residue in PR was identified by structure-based cysteine mutagenesis combined with a proton-pumping assay using E. coli cells overexpressing PR and PR proteoliposomes. The identified PR mutant represents the first light-driven proton pump that can be chemically switched on/off depending on the requirements of the molecular system. PMID:27294681

  14. CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations.

    PubMed

    Pan, Changtian; Ye, Lei; Qin, Li; Liu, Xue; He, Yanjun; Wang, Jie; Chen, Lifei; Lu, Gang

    2016-01-01

    The CRISPR/Cas9 system has successfully been used in various organisms for precise targeted gene editing. Although it has been demonstrated that CRISPR/Cas9 system can induce mutation in tomato plants, the stability of heredity in later generations and mutant specificity induced by the CRISPR/Cas9 system in tomato plants have not yet been elucidated in detail. In this study, two genes, SlPDS and SlPIF4, were used for testing targeted mutagenesis in tomato plants through an Agrobacterium tumefaciens-mediated transformation method. A high mutation frequency was observed in all tested targets in the T0 transgenic tomato plants, with an average frequency of 83.56%. Clear albino phenotypes were observed for the psd mutants. High frequencies of homozygous and biallelic mutants were detected even in T0 plants. The majority of the detected mutations were 1- to 3-nucleotide deletions, followed by 1-bp insertions. The target mutations in the T0 lines were stably transmitted to the T1 and T2 generations, without new modifications or revision. Off-target activities associated with SlPDS and SlPIF4 were also evaluated by sequencing the putative off-target sites, and no clear off-target events were detected. Our results demonstrate that the CRISPR/Cas9 system is an efficient tool for generating stable and heritable modifications in tomato plants. PMID:27097775

  15. PiggyBac Transposon-Mediated Mutagenesis in Rats Reveals a Crucial Role of Bbx in Growth and Male Fertility.

    PubMed

    Wang, Chieh-Ying; Tang, Ming-Chu; Chang, Wen-Chi; Furushima, Kenryo; Jang, Chuan-Wei; Behringer, Richard R; Chen, Chun-Ming

    2016-09-01

    Bobby sox homolog (Bbx) is an evolutionally conserved gene, but its biological function remains elusive. Here, we characterized defects of Bbx mutant rats that were created by PiggyBac-mediated insertional mutagenesis. Smaller body size and male infertility were the two major phenotypes of homozygous Bbx mutants. Bbx expression profile analysis showed that Bbx was more highly expressed in the testis and pituitary gland than in other organs. Histology and hormonal gene expression analysis of control and Bbx-null pituitary glands showed that loss of Bbx appeared to be dispensable for pituitary histogenesis and the expression of major hormones. BBX was localized in the nuclei of postmeiotic spermatids and Sertoli cells in wild-type testes, but absent in mutant testes. An increased presence of aberrant multinuclear giant cells and apoptotic cells was observed in mutant seminiferous tubules. TUNEL-positive cells costained with CREM (round spermatid marker), but not PLZF (spermatogonia marker), gammaH2Ax (meiotic spermatocyte marker), or GATA4 (Sertoli cell marker). Finally, there were drastically reduced numbers and motility of epididymal sperm from Bbx-null rats. These results suggest that loss of BBX induces apoptosis of postmeiotic spermatids and results in spermiogenesis defects and infertility. PMID:27465138

  16. CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations

    PubMed Central

    Pan, Changtian; Ye, Lei; Qin, Li; Liu, Xue; He, Yanjun; Wang, Jie; Chen, Lifei; Lu, Gang

    2016-01-01

    The CRISPR/Cas9 system has successfully been used in various organisms for precise targeted gene editing. Although it has been demonstrated that CRISPR/Cas9 system can induce mutation in tomato plants, the stability of heredity in later generations and mutant specificity induced by the CRISPR/Cas9 system in tomato plants have not yet been elucidated in detail. In this study, two genes, SlPDS and SlPIF4, were used for testing targeted mutagenesis in tomato plants through an Agrobacterium tumefaciens-mediated transformation method. A high mutation frequency was observed in all tested targets in the T0 transgenic tomato plants, with an average frequency of 83.56%. Clear albino phenotypes were observed for the psd mutants. High frequencies of homozygous and biallelic mutants were detected even in T0 plants. The majority of the detected mutations were 1- to 3-nucleotide deletions, followed by 1-bp insertions. The target mutations in the T0 lines were stably transmitted to the T1 and T2 generations, without new modifications or revision. Off-target activities associated with SlPDS and SlPIF4 were also evaluated by sequencing the putative off-target sites, and no clear off-target events were detected. Our results demonstrate that the CRISPR/Cas9 system is an efficient tool for generating stable and heritable modifications in tomato plants. PMID:27097775

  17. TALEN-Based Mutagenesis of Lipoxygenase LOX3 Enhances the Storage Tolerance of Rice (Oryza sativa) Seeds

    PubMed Central

    Li, Zhenwei; Zhang, Jianfu; Li, Xin; Dong, Jiangli; Wang, Tao

    2015-01-01

    The deterioration of rice grain reduces the quality of rice, resulting in serious economic losses for farmers. Lipoxygenases (LOXs) catalyze the dioxygenation of polyunsaturated fatty acids with at least one cis,cis-1,4-pentadiene to form hydroperoxide, which is a major factor influencing seed longevity and viability. Recently, genome editing, an essential tool employed in reverse genetics, has been used experimentally to investigate basic plant biology or to modify crop plants for the improvement of important agricultural traits. In this study, we performed targeted mutagenesis in rice using transcription activator-like effector nucleases (TALENs) to improve seed storability. A modified ligation-independent cloning method (LIC) was employed to allow for the quick and efficient directional insertion of TALEN monomer modules into destination vectors used in plants. We demonstrated the feasibility and flexibility of the technology by developing a set of modular vectors for genome editing. After construction and validation, the TALEN pairs were used to create stable transgenic rice lines via Agrobacterium-mediated transformation. One heterozygous mutant (4%) was recovered from 25 transgenic NPTII-resistant lines, and the mutation was transmitted to the next generation. Further molecular and protein level experiments verified LOX3 deficiency and demonstrated the improvement of seed storability. Our work provides a flexible genome editing tool for improving important agronomic traits, as well as direct evidence that Lox3 has only a limited impact on seed longevity. PMID:26641666

  18. Natural channel protein inserts and functions in a completely artificial, solid-supported bilayer membrane

    PubMed Central

    Zhang, Xiaoyan; Fu, Wangyang; Palivan, Cornelia G.; Meier, Wolfgang

    2013-01-01

    Reconstitution of membrane proteins in artificial membrane systems creates a platform for exploring their potential for pharmacological or biotechnological applications. Previously, we demonstrated amphiphilic block copolymers as promising building blocks for artificial membranes with long-term stability and tailorable structural parameters. However, the insertion of membrane proteins has not previously been realized in a large-area, stable, and solid-supported artificial membrane. Here, we show the first, preliminary model of a channel membrane protein that is functionally incorporated in a completely artificial polymer, tethered, solid-supported bilayer membrane (TSSBM). Unprecedented ionic transport characteristics that differ from previous results on protein insertion into planar, free-standing membranes, are identified. Our findings mark a change in understanding protein insertion and ion flow within natural channel proteins when inserted in an artificial TSSBM, thus holding great potential for numerous applications such as drug screening, trace analyzing, and biosensing. PMID:23846807

  19. Mechanism of Is1 Transposition in E. COLI: Choice between Simple Insertion and Cointegration

    PubMed Central

    Biel, Susan W.; Berg, Douglas E.

    1984-01-01

    Insertion element IS1 and IS1-based transposon Tn9 generate cointegrates (containing vector and target DNAs joined by duplicate copies of IS1 or Tn9) and simple insertions (containing IS1 or Tn9 detached from vector sequences). Based on studies of transposon Tn5 we had proposed a conservative (non-replicative) model for simple insertion. Others had proposed that all transposition is replicative, occurring in a rolling circle structure, and that the way DNA strands are joined when replication terminates determines whether a simple insertion or a cointegrate is formed.—We selected for the transposition of amp and cam resistance markers from pBR322::Tn9 plasmids to an F factor in recA- E. coli and identified products containing three and four copies of IS1, corresponding to true cointegrates (from monomeric plasmids), and simple insertions (from dimeric plasmids). The simple insertions with four copies of IS1 outnumbered those with three by a ratio of about 3:1, whereas true cointegrates containing three copies of IS1 were more numerous than those with four.—A straightforward rolling circle model had predicted that the simple insertions containing three copies of IS1 should be more frequent than those with four. Because we obtained the opposite result we propose that simple insertions only arise when the element fails to replicate or if replication starts but then terminates prematurely. The two classes of products, simple insertions and cointegrates, reflect alternative conservative and replicative fates, respectively, of an early intermediate in transposition. PMID:6094304

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

    PubMed

    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. PMID:25855067