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Sample records for mutant egfrviii significantly

  1. Aptamer targeting EGFRvIII mutant hampers its constitutive autophosphorylation and affects migration, invasion and proliferation of glioblastoma cells

    PubMed Central

    Camorani, Simona; Crescenzi, Elvira; Colecchia, David; Carpentieri, Andrea; Amoresano, Angela; Fedele, Monica; Chiariello, Mario; Cerchia, Laura

    2015-01-01

    Glioblastoma Multiforme (GBM) is the most common and aggressive human brain tumor, associated with very poor survival despite surgery, radiotherapy and chemotherapy. The epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor β (PDGFRβ) are hallmarks in GBM with driving roles in tumor progression. In approximately half of the tumors with amplified EGFR, the EGFRvIII truncated extracellular mutant is detected. EGFRvIII does not bind ligands, is highly oncogenic and its expression confers resistance to EGFR tyrosine kinase inhibitors (TKIs). It has been demonstrated that EGFRvIII-dependent cancers may escape targeted therapy by developing dependence on PDGFRβ signaling, thus providing a strong rationale for combination therapy aimed at blocking both EGFRvIII and PDGFRβ signaling. We have recently generated two nuclease resistant RNA aptamers, CL4 and Gint4.T, as high affinity ligands and inhibitors of the human wild-type EGFR (EGFRwt) and PDGFRβ, respectively. Herein, by different approaches, we demonstrate that CL4 aptamer binds to the EGFRvIII mutant even though it lacks most of the extracellular domain. As a consequence of binding, the aptamer inhibits EGFRvIII autophosphorylation and downstream signaling pathways, thus affecting migration, invasion and proliferation of EGFRvIII-expressing GBM cell lines. Further, we show that targeting EGFRvIII by CL4, as well as by EGFR-TKIs, erlotinib and gefitinib, causes upregulation of PDGFRβ. Importantly, CL4 and gefitinib cooperate with the anti-PDGFRβ Gint4.T aptamer in inhibiting cell proliferation. The proposed aptamer-based strategy could have impact on targeted molecular cancer therapies and may result in progresses against GBMs. PMID:26461476

  2. ZD6474, a Multitargeted Inhibitor for Receptor Tyrosine Kinases, Suppresses Growth of Gliomas Expressing an Epidermal Growth Factor Receptor Mutant, EGFRvIII, in the Brain

    PubMed Central

    Yiin, Jia-Jean; Hu, Bo; Schornack, Paul A.; Sengar, Raghvendra S.; Liu, Kun-wei; Feng, Haizhong; Lieberman, Frank S.; Chiou, Shih-Hwa; Sarkaria, Jann N.; Wiener, Erik C.; Ma, Hsin-I; Cheng, Shi-Yuan

    2010-01-01

    Epidermal growth factor receptor (EGFR) vIII is a mutated EGFR that is frequently overexpressed in glioblastomas and implicated in response to receptor tyrosine kinase inhibitors. In this study, we investigate the effect of ZD6474 (ZACTIMA, vandetanib), a dual inhibitor for vascular endothelial growth factor receptor 2 and EGFR on growth and angiogenesis of gliomas expressing EGFRvIII. We used two glioma xenograft models, U87MG cells overexpressing EGFRvIII and short-term cultured primary glioma GBM8 cells with EGFRvIII. ZD6474 inhibited tumor growth and angiogenesis and induced cell apoptosis in various brain gliomas. Moreover, significant inhibition of EGFRvIII-expressing U87MG and GBM8 gliomas was observed compared with their controls. Magnetic resonance imaging analysis using the apparent diffusion coefficient and three-dimensional T2*weighed measurements validated ZD6474 inhibition on tumor growth and angiogenesis in EGFRvIII-expressing GBM8 gliomas. Mechanistically, ZD6474 shows better inhibition of cell growth and survival of U87MG/EGFRvIII, GBM6, and GBM8 cells that express EGFRvIII than U87MG or GBM14 cells that have nondetectable EGFRvIII through attenuation of activated phosphorylation of signal transducer and activator of transcription 3, Akt, and Bcl-XL expression. Albeit in lesser extent, ZD6474 also displays suppressions of U87MG/EGFR and GBM12 cells that overexpress wild-type EGFR. Additionally, ZD6474 inhibits activation of extracellular signal-regulated kinase 1/2 in both types of cells, and expression of a constitutively active phosphoinositide 3-kinases partially rescued ZD6474 inhibition in U87MG/EGFRvIII cells. Taken together, these data show that ZD6474 significantly inhibited growth and angiogenesis of gliomas expressing EGFRvIII by specifically blocking EGFRvIII-activated signaling mediators, suggesting a potential application of ZD6474 in treatments for glioblastomas that overexpress EGFRvIII. PMID:20371720

  3. EGFRvIII stimulates glioma growth and invasion through PKA-dependent serine phosphorylation of Dock180.

    PubMed

    Feng, H; Hu, B; Vuori, K; Sarkaria, J N; Furnari, F B; Cavenee, W K; Cheng, S-Y

    2014-05-01

    Glioblastomas (GBMs), the most common and malignant brain tumors, are highly resistant to current therapies. The failure of targeted therapies against aberrantly activated oncogenic signaling, such as that of the EGFR-PI3K/Akt pathway, underscores the urgent need to understand alternative downstream pathways and to identify new molecular targets for the development of more effective treatments for gliomas. Here, we report that EGFRvIII (ΔEGFR/de2-7EGFR), a constitutively active EGFR mutant that is frequently co-overexpressed with EGFR in clinical GBM tumors, promotes glioma growth and invasion through protein kinase A (PKA)-dependent phosphorylation of Dock180, a bipartite guanine nucleotide exchange factor (GEF) for Rac1. We demonstrate that EGFRvIII induces serine phosphorylation of Dock180, stimulates Rac1 activation and glioma cell migration. Treatments of glioma cells using the PKA inhibitors H-89 and KT5720, overexpression of a PKA inhibitor (PKI), and in vitro PKA kinase assays show that EGFRvIII induction of serine phosphorylation of Dock180 is PKA-dependent. Significantly, PKA induces phosphorylation of Dock180 at amino acid residue S1250 that resides within its Rac1-activating DHR-2 domain. Expression of the Dock180(S1250L) mutant, but not wild type Dock180(WT), protein in EGFRvIII-expressing glioma cells inhibited receptor-stimulated cell proliferation, survival, migration in vitro and glioma tumor growth and invasion in vivo. Together, our findings describe a novel mechanism by which EGFRvIII drives glioma tumorigenesis and invasion through PKA-dependent phosphorylation of Dock180, thereby suggesting that targeting EGFRvIII-PKA-Dock180-Rac1 signaling axis could provide a novel pathway to develop potential therapeutic strategies for malignant gliomas. PMID:23728337

  4. CAR-Engineered NK Cells Targeting Wild-Type EGFR and EGFRvIII Enhance Killing of Glioblastoma and Patient-Derived Glioblastoma Stem Cells

    PubMed Central

    Han, Jianfeng; Chu, Jianhong; Keung Chan, Wing; Zhang, Jianying; Wang, Youwei; Cohen, Justus B.; Victor, Aaron; Meisen, Walter H.; Kim, Sung-hak; Grandi, Paola; Wang, Qi-En; He, Xiaoming; Nakano, Ichiro; Chiocca, E. Antonio; Glorioso III, Joseph C.; Kaur, Balveen; Caligiuri, Michael A.; Yu, Jianhua

    2015-01-01

    Glioblastoma (GB) remains the most aggressive primary brain malignancy. Adoptive transfer of chimeric antigen receptor (CAR)-modified immune cells has emerged as a promising anti-cancer approach, yet the potential utility of CAR-engineered natural killer (NK) cells to treat GB has not been explored. Tumors from approximately 50% of GB patients express wild-type EGFR (wtEGFR) and in fewer cases express both wtEGFR and the mutant form EGFRvIII; however, previously reported CAR T cell studies only focus on targeting EGFRvIII. Here we explore whether both wtEGFR and EGFRvIII can be effectively targeted by CAR-redirected NK cells to treat GB. We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells. EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-γ production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner. In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival. These findings support intracranial administration of NK-92-EGFR-CAR cells represents a promising clinical strategy to treat GB. PMID:26155832

  5. EGFRvIII mCAR-modified T-cell therapy cures mice with established intracerebral glioma and generates host immunity against tumor-antigen loss

    PubMed Central

    Sampson, John H.; Choi, Bryan D.; Sanchez-Perez, Luis; Suryadevara, Carter M.; Snyder, David J.; Flores, Catherine T.; Schmittling, Robert J.; Nair, Smita; Reap, Elizabeth A.; Norberg, Pamela K.; Herndon, James E.; Kuan, Chien-Tsun; Morgan, Richard A.; Rosenberg, Steven A.; Johnson, Laura A.

    2014-01-01

    Purpose Chimeric antigen receptor (CAR) transduced T cells represent a promising immune therapy that has been shown to successfully treat cancers in mice and humans. However, CARs targeting antigens expressed in both tumors and normal tissues have led to significant toxicity. Preclinical studies have been limited by the use of xenograft models that do not adequately recapitulate the immune system of a clinically relevant host. EGFRvIII is a constitutively activated mutant of the naturally occurring epidermal growth factor receptor and is antigenically identical in both human and mouse glioma, but is also completely absent from any normal tissues. Experimental Design We developed a third-generation, EGFRvIII-specific murine CAR (mCAR), and performed tests to determine its efficacy in a fully immune-competent mouse model of malignant glioma. Results At elevated doses, infusion with EGFRvIII mCAR T cells led to cures in all mice with brain tumors. Additionally, antitumor efficacy was found to be dependent on lymphodepletive host conditioning. Selective blockade with EGFRvIII soluble peptide significantly abrogated the activity of EGFRvIII mCAR T cells in vitro and in vivo, and may offer a novel strategy to enhance the safety profile for CAR-based therapy. Lastly, mCAR-treated, cured mice were resistant to rechallenge with EGFRvIIINEG tumors, suggesting generation of host immunity against additional tumor antigens. Conclusion All together, these data support that third-generation, EGFRvIII specific mCARs are effective against gliomas in the brain and highlight the importance of syngeneic, immune-competent models in the preclinical evaluation of tumor immunotherapies. PMID:24352643

  6. EGFRvIII does not affect radiosensitivity with or without gefitinib treatment in glioblastoma cells

    PubMed Central

    Struve, Nina; Riedel, Matthias; Schulte, Alexander; Rieckmann, Thorsten; Grob, Tobias J.; Gal, Andreas; Rothkamm, Kai; Lamszus, Katrin; Petersen, Cordula; Dikomey, Ekkehard; Kriegs, Malte

    2015-01-01

    Background Glioblastomas (GBM) are often characterized by an elevated expression of the epidermal growth factor receptor variant III (EGFRvIII). We used GBM cell lines with native EGFRvIII expression to determine whether this EGFR variant affects radiosensitivity with or without EGFR targeting. Methods Experiments were performed with GBM cell lines lacking (LN229, U87MG, U251, CAS-1) or endogenously expressing EGFRvIII (BS153, DKMG). The two latter cell lines were also used to establish sublines with a low (−) or a high proportion (+) of cells expressing EGFRvIII. EGFR signaling and the cell cycle were analyzed using Western blot and flow cytometry; cell survival was assessed by colony forming assay and double-strand break repair capacity by immunofluorescence. Results DKMG and BS153 parental cells with heterogeneous EGFRvIII expression were clearly more radiosensitive compared to other GBM cell lines without EGFRvIII expression. However, no significant difference was observed in cell proliferation, clonogenicity or radiosensitivity between the EGFRvIII− and + sublines derived from DKMG and BS153 parental cells. Expression of EGFRvIII was associated with decreased DSB repair capacity for BS153 but not for DKMG cells. The effects of EGFR targeting by gefitinib alone or in combination with irradiation were also found not to depend on EGFRvIII expression. Gefitinib was only observed to influence the proliferation of EGFRvIII− BS153 cells. Conclusion The data indicate that EGFRvIII does not alter radiosensitivity with or without anti-EGFR treatment. PMID:26418954

  7. EGFRvIII and c-Met pathway inhibitors synergize against PTEN-null/EGFRvIII+ glioblastoma xenografts

    PubMed Central

    Lal, Bachchu; Goodwin, Courtney R.; Sang, Yingying; Foss, Catherine A.; Cornet, Kathrine; Muzamil, Sameena; Pomper, Martin G.; Kim, Jin; Laterra, John

    2010-01-01

    Receptor tyrosine kinase (RTK) systems, such as hepatocyte growth factor (HGF) and its receptor c-Met, and EGFR, are responsible for the malignant progression of multiple solid tumors. Recent research shows that these RTK systems co-modulate overlapping and dynamically adaptable oncogenic downstream signaling pathways. This paper investigates how EGFRvIII, a constitutively active EGFR deletion mutant, alters tumor growth and signaling responses to RTK inhibition in PTEN-null/HGF+/c-Met+ glioma xenografts. We show that a neutralizing anti-HGF mAb (L2G7) potently inhibits tumor growth and the activation of Akt and MAPK in PTEN-null/HGF+/c-Met+/EGFRvIII−U87 glioma xenografts (U87wt). Isogenic EGFRvIII+ U87 xenografts (U87-EGFRvIII), which grew 5-times more rapidly than U87-wt xenografts, were unresponsive to EGFRvIII inhibition by erlotinib and were only minimally responsive to anti-HGF mAb. EGFRvIII-expression diminished the magnitude of Akt inhibition and completely prevented MAPK inhibition by L2G7. Despite the lack of response to L2G7 or erlotinib as single agents, their combination synergized to produce substantial anti-tumor effects (inhibited tumor cell proliferation, enhanced apoptosis, arrested tumor growth, prolonged animal survival), against subcutaneous and orthotopic U87-EGFRvIII xenografts. The dramatic response to combining HGF:c-Met and EGFRvIII pathway inhibitors in U87-EGFRvIII xenografts occurred in the absence of Akt and MAPK inhibition. These findings show that combining c-Met and EGFRvIII pathway inhibitors can generate potent anti-tumor effects in PTEN-null tumors. They also provide insights into how EGFRvIII and c-Met may alter signaling networks and reveal the potential limitations of certain biochemical biomarkers to predict the efficacy of RTK inhibition in genetically diverse cancers. PMID:19584231

  8. Nimotuzumab enhances temozolomide-induced growth suppression of glioma cells expressing mutant EGFR in vivo.

    PubMed

    Nitta, Yusuke; Shimizu, Saki; Shishido-Hara, Yukiko; Suzuki, Kaori; Shiokawa, Yoshiaki; Nagane, Motoo

    2016-03-01

    A mutant form of epidermal growth factor receptor (EGFR), EGFRvIII, is common in glioblastoma (GBM) and confers enhanced tumorigenic activity and drug resistance. Nimotuzumab, an anti-EGFR antibody, has shown preclinical and clinical activity to GBM, but its specific activity against EGFRvIII has not been fully investigated. Human glioma U87MG or LNZ308 cells overexpressing either wild-type (wt) EGFR or EGFRvIII were treated with nimotuzumab, temozolomide, or both. Expression and phosphorylation status of molecules were determined by Western blot analysis. Methylation status of promoter region of O(6) -methylguanine-DNA methyltransferase (MGMT) was detected by methylation-specific PCR. Antitumor activity was tested using nude mice bearing either subcutaneous or intracerebral xenografts along with analyses of EGFR phosphorylation status, proliferation, apoptosis, and vessel density. Nimotuzumab treatment resulted in reduction of EGFRvIII tyrosine phosphorylation with a decrease in Akt phosphorylation that was greater than that of wtEGFR. Correspondingly, antitumor effects, growth suppression and survival elongation, were more significant in mice bearing either subcutaneous or intracerebral tumor expressing EGFRvIII than in those expressing wtEGFR. These effects were markedly increased when temozolomide was combined with nimotuzumab. The post-treatment recurrent brain tumors exhibited a decrease in expression of the mismatch repair (MMR) proteins, MSH6 and MLH1, but their methylated MGMT status did not changed. Nimotuzumab has in vivo antitumor activity against GBM, especially those expressing EGFRvIII, when combined with temozolomide. This could provide a basis for preselection of patients with GBM by EGFR status who might benefit from the nimotuzumab and temozolomide combination therapy. PMID:26778701

  9. Mesenchymal Stem Cells Modified with a Single-Chain Antibody against EGFRvIII Successfully Inhibit the Growth of Human Xenograft Malignant Glioma

    PubMed Central

    Balyasnikova, Irina V.; Ferguson, Sherise D.; Sengupta, Sadhak; Han, Yu; Lesniak, Maciej S.

    2010-01-01

    Background Glioblastoma multiforme is the most lethal brain tumor with limited therapeutic options. Antigens expressed on the surface of malignant cells are potential targets for antibody-mediated gene/drug delivery. Principal Findings In this study, we investigated the ability of genetically modified human mesenchymal stem cells (hMSCs) expressing a single-chain antibody (scFv) on their surface against a tumor specific antigen, EGFRvIII, to enhance the therapy of EGFRvIII expressing glioma cells in vivo. The growth of U87-EGFRvIII was specifically delayed in co-culture with hMSC-scFvEGFRvIII. A significant down-regulation was observed in the expression of pAkt in EGFRvIII expressing glioma cells upon culture with hMSC-scFvEGFRvIII vs. controls as well as in EGFRvIII expressing glioma cells from brain tumors co-injected with hMSC-scFvEGFRvIII in vivo. hMSC expressing scFvEGFRvIII also demonstrated several fold enhanced retention in EGFRvIII expressing flank and intracranial glioma xenografts vs. control hMSCs. The growth of U87-EGFRvIII flank xenografts was inhibited by 50% in the presence of hMSC-scFvEGFRvIII (p<0.05). Moreover, animals co-injected with U87-EGFRvIII and hMSC-scFvEGFRvIII intracranially showed significantly improved survival compared to animals injected with U87-EGFRvIII glioma cells alone or with control hMSCs. This survival was further improved when the same animals received an additional dosage of hMSC-scFvEGFRvIII two weeks after initial tumor implantation. Of note, EGFRvIII expressing brain tumors co-injected with hMSCs had a lower density of CD31 expressing blood vessels in comparison with control tumors, suggesting a possible role in tumor angiogenesis. Conclusions/Significance The results presented in this study illustrate that genetically modified MSCs may function as a novel therapeutic vehicle for malignant brain tumors. PMID:20305783

  10. Clinical significance of hepatitis B surface antigen mutants

    PubMed Central

    Coppola, Nicola; Onorato, Lorenzo; Minichini, Carmine; Di Caprio, Giovanni; Starace, Mario; Sagnelli, Caterina; Sagnelli, Evangelista

    2015-01-01

    Hepatitis B virus (HBV) infection is a major public health problem in many countries, with nearly 300 million people worldwide carrying HBV chronic infection and over 1 million deaths per year due to cirrhosis and liver cancer. Several hepatitis B surface antigen (HBsAg) mutations have been described, most frequently due to a single amino acid substitution and seldom to a nucleotide deletion. The majority of mutations are located in the S region, but they have also been found in the pre-S1 and pre-S2 regions. Single amino acid substitutions in the major hydrophilic region of HBsAg, called the “a” determinant, have been associated with immune escape and the consequent failure of HBV vaccination and HBsAg detection, whereas deletions in the pre-S1 or pre-S2 regions have been associated with the development of hepatocellular carcinoma. This review article will focus on the HBsAg mutants and their biological and clinical implications. PMID:26644816

  11. F25P preproinsulin abrogates the secretion of pro-growth factors from EGFRvIII cells and suppresses tumor growth in an EGFRvIII/wt heterogenic model.

    PubMed

    Wei, Jian-Wei; Cui, Jing-Qiu; Zhou, Xuan; Fang, Chuan; Tan, Yan-Li; Chen, Lu-Yue; Yang, Chao; Liu, Ming; Kang, Chun-Sheng

    2016-09-28

    Extensive heterogeneity is a defining hallmark of glioblastoma multiforme (GBM) at the cellular and molecular levels. EGFRvIII, the most common EGFR mutant, is expressed in 24-67% of cases and strongly indicates a poor survival prognosis. By co-expressing EGFRvIII and EGFRwt, we established an EGFRvIII/wt heterogenic model. Using this approach, we confirmed that a mixture of EGFRvIII and EGFRwt at a certain ratio could clearly enhance tumor growth in vitro and in vivo compared with EGFRwt cells, thereby indicating that EGFRvIII cells promote tumor growth. Furthermore, we demonstrated that the EGFRvIII cells could support the growth of EGFRwt cells by secreting growth factors, thus acting as the principal source for maintaining tumor survival. F25P preproinsulin effectively reduced the concentrations of EGF, VEGF, and MMP-9 in the blood of tumor-bearing mice by competitively inhibiting the endoplasmic reticulum signal peptidase and increased the overall survival in orthotopic models. Taken together, our results provided an effective therapy of F25P preproinsulin in the EGFRvIII/wt heterogenic model. PMID:27317648

  12. Guanylate binding protein-1 mediates EGFRvIII and promotes glioblastoma growth in vivo but not in vitro

    PubMed Central

    Cheng, Yanwei; Mukasa, Akitaki; Ma, Jiawei; Hong, Lei; Yu, Shuye; Sun, Lili; Huang, Qiang; Purow, Benjamin; Li, Ming

    2016-01-01

    Glioblastoma multiforme (GBM) is the most common and deadly primary brain tumor in adults. Epidermal growth factor receptor (EGFR) is frequently amplified and mutated in GBM. We previously reported that Guanylate binding protein-1 (GBP1) is a novel transcriptional target gene of EGFR and plays a role in GBM invasion. Here we demonstrate that GBP1 can also be induced by EGFRvIII at the transcriptional level through the p38 MAPK/Yin Yang 1 (YY1) signaling pathway. Silencing of GBP1 by RNA interference significantly inhibits EGFRvIII-mediated GBM cell proliferation in vitro and in a mouse model. Overexpression of GBP1 has no obvious effect on glioblastoma cell proliferation in vitro. In contrast, in an orthotopic glioma mouse model GBP1 overexpression significantly promotes glioma growth and reduces survival rate of glioma-bearing mice by increasing cell proliferation and decreasing cell apoptosis in tumor. Clinically, GBP1 expression is elevated in human GBM tumors and positively correlates with EGFRvIII status in GBM specimens, and its expression is inversely correlated with the survival rate of GBM patients. Taken together, these results reveal that GBP1 may serve as a potential therapeutic target for GBMs with EGFRvIII mutation. PMID:26848767

  13. Head and Neck Squamous Cell Carcinomas Do Not Express EGFRvIII

    SciTech Connect

    Melchers, Lieuwe J.; Clausen, Martijn J.A.M.; Mastik, Mirjam F.; Slagter-Menkema, Lorian; Laan, Bernard F.A.M. van der; Roodenburg, Jan L.N.; Schuuring, Ed

    2014-10-01

    Purpose: To assess the prevalence of EGFRvIII, a specific variant of EGFR (epidermal growth factor receptor), in 3 well-defined cohorts of head and neck squamous cell carcinoma (HNSCC). Methods and Materials: Immunohistochemistry for the specific detection of EGFRvIII using the L8A4 antibody was optimized on formalin-fixed, paraffin-embedded tissue using glioblastoma tissue. It was compared with EGFR and EGFRvIII RNA expression using a specific reverse transcription–polymerase chain reaction also optimized for formalin-fixed, paraffin-embedded tissue. Tissue microarrays including 531 HNSCCs of various stages with complete clinicopathologic and follow-up data were tested for the presence of EGFRvIII. Results: None of the 531 cases showed EGFRvIII protein expression. Using an immunohistochemistry protocol reported by others revealed cytoplasmic staining in 8% of cases. Reverse transcription–polymerase chain reaction for the EGFRvIII transcript of the 28 highest cytoplasmic staining cases, as well as 69 negative cases, did not show expression in any of the tested cases, suggesting aspecific staining by a nonoptimal protocol. Conclusions: The EGFRvIII mutation is not present in HNSCC. Therefore, EGFRvIII does not influence treatment response in HNSCC and is not a usable clinical prognostic marker.

  14. EGFRvIII promotes glioma angiogenesis and growth through the NF-κB, interleukin-8 pathway

    PubMed Central

    Bonavia, R; Inda, MM; Vandenberg, S; Cheng, S-Y; Nagane, M; Hadwiger, P; Tan, P; Sah, DWY; Cavenee, WK; Furnari, FB

    2012-01-01

    Sustaining a high growth rate requires tumors to exploit resources in their microenvironment. One example of this is the extensive angiogenesis that is a typical feature of high-grade gliomas. Here, we show that expression of the constitutively active mutant epidermal growth factor receptor, ΔEGFR (EGFRvIII, EGFR*, de2-7EGFR) is associated with significantly higher expression levels of the pro-angiogenic factor interleukin (IL)-8 in human glioma specimens and glioma stem cells. Furthermore, the ectopic expression of ΔEGFR in different glioma cell lines caused up to 60-fold increases in the secretion of IL-8. Xenografts of these cells exhibit increased neovascularization, which is not elicited by cells overexpressing wildtype (wt)EGFR or ΔEGFR with an additional kinase domain mutation. Analysis of the regulation of IL-8 by site-directed mutagenesis of its promoter showed that ΔEGFR regulates its expression through the transcription factors nuclear factor (NF)-κB, activator protein 1 (AP-1) and CCAAT/enhancer binding protein (C/EBP). Glioma cells overexpressing ΔEGFR showed constitutive activation and DNA binding of NF-κB, overexpression of c-Jun and activation of its upstream kinase c-Jun N-terminal kinase (JNK) and overexpression of C/EBPβ. Selective pharmacological or genetic targeting of the NF-κB or AP-1 pathways efficiently blocked promoter activity and secretion of IL-8. Moreover, RNA interference-mediated knock-down of either IL-8 or the NF-κB subunit p65, in ΔEGFR-expressing cells attenuated their ability to form tumors and to induce angiogenesis when injected subcutaneously into nude mice. On the contrary, the overexpression of IL-8 in glioma cells lacking ΔEGFR potently enhanced their tumorigenicity and produced highly vascularized tumors, suggesting the importance of this cytokine and its transcription regulators in promoting glioma angiogenesis and tumor growth. PMID:22139077

  15. Nuclear EGFRvIII resists hypoxic microenvironment induced apoptosis via recruiting ERK1/2 nuclear translocation.

    PubMed

    Xie, Hui; Yang, Jinfeng; Xing, Wenjing; Dong, Yucui; Ren, Huan

    2016-02-01

    Glioblastoma (GBM) is the most aggressive type of primary brain tumor. Its interaction with the tumor microenvironment promotes tumor progression. Furthermore, GBM bearing expression of EGFRvIII displays more adaptation to tumor microenvironment related stress. But the mechanisms were poorly understood. Here, we presented evidence that in the human U87MG glioblastoma tumor model, EGFRvIII overexpression led aberrant kinase activation and nuclear translocation of EGFRvIII/ERK1/2 under hypoxia, which induced growth advantage by resisting apoptosis. Additionally, EGFRvIII defective in nuclear entry impaired this capacity in hypoxia adaptation, and partially interrupted ERK1/2 nuclear translocation. Pharmacology or genetic interference ERK1/2 decreased hypoxia resistance triggered by EGFRvIII expression, but not EGFRvIII nuclear translocation. In summary, this study identified a novel role for EGFRvIII in hypoxia tolerance, supporting an important link between hypoxia and subcellular localization alterations of the receptor. PMID:26742423

  16. Retargeted human avidin-CAR T cells for adoptive immunotherapy of EGFRvIII expressing gliomas and their evaluation via optical imaging

    PubMed Central

    Peng, Zhiping; Sun, Haojie; Zhang, Mingzhi; Zhang, Jianning; Liu, Shuang; Hao, Limin; Lu, Guoqiu; Zheng, Kangcheng; Gong, Xikui; Wu, Di; Wang, Fan; Shen, Li

    2015-01-01

    There has been significant progress in the design of chimeric antigen receptors (CAR) for adoptive immunotherapy targeting tumor-associated antigens. However, the challenge of monitoring the therapy in real time has been continually ignored. To address this issue, we developed optical molecular imaging approaches to evaluate a recently reported novel CAR strategy for adoptive immunotherapy against glioma xenografts expressing EGFRvIII. We initially biotinylated a novel anti-EGFRvIII monoclonal antibody (biotin-4G1) to pre-target EGFRvIII+ gliomas and then redirect activated avidin-CAR expressing T cells against the pre-targeted biotin-4G1. By optical imaging study and bio-distribution analysis, we confirmed the specificity of pre-target and target and determined the optimal time for T cells adoptive transfer in vivo. The results showed this therapeutic strategy offered efficient therapy effect to EGFRvIII+ glioma-bearing mice and implied that optical imaging is a highly useful tool in aiding in the instruction of clinical CAR-T cells adoptive transfer in future. PMID:26124178

  17. EGFRvIII expression and PTEN loss synergistically induce chromosomal instability and glial tumors

    PubMed Central

    Li, Li; Dutra, Amalia; Pak, Evgenia; Labrie, Joseph E.; Gerstein, Rachel M.; Pandolfi, Pier Paolo; Recht, Larry D.; Ross, Alonzo H.

    2009-01-01

    Glioblastomas often show activation of epidermal growth factor receptor (EGFR) and loss of PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor, but it is not known if these two genetic lesions act together to transform cells. To answer this question, we infected PTEN−/− neural precursor cells with a retrovirus encoding EGFRvIII, which is a constitutively activated receptor. EGFRvIII PTEN−/− cells formed highly mitotic tumors with nuclear pleomorphism, necrotic areas, and glioblastoma markers. The transformed cells showed increased cell proliferation, centrosome amplification, colony formation in soft agar, self-renewal, expression of the stem cell marker CD133, and resistance to oxidative stress and ionizing radiation. The RAS/mitogen-activated protein kinase (ERK) and phosphoinositide 3-kinase/protein kinase B (PI3K/ Akt) pathways were activated, and checkpoint kinase 1 (Chk1), the DNA damage regulator, was phosphorylated at S280 by Akt, suppressing Chk1 phosphorylation at S345 in response to ionizing irradiation. The PTEN−/− cells showed low levels of DNA damage in the absence of irradiation, which was increased by EGFRvIII expression. Finally, secondary changes occurred during tumor growth in mice. Cells from these tumors showed decreased tumor latencies and additional chromosomal aberrations. Most of these tumor lines showed translocations of mouse chromosome 15. Intracranial injections of one of these lines led to invasive, glial fibrillary acidic protein–positive, nestin-positive tumors. These results provide a molecular basis for the occurrence of these two genetic lesions in brain tumors and point to a role in induction of genomic instability. PMID:18812521

  18. DR-07TARGETING EGFR-VIII WITH DICHLOROACETATE IN TEMOZOLOMIDE RESISTANT GLIOBLASTOMA MODELS

    PubMed Central

    Velpula, Kiran Kumar; Asuthkar, Swapna; Lathia, Justin D.; Tsung, Andrew J.

    2014-01-01

    Epidermal growth factor receptor (EGFR) gene amplification and its subsequent over-expression is the most frequent genetic alteration associated with glioblastoma (GBM) approximating 40%. EGFR amplification is accompanied by a gene rearrangement that overexpresses the receptor variant III (EGFRvIII) characterized by an in-frame deletion of 801 bp of coding sequence from exons 2-7. Our in vitro preliminary studies conducted using U373 cell lines stably expressing EGFRvIII (U373vIII) conferred increased proliferation and invasiveness when compared to control. Temozolomide (TMZ) resistance in this context is a well-known concept, where high initial response is met with eventual failure and tumor propagation. We thus generated an aggressive TMZ resistance model for U373vIII cells (U373vIII-TMZ-R) by continual exposure to 150uM TMZ for 6 months. After the initial phase of cell death in the majority and prolonged lag in replication, the eventual surviving U373vIII-TMZ-R colonies showed identical morphology and proliferation potential of the parent U373vIII. This resulted in a cell line mimicking the actual growth potential of recurrent GBM. We have previously shown that dichloroacetic acid (DCA) reduces EGFR expression in GBMs. Since EGFRvIII expression displays an aggressive phenotype yet remains tumor specific, we hypothesize studying DCA alone or in combination with dasatinib, an inhibitor of EGFR subcellular translocation, in U373vIII and U373vIII-TMZ-R cell lines. By more accurately depicting the status of GBM at the time of recurrence after standard of care chemotherapy treatment, the investigation of DCA will augment and possibly redefine our current understanding of resistance mechanisms and establish novel methods of circumventing this pervasive phenomena.

  19. Genetic Modification of Mesenchymal Stem Cells to Express a Single-Chain Antibody Against EGFRvIII on the Cell Surface

    PubMed Central

    Balyasnikova, Irina V.; Franco-Gou, Rosa; Mathis, J. Michael; Lesniak, Maciej S.

    2010-01-01

    Human adult mesenchymal stem cells (hMSC) are under active investigation as cellular carriers for gene therapy. hMSC possess natural tropism toward tumors, however, the targeting of hMSC to specific cell populations within tumors is unexplored. In the case of glioblastoma multiforme (GBM), at least half of the tumors express EGFRvIII on the cell surface, an ideal target for antibody-mediated gene/drug delivery. In this study, we investigated the feasibility of genetically modifying hMSC to express a single-chain antibody (scFv) to EGFRvIII on their surface. Nucleofection was used to transfect hMSC with cDNA encoding scFv EGFRvIII fused with PDGFR or human B7-1 transmembrane domains. The expression of scFv EGFRvIII on the cell surface was assessed by FACS. A stable population of scFv EGFRvIII-expressing hMSC was selected based on antibiotic resistance and enriched using FACS. We found that nucleofection allows the efficient expression of scFv EGFRvIII on the cell surface of hMSC. hMSCs transfected with the construct encoding scFv EGFRvIII as a fusion with PDGFRtm showed scFv EGFRvIII expression in up to 86% of cells. Most importantly, human MSC expressing scFv against EGFRvIII demonstrated enhanced binding to U87-EGFRvIII cells in vitro and at least 7-fold increased retention in human U87-EGFRvIII expressing tumors in vivo. In summary, we provide the first conclusive evidence of genetic modification of hMSC with a single-chain antibody against an antigen expressed on the surface of tumor cells, thereby opening up a new venue for enhanced delivery of gene therapy applications in the context of malignant brain cancer. PMID:19937911

  20. Erlotinib resistance in EGFR-amplified glioblastoma cells is associated with upregulation of EGFRvIII and PI3Kp110δ

    PubMed Central

    Schulte, Alexander; Liffers, Katrin; Kathagen, Annegret; Riethdorf, Sabine; Zapf, Svenja; Merlo, Adrian; Kolbe, Katharina; Westphal, Manfred; Lamszus, Katrin

    2013-01-01

    Background The treatment efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors like erlotinib has not met expectations for glioblastoma therapy, even for EGFR-overexpressing tumors. We determined possible mechanisms of therapy resistance using the unique BS153 glioblastoma cell line, which has retained amplification of the egfr gene and expression of EGFR variant (v)III. Methods Functional effects of erlotinib, gefitinib, and cetuximab on BS153 proliferation, migration, and EGFR-dependent signal transduction were systematically compared in vitro. The tumor-initiating capacity of parental and treatment-resistant BS153 was studied in Naval Medical Research Institute/Foxn1nu mice. Potential mediators of resistance were knocked down using small interfering (si)RNA. Results Erlotinib and gefitinib inhibited proliferation and migration of BS153 in a dose-dependent manner, whereas cetuximab had no effect. BS153 developed resistance to erlotinib (BS153resE) but not to gefitinib. Resistance was associated with strong upregulation of EGFRvIII and subsequent activation of the phosphatidylinositol-3-OH kinase (PI3K) pathway in BS153resE and an increased expression of the regulatory 110-kDa delta subunit of PI3K (p110δ). Knockdown of EGFRvIII in BS153resE largely restored sensitivity to erlotinib. Targeting PI3K pharmacologically caused a significant decrease in cell viability, and specifically targeting p110δ by siRNA partially restored erlotinib sensitivity in BS153resE. In vivo, BS153 formed highly invasive tumors with an unusual growth pattern, displaying numerous satellites distant from the initial injection site. Erlotinib resistance led to delayed onset of tumor growth as well as prolonged overall survival of mice without changing tumor morphology. Conclusions EGFRvIII can mediate resistance to erlotinib in EGFR-amplified glioblastoma via an increase in PI3Kp110δ. Interfering with PI3Kp110δ can restore sensitivity toward the tyrosine kinase

  1. Substantially elevating the levels of αB-crystallin in spinal motor neurons of mutant SOD1 mice does not significantly delay paralysis or attenuate mutant protein aggregation

    PubMed Central

    Xu, Guilian; Fromholt, Susan; Ayers, Jacob I.; Brown, Hilda; Siemienski, Zoe; Crosby, Keith W.; Mayer, Christopher A.; Janus, Christopher; Borchelt, David R.

    2015-01-01

    There has been great interest in enhancing endogenous protein maintenance pathways such as the heat-shock chaperone response, as it is postulated that enhancing clearance of misfolded proteins could have beneficial disease modifying effects in ALS and other neurodegenerative disorders. In cultured cell models of mutant SOD1 aggregation, co-expression of αB-crystallin (αB-crys) has been shown to inhibit the formation of detergent-insoluble forms of mutant protein. Here, we describe the generation of a new line of transgenic mice that express αB-crys at >6-fold the normal level in spinal cord, with robust increases in immunoreactivity throughout the spinal cord grey matter and, specifically, in spinal motor neurons. Surprisingly, spinal cords of mice expressing αB-crys alone contained 20% more motor neurons per section than littermate controls. Raising αB-crys by these levels in mice transgenic for either G93A or L126Z mutant SOD1 had no effect on the age at which paralysis developed. In the G93A mice, which showed the most robust degree of motor neuron loss, the number of these cells declined by the same proportion as in mice expressing the mutant SOD1 alone. In paralyzed bigenic mice, the levels of detergent-insoluble, misfolded, mutant SOD1 were similar to those of mice expressing mutant SOD1 alone. These findings indicate that raising the levels of αB-crys in spinal motor neurons by 6-fold does not produce the therapeutic effects predicted by cell culture models of mutant SOD1 aggregation. PMID:25557022

  2. The evolution of the EGFRvIII (rindopepimut) immunotherapy for glioblastoma multiforme patients

    PubMed Central

    Paff, Michelle; Alexandru-Abrams, Daniela; Hsu, Frank P K; Bota, Daniela A

    2015-01-01

    Glioblastoma Multiforme (GBM) is the most common type of brain tumor and it is uniformly fatal. The community standard of treatment for this disease is gross or subtotal resection of the tumor, followed by radiation and temozolomide. At recurrence bevacizumab can be added for increased progression free survival. Many challenges are encountered while trying to devise new drugs to treat GBM, such as the presence of the blood brain barrier which is impermeable to most drugs. Therefore in the past few years attention was turned to immunological means for the treatment of this devastating disease. EGFRvIII targeting has proven a good way to attack glioblastoma cells by using the immune system. Although in still in development, this approach holds the promise as a great first step toward immune-tailored drugs for the treatment of brain cancers. PMID:25625931

  3. Detection of in vivo protein tyrosine nitration in petite mutant of Saccharomyces cerevisiae: consequence of its formation and significance.

    PubMed

    Panja, Chiranjit; Ghosh, Sanjay

    2014-09-01

    Protein tyrosine nitration (PTN) is a selective post-translational modification often associated with physiological and pathophysiological conditions. Tyrosine is modified in the 3-position of the phenolic ring through the addition of a nitro group. In our previous study we first time showed that PTN occurs in vivo in Saccharomyces cerevisiae. In the present study we observed occurrence of PTN in petite mutant of S. cerevisiae which indicated that PTN is not absolutely dependent on functional mitochondria. Nitration of proteins in S. cerevisiae was also first time confirmed in immunohistochemical study using spheroplasts. Using proteosomal mutants Rpn10Δ, Pre9Δ, we first time showed that the fate of protein nitration in S. cerevisiae was not dependent on proteosomal clearing and probably played vital role in modulating signaling cascades. From our study it is evident that protein tyrosine nitration is a normal physiological event of S. cerevisiae. PMID:25111815

  4. Pigment Epithelium-Derived Factor (PEDF) Expression Induced by EGFRvIII Promotes Self-renewal and Tumor Progression of Glioma Stem Cells.

    PubMed

    Yin, Jinlong; Park, Gunwoo; Kim, Tae Hoon; Hong, Jun Hee; Kim, Youn-Jae; Jin, Xiong; Kang, Sangjo; Jung, Ji-Eun; Kim, Jeong-Yub; Yun, Hyeongsun; Lee, Jeong Eun; Kim, Minkyung; Chung, Junho; Kim, Hyunggee; Nakano, Ichiro; Gwak, Ho-Shin; Yoo, Heon; Yoo, Byong Chul; Kim, Jong Heon; Hur, Eun-Mi; Lee, Jeongwu; Lee, Seung-Hoon; Park, Myung-Jin; Park, Jong Bae

    2015-05-01

    Epidermal growth factor receptor variant III (EGFRvIII) has been associated with glioma stemness, but the direct molecular mechanism linking the two is largely unknown. Here, we show that EGFRvIII induces the expression and secretion of pigment epithelium-derived factor (PEDF) via activation of signal transducer and activator of transcription 3 (STAT3), thereby promoting self-renewal and tumor progression of glioma stem cells (GSCs). Mechanistically, PEDF sustained GSC self-renewal by Notch1 cleavage, and the generated intracellular domain of Notch1 (NICD) induced the expression of Sox2 through interaction with its promoter region. Furthermore, a subpopulation with high levels of PEDF was capable of infiltration along corpus callosum. Inhibition of PEDF diminished GSC self-renewal and increased survival of orthotopic tumor-bearing mice. Together, these data indicate the novel role of PEDF as a key regulator of GSC and suggest clinical implications. PMID:25992628

  5. Prognostic significance of L1CAM expression and its association with mutant p53 expression in high-risk endometrial cancer.

    PubMed

    Van Gool, Inge C; Stelloo, Ellen; Nout, Remi A; Nijman, Hans W; Edmondson, Richard J; Church, David N; MacKay, Helen J; Leary, Alexandra; Powell, Melanie E; Mileshkin, Linda; Creutzberg, Carien L; Smit, Vincent T H B M; Bosse, Tjalling

    2016-02-01

    Studies in early-stage, predominantly low- and intermediate-risk endometrial cancer have demonstrated that L1 cell adhesion molecule (L1CAM) overexpression identifies patients at increased risk of recurrence, yet its prognostic significance in high-risk endometrial cancer is unclear. To evaluate this, its frequency, and the relationship of L1CAM with the established endometrial cancer biomarker p53, we analyzed the expression of both markers by immunohistochemistry in a pilot series of 116 endometrial cancers (86 endometrioid, 30 non-endometrioid subtype) with high-risk features (such as high tumor grade and deep myometrial invasion) and correlated results with clinical outcome. We used The Cancer Genome Atlas (TCGA) endometrial cancer series to validate our findings. Using the previously reported cutoff of 10% positive staining, 51/116 (44%) tumors were classified as L1CAM-positive, with no significant association between L1CAM positivity and the rate of distant metastasis (P=0.195). However, increasing the threshold for L1CAM positivity to 50% resulted in a reduction of the frequency of L1CAM-positive tumors to 24% (28/116), and a significant association with the rate of distant metastasis (P=0.018). L1CAM expression was strongly associated with mutant p53 in the high-risk and TCGA series (P<0.001), although a substantial fraction (36% of endometrioid, 10% of non-endometrioid morphology) of p53-mutant endometrial cancers displayed <10% L1CAM positivity. Moreover, 30% of p53-wild-type non-endometrioid endometrial cancers demonstrated diffuse L1CAM staining, suggesting p53-independent mechanisms of L1CAM overexpression. In conclusion, the previously proposed threshold for L1CAM positivity of >10% does not predict prognosis in high-risk endometrial cancer, whereas an alternative threshold (>50%) does. L1CAM expression is strongly, but not universally, associated with mutant p53, and may be strong enough for clinical implementation as prognostic marker in combination

  6. The Small Molecule Inhibitor G6 Significantly Reduces Bone Marrow Fibrosis and the Mutant Burden in a Mouse Model of Jak2-Mediated Myelofibrosis

    PubMed Central

    Kirabo, Annet; Park, Sung O.; Wamsley, Heather L.; Gali, Meghanath; Baskin, Rebekah; Reinhard, Mary K.; Zhao, Zhizhuang J.; Bisht, Kirpal S.; Keserű, György M.; Cogle, Christopher R.; Sayeski, Peter P.

    2013-01-01

    Philadelphia chromosome–negative myeloproliferative neoplasms, including polycythemia vera, essential thrombocytosis, and myelofibrosis, are disorders characterized by abnormal hematopoiesis. Among these myeloproliferative neoplasms, myelofibrosis has the most unfavorable prognosis. Furthermore, currently available therapies for myelofibrosis have little to no efficacy in the bone marrow and hence, are palliative. We recently developed a Janus kinase 2 (Jak2) small molecule inhibitor called G6 and found that it exhibits marked efficacy in a xenograft model of Jak2-V617F–mediated hyperplasia and a transgenic mouse model of Jak2-V617F–mediated polycythemia vera/essential thrombocytosis. However, its efficacy in Jak2-mediated myelofibrosis has not previously been examined. Here, we hypothesized that G6 would be efficacious in Jak2-V617F–mediated myelofibrosis. To test this, mice expressing the human Jak2-V617F cDNA under the control of the vav promoter were administered G6 or vehicle control solution, and efficacy was determined by measuring parameters within the peripheral blood, liver, spleen, and bone marrow. We found that G6 significantly reduced extramedullary hematopoiesis in the liver and splenomegaly. In the bone marrow, G6 significantly reduced pathogenic Jak/STAT signaling by 53%, megakaryocytic hyperplasia by 70%, and the Jak2 mutant burden by 68%. Furthermore, G6 significantly improved the myeloid to erythroid ratio and significantly reversed the myelofibrosis. Collectively, these results indicate that G6 is efficacious in Jak2-V617F–mediated myelofibrosis, and given its bone marrow efficacy, it may alter the natural history of this disease. PMID:22796437

  7. Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: comparison of wild-type protein and active-site mutant D27E.

    PubMed

    Ohmae, Eiji; Miyashita, Yurina; Tate, Shin-Ichi; Gekko, Kunihiko; Kitazawa, Soichiro; Kitahara, Ryo; Kuwajima, Kunihiro

    2013-12-01

    To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl2 concentration-dependence of the (1)H-(15)N HSQC spectra of the wild-type DHFR-folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increased Km and Kd values for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250MPa together with negative activation volumes of -4.0 or -4.8mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme. PMID:24140567

  8. EGFR and EGFRvIII interact with PUMA to inhibit mitochondrial translocalization of PUMA and PUMA-mediated apoptosis independent of EGFR kinase activity

    PubMed Central

    Zhu, Hu; Cao, Xinyu; Ali-Osman, Francis; Keir, Stephen; Lo, Hui-Wen

    2010-01-01

    EGFR and its constitutively activated variant EGFRvIII are linked to glioblastoma resistance to therapy, the mechanisms underlying this association, however, are still unclear. We report that in glioblastoma, EGFR/EGFRvIII paradoxically co-expresses with p53-upregulated modulator of apoptosis (PUMA), a proapoptotic member of the Bcl-2 family of proteins primarily located on the mitochondria. EGFR/EGFRvIII binds to PUMA constitutively and under apoptotic stress, and subsequently sequesters PUMA in the cytoplasm. The EGFR-PUMA interaction is independent of EGFR activation and is sustained under EGFR inhibition. A Bcl-2/Bcl-xL inhibitor that mimics PUMA activity sensitizes EGFR/EGFRvIII-expressing glioblastoma cells to Iressa. Collectively, we uncovered a novel kinase-independent function of EGFR/EGFRvIII that leads to tumor drug resistance. PMID:20153921

  9. Insights into significance of combined inhibition of MEK and m-TOR signalling output in KRAS mutant non-small-cell lung cancer

    PubMed Central

    Broutin, Sophie; Stewart, Adam; Thavasu, Parames; Paci, Angelo; Bidart, Jean-Michel; Banerji, Udai

    2016-01-01

    Background: We aimed to understand the dependence of MEK and m-TOR inhibition in EGFRWT/ALKnon-rearranged NSCLC cell lines. Methods: In a panel of KRASM and KRASWT NSCLC cell lines, we determined growth inhibition (GI) following maximal reduction in p-ERK and p-S6RP caused by trametinib (MEK inhibitor) and AZD2014 (m-TOR inhibitor), respectively. Results: GI caused by maximal m-TOR inhibition was significantly greater than GI caused by maximal MEK inhibition in the cell line panel (52% vs 18%, P<10−4). There was no significant difference in GI caused by maximal m-TOR compared with maximal m-TOR+MEK inhibition. However, GI caused by the combination was significantly greater in the KRASM cell lines (79% vs 61%, P=0.017). Conclusions: m-TOR inhibition was more critical to GI than MEK inhibition in EGFRWT/ALKnon-rearranged NSCLC cells. The combination of MEK and m-TOR inhibition was most effective in KRASM cells. PMID:27441499

  10. Inhibition of auxin transport and auxin signaling and treatment with far red light induces root coiling in the phospholipase-A mutant ppla-I-1. Significance for surface penetration?

    PubMed

    Perrineau, F; Wimalasekera, R; Effendi, Y; Scherer, G F E

    2016-06-01

    When grown on a non-penetretable at a surface angle of 45°, Arabidopsis roots form wave-like structures and, in wild type rarely, but in certain mutants the tip root even may form circles. These circles are called coils. The formation of coils depends on the complex interaction of circumnutation, gravitropism and negative thigmotropism where - at least - gravitropism is intimately linked to auxin transport and signaling. The knockout mutant of patatin-related phospholipase-AI-1 (pplaI-1) is an auxin-signaling mutant which forms moderately increased numbers of coils on tilted agar plates. We tested the effects of the auxin efflux transport inhibitor NPA (1-naphthylphtalamic acid) and of the influx transport inhibitor 1-NOA (1-naphthoxyacetic acid) which both further increased root coil formation. The pPLAI-1 inhibitors HELSS (haloenol lactone suicide substrate=E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one) and ETYA (eicosatetraynoic acid) which are auxin signaling inhibitors also increased coil formation. In addition, far red light treatment increased coil formation. The results point out that a disturbance of auxin transport and signaling is one potential cause for root coils. As we show that the mutant pplaI-1 penetrates horizontal agar plates better than wild type plants root movements may help penetrating the soil. PMID:27058428

  11. Identification of a compensatory mutant (lpg2-REV) of Leishmania major able to survive as amastigotes within macrophages without LPG2-dependent glycoconjugates and its significance to virulence and immunization strategies.

    PubMed

    Späth, Gerald F; Lye, Lon-Fye; Segawa, Hiroaki; Turco, Salvatore J; Beverley, Stephen M

    2004-06-01

    Different Leishmania species rely to different extents on abundant glycoconjugates, such as lipophosphoglycan (LPG) and related molecules, in mammalian infections. Previously, we showed that Leishmania major deletion mutants lacking the Golgi GDP-mannose transporter LPG2, which is required for assembly of the dominant phosphoglycan (PG) repeats of LPG, were unable to survive in macrophages. These lpg2- mutants, however, retained the ability to generate asymptomatic, persistent infections in mice. In contrast, Ilg and colleagues showed that Leishmania mexicana LPG2 mutants retained virulence for mice. Here we identified a partial revertant population of the L. major lpg2- mutants (designated lpg2(-)REV) that had regained the ability to replicate in macrophages and induce disease pathology through a compensatory change. Like the lpg2 parent, the lpg2(-)REV revertant was unable to synthesize LPG2-dependent PGs in the promastigote stage and thus remained highly attenuated in the ability to induce infection. However, after considerable delay lpg2(-)REV revertant-infected mice exhibited lesions, and amastigotes isolated from these lesions were able to replicate within macrophages despite the fact that they were unable to synthesize PGs. Thus, in some respects, the lpg2(-)REV amastigotes resemble L. mexicana amastigotes. Future studies of the gene(s) responsible may shed light on the mechanisms employed by L. major to survive in the absence of LPG2-dependent glycoconjugates and may also improve the potential of the lpg2- L. major line to serve as a live parasite vaccine by overcoming its tendency to revert toward virulence. PMID:15155672

  12. Molecular structure of large-scale extracted β-glucan from barley and oat: Identification of a significantly changed block structure in a high β-glucan barley mutant.

    PubMed

    Mikkelsen, Mette S; Jespersen, Birthe M; Larsen, Flemming H; Blennow, Andreas; Engelsen, Søren B

    2013-01-01

    Health effects of β-glucan are typically related to dose, size and viscosity without taking the specific molecular structure into account. High β-glucan mutant barley, mother barley and oat β-glucans were large-scale extracted by comparable protocols using hot water, enzyme assisted hydrolysis and ethanol precipitation leading to similar molecular masses (200-300kDa). Multivariate data analysis on all compositional, structural and functional features demonstrated that the main variance among the samples was primarily explained by block structural differences as determined by HPSEC-PAD. In particular the barley high β-glucan mutant proved to exhibit a unique block structure with DP3 and DP4 contributions of: 78.9% and 16.7% as compared to the barley mother (72.1% and 21.4%) and oat (66.1% and 29.1%). This unique block structure was further confirmed by the (1)H NMR determination of the β-1,4 to β-1,3 linkage ratio. Low solubility of the barley samples was potentially an effect of substructures consisting of longer repetitive cellotriosyl sequences. FT-Raman and NMR spectroscopy were useful in measuring sample impurities of α-glucans and prediction of β-linkage characteristics. PMID:23017403

  13. Nebulin binding impedes mutant desmin filament assembly

    PubMed Central

    Baker, Laura K.; Gillis, David C.; Sharma, Sarika; Ambrus, Andy; Herrmann, Harald; Conover, Gloria M.

    2013-01-01

    Desmin intermediate filaments (DIFs) form an intricate meshwork that organizes myofibers within striated muscle cells. The mechanisms that regulate the association of desmin to sarcomeres and their role in desminopathy are incompletely understood. Here we compare the effect nebulin binding has on the assembly kinetics of desmin and three desminopathy-causing mutant desmin variants carrying mutations in the head, rod, or tail domains of desmin (S46F, E245D, and T453I). These mutants were chosen because the mutated residues are located within the nebulin-binding regions of desmin. We discovered that, although nebulin M160–164 bound to both desmin tetrameric complexes and mature filaments, all three mutants exhibited significantly delayed filament assembly kinetics when bound to nebulin. Correspondingly, all three mutants displayed enhanced binding affinities and capacities for nebulin relative to wild-type desmin. Electron micrographs showed that nebulin associates with elongated normal and mutant DIFs assembled in vitro. Moreover, we measured significantly delayed dynamics for the mutant desmin E245D relative to wild-type desmin in fluorescence recovery after photobleaching in live-cell imaging experiments. We propose a mechanism by which mutant desmin slows desmin remodeling in myocytes by retaining nebulin near the Z-discs. On the basis of these data, we suggest that for some filament-forming desmin mutants, the molecular etiology of desminopathy results from subtle deficiencies in their association with nebulin, a major actin-binding filament protein of striated muscle. PMID:23615443

  14. ENIGMA--evidence-based network for the interpretation of germline mutant alleles: an international initiative to evaluate risk and clinical significance associated with sequence variation in BRCA1 and BRCA2 genes.

    PubMed

    Spurdle, Amanda B; Healey, Sue; Devereau, Andrew; Hogervorst, Frans B L; Monteiro, Alvaro N A; Nathanson, Katherine L; Radice, Paolo; Stoppa-Lyonnet, Dominique; Tavtigian, Sean; Wappenschmidt, Barbara; Couch, Fergus J; Goldgar, David E

    2012-01-01

    As genetic testing for predisposition to human diseases has become an increasingly common practice in medicine, the need for clear interpretation of the test results is apparent. However, for many disease genes, including the breast cancer susceptibility genes BRCA1 and BRCA2, a significant fraction of tests results in the detection of a genetic variant for which disease association is not known. The finding of an "unclassified" variant (UV)/variant of uncertain significance (VUS) complicates genetic test reporting and counseling. As these variants are individually rare, a large collaboration of researchers and clinicians will facilitate studies to assess their association with cancer predisposition. It was with this in mind that the ENIGMA consortium (www.enigmaconsortium.org) was initiated in 2009. The membership is both international and interdisciplinary, and currently includes more than 100 research scientists and clinicians from 19 countries. Within ENIGMA, there are presently six working groups focused on the following topics: analysis, clinical, database, functional, tumor histopathology, and mRNA splicing. ENIGMA provides a mechanism to pool resources, exchange methods and data, and coordinately develop and apply algorithms for classification of variants in BRCA1 and BRCA2. It is envisaged that the research and clinical application of models developed by ENIGMA will be relevant to the interpretation of sequence variants in other disease genes. PMID:21990146

  15. Incomplete flagellar structures in Escherichia coli mutants.

    PubMed Central

    Suzuki, T; Komeda, Y

    1981-01-01

    Escherichia coli mutants with defects in 29 flagellar genes identified so far were examined by electron microscopy for possession of incomplete flagellar structures in membrane-associated fractions. The results are discussed in consideration of the known transcriptional interaction of flagellar genes. Hook-basal body structures were detected in flaD, flaS, flaT, flbC, and hag mutants. The flaE mutant had a polyhook-basal body structure. An intact basal body appeared in flaK mutants. Putative precursors of the basal body were detected in mutants with defects in flaM, flaU, flaV, and flaY. No structures homologous to the flagellar basal body or its parts were detected in mutants with defects in flaA, flaB, flaC, flaG, flaH, flaI, flaL, flaN, flaO, flaP, flaQ, flaR, flaW, flaX, flbA, flbB, and flbD. One flaZ mutant had an incomplete flagellar basal body structure and another formed no significant structure, suggesting that flaZ is responsible for both basal body assembly and the transcription of the hag gene. Images PMID:7007337

  16. Mutant fatty acid desaturase

    DOEpatents

    Shanklin, John; Cahoon, Edgar B.

    2004-02-03

    The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.

  17. Connexin Mutants and Cataracts

    PubMed Central

    Beyer, Eric C.; Ebihara, Lisa; Berthoud, Viviana M.

    2013-01-01

    The lens is a multicellular, but avascular tissue that must stay transparent to allow normal transmission of light and focusing of it on the retina. Damage to lens cells and/or proteins can cause cataracts, opacities that disrupt these processes. The normal survival of the lens is facilitated by an extensive network of gap junctions formed predominantly of connexin46 and connexin50. Mutations of the genes that encode these connexins (GJA3 and GJA8) have been identified and linked to inheritance of cataracts in human families and mouse lines. In vitro expression studies of several of these mutants have shown that they exhibit abnormalities that may lead to disease. Many of the mutants reduce or modify intercellular communication due to channel alterations (including loss of function or altered gating) or due to impaired cellular trafficking which reduces the number of gap junction channels within the plasma membrane. However, the abnormalities detected in studies of other mutants suggest that they cause cataracts through other mechanisms including gain of hemichannel function (leading to cell injury and death) and formation of cytoplasmic accumulations (that may act as light scattering particles). These observations and the anticipated results of ongoing studies should elucidate the mechanisms of cataract development due to mutations of lens connexins and abnormalities of other lens proteins. They may also contribute to our understanding of the mechanisms of disease due to connexin mutations in other tissues. PMID:23596416

  18. The zebrafish early arrest mutants.

    PubMed

    Kane, D A; Maischein, H M; Brand, M; van Eeden, F J; Furutani-Seiki, M; Granato, M; Haffter, P; Hammerschmidt, M; Heisenberg, C P; Jiang, Y J; Kelsh, R N; Mullins, M C; Odenthal, J; Warga, R M; Nüsslein-Volhard, C

    1996-12-01

    This report describes mutants of the zebrafish having phenotypes causing a general arrest in early morphogenesis. These mutants identify a group of loci making up about 20% of the loci identified by mutants with visible morphological phenotypes within the first day of development. There are 12 Class I mutants, which fall into 5 complementation groups and have cells that lyse before morphological defects are observed. Mutants at three loci, speed bump, ogre and zombie, display abnormal nuclei. The 8 Class II mutants, which fall into 6 complementation groups, arrest development before cell lysis is observed. These mutants seemingly stop development in the late segmentation stages, and maintain a body shape similar to a 20 hour embryo. Mutations in speed bump, ogre, zombie, specter, poltergeist and troll were tested for cell lethality by transplanting mutant cells into wild-type hosts. With poltergeist, transplanted mutant cells all survive. The remainder of the mutants tested were autonomously but conditionally lethal: mutant cells, most of which lyse, sometimes survive to become notochord, muscles, or, in rare cases, large neurons, all cell types which become postmitotic in the gastrula. Some of the genes of the early arrest group may be necessary for progression though the cell cycle; if so, the survival of early differentiating cells may be based on having their terminal mitosis before the zygotic requirement for these genes. PMID:9007229

  19. Forward genetic screen for auxin-deficient mutants by cytokinin

    PubMed Central

    Wu, Lei; Luo, Pan; Di, Dong-Wei; Wang, Li; Wang, Ming; Lu, Cheng-Kai; Wei, Shao-Dong; Zhang, Li; Zhang, Tian-Zi; Amakorová, Petra; Strnad, Miroslav; Novák, Ondřej; Guo, Guang-Qin

    2015-01-01

    Identification of mutants with impairments in auxin biosynthesis and dynamics by forward genetic screening is hindered by the complexity, redundancy and necessity of the pathways involved. Furthermore, although a few auxin-deficient mutants have been recently identified by screening for altered responses to shade, ethylene, N-1-naphthylphthalamic acid (NPA) or cytokinin (CK), there is still a lack of robust markers for systematically isolating such mutants. We hypothesized that a potentially suitable phenotypic marker is root curling induced by CK, as observed in the auxin biosynthesis mutant CK-induced root curling 1 / tryptophan aminotransferase of Arabidopsis 1 (ckrc1/taa1). Phenotypic observations, genetic analyses and biochemical complementation tests of Arabidopsis seedlings displaying the trait in large-scale genetic screens showed that it can facilitate isolation of mutants with perturbations in auxin biosynthesis, transport and signaling. However, unlike transport/signaling mutants, the curled (or wavy) root phenotypes of auxin-deficient mutants were significantly induced by CKs and could be rescued by exogenous auxins. Mutants allelic to several known auxin biosynthesis mutants were re-isolated, but several new classes of auxin-deficient mutants were also isolated. The findings show that CK-induced root curling provides an effective marker for discovering genes involved in auxin biosynthesis or homeostasis. PMID:26143750

  20. Identification of novel attenuated Salmonella Enteritidis mutants.

    PubMed

    Chang, Jason; Pang, Ervinna; He, Haiqi; Kwang, Jimmy

    2008-06-01

    Salmonella Enteritidis is a major food-borne pathogen that causes nontyphoidal diarrhoea in humans. Infection of adult egg-laying hens usually results in symptomless carriage but in young chicks it may cause paratyphoid disease. It is not known whether S. Enteritidis requires genes additional to known virulence genes for systemic infection of young chickens. A transposon insertion library was created using S. Enteritidis 10/02, which yielded 1246 mutants. Of 384 mutants screened in chickens for attenuation (30.8% of insertion library), 12 (3.1%) had a 50% lethal dose at least 100 times that of the parental strain. Sequencing revealed insertions in genes involved in the biosynthesis of lipopolysaccharide, cell membrane, ATP biosynthesis, transcriptional regulation of virulence and the yhbC gene, which has an unknown function. Evaluation of in vitro virulence characteristics of a Delta yhbC mutant revealed that its ability to invade HeLa cells and survive within a chicken macrophage cell line (HD11) was significantly reduced. It was also less resistant to reactive oxygen and nitrogen intermediates and had a retarded growth rate. Chickens challenged with the Delta yhbC mutant cleared the organism from the liver and spleen 1 week faster than the parental strain and were able to develop specific serum IgG antibodies against the Delta yhbC mutant. PMID:18355292

  1. ECB deacylase mutants

    DOEpatents

    Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.

    2002-01-01

    A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

  2. [Pigment composition and photosynthetic activity of pea chlorophyll mutants].

    PubMed

    Ladygin, V G

    2003-01-01

    Pea chlorophyll mutants chlorotica 2004 and 2014 have been studied. The mutants differ from the initial form (pea cultivar Torsdag) in stem and leaf color (light green in the mutant 2004 and yellow-green in the mutant 2014), relative chlorophyll content (approximately 80 and 50%, respectively), and the composition of carotenoids: the mutant 2004 contains a significantly smaller amount of carotene but accumulates more lutein and violaxanthine; in the mutant 2014, the contents of all carotenoids are decreased proportionally to the decrease in chlorophyll content. It is shown that the rates of CO2 assimilation and oxygen production in the mutant chlorotica 2004 and 2014 plants are reduced. The quantum efficiency of photosynthesis in the mutants is 29-30% lower than in the control plants; in their hybrids, however, it is 1.5-2 higher. It is proposed that both the greater role of dark respiration in gas exchange and the reduced photosynthetic activity in chlorotica mutants are responsible for the decreased phytomass increment in these plants. On the basis of these results, the conclusion is drawn that the mutations chlorotica 2004 and 2014 affect the genes controlling the formation and functioning of various components of the photosynthetic apparatus. PMID:12942751

  3. Histological and Molecular Characterization of Grape Early Ripening Bud Mutant

    PubMed Central

    Yu, Yi-He; Xi, Fei-Fei; Shi, Yan-Yan; Zhang, Guo-Hai

    2016-01-01

    An early ripening bud mutant was analyzed based on the histological, SSR, and methylation-sensitive amplified polymorphism (MSAP) analysis and a layer-specific approach was used to investigate the differentiation between the bud mutant and its parent. The results showed that the thickness of leaf spongy tissue of mutant (MT) is larger than that of wild type (WT) and the differences are significant. The mean size of cell layer L2 was increased in the mutant and the difference is significant. The genetic background of bud mutant revealed by SSR analysis is highly uniform to its parent; just the variations from VVS2 SSR marker were detected in MT. The total methylation ratio of MT is lower than that of the corresponding WT. The outside methylation ratio in MT is much less than that in WT; the average inner methylation ratio in MT is larger than that in WT. The early ripening bud mutant has certain proportion demethylation in cell layer L2. All the results suggested that cell layer L2 of the early ripening bud mutant has changed from the WT. This study provided the basis for a better understanding of the characteristic features of the early ripening bud mutant in grape. PMID:27610363

  4. Histological and Molecular Characterization of Grape Early Ripening Bud Mutant.

    PubMed

    Guo, Da-Long; Yu, Yi-He; Xi, Fei-Fei; Shi, Yan-Yan; Zhang, Guo-Hai

    2016-01-01

    An early ripening bud mutant was analyzed based on the histological, SSR, and methylation-sensitive amplified polymorphism (MSAP) analysis and a layer-specific approach was used to investigate the differentiation between the bud mutant and its parent. The results showed that the thickness of leaf spongy tissue of mutant (MT) is larger than that of wild type (WT) and the differences are significant. The mean size of cell layer L2 was increased in the mutant and the difference is significant. The genetic background of bud mutant revealed by SSR analysis is highly uniform to its parent; just the variations from VVS2 SSR marker were detected in MT. The total methylation ratio of MT is lower than that of the corresponding WT. The outside methylation ratio in MT is much less than that in WT; the average inner methylation ratio in MT is larger than that in WT. The early ripening bud mutant has certain proportion demethylation in cell layer L2. All the results suggested that cell layer L2 of the early ripening bud mutant has changed from the WT. This study provided the basis for a better understanding of the characteristic features of the early ripening bud mutant in grape. PMID:27610363

  5. Significant Treasures.

    ERIC Educational Resources Information Center

    Andrews, Ian A.

    1999-01-01

    Provides a crossword puzzle with an answer key corresponding to the book entitled "Significant Treasures/Tresors Parlants" that is filled with color and black-and-white prints of paintings and artifacts from 131 museums and art galleries as a sampling of the 2,200 such Canadian institutions. (CMK)

  6. Nonchemotactic Mutants of Escherichia coli

    PubMed Central

    Armstrong, John B.; Adler, Julius; Dahl, Margaret M.

    1967-01-01

    We have isolated 40 mutants of Escherichia coli which are nonchemotactic as judged by their failure to swarm on semisolid tryptone plates or to make bands in capillary tubes containing tryptone broth. All the mutants have normal flagella, a fact shown by their shape and reaction with antiflagella serum. All are fully motile under the microscope and all are sensitive to the phage chi. Unlike its parent, one of the mutants, studied in greater detail, failed to show chemotaxis toward oxygen, glucose, serine, threonine, or aspartic acid. The failure to exhibit chemotaxis does not result from a failure to use the chemicals. The swimming of this mutant was shown to be random. The growth rate was normal under several conditions, and the growth requirements were unchanged. Images PMID:5335897

  7. Subunit-specific phenotypes of Salmonella typhimurium HU mutants.

    PubMed Central

    Hillyard, D R; Edlund, M; Hughes, K T; Marsh, M; Higgins, N P

    1990-01-01

    Salmonella hupA and hupB mutants were studied to determine the reasons for the high degree of conservation in HU structure in bacteria. We found one HU-1-specific effect; the F'128 plasmid was 25-fold less stable in hupB compared with hupA or wild-type cells. F' plasmids were 120-fold more unstable in hupA hupB double mutants compared with wild-type cells, and the double mutant also had a significant alteration in plasmid DNA structure. pBR322 DNA isolated from hupA hupB strains was deficient in supercoiling by 10 to 15% compared with wild-type cells, and the topoisomer distribution was significantly more heterogeneous than in wild-type or single-mutant strains. Other systems altered by HU inactivation included flagellar phase variation and phage Mu transposition. However, Mu transposition rates were only about fourfold lower in Salmonella HU double mutants. One reason that Salmonella HU double mutants may be less defective for Mu transposition than E. coli is the synthesis in double mutants of a new, small, basic heat-stable protein, which might partially compensate for the loss of HU. The results indicate that although either HU-1 or HU-2 subunit alone may accommodate the cellular need for general chromosomal organization, the selective pressure to conserve HU-1 and HU-2 structure during evolution could involve specialized roles of the individual subunits. Images PMID:2168381

  8. Motility mutants of Dictyostelium discoideum

    PubMed Central

    1982-01-01

    We describe six motility mutants of Dictyostelium discoideum in this report. They were identified among a group of temperature-sensitive growth (Tsg) mutants that had been previously isolated using an enrichment for phagocytosis-defective cells. The Tsg mutants were screened for their ability to produce tracks on gold-coated cover slips, and several strains were found that were temperature-sensitive for migration in this assay. Analysis of spontaneous Tsg+ revertants of 10 migration-defective strains identified six strains that co-reverted the Tsg and track formation phenotypes. Characterization of these six strains indicated that they were defective at restrictive temperature in track formation, phagocytosis of bacteria, and pseudopodial and filopodial activity, while retaining normal rates of oxygen consumption and viability. Because they had lost this group of motile capabilities, these strains were designated motility mutants. The Tsg+ revertants of these mutants, which coordinately recovered all of the motile activities, were found at frequencies consistent with single genetic events. Analysis of the motility mutants and their revertants suggests a relationship between the motility mutations in some of these strains and genes affecting axenic growth. PMID:7118999

  9. Differential analysis in Proteome of Space Induced Rice and Soybean Mutants

    NASA Astrophysics Data System (ADS)

    Wang, W.; Lu, B.; Gu, D.; Han, S.; Gao, Y.; Sun, Y.

    To investigate the change trends of proteome induced in space environment we chose 3 Rice mutants 2 Soybean mutants and the seeds which were selected as high yields high tillering rice blast resistance soybean insect pest resistance and wider leaf shape individually after abroad Recoverable Satellite JB-1 for 15 days in 1996 and their corresponding controls Two-dimensional gel electrophoresis 2-D with Coomassie Brilliant Blue staining and PDQuest TM software analysis found that In 6 rice samples 329 pm 35 protein spots were detected in controls whereas 298 pm 37 protein spots detected in mutants representing a 9 decrease 69 pm 27 protein spots were lost in mutants while 37 pm 14 protein spots appeared additionally showing 11 protein spots were lost in mutants 58 protein spots were significantly regulated in mutants with 16 pm 7 up- and 42 pm 18 down-regulated which occupied 5 and 14 of the total average mutants spots separately In 3 soybean leaf samples 263 pm 12 protein spots were detected in controls whereas 255 pm 20 protein spots detected in mutants representing a 3 decrease 49 pm 10 protein spots were lost in mutants while 36 pm 16 protein spots appeared additionally showing 5 protein spots lost in mutants 51 protein spots were significantly regulated in mutants with 25 pm 7 up- and 26 pm 15 down-regulated which occupied 9 8 and 10 2 of the total average mutants spots separately In 3 soybean seed samples 208 pm 41 protein spots were

  10. Phosphorodiamidate morpholino oligomers suppress mutant huntingtin expression and attenuate neurotoxicity.

    PubMed

    Sun, Xin; Marque, Leonard O; Cordner, Zachary; Pruitt, Jennifer L; Bhat, Manik; Li, Pan P; Kannan, Geetha; Ladenheim, Ellen E; Moran, Timothy H; Margolis, Russell L; Rudnicki, Dobrila D

    2014-12-01

    Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Disease pathogenesis derives, at least in part, from the long polyglutamine tract encoded by mutant HTT. Therefore, considerable effort has been dedicated to the development of therapeutic strategies that significantly reduce the expression of the mutant HTT protein. Antisense oligonucleotides (ASOs) targeted to the CAG repeat region of HTT transcripts have been of particular interest due to their potential capacity to discriminate between normal and mutant HTT transcripts. Here, we focus on phosphorodiamidate morpholino oligomers (PMOs), ASOs that are especially stable, highly soluble and non-toxic. We designed three PMOs to selectively target expanded CAG repeat tracts (CTG22, CTG25 and CTG28), and two PMOs to selectively target sequences flanking the HTT CAG repeat (HTTex1a and HTTex1b). In HD patient-derived fibroblasts with expanded alleles containing 44, 77 or 109 CAG repeats, HTTex1a and HTTex1b were effective in suppressing the expression of mutant and non-mutant transcripts. CTGn PMOs also suppressed HTT expression, with the extent of suppression and the specificity for mutant transcripts dependent on the length of the targeted CAG repeat and on the CTG repeat length and concentration of the PMO. PMO CTG25 reduced HTT-induced cytotoxicity in vitro and suppressed mutant HTT expression in vivo in the N171-82Q transgenic mouse model. Finally, CTG28 reduced mutant HTT expression and improved the phenotype of Hdh(Q7/Q150) knock-in HD mice. These data demonstrate the potential of PMOs as an approach to suppressing the expression of mutant HTT. PMID:25035419

  11. Mutant prevention concentration and mutant selection window for 10 antimicrobial agents against Rhodococcus equi.

    PubMed

    Berghaus, Londa J; Giguère, Steeve; Guldbech, Kristen

    2013-10-25

    The objectives of this study were to determine the mutant prevention concentration (MPC), time above the MPC and mutant selection window for 10 antimicrobial agents against Rhodococcus equi and to determine if the combination of a macrolide with rifampin would decrease emergence of resistant mutants. Antimicrobial agents investigated (erythromycin, clarithromycin, azithromycin, rifampin, amikacin, gentamicin, enrofloxacin, vancomycin, imipenem, and doxycycline) were selected based on in vitro activity and frequency of use in foals or people infected with R. equi. Each antimicrobial agent or combination of agents was evaluated against four virulent strains of R. equi. MPC were determined using an agar plate assay. Pharmacodynamic parameters were calculated using published plasma and pulmonary pharmacokinetic variables. There was a significant (P<0.001) effect of the type of antimicrobial agent on the MPC. The MPC of clarithromycin (1.0 μg/ml) was significantly lower and the MPC of rifampin and amikacin (512 and 384 μg/ml, respectively) were significantly higher than that of all other antimicrobial agents tested. Combining erythromycin, clarithromycin, or azithromycin with rifampin resulted in a significant (P≤0.005) decrease in MPC and MPC/MIC ratio. When MIC and MPC were combined with pharmacokinetic variables, only gentamicin and vancomycin were predicted to achieve plasma concentrations above the MPC for any given periods of time. Only clarithromycin and the combination clarithromycin-rifampin were predicted to achieve concentrations in bronchoalveolar cells and pulmonary epithelial lining fluid above the MPC for the entire dosing interval. In conclusion, the combination of a macrolide with rifampin considerably decreases the emergence of resistant mutants of R. equi. PMID:23915992

  12. Inhibition of citric acid accumulation by manganese ions in Aspergillus niger mutants with reduced citrate control of phosphofructokinase

    SciTech Connect

    Schreferl, G.; Kubicek, C.P.; Roehr, M.

    1986-03-01

    Mutant strains of Aspergillus niger with reduced citrate control of carbohydrate catabolism (cic mutants) grow faster than the parent strain on media containing 5% (wt/vol) citrate. The mutants tolerated a higher intracellular citrate concentration than the parent strain. One mutant (cic-7/3) contained phosphofructokinase activity significantly less sensitive towards citrate than the enzyme from the parent strain. When this mutant was grown under citrate accumulating conditions, acidogenesis was far less sensitive to inhibition by Mn/sup 2 +/ than in the parent strain. Some of the cic mutants also showed altered citrate inhibition of NADP-specific isocitrate dehydrogenase.

  13. Attenuated virulence of chitin-deficient mutants of Candida albicans.

    PubMed Central

    Bulawa, C E; Miller, D W; Henry, L K; Becker, J M

    1995-01-01

    We have analyzed the role of chitin, a cell-wall polysaccharide, in the virulence of Candida albicans. Mutants with a 5-fold reduction in chitin were obtained in two ways: (i) by selecting mutants resistant to Calcofluor, a fluorescent dye that binds to chitin and inhibits growth, and (ii) by disrupting CHS3, the C. albicans homolog of CSD2/CAL1/DIT101/KT12, a Saccharomyces cerevisiae gene required for synthesis of approximately 90% of the cell-wall chitin. Chitin-deficient mutants have no obvious alterations in growth rate, sugar assimilation, chlamydospore formation, or germ-tube formation in various media. When growing vegetatively in liquid media, the mutants tend to clump and display minor changes in morphology. Staining of cells with the fluorescent dye Calcofluor indicates that CHS3 is required for synthesis of the chitin rings found on the surface of yeast cells but not formation of septa in either yeast cells or germ tubes. Despite their relatively normal growth, the mutants are significantly less virulent than the parental strain in both immunocompetent and immunosuppressed mice; at 13 days after infection, survival was 95% in immunocompetent mice that received chs3/chs3 cells and 10% in immunocompetent mice that received an equal dose of chs3/CHS3 cells. Chitin-deficient strains can colonize the organs of infected mice, suggesting that the reduced virulence of the mutants is not due to accelerated clearing. Images Fig. 1 Fig. 2 PMID:7479842

  14. FTIR and EDXRF investigations of salt tolerant soybean mutants

    NASA Astrophysics Data System (ADS)

    Akyuz, Sevim; Akyuz, Tanil; Celik, Ozge; Atak, Cimen

    2013-07-01

    Molecular structure and elemental composition of soybean (Glycine max L. Merr.) seeds of S04-05 (Ustun-1) variety together with its salt tolerant mutants were investigated by Fourier transform infrared (FTIR) and energy dispersive X-ray fluorescence (EDXRF) spectrometry. Salt tolerant soybean mutants were in vivo and in vitro selected from the M2 generation of gamma irradiated S04-05 soybean variety. Examination of the secondary structure of proteins revealed the presence of some alterations in soybean mutants in comparison to those of the control groups. The difference IR spectra indicated that salt tolerant mutants (M2) have less protein but more lipid contents. Chemometric treatment of the FTIR data was performed and principle component analysis (PCA) revealed clear difference between control group of seeds and mutants. EDXRF analysis showed that salt tolerant mutants considerably contained more chlorine, copper and zinc elements when compared to the control group, although most of the trace elements concentrations were not significantly altered.

  15. Enhanced Symbiotic Performance by Rhizobium tropici Glycogen Synthase Mutants

    PubMed Central

    Marroquí, Silvia; Zorreguieta, Angeles; Santamaría, Carmen; Temprano, Francisco; Soberón, Mario; Megías, Manuel; Downie, J. Allan

    2001-01-01

    We isolated a Tn5-induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N-dimethyl-p-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes c1 and CycM and a small increase in the amount of cytochrome aa3. In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli: glycogen phosphorylase (glgP), glycogen branching enzyme (glgB), ADP glucose pyrophosphorylase (glgC), glycogen synthase (glgA), phosphoglucomutase (pgm), and glycogen debranching enzyme (glgX). All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently. Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in glgA. This mutant had a phenotype similar to that of the Tn5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene. PMID:11208782

  16. Isolation of prostrate turfgrass mutants via screening of dwarf phenotype and characterization of a perennial ryegrass prostrate mutant.

    PubMed

    Chen, Junmei; Thammina, Chandra; Li, Wei; Yu, Hao; Yer, Huseyin; El-Tanbouly, Rania; Marron, Manon; Katin-Grazzini, Lorenzo; Chen, Yongqin; Inguagiato, John; McAvoy, Richard J; Guillard, Karl; Zhang, Xian; Li, Yi

    2016-01-01

    Prostrate turf varieties are desirable because of their increased low mowing tolerance, heat resistance, traffic resistance and ground coverage compared with upright varieties. Mutation breeding may provide a powerful tool to create prostrate varieties, but there are no simple, straightforward methods to screen for such mutants. Elucidation of the molecular basis of the major 'green revolution' traits, dwarfism and semi-dwarfism, guided us to design a simple strategy for isolating dwarf mutants of perennial ryegrass (Lolium perenne L.). We have shown that gamma-ray-mediated dominant dwarf mutants can be easily screened for at the three-leaf stage. About 10% of dwarf mutant lines also displayed a prostrate phenotype at mature stages (>10 tillers). One prostrate line, Lowboy I, has been characterized in detail. Lowboy I had significantly shorter canopy, leaf blade and internode lengths compared with wild type. Lowboy I also exhibited greater tolerance to low mowing stress than wild type. Exogenous gibberellic acid (GA) restored Lowboy I to a wild-type phenotype, indicating that the dwarf and prostrate phenotypes were both due to GA deficiency. We further showed that phenotypes of Lowboy I were dominant and stably inherited through sexual reproduction. Prostrate turfgrass mutants are difficult to screen for because the phenotype is not observed at young seedling stages, therefore our method represents a simple strategy for easily isolating prostrate mutants. Furthermore, Lowboy I may provide an outstanding germplasm for breeding novel prostrate perennial ryegrass cultivars. PMID:26955481

  17. Isolation of prostrate turfgrass mutants via screening of dwarf phenotype and characterization of a perennial ryegrass prostrate mutant

    PubMed Central

    Chen, Junmei; Thammina, Chandra; Li, Wei; Yu, Hao; Yer, Huseyin; El-Tanbouly, Rania; Marron, Manon; Katin-Grazzini, Lorenzo; Chen, Yongqin; Inguagiato, John; McAvoy, Richard J.; Guillard, Karl; Zhang, Xian; Li, Yi

    2016-01-01

    Prostrate turf varieties are desirable because of their increased low mowing tolerance, heat resistance, traffic resistance and ground coverage compared with upright varieties. Mutation breeding may provide a powerful tool to create prostrate varieties, but there are no simple, straightforward methods to screen for such mutants. Elucidation of the molecular basis of the major ‘green revolution’ traits, dwarfism and semi-dwarfism, guided us to design a simple strategy for isolating dwarf mutants of perennial ryegrass (Lolium perenne L.). We have shown that gamma-ray-mediated dominant dwarf mutants can be easily screened for at the three-leaf stage. About 10% of dwarf mutant lines also displayed a prostrate phenotype at mature stages (>10 tillers). One prostrate line, Lowboy I, has been characterized in detail. Lowboy I had significantly shorter canopy, leaf blade and internode lengths compared with wild type. Lowboy I also exhibited greater tolerance to low mowing stress than wild type. Exogenous gibberellic acid (GA) restored Lowboy I to a wild-type phenotype, indicating that the dwarf and prostrate phenotypes were both due to GA deficiency. We further showed that phenotypes of Lowboy I were dominant and stably inherited through sexual reproduction. Prostrate turfgrass mutants are difficult to screen for because the phenotype is not observed at young seedling stages, therefore our method represents a simple strategy for easily isolating prostrate mutants. Furthermore, Lowboy I may provide an outstanding germplasm for breeding novel prostrate perennial ryegrass cultivars. PMID:26955481

  18. Mutant frequency of radiotherapy technicians appears to be associated with recent dose of ionizing radiation

    SciTech Connect

    Messing, K.; Ferraris, J.; Bradley, W.E.; Swartz, J.; Seifert, A.M. )

    1989-10-01

    The frequency of hypoxanthine phosphoribosyl transferase (HPRT) mutants among peripheral T-lymphocytes of radiotherapy technicians primarily exposed to 60Co was measured by the T-cell cloning method. Mutant frequencies of these technicians in 1984 and 1986 were significantly higher than those of physiotherapy technicians who worked in a neighboring service, and correlated significantly with thermoluminescence dosimeter readings recorded during the 6 mo preceding mutant frequency determination. Correlations decreased when related to dose recorded over longer time intervals. HPRT mutant frequency determination in peripheral lymphocytes is a good measure of recently received biologically effective radiation dose in an occupationally exposed population.

  19. Genome-Wide Screen for Oxalate-Sensitive Mutants of Saccharomyces cerevisiae▿ †

    PubMed Central

    Cheng, V.; Stotz, H. U.; Hippchen, K.; Bakalinsky, A. T.

    2007-01-01

    Oxalic acid is an important virulence factor produced by phytopathogenic filamentous fungi. In order to discover yeast genes whose orthologs in the pathogen may confer self-tolerance and whose plant orthologs may protect the host, a Saccharomyces cerevisiae deletion library consisting of 4,827 haploid mutants harboring deletions in nonessential genes was screened for growth inhibition and survival in a rich medium containing 30 mM oxalic acid at pH 3. A total of 31 mutants were identified that had significantly lower cell yields in oxalate medium than in an oxalate-free medium. About 35% of these mutants had not previously been detected in published screens for sensitivity to sorbic or citric acid. Mutants impaired in endosomal transport, the rgp1Δ, ric1Δ, snf7Δ, vps16Δ, vps20Δ, and vps51Δ mutants, were significantly overrepresented relative to their frequency among all verified yeast open reading frames. Oxalate exposure to a subset of five mutants, the drs2Δ, vps16Δ, vps51Δ, ric1Δ, and rib4Δ mutants, was lethal. With the exception of the rib4Δ mutant, all of these mutants are impaired in vesicle-mediated transport. Indirect evidence is provided suggesting that the sensitivity of the rib4Δ mutant, a riboflavin auxotroph, is due to oxalate-mediated interference with riboflavin uptake by the putative monocarboxylate transporter Mch5. PMID:17644632

  20. Arabidopsis mutants impaired in cosuppression.

    PubMed Central

    Elmayan, T; Balzergue, S; Béon, F; Bourdon, V; Daubremet, J; Guénet, Y; Mourrain, P; Palauqui, J C; Vernhettes, S; Vialle, T; Wostrikoff, K; Vaucheret, H

    1998-01-01

    Post-transcriptional gene silencing (cosuppression) results in the degradation of RNA after transcription. A transgenic Arabidopsis line showing post-transcriptional silencing of a 35S-uidA transgene and uidA-specific methylation was mutagenized using ethyl methanesulfonate. Six independent plants were isolated in which uidA mRNA accumulation and beta-glucuronidase activity were increased up to 3500-fold, whereas the transcription rate of the 35S-uidA transgene was increased only up to threefold. These plants each carried a recessive monogenic mutation that is responsible for the release of silencing. These mutations defined two genetic loci, called sgs1 and sgs2 (for suppressor of gene silencing). Transgene methylation was distinctly modified in sgs1 and sgs2 mutants. However, methylation of centromeric repeats was not affected, indicating that sgs mutants differ from ddm (for decrease in DNA methylation) and som (for somniferous) mutants. Indeed, unlike ddm and som mutations, sgs mutations were not able to release transcriptional silencing of a 35S-hpt transgene. Conversely, both sgs1 and sgs2 mutations were able to release cosuppression of host Nia genes and 35S-Nia2 transgenes. These results therefore indicate that sgs mutations act in trans to impede specifically transgene-induced post-transcriptional gene silencing. PMID:9761800

  1. EGFRvIII-mediated transactivation of receptor tyrosine kinases in glioma: mechanism and therapeutic implications.

    PubMed

    Greenall, S A; Donoghue, J F; Van Sinderen, M; Dubljevic, V; Budiman, S; Devlin, M; Street, I; Adams, T E; Johns, T G

    2015-10-01

    A truncation mutant of the epidermal growth factor receptor, EGFRvIII, is commonly expressed in glioma, an incurable brain cancer. EGFRvIII is tumorigenic, in part, through its transactivation of other receptor tyrosine kinases (RTKs). Preventing the effects of this transactivation could form part of an effective therapy for glioma; however, the mechanism by which the transactivation occurs is unknown. Focusing on the RTK MET, we show that MET transactivation in U87MG human glioma cells in vitro is proportional to EGFRvIII activity and involves MET heterodimerization associated with a focal adhesion kinase (FAK) scaffold. The transactivation of certain other RTKs was, however, independent of FAK. Simultaneously targeting EGFRvIII (with panitumumab) and the transactivated RTKs themselves (with motesanib) in an intracranial mouse model of glioma resulted in significantly greater survival than with either agent alone, indicating that cotargeting these RTKs has potent antitumor efficacy and providing a strategy for treating EGFRvIII-expressing gliomas, which are usually refractory to treatment. PMID:25659577

  2. Problem-Solving Test: Tryptophan Operon Mutants

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

  3. Isolation and characterization of symbiotic mutants of bradyrhizobium sp. (Arachis) strain NC92: mutants with host-specific defects in nodulation and nitrogen fixation.

    PubMed Central

    Wilson, K J; Anjaiah, V; Nambiar, P T; Ausubel, F M

    1987-01-01

    Random transposon Tn5 mutagenesis of Bradyrhizobium sp. (Arachis) strain NC92, a member of the cowpea cross-inoculation group, was carried out, and kanamycin-resistant transconjugants were tested for their symbiotic phenotype on three host plants: groundnut, siratro, and pigeonpea. Two nodulation (Nod- phenotype) mutants were isolated. One is unable to nodulate all three hosts and appears to contain an insertion in one of the common nodulation genes (nodABCD); the other is a host-specific nodulation mutant that fails to nodulate pigeonpea, elicits uninvaded nodules on siratro, and elicits normal, nitrogen-fixing nodules on groundnut. In addition, nine mutants defective in nitrogen fixation (Fix- phenotype) were isolated. Three fail to supply symbiotically fixed nitrogen to all three host plants. Surprisingly, nodules elicited by one of these mutants exhibit high levels of acetylene reduction activity, demonstrating the presence of the enzyme nitrogenase. Three more mutants have partially effective phenotypes (Fix +/-) in symbiosis with all three host plants. The remaining three mutants fail to supply fixed nitrogen to one of the host plants tested while remaining partially or fully effective on the other two hosts; two of these mutants are Fix- in pigeonpea and Fix +/- on groundnut and on siratro, whereas the other one is Fix- on groundnut but Fix+ on siratro and on pigeonpea. These latter mutants also retain significant nodule acetylene reduction activity, even in the ineffective symbioses. Such bacterial host-specific fixation (Hsf) mutants have not previously been reported. Images PMID:3032910

  4. Auditory development in progressive motor neuronopathy mouse mutants.

    PubMed

    Volkenstein, Stefan; Brors, Dominik; Hansen, Stefan; Berend, Achim; Mlynski, Robert; Aletsee, Christoph; Dazert, Stefan

    2009-11-01

    The present study was performed to elucidate the hearing development in the progressive motor neuronopathy (pmn) mouse mutant. This mouse has been used as a model for human motoneuron disease. A missense mutation in the tubulin-specific chaperon E (Tbce) gene on mouse chromosome 13 was localized as the underlying genetic defect. The protein encoded by the Tbce gene is essential for the formation of primary tubulin complexes. Studies on motoneurons show disorganization in microtubules and disturbed axonal transport, followed by retrograde degeneration of the motoneurons. A similar pathomechanism is also possible for hearing disorders where disrupted microtubules could cause functional deficits in spiral ganglion neurons or in cochlear hair cells. Click auditory brainstem response (ABR) audiometry in homozygous pmn mutants showed a normal onset of hearing, but an increasing hearing threshold from postnatal day 26 (P26) on to death, compared to heterozygous mutants and wild-type mice. Histological sections of the cochlea at different ages showed a regular morphology. Additionally, spiral ganglion explants from mutant and wild-type mice were cultured. The neurite length from pmn mutants was shorter than in wild-type mice, and the neurite number/explant was significantly decreased in pmn mutants. We show that the pmn mouse mutant is a model for a progressive rapid hearing loss from P26 on, after initially normal hearing development. Heterozygous mice are not affected by this defect. With the knowledge of the well-known pathomechanism of this defect in motoneurons, a dysfunction of cellular mechanisms regulating tubulin assembling suggests that tubulin assembling plays an essential role in hearing function and maintenance. PMID:19735697

  5. Salmonella typhimurium mutants defective in acetohydroxy acid synthases I and II.

    PubMed

    Shaw, K J; Berg, C M; Sobol, T J

    1980-03-01

    An analysis of transposon-induced mutants shows that Salmonella typhimurium possesses two major isozymes of acetohydroxy acid synthase, the enzymes which mediate the first common step in isoleucine and valine biosynthesis. A third (minor) acetohydroxy acid synthase is present, but its significance in isoleucine and valine synthesis may be negligible. Mutants defective in acetohydroxy acid synthase II (ilvG::Tn10) require isoleucine, alpha-ketobutyrate, or threonine for growth, a mutant defective in acetohydroxy acid synthase I (ilvB::Tn5) is a prototroph, and a double mutant (ilvG::Tn10 ilvB::Tn5) requires isoleucine plus valine for growth. PMID:6245063

  6. Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants*

    PubMed Central

    Sasaki, Yo; Margolin, Zachary; Borgo, Benjamin; Havranek, James J.; Milbrandt, Jeffrey

    2015-01-01

    Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD+ biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD+ synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome. PMID:26018082

  7. Altered etioplast development in phytochrome chromophore-deficient mutants.

    PubMed

    Terry, M J; Ryberg, M; Raitt, C E; Page, A M

    2001-12-01

    Inhibition of chromophore synthesis in the phytochrome-deficient aurea (au) and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum L.) results in a severe reduction of protochlorophyllide (Pchlide) accumulation in dark-grown hypocotyls. Experiments with apophytochrome-deficient mutants indicate that the inhibition of Pchlide accumulation results from two separate effects: one dependent on the activity of phytochromes A and B1 and one phytochrome-independent effect that is attributed to a feedback inhibition of the tetrapyrrole biosynthesis pathway. Cotyledons only show phytochrome-independent inhibition of Pchlide synthesis. Analysis of NADPH:protochlorophyllide oxidoreductase levels by western blotting showed that the reduction in Pchlide in au and yg-2 is accompanied by a correlative, but less substantial, decrease in NADPH:protochlorophyllide oxidoreductase. Consistent with this result, in vivo fluorescence spectra demonstrate that both mutants are primarily deficient in non-phototransformable Pchlide. Analysis of etioplast structure indicates that plastid development in au and yg-2 is retarded in hypocotyls and partially impaired in cotyledons, again correlating with the reduction in Pchlide. Since Pchlide synthesis is also reduced in chromophore-deficient mutants of pea (Pisum sativum L.) and Arabidopsis thaliana (L.) Heynh. (Landsberg erecta) these results may be significant for explaining aspects of the phenotype of this mutant class that are independent of the loss of phytochrome. PMID:11800397

  8. Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants.

    PubMed

    Sasaki, Yo; Margolin, Zachary; Borgo, Benjamin; Havranek, James J; Milbrandt, Jeffrey

    2015-07-10

    Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD(+) biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD(+) synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome. PMID:26018082

  9. Metabolism of Phenol and Cresols by Mutants of Pseudomonas putida

    PubMed Central

    Bayly, R. C.; Wigmore, G. J.

    1973-01-01

    Mutant strains of Pseudomonas putida strain U have been obtained which are deficient in enzymes of the degradative pathways of phenol and cresols. Mutant strains deficient in catechol 2, 3-oxygenase accumulated the appropriate catechol derivative from cresols. A mutant strain which would not grow on either phenol or a cresol was shown to be deficient in both 2-hydroxymuconic semialdehyde hydrolase and a nicotinamide adenine dinucleotide, oxidized form, (NAD+)-dependent aldehyde dehydrogenase. When this strain was grown in the presence of phenol or a cresol, the appropriate product of meta fission of these compounds accumulated in the growth medium. A partial revertant of this mutant strain, which was able to grow on ortho- and meta-cresol but not para-cresol, was shown to have regained only the hydrolase activity. This strain was used to show that the products of meta ring fission of the cresols and phenol are metabolized as follows: (i) ortho- and meta-cresol exclusively by a hydrolase; (ii) para-cresol exclusively by a NAD+-dependent aldehyde dehydrogenase; (iii) phenol by both a NAD+-dependent dehydrogenase and a hydrolase in the approximate ratio of 5 to 1. This conclusion is supported by the substrate specificity and enzymatic activity of the hydrolase and NAD+-dependent aldehyde dehydrogenase enzymes of the wild-type strain. The results are discussed in terms of the physiological significance of the pathway. Properties of some of the mutant strains isolated are discussed. PMID:4347965

  10. Nanoformulated cell-penetrating survivin mutant and its dual actions

    PubMed Central

    Sriramoju, Bhasker; Kanwar, Rupinder K; Kanwar, Jagat R

    2014-01-01

    In this study, we investigated the differential actions of a dominant-negative survivin mutant (SurR9-C84A) against cancerous SK-N-SH neuroblastoma cell lines and differentiated SK-N-SH neurons. In both the cases, the mutant protein displayed dual actions, where its effects were cytotoxic toward cancerous cells and proliferative toward the differentiated neurons. This can be explained by the fact that tumorous (undifferentiated SK-N-SH) cells have a high endogenous survivin pool and upon treatment with mutant SuR9-C84A causes forceful survivin expression. These events significantly lowered the microtubule dynamics and stability, eventually leading to apoptosis. In the case of differentiated SK-N-SH neurons that express negligible levels of wild-type survivin, the mutant indistinguishably behaved in a wild-type fashion. It also favored cell-cycle progression, forming the chromosome-passenger complex, and stabilized the microtubule-organizing center. Therefore, mutant SurR9-C84A represents a novel therapeutic with its dual actions (cytotoxic toward tumor cells and protective and proliferative toward neuronal cells), and hence finds potential applications against a variety of neurological disorders. In this study, we also developed a novel poly(lactic-co-glycolic acid) nanoparticulate formulation to surmount the hurdles associated with the delivery of SurR9-C84A, thus enhancing its effective therapeutic outcome. PMID:25045261

  11. TAE226, a Bis-Anilino Pyrimidine Compound, Inhibits the EGFR-Mutant Kinase Including T790M Mutant to Show Anti-Tumor Effect on EGFR-Mutant Non-Small Cell Lung Cancer Cells.

    PubMed

    Otani, Hiroki; Yamamoto, Hiromasa; Takaoka, Munenori; Sakaguchi, Masakiyo; Soh, Junichi; Jida, Masaru; Ueno, Tsuyoshi; Kubo, Takafumi; Asano, Hiroaki; Tsukuda, Kazunori; Kiura, Katsuyuki; Hatakeyama, Shinji; Kawahara, Eiji; Naomoto, Yoshio; Miyoshi, Shinichiro; Toyooka, Shinichi

    2015-01-01

    TAE226, a bis-anilino pyrimidine compound, has been developed as an inhibitor of focal adhesion kinase (FAK) and insulin-like growth factor-I receptor (IGF-IR). In this study, we investigated the effect of TAE226 on non-small-cell lung cancer (NSCLC), especially focusing on the EGFR mutational status. TAE226 was more effective against cells with mutant EGFR, including the T790M mutant, than against cells with wild-type one. TAE226 preferentially inhibited phospho-EGFR and its downstream signaling mediators in the cells with mutant EGFR than in those with wild-type one. Phosphorylation of FAK and IGF-IR was not inhibited at the concentration at which the proliferation of EGFR-mutant cells was inhibited. Results of the in vitro binding assay indicated significant differences in the affinity for TAE226 between the wild-type and L858R (or delE746_A750) mutant, and the reduced affinity of ATP to the L858R (or delE746_A750) mutant resulted in good responsiveness of the L858R (or delE746_A750) mutant cells to TAE226. Of interest, the L858R/T790M or delE746_A750/T790M mutant enhanced the binding affinity for TAE226 compared with the L858R or delE746_A750 mutant, resulting in the effectiveness of TAE226 against T790M mutant cells despite the T790M mutation restoring the ATP affinity for the mutant EGFR close to that for the wild-type. TAE226 also showed higher affinity of about 15-fold for the L858R/T790M mutant than for the wild-type one by kinetic interaction analysis. The anti-tumor effect against EGFR-mutant tumors including T790M mutation was confirmed in mouse models without any significant toxicity. In summary, we showed that TAE226 inhibited the activation of mutant EGFR and exhibited anti-proliferative activity against NSCLCs carrying EGFR mutations, including T790M mutation. PMID:26090892

  12. TAE226, a Bis-Anilino Pyrimidine Compound, Inhibits the EGFR-Mutant Kinase Including T790M Mutant to Show Anti-Tumor Effect on EGFR-Mutant Non-Small Cell Lung Cancer Cells

    PubMed Central

    Otani, Hiroki; Yamamoto, Hiromasa; Takaoka, Munenori; Sakaguchi, Masakiyo; Soh, Junichi; Jida, Masaru; Ueno, Tsuyoshi; Kubo, Takafumi; Asano, Hiroaki; Tsukuda, Kazunori; Kiura, Katsuyuki; Hatakeyama, Shinji; Kawahara, Eiji; Naomoto, Yoshio; Miyoshi, Shinichiro; Toyooka, Shinichi

    2015-01-01

    TAE226, a bis-anilino pyrimidine compound, has been developed as an inhibitor of focal adhesion kinase (FAK) and insulin-like growth factor-I receptor (IGF-IR). In this study, we investigated the effect of TAE226 on non-small-cell lung cancer (NSCLC), especially focusing on the EGFR mutational status. TAE226 was more effective against cells with mutant EGFR, including the T790M mutant, than against cells with wild-type one. TAE226 preferentially inhibited phospho-EGFR and its downstream signaling mediators in the cells with mutant EGFR than in those with wild-type one. Phosphorylation of FAK and IGF-IR was not inhibited at the concentration at which the proliferation of EGFR-mutant cells was inhibited. Results of the in vitro binding assay indicated significant differences in the affinity for TAE226 between the wild-type and L858R (or delE746_A750) mutant, and the reduced affinity of ATP to the L858R (or delE746_A750) mutant resulted in good responsiveness of the L858R (or delE746_A750) mutant cells to TAE226. Of interest, the L858R/T790M or delE746_A750/T790M mutant enhanced the binding affinity for TAE226 compared with the L858R or delE746_A750 mutant, resulting in the effectiveness of TAE226 against T790M mutant cells despite the T790M mutation restoring the ATP affinity for the mutant EGFR close to that for the wild-type. TAE226 also showed higher affinity of about 15-fold for the L858R/T790M mutant than for the wild-type one by kinetic interaction analysis. The anti-tumor effect against EGFR-mutant tumors including T790M mutation was confirmed in mouse models without any significant toxicity. In summary, we showed that TAE226 inhibited the activation of mutant EGFR and exhibited anti-proliferative activity against NSCLCs carrying EGFR mutations, including T790M mutation. PMID:26090892

  13. Clostridium acetobutylicum mutants that produce butyraldehyde and altered quantities of solvents

    SciTech Connect

    Rogers, P.; Palosaari, N.

    1987-12-01

    Spontaneous mutants of Clostridium acetobutylicum NRRL B643 that were resistant to allyl alcohol (AA) were selected and characterized. These mutants contained 10- to 100-fold reduced activities of butanol and ethanol alcohol dehydrogenase. The AA mutants formed two groups and produced no ethanol. Type 1 AA mutants produced significant amounts of a new solvent, butyraldehyde, and contained normal levels of the coenzyme A-dependent butyraldehyde dehydrogenase (BAD). Type 2 AA mutants produced no significant butyraldehyde and lower levels of all solvents, and they contained 45- to 100-fold lower activity levels of BAD. Following ethyl methanesulfonate mutagenesis, low-acid-producing (Acid/sup -/) mutants were selected and characterized as superinduced solvent producers, yielding more than 99% of theoretical glucose carbon as solvents and only small amounts of acetate and butyrate. Following ethyl methanesulfonate mutagenesis, 13 sporulation-negative (Spo/sup -/) mutants were characterized; and 3 were found to produce only butyrate and acetate, a minor amount of acetone, and no alcohols. These Spo/sup -/ mutants contained reduced butanol dehydrogenase activity and no BAD enzyme activity. The data support the view that the type 2 AA, the Acid/sup -/, and the Spo/sup -/ mutants somehow alter normal regulated expression of the solvent pathway in C. acetobutylicum.

  14. Clostridium acetobutylicum Mutants That Produce Butyraldehyde and Altered Quantities of Solvents

    PubMed Central

    Rogers, Palmer; Palosaari, Neil

    1987-01-01

    Spontaneous mutants of Clostridium acetobutylicum NRRL B643 that were resistant to allyl alcohol (AA) were selected and characterized. These mutants contained 10- to 100-fold reduced activities of butanol and ethanol alcohol dehydrogenase. The AA mutants formed two groups and produced no ethanol. Type 1 AA mutants produced significant amounts of a new solvent, butyraldehyde, and contained normal levels of the coenzyme A-dependent butyraldehyde dehydrogenase (BAD). Type 2 AA mutants produced no significant butyraldehyde and lower levels of all solvents, and they contained 45- to 100-fold lower activity levels of BAD. Following ethyl methanesulfonate mutagenesis, low-acid-producing (Acid−) mutants were selected and characterized as superinduced solvent producers, yielding more than 99% of theoretical glucose carbon as solvents and only small amounts of acetate and butyrate. Following ethyl methanesulfonate mutagenesis, 13 sporulation-negative (Spo−) mutants were characterized; and 3 were found to produce only butyrate and acetate, a minor amount of acetone, and no alcohols. These Spo− mutants contained reduced butanol dehydrogenase activity and no BAD enzyme activity. The data support the view that the type 2 AA, the Acid−, and the Spo− mutants somehow alter normal regulated expression of the solvent pathway in C. acetobutylicum. PMID:16347493

  15. Modifiers of mutant huntingtin aggregation

    PubMed Central

    Teuling, Eva; Bourgonje, Annika; Veenje, Sven; Thijssen, Karen; de Boer, Jelle; van der Velde, Joeri; Swertz, Morris; Nollen, Ellen

    2011-01-01

    Protein aggregation is a common hallmark of a number of age-related neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and polyglutamine-expansion disorders such as Huntington’s disease, but how aggregation-prone proteins lead to pathology is not known. Using a genome-wide RNAi screen in a C. elegans-model for polyglutamine aggregation, we previously identified 186 genes that suppress aggregation. Using an RNAi screen for human orthologs of these genes, we here present 26 human genes that suppress aggregation of mutant huntingtin in a human cell line. Among these are genes that have not been previously linked to mutant huntingtin aggregation. They include those encoding eukaryotic translation initiation, elongation and translation factors, and genes that have been previously associated with other neurodegenerative diseases, like the ATP-ase family gene 3-like 2 (AFG3L2) and ubiquitin-like modifier activating enzyme 1 (UBA1). Unravelling the role of these genes will broaden our understanding of the pathogenesis of Huntington’s disease. PMID:21915392

  16. Allele Specific p53 Mutant Reactivation

    PubMed Central

    Yu, Xin; Vazquez, Alexei; Levine, Arnold J.; Carpizo, Darren R.

    2012-01-01

    Summary Rescuing the function of mutant p53 protein is an attractive cancer therapeutic strategy. Using the NCI anticancer drug screen data, we identified two compounds from the thiosemicarbazone family that manifest increased growth inhibitory activity in mutant p53 cells, particularly for the p53R175 mutant. Mechanistic studies reveal that NSC319726 restores WT structure and function to the p53R175 mutant. This compound kills p53R172H knock-in mice with extensive apoptosis and inhibits xenograft tumor growth in a 175-allele specific mutant p53 dependent manner. This activity depends upon the zinc ion chelating properties of the compound as well as redox changes. These data identify NSC319726 as a p53R175 mutant reactivator and as a lead compound for p53 targeted drug development. PMID:22624712

  17. Abnormal lignin in a loblolly pine mutant

    SciTech Connect

    Ralph, J.; MacKay, J.J.; Hatfield, R.D.

    1997-07-11

    Novel lignin is formed in a mutant loblolly pine (Pinus taeda L.) severely depleted in cinnamyl alcohol dehydrogenase (E.C. 1.1.1.195), which converts coniferaldehyde to coniferyl alcohol, the primary lignin precursor in pines. Dihydroconiferyl alcohol, a monomer not normally associated with the lignin biosynthetic pathway, is the major component of the mutant`s lignin, accounting for {approximately}30 percent (versus {approximately}3 percent in normal pine) of the units. The level of aldehydes, including new 2-methoxybenzaldehydes, is also increased. The mutant pines grew normally indicating that, even within a species, extensive variations in lignin composition need not disrupt the essential functions of lignin.

  18. Identifying representative drug resistant mutants of HIV

    PubMed Central

    2015-01-01

    Background Drug resistance is one of the most important causes for failure of anti-AIDS treatment. During therapy, multiple mutations accumulate in the HIV genome, eventually rendering the drugs ineffective in blocking replication of the mutant virus. The huge number of possible mutants precludes experimental analysis to explore the molecular mechanisms of resistance and develop improved antiviral drugs. Results In order to solve this problem, we have developed a new algorithm to reveal the most representative mutants from the whole drug resistant mutant database based on our newly proposed unified protein sequence and 3D structure encoding method. Mean shift clustering and multiple regression analysis were applied on genotype-resistance data for mutants of HIV protease and reverse transcriptase. This approach successfully chooses less than 100 mutants with the highest resistance to each drug out of about 10K in the whole database. When considering high level resistance to multiple drugs, the numbers reduce to one or two representative mutants. Conclusion This approach for predicting the most representative mutants for each drug has major importance for experimental verification since the results provide a small number of representative sequences, which will be amenable for in vitro testing and characterization of the expressed mutant proteins. PMID:26678327

  19. Construction and pilot screening of a signature-tagged mutant library of Sinorhizobium fredii.

    PubMed

    Wang, Dan; Wang, Yuan Chun; Wu, Li Juan; Liu, Jian Xin; Zhang, Pan; Jiao, Jian; Yan, Hui; Liu, Tao; Tian, Chang Fu; Chen, Wen Xin

    2016-03-01

    Sinorhizobium fredii is well known for its ability to establish symbiosis with diverse legumes such as Glycine max (soybean, determinate nodules) and Cajanus cajan (pigeon pea, indeterminate nodules). In order to make screening of S. fredii genes related to symbiosis cost-effective, we constructed a large Tn5 insertion mutant library of S. fredii CCBAU45436 using the signature-tagged mutagenesis (STM) technique. This STM library contains a total of 25,500 independent mutants distributed in 17 sublibraries tagged by corresponding distinct DNA bar-code sequences. After the pilot screening of 255 mutants in 15 batches, Tag85-4, Tag4-17, Tag4-11 and Tag10-13 were found to have attenuated competitiveness (0-30 % in nodule occupation) compared to the wild-type strain when inoculated on soybean. Further characterization of these mutants suggests that Tag4-11 (a pyrC mutant) and Tag10-13 (a nrdJ mutant) are defective in establishing symbiosis with soybean. The pyrC mutant induced uninfected pseudonodules while the nrdJ mutant formed significantly more nodules containing bacteroids with poor persistence ability. When these two mutants were tested on pigeon pea, host-specific symbiotic defects were found. These results demonstrated the STM library as a valuable resource for identifying S. fredii genes relevant to symbiosis. PMID:26472206

  20. New Infestin-4 Mutants with Increased Selectivity against Factor XIIa

    PubMed Central

    Vuimo, Tatiana A.; Surov, Stepan S.; Ovsepyan, Ruzanna A.; Korneeva, Vera A.; Vorobiev, Ivan I.; Orlova, Nadezhda A.; Minakhin, Leonid; Kuznedelov, Konstantin; Severinov, Konstantin V.; Ataullakhanov, Fazoil I.; Panteleev, Mikhail A.

    2015-01-01

    Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in ‘global’ assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its Ki for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5–20 μM, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics. PMID:26670620

  1. Screening of Bacillus subtilis transposon mutants with altered riboflavin production.

    PubMed

    Tännler, Simon; Zamboni, Nicola; Kiraly, Csilla; Aymerich, Stéphane; Sauer, Uwe

    2008-09-01

    To identify novel targets for metabolic engineering of riboflavin production, we generated about 10,000 random, transposon-tagged mutants of an industrial, riboflavin-producing strain of Bacillus subtilis. Process-relevant screening conditions were established by developing a 96-deep-well plate method with raffinose as the carbon source, which mimics, to some extent, carbon limitation in fed batch cultures. Screening in raffinose and complex LB medium identified more efficiently riboflavin overproducing and underproducing mutants, respectively. As expected for a "loss of function" analysis, most identified mutants were underproducers. Insertion mutants in two genes with yet unknown function, however, were found to attain significantly improved riboflavin titers and yields. These genes and possibly further ones that are related to them are promising candidates for metabolic engineering. While causal links to riboflavin production were not obvious for most underproducers, we demonstrated for the gluconeogenic glyceraldehyde-3-phosphate dehydrogenase GapB how a novel, non-obvious metabolic engineering strategy can be derived from such underproduction mutations. Specifically, we improved riboflavin production on various substrates significantly by deregulating expression of the gluconeogenic genes gapB and pckA through knockout of their genetic repressor CcpN. This improvement was also verified under the more process-relevant conditions of a glucose-limited fed-batch culture. PMID:18582593

  2. Towards an informative mutant phenotype for every bacterial gene

    DOE PAGESBeta

    Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly M.; Tarjan, Daniel R.; Xu, Zhuchen; Shao, Wenjen; Leon, Dacia; Arkin, Adam P.; Skerker, Jeffrey M.

    2014-08-11

    Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, inmore » Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness.« less

  3. Towards an informative mutant phenotype for every bacterial gene

    SciTech Connect

    Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly M.; Tarjan, Daniel R.; Xu, Zhuchen; Shao, Wenjen; Leon, Dacia; Arkin, Adam P.; Skerker, Jeffrey M.

    2014-08-11

    Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, in Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness.

  4. Genetic control of chromosome breakage and rejoining in Drosophila melanogaster: spontaneous chromosome aberrations in X-linked mutants defective in DNA metabolism.

    PubMed Central

    Gatti, M

    1979-01-01

    Eight X-linked recombination-defective meiotic mutants (representing five loci) and 12 X-linked mutagen-sensitive mutants (representing seven loci) of Drosophila melanogaster have been examined cytologically in neuroblast metaphases for their effects on the frequencies and types of spontaneous chromosome aberrations. Twelve mutants, representing five loci, significantly increase the frequency of chromosomal aberrations. The mutants at these five loci, however, differ markedly both in the types of aberrations produced and the localization of their effects along the chromosome. According to these criteria, the mutants can be assigned to four groups: (i) mutants producing almost exclusively chromatid breaks in both euchromatin and heterochromatin; (ii) mutants producing chromatid and isochromatid breaks in both euchromatin and heterochromatin; (iii) mutants producing chromatid mutants producing chromatid and isochromatid breaks clustered in the heterochromatin. Images PMID:108678

  5. Regulation of Mutant p53 Protein Expression

    PubMed Central

    Vijayakumaran, Reshma; Tan, Kah Hin; Miranda, Panimaya Jeffreena; Haupt, Sue; Haupt, Ygal

    2015-01-01

    For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation. PMID:26734569

  6. Uncaging Mutants: Moving From Menageries to Menages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The thousands of mutants of maize are a remarkable resource for study of plant physiology, phylogeny, cell biology, biochemistry, development, and molecular biology. Mutants are most often applied in research studies as "members of collections" rather than as select families of members relevant to ...

  7. Mutants of thermotaxis in Dictyostelium discoideum

    SciTech Connect

    Schneider, M.J.; Fontana, D.R.; Poff, K.L.

    1982-08-01

    Amoebae of Dictyostelium discoideum, strain HL50 were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, cloned, allowed to form pseudoplasmodia and screened for aberrant positive and negative thermotaxis. Three types of mutants were found. Mutant HO428 exhibits only positive thermotaxis over the entire temperature range (no negative thermotaxis). HO596 and HO813 exhibit weakened positive thermotaxis and normal negative thermotaxis. The weakened positive thermotactic response results in a shift toward warmer temperatures in the transition temperature from negative to positive thermotaxis. Mutant HO209 exhibits weakened positive and negative thermotactic responses and has a transition temperature similar to the 'wild type' (HL50).The two types of mutants represented by HO428, HO596 and HO813 support the model that positive and negative thermotaxis have separate pathways for temperature sensing. The type of mutants which contains HO209 suggests that those two pathways converge at some point before the response.

  8. Estimating the Degree of Saturation in Mutant Screens

    PubMed Central

    Pollock, David D.; Larkin, John C.

    2004-01-01

    Large-scale screens for loss-of-function mutants have played a significant role in recent advances in developmental biology and other fields. In such mutant screens, it is desirable to estimate the degree of “saturation” of the screen (i.e., what fraction of the possible target genes has been identified). We applied Bayesian and maximum-likelihood methods for estimating the number of loci remaining undetected in large-scale screens and produced credibility intervals to assess the uncertainty of these estimates. Since different loci may mutate to alleles with detectable phenotypes at different rates, we also incorporated variation in the degree of mutability among genes, using either gamma-distributed mutation rates or multiple discrete mutation rate classes. We examined eight published data sets from large-scale mutant screens and found that credibility intervals are much broader than implied by previous assumptions about the degree of saturation of screens. The likelihood methods presented here are a significantly better fit to data from published experiments than estimates based on the Poisson distribution, which implicitly assumes a single mutation rate for all loci. The results are reasonably robust to different models of variation in the mutability of genes. We tested our methods against mutant allele data from a region of the Drosophila melanogaster genome for which there is an independent genomics-based estimate of the number of undetected loci and found that the number of such loci falls within the predicted credibility interval for our models. The methods we have developed may also be useful for estimating the degree of saturation in other types of genetic screens in addition to classical screens for simple loss-of-function mutants, including genetic modifier screens and screens for protein-protein interactions using the yeast two-hybrid method. PMID:15454559

  9. Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models

    PubMed Central

    Amorim-Vaz, Sara; Delarze, Eric; Ischer, Françoise; Sanglard, Dominique; Coste, Alix T

    2015-01-01

    The aim of the present study was to identify Candida albicans transcription factors (TFs) involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens (FBs) quantified in kidneys. Mutants of unannotated genes which generated a kidney FB significantly different from that of wild-type were selected and individually examined in Galleria mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25% of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects), a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching FB phenotypes were observed in 50% of the cases, highlighting the bias due to host effects. In contrast, 33.4% concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the “pool effect.” After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adapt PMID:25999923

  10. Isolation and characterization of Schizosaccharomyces pombe fragile mutants.

    PubMed

    Belda, F; Zárate, V

    1996-05-01

    Three Schizosaccharomyces pombe fragile mutants requiring the presence of an osmotic stabilizer to grow, that lyse when transferred into hypotonic solutions and that secrete to the extracellular medium more protein than the parental strain were isolated. In the three mutants, the fragile phenotype segregated in a Mendelian fashion, indicating a single chromosomal gene mutation, and behaved as a recessive character. By complementation analysis, the three fragile mutants fell in a single complementation group, defining the same gene (SRB1). Mutations of this gene are responsible for alterations in the cells such as fragile character, increase in the cell wall porosity, changes in the cell morphology and floc-forming ability. The study of the three srb1 alleles indicated that the degree of these alterations is proportional to a significant decrease in the galactomannan fraction of the mutants cell wall. The data presented in this report suggest that the product of the SRB1 gene is critical for the maintenance of the integrity and structure of Sz. pombe cell wall. PMID:8771710

  11. Insulator dysfunction and oncogene activation in IDH mutant gliomas

    PubMed Central

    Flavahan, William A.; Drier, Yotam; Liau, Brian B.; Gillespie, Shawn M.; Venteicher, Andrew S.; Stemmer-Rachamimov, Anat O.; Suvà, Mario L.; Bernstein, Bradley E.

    2015-01-01

    Gain-of-function IDH mutations are initiating events that define major clinical and prognostic classes of gliomas1,2. Mutant IDH protein produces a novel onco-metabolite, 2-hydroxyglutarate (2-HG), that interferes with iron-dependent hydroxylases, including the TET family of 5′-methylcytosine hydroxylases3–7. TET enzymes catalyze a key step in the removal of DNA methylation8,9. IDH mutant gliomas thus manifest a CpG island methylator phenotype (G-CIMP)10,11, though the functional significance of this altered epigenetic state remains unclear. Here we show that IDH mutant gliomas exhibit hyper-methylation at CTCF binding sites, compromising binding of this methylation-sensitive insulator protein. Reduced CTCF binding is associated with loss of insulation between topological domains and aberrant gene activation. We specifically demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to aberrantly interact with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Treatment of IDH mutant gliomaspheres with demethylating agent partially restores insulator function and down-regulates PDGFRA. Conversely, CRISPR-mediated disruption of the CTCF motif in IDH wildtype gliomaspheres up-regulates PDGFRA and increases proliferation. Our study suggests that IDH mutations promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression. PMID:26700815

  12. Factors influencing maternal behavior in the hubb/hubb mutant mouse.

    PubMed

    Alston-Mills, B; Parker, A C; Eisen, E J; Wilson, R; Fletcher, S

    We examined the maternal behavior of hubb/hubb mutant mice and normal control (+/hubb) siblings. From previous observations we noted that mutants groom their pups less, suckle less than normal, and often cannibalize the young. To date, these observations had not been quantified. Although prolactin (PRL) is linked to maternal behavior, it was difficult to measure because of the hyperirratibility of the mutant mice. Consequently, dopamine (DA) and its metabolite, dihydroxyphenylacetic acid (DOPAC), were measured in the median eminence in brains of both normal and mutant mice. Tyrosine hydroxylase, the rate-determining step in dopamine synthesis, was localized in the brain by immunohistochemistry. Five mutant and nine normal dams were observed for pup retrieval and crouching. Mean time for pup retrieval was slower (p < 0.06) for mutants (28.09 s) than for normal dams (18.49 s). Crouching was the same for both strains. Mutant pups were cold to the touch, and not well groomed. Brains from both strains were examined at Day 11 and Day 18 of gestation and Day 2 and Day 11 of lactation. Qualitatively, tyrosine hydroxylase localization in the arcuate nucleus and median eminence was the same in both strains for the gestation samples. The decrease in staining observed from gestation to lactation in the normal mice was increased in the mutants. Dopamine was similar in both strains at all stages, but DOPAC was significantly higher at early lactation in the mutants. We do not assume an absolute inverse relationship between dopaminergic activities and prolactin, but it is likely that the increase in DOPAC in the mutant reflects a decrease in prolactin, which could contribute to the diminished maternal care in the mutants. PMID:10627055

  13. Reduced gravitropic sensitivity in roots of a starch-deficient mutant of Nicotiana sylvestris

    NASA Technical Reports Server (NTRS)

    Kiss, J. Z.; Sack, F. D.

    1989-01-01

    Gravitropism was studied in seedlings of Nicotiana sylvestris Speg. et Comes wild-type (WT) and mutant NS 458 which has a defective plastid phosphoglucomutase (EC 2.7.5.1.). Starch was greatly reduced in NS 458 compared to the WT, but small amounts of starch were detected in rootcap columella cells in NS 458 by light and electron microscopy. The roots of WT are more sensitive to gravity than mutant NS 458 roots since: (1) in mutant roots, curvature was reduced and delayed in the time course of curvature; (2) curvature of mutant roots was 24-56% that of WT roots over the range of induction periods tested; (3) in intermittent-stimulation experiments, curvature of mutant roots was 37% or less than that of WT roots in all treatments tested. The perception time, determined by intermittent-stimulation experiments, was < or = 5 s for WT roots and 30-60 s for mutant roots. The growth rates for WT and NS 458 roots were essentially equal. These results and our previous results with WT and starchless mutant Arabidopsis roots (Kiss et al. 1989, Planta 177, 198-206) support the conclusions that a full complement of starch is necessary for full gravitropic sensitivity and that amyloplasts function in gravity perception. Since a presumed relatively small increase in plastid buoyant mass (N. sylvestris mutant versus Arabidopsis mutant) significantly improves the orientation of the N. sylvestris mutant roots, we suggest that plastids are the likeliest candidates to be triggering gravity perception in roots of both mutants.

  14. Generation and Evaluation of High β-Glucan Producing Mutant Strains of Sparassis crispa

    PubMed Central

    Kim, Seung-Rak; Kang, Hyeon-Woo

    2013-01-01

    A chemical mutagenesis technique was employed for development of mutant strains of Sparassis crispa targeting the shortened cultivation time and the high β-glucan content. The homogenized mycelial fragments of S. crispa IUM4010 strain were treated with 0.2 vol% methyl methanesulfonate, an alkylating agent, yielding 199 mutant strains. Subsequent screening in terms of growth and β-glucan content yielded two mutant strains, B4 and S7. Both mutants exhibited a significant increase in β-glucan productivity by producing 0.254 and 0.236 mg soluble β-glucan/mg dry cell weight for the B4 and S7 strains, respectively, whereas the wild type strain produced 0.102 mg soluble β-glucan/mg dry cell weight. The results demonstrate the usefulness of chemical mutagenesis for generation of mutant mushroom strains. PMID:24198672

  15. Biochemical Studies of Paraquat-Tolerant Mutants of the Fern Ceratopteris richardii1

    PubMed Central

    Carroll, Elizabeth W.; Schwarz, Otto J.; Hickok, Leslie G.

    1988-01-01

    Enzymes and metabolites associated with mitigation of paraquat toxicity were compared in two paraquat-tolerant mutants and a sensitive wild-type strain of the fern Ceratopteris richardii Brongn. In 21-day-old gametophytes, the specific activities of superoxide dismutase, catalase, peroxidase, glutathione reductase, dehydroascorbate reductase, and ascorbate peroxidase showed no differences that would explain mutant tolerance. Constitutive levels of ascorbate and glutathione also did not differ significantly in the three strains. An experiment testing the inducibility of paraquat tolerance revealed no change in the dose response of mutant or wild type gametophytes after exposure to sublethal concentrations of the herbicide. Uptake of paraquat by whole gametophytes was also equivalent in mutants and wild type. These data suggest that the physiological basis for tolerance in these mutants, unlike several other tolerant biotypes reported, does not lie in the oxygen radical scavenging system, in an inducible stress response, or in a block to whole-plant uptake. PMID:16666201

  16. Azide-resistant mutants of Azorhizobium caulinodans with enhanced symbiotic effectiveness.

    PubMed

    Saini, I; Sindhu, S S; Dadarwal, K R

    2001-01-01

    Azide-resistant mutants of Azorhizobium caulinodans strains Sb3, S78, SrR13 and SrS8 were isolated and screened for nitrate reductase activity. Selected nitrate reductase negative mutants were inoculated on Sesbania bispinosa and S. rostrata under sterile conditions in chillum jars to study their symbiotic behavior. Azide-resistant mutants exhibited either similar or higher symbiotic effectiveness than the parent strain after 30 d of plant growth. Nodule mass, nitrogenase activity and uptake hydrogenase activity of the mutants varied depending on the host as well as on the plant growth stage. In comparison to wild-type parent strains, four azide-resistant mutants, Sb3Az18, S78Az21, SrR13Az17 and SrS8Az6 showed significant increase in nodulation and nitrogen fixation as well as shoot dry mass of the inoculated plants. PMID:11702406

  17. Kasugamycin-dependent mutants of Escherichia coli.

    PubMed Central

    Dabbs, E R

    1978-01-01

    Kasugamycin-dependent mutants have been isolated from Escherichia coli B. They were obtained through mutagenesis with ethyl methane sulfonate or nitrosoguanidine in conjunction with an antibiotic underlay technique. In the case of nitrosoguanidine, dependent mutants were obtained at a frequency of about 3% of survivors growing up in the selection. In the case of ethyl methane sulfonate, the corresponding value was 1%. Nineteen mutants showing a kasugamycin-dependent phenotype were studied. In terms of response to various temperatures and antibiotic concentrations, they were very heterogeneous, although most fell into two general classes. Genetic analysis indicated that in at least some cases, the kasugamycin-dependent phenotype was the product of two mutations. Two-dimensional gel electropherograms revealed alterations in the ribosomal proteins of seven mutants. One mutant had an alteration in protein S13, and one had an alteration in protein L14. Three showed changes in protein S9. Each of two mutants had changes in two proteins, S18 and L11. Three of these mutants additionally had protein S18 occurring in a partly altered, partly unaltered form. Images PMID:363701

  18. Phenotypic comparison of samdc and spe mutants reveals complex relationships of polyamine metabolism in Ustilago maydis.

    PubMed

    Valdés-Santiago, Laura; Cervantes-Chávez, José Antonio; Winkler, Robert; León-Ramírez, Claudia G; Ruiz-Herrera, José

    2012-03-01

    Synthesis of spermidine involves the action of two enzymes, spermidine synthase (Spe) and S-adenosylmethionine decarboxylase (Samdc). Previously we cloned and disrupted the gene encoding Spe as a first approach to unravel the biological function of spermidine in Ustilago maydis. With this background, the present study was designed to provide a better understanding of the role played by Samdc in the regulation of the synthesis of this polyamine. With this aim we proceeded to isolate and delete the gene encoding Samdc from U. maydis, and made a comparative analysis of the phenotypes of samdc and spe mutants. Both spe and samdc mutants behaved as spermidine auxotrophs, and were more sensitive than the wild-type strain to different stress conditions. However, the two mutants displayed significant differences: in contrast to spe mutants, samdc mutants were more sensitive to LiCl stress, high spermidine concentrations counteracted their dimorphic deficiency, and they were completely avirulent. It is suggested that these differences are possibly related to differences in exogenous spermidine uptake or the differential location of the respective enzymes in the cell. Alternatively, since samdc mutants accumulate higher levels of S-adenosylmethionine (SAM), whereas spe mutants accumulate decarboxylated SAM, the known opposite roles of these metabolites in the processes of methylation and differentiation offer an additional attractive hypothesis to explain the phenotypic differences of the two mutants, and provide insights into the additional roles of polyamine metabolism in the physiology of the cell. PMID:22222500

  19. Correction of Hair Shaft Defects through Allele-Specific Silencing of Mutant Krt75.

    PubMed

    Liu, Ying; Snedecor, Elizabeth R; Zhang, Xu; Xu, Yanfeng; Huang, Lan; Jones, Evan C; Zhang, Lianfeng; Clark, Richard A; Roop, Dennis R; Qin, Chuan; Chen, Jiang

    2016-01-01

    Dominant mutations in keratin genes can cause a number of inheritable skin disorders characterized by intraepidermal blistering, epidermal hyperkeratosis, or abnormalities in skin appendages, such as nail plate dystrophy and structural defects in hair. Allele-specific silencing of mutant keratins through RNA interference is a promising therapeutic approach for suppressing the expression of mutant keratins and related phenotypes in the epidermis. However, its effectiveness on skin appendages remains to be confirmed in vivo. In this study, we developed allele-specific small interfering RNAs capable of selectively suppressing the expression of a mutant Krt75, which causes hair shaft structural defects characterized by the development of blebs along the hair shaft in mice. Hair regenerated from epidermal keratinocyte progenitor cells isolated from mutant Krt75 mouse models reproduced the blebbing phenotype when grafted in vivo. In contrast, mutant cells manipulated with a lentiviral vector expressing mutant Krt75-specific short hairpin RNA (shRNA) persistently suppressed this phenotype. The phenotypic correction was associated with a significant reduction of mutant Krt75 mRNA in the skin grafts. Thus, data obtained from this study demonstrated the feasibility of utilizing RNA interference to achieve durable correction of hair structural phenotypes through allele-specific silencing of mutant keratin genes. PMID:26763422

  20. Correction of hair shaft defects through allele-specific silencing of mutant Krt75

    PubMed Central

    Liu, Ying; Snedecor, Elizabeth R.; Zhang, Xu; Xu, Yan-Feng; Huang, Lan; Jones, Evan; Zhang, Lianfeng; Clark, Richard A.; Roop, Dennis R.; Qin, Chuan; Chen, Jiang

    2015-01-01

    Dominant mutations in keratin genes can cause a number of inheritable skin disorders characterized by intraepidermal blistering, epidermal hyperkeratosis, or abnormalities in skin appendages, such as nail plate dystrophy and structural defects in hair. Allele-specific silencing of mutant keratins through RNA interference is a promising therapeutic approach for suppressing the expression of mutant keratins and related phenotypes in the epidermis. However, its effectiveness on skin appendages remains to be confirmed in vivo. In this study, we developed allele specific siRNAs capable of selectively suppressing the expression of a mutant Krt75, which causes hair shaft structural defects characterized by the development of blebs along the hair shaft in mice. Hair regenerated from epidermal keratinocyte progenitor cells isolated from mutant Krt75 mouse models reproduced the blebbing phenotype when grafted in vivo. In contrast, mutant cells manipulated with a lentiviral vector expressing mutant Krt75-specific shRNA persistently suppressed this phenotype. The phenotypic correction was associated with significant reduction of mutant Krt75 mRNA in the skin grafts. Thus, data obtained from this study demonstrated the feasibility of utilizing RNA interference to achieve durable correction of hair structural phenotypes through allele-specific silencing of the mutant keratin genes. PMID:26763422

  1. Mutant HNF-1{alpha} and mutant HNF-1{beta} identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

    SciTech Connect

    Gu Ning; Adachi, Tetsuya; Matsunaga, Tetsuro; Takeda, Jun; Tsujimoto, Gozoh; Ishihara, Akihiko; Yasuda, Koichiro; Tsuda, Kinsuke . E-mail: jinkan@tom.life.h.kyoto-u.ac.jp

    2006-08-04

    Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1{alpha} and HNF-1{beta}, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1{alpha} and mutant HNF-1{beta} in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1{alpha} and 13 mutant HNF-1{alpha}, as well as wild HNF-1{beta} and 2 mutant HNF-1{beta}, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1{alpha} and wild HNF-1{beta} significantly transactivated DPP-IV promoter, but mutant HNF-1{alpha} and mutant HNF-1{beta} exhibited low transactivation activity. Moreover, to study whether mutant HNF-1{alpha} and mutant HNF-1{beta} change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1{alpha} or wild HNF-1{beta}, or else respective dominant-negative mutant HNF-1{alpha}T539fsdelC or dominant-negative mutant HNF-1{beta}R177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1{alpha} cells and wild HNF-1{beta} cells, whereas they decreased in HNF-1{alpha}T539fsdelC cells and HNF-1{beta}R177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1{alpha} and wild HNF-1{beta} have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1{alpha} and mutant HNF-1{beta} attenuate the stimulatory effect.

  2. Swimming activity in dystonia musculorum mutant mice.

    PubMed

    Lalonde, R; Joyal, C C; Cote, C

    1993-07-01

    Dystonia musculorum (dt) mutant mice, characterized by degeneration of spinocerebellar fibers, were evaluated in a visible platform swim test. It was found that dt mutants were slower to reach the platform than normal mice. However, the number of quadrants traversed was not higher in dt mutants. It is concluded that spinocerebellar fibers to the vermis are important in limb control during swimming but not in visuo-motor guidance (navigational skills) of the animal towards a visible goal, at least in regard to the quadrant measure. It is not excluded that a measure tracing their path may find a mild deviation from the goal. PMID:8327590

  3. Molecular characterization of an atl null mutant of Staphylococcus aureus.

    PubMed

    Takahashi, Junko; Komatsuzawa, Hitoshi; Yamada, Sakuo; Nishida, Tetsuya; Labischinski, Harald; Fujiwara, Tamaki; Ohara, Masaru; Yamagishi, Jun-ichi; Sugai, Motoyuki

    2002-01-01

    atl is a gene encoding a bifunctional peptidoglycan hydrolase of Staphylococcus aureus. The gene product of atl is a 138 kDa protein that has an amidase domain and a glucosaminidase domain, and undergoes processing to generate two major peptidoglycan hydrolases, a 51 kDa glucosaminidase and a 62 kDa amidase in culture supernatant. An atl null mutant was isolated by allelic replacement and characterized. The mutant grew in clusters and sedimented when grown in broth culture. Analysis of peptidoglycan prepared from the wild type and the mutant revealed that there were no differences in muropeptide composition or in glycan chain length distribution. On the other hand, the atl mutation resulted in pleiotropic effects on cell surface nature. The mutant cells showed complete inhibition of metabolic turnover of cell wall peptidoglycan and revealed a rough outer cell wall surface. The mutation also decreased the amount of protein non-covalently bound to the cell surface and altered the protein profile, but did not affect proteins covalently associated with the cell wall. Lysis of growing cells treated with otherwise lytic concentration of penicillin G was completely inhibited in the mutant, but that of non-growing cells was not affected by the mutation. The atl mutation did not significantly affect the ability of S. aureus to provoke an acute infection when inoculated intraperitoneally in a mouse sepsis model. These results further support the supposition that atl gene products are involved in cell separation, cell wall turnover and penicillin-induced lysis of the cells. PMID:12437027

  4. Fatty acid composition analyses of the DCMU resistant mutants of Nannochloropsis oculata (eustigmatophyceae)

    NASA Astrophysics Data System (ADS)

    Jimin, Zhang; Shuang, Liu; Xue, Sun; Guanpin, Yang; Xuecheng, Zhang; Zhenhui, Gao

    2003-04-01

    Ultraviolet mutagenesis was applied to Nannochloropsis oculata and three mutants resistant to 3-(3, 4-dichlorophenyl)-1,1-dimethylurea (DCMU) were isolated. The cellular chlorophyll a and total lipid content of the wild are higher in the medium supplemented with DCMU than in the control without DCMU. Without DCMU, the growth rates and chlorophyll a contents of the mutants are similar to those of the wild. Significant changes of fatty acid content and composition have occurred in DCMU-resistant mutants growing in the medium supplemented with DCMU. The total lipid, palmitic acid (16:0), palmitoleic acid (16:1ω9) and oleic (18:1ω9) contents decrease significantly, while the vaccenic acid (18:1ω11) increases significantly and the EPA content of dried powder increases slightly in the mutants. The study may provide a basis to improve EPA content in Nannochloropsis oculata in the future.

  5. Phosphoglucomutase Mutants of Escherichia coli K-12

    PubMed Central

    Adhya, Sankar; Schwartz, Maxime

    1971-01-01

    Bacteria with strongly depressed phosphoglucomutase (EC 2.7.5.1) activity are found among the mutants of Escherichia coli which, when grown on maltose, accumulate sufficient amylose to be detectable by iodine staining. These pgm mutants grow poorly on galactose but also accumulate amylose on this carbon source. Growth on lactose does not produce high amylose but, instead, results in the induction of the enzymes of maltose metabolism, presumably by accumulation of maltose. These facts suggest that the catabolism of glucose-1-phosphate is strongly depressed in pgm mutants, although not completely abolished. Anabolism of glucose-1-phosphate is also strongly depressed, since amino acid- or glucose-grown pgm mutants are sensitive to phage C21, indicating a deficiency in the biosynthesis of uridine diphosphoglucose or uridine diphosphogalactose, or both. All pgm mutations isolated map at about 16 min on the genetic map, between purE and the gal operon. PMID:4942754

  6. Cooperative Interaction Within RNA Virus Mutant Spectra.

    PubMed

    Shirogane, Yuta; Watanabe, Shumpei; Yanagi, Yusuke

    2016-01-01

    RNA viruses usually consist of mutant spectra because of high error rates of viral RNA polymerases. Growth competition occurs among different viral variants, and the fittest clones predominate under given conditions. Individual variants, however, may not be entirely independent of each other, and internal interactions within mutant spectra can occur. Examples of cooperative and interfering interactions that exert enhancing and suppressing effects on replication of the wild-type virus, respectively, have been described, but their underlying mechanisms have not been well defined. It was recently found that the cooperation between wild-type and variant measles virus genomes produces a new phenotype through the heterooligomer formation of a viral protein. This observation provides a molecular mechanism underlying cooperative interactions within mutant spectra. Careful attention to individual sequences, in addition to consensus sequences, may disclose further examples of internal interactions within mutant spectra. PMID:26162566

  7. Mutant p53: one name, many proteins

    PubMed Central

    Freed-Pastor, William A.; Prives, Carol

    2012-01-01

    There is now strong evidence that mutation not only abrogates p53 tumor-suppressive functions, but in some instances can also endow mutant proteins with novel activities. Such neomorphic p53 proteins are capable of dramatically altering tumor cell behavior, primarily through their interactions with other cellular proteins and regulation of cancer cell transcriptional programs. Different missense mutations in p53 may confer unique activities and thereby offer insight into the mutagenic events that drive tumor progression. Here we review mechanisms by which mutant p53 exerts its cellular effects, with a particular focus on the burgeoning mutant p53 transcriptome, and discuss the biological and clinical consequences of mutant p53 gain of function. PMID:22713868

  8. Identification of amylase inhibitor deficient mutants in pigeonpea (Cajanus cajan (L.) Millisp.).

    PubMed

    Chougule, N P; Giri, A P; Hivrale, V K; Chhabda, P J; Kachole, M S

    2004-06-01

    We have developed and analyzed several mutant lines (M6 generation) of pigeonpea (Cajanus cajan (L.) Millsp.) for the content of defensive proteins and antinutritional factors. Inhibitors of proteinase and of amylase, lectins, and raffinose family oligosaccharides were analyzed in mature seeds of different pigeonpea accessions (untreated) and compared with mutant lines. Proteinase inhibitor profiles were similar in terms of number and intensities of activity bands but they differ marginally in the activity units in pigeonpea accessions and mutants. Pigeonpea mutants showed significant differences in amylase inhibitor profiles as well as activity units from those of pigeonpea accessions. Interestingly, two mutants (A6-5-1 and A7-3-2) were identified to have absence of amylase inhibitor isoforms. Hemagglutinating activity and raffinose family oligosaccharides content were found to be significantly higher in mutants than in accessions. It is evident from the results that proteinase inhibitors of pigeonpea are stable while amylase inhibitors, lectins, and raffinose family oligosaccharides show altered expression upon mutagen treatments. These mutants will be ideal candidates for further evaluation. PMID:15260142

  9. Ipsen 5i is a Novel Potent Pharmacoperone for Intracellularly Retained Melanocortin-4 Receptor Mutants

    PubMed Central

    Tao, Ya-Xiong; Huang, Hui

    2014-01-01

    Inactivating mutations of the melanocortin-4 receptor (MC4R) cause early-onset severe obesity in humans. Comprehensive functional studies show that most of the inactivating mutants of the MC4R are retained intracellularly. In the present study, we investigated whether a small molecule inverse agonist of the MC4R, Ipsen 5i, could act as a pharmacoperone and correct the cell surface expression and function of intracellularly retained mutant MC4Rs using multiple cell lines, including HEK293 and two neuronal cell lines. We showed that Ipsen 5i rescued the cell surface expression of all 11 intracellularly retained mutant MC4Rs studied herein in at least one cell line. Ipsen 5i functionally rescued seven mutants in all cell lines used. One mutant (Y157S) was functionally rescued in HEK293 cells but not in the two neuronal cell lines. Ipsen 5i increased cell surface expression of three mutants (S58C, G98R, and F261S) but did not affect signaling. Ipsen 5i had no effect on mutant MC4Rs with other defects (Δ88-92, D90N, I102S) or no defect (N274S). It also did not affect trafficking of a misrouted MC3R mutant (I335S). Cell impermeable peptide ligands of the MC4R or cell permeable small molecule ligand of δ opioid receptor could not rescue misrouted mutant MC4R. In summary, we demonstrated that Ipsen 5i was a novel potent pharmacoperone of the MC4R, correcting trafficking and signaling of a significant portion (73%) of intracellularly retained mutants. Additional studies are needed to demonstrate its in vivo efficacy. PMID:25136332

  10. Arabidopsis mutants with a reduced seed dormancy.

    PubMed Central

    Léon-Kloosterziel, K M; van de Bunt, G A; Zeevaart, J A; Koornneef, M

    1996-01-01

    The development of seed dormancy is an aspect of seed maturation, the last stage of seed development. To isolate mutants of Arabidopsis thaliana that are affected in this process, we selected directly for the absence of dormancy among freshly harvested M2 seeds. The screen yielded two mutants exhibiting a reduced dormancy, rdo1 and rdo2, that are specifically affected in dormancy determined by the embryo. The rdo1 and rdo2 mutants show normal levels of abscisic acid and the same sensitivity to abscisic acid, ethylene, auxin, and cytokinin as the wild type. The rdo2 mutant but not the rdo1 mutant has a reduced sensitivity to the gibberellin biosynthesis inhibitor tetcyclacis. Double-mutant analysis suggested that the RDO1 and RDO2 genes are involved in separate pathways leading to the development of dormancy. We assume that the RDO2 gene controls a step in the induction of dormancy that is most likely induced by abscisic acid and is expressed as an increase of the gibberellin requirement for germination. PMID:8587986

  11. Dynamic void distribution in myoglobin and five mutants.

    PubMed

    Jiang, Yingying; Kirmizialtin, Serdal; Sanchez, Isaac C

    2014-01-01

    Globular proteins contain cavities/voids that play specific roles in controlling protein function. Elongated cavities provide migration channels for the transport of ions and small molecules to the active center of a protein or enzyme. Using Monte Carlo and Molecular Dynamics on fully atomistic protein/water models, a new computational methodology is introduced that takes into account the protein's dynamic structure and maps all the cavities in and on the surface. To demonstrate its utility, the methodology is applied to study cavity structure in myoglobin and five of its mutants. Computed cavity and channel size distributions reveal significant differences relative to the wild type myoglobin. Computer visualization of the channels leading to the heme center indicates restricted ligand access for the mutants consistent with the existing interpretations. The new methodology provides a quantitative measure of cavity structure and distributions and can become a valuable tool for the structural characterization of proteins. PMID:24500195

  12. Dominant-and-recessive epistasis in a homeotic mosquito mutant.

    PubMed

    Bhalla, S C

    1976-12-01

    Following selection for 15 generations a pure strain of a homeotic mutant spur was isolated from a Brazilian population of the mosquito Culex pipiens fatigans. Monohybrid crosses showed a 13:3 segregation indicating dominant-and-recessive epistasis for wild-type vs. spur. This implies that a dominant allele at one locus and a recessive at the other interact to produce the mutant phenotype. Dihybrid crosses with linkage group II markers yellow and ruby gave 39:13:9:3 ratios indicating independent segregation. However, the dihybrid cross with linkage group I marker maroon showed a highly significant departure from 39:13:9:3 ratio. Data available indicate that the phenotype spur is controlled by a dominant epistat in linkage group III and a recessive epistat (approximately 31.9 crossover units from maroon) in linkage group I. PMID:1022329

  13. Trans-dominant negative mutants of Fos and Jun.

    PubMed Central

    Ransone, L J; Visvader, J; Wamsley, P; Verma, I M

    1990-01-01

    Jun and Fos nuclear oncoproteins form a complex that regulates transcription from promoters containing activator protein AP-1 binding sites. The leucine-zipper and basic-region domains of both Fos and Jun are necessary for formation of the heterodimer that binds to DNA. Reciprocal mutations in the basic region of Fos or Jun can influence the binding of the heterodimer to DNA, implying a symmetrical binding site. DNA-binding mutants of Jun exhibit increased affinity for Fos and are capable of suppressing wild-type Fos-Jun DNA-binding activity. In contrast, mutations in the basic domain of Fos, which prevent binding to DNA in association with Jun, do not significantly diminish the ability of the wild-type heterodimer to bind to DNA. These dominant negative mutants are functional in vivo and can be exploited to study the role of Fos and Jun in normal and transformed cells. Images PMID:2111017

  14. Pleiotropic effects of hemagglutinin amino acid substitutions of H5 influenza escape mutants

    SciTech Connect

    Rudneva, Irina A.; Timofeeva, Tatiana A.; Ignatieva, Anna V.; Shilov, Aleksandr A.; Krylov, Petr S.; Ilyushina, Natalia A.; Kaverin, Nikolai V.

    2013-12-15

    In the present study we assessed pleiotropic characteristics of the antibody-selected mutations. We examined pH optimum of fusion, temperatures of HA heat inactivation, and in vitro and in vivo replication kinetics of the previously obtained influenza H5 escape mutants. Our results showed that HA1 N142K mutation significantly lowered the pH of fusion optimum. Mutations of the escape mutants located in the HA lateral loop significantly affected H5 HA thermostability (P<0.05). HA changes at positions 131, 144, 145, and 156 and substitutions at positions 131, 142, 145, and 156 affected the replicative ability of H5 escape mutants in vitro and in vivo, respectively. Overall, a co-variation between antigenic specificity and different HA phenotypic properties has been demonstrated. We believe that the monitoring of pleiotropic effects of the HA mutations found in H5 escape mutants is essential for accurate prediction of mutants with pandemic potential. - Highlights: • HA1 N142K mutation significantly lowered the pH of fusion optimum. • Mutations located in the HA lateral loop significantly affected H5 HA thermostability. • HA changes at positions 131, 142, 144, 145, and 156 affected the replicative ability of H5 mutants. • Acquisition of glycosylation site could lead to the emergence of multiple pleiotropic effects.

  15. Isolation and characterization of hypertoxinogenic (htx) mutants of Escherichia coli KL320(pCG86).

    PubMed Central

    Bramucci, M G; Twiddy, E M; Baine, W B; Holmes, R K

    1981-01-01

    The structural genes for heat-labile enterotoxin (LT) are present on plasmid pCG86. Escherichia coli KL320(pCG86), LT was found to be cell associated. LT was present as a soluble protein in sonic lysates of KL320(pCG86). Thirty-one mutants of KL320(pCG86) that produced increased amounts of extracellular LT were isolated. These hypertoxinogenic (htx) mutants were assigned to four phenotypically distinct classes based on the amounts of cell-associated and extracellular LT in early-stationary-phase cultures. Type 1 and type 2 htx mutants produced significantly increased amounts of cell-associated LT. Type 3 and type 4 htx mutants produced normal or decreased amounts of cell-associated LT was similar to that of the wild type. In the mutants of types 1, 3, and 4, the ratios of extracellular to cell-associated LT were higher than that of the wild type and were characteristic for each strain. Cell lysis or leakage of macromolecular cytoplasmic constituents appeared to be significant for release of LT by mutants of types 1, 3, and 4, because supernatants from cultures of these mutants also contained increased amounts of protein and of the cytoplasmic enzyme glucose 6-phosphate dehydrogenase. In all four representative htx mutants, the hypertoxinogenic phenotypes were dependent on chromosomal mutations. The resident pCG86 plasmids were eliminated from the htx mutants of types 2 and 3. After wild-type plasmid pCG86 was introduced into the cured strains by conjugation, their hypertoxinogenic phenotypes were restored. We conclude that chromosomal loci in E. coli KL320 are important in regulating expression of the LT structural genes of plasmid pCG86. Images PMID:7019086

  16. Biological Significance of Mutant Isocitrate Dehydrogenase 1 and 2 in Gliomagenesis

    PubMed Central

    OHBA, Shigeo; HIROSE, Yuichi

    2016-01-01

    Mutations of the isocitrate dehydrogenase (IDH) genes are considered an important event that occurs at an early stage during gliomagenesis. The mutations often occur in grade 2 or 3 gliomas and secondary glioblastomas. Most IDH mutations are associated with codon 132 and 172 in IDH1 and IDH2 in gliomas, respectively. While IDH1 and IDH2 catalyze the oxidative decarboxylation of isocitrate to form α-ketoglutarate (α-KG), IDH1 and IDH2 mutations convert α-KG to 2-hydroxyglutarate (2-HG). The accumulation of oncometabolite 2-HG is believed to lead progenitor cells into gliomas, inhibiting several α-KG-dependent enzymes, including ten-eleven translocation enzymes, histone demethylases, and prolyl hydroxylases, although the mechanisms have not been fully revealed. Herein, we review the contribution of IDH1 and IDH2 mutations to gliomagenesis. PMID:26960449

  17. Biological Significance of Mutant Isocitrate Dehydrogenase 1 and 2 in Gliomagenesis.

    PubMed

    Ohba, Shigeo; Hirose, Yuichi

    2016-04-15

    Mutations of the isocitrate dehydrogenase (IDH) genes are considered an important event that occurs at an early stage during gliomagenesis. The mutations often occur in grade 2 or 3 gliomas and secondary glioblastomas. Most IDH mutations are associated with codon 132 and 172 in IDH1 and IDH2 in gliomas, respectively. While IDH1 and IDH2 catalyze the oxidative decarboxylation of isocitrate to form α-ketoglutarate (α-KG), IDH1 and IDH2 mutations convert α-KG to 2-hydroxyglutarate (2-HG). The accumulation of oncometabolite 2-HG is believed to lead progenitor cells into gliomas, inhibiting several α-KG-dependent enzymes, including ten-eleven translocation enzymes, histone demethylases, and prolyl hydroxylases, although the mechanisms have not been fully revealed. Herein, we review the contribution of IDH1 and IDH2 mutations to gliomagenesis. PMID:26960449

  18. Isolation and characterization of butanol-resistant mutants of Clostridium acetobutylicum

    SciTech Connect

    Hermann, M.; Fayolle, f.; Marchal, R.; Podvin, L.; Sebald, M.; Vandecasteele, J.P.

    1985-11-01

    In a wild-type strain of Clostridium acetobutylicum isolated from soil, solvent production appeared limited by butanol toxicity. Butanol-resistant mutants have been obtained which produced significantly higher solvent concentrations (about 30%) than the wild-type strain. Some other physiological differences were observed between a selected resistant mutant and the wild-type strain at the level of solvent resistance and sporulation.

  19. Diminished virulence of a sar-/agr- mutant of Staphylococcus aureus in the rabbit model of endocarditis.

    PubMed Central

    Cheung, A L; Eberhardt, K J; Chung, E; Yeaman, M R; Sullam, P M; Ramos, M; Bayer, A S

    1994-01-01

    Microbial pathogenicity in Staphylococcus aureus is a complex process involving a number of virulence genes that are regulated by global regulatory systems including sar and agr. To evaluate the roles of these two loci in virulence, we constructed sar-/agr- mutants of strains RN6390 and RN450 and compared their phenotypic profiles to the corresponding single sar- and agr- mutants and parents. The secretion of all hemolysins was absent in the sar-/agr- mutants while residual beta-hemolysin activity remained in single agr- mutants. The fibronectin binding capacity was significantly diminished in both single sar- mutants and double mutants when compared with parents while the reduction in fibrinogen binding capacity in the double mutants was modest. In the rabbit endocarditis model, there was a significant decrease in both infectivity rates and intravegetation bacterial densities with the double mutant as compared to the parent (RN6390) at 10(3)-10(6) CFU inocula despite comparable levels of early bacteremia among various challenge groups. Notably, fewer bacteria in the double mutant group adhered to valvular vegetations at 30 min after challenge (10(6) CFU) than the parent group. These studies suggest that both the sar and agr loci are involved in initial valvular adherence, intravegetation persistence and multiplication of S. aureus in endocarditis. Images PMID:7962526

  20. Structural stability of Bacillus thuringiensis delta-endotoxin homolog-scanning mutants determined by susceptibility to proteases.

    PubMed Central

    Almond, B D; Dean, D H

    1993-01-01

    Forty homolog-scanning (double-reciprocal-crossover) mutant proteins of two Bacillus thuringiensis delta-endotoxin genes (cryIAa and cryIAc) were examined for potential structural alterations by a series of proteolytic assays. Three groups of mutants could be identified. Group 1, consisting of 13 mutants, showed no delta-endotoxin present during overexpression conditions in Escherichia coli (48 h at 37 degrees C, with a ptac promoter). These mutants produced full-sized delta-endotoxin detectable by polyacrylamide gel electrophoresis with Coomassie blue staining or Western immunoanalysis after 24 h of growth but not after 48 h, suggesting sensitivity to intracellular proteases. Group 2 consisted of 13 mutants that produced stable delta-endotoxins that were completely digested by 2% bovine trypsin. In contrast, native delta-endotoxin produces a 65,000-Da trypsin-resistant peptide, which is the active toxin. Group 3 mutants expressed delta-endotoxin and trypsin-stable toxins, similar to the wild type. In this study, 12 group 3 mutant toxins were compared with wild type toxins by thermolysin digestion at a range of temperatures. The two wild-type toxins exhibited significant differences in thermolysin digestion midpoints. Among the group 3 mutants, most possessed significantly different protein stabilities relative to their parental toxins. Two of the group 3 mutants were observed to have exchanged the thermolysin sensitivity properties of the parental toxins. Images PMID:8368834

  1. Effect of light quality and vernalization on late-flowering mutants of Arabidopsis thaliana

    SciTech Connect

    Martinez-Zapater, J.M. ); Somerville, C.R. )

    1990-03-01

    We have analyzed the response to vernalization and light quality of six classes of late-flowering mutants (fb, fca, fe, fg, ft, and fy) previously isolated following mutagenesis of the early Landsberg race of Arabidopsis thaliana (L.) Heynh. When grown in continuous fluorescent illumination, four mutants (fca, fe, ft, and fy) and the Landsberg wild type exhibited a reduction in both flowering time and leaf number following 6 weeks of vernalization. A significant decrease in flowering time was also observed for all the mutants and the wild type when constant fluorescent illumination was supplemented with irradiation enriched in the red and far red regions of the spectrum. In the most extreme case, the late-flowering phenotype of the fca mutant was completely suppressed by vernalization, suggesting that this mutation has a direct effect on flowering. The fe and fy mutants also showed a more pronounced response than wild type to both vernalization and incandescent supplementation. The ft mutant showed a similar response to that of the wild type. The fb and fg mutants were substantially less sensitive to these treatments. These results are interpreted in the context of a multifactorial pathway for induction of flowering, in which the various mutations affect different steps of the pathway.

  2. Characterization and mapping of a spotted leaf mutant in rice (Oryza sativa).

    PubMed

    Xu, Xue; Zhang, Lili; Liu, Binmei; Ye, Yafeng; Wu, Yuejin

    2014-06-01

    Spotted leaf mutant belongs to a class of mutants that can produce necrotic lesions spontaneously in plants without any attack by pathogens. These mutants have no beneficial effect on plant productivity but provide a unique opportunity to study programmed cell death in plant defense responses. A novel rice spotted leaf mutant (spl30) was isolated through low-energy heavy ion irradiation. Lesion expression was sensitive to light and humidity. The spl30 mutant caused a decrease in chlorophyll and soluble protein content, with marked accumulation of reactive oxygen species (ROS) around the lesions. In addition, the spl30 mutant significantly enhanced resistance to rice bacterial blight (X. oryzae pv. oryzae) from China (C1-C7). The use of SSR markers showed that the spl30 gene was located between markers XSN2 and XSN4. The genetic distance between the spl30 gene and XSN2 and between spl30 and XSN4 was 1.7 cM and 0.2 cM, respectively. The spl30 gene is a new gene involved in lesion production and may be related to programmed cell death in rice. The ability of this mutant to confer broad resistance to bacterial blight provides a model for studying the interaction between plants and pathogenic bacteria. PMID:25071406

  3. Functional rescue of a kidney anion exchanger 1 trafficking mutant in renal epithelial cells.

    PubMed

    Chu, Carmen Y S; King, Jennifer C; Berrini, Mattia; Alexander, R Todd; Cordat, Emmanuelle

    2013-01-01

    Mutations in the SLC4A1 gene encoding the anion exchanger 1 (AE1) can cause distal renal tubular acidosis (dRTA), a disease often due to mis-trafficking of the mutant protein. In this study, we investigated whether trafficking of a Golgi-retained dRTA mutant, G701D kAE1, or two dRTA mutants retained in the endoplasmic reticulum, C479W and R589H kAE1, could be functionally rescued to the plasma membrane of Madin-Darby Canine Kidney (MDCK) cells. Treatments with DMSO, glycerol, the corrector VX-809, or low temperature incubations restored the basolateral trafficking of G701D kAE1 mutant. These treatments had no significant rescuing effect on trafficking of the mis-folded C479W or R589H kAE1 mutants. DMSO was the only treatment that partially restored G701D kAE1 function in the plasma membrane of MDCK cells. Our experiments show that trafficking of intracellularly retained dRTA kAE1 mutants can be partially restored, and that one chemical treatment rescued both trafficking and function of a dRTA mutant. These studies provide an opportunity to develop alternative therapeutic solutions for dRTA patients. PMID:23460825

  4. Functional Rescue of a Kidney Anion Exchanger 1 Trafficking Mutant in Renal Epithelial Cells

    PubMed Central

    Chu, Carmen Y. S.; King, Jennifer C.; Berrini, Mattia; Alexander, R. Todd; Cordat, Emmanuelle

    2013-01-01

    Mutations in the SLC4A1 gene encoding the anion exchanger 1 (AE1) can cause distal renal tubular acidosis (dRTA), a disease often due to mis-trafficking of the mutant protein. In this study, we investigated whether trafficking of a Golgi-retained dRTA mutant, G701D kAE1, or two dRTA mutants retained in the endoplasmic reticulum, C479W and R589H kAE1, could be functionally rescued to the plasma membrane of Madin-Darby Canine Kidney (MDCK) cells. Treatments with DMSO, glycerol, the corrector VX-809, or low temperature incubations restored the basolateral trafficking of G701D kAE1 mutant. These treatments had no significant rescuing effect on trafficking of the mis-folded C479W or R589H kAE1 mutants. DMSO was the only treatment that partially restored G701D kAE1 function in the plasma membrane of MDCK cells. Our experiments show that trafficking of intracellularly retained dRTA kAE1 mutants can be partially restored, and that one chemical treatment rescued both trafficking and function of a dRTA mutant. These studies provide an opportunity to develop alternative therapeutic solutions for dRTA patients. PMID:23460825

  5. Herpes simplex ICP27 mutant viruses exhibit reduced expression of specific DNA replication genes.

    PubMed Central

    Uprichard, S L; Knipe, D M

    1996-01-01

    Herpes simplex virus type 1 mutants with certain lesions in the ICP27 gene show a 5- to 10-fold reduction in viral DNA synthesis. To determine how ICP27 promotes amplification of viral DNA, we examined the synthesis, accumulation, and stability of the essential viral replication proteins and steady-state levels of the replication gene transcripts throughout the course of ICP27 mutant virus infections. These studies reveal that in the absence of ICP27, expression of the UL5, UL8, UL52, UL9, UL42, and UL30 genes is significantly reduced at the level of mRNA accumulation. In contrast to that of these beta genes, ICP8 expression is unaltered in mutant virus-infected cells, indicating that ICP27 selectively stimulates only a subset of herpes simplex virus beta genes. Analysis of multiple ICP27 mutant viruses indicates a quantitative correlation between the ability of these mutants to replicate viral DNA and the level of replication proteins produced by each mutant. Therefore, we conclude that the primary defect responsible for restricted viral DNA synthesis in cells infected with ICP27 mutants is insufficient expression of most of the essential replication genes. Of further interest, this analysis also provides new information about the structure of the UL52 gene transcripts. PMID:8627723

  6. Phenome Analysis in Plant Species Using Loss-of-Function and Gain-of-Function Mutants

    PubMed Central

    Kuromori, Takashi; Takahashi, Shinya; Kondou, Youichi; Shinozaki, Kazuo; Matsui, Minami

    2009-01-01

    Analysis of genetic mutations is one of the most effective ways to investigate gene function. We now have methods that allow for mass production of mutant lines and cells in a variety of model species. Recently, large numbers of mutant lines have been generated by both ‘loss-of-function’ and ‘gain-of-function’ techniques. In parallel, phenotypic information covering various mutant resources has been acquired and released in web-based databases. As a result, significant progress in comprehensive pheno-type analysis is being made through the use of these tools. Arabidopsis and rice are two major model plant species in which genome sequencing projects have been completed. Arabidopsis is the most widely used experimental plant, with a large number of mutant resources and several examples of systematic phenotype analysis. Rice is a major crop species and is used as a model plant, with an increasing number of mutant resources. Other plant species are also being employed in functional genetics research. In this review, the present status of mutant resources for large-scale studies of gene function in plant research and the current perspective on using loss-of-function and gain-of-function mutants in phenome research will be discussed. PMID:19502383

  7. Endoplasmic Reticulum-associated Degradation (ERAD) and Autophagy Cooperate to Degrade Polymerogenic Mutant Serpins*

    PubMed Central

    Kroeger, Heike; Miranda, Elena; MacLeod, Ian; Pérez, Juan; Crowther, Damian C.; Marciniak, Stefan J.; Lomas, David A.

    2009-01-01

    The serpinopathies are a family of diseases characterized by the accumulation of ordered polymers of mutant protein within the endoplasmic reticulum. They are a diverse group including α1-antitrypsin deficiency and the inherited dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. We have used transient transfection of COS7 cells and mouse embryonic fibroblasts, PC12 cell lines that conditionally express wild type and mutant neuroserpin and fly models of FENIB to assess the cellular handling of wild type and mutant serpins. By using a polymer-specific monoclonal antibody, we show that mutant neuroserpin forms polymers after a delay of at least 30 min and that polymers can be cleared in PC12 cell lines and from the brain in a fly model of FENIB. At steady state, the fractions of intracellular polymerogenic G392E mutant neuroserpin in the monomeric and polymeric states are comparable. Inhibition of the proteasome with MG132 reveals that both mutant neuroserpin and α1-antitrypsin are degraded predominantly by endoplasmic reticulum-associated degradation (ERAD). Pharmacological and genetic inhibitions demonstrate that autophagy is responsible for bulk turnover of wild type and mutant serpins, but can be stimulated by rapamycin to compensate for proteasome inhibition. The significance of these findings to the treatment of serpinopathies is discussed. PMID:19549782

  8. Establishment of permanent chimerism in a lactate dehydrogenase-deficient mouse mutant with hemolytic anemia

    SciTech Connect

    Datta, T.; Doermer, P.

    1987-12-01

    Pluripotent hemopoietic stem cell function was investigated in the homozygous muscle type lactate dehydrogenase (LDH-A) mutant mouse using bone marrow transplantation experiments. Hemopoietic tissues of LDH-A mutants showed a marked decreased in enzyme activity that was associated with severe hemolytic anemia. This condition proved to be transplantable into wild type mice (+/+) through total body irradiation (TBI) at a lethal dose of 8.0 Gy followed by engraftment of mutant bone marrow cells. Since the mutants are extremely radiosensitive (lethal dose50/30 4.4 Gy vs 7.3 Gy in +/+ mice), 8.0-Gy TBI followed by injection of even high numbers of normal bone marrow cells did not prevent death within 5-6 days. After a nonlethal dose of 4.0 Gy and grafting of normal bone marrow cells, a transient chimerism showing peripheral blood characteristics of the wild type was produced that returned to the mutant condition within 12 weeks. The transfusion of wild type red blood cells prior to and following 8.0-Gy TBI and reconstitution with wild type bone marrow cells prevented the early death of the mutants and permanent chimerism was achieved. The chimeras showed all hematological parameters of wild type mice, and radiosensitivity returned to normal. It is concluded that the mutant pluripotent stem cells are functionally comparable to normal stem cells, emphasizing the significance of this mouse model for studies of stem cell regulation.

  9. Inositol-Requiring Mutants of SACCHAROMYCES CEREVISIAE

    PubMed Central

    Culbertson, Michael R.; Henry, Susan A.

    1975-01-01

    Fifty-two inositol-requiring mutants of Saccharomyces cerevisiae were isolated following mutagenesis with ethyl methanesulfonate. Complementation and tetrad analysis revealed ten major complementation classes, representing ten independently segregating loci (designated ino1 through ino10) which recombined freely with their respective centromeres. Members of any given complementation class segregated as alleles of a single locus. Thirteen complementation subclasses were identified among thirty-six mutants which behaved as alleles of the ino1 locus. The complementation map for these mutants was circular.—Dramatic cell viability losses indicative of unbalanced growth were observed in liquid cultures of representative mutants under conditions of inositol starvation. Investigation of the timing, kinetics, and extent of cell death revealed that losses in cell viability in the range of 2-4 log orders could be prevented by the addition of inositol to the medium or by disruption of protein synthesis with cycloheximide. Mutants defective in nine of the ten loci identified in this study displayed these unusual characteristics. The results suggest an important physiological role for inositol that may be related to its cellular localization and function in membrane phospholipids. The possibility is discussed that inositol deficiency initiates the process of unbalanced growth leading to cell death through the loss of normal assembly, function, or integrity of biomembranes.—Part of this work has been reported in preliminary form (Culbertson and Henry 1974). PMID:1093935

  10. Escherichia coli mutants deficient in deoxyuridine triphosphatase.

    PubMed Central

    Hochhauser, S J; Weiss, B

    1978-01-01

    Mutants deficient in deoxyuridine triphosphatase (dUTPase) were identified by enzyme assays of randomly chosen heavily mutagenized clones. Five mutants of independent origin were obtained. One mutant produced a thermolabile enzyme, and it was presumed to have a mutation in the structural gene for dUTPase, designated dut. The most deficient mutant had the following associated phenotypes: less than 1% of parental dUTPase activity, prolonged generation time, increased sensitivity to 5'-fluorodeoxyuridine, increased rate of spontaneous mutation, increased rate of recombination (hyper-Rec), an inhibition of growth in the presence of 2 mM uracil, and a decreased ability to support the growth of phage P1 (but not T4 or lambda). This mutation also appeared to be incompatible with pyrE mutations. A revertant selected by its faster growth had regained dUTPase activity and lost its hyper-Rec phenotype. Many of the properties of the dut mutants are compatible with their presumed increased incorporation of uracil into DNA and the subsequent transient breakage of the DNA by excision repair. PMID:148458

  11. Phanerochaete mutants with enhanced ligninolytic activity

    SciTech Connect

    Kakar, S.N.; Perez, A.; Gonzales, J.

    1993-06-01

    In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organopollutants in soils and aqueous media. Although some of the organic compounds are degraded under nonligninolytic conditions, most are degraded under ligninolytic conditions with the involvement of the extracellular enzymes, lignin peroxidases, and manganese-dependent peroxidases, which are produced as secondary metabolites triggered by conditions of nutrient starvation (e.g., nitrogen limitation). The fungus and its enzymes can thus provide alternative technologies for bioremediation, biopulping, biobleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated or are hyperproducers or supersecretors of key enzymes under enriched conditions. Through ultraviolet-light and gamma-rays mutagenesis we have isolated a variety of mutants, some of which produce key enzymes of the ligninolytic system under high-nitrogen growth conditions. One of the mutants produced 272 units (U) of lignin peroxidases enzyme activity per liter after nine days under high nitrogen. The mutant and the parent strains produced up to 54 U/L and 62 U/L, respectively, of the enzyme activity under low-nitrogen growth conditions during this period. In some experiments the mutant showed 281 U/L of enzyme activity under high nitrogen after 17 days.

  12. Functional verification of a porcine myostatin propeptide mutant.

    PubMed

    Ma, Dezun; Jiang, Shengwang; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Xiao, Gaojun; Yang, Jinzeng; Cui, Wentao

    2015-10-01

    Myostatin is a member of TGF-β superfamily that acts as a key negative regulator in development and growth of embryonic and postnatal muscles. In this study, the inhibitory activities of recombinant porcine myostatin propeptide and its mutated form (at the cleavage site of metalloproteinases of BMP-1/TLD family) against murine myostatin was evaluated in vivo by intraperitoneal injection into mice. Results showed that both wild type and mutated form of porcine propeptide significantly inhibited myostatin activity in vivo. The average body weight of mice receiving wild type propeptide or its mutated form increased by 12.5 % and 24.14%, respectively, compared to mice injected with PBS, implying that the in vivo efficacy of porcine propeptide mutant is greater than its wild type propeptide. Transgenic mice expressing porcine myostatin propeptide mutant were generated to further verify the results obtained from mice injected with recombinant porcine propeptide mutant. Compared with wild type (non-transgenic) mice, relative weight of gastrocnemius, rectusfemoris, and tibialis anterior increased by 22.14 %, 34.13 %, 25.37%, respectively, in transgenic male mice, and by 19.90 %, 42.47 %, 45.61%, respectively, in transgenic female mice. Our data also demonstrated that the mechanism by which muscle growth enhancement is achieved by these propeptides is due to an increase in fiber sizes, not by an increase in number of fiber cells. PMID:26174475

  13. Arabidopsis AMY1 expressions and early flowering mutant phenotype.

    PubMed

    Jie, Wang; Dashi, Yu; XinHong, Guo; Xuanming, Liu

    2009-02-28

    The homozygous T-DNA mutant of the AMY1 gene in Arabidopsis was identified and importantly, shown to cause an early flowering phenotype. We found that the disruption of AMY1 enhanced expression of CO and FT. The expression analyses of genes related to starch metabolism revealed that expression of the AGPase small subunit APS1 in the wild type was higher than in the amy1 mutant. However, there were no significant differences in expression levels of the AGPase large subunit genes ApL1, AMY2, or AMY3 between wild type and the amy1 mutant. Expression profiling showed that AMY1 was highly expressed in leaves, stems, and flowers, and expressed less in leafstalks and roots. Furthermore, the level of AMY1 mRNA was highly elevated with age and in senescing leaves. RT-PCR analyses showed that the expression of AMY1 was induced by heat shock, GA, and ABA, while salt stress had no apparent effect on its expression. PMID:19250611

  14. Paracoccus denitrificans cytochrome c1 gene replacement mutants.

    PubMed Central

    Gerhus, E; Steinrücke, P; Ludwig, B

    1990-01-01

    We describe the construction and characterization of gene replacement mutants for the respiratory chain component cytochrome c1 in the bacterium Paracoccus denitrificans. Its structural gene (fbcC) was inactivated by insertion of the kanamycin resistance gene, introduced into a suicide vector, and conjugated into Paracoccus; chromosomal mutants obtained by homologous recombination were selected by antibiotic resistance screening and further characterized biochemically. They showed the complete spectral, enzymatic, and immunological loss of the fbcC gene product together with a serious defect in the assembly of the two other gene products of the fbc operon, cytochrome b and the FeS protein. A possible role of the cytochrome c1 in the assembly process for the enzyme complex is discussed. A functional restoration to wild-type phenotype was achieved by complementing in trans with a newly constructed broad-host-range vector carrying the fbcC gene cassette. When the complete fbc operon was present on this vector, overexpression of complex III subunits was observed. Apart from their physiological significance, such mutants are a prerequisite for probing structure-function relationships by site-directed mutagenesis in order to understand molecular details of electron transport and energy transduction processes of this respiratory enzyme in bacteria and in mitochondria. Images PMID:2158969

  15. Construction of a large-scale Burkholderia cenocepacia J2315 transposon mutant library

    NASA Astrophysics Data System (ADS)

    Wong, Yee-Chin; Pain, Arnab; Nathan, Sheila

    2014-09-01

    Burkholderia cenocepacia, a pathogenic member of the Burkholderia cepacia complex (Bcc), has emerged as a significant threat towards cystic fibrosis patients, where infection often leads to the fatal clinical manifestation known as cepacia syndrome. Many studies have investigated the pathogenicity of B. cenocepacia as well as its ability to become highly resistant towards many of the antibiotics currently in use. In addition, studies have also been undertaken to understand the pathogen's capacity to adapt and survive in a broad range of environments. Transposon based mutagenesis has been widely used in creating insertional knock-out mutants and coupled with recent advances in sequencing technology, robust tools to study gene function in a genome-wide manner have been developed based on the assembly of saturated transposon mutant libraries. In this study, we describe the construction of a large-scale library of B. cenocepacia transposon mutants. To create transposon mutants of B. cenocepacia strain J2315, electrocompetent bacteria were electrotransformed with the EZ-Tn5 transposome. Tetracyline resistant colonies were harvested off selective agar and pooled. Mutants were generated in multiple batches with each batch consisting of ˜20,000 to 40,000 mutants. Transposon insertion was validated by PCR amplification of the transposon region. In conclusion, a saturated B. cenocepacia J2315 transposon mutant library with an estimated total number of 500,000 mutants was successfully constructed. This mutant library can now be further exploited as a genetic tool to assess the function of every gene in the genome, facilitating the discovery of genes important for bacterial survival and adaptation, as well as virulence.

  16. Structure of mutant human oncogene protein determined

    SciTech Connect

    Baum, R.

    1989-01-16

    The protein encoded by a mutant human oncogene differs only slightly in structure from the native protein that initiates normal cell division, a finding that may complicate efforts to develop inhibitors of the mutant protein. Previously, the x-ray structure of the protein encoded by the normal c-Ha-ras gene, a protein believed to signal cells to start or stop dividing through its interaction with guanosine triphosphate (GTP), was reported. The structure of the protein encoded by a transforming c-Ha-ras oncogene, in which a valine codon replaces the normal glycine codon at position 12 in the gene, has now been determined. The differences in the structures of the mutant and normal proteins are located primarily in a loop that interacts with the /beta/-phosphate of a bound guanosine diphosphate (GDP) molecule.

  17. Amphid defective mutant of Caenorhabditis elegans.

    PubMed

    De Riso, L; Ristoratore, F; Sebastiano, M; Bazzicalupo, P

    1994-01-01

    Studies are reported on a chemoreception mutant which arose in a mutator strain. The mutant sensory neurons do not stain with fluoresceine isothiocyanate (Dyf phenotype), hence the name, dyf-1, given to the gene it identifies. The gene maps on LGI, 0.4 map units from dpy-5 on the unc-11 side. The response of mutant worms to various repellents has been studied and shown to be partially altered. Other chemoreception based behaviors are less affected. The cilia of the sensory neurons of the amphid are shorter than normal and the primary defect may be in the capacity of the sheath cells to secrete the matrix material that fills the space between cilia in the amphid channel. Progress toward the molecular cloning of the gene is also reported. Relevant results from other laboratories are briefly reviewed. PMID:7896139

  18. High Persister Mutants in Mycobacterium tuberculosis.

    PubMed

    Torrey, Heather L; Keren, Iris; Via, Laura E; Lee, Jong Seok; Lewis, Kim

    2016-01-01

    Mycobacterium tuberculosis forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in M. tuberculosis is not well understood. In this study, we selected for high persister (hip) mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the hip mutants obtained in vitro with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical hip isolates exhibited greater ex vivo survival than the low persister isolates. Our data suggest that M. tuberculosis persister formation involves multiple pathways, and hip mutants may contribute to the recalcitrance of the infection. PMID:27176494

  19. High Persister Mutants in Mycobacterium tuberculosis

    PubMed Central

    Torrey, Heather L.; Keren, Iris; Via, Laura E.; Lee, Jong Seok; Lewis, Kim

    2016-01-01

    Mycobacterium tuberculosis forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in M. tuberculosis is not well understood. In this study, we selected for high persister (hip) mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the hip mutants obtained in vitro with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical hip isolates exhibited greater ex vivo survival than the low persister isolates. Our data suggest that M. tuberculosis persister formation involves multiple pathways, and hip mutants may contribute to the recalcitrance of the infection. PMID:27176494

  20. TOMATOMA: a novel tomato mutant database distributing Micro-Tom mutant collections.

    PubMed

    Saito, Takeshi; Ariizumi, Tohru; Okabe, Yoshihiro; Asamizu, Erika; Hiwasa-Tanase, Kyoko; Fukuda, Naoya; Mizoguchi, Tsuyoshi; Yamazaki, Yukiko; Aoki, Koh; Ezura, Hiroshi

    2011-02-01

    The tomato is an excellent model for studies of plants bearing berry-type fruits and for experimental studies of the Solanaceae family of plants due to its conserved genetic organization. In this study, a comprehensive mutant tomato population was generated in the background of Micro-Tom, a dwarf, rapid-growth variety. In this and previous studies, a family including 8,598 and 6,422 M(2) mutagenized lines was produced by ethylmethane sulfonate (EMS) mutagenesis and γ-ray irradiation, and this study developed and investigated these M(2) plants for alteration of visible phenotypes. A total of 9,183 independent M(2) families comprising 91,830 M(2) plants were inspected for phenotypic alteration, and 1,048 individual mutants were isolated. Subsequently, the observed mutant phenotypes were classified into 15 major categories and 48 subcategories. Overall, 1,819 phenotypic categories were found in 1,048 mutants. Of these mutants, 549 were pleiotropic, whereas 499 were non-pleiotropic. Multiple different mutant alleles per locus were found in the mutant libraries, suggesting that the mutagenized populations were nearly saturated. Additionally, genetic analysis of backcrosses indicated the successful inheritance of the mutations in BC(1)F(2) populations, confirming the reproducibility in the morphological phenotyping of the M(2) plants. To integrate and manage the visible phenotypes of mutants and other associated data, we developed the in silico database TOMATOMA, a relational system interfacing modules between mutant line names and phenotypic categories. TOMATOMA is a freely accessible database, and these mutant recourses are available through the TOMATOMA (http://tomatoma.nbrp.jp/index.jsp). PMID:21258066

  1. TOMATOMA: A Novel Tomato Mutant Database Distributing Micro-Tom Mutant Collections

    PubMed Central

    Saito, Takeshi; Ariizumi, Tohru; Okabe, Yoshihiro; Asamizu, Erika; Hiwasa-Tanase, Kyoko; Fukuda, Naoya; Mizoguchi, Tsuyoshi; Yamazaki, Yukiko; Aoki, Koh; Ezura, Hiroshi

    2011-01-01

    The tomato is an excellent model for studies of plants bearing berry-type fruits and for experimental studies of the Solanaceae family of plants due to its conserved genetic organization. In this study, a comprehensive mutant tomato population was generated in the background of Micro-Tom, a dwarf, rapid-growth variety. In this and previous studies, a family including 8,598 and 6,422 M2 mutagenized lines was produced by ethylmethane sulfonate (EMS) mutagenesis and γ-ray irradiation, and this study developed and investigated these M2 plants for alteration of visible phenotypes. A total of 9,183 independent M2 families comprising 91,830 M2 plants were inspected for phenotypic alteration, and 1,048 individual mutants were isolated. Subsequently, the observed mutant phenotypes were classified into 15 major categories and 48 subcategories. Overall, 1,819 phenotypic categories were found in 1,048 mutants. Of these mutants, 549 were pleiotropic, whereas 499 were non-pleiotropic. Multiple different mutant alleles per locus were found in the mutant libraries, suggesting that the mutagenized populations were nearly saturated. Additionally, genetic analysis of backcrosses indicated the successful inheritance of the mutations in BC1F2 populations, confirming the reproducibility in the morphological phenotyping of the M2 plants. To integrate and manage the visible phenotypes of mutants and other associated data, we developed the in silico database TOMATOMA, a relational system interfacing modules between mutant line names and phenotypic categories. TOMATOMA is a freely accessible database, and these mutant recourses are available through the TOMATOMA (http://tomatoma.nbrp.jp/index.jsp). PMID:21258066

  2. Escherichia coli mutants deficient in exonuclease VII.

    PubMed Central

    Chase, J W; Richardson, C C

    1977-01-01

    Mutants of Escherichia coli having reduced levels of exonuclease VII activity have been isolated by a mass screening procedure. Nine mutants, five of which are known to be of independent origin, were obtained and designated xse. The defects in these strains lie at two or more loci. One of these loci, xseA, lies in the interval between purG and purC; it is 93 to 97% co-transducible with guaA. The order of the genes in this region is purG-xseA guaA,B-purC. The available data do not allow xseA to be ordered with respect to guaA,B. Exonuclease VII purified from E. coli KLC3 xseA3 is more heat labile than exonuclease VII purified from the parent, E. coli PA610 xse+. Therefore, xseA is the structural gene for exonuclease VII. Mutants with defects in the xseA gene show increased sensitivity to nalidixic acid and have an abnormally high frequency of recombination (hyper-Rec phenotype) as measured by the procedure of Konrad and Lehlman (1974). The hyper-Rec character of xseA strains is approximately one-half that of the polAex1 mutant defective in the 5' leads to 3' hydrolytic activity of deoxyribonucleic acid polymerase I. The double mutant, polAex1 xseA7, is twice as hyper-Rec as the polAex1 mutant alone. The xseA- strains are slightly more sensitive to ultraviolet irradiation than the parent strain. Bacteriophages T7, fd, and lambdared grow normally in xseA- strains. Images PMID:320198

  3. Stability analysis of a high fibre yield and low lignin content "thick stem" mutant in tossa jute (Corchorus olitorius L.).

    PubMed

    Mandal, Aninda; Datta, Animesh K

    2014-01-01

    A "thick stem" mutant of Corchorus olitorius L. was induced at M2 (0.50%, 4 h, EMS) and the true breeding mutant is assessed across generations (M5 to M7) considering morphometric traits as well as SEM analysis of pollen grains and raw jute fibres, stem anatomy, cytogenetical attributes, and lignin content in relation to control. Furthermore, single fibre diameter and tensile strength are also analysed. The objective is to assess the stability of mutant for its effective exploration for raising a new plant type in tossa jute for commercial exploitation and efficient breeding. The mutant trait is monogenic recessive to normal. Results indicate that "thick stem" mutant is stable across generations (2n = 14) with distinctive high seed and fibre yield and significantly low lignin content. Stem anatomy of the mutant shows significant enhancement in fibre zone, number of fibre pyramids and fibre bundles per pyramid, and diameter of fibre cell in relation to control. Moreover, tensile strength of mutant fibre is significantly higher than control fibre and the trait is inversely related to fibre diameter. However the mutant is associated with low germination frequency, poor seed viability, and high pollen sterility, which may be eliminated through mutational approach followed by rigorous selection and efficient breeding. PMID:24860822

  4. Neurospora crassa mutants deficient in asparagine synthetase.

    PubMed Central

    MacPhee, K G; Nelson, R E; Schuster, S M

    1983-01-01

    Neurospora crassa mutants deficient in asparagine synthetase were selected by using the procedure of inositol-less death. Complementation tests among the 100 mutants isolated suggested that their alterations were genetically allelic. Recombination analysis with strain S1007t, an asparagine auxotroph, indicated that the mutations were located near or within the asn gene on linkage group V. In vitro assays with a heterokaryon indicated that the mutation was dominant. Thermal instability of cell extracts from temperature-sensitive strains in an in vitro asparagine synthetase assay determined that the mutations were in the structural gene(s) for asparagine synthetase. PMID:6137480

  5. Evolutionary Mutant Models for Human Disease

    PubMed Central

    Albertson, R. Craig; Cresko, William; Detrich, H. William; Postlethwait, John H.

    2010-01-01

    Although induced mutations in traditional laboratory animals have been valuable as models for human diseases, they have some important limitations. Here we propose a complementary approach to discover genes and mechanisms that might contribute to human disorders: the analysis of evolutionary mutant models whose adaptive phenotypes mimic maladaptive human diseases. If the type and mode of action of mutations favored by natural selection in wild populations are similar to those that contribute to human diseases, then studies in evolutionary mutant models have the potential to identify novel genetic factors and gene-by-environment interactions that affect human health and underlie human disease. PMID:19108930

  6. The Rec102 Mutant of Yeast Is Defective in Meiotic Recombination and Chromosome Synapsis

    PubMed Central

    Bhargava, J.; Engebrecht, J. A.; Roeder, G. S.

    1992-01-01

    A mutation at the REC102 locus was identified in a screen for yeast mutants that produce inviable spores. rec102 spore lethality is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The rec102 mutation completely eliminates meiotically induced gene conversion and crossing over but has no effect on mitotic recombination frequencies. Cytological studies indicate that the rec102 mutant makes axial elements (precursors to the synaptonemal complex), but homologous chromosomes fail to synapse. In addition, meiotic chromosome segregation is significantly delayed in rec102 strains. Studies of double and triple mutants indicate that the REC102 protein acts before the RAD52 gene product in the meiotic recombination pathway. The REC102 gene was cloned based on complementation of the mutant defect and the gene was mapped to chromosome XII between CDC25 and STE11. PMID:1732169

  7. A fluorescence-activated cell sorting-based strategy for rapid isolation of high-lipid Chlamydomonas mutants

    PubMed Central

    Terashima, Mia; Freeman, Elizabeth S; Jinkerson, Robert E; Jonikas, Martin C

    2015-01-01

    There is significant interest in farming algae for the direct production of biofuels and valuable lipids. Chlamydomonas reinhardtii is the leading model system for studying lipid metabolism in green algae, but current methods for isolating mutants of this organism with a perturbed lipid content are slow and tedious. Here, we present the Chlamydomonas high-lipid sorting (CHiLiS) strategy, which enables enrichment of high-lipid mutants by fluorescence-activated cell sorting (FACS) of pooled mutants stained with the lipid-sensitive dye Nile Red. This method only takes 5 weeks from mutagenesis to mutant isolation. We developed a staining protocol that allows quantification of lipid content while preserving cell viability. We improved separation of high-lipid mutants from the wild type by using each cell's chlorophyll fluorescence as an internal control. We initially demonstrated 20-fold enrichment of the known high-lipid mutant sta1 from a mixture of sta1 and wild-type cells. We then applied CHiLiS to sort thousands of high-lipid cells from a pool of about 60 000 mutants. Flow cytometry analysis of 24 individual mutants isolated by this approach revealed that about 50% showed a reproducible high-lipid phenotype. We further characterized nine of the mutants with the highest lipid content by flame ionization detection and mass spectrometry lipidomics. All mutants analyzed had a higher triacylglycerol content and perturbed whole-cell fatty acid composition. One arbitrarily chosen mutant was evaluated by microscopy, revealing larger lipid droplets than the wild type. The unprecedented throughput of CHiLiS opens the door to a systems-level understanding of green algal lipid biology by enabling genome-saturating isolation of mutants in key genes. PMID:25267488

  8. Proteomic profiling of maize opaque endosperm mutants reveals selective accumulation of lysine-enriched proteins

    PubMed Central

    Morton, Kyla J.; Jia, Shangang; Zhang, Chi; Holding, David R.

    2016-01-01

    Reduced prolamin (zein) accumulation and defective endoplasmic reticulum (ER) body formation occurs in maize opaque endosperm mutants opaque2 (o2), floury2 (fl2), defective endosperm*B30 (DeB30), and Mucronate (Mc), whereas other opaque mutants such as opaque1 (o1) and floury1 (fl1) are normal in these regards. This suggests that other factors contribute to kernel texture. A liquid chromatography approach coupled with tandem mass spectrometry (LC-MS/MS) proteomics was used to compare non-zein proteins of nearly isogenic opaque endosperm mutants. In total, 2762 proteins were identified that were enriched for biological processes such as protein transport and folding, amino acid biosynthesis, and proteolysis. Principal component analysis and pathway enrichment suggested that the mutants partitioned into three groups: (i) Mc, DeB30, fl2 and o2; (ii) o1; and (iii) fl1. Indicator species analysis revealed mutant-specific proteins, and highlighted ER secretory pathway components that were enriched in selected groups of mutants. The most significantly changed proteins were related to stress or defense and zein partitioning into the soluble fraction for Mc, DeB30, o1, and fl1 specifically. In silico dissection of the most significantly changed proteins revealed novel qualitative changes in lysine abundance contributing to the overall lysine increase and the nutritional rebalancing of the o2 and fl2 endosperm. PMID:26712829

  9. Molecular characterization of seipin and its mutants: implications for seipin in triacylglycerol synthesis[S

    PubMed Central

    Fei, Weihua; Li, Hui; Shui, Guanghou; Kapterian, Tamar S.; Bielby, Christopher; Du, Ximing; Brown, Andrew J.; Li, Peng; Wenk, Markus R.; Liu, Pingsheng; Yang, Hongyuan

    2011-01-01

    The human lipodystrophy gene product Berardinelli-Seip congenital lipodystrophy 2/seipin has been implicated in adipocyte differentiation, lipid droplet (LD) formation, and motor neuron development. However, the molecular function of seipin and its disease-causing mutants remains to be elucidated. Here, we characterize seipin and its mis-sense mutants: N88S/S90L (both linked to motoneuron disorders) and A212P (linked to lipodystrophy) in cultured mammalian cells. Knocking down seipin significantly increases oleate incorporation into triacylglycerol (TAG) and the steady state level of TAG, and induces the proliferation and clustering of small LDs. By contrast, overexpression of seipin reduces TAG synthesis, leading to decreased formation of LDs. Expression of the A212P mutant, however, had little effect on LD biogenesis. Surprisingly, expression of N88S or S90L causes the formation of many small LDs reminiscent of seipin deficient cells. This dominant-negative effect may be due to the N88S/S90L-induced formation of inclusions where wild-type seipin can be trapped. Importantly, coexpression of wild-type seipin and the N88S or S90L mutant can significantly reduce the formation of inclusions. Finally, we demonstrate that seipin can interact with itself and its mutant forms. Our results provide important insights into the biochemical characteristics of seipin and its mis-sense mutants, and suggest that seipin may function to inhibit lipogenesis. PMID:21957196

  10. Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms.

    PubMed

    Araki, Marito; Yang, Yinjie; Masubuchi, Nami; Hironaka, Yumi; Takei, Hiraku; Morishita, Soji; Mizukami, Yoshihisa; Kan, Shin; Shirane, Shuichi; Edahiro, Yoko; Sunami, Yoshitaka; Ohsaka, Akimichi; Komatsu, Norio

    2016-03-10

    Recurrent somatic mutations of calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains elusive. Here, we found that the expression of mutant but not wild-type CALR induces the thrombopoietin (TPO)-independent growth of UT-7/TPO cells. We demonstrated that c-MPL, the TPO receptor, is required for this cytokine-independent growth of UT-7/TPO cells. Mutant CALR preferentially associates with c-MPL that is bound to Janus kinase 2 (JAK2) over the wild-type protein. Furthermore, we demonstrated that the mutant-specific carboxyl terminus portion of CALR interferes with the P-domain of CALR to allow the N-domain to interact with c-MPL, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induces the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked by JAK2 inhibitor treatment. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in induced pluripotent stem cell-derived hematopoietic stem cells harboring the CALR mutation. These findings imply that mutant CALR activates the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes myeloproliferative neoplasm development by activating c-MPL and its downstream pathway. PMID:26817954

  11. Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks.

    PubMed

    Yang, Hee-Jeong; Bogomolnaya, Lydia M; Elfenbein, Johanna R; Endicott-Yazdani, Tiana; Reynolds, M Megan; Porwollik, Steffen; Cheng, Pui; Xia, Xiao-Qin; McClelland, Michael; Andrews-Polymenis, Helene

    2016-04-01

    Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used bySalmonellato colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence ofSalmonella entericaserotype Typhimurium in chickens. A library of 182S.Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks.STM0580,STM1295,STM1297,STM3612,STM3615, andSTM3734are needed forSalmonellato colonize and persist in chicks and were not previously associated with this ability. One of these key genes,STM1297(selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection. PMID:26857572

  12. Ethanol production using engineered mutant E. coli

    DOEpatents

    Ingram, Lonnie O.; Clark, David P.

    1991-01-01

    The subject invention concerns novel means and materials for producing ethanol as a fermentation product. Mutant E. coli are transformed with a gene coding for pyruvate decarboxylase activity. The resulting system is capable of producing relatively large amounts of ethanol from a variety of biomass sources.

  13. Phenotypic mutant library: potential for gene discovery

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rapid development of high throughput and affordable Next- Generation Sequencing (NGS) techniques has renewed interest in gene discovery using forward genetics. The conventional forward genetic approach starts with isolation of mutants with a phenotype of interest, mapping the mutation within a s...

  14. Rapid Antibiotic Resistance Evolution of GASP Mutants

    NASA Astrophysics Data System (ADS)

    Zhang, Qiucen; Kim, Hyunsung; Pourmand, Nader; Austin, Robert

    2012-02-01

    The GASP phenotype in bacteria is due to a mutation which enables the bacteria to grow under high stress conditions where other bacteria stop growing. We probe using our Death Galaxy microenvironment how rapidly the GASP mutant can evolve resistance to mutagenic antibiotics compared to wild-type bacteria, and explore the genomic landscape changes due to the evolution of resistance.

  15. Yeast mutants overproducing iso-cytochromes c

    SciTech Connect

    Sherman, F.; Cardillo, T.S.; Errede, B.; Friedman, L.; McKnight, G.; Stiles, J.I.

    1980-01-01

    For over 15 years, the iso-cytochrome c system in the yeast Saccharomyces cerevisiae has been used to investigate a multitude of problems in genetics and molecular biology. More recently, attention has been focused on using mutants for examining translation and transcriptional processes and for probing regulatory regions governing gene expression. In an effort to explore regulatory mechanisms and to investigate mutational alterations that lead to increased levels of gene products, we have isolated and characterized mutants that overproduce cytochrome c. In this paper we have briefly summarized background information of some essential features of the iso-cytochrome c system and we have described the types of mutants that overproduce iso-1-cytochrome c or iso-2-cytochrome c. Genetic procedures and recombinant DNA procedures were used to demonstrate that abnormally high amounts of gene products occur in mutants as result of duplications of gene copies or of extended alteration of regulatory regions. The results summarized in this paper point out the requirements of gross mutational changes or rearrangements of chromosomal segments for augmenting gene products.

  16. Genotyping-by-sequencing of glossy mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glossy mutants are a common occurrence in Brassica oleracea L. and they have been documented in most crop varieties of the species including cabbage, kale, broccoli, and collard. Glossy phenotypes have been of particular interest to researchers due to observations that they influence insect behavior...

  17. Quantitative genetics and utilization of mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The relatively low level of genetic variability currently available in cotton makes mutagenesis attractive to overcome this problem. Mutations can occur either spontaneously or be induced. The majority of the genes we use today are spontaneous mutants that developed over a long period of time. Induc...

  18. Applications of mutant yeast strains with low glycogen storage capability

    NASA Technical Reports Server (NTRS)

    Petersen, G. R.; Schubert, W. W.; Stokes, B. O.

    1981-01-01

    Several strains of Hansenula polymorpha were selected for possible low glycogen storage characteristics based on a selective I2 staining procedure. The levels of storage carbohydrates in the mutant strains were found to be 44-70% of the levels in the parent strain for cultures harvested in stationary phase. Similar differences generally were not found for cells harvested in exponential phase. Yeast strains deficient in glycogen storage capability are valuable in increasing the relative protein value of microbial biomass and also may provide significant cost savings in substrate utilization in fermentative processes.

  19. A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis

    PubMed Central

    Subramanian, M; Francis, P; Bilke, S; Li, XL; Hara, T; Lu, X; Jones, MF; Walker, RL; Zhu, Y; Pineda, M; Lee, C; Varanasi, L; Yang, Y; Martinez, LA; Luo, J; Ambs, S; Sharma, S; Wakefield, LM; Meltzer, PS; Lal, A

    2015-01-01

    Most p53 mutations in human cancers are missense mutations resulting in a full-length mutant p53 protein. Besides losing tumor suppressor activity, some hotspot p53 mutants gain oncogenic functions. This effect is mediated in part, through gene expression changes due to inhibition of p63 and p73 by mutant p53 at their target gene promoters. Here, we report that the tumor suppressor microRNA let-7i is downregulated by mutant p53 in multiple cell lines expressing endogenous mutant p53. In breast cancer patients, significantly decreased let-7i levels were associated with missense mutations in p53. Chromatin immunoprecipitation and promoter luciferase assays established let-7i as a transcriptional target of mutant p53 through p63. Introduction of let-7i to mutant p53 cells significantly inhibited migration, invasion and metastasis by repressing a network of oncogenes including E2F5, LIN28B, MYC and NRAS. Our findings demonstrate that repression of let-7i expression by mutant p53 has a key role in enhancing migration, invasion and metastasis. PMID:24662829

  20. Field evaluation of mint mutant and hybrid lines for resistance to Verticillium wilt and yield

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Severity of Verticillium wilt varied significantly among mint lines and cultivars in the inoculated and non-inoculated sub-plots in two field trials. Verticillium wilt was significantly less severe for mutant lines 87M0109-1, 84M0107-7, and M90-11 than for Black Mitcham in 2002 and 2003. Verticilli...

  1. A Small Indel Mutant Mouse Model of Epidermolytic Palmoplantar Keratoderma and Its Application to Mutant-specific shRNA Therapy.

    PubMed

    Lyu, Ya-Su; Shi, Pei-Liang; Chen, Xiao-Ling; Tang, Yue-Xiao; Wang, Yan-Fang; Liu, Rong-Rong; Luan, Xiao-Rui; Fang, Yu; Mei, Ru-Huan; Du, Zhen-Fang; Ke, Hai-Ping; Matro, Erik; Li, Ling-En; Lin, Zhao-Yu; Zhao, Jing; Gao, Xiang; Zhang, Xian-Ning

    2016-01-01

    Epidermolytic palmoplantar keratoderma (EPPK) is a relatively common autosomal-dominant skin disorder caused by mutations in the keratin 9 gene (KRT9), with few therapeutic options for the affected so far. Here, we report a knock-in transgenic mouse model that carried a small insertion-deletion (indel) mutant of Krt9, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), corresponding to the human mutation KRT9/c.500delAinsGGCT (p.Tyr167delinsTrpLeu), which resulted in a human EPPK-like phenotype in the weight-stress areas of the fore- and hind-paws of both Krt9(+/mut) and Krt9(mut/mut) mice. The phenotype confirmed that EPPK is a dominant-negative condition, such that mice heterozygotic for the K9-mutant allele (Krt9(+/mut)) showed a clear EPPK-like phenotype. Then, we developed a mutant-specific short hairpin RNA (shRNA) therapy for EPPK mice. Mutant-specific shRNAs were systematically identified in vitro using a luciferase reporter gene assay and delivered into Krt9(+/mut) mice. shRNA-mediated knockdown of mutant protein resulted in almost normal morphology and functions of the skin, whereas the same shRNA had a negligible effect in wild-type K9 mice. Our results suggest that EPPK can be treated by gene therapy, and this has significant implications for future clinical application. PMID:27003758

  2. Mutant p53 oncogenic functions are sustained by Plk2 kinase through an autoregulatory feedback loop.

    PubMed

    Valenti, Fabio; Fausti, Francesca; Biagioni, Francesca; Shay, Tal; Fontemaggi, Giulia; Domany, Eytan; Yaffe, Michael B; Strano, Sabrina; Blandino, Giovanni; Di Agostino, Silvia

    2011-12-15

    Aberrant activation of kinases has emerged to be a key event along with tumor progression, maintenance of tumor phenotype and response to anticancer treatments. This study documents the existence of an oncogenic auto-regulatory feedback loop that includes the Polo-like kinase-2 (Snk/Plk2) and mutant p53 proteins. Plk2 protein binds to and phosphorylates mutant p53, thereby potentiating its oncogenic activities. Phosphorylated mutant p53 binds more efficiently to p300 consequently strengthening its own transcriptional activity. Plk2 gene is regulated at a transcriptional level by both wt- and mutant p53 proteins. This leads to growth suppression or enhanced cell proliferation and chemo-resistance, respectively. In turn, the siRNA-mediated knock down of either mutant p53 or Plk2 proteins significantly curtails the growth properties of tumor cells and their chemo-resistance to anticancer treatments. Therefore, this paper identifies a novel tumor network including Plk2 and mutant p53 proteins whose triggering in response to DNA damage might disclose important implications for the treatment of human cancers. PMID:22134238

  3. Functional, histological and biomechanical characterization of wheat water-mutant leaves.

    PubMed

    Rascio, Agata; Rascio, Nicoletta; Rinaldi, Michele; Valentini, Massimiliano

    2015-06-01

    A wheat (Triticum turgidum subsp. durum) mutant, generated with sodium azide from wild-type (WT) cv. 'Trinakria', differs in its water affinity of dry leaves, and was designated as a water-mutant. Compared with the WT, water-mutant leaves have lower rates of water uptake, while stomatal and cuticular transpiration do not differ. The nuclear magnetic resonance proton signals used for image reconstruction of leaf cross sections showed differences between these genotypes for the T1 proton spin-density and the T2 proton spin-spin relaxation time. Structural and histochemical analyses at midrib level showed that the water-mutant has thinner leaves, with more and smaller cells per unit area of mesophyll and sclerenchyma, and has altered staining patterns of lignin and pectin-like substances. Stress-strain curves to examine the rheological properties of the leaves showed a biphasic trend, which reveals that the tensile strength at break load and the elastic modulus of the second phase of the water-mutant are significantly higher than for the WT. These data support the proposal of interrelationships among local biophysical properties of the leaf, the microscopic water structure, the rheological properties and the water flux rate across the leaf. This water-mutant can be used for analysis of the genetic basis of these differences, and for identification of gene(s) that govern these traits. PMID:25212239

  4. Fluoride-Tolerant Mutants of Aspergillus niger Show Enhanced Phosphate Solubilization Capacity

    PubMed Central

    Silva, Ubiana de Cássia; Mendes, Gilberto de Oliveira; Silva, Nina Morena R. M.; Duarte, Josiane Leal; Silva, Ivo Ribeiro; Tótola, Marcos Rogério; Costa, Maurício Dutra

    2014-01-01

    P-solubilizing microorganisms are a promising alternative for a sustainable use of P against a backdrop of depletion of high-grade rock phosphates (RPs). Nevertheless, toxic elements present in RPs, such as fluorine, can negatively affect microbial solubilization. Thus, this study aimed at selecting Aspergillus niger mutants efficient at P solubilization in the presence of fluoride (F−). The mutants were obtained by exposition of conidia to UV light followed by screening in a medium supplemented with Ca3(PO4)2 and F−. The mutant FS1-555 showed the highest solubilization in the presence of F−, releasing approximately 70% of the P contained in Ca3(PO4)2, a value 1.7 times higher than that obtained for the wild type (WT). The mutant FS1-331 showed improved ability of solubilizing fluorapatites, increasing the solubilization of Araxá, Catalão, and Patos RPs by 1.7, 1.6, and 2.5 times that of the WT, respectively. These mutants also grew better in the presence of F−, indicating that mutagenesis allowed the acquisition of F− tolerance. Higher production of oxalic acid by FS1-331 correlated with its improved capacity for RP solubilization. This mutant represents a significant improvement and possess a high potential for application in solubilization systems with fluoride-rich phosphate sources. PMID:25310310

  5. Functional Loss of Bmsei Causes Thermosensitive Epilepsy in Contractile Mutant Silkworm, Bombyx mori

    NASA Astrophysics Data System (ADS)

    Nie, Hongyi; Cheng, Tingcai; Huang, Xiaofeng; Zhou, Mengting; Zhang, Yinxia; Dai, Fangyin; Mita, Kazuei; Xia, Qingyou; Liu, Chun

    2015-07-01

    The thermoprotective mechanisms of insects remain largely unknown. We reported the Bombyx mori contractile (cot) behavioral mutant with thermo-sensitive seizures phenotype. At elevated temperatures, the cot mutant exhibit seizures associated with strong contractions, rolling, vomiting, and a temporary lack of movement. We narrowed a region containing cot to ~268 kb by positional cloning and identified the mutant gene as Bmsei which encoded a potassium channel protein. Bmsei was present in both the cell membrane and cytoplasm in wild-type ganglia but faint in cot. Furthermore, Bmsei was markedly decreased upon high temperature treatment in cot mutant. With the RNAi method and injecting potassium channel blockers, the wild type silkworm was induced the cot phenotype. These results demonstrated that Bmsei was responsible for the cot mutant phenotype and played an important role in thermoprotection in silkworm. Meanwhile, comparative proteomic approach was used to investigate the proteomic differences. The results showed that the protein of Hsp-1 and Tn1 were significantly decreased and increased on protein level in cot mutant after thermo-stimulus, respectively. Our data provide insights into the mechanism of thermoprotection in insect. As cot phenotype closely resembles human epilepsy, cot might be a potential model for the mechanism of epilepsy in future.

  6. C. elegans and mutants with chronic nicotine exposure as a novel model of cancer phenotype.

    PubMed

    Kanteti, Rajani; Dhanasingh, Immanuel; El-Hashani, Essam; Riehm, Jacob J; Stricker, Thomas; Nagy, Stanislav; Zaborin, Alexander; Zaborina, Olga; Biron, David; Alverdy, John C; Im, Hae Kyung; Siddiqui, Shahid; Padilla, Pamela A; Salgia, Ravi

    2016-01-01

    We previously investigated MET and its oncogenic mutants relevant to lung cancer in C. elegans. The inactive orthlogues of the receptor tyrosine kinase Eph and MET, namely vab-1 and RB2088 respectively, the temperature sensitive constitutively active form of KRAS, SD551 (let-60; GA89) and the inactive c-CBL equivalent mutants in sli-1 (PS2728, PS1258, and MT13032) when subjected to chronic exposure of nicotine resulted in a significant loss in egg-laying capacity and fertility. While the vab-1 mutant revealed increased circular motion in response to nicotine, the other mutant strains failed to show any effect. Overall locomotion speed increased with increasing nicotine concentration in all tested mutant strains except in the vab-1 mutants. Moreover, chronic nicotine exposure, in general, upregulated kinases and phosphatases. Taken together, these studies provide evidence in support of C. elegans as initial in vivo model to study nicotine and its effects on oncogenic mutations identified in humans. PMID:26574927

  7. Characterization of a Bradyrhizobium japonicum ferrochelatase mutant and isolation of the hemH gene.

    PubMed Central

    Frustaci, J M; O'Brian, M R

    1992-01-01

    A Tn5-induced mutant of Bradyrhizobium japonicum, strain LORBF1, was isolated on the basis of the formation of fluorescent colonies, and stable derivatives were constructed in backgrounds of strains LO and I110. The stable mutant strains LOek4 and I110ek4 were strictly dependent upon the addition of exogenous hemin for growth in liquid culture and formed fluorescent colonies. The fluorescent compound was identified as protoporphyrin IX, the immediate precursor of protoheme. Cell extracts of strains LOek4 and I110ek4 were deficient in ferrochelatase activity, the enzyme which catalyzes the incorporation of ferrous iron into protoporphyrin IX to produce protoheme. Mutant strain I110ek4 could take up 55Fe from the growth medium, but, unlike the parent strain, no significant incorporation of radiolabel into heme was found. This observation shows that heme was not synthesized in mutant strain I110ek4 and that the heme found in those cells was derived from exogenous hemin in the growth medium. The putative protein encoded by the gene disrupted in strain LORBF1 and its derivatives was homologous to ferrochelatases from eukaryotic organisms. This homology, along with the described mutant phenotype, provides strong evidence that the disrupted gene is hemH, that which encodes ferrochelatase. Mutant strain I110ek4 incited nodules on soybean that did not fix nitrogen, contained few viable bacteria, and did not express leghemoglobin heme or apoprotein. The data show that B. japonicum ferrochelatase is essential for normal nodule development. PMID:1624416

  8. Nodulating Competitiveness of a Nonmotile Tn7 Mutant of Bradyrhizobium japonicum in Nonsterile Soil †

    PubMed Central

    Liu, Ruilong; Tran, Van Mai; Schmidt, E. L.

    1989-01-01

    A nonmotile mutant of Bradyrhizobium japonicum serogroup 127 was generated by Tn7 mutagenesis and matched with the wild type against a common competitor in studies of soybean nodulation in nonsterile soil. The Tn7 mutant was very similar to the wild type in growth rate in culture, soybean lectin-binding ability, flagellar morphology, and nodulating capability, but it had a longer lag phase. Competing strains were distributed uniformly in soil in various ratios and at different population densities prior to planting. Mutant and wild type were equally prevalent in the seedling rhizosphere at about the time of nodule initiation, suggesting that motility conferred no advantage in rhizosphere colonization. Nodulation success of the Tn7 mutant was lower than that of the wild type under all test conditions. Differences were greatest at low soil populations of competitors and much less pronounced at initial populations of 107 g−1. The longer lag phase of the Tn7 mutant may have contributed to its decreased competitiveness, especially at the higher inoculation levels. The antibiotic and motility markers were stable, and the rifampin resistance derived from the parent did not affect adversely the competitiveness of the Tn7 mutant. We found motility to be of limited importance to the competitiveness of a strain in normal nonsterile soil, where the significance, if any, of this ability may be in migration at the immediate root surface in soils sparsely populated with rhizobial symbionts. PMID:16347986

  9. Morphological characterization and assessment of genetic variability, character association, and divergence in soybean mutants.

    PubMed

    Malek, M A; Rafii, Mohd Y; Shahida Sharmin Afroz, Most; Nath, Ujjal Kumar; Mondal, M Monjurul Alam

    2014-01-01

    Genetic diversity is important for crop improvement. An experiment was conducted during 2011 to study genetic variability, character association, and genetic diversity among 27 soybean mutants and four mother genotypes. Analysis of variance revealed significant differences among the mutants and mothers for nine morphological traits. Eighteen mutants performed superiorly to their mothers in respect to seed yield and some morphological traits including yield attributes. Narrow differences between phenotypic and genotypic coefficients of variation (PCV and GCV) for most of the characters revealed less environmental influence on their expression. High values of heritability and genetic advance with high GCV for branch number, plant height, pod number, and seed weight can be considered as favorable attributes for soybean improvement through phenotypic selection and high expected genetic gain can be achieved. Pod and seed number and maturity period appeared to be the first order traits for higher yield and priority should be given in selection due to their strong associations and high magnitudes of direct effects on yield. Cluster analysis grouped 31 genotypes into five groups at the coefficient value of 235. The mutants/genotypes from cluster I and cluster II could be used for hybridization program with the mutants of clusters IV and V in order to develop high yielding mutant-derived soybean varieties for further improvement. PMID:25197722

  10. Morphological Characterization and Assessment of Genetic Variability, Character Association, and Divergence in Soybean Mutants

    PubMed Central

    Malek, M. A.; Rafii, Mohd Y.; Shahida Sharmin Afroz, Most.; Nath, Ujjal Kumar; Mondal, M. Monjurul Alam

    2014-01-01

    Genetic diversity is important for crop improvement. An experiment was conducted during 2011 to study genetic variability, character association, and genetic diversity among 27 soybean mutants and four mother genotypes. Analysis of variance revealed significant differences among the mutants and mothers for nine morphological traits. Eighteen mutants performed superiorly to their mothers in respect to seed yield and some morphological traits including yield attributes. Narrow differences between phenotypic and genotypic coefficients of variation (PCV and GCV) for most of the characters revealed less environmental influence on their expression. High values of heritability and genetic advance with high GCV for branch number, plant height, pod number, and seed weight can be considered as favorable attributes for soybean improvement through phenotypic selection and high expected genetic gain can be achieved. Pod and seed number and maturity period appeared to be the first order traits for higher yield and priority should be given in selection due to their strong associations and high magnitudes of direct effects on yield. Cluster analysis grouped 31 genotypes into five groups at the coefficient value of 235. The mutants/genotypes from cluster I and cluster II could be used for hybridization program with the mutants of clusters IV and V in order to develop high yielding mutant-derived soybean varieties for further improvement. PMID:25197722

  11. Kinetics of ethanolic fermentation of D-xylose by Klebsiella pneumoniae and its mutants.

    PubMed Central

    Banerjee, M

    1989-01-01

    The microbial production of ethanol from D-xylose by a new soil isolate of Klebsiella pneumoniae and the mutants K. pneumoniae MB-16 and MB-16-1048 was studied. Kinetic and physiological properties of the mutants were compared with those of the original isolate. The volumetric rates of ethanol formation by mutants MB-16-1048 and MB-16 and the original isolate were 1.58, 0.50, and 0.06 g liter-1 h-1, respectively. The cultivation times of mutants MB-16-1048 and MB-16 were 20 and 18 h, respectively, and that of the original isolate was 118 h. Both the mutants exhibited metabolic similarities with the original isolate. Ethanol was the major end product of fermentation in all three strains. Acetic acid and carbon dioxide were the other two important by-products of fermentation. Pyruvic acid was accumulated in significant proportions as an intermediate. The proportion of pyruvate in the original isolate was 54% of the total D-xylose utilized, whereas for MB-16 and MB-16-1048 the values were about 42 and 22%, respectively. The lower fractions of pyruvate in mutants MB-16 and MB-16-1048 showed up as a 41 and 82% improvement, respectively, over the original isolate in terms of the ethanol yield. PMID:2502944

  12. Isolation and partial characterization of mutants with elevated lipid content in Chlorella sorokiniana and Scenedesmus obliquus.

    PubMed

    Vigeolas, Hélène; Duby, Francéline; Kaymak, Esra; Niessen, Guillaume; Motte, Patrick; Franck, Fabrice; Remacle, Claire

    2012-11-30

    This paper describes the isolation and partial biomass characterization of high triacylglycerol (TAG) mutants of Chlorella sorokiniana and Scenedesmus obliquus, two algal species considered as potential source of biodiesel. Following UV mutagenesis, 2000 Chlorella and 2800 Scenedesmus colonies were screened with a method based on Nile Red fluorescence. Several mutants with high Nile Red fluorescence were selected by this high-throughput method in both species. Growth and biomass parameters of the strongest mutants were analyzed in detail. All of the four Chlorella mutants showed no significant changes in growth rate, cell weight, cell size, protein and chlorophyll contents on a per cell basis. Whereas all contained elevated total lipid and TAG content per unit of dry weight, two of them were also affected for starch metabolism, suggesting a change in biomass/storage carbohydrate composition. Two Scenedesmus mutants showed a 1.5 and 2-fold increased cell weight and larger cells compared to the wild type, which led to a general increase of biomass including total lipid and TAG content on a per cell basis. Such mutants could subsequently be used as commercial oleaginous algae and serve as an alternative to conventional petrol. PMID:22480533

  13. Improving the synthesis of phenolic polymer using Coprinus cinereus peroxidase mutant Phe230Ala.

    PubMed

    Kim, Su Jin; Joo, Jeong Chan; Song, Bong Keun; Yoo, Young Je; Kim, Yong Hwan

    2016-06-01

    The F230A mutant of Coprinus cinereus peroxidase (CiP), which has a high stability against radical-inactivation, was previously reported. In the present study, the radical-robust F230A mutant was applied to the oxidative polymerization of phenol. The F230A mutant exhibited better polymerization activities than the wild-type CiP in the presence of water-miscible alcohols i.e., methanol, ethanol, and isopropanol despite its lower stability against alcohols. In particular, the F230A mutant showed a higher consumption of phenol (40%) and yielded phenolic polymer of larger molecular weight (8850Da) in a 50% (v/v) isopropanol-buffer mixture compared with the wild-type CiP (2% and 1519Da, respectively). In addition, the wild-type CiP and F230A mutant had no significant differences in enzyme inactivation by physical adsorption on the polymeric products or by heat incubation, and showed comparable kinetic parameters. These results indicate that high radical stability of the F230A mutant and improved solubility of phenolic polymers in alcohol-water cosolvent systems may synergistically contribute to the production of the high molecular weight phenolic polymer. PMID:27178793

  14. Differential effects of lesion mimic mutants in barley on disease development by facultative pathogens

    PubMed Central

    McGrann, Graham R. D.; Steed, , Andrew; Burt, Christopher; Nicholson, Paul; Brown, James K. M.

    2015-01-01

    Lesion mimic mutants display spontaneous necrotic spots and chlorotic leaves as a result of mis-regulated cell death programmes. Typically these mutants have increased resistance to biotrophic pathogens but their response to facultative fungi that cause necrotrophic diseases is less well studied. The effect of altered cell death regulation on the development of disease caused by Ramularia collo-cygni, Fusarium culmorum and Oculimacula yallundae was explored using a collection of barley necrotic (nec) lesion mimic mutants. nec8 mutants displayed lower levels of all three diseases compared to nec9 mutants, which had increased R. collo-cygni but decreased F. culmorum disease symptoms. nec1 mutants reduced disease development caused by both R. collo-cygni and F. culmorum. The severity of the nec1-induced lesion mimic phenotype and F. culmorum symptom development was reduced by mutation of the negative cell death regulator MLO. The significant reduction in R. collo-cygni symptoms caused by nec1 was completely abolished in the presence of the mlo-5 allele and both symptoms and fungal biomass were greater than in the wild-type. These results indicate that physiological pathways involved in regulation of cell death interact with one another in their effects on different fungal pathogens. PMID:25873675

  15. Functional Loss of Bmsei Causes Thermosensitive Epilepsy in Contractile Mutant Silkworm, Bombyx mori

    PubMed Central

    Nie, Hongyi; Cheng, Tingcai; Huang, Xiaofeng; Zhou, Mengting; Zhang, Yinxia; Dai, Fangyin; Mita, Kazuei; Xia, Qingyou; Liu, Chun

    2015-01-01

    The thermoprotective mechanisms of insects remain largely unknown. We reported the Bombyx mori contractile (cot) behavioral mutant with thermo-sensitive seizures phenotype. At elevated temperatures, the cot mutant exhibit seizures associated with strong contractions, rolling, vomiting, and a temporary lack of movement. We narrowed a region containing cot to ~268 kb by positional cloning and identified the mutant gene as Bmsei which encoded a potassium channel protein. Bmsei was present in both the cell membrane and cytoplasm in wild-type ganglia but faint in cot. Furthermore, Bmsei was markedly decreased upon high temperature treatment in cot mutant. With the RNAi method and injecting potassium channel blockers, the wild type silkworm was induced the cot phenotype. These results demonstrated that Bmsei was responsible for the cot mutant phenotype and played an important role in thermoprotection in silkworm. Meanwhile, comparative proteomic approach was used to investigate the proteomic differences. The results showed that the protein of Hsp-1 and Tn1 were significantly decreased and increased on protein level in cot mutant after thermo-stimulus, respectively. Our data provide insights into the mechanism of thermoprotection in insect. As cot phenotype closely resembles human epilepsy, cot might be a potential model for the mechanism of epilepsy in future. PMID:26198671

  16. Cellular Adherence, Glucosyltransferase Adsorption, and Glucan Synthesis of Streptococcus mutans AHT Mutants

    PubMed Central

    Koga, Toshihiko; Inoue, Masakazu

    1978-01-01

    Streptococcus mutans AHT mutants M1, M2, and M13 failed to adhere to a glass surface, whereas mutants M9 and M35 exhibited decreased and increased adherence, respectively, as compared with the parent strain, when grown in sucrose broth. Extracellular glucosyltransferase prepared from glucose-grown cultures of the adherent strains (wild type, M9, and M35) induced adherence of heat-killed cells of the homologous and heterologous streptococcal strains as well as of Escherichia coli K-12 and uncoated resin particles. The glucosyltransferase was adsorbed on all the streptococcal cells and glucan-coated resins, but not on E. coli cells and the uncoated resins. Glucosyltransferase from the nonadhering mutants (M1, M2, M13) neither was significantly adsorbed on nor induced adherence of any of the cells and resins. Cell-free enzymes from the glucose-grown adherent strains produced water-soluble and water-insoluble glucans, whereas those from the nonadhering mutants produced only water-soluble glucans. Small amounts of alkali-soluble, cell-associated glucan were recovered from the sucrose-grown nonadhering mutants. Thus, the relative proportions of glucosyltransferase isozymes elaborated by the S. mutans mutants, insofar as they affect the physico-chemical properties of the glucans produced, seem to determine the adherence abilities of the cells. The adsorption of glucosyltransferase on glucan molecules on the cell surface is not required for the adherence of S. mutans, but de novo glucan synthesis is important in the adherence process. PMID:631879

  17. Targeting the mTOR Complex by Everolimus in NRAS Mutant Neuroblastoma

    PubMed Central

    Kiessling, Michael K.; Curioni-Fontecedro, Alessandra; Samaras, Panagiotis; Lang, Silvia; Scharl, Michael; Aguzzi, Adriano; Oldrige, Derek A.; Maris, John M.; Rogler, Gerhard

    2016-01-01

    High-risk neuroblastoma remains lethal in about 50% of patients despite multimodal treatment. Recent attempts to identify molecular targets for specific therapies have shown that Neuroblastoma RAS (NRAS) is significantly mutated in a small number of patients. However, few inhibitors for the potential treatment for NRAS mutant neuroblastoma have been investigated so far. In this in-vitro study, we show that MEK inhibitors AZD6244, MEK162 and PD0325901 block cell growth in NRAS mutant neuroblastoma cell lines but not in NRAS wild-type cell lines. Several studies show that mutant NRAS leads to PI3K pathway activation and combined inhibitors of PI3K/mTOR effectively block cell growth. However, we observed the combination of MEK inhibitors with PI3K or AKT inhibitors did not show synergestic effects on cell growth. Thus, we tested single mTOR inhibitors Everolimus and AZD8055. Interestingly, Everolimus and AZD8055 alone were sufficient to block cell growth in NRAS mutant cell lines but not in wild-type cell lines. We found that Everolimus alone induced apoptosis in NRAS mutant neuroblastoma. Furthermore, the combination of mTOR and MEK inhibitors resulted in synergistic growth inhibition. Taken together, our results show that NRAS mutant neuroblastoma can be targeted by clinically available Everolimus alone or in combination with MEK inhibitors which could impact future clinical studies. PMID:26821351

  18. Surface Polysaccharide Mutants Reveal that Absence of O Antigen Reduces Biofilm Formation of Actinobacillus pleuropneumoniae

    PubMed Central

    Hathroubi, S.; Hancock, M. A.; Langford, P. R.; Tremblay, Y. D. N.; Labrie, J.

    2015-01-01

    Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides, including lipopolysaccharides (LPS) and capsular polysaccharides (CPS), are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement for these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well-characterized mutants were used: an O-antigen LPS mutant, a truncated core LPS mutant with an intact O antigen, a capsule mutant, and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the findings for the biofilm of the parental or other strains, the biofilm of the O antigen and the PGA mutants was dramatically reduced, and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR, and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to that in the biofilm cells of the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance, but the lack of O antigen did not abolish these interactions. In conclusion, the absence of the O antigen reduces the ability of A. pleuropneumoniae to form a biofilm, and this is associated with the reduced expression and production of PGA. PMID:26483403

  19. The phenotype of a phospholipase C (plc-1) mutant in a filamentous fungus, Neurospora crassa.

    PubMed

    Lew, Roger R; Giblon, Rachel E; Lorenti, Miranda S H

    2015-09-01

    In the filamentous fungus Neurospora crassa, phospholipase C may play a role in hyphal extension at the growing tips as part of a growth-sensing mechanism that activates calcium release from internal stores to mediate continued expansion of the hyphal tip. One candidate for a tip-localized phospholipase C is PLC-1. We characterized morphology and growth characteristics of a knockout mutant (KO plc-1) and a RIP mutated strain (RIP plc-1) (missense mutations and a nonsense mutation render the gene product non-functional). Growth and hyphal cytology of wildtype and KO plc-1 were similar, but the RIP plc-1 mutant grew slower and exhibited abnormal membrane structures at the hyphal tip, imaged using the fluorescence dye FM4-64. To test for causes of the slower growth of the RIP plc-1 mutant, we examined its physiological poise compared to wildtype and the KO plc-1 mutant. The electrical properties of all three strains and the electrogenic contribution of the plasma membrane H(+)-ATPase (identified by cyanide inhibition) were the same. Responses to high osmolarity were also similar. However, the RIP plc-1 mutant had a significantly lower turgor, a possible cause of its slower growth. While growth of all three strains was inhibited by the phospholipase C inhibitor 3-nitrocoumarin, the RIP plc-1 mutant did not exhibit hyphal bursting after addition of the inhibitor, observed in both wildtype and the KO plc-1 mutant. Although the plc-1 gene is not obligatory for tip growth, the phenotype of the RIP plc-1 mutant - abnormal tip cytology, lower turgor and resistance to inhibitor-induced hyphal bursting - suggest it does play a role in tip growth. The expression of a dysfunctional plc-1 gene may cause a shift to alternative mechanism(s) of growth sensing in hyphal extension. PMID:26212074

  20. Akt mediated ROS-dependent selective targeting of mutant KRAS tumors.

    PubMed

    Iskandar, Kartini; Rezlan, Majidah; Pervaiz, Shazib

    2014-10-01

    Reactive oxygen species (ROS) play a critical role in a variety of cellular processes, ranging from cell survival and proliferation to cell death. Previously, we reported the ability of a small molecule compound, C1, to induce ROS dependent autophagy associated apoptosis in human cancer cell lines and primary tumor cells (Wong C. et al. 2010). Our ongoing investigations have unraveled a hitherto undefined novel signaling network involving hyper-phosphorylation of Akt and Akt-mediated ROS production in cancer cell lines. Interestingly, drug-induced Akt activation is selectively seen in cell lines that carry mutant KRAS; HCT116 cells that carry the V13D KRAS mutation respond favorably to C1 while HT29 cells expressing wild type KRAS are relatively resistant. Of note, not only does the compound target mutant KRAS expressing cells but also induces RAS activation as evidenced by the PAK pull down assay. Corroborating this, pharmacological inhibition as well as siRNA mediated silencing of KRAS or Akt, blocked C1-induced ROS production and rescued tumor colony forming ability in HCT116 cells. To further confirm the involvement of KRAS, we made use of mutant KRAS transformed RWPE-1 prostate epithelial cells. Notably, drug-induced ROS generation and death sensitivity was significantly higher in RWPE-1-KRAS cells than the RWPE-1-vector cells, thus confirming the results obtained with mutant KRAS colorectal carcinoma cell line. Lastly, we made use of HCT116 mutant KRAS knockout cells (KO) where the mutant KRAS allele had been deleted, thus expressing a single wild-type KRAS allele. Exposure of the KO cells to C1 failed to induce Akt activation and mitochondrial ROS production. Taken together, results show the involvement of activated Akt in ROS-mediated selective targeting of mutant KRAS expressing tumors, which could have therapeutic implications given the paucity of chemotherapeutic strategies specifically targeting KRAS mutant cancers. PMID:26461287

  1. A γA-Crystallin Mouse Mutant Secc with Small Eye, Cataract and Closed Eyelid.

    PubMed

    Cheng, Man Hei; Tam, Chung Nga; Choy, Kwong Wai; Tsang, Wai Hung; Tsang, Sze Lan; Pang, Chi Pui; Song, You Qiang; Sham, Mai Har

    2016-01-01

    Cataract is the most common cause of visual loss in humans. A spontaneously occurred, autosomal dominant mouse mutant Secc, which displayed combined features of small eye, cataract and closed eyelid was discovered in our laboratory. In this study, we identified the mutation and characterized the cataract phenotype of this novel Secc mutant. The Secc mutant mice have eyelids that remain half-closed throughout their life. The mutant lens has a significant reduction in size and with opaque spots clustered in the centre. Histological analysis showed that in the core region of the mutant lens, the fiber cells were disorganized and clefts and vacuoles were observed. The cataract phenotype was evident from new born stage. We identified the Secc mutation by linkage analysis using whole genome microsatellite markers and SNP markers. The Secc locus was mapped at chromosome 1 flanked by SNPs rs3158129 and rs13475900. Based on the chromosomal position, the candidate cataract locus γ-crystallin gene cluster (Cryg) was investigated by sequencing. A single base deletion (299delG) in exon 3 of Cryga which led to a frame-shift of amino acid sequence from position 91 was identified. As a result of this mutation, the sequences of the 3rd and 4th Greek-key motifs of the γA-crystallin are replaced with an unrelated C-terminal peptide of 75 residues long. Coincidentally, the point mutation generated a HindIII restriction site, allowing the identification of the CrygaSecc mutant allele by RFLP. Western blot analysis of 3-week old lenses showed that the expression of γ-crystallins was reduced in the CrygaSecc mutant. Furthermore, in cell transfection assays using CrygaSecc mutant cDNA expression constructs in 293T, COS-7 and human lens epithelial B3 cell lines, the mutant γA-crystallins were enriched in the insoluble fractions and appeared as insoluble aggregates in the transfected cells. In conclusion, we have demonstrated that the Secc mutation leads to the generation of Cryga

  2. Enhanced antiinflammatory capacity of a Lactobacillus plantarum mutant synthesizing modified teichoic acids

    PubMed Central

    Grangette, Corinne; Nutten, Sophie; Palumbo, Emmanuelle; Morath, Siegfried; Hermann, Corinna; Dewulf, Joelle; Pot, Bruno; Hartung, Thomas; Hols, Pascal; Mercenier, Annick

    2005-01-01

    Teichoic acids (TAs), and especially lipoteichoic acids (LTAs), are one of the main immunostimulatory components of pathogenic Gram-positive bacteria. Their contribution to the immunomodulatory properties of commensal bacteria and especially of lactic acid bacteria has not yet been investigated in detail. To evaluate the role of TAs in the interaction between lactic acid bacteria and the immune system, we analyzed the antiinflammatory properties of a mutant of Lactobacillus plantarum NCIMB8826 affected in the TA biosynthesis pathway both in vitro (mononuclear cells stimulation) and in vivo (murine model of colitis). This Dlt- mutant was found to incorporate much less d-Ala in its TAs than the WT strain. This defect significantly impacted the immunomodulation reactions induced by the bacterium, as shown by a dramatically reduced secretion of proinflammatory cytokines by peripheral blood mononuclear cells and monocytes stimulated by the Dlt- mutant as compared with the parental strain. Concomitantly, a significant increase in IL-10 production was stimulated by the Dlt- mutant in comparison with the WT strain. Moreover, the proinflammatory capacity of L. plantarum-purified LTA was found to be Toll-like receptor 2-dependent. Consistent with the in vitro results, the Dlt- mutant was significantly more protective in a murine colitis model than its WT counterpart. The results indicated that composition of LTA within the whole-cell context of L. plantarum can modulate proinflammatory or antiinflammatory immune responses. PMID:15985548

  3. Zebrafish Mutants calamity and catastrophe Define Critical Pathways of Gene–Nutrient Interactions in Developmental Copper Metabolism

    PubMed Central

    Madsen, Erik C.; Gitlin, Jonathan D.

    2008-01-01

    Nutrient availability is an important environmental variable during development that has significant effects on the metabolism, health, and viability of an organism. To understand these interactions for the nutrient copper, we used a chemical genetic screen for zebrafish mutants sensitive to developmental copper deficiency. In this screen, we isolated two mutants that define subtleties of copper metabolism. The first contains a viable hypomorphic allele of atp7a and results in a loss of pigmentation when exposed to mild nutritional copper deficiency. This mutant displays incompletely penetrant skeletal defects affected by developmental copper availability. The second carries an inactivating mutation in the vacuolar ATPase that causes punctate melanocytes and embryonic lethality. This mutant, catastrophe, is sensitive to copper deprivation revealing overlap between ion metabolic pathways. Together, the two mutants illustrate the utility of chemical genetic screens in zebrafish to elucidate the interaction of nutrient availability and genetic polymorphisms in cellular metabolism. PMID:19008952

  4. Mutant IDH1 Downregulates ATM and Alters DNA Repair and Sensitivity to DNA Damage Independent of TET2.

    PubMed

    Inoue, Satoshi; Li, Wanda Y; Tseng, Alan; Beerman, Isabel; Elia, Andrew J; Bendall, Sean C; Lemonnier, François; Kron, Ken J; Cescon, David W; Hao, Zhenyue; Lind, Evan F; Takayama, Naoya; Planello, Aline C; Shen, Shu Yi; Shih, Alan H; Larsen, Dana M; Li, Qinxi; Snow, Bryan E; Wakeham, Andrew; Haight, Jillian; Gorrini, Chiara; Bassi, Christian; Thu, Kelsie L; Murakami, Kiichi; Elford, Alisha R; Ueda, Takeshi; Straley, Kimberly; Yen, Katharine E; Melino, Gerry; Cimmino, Luisa; Aifantis, Iannis; Levine, Ross L; De Carvalho, Daniel D; Lupien, Mathieu; Rossi, Derrick J; Nolan, Garry P; Cairns, Rob A; Mak, Tak W

    2016-08-01

    Mutations in the isocitrate dehydrogenase-1 gene (IDH1) are common drivers of acute myeloid leukemia (AML) but their mechanism is not fully understood. It is thought that IDH1 mutants act by inhibiting TET2 to alter DNA methylation, but there are significant unexplained clinical differences between IDH1- and TET2-mutant diseases. We have discovered that mice expressing endogenous mutant IDH1 have reduced numbers of hematopoietic stem cells (HSCs), in contrast to Tet2 knockout (TET2-KO) mice. Mutant IDH1 downregulates the DNA damage (DD) sensor ATM by altering histone methylation, leading to impaired DNA repair, increased sensitivity to DD, and reduced HSC self-renewal, independent of TET2. ATM expression is also decreased in human IDH1-mutated AML. These findings may have implications for treatment of IDH-mutant leukemia. PMID:27424808

  5. Impairment in motor learning of somatostatin null mutant mice.

    PubMed

    Zeyda, T; Diehl, N; Paylor, R; Brennan, M B; Hochgeschwender, U

    2001-07-01

    Somatostatin was first identified as a hypothalamic factor which inhibits the release of growth hormone from the anterior pituitary (somatotropin release inhibitory factor, SRIF). Both SRIF and its receptors were subsequently found widely distributed within and outside the nervous system, in the adult as well as in the developing organism. Reflecting this wide distribution, somatostatin has been implicated regulating a diverse array of biological processes. These include body growth, homeostasis, sensory perception, autonomous functions, rate of intestinal absorption, behavior, including cognition and memory, and developmental processes. We produced null mutant mice lacking somatostatin through targeted mutagenesis. The mutant mice are healthy, fertile, and superficially indistinguishable from their heterozygous and wildtype littermates. A 'first round' phenotype screen revealed that mice lacking somatostatin have elevated plasma growth hormone levels, despite normal body size, and have elevated basal plasma corticosterone levels. In order to uncover subtle and unexpected differences, we carried out a systematic behavioral phenotype screen which identified a significant impairment in motor learning revealed when increased demands were made on motor coordination. Motor coordination and motor learning require an intact cerebellum. While somatostatin is virtually absent from the adult cerebellum, the ligand and its receptor(s) are transiently expressed at high levels in the developing cerebellum. This result suggests the functional significance of transient expression of SRIF and its receptors in the development of the cerebellum. PMID:11430867

  6. Mutant p53: One, No One, and One Hundred Thousand

    PubMed Central

    Walerych, Dawid; Lisek, Kamil; Del Sal, Giannino

    2015-01-01

    Encoded by the mutated variants of the TP53 tumor suppressor gene, mutant p53 proteins are getting an increased experimental support as active oncoproteins promoting tumor growth and metastasis. p53 missense mutant proteins are losing their wild-type tumor suppressor activity and acquire oncogenic potential, possessing diverse transforming abilities in cell and mouse models. Whether various mutant p53s differ in their oncogenic potential has been a matter of debate. Recent discoveries are starting to uncover the existence of mutant p53 downstream programs that are common to different mutant p53 variants. In this review, we discuss a number of studies on mutant p53, underlining the advantages and disadvantages of alternative experimental approaches that have been used to describe the numerous mutant p53 gain-of-function activities. Therapeutic possibilities are also discussed, taking into account targeting either individual or multiple mutant p53 proteins in human cancer. PMID:26734571

  7. Intact Interval Timing in Circadian CLOCK Mutants

    PubMed Central

    Cordes, Sara; Gallistel, C. R.

    2008-01-01

    While progress has been made in determining the molecular basis for the circadian clock, the mechanism by which mammalian brains time intervals measured in seconds to minutes remains a mystery. An obvious question is whether the interval timing mechanism shares molecular machinery with the circadian timing mechanism. In the current study, we trained circadian CLOCK +/− and −/− mutant male mice in a peak-interval procedure with 10 and 20-s criteria. The mutant mice were more active than their wild-type littermates, but there were no reliable deficits in the accuracy or precision of their timing as compared with wild-type littermates. This suggests that expression of the CLOCK protein is not necessary for normal interval timing. PMID:18602902

  8. Oxygen sensitivity of an Escherichia coli mutant.

    PubMed

    Adler, H; Mural, R; Suttle, B

    1992-04-01

    Genetic evidence indicates that Oxys-6, an oxygen-sensitive mutant of Escherichia coli AB1157, is defective in the region of the hemB locus. Oxys-6 is capable of growth under aerobic conditions only if cultures are initiated at low-inoculum levels. Aerobic liquid cultures are limited to a cell density of 10(7) cells per ml by the accumulation of a metabolically produced, low-molecular-weight, heat-stable material in complex organic media. Both Oxys-6 and AB1157 cells produce the material, but only aerobic cultures of the mutant are inhibited by it. The material is produced by both intact cells and cell extracts in complex media. This reaction also occurs when the amino acid L-lysine is substituted for complex media. PMID:1551829

  9. Oxygen sensitivity of an Escherichia coli mutant.

    PubMed Central

    Adler, H; Mural, R; Suttle, B

    1992-01-01

    Genetic evidence indicates that Oxys-6, an oxygen-sensitive mutant of Escherichia coli AB1157, is defective in the region of the hemB locus. Oxys-6 is capable of growth under aerobic conditions only if cultures are initiated at low-inoculum levels. Aerobic liquid cultures are limited to a cell density of 10(7) cells per ml by the accumulation of a metabolically produced, low-molecular-weight, heat-stable material in complex organic media. Both Oxys-6 and AB1157 cells produce the material, but only aerobic cultures of the mutant are inhibited by it. The material is produced by both intact cells and cell extracts in complex media. This reaction also occurs when the amino acid L-lysine is substituted for complex media. Images PMID:1551829

  10. Functional analysis and drug response to zinc and D-penicillamine in stable ATP7B mutant hepatic cell lines

    PubMed Central

    Chandhok, Gursimran; Horvath, Judit; Aggarwal, Annu; Bhatt, Mohit; Zibert, Andree; Schmidt, Hartmut HJ

    2016-01-01

    AIM: To study the effect of anti-copper treatment for survival of hepatic cells expressing different ATP7B mutations in cell culture. METHODS: The most common Wilson disease (WD) mutations p.H1069Q, p.R778L and p.C271*, found in the ATP7B gene encoding a liver copper transporter, were studied. The mutations represent major genotypes of the United States and Europe, China, and India, respectively. A human hepatoma cell line previously established to carry a knockout of ATP7B was used to stably express WD mutants. mRNA and protein expression of mutant ATP7B, survival of cells, apoptosis, and protein trafficking were determined. RESULTS: Low temperature increased ATP7B protein expression in several mutants. Intracellular ATP7B localization was significantly impaired in the mutants. Mutants were classified as high, moderate, and no survival based on their viability on exposure to toxic copper. Survival of mutant p.H1069Q and to a lesser extent p.C271* improved by D-penicillamine (DPA) treatment, while mutant p.R778L showed a pronounced response to zinc (Zn) treatment. Overall, DPA treatment resulted in higher cell survival as compared to Zn treatment; however, only combined Zn + DPA treatment fully restored cell viability. CONCLUSION: The data indicate that the basic impact of a genotype might be characterized by analysis of mutant hepatic cell lines. PMID:27122662

  11. Classification and identification of Arabidopsis cell wall mutants using Fourier-Transform InfraRed (FT-IR) microspectroscopy.

    PubMed

    Mouille, Grégory; Robin, Stéphane; Lecomte, Mannaïg; Pagant, Silvère; Höfte, Herman

    2003-08-01

    We have developed a novel procedure for the rapid classification and identification of Arabidopsis mutants with altered cell wall architecture based on Fourier-Transform Infrared (FT-IR) microspectroscopy. FT-IR transmission spectra were sampled from native 4-day-old dark-grown hypocotyls of 46 mutants and the wild type treated with various drugs. The Mahalanobis distance between mutants, calculated from the spectral information after compression with the Discriminant Variables Selection procedure, was used for alpha hierarchical cluster analysis. Despite the completely unsupervised nature of the classification procedure, we show that all mutants with cellulose defects appeared in the same cluster. In addition, mutant alleles of similar strength for several unrelated loci were also clustered, which demonstrates the sensitivity of the method to detect a wide array of cell wall defects. Comparing the cellulose-deficient cluster with the cluster that contained wild-type controls led to the identification of wave numbers that were diagnostic for altered cellulose content in the context of an intact cell wall. The results show that FT-IR spectra can be used to identify different classes of mutants and to characterize cell wall changes at a microscopic level in unknown mutants. This procedure significantly accelerates the identification and classification of cell wall mutants, which makes cell wall polysaccharides more accessible to functional genomics approaches. PMID:12887590

  12. In vitro characterization of felid herpesvirus 1 (FHV-1) mutants generated by recombineering in a recombinant BAC vector.

    PubMed

    Tai, S-H Sheldon; Holz, Carine; Engstrom, Michael D; Cheng, Hans H; Maes, Roger K

    2016-08-01

    Felid herpesvirus 1 (FHV-1) mutants were constructed using two-step Red-mediated recombination techniques based on a virulent full-length FHV-1 BAC clone. The individual mutant viruses generated were deficient in glycoprotein C (gC), glycoprotein E (gE), US3 serine/threonine protein kinase (PK), or both gE and thymidine kinase (TK). The gC- mutant virus produced plaques that were similar in size to those resulting from infection with the C-27 parent strain. In contrast, the gE(-), PK(-), and gE(-)PK(-) deletion mutants produced plaques that were significantly smaller. Multistep in vitro growth kinetics of the gE(-), PK(-), and gE(-)PK(-) viruses were slightly delayed compared to those of the C-27 parent strain. Peak progeny titers of these three mutants were approximately 10-fold lower than those generated with the C-27 strain. There was no delay in the growth kinetics of the gC- mutant, but the progeny virus titer obtained with this mutant was at least 3 logs lower compared to the parental strain titer. Based upon their in vitro characteristics, these mutants will be useful for the development of novel immunization strategies against this important feline pathogen. PMID:27157860

  13. Recombination-deficient mutant of Streptococcus faecalis

    SciTech Connect

    Yagi, Y.; Clewell, D.B.

    1980-08-01

    An ultraviolet radiation-sensitive derivative of Streptococcus faecalis strain JH2-2 was isolated and found to be deficient in recombination, using a plasmid-plasmid recombination system. The strain was sensitive to chemical agents which interact with deoxyribonucleic acid and also underwent deoxyribonucleic acid degradation after ultraviolet irradiation. Thus, the mutant has properties similar to those of recA strains of Escherichia coli.

  14. Induced Dwarf Mutant in Catharanthus roseus with Enhanced Antibacterial Activity

    PubMed Central

    Verma, A. K.; Singh, R. R.

    2010-01-01

    Evaluation of an ethyl methane sulphonate-induced dwarf mutant of Catharanthus roseus (L.) G. Don revealed that the mutant exhibited marked variation in morphometric parameters. The in vitro antibacterial activity of the aqueous and alcoholic leaf extracts of the mutant and control plants was investigated against medically important bacteria. The mutant leaf extracts showed enhanced antibacterial activity against all the tested bacteria except Bacillus subtilis. PMID:21695004

  15. Sleep restores behavioral plasticity to Drosophila mutants

    PubMed Central

    Dissel, Stephane; Angadi, Veena; Kirszenblat, Leonie; Suzuki, Yasuko; Donlea, Jeff; Klose, Markus; Koch, Zachary; English, Denis; Winsky-Sommerer, Raphaelle; van Swinderen, Bruno; Shaw, Paul J.

    2015-01-01

    SUMMARY Given the role that sleep plays in modulating plasticity, we hypothesized that increasing sleep would restore memory to canonical memory mutants without specifically rescuing the causal molecular-lesion. Sleep was increased using three independent strategies: activating the dorsal Fan Shaped Body (FB), increasing the expression of Fatty acid binding protein (dFabp) or by administering the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP). Short-term memory (STM) or Long-term memory (LTM) was evaluated in rutabaga (rut) and dunce (dnc) mutants using Aversive Phototaxic Suppression (APS) and courtship conditioning. Each of the three independent strategies increased sleep and restored memory to rut and dnc mutants. Importantly, inducing sleep also reverses memory defects in a Drosophila model of Alzheimer’s disease. Together these data demonstrate that sleep plays a more fundamental role in modulating behavioral plasticity than previously appreciated and suggests that increasing sleep may benefit patients with certain neurological disorders. PMID:25913403

  16. Mutant Sodium Channel for Tumor Therapy

    PubMed Central

    Tannous, Bakhos A; Christensen, Adam P; Pike, Lisa; Wurdinger, Thomas; Perry, Katherine F; Saydam, Okay; Jacobs, Andreas H; García-Añoveros, Jaime; Weissleder, Ralph; Sena-Esteves, Miguel; Corey, David P; Breakefield, Xandra O

    2009-01-01

    Viral vectors have been used to deliver a wide range of therapeutic genes to tumors. In this study, a novel tumor therapy was achieved by the delivery of a mammalian brain sodium channel, ASIC2a, carrying a mutation that renders it constitutively open. This channel was delivered to tumor cells using a herpes simplex virus-1/Epstein–Barr virus (HSV/EBV) hybrid amplicon vector in which gene expression was controlled by a tetracycline regulatory system (tet-on) with silencer elements. Upon infection and doxycycline induction of mutant channel expression in tumor cells, the open channel led to amiloride-sensitive sodium influx as assessed by patch clamp recording and sodium imaging in culture. Within hours, tumor cells swelled and died. In addition to cells expressing the mutant channel, adjacent, noninfected cells connected by gap junctions also died. Intratumoral injection of HSV/EBV amplicon vector encoding the mutant sodium channel and systemic administration of doxycycline led to regression of subcutaneous tumors in nude mice as assessed by in vivo bioluminescence imaging. The advantage of this direct mode of tumor therapy is that all types of tumor cells become susceptible and death is rapid with no time for the tumor cells to become resistant. PMID:19259066

  17. Induction of sarcomas by mutant IDH2

    PubMed Central

    Lu, Chao; Venneti, Sriram; Akalin, Altuna; Fang, Fang; Ward, Patrick S.; DeMatteo, Raymond G.; Intlekofer, Andrew M.; Chen, Chong; Ye, Jiangbin; Hameed, Meera; Nafa, Khedoudja; Agaram, Narasimhan P.; Cross, Justin R.; Khanin, Raya; Mason, Christopher E.; Healey, John H.; Lowe, Scott W.; Schwartz, Gary K.; Melnick, Ari; Thompson, Craig B.

    2013-01-01

    More than 50% of patients with chondrosarcomas exhibit gain-of-function mutations in either isocitrate dehydrogenase 1 (IDH1) or IDH2. In this study, we performed genome-wide CpG methylation sequencing of chondrosarcoma biopsies and found that IDH mutations were associated with DNA hypermethylation at CpG islands but not other genomic regions. Regions of CpG island hypermethylation were enriched for genes implicated in stem cell maintenance/differentiation and lineage specification. In murine 10T1/2 mesenchymal progenitor cells, expression of mutant IDH2 led to DNA hypermethylation and an impairment in differentiation that could be reversed by treatment with DNA-hypomethylating agents. Introduction of mutant IDH2 also induced loss of contact inhibition and generated undifferentiated sarcomas in vivo. The oncogenic potential of mutant IDH2 correlated with the ability to produce 2-hydroxyglutarate. Together, these data demonstrate that neomorphic IDH2 mutations can be oncogenic in mesenchymal cells. PMID:24065766

  18. Biochemical and Structural Characterization of HDAC8 Mutants Associated with Cornelia de Lange Syndrome Spectrum Disorders

    PubMed Central

    2015-01-01

    Cornelia de Lange Syndrome (CdLS) spectrum disorders are characterized by multiple organ system congenital anomalies that result from mutations in genes encoding core cohesin proteins SMC1A, SMC3, and RAD21, or proteins that regulate cohesin function such as NIPBL and HDAC8. HDAC8 is the Zn2+-dependent SMC3 deacetylase required for cohesin recycling during the cell cycle, and 17 different HDAC8 mutants have been identified to date in children diagnosed with CdLS. As part of our continuing studies focusing on aberrant HDAC8 function in CdLS, we now report the preparation and biophysical evaluation of five human HDAC8 mutants: P91L, G117E, H180R, D233G, and G304R. Additionally, the double mutants D233G–Y306F and P91L–Y306F were prepared to enable cocrystallization of intact enzyme–substrate complexes. X-ray crystal structures of G117E, P91L–Y306F, and D233G–Y306F HDAC8 mutants reveal that each CdLS mutation causes structural changes that compromise catalysis and/or thermostability. For example, the D233G mutation disrupts the D233–K202–S276 hydrogen bond network, which stabilizes key tertiary structure interactions, thereby significantly compromising thermostability. Molecular dynamics simulations of H180R and G304R HDAC8 mutants suggest that the bulky arginine side chain of each mutant protrudes into the substrate binding site and also causes active site residue Y306 to fluctuate away from the position required for substrate activation and catalysis. Significantly, the catalytic activities of most mutants can be partially or fully rescued by the activator N-(phenylcarbamothioyl)-benzamide, suggesting that HDAC8 activators may serve as possible leads in the therapeutic management of CdLS. PMID:26463496

  19. Rubisco mutants of Chlamydomonas reinhardtii enhance photosynthetic hydrogen production.

    PubMed

    Pinto, T S; Malcata, F X; Arrabaça, J D; Silva, J M; Spreitzer, R J; Esquível, M G

    2013-06-01

    Molecular hydrogen (H2) is an ideal fuel characterized by high enthalpy change and lack of greenhouse effects. This biofuel can be released by microalgae via reduction of protons to molecular hydrogen catalyzed by hydrogenases. The main competitor for the reducing power required by the hydrogenases is the Calvin cycle, and rubisco plays a key role therein. Engineered Chlamydomonas with reduced rubisco levels, activity and stability was used as the basis of this research effort aimed at increasing hydrogen production. Biochemical monitoring in such metabolically engineered mutant cells proceeded in Tris/acetate/phosphate culture medium with S-depletion or repletion, both under hypoxia. Photosynthetic activity, maximum photochemical efficiency, chlorophyll and protein levels were all measured. In addition, expression of rubisco, hydrogenase, D1 and Lhcb were investigated, and H2 was quantified. At the beginning of the experiments, rubisco increased followed by intense degradation. Lhcb proteins exhibited monomeric isoforms during the first 24 to 48 h, and D1 displayed sensitivity under S-depletion. Rubisco mutants exhibited a significant decrease in O2 evolution compared with the control. Although the S-depleted medium was much more suitable than its complete counterpart for H2 production, hydrogen release was observed also in sealed S-repleted cultures of rubisco mutated cells under low-moderate light conditions. In particular, the rubisco mutant Y67A accounted for 10-15-fold higher hydrogen production than the wild type under the same conditions and also displayed divergent metabolic parameters. These results indicate that rubisco is a promising target for improving hydrogen production rates in engineered microalgae. PMID:23649352

  20. Registration of two allelic erect leaf mutants of sorghum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two allelic sorghum [Sorghum bicolor (L.) Moench] erect leaf (erl) mutants were isolated from an Annotated Individually-pedigreed Mutagenized Sorghum (AIMS) mutant library developed at the Plant Stress and Germplasm Development Unit, at Lubbock, Texas. The two mutants, erl1-1 and erl1-2, were isol...

  1. Targeting Oncogenic Mutant p53 for Cancer Therapy

    PubMed Central

    Parrales, Alejandro; Iwakuma, Tomoo

    2015-01-01

    Among genetic alterations in human cancers, mutations in the tumor suppressor p53 gene are the most common, occurring in over 50% of human cancers. The majority of p53 mutations are missense mutations and result in the accumulation of dysfunctional p53 protein in tumors. These mutants frequently have oncogenic gain-of-function activities and exacerbate malignant properties of cancer cells, such as metastasis and drug resistance. Increasing evidence reveals that stabilization of mutant p53 in tumors is crucial for its oncogenic activities, while depletion of mutant p53 attenuates malignant properties of cancer cells. Thus, mutant p53 is an attractive druggable target for cancer therapy. Different approaches have been taken to develop small-molecule compounds that specifically target mutant p53. These include compounds that restore wild-type conformation and transcriptional activity of mutant p53, induce depletion of mutant p53, inhibit downstream pathways of oncogenic mutant p53, and induce synthetic lethality to mutant p53. In this review article, we comprehensively discuss the current strategies targeting oncogenic mutant p53 in cancers, with special focus on compounds that restore wild-type p53 transcriptional activity of mutant p53 and those reducing mutant p53 levels. PMID:26732534

  2. The Contrived Mutant p53 Oncogene – Beyond Loss of Functions

    PubMed Central

    Sabapathy, Kanaga

    2015-01-01

    Mutations in p53 are almost synonymous with cancer – be it susceptibility to the disease or response to treatment – and therefore, are a critical determinant of overall survival. As most of these mutations occur in the DNA-binding domain of p53, many of the clinical correlations with mutant p53 have been initially relegated to the loss of its transcription-dependent activities as a tumor suppressor. However, significant efforts over the last two decades have led to the vast knowledge on the potential functions of the mutated p53 protein, which have been attributed to the physical presence of the mutant protein rather than the loss of its wild-type (WT) functions. Beyond the inhibitory effects of mutant p53 on the remaining WT protein that leads to the dominant-negative effect in the heterozygous state, mutant p53’s presence has also been significantly attributed to novel gain-of-functions that lead to addiction of cancer cells to its presence for survival, as well as for their ability to invade and metastasize, elevating it to a contrived oncogene that drives the cancer cells forward. This review will summarize the functional consequences of the presence of mutant p53 protein on cellular and organismal physiology. PMID:26697411

  3. Characterization of the mutant spectra of a fish RNA virus within individual hosts during natural infections

    USGS Publications Warehouse

    Emmenegger, Eveline J.; Troyer, Ryan M.; Kurath, Gael

    2003-01-01

    Infectious hematopoietic necrosis virus (IHNV) is an RNA virus that causes significant mortalities of salmonids in the Pacific Northwest of North America. RNA virus populations typically contain genetic variants that form a heterogeneous virus pool, referred to as a quasispecies or mutant spectrum. This study characterized the mutant spectra of IHNV populations within individual fish reared in different environmental settings by RT–PCR of genomic viral RNA and determination of partial glycoprotein gene sequences of molecular clones. The diversity of the mutant spectra from ten in vivo populations was low and the average mutation frequencies of duplicate populations did not significantly exceed the background mutation level expected from the methodology. In contrast, two in vitro populations contained variants with an identical mutational hot spot. These results indicated that the mutant spectra of natural IHNV populations is very homogeneous, and does not explain the different magnitudes of genetic diversity observed between the different IHNV genogroups. Overall the mutant frequency of IHNV within its host is one of the lowest reported for RNA viruses.

  4. Significant lexical relationships

    SciTech Connect

    Pedersen, T.; Kayaalp, M.; Bruce, R.

    1996-12-31

    Statistical NLP inevitably deals with a large number of rare events. As a consequence, NLP data often violates the assumptions implicit in traditional statistical procedures such as significance testing. We describe a significance test, an exact conditional test, that is appropriate for NLP data and can be performed using freely available software. We apply this test to the study of lexical relationships and demonstrate that the results obtained using this test are both theoretically more reliable and different from the results obtained using previously applied tests.

  5. Mutant prevention concentration and phenotypic and molecular basis of fluoroquinolone resistance in clinical isolates and in vitro-selected mutants of Escherichia coli from dogs.

    PubMed

    Gebru, Elias; Damte, Dereje; Choi, Myung-Jin; Lee, Seung-Jin; Kim, Young-Hoan; Park, Seung Chun

    2012-01-27

    The antibacterial activity, selection of Escherichia coli (E. coli) mutants and mechanisms of fluoroquinolone resistance were investigated by integrating the minimum inhibitory concentration (MIC), mutant prevention concentration (MPC) and in vitro dynamic model approaches. Difloxacin and orbifloxacin, for which the above information has been scarce, were used. A range of area under curve over a 24h interval (AUC(24h))/MIC ratios and selected E. coli strains were investigated using the dynamic models. Continuous incubation for three days in the presence of difloxacin or orbifloxacin resulted in losses in E. coli susceptibility. An AUC(24h)/MIC (AUC(24h)/MPC)-dependent fluoroquinolone activity and selection of E. coli mutants was confirmed. Maximum losses in susceptibility occurred at AUC(24h)/MIC ratios of 54 (orbifloxacin) and 57.3 (difloxacin). AUC(24h)/MIC ratios of 169.8 (orbifloxacin) and 199.5 (difloxacin) were estimated to be protective against the selection of E. coli mutants, and the corresponding ratios based on AUC(24h)/MPC predictions were 34 (orbifloxacin) and 36.3 (difloxacin). When integrating our in vitro data with pharmacokinetic data in dogs, the conventional clinical doses of both drugs were found to be inadequate to attain the above protective values for 90% of the mutant subpopulation (AUC(24h)/MPC(90)). Both target mutations, esp. at codon 83 (Ser to Leu) of gyrA, and overexpression of efflux pumps contributed to resistance development, with mutants also showing decreased susceptibility to enrofloxacin and marbofloxacin. Additional studies would determine the role of mutations found outside the QRDR, at codon 24 of gyrA, and at codon 116 of parC, and establish the significance of these observations in vivo. PMID:21893387

  6. Statistical Significance Testing.

    ERIC Educational Resources Information Center

    McLean, James E., Ed.; Kaufman, Alan S., Ed.

    1998-01-01

    The controversy about the use or misuse of statistical significance testing has become the major methodological issue in educational research. This special issue contains three articles that explore the controversy, three commentaries on these articles, an overall response, and three rejoinders by the first three authors. They are: (1)…

  7. Lack of Statistical Significance

    ERIC Educational Resources Information Center

    Kehle, Thomas J.; Bray, Melissa A.; Chafouleas, Sandra M.; Kawano, Takuji

    2007-01-01

    Criticism has been leveled against the use of statistical significance testing (SST) in many disciplines. However, the field of school psychology has been largely devoid of critiques of SST. Inspection of the primary journals in school psychology indicated numerous examples of SST with nonrandom samples and/or samples of convenience. In this…

  8. In vitro susceptibility to pyrimethamine of DHFR I164L single mutant Plasmodium falciparum

    PubMed Central

    2011-01-01

    Background Recently, Plasmodium falciparum parasites bearing Pfdhfr I164L single mutation were found in Madagascar. These new mutants may challenge the use of antifolates for the intermittent preventive treatment of malaria during pregnancy (IPTp). Assays with transgenic bacteria suggested that I164L parasites have a wild-type phenotype for pyrimethamine but it had to be confirmed by testing the parasites themselves. Methods Thirty Plasmodium falciparum clinical isolates were collected in 2008 in the south-east of Madagascar. A part of Pfdhfr gene encompassing codons 6 to 206 was amplified by PCR and the determination of the presence of single nucleotide polymorphisms was performed by DNA sequencing. The multiplicity of infection was estimated by using an allelic family-specific nested PCR. Isolates that appeared monoclonal were submitted to culture adaptation. Determination of IC50s to pyrimethamine was performed on adapted isolates. Results Four different Pfdhfr alleles were found: the 164L single mutant-type (N = 13), the wild-type (N = 7), the triple mutant-type 51I/59R/108N (N = 9) and the double mutant-type 108N/164L (N = 1). Eleven out 30 (36.7%) of P. falciparum isolates were considered as monoclonal infection. Among them, five isolates were successfully adapted in culture and tested for pyrimethamine in vitro susceptibility. The wild-type allele was the most susceptible with a 50% inhibitory concentration (IC50) < 10 nM. The geometric mean of IC50 of the three I164L mutant isolates was 6-fold higher than the wild-type with 61.3 nM (SD = 3.2 nM, CI95%: 53.9-69.7 nM). These values remained largely below the IC50 of the triple mutant parasite (13,804 nM). Conclusion The IC50s of the I164L mutant isolates were significantly higher than those of the wild-type (6-fold higher) and close from those usually reported for simple mutants S108N (roughly10-fold higher than wild type). Given the observed values, the determination of IC50s directly on parasites did not

  9. New therapeutic strategies for BRAF mutant colorectal cancers

    PubMed Central

    2015-01-01

    Oncogenic BRAF mutations are found in ~10% of colorectal cancers (CRCs) and predict poor prognosis. Although BRAF inhibitors have demonstrated striking efficacy in BRAF mutant melanomas, BRAF inhibitor monotherapy is ineffective in BRAF mutant CRC. Over the past few years, studies have begun to define the molecular mechanisms underlying the relative resistance of BRAF mutant CRC to BRAF inhibitors, leading to the development of novel therapeutic strategies that are showing promising clinical activity in initial clinical trials. Our current understanding of the mechanisms of BRAF inhibitor resistance in BRAF mutant CRC and the therapeutic approaches currently in clinical trials for BRAF mutant CRC are reviewed herein. PMID:26697198

  10. Mutant Profilin Suppresses Mutant Actin-dependent Mitochondrial Phenotype in Saccharomyces cerevisiae*

    PubMed Central

    Wen, Kuo-Kuang; McKane, Melissa; Stokasimov, Ema; Rubenstein, Peter A.

    2011-01-01

    In the Saccharomyces cerevisiae actin-profilin interface, Ala167 of the actin barbed end W-loop and His372 near the C terminus form a clamp around a profilin segment containing residue Arg81 and Tyr79. Modeling suggests that altering steric packing in this interface regulates actin activity. An actin A167E mutation could increase interface crowding and alter actin regulation, and A167E does cause growth defects and mitochondrial dysfunction. We assessed whether a profilin Y79S mutation with its decreased mass could compensate for actin A167E crowding and rescue the mutant phenotype. Y79S profilin alone caused no growth defect in WT actin cells under standard conditions in rich medium and rescued the mitochondrial phenotype resulting from both the A167E and H372R actin mutations in vivo consistent with our model. Rescue did not result from effects of profilin on actin nucleotide exchange or direct effects of profilin on actin polymerization. Polymerization of A167E actin was less stimulated by formin Bni1 FH1-FH2 fragment than was WT actin. Addition of WT profilin to mixtures of A167E actin and formin fragment significantly altered polymerization kinetics from hyperbolic to a decidedly more sigmoidal behavior. Substitution of Y79S profilin in this system produced A167E behavior nearly identical to that of WT actin. A167E actin caused more dynamic actin cable behavior in vivo than observed with WT actin. Introduction of Y79S restored cable movement to a more normal phenotype. Our studies implicate the importance of the actin-profilin interface for formin-dependent actin and point to the involvement of formin and profilin in the maintenance of mitochondrial integrity and function. PMID:21956104

  11. Bacillus pumilus Cyanide Dihydratase Mutants with Higher Catalytic Activity.

    PubMed

    Crum, Mary A; Sewell, B Trevor; Benedik, Michael J

    2016-01-01

    Cyanide degrading nitrilases are noted for their potential to detoxify industrial wastewater contaminated with cyanide. However, such application would benefit from an improvement to characteristics such as their catalytic activity and stability. Following error-prone PCR for random mutagenesis, several cyanide dihydratase mutants from Bacillus pumilus were isolated based on improved catalysis. Four point mutations, K93R, D172N, A202T, and E327K were characterized and their effects on kinetics, thermostability and pH tolerance were studied. K93R and D172N increased the enzyme's thermostability whereas E327K mutation had a less pronounced effect on stability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 but lowered its k cat. However, the A202T mutation, located in the dimerization or the A surface, destabilized the protein and abolished its activity. No significant effect on activity at alkaline pH was observed for any of the purified mutants. These mutations help confirm the model of CynD and are discussed in the context of the protein-protein interfaces leading to the protein quaternary structure. PMID:27570524

  12. Functionalities of expressed messenger RNAs revealed from mutant phenotypes.

    PubMed

    Liao, Ben-Yang; Weng, Meng-Pin

    2016-07-01

    Total messenger RNAs mRNAs that are produced from a given gene under a certain set of conditions include both functional and nonfunctional transcripts. The high prevalence of nonfunctional mRNAs that have been detected in cells has raised questions regarding the functional implications of mRNA expression patterns and divergences. Phenotypes that result from the mutagenesis of protein-coding genes have provided the most straightforward descriptions of gene functions, and such data obtained from model organisms have facilitated investigations of the functionalities of expressed mRNAs. Mutant phenotype data from mouse tissues have revealed various attributes of functional mRNAs, including tissue-specificity, strength of expression, and evolutionary conservation. In addition, the role that mRNA expression evolution plays in driving morphological evolution has been revealed from studies designed to exploit morphological and physiological phenotypes of mouse mutants. Investigations into yeast essential genes (defined by an absence of colony growth after gene deletion) have further described gene regulatory strategies that reduce protein expression noise by mediating the rates of transcription and translation. In addition to the functional significance of expressed mRNAs as described in the abovementioned findings, the functionalities of other type of RNAs (i.e., noncoding RNAs) remain to be characterized with systematic mutations and phenotyping of the DNA regions that encode these RNA molecules. WIREs RNA 2016, 7:416-427. doi: 10.1002/wrna.1329 For further resources related to this article, please visit the WIREs website. PMID:26748449

  13. Bacillus pumilus Cyanide Dihydratase Mutants with Higher Catalytic Activity

    PubMed Central

    Crum, Mary A.; Sewell, B. Trevor; Benedik, Michael J.

    2016-01-01

    Cyanide degrading nitrilases are noted for their potential to detoxify industrial wastewater contaminated with cyanide. However, such application would benefit from an improvement to characteristics such as their catalytic activity and stability. Following error-prone PCR for random mutagenesis, several cyanide dihydratase mutants from Bacillus pumilus were isolated based on improved catalysis. Four point mutations, K93R, D172N, A202T, and E327K were characterized and their effects on kinetics, thermostability and pH tolerance were studied. K93R and D172N increased the enzyme’s thermostability whereas E327K mutation had a less pronounced effect on stability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 but lowered its kcat. However, the A202T mutation, located in the dimerization or the A surface, destabilized the protein and abolished its activity. No significant effect on activity at alkaline pH was observed for any of the purified mutants. These mutations help confirm the model of CynD and are discussed in the context of the protein–protein interfaces leading to the protein quaternary structure. PMID:27570524

  14. Olesoxime suppresses calpain activation and mutant huntingtin fragmentation in the BACHD rat.

    PubMed

    Clemens, Laura E; Weber, Jonasz J; Wlodkowski, Tanja T; Yu-Taeger, Libo; Michaud, Magali; Calaminus, Carsten; Eckert, Schamim H; Gaca, Janett; Weiss, Andreas; Magg, Janine C D; Jansson, Erik K H; Eckert, Gunter P; Pichler, Bernd J; Bordet, Thierry; Pruss, Rebecca M; Riess, Olaf; Nguyen, Huu P

    2015-12-01

    Huntington's disease is a fatal human neurodegenerative disorder caused by a CAG repeat expansion in the HTT gene, which translates into a mutant huntingtin protein. A key event in the molecular pathogenesis of Huntington's disease is the proteolytic cleavage of mutant huntingtin, leading to the accumulation of toxic protein fragments. Mutant huntingtin cleavage has been linked to the overactivation of proteases due to mitochondrial dysfunction and calcium derangements. Here, we investigated the therapeutic potential of olesoxime, a mitochondria-targeting, neuroprotective compound, in the BACHD rat model of Huntington's disease. BACHD rats were treated with olesoxime via the food for 12 months. In vivo analysis covered motor impairments, cognitive deficits, mood disturbances and brain atrophy. Ex vivo analyses addressed olesoxime's effect on mutant huntingtin aggregation and cleavage, as well as brain mitochondria function. Olesoxime improved cognitive and psychiatric phenotypes, and ameliorated cortical thinning in the BACHD rat. The treatment reduced cerebral mutant huntingtin aggregates and nuclear accumulation. Further analysis revealed a cortex-specific overactivation of calpain in untreated BACHD rats. Treated BACHD rats instead showed significantly reduced levels of mutant huntingtin fragments due to the suppression of calpain-mediated cleavage. In addition, olesoxime reduced the amount of mutant huntingtin fragments associated with mitochondria, restored a respiration deficit, and enhanced the expression of fusion and outer-membrane transport proteins. In conclusion, we discovered the calpain proteolytic system, a key player in Huntington's disease and other neurodegenerative disorders, as a target of olesoxime. Our findings suggest that olesoxime exerts its beneficial effects by improving mitochondrial function, which results in reduced calpain activation. The observed alleviation of behavioural and neuropathological phenotypes encourages further

  15. Characterization and Protective Property of Brucella abortus cydC and looP Mutants

    PubMed Central

    Truong, Quang Lam; Cho, Youngjae; Barate, Abhijit Kashinath; Kim, Suk

    2014-01-01

    Brucella abortus readily multiplies in professional or nonprofessional phagocytes in vitro and is highly virulent in mice. Isogenic mutants of B. abortus biovar 1 strain IVKB9007 lacking the ATP/GDP-binding protein motif A (P-loop) (named looP; designated here the IVKB9007 looP::Tn5 mutant) and the ATP-binding/permease protein (cydC; designated here the IVKB9007 cydC::Tn5 mutant) were identified and characterized by transposon mutagenesis using the mini-Tn5Km2 transposon. Both mutants were found to be virtually incapable of intracellular replication in both murine macrophages (RAW264.7) and the HeLa cell line, and their virulence was significantly impaired in BALB/c mice. Respective complementation of the IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants restored their ability to survive in vitro and in vivo to a level comparable with that of the wild type. These findings indicate that the cydC and looP genes play important roles in the virulence of B. abortus. In addition, intraperitoneal immunization of mice with a dose of the live IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants provided a high degree of protection against challenge with pathogenic B. abortus strain 544. Both mutants should be evaluated further as a live attenuated vaccine against bovine brucellosis for their ability to stimulate a protective immune response. PMID:25253663

  16. A tomato strigolactone-impaired mutant displays aberrant shoot morphology and plant interactions.

    PubMed

    Koltai, Hinanit; LekKala, Sivarama P; Bhattacharya, Chaitali; Mayzlish-Gati, Einav; Resnick, Nathalie; Wininger, Smadar; Dor, Evgenya; Yoneyama, Kaori; Yoneyama, Koichi; Hershenhorn, Joseph; Joel, Daniel M; Kapulnik, Yoram

    2010-06-01

    Strigolactones are considered a new group of plant hormones. Their role as modulators of plant growth and signalling molecules for plant interactions first became evident in Arabidopsis, pea, and rice mutants that were flawed in strigolactone production, release, or perception. The first evidence in tomato (Solanum lycopersicon) of strigolactone deficiency is presented here. Sl-ORT1, previously identified as resistant to the parasitic plant Orobanche, had lower levels of arbuscular mycorrhizal fungus (Glomus intraradices) colonization, possibly as a result of its reduced ability to induce mycorrhizal hyphal branching. Biochemical analysis of mutant root extracts suggested that it produces only minute amounts of two of the tomato strigolactones: solanacol and didehydro-orobanchol. Accordingly, the transcription level of a key enzyme (CCD7) putatively involved in strigolactone synthesis in tomato was reduced in Sl-ORT1 compared with the wild type (WT). Sl-ORT1 shoots exhibited increased lateral shoot branching, whereas exogenous application of the synthetic strigolactone GR24 to the mutant restored the WT phenotype by reducing the number of lateral branches. Reduced lateral shoot branching was also evident in grafted plants which included a WT interstock, which was grafted between the mutant rootstock and the scion. In roots of these grafted plants, the CCD7 transcription level was not significantly induced, nor was mycorrhizal sensitivity restored. Hence, WT-interstock grafting, which restores mutant shoot morphology to WT, does not restore mutant root properties to WT. Characterization of the first tomato strigolactone-deficient mutant supports the putative general role of strigolactones as messengers of suppression of lateral shoot branching in a diversity of plant species. PMID:20194924

  17. The yeast complex I equivalent NADH dehydrogenase rescues pink1 mutants.

    PubMed

    Vilain, Sven; Esposito, Giovanni; Haddad, Dominik; Schaap, Onno; Dobreva, Mariya P; Vos, Melissa; Van Meensel, Stefanie; Morais, Vanessa A; De Strooper, Bart; Verstreken, Patrik

    2012-01-01

    Pink1 is a mitochondrial kinase involved in Parkinson's disease, and loss of Pink1 function affects mitochondrial morphology via a pathway involving Parkin and components of the mitochondrial remodeling machinery. Pink1 loss also affects the enzymatic activity of isolated Complex I of the electron transport chain (ETC); however, the primary defect in pink1 mutants is unclear. We tested the hypothesis that ETC deficiency is upstream of other pink1-associated phenotypes. We expressed Saccaromyces cerevisiae Ndi1p, an enzyme that bypasses ETC Complex I, or sea squirt Ciona intestinalis AOX, an enzyme that bypasses ETC Complex III and IV, in pink1 mutant Drosophila and find that expression of Ndi1p, but not of AOX, rescues pink1-associated defects. Likewise, loss of function of subunits that encode for Complex I-associated proteins displays many of the pink1-associated phenotypes, and these defects are rescued by Ndi1p expression. Conversely, expression of Ndi1p fails to rescue any of the parkin mutant phenotypes. Additionally, unlike pink1 mutants, fly parkin mutants do not show reduced enzymatic activity of Complex I, indicating that Ndi1p acts downstream or parallel to Pink1, but upstream or independent of Parkin. Furthermore, while increasing mitochondrial fission or decreasing mitochondrial fusion rescues mitochondrial morphological defects in pink1 mutants, these manipulations fail to significantly rescue the reduced enzymatic activity of Complex I, indicating that functional defects observed at the level of Complex I enzymatic activity in pink1 mutant mitochondria do not arise from morphological defects. Our data indicate a central role for Complex I dysfunction in pink1-associated defects, and our genetic analyses with heterologous ETC enzymes suggest that Ndi1p-dependent NADH dehydrogenase activity largely acts downstream of, or in parallel to, Pink1 but upstream of Parkin and mitochondrial remodeling. PMID:22242018

  18. Significant Tsunami Events

    NASA Astrophysics Data System (ADS)

    Dunbar, P. K.; Furtney, M.; McLean, S. J.; Sweeney, A. D.

    2014-12-01

    Tsunamis have inflicted death and destruction on the coastlines of the world throughout history. The occurrence of tsunamis and the resulting effects have been collected and studied as far back as the second millennium B.C. The knowledge gained from cataloging and examining these events has led to significant changes in our understanding of tsunamis, tsunami sources, and methods to mitigate the effects of tsunamis. The most significant, not surprisingly, are often the most devastating, such as the 2011 Tohoku, Japan earthquake and tsunami. The goal of this poster is to give a brief overview of the occurrence of tsunamis and then focus specifically on several significant tsunamis. There are various criteria to determine the most significant tsunamis: the number of deaths, amount of damage, maximum runup height, had a major impact on tsunami science or policy, etc. As a result, descriptions will include some of the most costly (2011 Tohoku, Japan), the most deadly (2004 Sumatra, 1883 Krakatau), and the highest runup ever observed (1958 Lituya Bay, Alaska). The discovery of the Cascadia subduction zone as the source of the 1700 Japanese "Orphan" tsunami and a future tsunami threat to the U.S. northwest coast, contributed to the decision to form the U.S. National Tsunami Hazard Mitigation Program. The great Lisbon earthquake of 1755 marked the beginning of the modern era of seismology. Knowledge gained from the 1964 Alaska earthquake and tsunami helped confirm the theory of plate tectonics. The 1946 Alaska, 1952 Kuril Islands, 1960 Chile, 1964 Alaska, and the 2004 Banda Aceh, tsunamis all resulted in warning centers or systems being established.The data descriptions on this poster were extracted from NOAA's National Geophysical Data Center (NGDC) global historical tsunami database. Additional information about these tsunamis, as well as water level data can be found by accessing the NGDC website www.ngdc.noaa.gov/hazard/

  19. Poliovirus: Generation and Characterization of Mutants

    PubMed Central

    Burrill, Cecily P.; Strings, Vanessa R.; Schulte, Michael B.; Andino, Raul

    2016-01-01

    Poliovirus (PV) is the prototypical picornavirus. It is a non-enveloped RNA virus with a small (~7.5 kb) genome of positive polarity. cDNA clones of several strains are available, and infectious virus can be produced by the transfection of in vitro transcribed viral genomes into an appropriate host cell. The ease of genetic studies in poliovirus is a primary reason that it has long served as a model to study RNA virus biology, pathogenesis, and evolution. Protocols for the generation and characterization of PV mutants are presented. A separate unit concerning the production, propagation, quantification, and purification of PV will also be presented. PMID:23686829

  20. An Arabidopsis mutant hypersensitive to red and far-red light signals.

    PubMed Central

    Genoud, T; Millar, A J; Nishizawa, N; Kay, S A; Schäfer, E; Nagatani, A; Chua, N H

    1998-01-01

    A new mutant called psi2 (for phytochrome signaling) was isolated by screening for elevated activity of a chlorophyll a/b binding protein-luciferase (CAB2-LUC) transgene in Arabidopsis. This mutant exhibited hypersensitive induction of CAB1, CAB2, and the small subunit of ribulose-1,5-bisphosphate carboxylase (RBCS) promoters in the very low fluence range of red light and a hypersensitive response in hypocotyl growth in continuous red light of higher fluences. In addition, at high- but not low-light fluence rates, the mutant showed light-dependent superinduction of the pathogen-related protein gene PR-1a and developed spontaneous necrotic lesions in the absence of any pathogen. Expression of genes responding to various hormone and environmental stress pathways in the mutant was not significantly different from that of the wild type. Analysis of double mutants demonstrated that the effects of the psi2 mutation are dependent on both phytochromes phyA and phyB. The mutation is recessive and maps to the bottom of chromosome 5. Together, our results suggest that PSI2 specifically and negatively regulates both phyA and phyB phototransduction pathways. The induction of cell death by deregulated signaling pathways observed in psi2 is reminiscent of retinal degenerative diseases in animals and humans. PMID:9634578

  1. Metabolic phenotypes of Saccharomyces cerevisiae mutants with altered trehalose 6-phosphate dynamics.

    PubMed

    Walther, Thomas; Mtimet, Narjes; Alkim, Ceren; Vax, Amélie; Loret, Marie-Odile; Ullah, Azmat; Gancedo, Carlos; Smits, Gertien J; François, Jean Marie

    2013-09-01

    In Saccharomyces cerevisiae, synthesis of T6P (trehalose 6-phosphate) is essential for growth on most fermentable carbon sources. In the present study, the metabolic response to glucose was analysed in mutants with different capacities to accumulate T6P. A mutant carrying a deletion in the T6P synthase encoding gene, TPS1, which had no measurable T6P, exhibited impaired ethanol production, showed diminished plasma membrane H⁺-ATPase activation, and became rapidly depleted of nearly all adenine nucleotides which were irreversibly converted into inosine. Deletion of the AMP deaminase encoding gene, AMD1, in the tps1 strain prevented inosine formation, but did not rescue energy balance or growth on glucose. Neither the 90%-reduced T6P content observed in a tps1 mutant expressing the Tps1 protein from Yarrowia lipolytica, nor the hyperaccumulation of T6P in the tps2 mutant had significant effects on fermentation rates, growth on fermentable carbon sources or plasma membrane H⁺-ATPase activation. However, intracellular metabolite dynamics and pH homoeostasis were strongly affected by changes in T6P concentrations. Hyperaccumulation of T6P in the tps2 mutant caused an increase in cytosolic pH and strongly reduced growth rates on non-fermentable carbon sources, emphasizing the crucial role of the trehalose pathway in the regulation of respiratory and fermentative metabolism. PMID:23763276

  2. Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation

    PubMed Central

    Kirov, Julia V.; Adkisson, Michael; Nava, A. J.; Cipollone, Andreana; Willis, Brandon; Engelhard, Eric K.; Lloyd, K. C. Kent; de Jong, Pieter; West, David B.

    2015-01-01

    Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown. PMID:26275310

  3. Mutant α-synuclein exacerbates age-related decrease of neurogenesis

    PubMed Central

    Winner, Beate; Rockenstein, Edward; Lie, D. Chichung; Aigner, Robert; Mante, Michael; Bogdahn, Ulrich; Couillard-Despres, Sebastien; Masliah, Eliezer; Winkler, Jürgen

    2010-01-01

    In Parkinson disease, wild-type α-synuclein accumulates during aging, whereas α-synuclein mutations lead to an early onset and accelerated course of the disease. The generation of new neurons is decreased in regions of neurogenesis in adult mice overexpressing wild-type human α-synuclein. We examined the subventricular zone/olfactory bulb neurogenesis in aged mice expressing either wild-type human or A53T mutant α-synuclein. Aging wild-type and mutant α-synuclein-expressing animals generated significantly fewer new neurons than their non-transgenic littermates. This decreased neurogenesis was caused by a reduction in cell proliferation within the subventricular zone of mutant α-synuclein mice. In contrast, no difference was detected in mice overexpressing the wild-type allele. Also, more TUNEL-positive profiles were detected in the subventricular zone, following mutant α-synuclein expression and in the olfactory bulb, following wild-type and mutant α-synuclein expression. The impaired neurogenesis in the olfactory bulb of different transgenic α-synuclein mice during aging highlights the need to further explore the interplay between olfactory dysfunction and neurogenesis in Parkinson disease. PMID:17275140

  4. Adaptation of Chlamydomonas reinhardtii high-CO sub 2 -requiring mutants to limiting CO sub 2

    SciTech Connect

    Suzuki, K.; Spalding, M.H. )

    1989-07-01

    Photosynthetic characteristics of four high-CO{sub 2}-requiring mutants of Chlamydomonas reinhardtii were compared to those of wild type before and after a 24-hour exposure to limiting CO{sub 2} concentrations. The four mutants represent two loci involved in the CO{sub 2}-concentrating system of this unicellular alga. All mutants had a lower photosynthetic affinity for inorganic carbon than did the wild type when grown at an elevated CO{sub 2} concentration, indicating that the genetic lesion in each is expressed even at elevated CO{sub 2} concentrations. Wild type and all four mutants exhibited adaptive responses to limiting CO{sub 2} characteristic of the induction of the CO{sub 2}-concentrating system, resulting in an increased affinity for inorganic carbon only in wild type. Although other components of the CO{sub 2}-concentrating system were induced in these mutants, the defective component in each was sufficient to prevent any increase in the affinity for inorganic carbon. It was concluded that the genes corresponding to the ca-1 and pmp-1 loci exhibit at least partially constitutive expression and that all components of the CO{sub 2}-concentrating system may be required to significantly affect the photosynthetic affinity for inorganic carbon.

  5. Mutants of Salmonella typhimurium with an Altered Leucyl-Transfer Ribonucleic Acid Synthetase

    PubMed Central

    Alexander, Renee R.; Calvo, J. M.; Freundlich, M.

    1971-01-01

    Two trifluoroleucine-resistant mutants of Salmonella typhimurium, strains CV69 and CV117, had an altered leucyl-transfer ribonucleic acid (tRNA) synthetase. The mutant enzymes had higher apparent Km values for leucine (ca. 10-fold) and lower specific activities (ca. twofold) than the parent enzyme when tested in crude extracts. Preparations of synthetase purified ca. 60-fold from the parent and strain CV117 differed sixfold in their leucine Km values. In addition, the mutant enzyme was inactivated faster than the parent enzyme at 50 C. The growth rates of strains CV69 and CV117 at 37 C were not significantly different from that of the parent, whereas at 42 C strain CV69 grew more slowly than the parent. Leucine-, valine-, and isoleucine-forming enzymes were partially derepressed when the mutants were grown in minimal medium; the addition of leucine repressed these enzymes to wild-type levels. During growth in minimal medium, the proportion of leucine tRNA that was charged in the mutants was about 75% of that in the parent. The properties of strain CV117 were shown to result from a single mutation located near gal at minute 18 on the genetic map. These studies suggest that leucyl-tRNA synthetase is involved in repression of the enzymes required for the synthesis of branched-chain amino acids. PMID:4928008

  6. Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation.

    PubMed

    Kirov, Julia V; Adkisson, Michael; Nava, A J; Cipollone, Andreana; Willis, Brandon; Engelhard, Eric K; Lloyd, K C Kent; de Jong, Pieter; West, David B

    2015-01-01

    Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown. PMID:26275310

  7. Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants

    PubMed Central

    Klein, Pontus; Müller-Rischart, Anne Kathrin; Motori, Elisa; Schönbauer, Cornelia; Schnorrer, Frank; Winklhofer, Konstanze F; Klein, Rüdiger

    2014-01-01

    Parkinson's disease (PD)-associated Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking. Glial cell line-derived neurotrophic factor (GDNF) and its signaling receptor Ret are neuroprotective in toxin-based animal models of PD. However, the mechanism by which GDNF/Ret protects cells from degenerating remains unclear. We investigated whether the Drosophila homolog of Ret can rescue Pink1 and park mutant phenotypes. We report that a signaling active version of Ret (RetMEN2B) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants. Interestingly, corresponding phenotypes of park mutants were not rescued, suggesting that the phenotypes of Pink1 and park mutants have partially different origins. In human neuroblastoma cells, GDNF treatment rescues morphological defects of PINK1 knockdown, without inducing mitophagy or Parkin recruitment. GDNF also rescues bioenergetic deficits of PINK knockdown cells. Furthermore, overexpression of RetMEN2B significantly improves electron transport chain complex I function in Pink1 mutant Drosophila. These results provide a novel mechanism underlying Ret-mediated cell protection in a situation relevant for human PD. PMID:24473149

  8. Increasing the Triacylglycerol Content in Dunaliella tertiolecta through Isolation of Starch-Deficient Mutants.

    PubMed

    Sirikhachornkit, Anchalee; Vuttipongchaikij, Supachai; Suttangkakul, Anongpat; Yokthongwattana, Kittisak; Juntawong, Piyada; Pokethitiyook, Prayad; Kangvansaichol, Kunn; Meetam, Metha

    2016-05-28

    The production cost of biodiesel from microalgae is still not competitive, compared with that of petroleum fuels. The genetic improvement of microalgal strains to increase triacylglycerol (TAG) accumulation is one way to reduce production costs. One of the most promising approaches is the isolation of starch-deficient mutants, which have been reported to successfully increase TAG yields. To date, such a stable mutant is not available in an oleaginous marine microalga, despite several advantages of using marine species for biodiesel production. Algae in the genus Dunaliella are known to tolerate high salt concentration and other environmental stresses. In addition, the cultivation processes for large-scale outdoor commercialization have been well established for this genus. In this study, Dunaliella tertiolecta was used to screen for starch-deficient mutants, using an iodine vapor-staining method. Four out of 20,016 UV-mutagenized strains showed a substantial reduction of starch content. A significantly higher TAG content, up to 3-fold of the wild-type level, was observed in three of the mutants upon induction by nitrogen depletion. The carotenoid production and growth characteristics of these mutants, under both normal and oxidative stress conditions, were not compromised, suggesting that these processes are not necessarily affected by starch deficiency. The results from this work open up new possibilities for exploring Dunaliella for biodiesel production. PMID:26869603

  9. Overexpression of a glucokinase point mutant in the treatment of diabetes mellitus

    PubMed Central

    Lu, G; Teng, X; Zheng, Z; Zhang, R; Peng, L; Zheng, F; Liu, J; Huang, H; Xiong, H

    2016-01-01

    Glucokinase (GCK) is an important enzyme critical for glucose metabolism, and has been targeted as such in the pursuit of a cure for diabetes mellitus. We show that streptozotocin (STZ)-induced diabetic murine model exhibits low GCK expression with high blood glucose levels; moreover, aggravated glomerulonephritis is observed in the model when there is IL10 deficiency. Although T cells infiltrate into the liver and pancreas in STZ-induced diabetes mice, T helper 1 (Th1) and T helper 17 (Th17) cells decrease significantly with STZ addition in in vitro polarization. Using a mutant GCK gene (GCK 262) with a knocked out cytosine at position 2643 results in lower protein expression and more ubiquitination-led protein degradation compared with wild-type GCK (GCK 261). We further observed that hsa-mir-1302 can bind to 3′-untranslated region of mutant GCK, which can decrease GCK mRNA translation. Finally, delivery of mutant GCK by subcutaneous injection is more effective at decreasing blood glucose in the STZ-treated (STZ) murine diabetes model than insulin treatment alone. Similarly, mutant GCK consistently and moderately decreases blood glucose levels in GK rats over a period of 12 and 70 days without inducing hypoglycemia, whereas insulin is only effective over 12 h. These results suggest that mutant GCK may be a future cure for diabetes. PMID:26752353

  10. Vaccination with an Attenuated Ferritin Mutant Protects Mice against Virulent Mycobacterium tuberculosis

    PubMed Central

    Subbian, Selvakumar; Pandey, Ruchi; Soteropoulos, Patricia; Rodriguez, G. Marcela

    2015-01-01

    Mycobacterium tuberculosis the causative agent of tuberculosis affects millions of people worldwide. New tools for treatment and prevention of tuberculosis are urgently needed. We previously showed that a ferritin (bfrB) mutant of M. tuberculosis has altered iron homeostasis and increased sensitivity to antibiotics and to microbicidal effectors produced by activated macrophages. Most importantly, M. tuberculosis lacking BfrB is strongly attenuated in mice, especially, during the chronic phase of infection. In this study, we examined whether immunization with a bfrB mutant could confer protection against subsequent infection with virulent M. tuberculosis in a mouse model. The results show that the protection elicited by immunization with the bfrB mutant is comparable to BCG vaccination with respect to reduction of bacterial burden. However, significant distinctions in the disease pathology and host genome-wide lung transcriptome suggest improved containment of Mtb infection in animals vaccinated with the bfrB mutant, compared to BCG. We found that downmodulation of inflammatory response and enhanced fibrosis, compared to BCG vaccination, is associated with the protective response elicited by the bfrB mutant. PMID:26339659

  11. Mutant K-RAS Promotes Invasion and Metastasis in Pancreatic Cancer Through GTPase Signaling Pathways

    PubMed Central

    Padavano, Julianna; Henkhaus, Rebecca S; Chen, Hwudaurw; Skovan, Bethany A; Cui, Haiyan; Ignatenko, Natalia A

    2015-01-01

    Pancreatic ductal adenocarcinoma is one of the most aggressive malignancies, characterized by the local invasion into surrounding tissues and early metastasis to distant organs. Oncogenic mutations of the K-RAS gene occur in more than 90% of human pancreatic cancers. The goal of this study was to investigate the functional significance and downstream effectors of mutant K-RAS oncogene in the pancreatic cancer invasion and metastasis. We applied the homologous recombination technique to stably disrupt K-RAS oncogene in the human pancreatic cell line MiaPaCa-2, which carries the mutant K-RASG12C oncogene in both alleles. Using in vitro assays, we found that clones with disrupted mutant K-RAS gene exhibited low RAS activity, reduced growth rates, increased sensitivity to the apoptosis inducing agents, and suppressed motility and invasiveness. In vivo assays showed that clones with decreased RAS activity had reduced tumor formation ability in mouse xenograft model and increased survival rates in the mouse orthotopic pancreatic cancer model. We further examined molecular pathways downstream of mutant K-RAS and identified RhoA GTP activating protein 5, caveolin-1, and RAS-like small GTPase A (RalA) as key effector molecules, which control mutant K-RAS-dependent migration and invasion in MiaPaCa-2 cells. Our study provides rational for targeting RhoA and RalA GTPase signaling pathways for inhibition of pancreatic cancer metastasis. PMID:26512205

  12. Live four-dimensional optical coherence tomography reveals embryonic cardiac phenotype in mouse mutant

    NASA Astrophysics Data System (ADS)

    Lopez, Andrew L., III; Wang, Shang; Larin, Kirill V.; Overbeek, Paul A.; Larina, Irina V.

    2015-09-01

    Efficient phenotyping of developmental defects in model organisms is critical for understanding the genetic specification of normal development and congenital abnormalities in humans. We previously reported that optical coherence tomography (OCT) combined with live embryo culture is a valuable tool for mouse embryo imaging and four-dimensional (4-D) cardiodynamic analysis; however, its capability for analysis of mouse mutants with cardiac phenotypes has not been previously explored. Here, we report 4-D (three-dimensional+time) OCT imaging and analysis of the embryonic heart in a Wdr19 mouse mutant, revealing a heart looping defect. Quantitative analysis of cardiac looping revealed a statistically significant difference between mutant and control embryos. Our results indicate that live 4-D OCT imaging provides a powerful phenotyping approach to characterize embryonic cardiac function in mouse models.

  13. Yeast dom34 mutants are defective in multiple developmental pathways and exhibit decreased levels of polyribosomes.

    PubMed Central

    Davis, L; Engebrecht, J

    1998-01-01

    The DOM34 gene of Saccharomyces cerevisiae is similar to genes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminisehd. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34delta growth defect. dom34delta mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34delata mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family. PMID:9584085

  14. Mutant IDH1 is required for IDH1 mutated tumor cell growth.

    PubMed

    Jin, Genglin; Pirozzi, Christopher J; Chen, Lee H; Lopez, Giselle Y; Duncan, Christopher G; Feng, Jie; Spasojevic, Ivan; Bigner, Darell D; He, Yiping; Yan, Hai

    2012-08-01

    Frequent somatic hotspot mutations in isocitrate dehydrogenase 1 (IDH1) have been identified in gliomas, acute myeloid leukemias, chondrosarcomas, and other cancers, providing a likely avenue for targeted cancer therapy. However, whether mutant IDH1 protein is required for maintaining IDH1 mutated tumor cell growth remains unknown. Here, using a genetically engineered inducible system, we report that selective suppression of endogenous mutant IDH1 expression in HT1080, a fibrosarcoma cell line with a native IDH1(R132C) heterozygous mutation, significantly inhibits cell proliferation and decreases clonogenic potential. Our findings offer insights into changes that may contribute to the inhibition of cell proliferation and offer a strong preclinical rationale for utilizing mutant IDH1 as a valid therapeutic target. PMID:22885298

  15. Characterisation of in vitro-selected mutants of Ureaplasma parvum resistant to macrolides and related antibiotics.

    PubMed

    Pereyre, S; Métifiot, M; Cazanave, C; Renaudin, H; Charron, A; Bébéar, C; Bébéar, C M

    2007-02-01

    Resistant mutants of Ureaplasma parvum were selected by serial passages of a susceptible strain in subinhibitory concentrations of different macrolides and related antibiotics (erythromycin, azithromycin, josamycin, quinupristin, quinupristin/dalfopristin, pristinamycin and telithromycin). Mechanisms of resistance were characterised by sequencing portions of genes encoding 23S rRNA and ribosomal proteins L4 and L22. Mutants with significantly increased minimum inhibitory concentrations could be selected with all the selector antibiotics, except quinupristin and pristinamycin. Mutants harboured mutations in domain V of the 23S rRNA gene at nucleotides G2056, G2057 or A2058 (Escherichia coli numbering) and in conserved portions of ribosomal proteins L4 and L22. Most of the mutations were associated with complete loss of macrolide and ketolide activity, whereas streptogramin combinations were less affected. PMID:17196370

  16. Yeast dom34 mutants are defective in multiple developmental pathways and exhibit decreased levels of polyribosomes.

    PubMed

    Davis, L; Engebrecht, J

    1998-05-01

    The DOM34 gene of Saccharomyces cerevisiae is similar to genes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminisehd. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34delta growth defect. dom34delta mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34delata mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family. PMID:9584085

  17. Statistical or biological significance?

    PubMed

    Saxon, Emma

    2015-01-01

    Oat plants grown at an agricultural research facility produce higher yields in Field 1 than in Field 2, under well fertilised conditions and with similar weather exposure; all oat plants in both fields are healthy and show no sign of disease. In this study, the authors hypothesised that the soil microbial community might be different in each field, and these differences might explain the difference in oat plant growth. They carried out a metagenomic analysis of the 16 s ribosomal 'signature' sequences from bacteria in 50 randomly located soil samples in each field to determine the composition of the bacterial community. The study identified >1000 species, most of which were present in both fields. The authors identified two plant growth-promoting species that were significantly reduced in soil from Field 2 (Student's t-test P < 0.05), and concluded that these species might have contributed to reduced yield. PMID:26541972

  18. Auxin physiology of the tomato mutant diageotropica

    NASA Technical Reports Server (NTRS)

    Daniel, S. G.; Rayle, D. L.; Cleland, R. E.

    1989-01-01

    The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.

  19. Auxin physiology of the tomato mutant diageotropical

    SciTech Connect

    Daniel, S.G.; Rayle, D.L. ); Cleland, R.E. )

    1989-11-01

    The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.

  20. Mutants of Arabidopsis thaliana with altered phototropism

    NASA Technical Reports Server (NTRS)

    Khurana, J. P.; Poff, K. L.

    1989-01-01

    Thirty five strains of Arabidopsis thaliana (L.) Heynh. have been identified with altered phototropic responses to 450-nm light. Four of these mutants have been more thoroughly characterized. Strain JK224 shows normal gravitropism and "second positive" phototropism. However, while the amplitude for "first positive" phototropism is the same as that in the wild-type, the threshold and fluence for the maximum response in "first positive" phototropism are shifted to higher fluence by a factor of 20-30. This mutant may represent an alteration in the photoreceptor pigment for phototropism. Strain JK218 exhibits no curvature to light at any fluence from 1 micromole m-2 to 2700 micromoles m-2, but shows normal gravitropism. Strain JK345 shows no "first positive" phototropism, and reduced gravitropism and "second positive" phototropism. Strain JK229 shows no measurable "first positive" phototropism, but normal gravitropism and "second positive" phototropism. Based on these data, it is suggested that: 1. gravitropism and phototropism contain at least one common element; 2. "first positive" and "second positive" phototropism contain at least one common element; and 3. "first positive" phototropism can be substantially altered without any apparent alteration of "second positive" phototropism.

  1. Method for rapid isolation of sensitive mutants

    DOEpatents

    Freyer, James P.

    1997-01-01

    Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned.

  2. Method for rapid isolation of sensitive mutants

    DOEpatents

    Freyer, J.P.

    1997-07-29

    Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned. 15 figs.

  3. Isolation of mouse cell proteoglycan mutants

    SciTech Connect

    Keller, K.M.; Keller, J.M.

    1986-05-01

    The sulfated proteoglycans on the surface of cultured mammalian cells have been implicated in a variety of phenomena. To obtain more direct evidence for the role of these molecules in specific cellular functions, they are isolating mutants that produce altered sulfated proteoglycans from a cloned line of Swiss mouse 3T3 cells. This cell type was selected because it exhibits contact inhibition of growth and there is extensive information on its' cell surface and extracellular proteoglycans and other glycoproteins. Cells were chemically mutagenized and subjected to one or more cycles of radiation suicide in the presence of /sup 35/S-sulfate. By replica plating, 150 clones, which appear to incorporate abnormal amounts of /sup 35/S-sulfate, have been selected. After recloning three times via the replica plating technique, the proteoglycans of 29 clones have thus far been analyzed. They have identified four clones which appear to make altered amounts of either cell surface heparan sulfate or chondroitin sulfate. The biochemical bases for the altered levels of the proteoglycans are under study. Of particular interest, however, is the fact that in this limited collection of mutants the chemical alterations correlate with specific altered cellular morphologies.

  4. Too Many Mutants with Multiple Mutations

    PubMed Central

    Drake, John W.

    2007-01-01

    It has recently become clear that the classical notion of the random nature of mutation does not hold for the distribution of mutations among genes: most collections of mutants contain more isolates with two or more mutations than predicted by the mutant frequency on the assumption of a random distribution of mutations. Excesses of multiples are seen in a wide range of organisms, including riboviruses, DNA viruses, prokaryotes, yeasts, and higher eukaryotic cell lines and tissues. In addition, such excesses are produced by DNA polymerases in vitro. These “multiples” appear to be generated by transient, localized hypermutation rather than by heritable mutator mutations. The components of multiples are sometimes scattered at random and sometimes display an excess of smaller distances between mutations. As yet, almost nothing is known about the mechanisms that generate multiples, but such mutations have the capacity to accelerate those evolutionary pathways that require multiple mutations where the individual mutations are neutral or deleterious. Examples that impinge on human health may include carcinogenesis and the adaptation of microbial pathogens as they move between individual hosts. PMID:17687667

  5. Anthropological significance of phenylketonuria.

    PubMed

    Saugstad, L F

    1975-01-01

    The highest incidence rates of phenylketonuria (PKU) have been observed in Ireland and Scotlant. Parents heterozygous for PKU in Norway differ significantly from the general population in the Rhesus, Kell and PGM systems. The parents investigated showed an excess of Rh negative, Kell plus and PGM type 1 individuals, which makes them similar to the present populations in Ireland and Scotlant. It is postulated that the heterozygotes for PKU in Norway are descended from a completely assimilated sub-population of Celtic origin, who came or were brought here, 1ooo years ago. Bronze objects of Western European (Scottish, Irish) origin, found in Viking graves widely distributed in Norway, have been taken as evidence of Vikings returning with loot (including a number of Celts) from Western Viking settlements. The continuity of residence since the Viking age in most habitable parts of Norway, and what seems to be a nearly complete regional relationship between the sites where Viking graves contain western imported objects and the birthplaces of grandparents of PKUs identified in Norway, lend further support to the hypothesis that the heterozygotes for PKU in Norway are descended from a completely assimilated subpopulation. The remarkable resemblance between Iceland and Ireland, in respect of several genetic markers (including the Rhesus, PGM and Kell systems), is considered to be an expression of a similar proportion of people of Celtic origin in each of the two countries. Their identical, high incidence rates of PKU are regarded as further evidence of this. The significant decline in the incidence of PKU when one passes from Ireland, Scotland and Iceland, to Denmark and on to Norway and Sweden, is therefore explained as being related to a reduction in the proportion of inhabitants of Celtic extraction in the respective populations. PMID:803884

  6. Studies on sodium bypass flow in lateral rootless mutants lrt1 and lrt2, and crown rootless mutant crl1 of rice (Oryza sativa L.).

    PubMed

    Faiyue, Bualuang; Vijayalakshmi, Chenniappan; Nawaz, Shafqat; Nagato, Yasuo; Taketa, Shin; Ichii, Masahiko; Al-Azzawi, Mohammed J; Flowers, Timothy J

    2010-05-01

    An apoplastic pathway, the so-called bypass flow, is important for Na+ uptake in rice (Oryza sativa L.) under saline conditions; however, the precise site of entry is not yet known. We report the results of our test of the hypothesis that bypass flow of Na+ in rice occurs at the site where lateral roots emerge from the main roots. We investigated Na+ uptake and bypass flow in lateral rootless mutants (lrt1, lrt2), a crown rootless mutant (crl1), their wild types (Oochikara, Nipponbare and Taichung 65, respectively) and in seedlings of rice cv. IR36. The results showed that shoot Na+ concentration in lrt1, lrt2 and crl1 was lower (by 20-23%) than that of their wild types. In contrast, the bypass flow quantified using trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS) was significantly increased in the mutants, from an average of 1.1% in the wild types to 3.2% in the mutants. Similarly, bypass flow in shoots of IR36 where the number of lateral and crown roots had been reduced through physical and hormonal manipulations was dramatically increased (from 5.6 to 12.5%) as compared to the controls. The results suggest that the path of bypass flow in rice is not at the sites of lateral root emergence. PMID:19930131

  7. Mutagenesis in the lacI gene target of E. coli: improved analysis for lacI(d) and lacO mutants.

    PubMed

    Swerdlow, Sarah J; Schaaper, Roel M

    2014-12-01

    The lacI gene of Escherichia coli has been a highly useful target for studies of mutagenesis, particularly for analysis of the specificity (spectrum) of mutations generated under a variety of conditions and in various genetic backgrounds. The gene encodes the repressor of the lac operon, and lacI-defective mutants displaying constitutive expression of the operon are readily selected. DNA sequencing of the lacI mutants has often been confined to the N-terminal region of the protein, as it presents a conveniently short target with a high density of detectably mutable sites. Mutants in this region are easily selected due to their dominance in a genetic complementation test (lacI(d) mutants). A potential complication in these studies is that constitutive expression of lac may also arise due to mutations in the lac operator (lacO mutants). Under some conditions, for example when analyzing spontaneous mutations, lacO mutants can comprise a very high fraction of the constitutive mutants due to a strong base-substitution hotspot in the lac operator. Such mutational hot spots diminish the return of the sequencing effort and do not yield significant new information. For this reason, a procedure to eliminate the lacO mutants prior to DNA sequencing is desirable. Here, we report a simple method that allows screening out of lacO mutants. This method is based on the lack of resistance of lacO mutants to kanamycin under conditions when the kan gene is expressed from a plasmid under control of the lac promoter-operator (lacPO). We show data validating the new approach with sets of known lacI(d) and lacO mutants, and further apply it to the generation of a new collection of spontaneous mutations, where lacO mutants have historically been a significant contributor. PMID:25771873

  8. 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.

  9. Synaptic transmission deficits in Caenorhabditis elegans synaptobrevin mutants.

    PubMed

    Nonet, M L; Saifee, O; Zhao, H; Rand, J B; Wei, L

    1998-01-01

    Synaptobrevins are vesicle-associated proteins implicated in neurotransmitter release by both biochemical studies and perturbation experiments that use botulinum toxins. To test these models in vivo, we have isolated and characterized the first synaptobrevin mutants in metazoans and show that neurotransmission is severely disrupted in mutant animals. Mutants lacking snb-1 die just after completing embryogenesis. The dying animals retain some capability for movement, although they are extremely uncoordinated and incapable of feeding. We also have isolated and characterized several hypomorphic snb-1 mutants. Although fully viable, these mutants exhibit a variety of behavioral abnormalities that are consistent with a general defect in the efficacy of synaptic transmission. The viable mutants are resistant to the acetylcholinesterase inhibitor aldicarb, indicating that cholinergic transmission is impaired. Extracellular recordings from pharyngeal muscle also demonstrate severe defects in synaptic transmission in the mutants. The molecular lesions in the hypomorphic alleles reside on the hydrophobic face of a proposed amphipathic-helical region implicated biochemically in interacting with the t-SNAREs syntaxin and SNAP-25. Finally, we demonstrate that double mutants lacking both the v-SNAREs synaptotagmin and snb-1 are phenotypically similar to snb-1 mutants and less severe than syntaxin mutants. Our work demonstrates that synaptobrevin is essential for viability and is required for functional synaptic transmission. However, our analysis also suggests that transmitter release is not completely eliminated by removal of either one or both v-SNAREs. PMID:9412487

  10. Mutants of Downy Mildew Resistance in Lactuca Sativa (Lettuce)

    PubMed Central

    Okubara, P. A.; Anderson, P. A.; Ochoa, O. E.; Michelmore, R. W.

    1994-01-01

    As part of our investigation of disease resistance in lettuce, we generated mutants that have lost resistance to Bremia lactucae, the casual fungus of downy mildew. Using a rapid and reliable screen, we identified 16 distinct mutants of Latuca sativa that have lost activity of one of four different downy mildew resistance genes (Dm). In all mutants, only a single Dm specificity was affected. Genetic analysis indicated that the lesions segregated as single, recessive mutations at the Dm loci. Dm3 was inactivated in nine of the mutants. One of five Dm1 mutants was selected from a population of untreated seeds and therefore carried a spontaneous mutation. All other Dm1, Dm3, Dm5/8 and Dm7 mutants were derived from γ- or fast neutron-irradiated seed. In two separate Dm1 mutants and in each of the eight Dm3 mutants analyzed, at least one closely linked molecular marker was absent. Also, high molecular weight genomic DNA fragments that hybridized to a tightly linked molecular marker in wild type were either missing entirely or were truncated in two of the Dm3 mutants, providing additional evidence that deletions had occurred in these mutants. Absence of mutations at loci epistatic to the Dm genes suggested that such loci were either members of multigene families, were critical for plant survival, or encoded components of duplicated pathways for resistance; alternatively, the genes determining downy mildew resistance might be limited to the Dm loci. PMID:8088530

  11. Mutants of Saccharomyces Cerevisiae with Defects in Acetate Metabolism: Isolation and Characterization of Acn(-) Mutants

    PubMed Central

    McCammon, M. T.

    1996-01-01

    The two carbon compounds, ethanol and acetate, can be oxidatively metabolized as well as assimilated into carbohydrate in the yeast Saccharomyces cerevisiae. The distribution of acetate metabolic enzymes among several cellular compartments, mitochondria, peroxisomes, and cytoplasm makes it an intriguing system to study complex metabolic interactions. To investigate the complex process of carbon catabolism and assimilation, mutants unable to grow on acetate were isolated. One hundred five Acn(-) (``ACetate Nonutilizing'') mutants were sorted into 21 complementation groups with an additional 20 single mutants. Five of the groups have defects in TCA cycle enzymes: MDH1, CIT1, ACO1, IDH1, and IDH2. A defect in RTG2, involved in the retrograde communication between the mitochondrion and the nucleus, was also identified. Four genes encode enzymes of the glyoxylate cycle and gluconeogenesis: ICL1, MLS1, MDH2, and PCK1. Five other genes appear to be defective in regulating metabolic activity since elevated levels of enzymes in several metabolic pathways, including the glyoxylate cycle, gluconeogenesis, and acetyl-CoA metabolism, were detected in these mutants: ACN8, ACN9, ACN17, ACN18, and ACN42. In summary, this analysis has identified at least 22 and as many as 41 different genes involved in acetate metabolism. PMID:8878673

  12. Sim2 mutants have developmental defects not overlapping with those of Sim1 mutants.

    PubMed

    Goshu, Eleni; Jin, Hui; Fasnacht, Rachel; Sepenski, Mike; Michaud, Jacques L; Fan, Chen-Ming

    2002-06-01

    The mouse genome contains two Sim genes, Sim1 and Sim2. They are presumed to be important for central nervous system (CNS) development because they are homologous to the Drosophila single-minded (sim) gene, mutations in which cause a complete loss of CNS midline cells. In the mammalian CNS, Sim2 and Sim1 are coexpressed in the paraventricular nucleus (PVN). While Sim1 is essential for the development of the PVN (J. L. Michaud, T. Rosenquist, N. R. May, and C.-M. Fan, Genes Dev. 12:3264-3275, 1998), we report here that Sim2 mutant has a normal PVN. Analyses of the Sim1 and Sim2 compound mutants did not reveal obvious genetic interaction between them in PVN histogenesis. However, Sim2 mutant mice die within 3 days of birth due to lung atelectasis and breathing failure. We attribute the diminished efficacy of lung inflation to the compromised structural components surrounding the pleural cavity, which include rib protrusions, abnormal intercostal muscle attachments, diaphragm hypoplasia, and pleural mesothelium tearing. Although each of these structures is minimally affected, we propose that their combined effects lead to the mechanical failure of lung inflation and death. Sim2 mutants also develop congenital scoliosis, reflected by the unequal sizes of the left and right vertebrae and ribs. The temporal and spatial expression patterns of Sim2 in these skeletal elements suggest that Sim2 regulates their growth and/or integrity. PMID:12024028

  13. Glycosynthase Mutants of Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate Specificity for Antibody Glycosylation Remodeling.

    PubMed

    Li, Tiezheng; Tong, Xin; Yang, Qiang; Giddens, John P; Wang, Lai-Xi

    2016-08-01

    Glycosylation can exert a profound impact on the structures and biological functions of antibodies. Glycosylation remodeling using the endoglycosidase-catalyzed deglycosylation and transglycosylation approach is emerging as a promising platform to produce homogeneous glycoforms of antibodies, but the broad application of this method will require the availability of highly efficient glycosynthase mutants. We describe in this paper a systematic site-directed mutagenesis of an endoglycosidase from Streptococcus pyogenes of serotype M49 (Endo-S2) and the evaluation of the resulting mutants for their hydrolysis and transglycosylation activities. We found that mutations at the Asp-184 residue gave mutants that demonstrated significantly different properties, some possessed potent transglycosylation activity with diminished hydrolysis activity but others did not, which would be otherwise difficult to predict without the comparative study. In contrast to the previously reported Endo-S mutants that are limited to action on complex type N-glycans, the Endo-S2 glycosynthases described here, including D184M and D184Q, were found to have remarkably relaxed substrate specificity and were capable of transferring three major types (complex, high-mannose, and hybrid type) of N-glycans for antibody glycosylation remodeling. In addition, the Endo-S2 glycosynthase mutants were found to be much more active in general than the Endo-S mutants for transglycosylation. The usefulness of these Endo-S2 glycosynthase mutants was exemplified by an efficient glycosylation remodeling of two therapeutic monoclonal antibodies, rituximab and trastuzumab (Herceptin). PMID:27288408

  14. Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase

    SciTech Connect

    Catalanotti, C.; Dubini, A.; Subramanian, V.; Yang, W. Q.; Magneschi, L.; Mus, F.; Seibert, M.; Posewitz, M. C.; Grossman, A. R.

    2012-02-01

    Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.

  15. Combined inhibition of MEK and Plk1 has synergistic anti-tumor activity in NRAS mutant melanoma

    PubMed Central

    Vujic, I; Sanlorenzo, M; Ma, J; Kim, ST; Kleffel, S; Schatton, T; Rappersberger, K; Gutteridge, R; Ahmad, N; Ortiz/Urda, S

    2015-01-01

    About one third of cancers harbor activating mutations in rat sarcoma viral oncogene homolog (RAS) oncogenes. In melanoma, aberrant neuroblastoma-RAS (NRAS) signaling fuels tumor progression in about 20% of patients. Current therapeutics for NRAS driven malignancies barely impact overall survival. To date, pathway interference downstream of mutant NRAS seems to be the most promising approach. In this study, data revealed that mutant NRAS induced Plk1 expression, and pharmacologic inhibition of Plk1 stabilized the size of NRAS mutant melanoma xenografts. The combination of MEK and Plk1 inhibitors resulted in a significant growth reduction of NRAS mutant melanoma cells in vitro, and regression of xenografted NRAS mutant melanoma in vivo. Independent cell cycle arrest and increased induction of apoptosis underlies the synergistic effect of this combination. Data further suggest that the p53 signaling pathway is of key importance to the observed therapeutic efficacy. This study provides in vitro, in vivo and first mechanistic data, that a MEK/Plk1 inhibitor combination might be a promising treatment approach for patients with NRAS driven melanoma. Since mutant NRAS signaling is similar across different malignancies, this inhibitor combination could also offer a previously unreported treatment modality for NRAS mutant tumors of other cell origins. PMID:26016894

  16. Rhodobacter sphaeroides mutants which accumulate 5-aminolevulinic acid under aerobic and dark conditions.

    PubMed

    Nishikawa, S; Watanabe, K; Tanaka, T; Miyachi, N; Hotta, Y; Murooka, Y

    1999-01-01

    The photosynthetic bacterium Rhodobacter sphaeroides accumulates 5-aminolevulinic acid (ALA), which is a precursor in tetrapyrrole biosynthesis, under light illumination and upon addition of levulinic acid as an inhibitor of ALA dehydratase. To generate an industrial strain which produces ALA in the absence of light, we sequentially mutated R. sphaeroides CR-286 using N-methyl-N'-nitro-N-nitrosoguanidine (NTG). The mutant strains were screened by cultivating in the absence of light and assayed for ALA by the Ehrlich reaction in a 96-well microtiter plate. The mutant strain CR-386, derived from R. sphaeroides CR-286, was selected as a mutant that exhibited significant ALA accumulation. While CR-286 required light illumination for ALA production, CR-386 was able to accumulate 1.5 mM ALA in the presence of 50 mM glucose, 60 mM glycine, 15 mM levulinic acid and 1.0% (w/v) yeast extract under conditions of agitation in the absence of light. The mutant strain CR-450, derived from strain CR-386, was selected further as a mutant that exhibited significant ALA accumulation but no accumulation of aminoacetone, analogue of ALA. CR-450 accumulated 3.8 mM ALA under the same conditions. In the presence of 50 mM glucose, 60 mM glycine, 5 mM levulinic acid and 1.0% (w/v) yeast extract, the mutant strain CR-520, derived from strain CR-450, and strain CR-606, derived from strain CR-520, accumulated 8.1 mM and 11.2 mM ALA, respectively. In batch fermentation, the strain CR-606 accumulated 20 mM ALA over 18 h after the addition of glycine, levulinic acid, glucose and yeast extract. PMID:16232557

  17. Differential and limited expression of mutant alleles in multiple myeloma

    PubMed Central

    Rashid, Naim U.; Sperling, Adam S.; Bolli, Niccolo; Wedge, David C.; Van Loo, Peter; Tai, Yu-Tzu; Shammas, Masood A.; Fulciniti, Mariateresa; Samur, Mehmet K.; Richardson, Paul G.; Magrangeas, Florence; Minvielle, Stephane; Futreal, P. Andrew; Anderson, Kenneth C.; Avet-Loiseau, Herve; Parmigiani, Giovanni

    2014-01-01

    Recent work has delineated mutational profiles in multiple myeloma and reported a median of 52 mutations per patient, as well as a set of commonly mutated genes across multiple patients. In this study, we have used deep sequencing of RNA from a subset of these patients to evaluate the proportion of expressed mutations. We find that the majority of previously identified mutations occur within genes with very low or no detectable expression. On average, 27% (range, 11% to 47%) of mutated alleles are found to be expressed, and among mutated genes that are expressed, there often is allele-specific expression where either the mutant or wild-type allele is suppressed. Even in the absence of an overall change in gene expression, the presence of differential allelic expression within malignant cells highlights the important contribution of RNA-sequencing in identifying clinically significant mutational changes relevant to our understanding of myeloma biology and also for therapeutic applications. PMID:25237203

  18. Antiphase Synchronization in a Flagellar-Dominance Mutant of Chlamydomonas

    NASA Astrophysics Data System (ADS)

    Leptos, Kyriacos C.; Wan, Kirsty Y.; Polin, Marco; Tuval, Idan; Pesci, Adriana I.; Goldstein, Raymond E.

    2013-10-01

    Groups of beating flagella or cilia often synchronize so that neighboring filaments have identical frequencies and phases. A prime example is provided by the unicellular biflagellate Chlamydomonas reinhardtii, which typically displays synchronous in-phase beating in a low-Reynolds number version of breaststroke swimming. We report the discovery that ptx1, a flagellar-dominance mutant of C. reinhardtii, can exhibit synchronization in precise antiphase, as in the freestyle swimming stroke. High-speed imaging shows that ptx1 flagella switch stochastically between in-phase and antiphase states, and that the latter has a distinct waveform and significantly higher frequency, both of which are strikingly similar to those found during phase slips that stochastically interrupt in-phase beating of the wild-type. Possible mechanisms underlying these observations are discussed.

  19. Differential and limited expression of mutant alleles in multiple myeloma.

    PubMed

    Rashid, Naim U; Sperling, Adam S; Bolli, Niccolo; Wedge, David C; Van Loo, Peter; Tai, Yu-Tzu; Shammas, Masood A; Fulciniti, Mariateresa; Samur, Mehmet K; Richardson, Paul G; Magrangeas, Florence; Minvielle, Stephane; Futreal, P Andrew; Anderson, Kenneth C; Avet-Loiseau, Herve; Campbell, Peter J; Parmigiani, Giovanni; Munshi, Nikhil C

    2014-11-13

    Recent work has delineated mutational profiles in multiple myeloma and reported a median of 52 mutations per patient, as well as a set of commonly mutated genes across multiple patients. In this study, we have used deep sequencing of RNA from a subset of these patients to evaluate the proportion of expressed mutations. We find that the majority of previously identified mutations occur within genes with very low or no detectable expression. On average, 27% (range, 11% to 47%) of mutated alleles are found to be expressed, and among mutated genes that are expressed, there often is allele-specific expression where either the mutant or wild-type allele is suppressed. Even in the absence of an overall change in gene expression, the presence of differential allelic expression within malignant cells highlights the important contribution of RNA-sequencing in identifying clinically significant mutational changes relevant to our understanding of myeloma biology and also for therapeutic applications. PMID:25237203

  20. Functional Rescue of Retinal Degeneration-Associated Mutant RPE65 Proteins.

    PubMed

    Jin, Minghao; Li, Songhua; Hu, Jane; Jin, Heather H; Jacobson, Samuel G; Bok, Dean

    2016-01-01

    More than 100 different mutations in the RPE65 gene are associated with inherited retinal degeneration. Although some missense mutations have been shown to abolish isomerase activity of RPE65, the molecular bases leading to loss of function and retinal degeneration remain incompletely understood. Here we show that several missense mutations resulted in significant decrease in expression level of RPE65 in the human retinal pigment epithelium cells. The 26S proteasome non-ATPase regulatory subunit 13, a newly identified negative regulator of RPE65, mediated degradation of mutant RPE65s, which were misfolded and formed aggregates in the cells. Many mutations, including L22P, T101I, and L408P, were mapped on nonactive sites of RPE65. Enzyme activities of these mutant RPE65s were significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. 4-phenylbutyrate (PBA) displayed a significant synergistic effect on the low temperature-mediated rescue of the mutant RPE65s. Our results suggest that a low temperature eye mask and PBA, a FDA-approved oral medicine, may provide a promising "protein repair therapy" that can enhance the efficacy of gene therapy for delaying retinal degeneration caused by RPE65 mutations. PMID:26427455

  1. Mutant Amyloid Precursor Protein Differentially Alters Adipose Biology under Obesogenic and Non-Obesogenic Conditions

    PubMed Central

    Freeman, Linnea R.; Zhang, Le; Dasuri, Kalavathi; Fernandez-Kim, Sun-Ok; Bruce-Keller, Annadora J.; Keller, Jeffrey N.

    2012-01-01

    Mutations in amyloid precursor protein (APP) have been most intensely studied in brain tissue for their link to Alzheimer’s disease (AD) pathology. However, APP is highly expressed in a variety of tissues including adipose tissue, where APP is also known to exhibit increased expression in response to obesity. In our current study, we analyzed the effects of mutant APP (E693Q, D694N, K670N/M671L) expression toward multiple aspects of adipose tissue homeostasis. These data reveal significant hypoleptinemia, decreased adiposity, and reduced adipocyte size in response to mutant APP, and this was fully reversed upon high fat diet administration. Additionally, mutant APP was observed to significantly exacerbate insulin resistance, triglyceride elevations, and macrophage infiltration of adipose tissue in response to a high fat diet. Taken together, these data have significant implications for linking mutant APP expression to adipose tissue dysfunction and global changes in endocrine and metabolic function under both obesogenic and non-obesogenic conditions. PMID:22912823

  2. Significant Radionuclides Determination

    SciTech Connect

    Jo A. Ziegler

    2001-07-31

    The purpose of this calculation is to identify radionuclides that are significant to offsite doses from potential preclosure events for spent nuclear fuel (SNF) and high-level radioactive waste expected to be received at the potential Monitored Geologic Repository (MGR). In this calculation, high-level radioactive waste is included in references to DOE SNF. A previous document, ''DOE SNF DBE Offsite Dose Calculations'' (CRWMS M&O 1999b), calculated the source terms and offsite doses for Department of Energy (DOE) and Naval SNF for use in design basis event analyses. This calculation reproduces only DOE SNF work (i.e., no naval SNF work is included in this calculation) created in ''DOE SNF DBE Offsite Dose Calculations'' and expands the calculation to include DOE SNF expected to produce a high dose consequence (even though the quantity of the SNF is expected to be small) and SNF owned by commercial nuclear power producers. The calculation does not address any specific off-normal/DBE event scenarios for receiving, handling, or packaging of SNF. The results of this calculation are developed for comparative analysis to establish the important radionuclides and do not represent the final source terms to be used for license application. This calculation will be used as input to preclosure safety analyses and is performed in accordance with procedure AP-3.12Q, ''Calculations'', and is subject to the requirements of DOE/RW-0333P, ''Quality Assurance Requirements and Description'' (DOE 2000) as determined by the activity evaluation contained in ''Technical Work Plan for: Preclosure Safety Analysis, TWP-MGR-SE-000010'' (CRWMS M&O 2000b) in accordance with procedure AP-2.21Q, ''Quality Determinations and Planning for Scientific, Engineering, and Regulatory Compliance Activities''.

  3. Fungi producing significant mycotoxins.

    PubMed

    2012-01-01

    Mycotoxins are secondary metabolites of microfungi that are known to cause sickness or death in humans or animals. Although many such toxic metabolites are known, it is generally agreed that only a few are significant in causing disease: aflatoxins, fumonisins, ochratoxin A, deoxynivalenol, zearalenone, and ergot alkaloids. These toxins are produced by just a few species from the common genera Aspergillus, Penicillium, Fusarium, and Claviceps. All Aspergillus and Penicillium species either are commensals, growing in crops without obvious signs of pathogenicity, or invade crops after harvest and produce toxins during drying and storage. In contrast, the important Fusarium and Claviceps species infect crops before harvest. The most important Aspergillus species, occurring in warmer climates, are A. flavus and A. parasiticus, which produce aflatoxins in maize, groundnuts, tree nuts, and, less frequently, other commodities. The main ochratoxin A producers, A. ochraceus and A. carbonarius, commonly occur in grapes, dried vine fruits, wine, and coffee. Penicillium verrucosum also produces ochratoxin A but occurs only in cool temperate climates, where it infects small grains. F. verticillioides is ubiquitous in maize, with an endophytic nature, and produces fumonisins, which are generally more prevalent when crops are under drought stress or suffer excessive insect damage. It has recently been shown that Aspergillus niger also produces fumonisins, and several commodities may be affected. F. graminearum, which is the major producer of deoxynivalenol and zearalenone, is pathogenic on maize, wheat, and barley and produces these toxins whenever it infects these grains before harvest. Also included is a short section on Claviceps purpurea, which produces sclerotia among the seeds in grasses, including wheat, barley, and triticale. The main thrust of the chapter contains information on the identification of these fungi and their morphological characteristics, as well as factors

  4. Mutant p53 in cell adhesion and motility.

    PubMed

    Yeudall, W Andrew; Wrighton, Katharine H; Deb, Sumitra

    2013-01-01

    Pro-oncogenic properties of mutant p53 were investigated with the aid of migration assays, adhesion assays, and soft agar growth assays using cells stably expressing gain-of-function p53 mutants. To determine cell migration, "wound-healing" (scratch) assays and haptotactic (chamber) assays were used. H1299 cells expressing mutant p53 were found to migrate more rapidly than cells transfected with empty vector alone. Results from both types of migration assay were broadly similar. Migratory ability differed for different p53 mutants, suggesting allele-specific effects. Cells expressing p53 mutants also showed enhanced adhesion to extracellular matrix compare to controls. Furthermore, stable transfection of mutant p53-H179L into NIH3T3 fibroblasts was sufficient to allow anchorage-independent growth in soft agar. PMID:23150443

  5. Characterization of Pseudomonas aeruginosa mutants with altered piliation.

    PubMed Central

    Johnson, K; Lory, S

    1987-01-01

    The pilus-specific Pseudomonas aeruginosa bacteriophage P04 was used to select spontaneous mutants of strain PAK which have altered piliation. The largest class of phage-resistant mutants synthesized the pilin polypeptide, but did not assemble pili. These mutants are likely to contain mutations in genes required for pilus assembly and not mutations in the pilin structural gene, as they could not be complemented by a normal copy of the pilin gene. In addition, two alterations in pilin gene transcription were found among the mutants--hyperpiliated mutants which overproduce pilin mRNA, and a mutant with temperature-sensitive pilin gene transcription. We also present a model for the regulation of pilin gene transcription by a feedback mechanism sensitive to the relative rates of pilus assembly and disassembly. Images PMID:2445731

  6. Mutant p53: Multiple Mechanisms Define Biologic Activity in Cancer

    PubMed Central

    Kim, Michael Paul; Zhang, Yun; Lozano, Guillermina

    2015-01-01

    The functional importance of p53 as a tumor suppressor gene is evident through its pervasiveness in cancer biology. The p53 gene is the most commonly altered gene in human cancer; however, not all genetic alterations are biologically equivalent. The majority of alterations involve p53 missense mutations that result in the production of mutant p53 proteins. Such mutant p53 proteins lack normal p53 function and may concomitantly gain novel functions, often with deleterious effects. Here, we review characterized mechanisms of mutant p53 gain of function in various model systems. In addition, we review mutant p53 addiction as emerging evidence suggests that tumors may depend on sustained mutant p53 activity for continued growth. We also discuss the role of p53 in stromal elements and their contribution to tumor initiation and progression. Lastly, current genetic mouse models of mutant p53 in various organ systems are reviewed and their limitations discussed. PMID:26618142

  7. Mutants affecting nucleotide recognition by T7 DNA polymerase.

    PubMed

    Donlin, M J; Johnson, K A

    1994-12-13

    Analysis of two mutations affecting nucleotide selection by the DNA polymerase from bacteriophage T7 is reported here. Two conserved residues (Glu480 and Tyr530) in the polymerase active site of an exonuclease deficient (exo-) T7 DNA polymerase were mutated using site-directed mutagenesis (Glu480-Asp and Tyr530-Phe). The kinetic and equilibrium constants governing DNA binding, nucleotide incorporation, and pyrophosphorolysis were measured with the mutants E480D(exo-) and Y530F(exo-) in single-turnover experiments using rapid chemical quench-flow methods. Both mutants have slightly lower Kd values for DNA binding compared to that of wild-type(exo-). With Y530F(exo-) the ground state nucleotide binding affinity was unchanged from wild-type for dGTP and dCTP, was 2-fold lower for dATP and 8-10-fold lower for dTTP binding. With E480D(exo-), the binding constants were 5-6-fold lower for dATP, dGTP, and dCTP and 40-fold lower for dTTP binding compared to those constants for wild-type(exo-). The significance of a specific destabilization of dTTP binding by these amino acids was examined using a dGTP analog, deoxyinosine triphosphate, which mimics the placement and number of hydrogen bonds of an A:T base pair. The Kd for dCTP opposite inosine was unchanged with wild-type(exo-) (197 microM) but higher with Y530F(exo-) (454 microM) and with E480D(exo-) (1 mM). The Kd for dITP was the same with wild-type(exo-) (180 microM) and Y530F(exo-) (229 microM), but significantly higher with E480D(exo-) (3.2 mM). These data support the suggestion that E480 selectively stabilizes dTTP in the wild-type enzyme, perhaps by hydrogen bonding to the unbonded carbonyl. Data on the incorporation of dideoxynucleotide analogs were consistent with the observation of a selective stabilization of dTTP by both residues. Pyrophosphorolysis experiments revealed that neither mutation had a significant effect on the chemistry of polymerization. The fidelity of the mutants were examined in

  8. Sequestosome 1/p62 links familial ALS mutant SOD1 to LC3 via an ubiquitin-independent mechanism

    PubMed Central

    Gal, Jozsef; Strom, Anna-Lena; Kwinter, David M.; Kilty, Renee; Zhang, Jiayu; Shi, Ping; Fu, Weisi; Wooten, Marie W.; Zhu, Haining

    2009-01-01

    The p62/sequestosome 1 protein has been identified as a component of pathological protein inclusions in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). P62 has also been implicated in autophagy, a process of mass degradation of intracellular proteins and organelles. Autophagy is a critical pathway for degrading misfolded and/or damaged proteins, including the copper-zinc superoxide dismutase (SOD1) mutants linked to familial ALS. We previously reported that p62 interacted with ALS mutants of SOD1 and that the ubiquitin-association (UBA) domain of p62 was dispensable for the interaction. In this study, we identified two distinct regions of p62 that were essential to its binding to mutant SOD1: the N-terminal PB1 domain (residues 1-104) and a separate internal region (residues 178–224) termed here as SOD1 mutant interaction region (SMIR). The PB1 domain is required for appropriate oligomeric status of p62 and the SMIR is the actual region interacting with mutant SOD1. Within the SMIR, the conserved W184, H190 and positively charged R183, R186, K187 and K189 residues are critical to the p62-mutant SOD1 interaction since substitution of these residues with alanine resulted in significantly abolished binding. In addition, SMIR and the p62 sequence responsible for the interaction with LC3, a protein essential for autophagy activation, are independent of each other. In cells lacking p62, the existence of mutant SOD1 in acidic autolysosomes decreased, suggesting that p62 can function as an adaptor between mutant SOD1 and the autophagy machinery. This study provides a novel molecular mechanism by which mutant SOD1 can be recognized by p62 in an ubiquitin-independent fashion and targeted for the autophagy-lysosome degradation pathway. PMID:19765191

  9. Grain Development Mutants of Barley ([alpha]-Amylase Production during Grain Maturation and Its Relation to Endogenous Gibberellic Acid Content).

    PubMed Central

    Green, L. S.; Faergestad, E. M.; Poole, A.; Chandler, P. M.

    1997-01-01

    Barley (Hordeum vulgare L. Himalaya) mutants with altered grain morphology were isolated to investigate whether defects in grain development, possibly involving gibberellins (GAs) and abscisic acid, would lead to altered patterns of [alpha]-amylase gene expression. Following treatment with sodium azide, 75 mutants, typically showing grain shriveling, were identified. At grain maturity 15 of the 75 mutants had higher [alpha]-amylase activities in shriveled grains compared with either phenotypically normal grains that developed on the same heterozygous plant or with grains of cv Himalaya. Studies of four of these mutants demonstrated increased levels of both high- and low-isoelectric point [alpha]-amylase isozymes midway through grain development. This category of mutant has been designated pga, for premature grain [alpha]-amylase. One such mutant (M326) showed an endosperm-determined inheritance pattern. When crossed into a (GA-deficient) dwarfing background there was a 10- to 20-fold reduction in [alpha]-amylase activity, suggesting a requirement for GA biosynthesis. Endogenous GAs and abscisic acid were quantified by combined gas chromatography-specific ion monitoring in normal and mutant grains of heterozygous M326 plants during the period of [alpha]-amylase accumulation. Mutant grains had significantly higher (5.8-fold) levels of the bioactive GA1 compared with normal grains but much lower (approximately 10-fold) levels of the 2[beta]-hydroxylated ("inactive") GAs, typical of developing barley grains (e.g. GA8, GA34, GA48). We propose that a reduced extent of 2[beta]-hydroxylation in the mutant grains results in an increased level of GA1, which is responsible for premature [alpha]-amylase gene expression. PMID:12223700

  10. Optimized cell transplantation using adult rag2 mutant zebrafish

    PubMed Central

    Tang, Qin; Abdelfattah, Nouran S.; Blackburn, Jessica S.; Moore, John C.; Martinez, Sarah A.; Moore, Finola E.; Lobbardi, Riadh; Tenente, Inês M.; Ignatius, Myron S.; Berman, Jason N.; Liwski, Robert S.; Houvras, Yariv; Langenau, David M.

    2014-01-01

    Cell transplantation into adult zebrafish has lagged behind mouse due to the lack of immune compromised models. Here, we have created homozygous rag2E450fs mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft zebrafish muscle, blood stem cells, and cancers. rag2E450fs mutant zebrafish are the first immune compromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer. PMID:25042784

  11. Isolation and characterization of Klebsiella pneumoniae unencapsulated mutants

    SciTech Connect

    Benedi, V.J.; Ciurana, B.; Tomas, J.M.

    1989-01-01

    Klebsiella pneumoniae mutants were obtained after UV irradiation and negative selection with anticapsular serum. Unencapsulation, rather than expression of a structurally altered capsule, was found in the mutants. The mutant strains showed no alterations in their outer membrane proteins and lipopolysaccharide, and a great similarity with the wild type in the properties tested (serum resistance, antimicrobial sensitivity, and lipopolysaccharide-specific bacteriophage sensitivity), with the exception of a higher cell surface hydrophobicity and resistance to bacteriophage FC3-9.

  12. Modulation of cellular and viral promoters by mutant human p53 proteins found in tumor cells.

    PubMed Central

    Deb, S; Jackson, C T; Subler, M A; Martin, D W

    1992-01-01

    Wild-type p53 has recently been shown to repress transcription from several cellular and viral promoters. Since p53 mutations are the most frequently reported genetic defects in human cancers, it becomes important to study the effects of mutations of p53 on promoter functions. We, therefore, have studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen (PCNA) promoter and on several viral promoters, including the herpes simplex virus type 1 UL9 promoter, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and a plasmid containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. As expected, expression of the wild-type p53 inhibited promoter function. Expression of a p53 with a mutation at any one of the four amino acid positions 175, 248, 273, or 281, however, correlated with a significant increase of the PCNA promoter activity (2- to 11-fold). The viral promoters were also activated, although to a somewhat lesser extent. We also showed that activation by a mutant p53 requires a minimal promoter containing a lone TATA box. A more significant increase (25-fold) in activation occurs when the promoter contains a binding site for the activating transcription factor or cyclic AMP response element-binding protein. Using Saos-2 cells that do not express p53, we showed that activation by a mutant p53 was a direct enhancement. The mutant forms of p53 used in this study are found in various cancer cells. The activation of PCNA by mutant p53s may indicate a way to increase cell proliferation by the mutant p53s. Thus, our data indicate a possible functional role for the mutants of p53 found in cancer cells in activating several important loci, including PCNA. Images PMID:1356162

  13. Genomic analysis of cichlid fish 'natural mutants'.

    PubMed

    Kuraku, Shigehiro; Meyer, Axel

    2008-12-01

    In the lakes of East Africa, cichlid fishes have formed adaptive radiations that are each composed of hundreds of endemic, morphologically stunningly diverse, but genetically extremely similar species. In the past 20 years, it became clear that their extreme phenotypic diversity arose within very short time spans, and that phenotypically radically different species are exceptionally similar genetically; hence, they could be considered to be 'natural mutants'. Many species can be hybridized and, therefore, provide a unique opportunity to study the genetic underpinnings of phenotypic diversification. Comparative large-scale genomic analyses are beginning to unravel the patterns and processes that led to the formation of the cichlid species flocks. Cichlids are an emerging evolutionary genomic model system for fundamental questions on the origin of phenotypic diversity. PMID:19095433

  14. Computational approaches for predicting mutant protein stability.

    PubMed

    Kulshreshtha, Shweta; Chaudhary, Vigi; Goswami, Girish K; Mathur, Nidhi

    2016-05-01

    Mutations in the protein affect not only the structure of protein, but also its function and stability. Prediction of mutant protein stability with accuracy is desired for uncovering the molecular aspects of diseases and design of novel proteins. Many advanced computational approaches have been developed over the years, to predict the stability and function of a mutated protein. These approaches based on structure, sequence features and combined features (both structure and sequence features) provide reasonably accurate estimation of the impact of amino acid substitution on stability and function of protein. Recently, consensus tools have been developed by incorporating many tools together, which provide single window results for comparison purpose. In this review, a useful guide for the selection of tools that can be employed in predicting mutated proteins' stability and disease causing capability is provided. PMID:27160393

  15. Stability Analysis of a High Fibre Yield and Low Lignin Content “Thick Stem” Mutant in Tossa Jute (Corchorus olitorius L.)

    PubMed Central

    Mandal, Aninda; Datta, Animesh K.

    2014-01-01

    A “thick stem” mutant of Corchorus olitorius L. was induced at M2 (0.50%, 4 h, EMS) and the true breeding mutant is assessed across generations (M5 to M7) considering morphometric traits as well as SEM analysis of pollen grains and raw jute fibres, stem anatomy, cytogenetical attributes, and lignin content in relation to control. Furthermore, single fibre diameter and tensile strength are also analysed. The objective is to assess the stability of mutant for its effective exploration for raising a new plant type in tossa jute for commercial exploitation and efficient breeding. The mutant trait is monogenic recessive to normal. Results indicate that “thick stem” mutant is stable across generations (2n = 14) with distinctive high seed and fibre yield and significantly low lignin content. Stem anatomy of the mutant shows significant enhancement in fibre zone, number of fibre pyramids and fibre bundles per pyramid, and diameter of fibre cell in relation to control. Moreover, tensile strength of mutant fibre is significantly higher than control fibre and the trait is inversely related to fibre diameter. However the mutant is associated with low germination frequency, poor seed viability, and high pollen sterility, which may be eliminated through mutational approach followed by rigorous selection and efficient breeding. PMID:24860822

  16. Sphingolipid synthesis deficiency in a mutant of Bacteroides levii

    SciTech Connect

    Brumleve, B.; Lev, M.

    1986-05-01

    Bacteroides levii, an anaerobic bacterium, synthesizes two sphingolipids; the sphingomyelin analogue, ceramide phosphorylethanolamine (CPE), and also ceramide phosphorylglycerol (CPG). The first enzyme in the sphingolipid pathway, 3-ketodihydro-sphingosine (3KDS) synthase, has been partially purified previously. To study subsequent steps in the pathways, mutants defective in sphingolipid synthesis were derived by ethyl methanesulfonate and nitrosoguanidine mutagenesis. Extracts of the mutant, 1075BB, show synthase activity although the cells do not synthesize CPE or CPG. The mutant differs from the wild type in that: (1) synthase activity was much diminished in the mutant, (2) sphingolipid synthesis does not occur in the mutant as evidenced by the absence of spots at sites where CPE and CPG migrate following two-dimensional thin layer chromatography, (3) incorporation of uniformly-labelled (/sup 14/C)serine carbon or (/sup 14/C)3KDS into sphingolipids was not observed in the mutant, (4) following incubation with (/sup 14/C)3KDS, radioactivity corresponding to dihydrosphingosine (DHS) and ceramide were observed in the mutant; no (/sup 14/C)DHS was detected in the wild type, and (5) enhanced incorporation of (/sup 14/C)serine carbon into two lipids not containing phosphorus was found in the mutant. The authors conclude, therefore, that this mutant, 1075BB, has a metabolic block at the terminal biosynthetic steps of sphingolipid synthesis.

  17. Growth and development of maize that contains mutant tubulin genes

    SciTech Connect

    Susan M. Wick

    2004-07-23

    Mutant maize plants containing a Mu transposon disrupting one of the five beta tubulin genes of interest were followed for several generations and hybridized with each other to produce plants containing disruptions in both copies of a single gene or disruption of more than one tubulin gene. Seedlings of some of these plants were grown under chilling conditions for a few weeks. After DOE funding ended, plants have been assessed to see whether mutant are more or less tolerant to chilling. Other mutant plants will be assessed for their male and female fertility relative to non-mutant siblings or other close relatives.

  18. Identification of mutant monoclonal antibodies with increased antigen binding.

    PubMed Central

    Pollock, R R; French, D L; Gefter, M L; Scharff, M D

    1988-01-01

    Sib selection and an ELISA have been used to isolate hybridoma subclones producing mutant antibodies that bind antigen better than the parental monoclonal antibody. Such mutants arise spontaneously in culture at frequencies of 2.5-5 X 10(-5). The sequences of the heavy and light chain variable regions of the mutant antibodies are identical to that of the parent and the Ka values of the mutants and the parent are the same. The increase in binding is associated with abnormalities of the constant region polypeptide and probably reflect changes in avidity of these antibodies. Images PMID:3267219

  19. Analysis of canthaxanthin and related pigments from Gordonia jacobaea mutants.

    PubMed

    de Miguel, T; Sieiro, C; Poza, M; Villa, T G

    2001-03-01

    A collection of 43 mutant strains of the bacterium Gordonia jacobaea was obtained by means of ethyl methanesulfonate treatment, and the strains were selected for their different pigmentation with respect to the wild-type strain. None of the mutants showed auxotrophy. They all showed good genetic stability and a growth rate similar to that of the parental strain. Canthaxanthin and other carotenoids from these mutants were extracted with acetone and ethanol and separated by high-performance liquid chromatography (HPLC). These HPLC analyses, together with spectrophotometric detection at 480 nm, revealed variations in the pigment contents of the different mutant strains. PMID:11312835

  20. The utilization of fructose by Escherichia coli. Properties of a mutant defective in fructose 1-phosphate kinase activity.

    PubMed

    Ferenci, T; Kornberg, H L

    1973-02-01

    1. The isolation and properties of a mutant of Escherichia coli devoid of fructose 1-phosphate kinase activity are described. 2. This mutant grew in media containing any one of a variety of substances, including hexoses, hexose 6-phosphates, sugar acids and glucogenic substrates, at rates not significantly different from those at which the parent organism grew on these substrates. However, only the parent grew on fructose or fructose 1-phosphate. 3. Fructose and fructose 1-phosphate inhibit the growth of the mutant, but not of its parent, on other carbon sources. 4. Even though not previously exposed to fructose, the mutant took up [(14)C]fructose rapidly but to only a small extent: [(14)C]fructose 1-phosphate was identified as the predominant labelled product. In contrast, the equally rapid but more extensive uptake of [(14)C]fructose by the parent organism required prior growth in the presence of fructose. PMID:4579702

  1. Suppression of KRas-mutant cancer through the combined inhibition of KRAS with PLK1 and ROCK

    PubMed Central

    Wang, Jieqiong; Hu, Kewen; Guo, Jiawei; Cheng, Feixiong; Lv, Jing; Jiang, Wenhao; Lu, Weiqiang; Liu, Jinsong; Pang, Xiufeng; Liu, Mingyao

    2016-01-01

    No effective targeted therapies exist for cancers with somatic KRAS mutations. Here we develop a synthetic lethal chemical screen in isogenic KRAS-mutant and wild-type cells to identify clinical drug pairs. Our results show that dual inhibition of polo-like kinase 1 and RhoA/Rho kinase (ROCK) leads to the synergistic effects in KRAS-mutant cancers. Microarray analysis reveals that this combinatory inhibition significantly increases transcription and activity of cyclin-dependent kinase inhibitor p21WAF1/CIP1, leading to specific G2/M phase blockade in KRAS-mutant cells. Overexpression of p21WAF1/CIP1, either by cDNA transfection or clinical drugs, preferentially impairs the growth of KRAS-mutant cells, suggesting a druggable synthetic lethal interaction between KRAS and p21WAF1/CIP1. Co-administration of BI-2536 and fasudil either in the LSL-KRASG12D mouse model or in a patient tumour explant mouse model of KRAS-mutant lung cancer suppresses tumour growth and significantly prolongs mouse survival, suggesting a strong synergy in vivo and a potential avenue for therapeutic treatment of KRAS-mutant cancers. PMID:27193833

  2. Altered chloroplast structure and function in a mutant of Arabidopsis deficient in plastid glycerol-3-phosphate acyltransferase activity

    SciTech Connect

    Kunst, L.; Somerville, C. ); Browse, J. )

    1989-07-01

    Mutants of Arabidopsis thaliana deficient in plastid glycerol-3-phosphate acyltransferase activity have altered chloroplast membrane lipid composition. This caused an increase in the number of regions of appressed membrane per chloroplast and a decrease in the average number of thylakoid membranes in the appressed regions. The net effect was a significant decrease in the ratio of appressed to nonappressed membranes. A comparison of 77 K fluorescence emission spectra of thylakoid membranes from the mutant and wild type indicated that the ultrastructural changes were associated with an altered distribution of excitation energy transfer from antenna chlorophyll to photosystem II and photosystem I in the mutant. The changes in leaf lipid composition did not significantly affect growth or development of the mutant under standard conditions. However, at temperatures above 28{degree}C the mutant grew slightly more rapidly than the wild type, and measurements of temperature-induced fluorescence yield enhancement suggested an increased thermal stability of the photosynthetic apparatus of the mutant. These effects are consistent with other evidence suggesting that membrane lipid composition is an important determinant of chloroplast structure but has relatively minor direct effects on the function of the membrane proteins associated with photosynthetic electron transport.

  3. Symbiotic effectiveness of antibiotic-resistant mutants of fast- and slow-growing strains of Rhizobium nodulating Lotus species.

    PubMed

    Pankhurst, C E

    1977-08-01

    Mutants resistant ot 16 individual antibiotics were isolated from two fast-growing and two slow-growing strains of Lotus rhizobia and their symbiotic effectiveness on Lotus pedunculatus evaluated. Resistance to streptomycin, spectinomycin, chloramphenicol, and tetracycline (inhibitors of protein synthesis) was associated with little or no loss of effectiveness with all four strains but resistance to nalidixic acid and rifampicin (inhibitors of nucleic acid synthesis), and to D-cycloserine, novobiocin, and penicillin (inhibitors of cell wall-cell membrane synthesis) was associated with significant loss of effectiveness in 20-100% of the mutants. Resistance to viomycin, neomycin, kanamycin, and vibramycin was associated with loss of effectiveness with mutants of the two fast-growing strains but not with mutants of the two slow-growing strains. When tested on four alternate host legumes individual mutants of a slow-growing strain showed significantly different levels of effectiveness. The results suggest that both the inherent characteristics of the bacterium and of the host plant will influence the symbiotic effectiveness of antibiotic-resistant mutants of Rhizobium. PMID:890601

  4. Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation

    SciTech Connect

    Marti, T.; Otto, H.; Mogi, T.; Roesselet, S.J.H.; Heyn, M.P.; Khorana, H.G. )

    1991-04-15

    To study their role in proton translocation by bacteriorhodopsin, 22 serine and threonine residues presumed to be located within and near the border of the transmembrane segments have been individually replaced by alanine or valine, respectively. Thr-89 was substituted by alanine, valine, and aspartic acid, and Ser-141 by alanine and cysteine. Most of the mutants showed essentially wild-type phenotype with regard to chromophore regeneration and absorption spectrum. However, replacement of Thr-89 by Val and of Ser-141 by Cys caused striking blue shifts of the chromophore by 100 and 80 nm, respectively. All substitutions of Thr-89 regenerated the chromophore at least 10-fold faster with 13-cis retinal than with all-trans retinal. The substitutions at positions 89, 90, and 141 also showed abnormal dark-light adaptation, suggesting interactions between these residues and the retinylidene chromophore. Proton pumping measurements revealed 60-75% activity for mutants of Thr-46, -89, -90, -205, and Ser-226, and about 20% for Ser-141----Cys, whereas the remaining mutants showed normal pumping. Kinetic studies of the photocycle and of proton release and uptake for mutants in which proton pumping was reduced revealed generally little alterations. The reduced activity in several of these mutants is most likely due to a lower percentage of all-trans retinal in the light-adapted state. In the mutants Thr-46----Val and Ser-226----Ala the decay of the photointer-mediate M was significantly accelerated, indicating an interaction between these residues and Asp-96 which reprotonates the Schiff base. Our results show that no single serine or threonine residue is obligatory for proton pumping.

  5. myosin 7aa(-/-) mutant zebrafish show mild photoreceptor degeneration and reduced electroretinographic responses.

    PubMed

    Wasfy, Meagan M; Matsui, Jonathan I; Miller, Jessica; Dowling, John E; Perkins, Brian D

    2014-05-01

    Mutations in myosin VIIa (MYO7A) cause Usher Syndrome 1B (USH1B), a disease characterized by the combination of sensorineural hearing loss and visual impairment termed retinitis pigmentosa (RP). Although the shaker-1 mouse model of USH1B exists, only minor defects in the retina have been observed during its lifespan. Previous studies of the zebrafish mariner mutant, which also carries a mutation in myo7aa, revealed balance and hearing defects in the mutants but the retinal phenotype has not been described. We found elevated cell death in the outer nuclear layer (ONL) of myo7aa(-/-) mutants. While myo7aa(-/-) mutants retained visual behaviors in the optokinetic reflex (OKR) assay, electroretinogram (ERG) recordings revealed a significant decrease in both a- and b-wave amplitudes in mutant animals, but not a change in ERG threshold sensitivity. Immunohistochemistry showed mislocalization of rod and blue cone opsins and reduced expression of rod-specific markers in the myo7aa(-/-) ONL, providing further evidence that the photoreceptor degeneration observed represents the initial stages of the RP. Further, constant light exposure resulted in widespread photoreceptor degeneration and the appearance of large holes in the retinal pigment epithelium (RPE). No differences were observed in the retinomotor movements of the photoreceptors or in melanosome migration within the RPE, suggesting that myo7aa(-/-) does not function in these processes in teleosts. These results indicate that the zebrafish myo7aa(-/-) mutant is a useful animal model for the RP seen in humans with USH1B. PMID:24698764

  6. myosin 7aa−/− mutant zebrafish show mild photoreceptor degeneration and reduced electroretinographic responses

    PubMed Central

    Wasfy, Meagan M.; Matsui, Jonathan I.; Miller, Jessica; Dowling, John E.; Perkins, Brian D.

    2014-01-01

    Mutations in myosin VIIa (MYO7A) cause Usher syndrome 1B (USH1B), a disease characterized by the combination of sensorineural hearing loss and visual impairment termed retinitis pigmentosa (RP). Although the shaker-1 mouse model of USH1B exists, only minor defects in the retina have been observed during its lifespan. Previous studies of the zebrafish mariner mutant, which also carries a mutation in myo7aa, revealed balance and hearing defects in the mutants but the retinal phenotype has not been described. We found elevated cell death in the outer nuclear layer (ONL) of myo7aa−/− mutants. While myo7aa−/− mutants retained visual behaviors in the optokinetic reflex (OKR) assay, electroretinogram (ERG) recordings revealed a significant decrease in both a- and b-wave amplitudes in mutant animals, but not a change in ERG threshold sensitivity. Immunohistochemistry showed mislocalization of rod and blue cone opsins and reduced expression of rod-specific markers in the myo7aa−/− ONL, providing further evidence that the photoreceptor degeneration observed represents the initial stages of the RP. Further, constant light exposure resulted in widespread photoreceptor degeneration and the appearance of large holes in the retinal pigment epithelium (RPE). No differences were observed in the retinomotor movements of the photoreceptors or in melanosome migration within the RPE, suggesting that myo7aa−/− does not function in these processes in teleosts. These results indicate that the zebrafish myo7aa−/− mutant is a useful animal model for the RP seen in humans with USH1B. PMID:24698764

  7. Erwinia amylovora pyrC mutant causes fire blight despite pyrimidine auxotrophy.

    PubMed

    Ramos, L S; Sinn, J P; Lehman, B L; Pfeufer, E E; Peter, K A; McNellis, T W

    2015-06-01

    Erwinia amylovora bacteria cause fire blight disease, which affects apple and pear production worldwide. The Erw. amylovora pyrC gene encodes a predicted dihydroorotase enzyme involved in pyrimidine biosynthesis. Here, we discovered that the Erw. amylovora pyrC244::Tn5 mutant was a uracil auxotroph. Unexpectedly, the Erw. amylovora pyrC244::Tn5 mutant grew as well as the wild-type in detached immature apple and pear fruits. Fire blight symptoms caused by the pyrC244::Tn5 mutant in immature apple and pear fruits were attenuated compared to those caused by the wild-type. The pyrC244::Tn5 mutant also caused severe fire blight symptoms in apple tree shoots. A plasmid-borne copy of the wild-type pyrC gene restored prototrophy and symptom induction in apple and pear fruit to the pyrC244::Tn5 mutant. These results suggest that Erw. amylovora can obtain sufficient pyrimidine from the host to support bacterial growth and fire blight disease development, although de novo pyrimidine synthesis by Erw. amylovora is required for full symptom development in fruits. Significance and impact of the study: This study provides information about the fire blight host-pathogen interaction. Although the Erwinia amylovora pyrC mutant was strictly auxotrophic for pyrimidine, it grew as well as the wild-type in immature pear and apple fruits and caused severe fire blight disease in apple trees. This suggests that Erw. amylovora can obtain sufficient pyrimidines from host tissue to support growth and fire blight disease development. This situation contrasts with findings in some human bacterial pathogens, which require de novo pyrimidine synthesis for growth in host blood, for example. PMID:25789570

  8. Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms

    PubMed Central

    Pietra, D; Rumi, E; Ferretti, V V; Buduo, C A Di; Milanesi, C; Cavalloni, C; Sant'Antonio, E; Abbonante, V; Moccia, F; Casetti, I C; Bellini, M; Renna, M C; Roncoroni, E; Fugazza, E; Astori, C; Boveri, E; Rosti, V; Barosi, G; Balduini, A; Cazzola, M

    2016-01-01

    A quarter of patients with essential thrombocythemia or primary myelofibrosis carry a driver mutation of CALR, the calreticulin gene. A 52-bp deletion (type 1) and a 5-bp insertion (type 2 mutation) are the most frequent variants. These indels might differentially impair the calcium binding activity of mutant calreticulin. We studied the relationship between mutation subtype and biological/clinical features of the disease. Thirty-two different types of CALR variants were identified in 311 patients. Based on their predicted effect on calreticulin C-terminal, mutations were classified as: (i) type 1-like (65%); (ii) type 2-like (32%); and (iii) other types (3%). Corresponding CALR mutants had significantly different estimated isoelectric points. Patients with type 1 mutation, but not those with type 2, showed abnormal cytosolic calcium signals in cultured megakaryocytes. Type 1-like mutations were mainly associated with a myelofibrosis phenotype and a significantly higher risk of myelofibrotic transformation in essential thrombocythemia. Type 2-like CALR mutations were preferentially associated with an essential thrombocythemia phenotype, low risk of thrombosis despite very-high platelet counts and indolent clinical course. Thus, mutation subtype contributes to determining clinical phenotype and outcomes in CALR-mutant myeloproliferative neoplasms. CALR variants that markedly impair the calcium binding activity of mutant calreticulin are mainly associated with a myelofibrosis phenotype. PMID:26449662

  9. Excretion of ammonium by a nifL mutant of Azotobacter vinelandii fixing nitrogen.

    PubMed Central

    Bali, A; Blanco, G; Hill, S; Kennedy, C

    1992-01-01

    A mutation in the gene upstream of nifA in Azotobacter vinelandii was introduced into the chromosome to replace the corresponding wild-type region. The resulting mutant, MV376, produced nitrogenase constitutively in the presence of 15 mM ammonium. When introduced into a nifH-lacZ fusion strain, the mutation permitted beta-galactosidase production in the presence of ammonium. The gene upstream of nifA is therefore designated nifL because of its similarity to the Klebsiella pneumoniae nifL gene in proximity to nifA, in mutant phenotype, and in amino acid sequence of the gene product. The A. vinelandii nifL mutant MV376 excreted significant quantities of ammonium (approximately 10 mM) during diazotrophic growth. In contrast, ammonium excretion during diazotrophy was much lower in a K. pneumoniae nifL deletion mutant (maximum, 0.15 mM) but significantly higher than in NifL+ K. pneumoniae. The expression of the A. vinelandii nifA gene, unlike that of K. pneumoniae, was not repressed by ammonium. PMID:1622243

  10. Overexpression of Swedish mutant APP in aged astrocytes attenuates excitatory synaptic transmission.

    PubMed

    Katsurabayashi, Shutaro; Kawano, Hiroyuki; Ii, Miyuki; Nakano, Sachiko; Tatsumi, Chihiro; Kubota, Kaori; Takasaki, Kotaro; Mishima, Kenichi; Fujiwara, Michihiro; Iwasaki, Katsunori

    2016-01-01

    Amyloid precursor protein (APP), a type I transmembrane protein, has different aspects, namely, performs essential physiological functions and produces β-amyloid peptide (Aβ). Overexpression of neuronal APP is responsible for synaptic dysfunction. In the central nervous system, astrocytes - a major glial cell type - have an important role in the regulation of synaptic transmission. Although APP is expressed in astrocytes, it remains unclear whether astrocytic overexpression of mutant APP affects synaptic transmission. In this study, the effect of astrocytic overexpression of a mutant APP on the excitatory synaptic transmission was investigated using coculture system of the transgenic (Tg) cortical astrocytes that express the human APP695 polypeptide with the double mutation K670N + M671L found in a large Swedish family with early onset Alzheimer's disease, and wild-type hippocampal neuron. Significant secretion of Aβ 1-40 and 1-42 was observed in cultured cortical astrocytes from the Tg2576 transgenic mouse that genetically overexpresses Swedish mutant APP. Under the condition, Tg astrocytes did not affect excitatory synaptic transmission of cocultured wild-type neurons. However, aged Tg astrocytes cultured for 9 weeks elicited a significant decrease in excitatory synaptic transmission in cocultured neurons. Moreover, a reduction in the number of readily releasable synaptic vesicles accompanied a decrease in the number of excitatory synapses in neurons cocultured with aged Tg astrocytes. These observations indicate that astrocytic expression of the mutant APP is involved in the downregulation of synaptic transmission with age. PMID:26733247

  11. Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms.

    PubMed

    Pietra, D; Rumi, E; Ferretti, V V; Di Buduo, C A; Milanesi, C; Cavalloni, C; Sant'Antonio, E; Abbonante, V; Moccia, F; Casetti, I C; Bellini, M; Renna, M C; Roncoroni, E; Fugazza, E; Astori, C; Boveri, E; Rosti, V; Barosi, G; Balduini, A; Cazzola, M

    2016-02-01

    A quarter of patients with essential thrombocythemia or primary myelofibrosis carry a driver mutation of CALR, the calreticulin gene. A 52-bp deletion (type 1) and a 5-bp insertion (type 2 mutation) are the most frequent variants. These indels might differentially impair the calcium binding activity of mutant calreticulin. We studied the relationship between mutation subtype and biological/clinical features of the disease. Thirty-two different types of CALR variants were identified in 311 patients. Based on their predicted effect on calreticulin C-terminal, mutations were classified as: (i) type 1-like (65%); (ii) type 2-like (32%); and (iii) other types (3%). Corresponding CALR mutants had significantly different estimated isoelectric points. Patients with type 1 mutation, but not those with type 2, showed abnormal cytosolic calcium signals in cultured megakaryocytes. Type 1-like mutations were mainly associated with a myelofibrosis phenotype and a significantly higher risk of myelofibrotic transformation in essential thrombocythemia. Type 2-like CALR mutations were preferentially associated with an essential thrombocythemia phenotype, low risk of thrombosis despite very-high platelet counts and indolent clinical course. Thus, mutation subtype contributes to determining clinical phenotype and outcomes in CALR-mutant myeloproliferative neoplasms. CALR variants that markedly impair the calcium binding activity of mutant calreticulin are mainly associated with a myelofibrosis phenotype. PMID:26449662

  12. Excretion of ammonium by a nifL mutant of Azotobacter vinelandii fixing nitrogen.

    PubMed

    Bali, A; Blanco, G; Hill, S; Kennedy, C

    1992-05-01

    A mutation in the gene upstream of nifA in Azotobacter vinelandii was introduced into the chromosome to replace the corresponding wild-type region. The resulting mutant, MV376, produced nitrogenase constitutively in the presence of 15 mM ammonium. When introduced into a nifH-lacZ fusion strain, the mutation permitted beta-galactosidase production in the presence of ammonium. The gene upstream of nifA is therefore designated nifL because of its similarity to the Klebsiella pneumoniae nifL gene in proximity to nifA, in mutant phenotype, and in amino acid sequence of the gene product. The A. vinelandii nifL mutant MV376 excreted significant quantities of ammonium (approximately 10 mM) during diazotrophic growth. In contrast, ammonium excretion during diazotrophy was much lower in a K. pneumoniae nifL deletion mutant (maximum, 0.15 mM) but significantly higher than in NifL+ K. pneumoniae. The expression of the A. vinelandii nifA gene, unlike that of K. pneumoniae, was not repressed by ammonium. PMID:1622243

  13. Comparative Transcriptome Analysis of Anthurium “Albama” and Its Anthocyanin-Loss Mutant

    PubMed Central

    Zhang, Xuequan; Xu, Li

    2015-01-01

    Anthurium is one of the most important tropical ornamental plants in the world. The traded value of anthurium is second only to that of tropical orchids among the tropical flowers. The spathe is the main ornamental organ and its color variation mainly arises from anthocyanin contents. Understanding the molecular regulation of spathe color will accelerate new variety creation of anthurium. To announce gene expression differences between Anthurium andraeanum ‘Albama’ and its one unique anthocyanin-loss mutant, we collected spathes of the wild-type and the mutant from two stages in spathe development (the flower separates protrude from the sheath and the spathe is fully expanded) and extracted total RNAs for transcriptome profiling. Using short read sequencing technology (Illumina), 51,955,564, 53,822,224, 54,221,990 and 52,276,418 sequencing raw reads, respectively, for wild-type and mutant in the two stages were assembled de novo into 111,268 unique sequences (unigenes) with a mean length of 652 bp. 47,563 unigenes had significant hits to the sequences in the Nr database, and 32,768 unigenes showed significant similarity to known proteins in the Swiss-Prot database. 28,350 and 19,293 unigenes had significant similarity to existing sequences in the KEGG and COG databases, respectively. Further, analysis of differentially expressed genes in the comparison between wild-type and mutant and between the two different developmental stages was carried out, indicating that the expression of an extensive set of genes changed as the result of mutation. Taken together, these data demonstrated that the Illumina sequencing allowed de novo transcriptome assembly and could obtain differentially expressed genes between A. andraeanum wild-type and the anthocyanin-loss mutant. The expression differences of AN2 and UFGT might cause the anthocyanin-loss mutation. PMID:25781998

  14. Yeast meiotic mutants proficient for the induction of ectopic recombination.

    PubMed Central

    Engebrecht, J; Masse, S; Davis, L; Rose, K; Kessel, T

    1998-01-01

    A screen was designed to identify Saccharomyces cerevisiae mutants that were defective in meiosis yet proficient for meiotic ectopic recombination in the return-to-growth protocol. Seven mutants alleles were isolated; two are important for chromosome synapsis (RED1, MEK1) and five function independently of recombination (SPO14, GSG1, SPOT8/MUM2, 3, 4). Similar to the spoT8-1 mutant, mum2 deletion strains do not undergo premeiotic DNA synthesis, arrest prior to the first meiotic division and fail to sporulate. Surprisingly, although DNA replication does not occur, mum2 mutants are induced for high levels of ectopic recombination. gsg1 diploids are reduced in their ability to complete premeiotic DNA synthesis and the meiotic divisions, and a small percentage of cells produce spores. mum3 mutants sporulate poorly and the spores produced are inviable. Finally, mum4-1 mutants produce inviable spores. The meiotic/sporulation defects of gsg1, mum2, and mum3 are not relieved by spo11 or spo13 mutations, indicating that the mutant defects are not dependent on the initiation of recombination or completion of both meiotic divisions. In contrast, the spore inviability of the mum4-1 mutant is rescued by the spo13 mutation. The mum4-1 spo13 mutant undergoes a single, predominantly equational division, suggesting that MUM4 functions at or prior to the first meiotic division. Although recombination is variably affected in the gsg1 and mum mutants, we hypothesize that these mutants define genes important for aspects of meiosis not directly related to recombination. PMID:9504908

  15. Quorum-sensing-negative (lasR) mutants of Pseudomonas aeruginosa avoid cell lysis and death.

    PubMed

    Heurlier, Karin; Dénervaud, Valérie; Haenni, Marisa; Guy, Lionel; Krishnapillai, Viji; Haas, Dieter

    2005-07-01

    In Pseudomonas aeruginosa, N-acylhomoserine lactone signals regulate the expression of several hundreds of genes, via the transcriptional regulator LasR and, in part, also via the subordinate regulator RhlR. This regulatory network termed quorum sensing contributes to the virulence of P. aeruginosa as a pathogen. The fact that two supposed PAO1 wild-type strains from strain collections were found to be defective for LasR function because of independent point mutations in the lasR gene led to the hypothesis that loss of quorum sensing might confer a selective advantage on P. aeruginosa under certain environmental conditions. A convenient plate assay for LasR function was devised, based on the observation that lasR mutants did not grow on adenosine as the sole carbon source because a key degradative enzyme, nucleoside hydrolase (Nuh), is positively controlled by LasR. The wild-type PAO1 and lasR mutants showed similar growth rates when incubated in nutrient yeast broth at pH 6.8 and 37 degrees C with good aeration. However, after termination of growth during 30 to 54 h of incubation, when the pH rose to > or = 9, the lasR mutants were significantly more resistant to cell lysis and death than was the wild type. As a consequence, the lasR mutant-to-wild-type ratio increased about 10-fold in mixed cultures incubated for 54 h. In a PAO1 culture, five consecutive cycles of 48 h of incubation sufficed to enrich for about 10% of spontaneous mutants with a Nuh(-) phenotype, and five of these mutants, which were functionally complemented by lasR(+), had mutations in lasR. The observation that, in buffered nutrient yeast broth, the wild type and lasR mutants exhibited similar low tendencies to undergo cell lysis and death suggests that alkaline stress may be a critical factor providing a selective survival advantage to lasR mutants. PMID:15995202

  16. A Mutant Hunt Using the C-Fern (Ceratopteris Richardii)

    ERIC Educational Resources Information Center

    Calie, Patrick J.

    2005-01-01

    A modification of the popular C-Fern system, the tropical fern Ceratopteris richardii is developed in which students plate out a genetically mixed set of fern spores and then select for specific mutants. This exercise can provide students with an experience in plant mutant selection and can be used as a platform to expose students to a diverse…

  17. Structurally altered capsular polysaccharides produced by mutant bacteria

    NASA Technical Reports Server (NTRS)

    Kern, Roger G. (Inventor); Petersen, Gene R. (Inventor); Richards, Gil F. (Inventor)

    1995-01-01

    Structurally altered capsular polysaccharides are produced by mutant bacteria. These polysaccharides are isolated by selecting a wild type bacterial strain and a phage producing degradative enzymes that have substrate specificity for the capsular polysaccharides produced by the wild type bacteria. Phage-resistant mutants producing capsular polysaccharides are selected and the structurally altered capsular polysaccharide is isolated therefrom.

  18. Isolation of Temperature-Sensitive Mutants of L-Cells*

    PubMed Central

    Thompson, L. H.; Mankovitz, R.; Baker, R. M.; Till, J. E.; Siminovitch, L.; Whitmore, G. F.

    1970-01-01

    Procedures are described for the isolation of conditional lethal mutants of mouse L-60T cells. The mutant lines were temperature sensitive by the following criteria: (a) colony-forming ability, (b) growth in suspension culture, and (c) rate of uptake of tritiated-thymidine. Images PMID:5271170

  19. Mutant strain of C. acetobutylicum and process for making butanol

    DOEpatents

    Jain, Mahendra K.; Beacom, Daniel; Datta, Rathin

    1993-01-01

    A biologically pure asporogenic mutant of Clostridium acetobutylicum is produced by growing sporogenic C. acetobutylicum ATCC 4259 and treating the parent strain with ethane methane sulfonate. The mutant which as been designated C. acetobutylicum ATCC 55025 is useful in an improved ABE fermentation process, and produces high concentrations of butanol and total solvents.

  20. Isolation of Mutants of the Nitrogen-Fixing Actinomycete Frankia

    PubMed Central

    Kakoi, Kentaro; Yamaura, Masatoshi; Kamiharai, Toshihito; Tamari, Daiki; Abe, Mikiko; Uchiumi, Toshiki; Kucho, Ken-Ichi

    2014-01-01

    Frankia is a nitrogen (N)-fixing multicellular actinomycete which establishes root-nodule symbiosis with actinorhizal plants. Several aspects of Frankia N fixation and symbiosis are distinct, but genes involved in the specific features are largely unknown because of the lack of an efficient mutant screening method. In this study, we isolated mutants of Frankia sp. strain CcI3 using hyphae fragments mutagenized by chemical mutagens. Firstly, we isolated uracil auxotrophs as gain-of-function mutants resistant to 5-fluoroorotic acid (5-FOA). We obtained seven 5-FOA resistant mutants, all of which required uracil for growth. Five strains carried a frame shift mutation in orotidine-5′-phosphate decarboxylase gene and two carried an amino acid substitution in the orotate phosphoribosyltransferase gene. Secondly, we isolated mutants showing loss-of-function phenotypes. Mutagenized hyphae were fragmented by ultrasound and allowed to multiply at their tips. Hyphae were fragmented again and short fragments were enriched by filtration through 5 μm pores filters. Next-generation and Sanger sequencing revealed that colonies formed from the short hyphae fragments consisted of cells with an identical genotype. From the mutagenized colony population, we isolated three pigmentation mutants and a mutant with reduced N-fixation activity. These results indicate that our procedure is useful for the isolation of loss-of-function mutants using hyphae of Frankia. PMID:24389412

  1. Sorghum Brown Midrib Mutants, Tools to Improve Biomass for Biofuels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To improve sorghum for cellulosic bioenergy uses, brown midrib mutants are being investigated for their ability to increase the conversion efficiency of biomass. brown midrib 6 and 12 (bmr6 and 12) mutants affect monolignol biosynthesis resulting in reduced lignin content and altered lignin composi...

  2. Gravitropism in roots of intermediate-starch mutants of Arabidopsis

    NASA Technical Reports Server (NTRS)

    Kiss, J. Z.; Wright, J. B.; Caspar, T.

    1996-01-01

    Gravitropism was studied in roots of wild type (WT) Arabidopsis thaliana (L.) Heynh. (strain Wassilewskija) and three starch-deficient mutants that were generated by T-DNA insertional mutagenesis. One of these mutants was starchless while the other two were intermediate mutants, which had 51% and 60%, respectively, of the WT amount of starch as determined by light and electron microscopy. The four parameters used to assay gravitropism were: orientation during vertical growth, time course of curvature, induction, and intermittent stimulation experiments. WT roots were much more responsive to gravity than were roots of the starchless mutant, and the intermediate starch mutants exhibited an intermediate graviresponse. Our data suggest that lowered starch content in the mutants primarily affects gravitropism rather than differential growth because both phototropic curvature and growth rates were approximately equal among all four genotypes. Since responses of intermediate-starch mutants were closer to the WT response than to the starchless mutant, it appears that 51-60% of the WT level of starch is near the threshold amount needed for full gravitropic sensitivity. While other interpretations are possible, the data are consistent with the starch statolith hypothesis for gravity perception in that the degree of graviresponsiveness is proportional to the total mass of plastids per cell.

  3. Mutant maize variety containing the glt1-1 allele

    DOEpatents

    Nelson, O.E.; Pan, D.

    1994-07-19

    A maize plant has in its genome a non-mutable form of a mutant allele designated vitX-8132. The allele is located at a locus designated as glt which conditions kernels having an altered starch characteristic. Maize plants including such a mutant allele produce a starch that does not increase in viscosity on cooling, after heating. 2 figs.

  4. Mutant maize variety containing the glt1-1 allele

    DOEpatents

    Nelson, Oliver E.; Pan, David

    1994-01-01

    A maize plant has in its genome a non-mutable form of a mutant allele designated vitX-8132. The allele is located at a locus designated as glt which conditions kernels having an altered starch characteristic. Maize plants including such a mutant allele produce a starch that does not increase in viscosity on cooling, after heating.

  5. Isolation of mutants of the nitrogen-fixing actinomycete Frankia.

    PubMed

    Kakoi, Kentaro; Yamaura, Masatoshi; Kamiharai, Toshihito; Tamari, Daiki; Abe, Mikiko; Uchiumi, Toshiki; Kucho, Ken-Ichi

    2014-01-01

    Frankia is a nitrogen (N)-fixing multicellular actinomycete which establishes root-nodule symbiosis with actinorhizal plants. Several aspects of Frankia N fixation and symbiosis are distinct, but genes involved in the specific features are largely unknown because of the lack of an efficient mutant screening method. In this study, we isolated mutants of Frankia sp. strain CcI3 using hyphae fragments mutagenized by chemical mutagens. Firstly, we isolated uracil auxotrophs as gain-of-function mutants resistant to 5-fluoroorotic acid (5-FOA). We obtained seven 5-FOA resistant mutants, all of which required uracil for growth. Five strains carried a frame shift mutation in orotidine-5'-phosphate decarboxylase gene and two carried an amino acid substitution in the orotate phosphoribosyltransferase gene. Secondly, we isolated mutants showing loss-of-function phenotypes. Mutagenized hyphae were fragmented by ultrasound and allowed to multiply at their tips. Hyphae were fragmented again and short fragments were enriched by filtration through 5 μm pores filters. Next-generation and Sanger sequencing revealed that colonies formed from the short hyphae fragments consisted of cells with an identical genotype. From the mutagenized colony population, we isolated three pigmentation mutants and a mutant with reduced N-fixation activity. These results indicate that our procedure is useful for the isolation of loss-of-function mutants using hyphae of Frankia. PMID:24389412

  6. Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis

    PubMed Central

    Hurst, Sheldon; Rowedder, Holli; Michaels, Brandye; Bullock, Hannah; Jackobeck, Ryan; Abebe-Akele, Feseha; Durakovic, Umjia; Gately, Jon; Janicki, Erik

    2015-01-01

    ABSTRACT The entomopathogenic nematode Heterorhabditis bacteriophora forms a specific mutualistic association with its bacterial partner Photorhabdus temperata. The microbial symbiont is required for nematode growth and development, and symbiont recognition is strain specific. The aim of this study was to sequence the genome of P. temperata and identify genes that plays a role in the pathogenesis of the Photorhabdus-Heterorhabditis symbiosis. A draft genome sequence of P. temperata strain NC19 was generated. The 5.2-Mb genome was organized into 17 scaffolds and contained 4,808 coding sequences (CDS). A genetic approach was also pursued to identify mutants with altered motility. A bank of 10,000 P. temperata transposon mutants was generated and screened for altered motility patterns. Five classes of motility mutants were identified: (i) nonmotile mutants, (ii) mutants with defective or aberrant swimming motility, (iii) mutant swimmers that do not require NaCl or KCl, (iv) hyperswimmer mutants that swim at an accelerated rate, and (v) hyperswarmer mutants that are able to swarm on the surface of 1.25% agar. The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis. The motility-defective mutant P13-7 had an insertion in the RNase II gene and showed reduced virulence and production of extracellular factors. Genetic complementation of this mutant restored wild-type activity. These results demonstrate a role for RNA turnover in insect pathogenesis and other physiological functions. IMPORTANCE The relationship between Photorhabdus and entomopathogenic nematode Heterorhabditis represents a well-known mutualistic system that has potential as a biological control agent. The elucidation of the genome of the bacterial partner and role that RNase II plays in its life cycle has provided a greater understanding of Photorhabdus as both an insect pathogen and a nematode symbiont. PMID

  7. Bacillus sphaericus asporogenous mutants: morphology, protein pattern and larvicidal activity.

    PubMed

    Charles, J F; Kalfon, A; Bourgouin, C; de Barjac, H

    1988-01-01

    Asporogenous mutants from Bacillus sphaericus strains 2297 and 1593-4, blocked at different stages of the sporulation process, were isolated. Two mutants (2297 Aspo30A and 2297 Aspo34) which were blocked early in sporulation did not possess any crystalline inclusions and were poorly toxic to Culex pipiens mosquito larvae. Other mutants (2297 Aspo115, 2297 Aspo24 and 1593-4 Aspo12) which were blocked at later stages synthesized crystal-like inclusions next to the forespores, and were highly toxic to mosquito larvae. Electrophoretic protein analysis of alkali extracts from mutants and wild type strains confirmed the absence of toxic crystal-related proteins in early-blocked mutants and their presence in later ones. Western blots with antisera directed against the crystal proteins confirmed those observations. PMID:3408593

  8. Viable deletion mutants in the simian virus 40 early region.

    PubMed Central

    Feunteun, J; Kress, M; Gardes, M; Monier, R

    1978-01-01

    For the purpose of isolating hr-t-like mutants of simian virus 40, we have constructed variants that have lost the unique site for the restriction enzyme Taq I at 0.565. Five mutants have been isolated and characterized by restriction enzyme analysis. All of them produce a normal size T antigen. Four produce a t antigen reduced in size as well as in amount; the fifth one does not seem to make any t antigen at all. The ability of these mutants to transform mouse cells in vitro, as tested by anchorage dependence, is clearly altered; however, the defect is only partial. In the same test, the mutants can complement a tsA mutant for transformation and therefore define a second complementation group in the simian virus 40 early region. Images PMID:212752

  9. Huntington's disease cerebrospinal fluid seeds aggregation of mutant huntingtin

    PubMed Central

    Tan, Z; Dai, W; van Erp, T G M; Overman, J; Demuro, A; Digman, M A; Hatami, A; Albay, R; Sontag, E M; Potkin, K T; Ling, S; Macciardi, F; Bunney, W E; Long, J D; Paulsen, J S; Ringman, J M; Parker, I; Glabe, C; Thompson, L M; Chiu, W; Potkin, S G

    2015-01-01

    Huntington's disease (HD), a progressive neurodegenerative disease, is caused by an expanded CAG triplet repeat producing a mutant huntingtin protein (mHTT) with a polyglutamine-repeat expansion. Onset of symptoms in mutant huntingtin gene-carrying individuals remains unpredictable. We report that synthetic polyglutamine oligomers and cerebrospinal fluid (CSF) from BACHD transgenic rats and from human HD subjects can seed mutant huntingtin aggregation in a cell model and its cell lysate. Our studies demonstrate that seeding requires the mutant huntingtin template and may reflect an underlying prion-like protein propagation mechanism. Light and cryo-electron microscopy show that synthetic seeds nucleate and enhance mutant huntingtin aggregation. This seeding assay distinguishes HD subjects from healthy and non-HD dementia controls without overlap (blinded samples). Ultimately, this seeding property in HD patient CSF may form the basis of a molecular biomarker assay to monitor HD and evaluate therapies that target mHTT. PMID:26100538

  10. Architectural phenotypes in the transparent testa mutants of Arabidopsis thaliana

    PubMed Central

    Buer, Charles S.; Djordjevic, Michael A.

    2009-01-01

    Flavonoids are low molecular weight secondary plant metabolites with a myriad of functions. As flavonoids affect auxin transport (an important growth-controlling hormone) and are biologically active in eukaryotes, flavonoid mutants were expected to have undescribed architectural phenotypes. The Arabidopsis thaliana transparent testa (tt) mutants are compromised in the enzymatic steps or transcriptional regulators affecting flavonoid synthesis. tt mutant seedlings were grown on hard-slanted agar (a stress condition), under varying light conditions, and in soil to examine the resulting growth patterns. These tt mutants revealed a wide variety of architectural phenotypes in root and aerial tissues. Mutants with increased inflorescences, siliques, and lateral root density or reduced stature are traits that could affect plant yield or performance under certain environmental conditions. The regulatory genes affected in architectural traits may provide useful molecular targets for examination in other plants. PMID:19129166

  11. Life without complex I: proteome analyses of an Arabidopsis mutant lacking the mitochondrial NADH dehydrogenase complex.

    PubMed

    Fromm, Steffanie; Senkler, Jennifer; Eubel, Holger; Peterhänsel, Christoph; Braun, Hans-Peter

    2016-05-01

    The mitochondrial NADH dehydrogenase complex (complex I) is of particular importance for the respiratory chain in mitochondria. It is the major electron entry site for the mitochondrial electron transport chain (mETC) and therefore of great significance for mitochondrial ATP generation. We recently described an Arabidopsis thaliana double-mutant lacking the genes encoding the carbonic anhydrases CA1 and CA2, which both form part of a plant-specific 'carbonic anhydrase domain' of mitochondrial complex I. The mutant lacks complex I completely. Here we report extended analyses for systematically characterizing the proteome of the ca1ca2 mutant. Using various proteomic tools, we show that lack of complex I causes reorganization of the cellular respiration system. Reduced electron entry into the respiratory chain at the first segment of the mETC leads to induction of complexes II and IV as well as alternative oxidase. Increased electron entry at later segments of the mETC requires an increase in oxidation of organic substrates. This is reflected by higher abundance of proteins involved in glycolysis, the tricarboxylic acid cycle and branched-chain amino acid catabolism. Proteins involved in the light reaction of photosynthesis, the Calvin cycle, tetrapyrrole biosynthesis, and photorespiration are clearly reduced, contributing to the significant delay in growth and development of the double-mutant. Finally, enzymes involved in defense against reactive oxygen species and stress symptoms are much induced. These together with previously reported insights into the function of plant complex I, which were obtained by analysing other complex I mutants, are integrated in order to comprehensively describe 'life without complex I'. PMID:27122571

  12. Life without complex I: proteome analyses of an Arabidopsis mutant lacking the mitochondrial NADH dehydrogenase complex

    PubMed Central

    Fromm, Steffanie; Senkler, Jennifer; Eubel, Holger; Peterhänsel, Christoph; Braun, Hans-Peter

    2016-01-01

    The mitochondrial NADH dehydrogenase complex (complex I) is of particular importance for the respiratory chain in mitochondria. It is the major electron entry site for the mitochondrial electron transport chain (mETC) and therefore of great significance for mitochondrial ATP generation. We recently described an Arabidopsis thaliana double-mutant lacking the genes encoding the carbonic anhydrases CA1 and CA2, which both form part of a plant-specific ‘carbonic anhydrase domain’ of mitochondrial complex I. The mutant lacks complex I completely. Here we report extended analyses for systematically characterizing the proteome of the ca1ca2 mutant. Using various proteomic tools, we show that lack of complex I causes reorganization of the cellular respiration system. Reduced electron entry into the respiratory chain at the first segment of the mETC leads to induction of complexes II and IV as well as alternative oxidase. Increased electron entry at later segments of the mETC requires an increase in oxidation of organic substrates. This is reflected by higher abundance of proteins involved in glycolysis, the tricarboxylic acid cycle and branched-chain amino acid catabolism. Proteins involved in the light reaction of photosynthesis, the Calvin cycle, tetrapyrrole biosynthesis, and photorespiration are clearly reduced, contributing to the significant delay in growth and development of the double-mutant. Finally, enzymes involved in defense against reactive oxygen species and stress symptoms are much induced. These together with previously reported insights into the function of plant complex I, which were obtained by analysing other complex I mutants, are integrated in order to comprehensively describe ‘life without complex I’. PMID:27122571

  13. Photoproduction of Hydrogen by Sulfur-Deprived Chlamydomonas reinhardtii Mutants with Impaired Photosystem II Photochemical Activity

    SciTech Connect

    Makarova, V. V.; Kosourov, S.; Krendeleva, T. E.; Semin, B. K.; Kukarskikh, G. P.; Rubin, A. B.; Sayre, R. T.; Ghirardi, M. L.; Seibert, M.

    2007-01-01

    Photoproduction of H2 was examined in a series of sulfur-deprived Chlamydomonas reinhardtii D1-R323 mutants with progressively impaired PSII photochemical activity. In the R323H, R323D, and R323E D1 mutants, replacement of arginine affects photosystem II (PSII) function, as demonstrated by progressive decreases in O2-evolving activity and loss of PSII photochemical activity. Significant changes in PSII activity were found when the arginine residue was replaced by negatively charged amino acid residues (R323D and R323E). However, the R323H (positively charged or neutral, depending on the ambient pH) mutant had minimal changes in PSII activity. The R323H, R323D, and R323E mutants and the pseudo-wild-type (pWt) with restored PSII function were used to study the effects of sulfur deprivation on H2-production activity. All of these mutants exhibited significant changes in the normal parameters associated with the H2-photoproduction process, such as a shorter aerobic phase, lower accumulation of starch, a prolonged anaerobic phase observed before the onset of H2-production, a shorter duration of H2-production, lower H2 yields compared to the pWt control, and slightly higher production of dark fermentation products such as acetate and formate. The more compromised the PSII photochemical activity, the more dramatic was the effect of sulfur deprivation on the H2-production process, which depends both on the presence of residual PSII activity and the amount of stored starch.

  14. Impaired Cellular Bioenergetics Causes Mitochondrial Calcium Handling Defects in MT-ND5 Mutant Cybrids

    PubMed Central

    Duchen, Michael R.

    2016-01-01

    Mutations in mitochondrial DNA (mtDNA) can cause mitochondrial disease, a group of metabolic disorders that affect both children and adults. Interestingly, individual mtDNA mutations can cause very different clinical symptoms, however the factors that determine these phenotypes remain obscure. Defects in mitochondrial oxidative phosphorylation can disrupt cell signaling pathways, which may shape these disease phenotypes. In particular, mitochondria participate closely in cellular calcium signaling, with profound impact on cell function. Here, we examined the effects of a homoplasmic m.13565C>T mutation in MT-ND5 on cellular calcium handling using transmitochondrial cybrids (ND5 mutant cybrids). We found that the oxidation of NADH and mitochondrial membrane potential (Δψm) were significantly reduced in ND5 mutant cybrids. These metabolic defects were associated with a significant decrease in calcium uptake by ND5 mutant mitochondria in response to a calcium transient. Inhibition of glycolysis with 2-deoxy-D-glucose did not affect cytosolic calcium levels in control cybrids, but caused an increase in cytosolic calcium in ND5 mutant cybrids. This suggests that glycolytically-generated ATP is required not only to maintain Δψm in ND5 mutant mitochondria but is also critical for regulating cellular calcium homeostasis. We conclude that the m.13565C>T mutation in MT-ND5 causes defects in both mitochondrial oxidative metabolism and mitochondrial calcium sequestration. This disruption of mitochondrial calcium handling, which leads to defects in cellular calcium homeostasis, may be an important contributor to mitochondrial disease pathogenesis. PMID:27110715

  15. Rapid Quantification of Mutant Fitness in Diverse Bacteria by Sequencing Randomly Bar-Coded Transposons

    PubMed Central

    Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.; Lamson, Jacob S.; He, Jennifer; Hoover, Cindi A.; Blow, Matthew J.; Bristow, James; Butland, Gareth

    2015-01-01

    ABSTRACT Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative d-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. PMID:25968644

  16. Photoproduction of hydrogen by sulfur-deprived C. reinhardtii mutants with impaired photosystem II photochemical activity.

    PubMed

    Makarova, Valeria V; Kosourov, Sergey; Krendeleva, Tatiana E; Semin, Boris K; Kukarskikh, Galina P; Rubin, Andrei B; Sayre, Richard T; Ghirardi, Maria L; Seibert, Michael

    2007-10-01

    Photoproduction of H2 was examined in a series of sulfur-deprived Chlamydomonas reinhardtii D1-R323 mutants with progressively impaired PSII photochemical activity. In the R323H, R323D, and R323E D1 mutants, replacement of arginine affects photosystem II (PSII) function, as demonstrated by progressive decreases in O2-evolving activity and loss of PSII photochemical activity. Significant changes in PSII activity were found when the arginine residue was replaced by negatively charged amino acid residues (R323D and R323E). However, the R323H (positively charged or neutral, depending on the ambient pH) mutant had minimal changes in PSII activity. The R323H, R323D, and R323E mutants and the pseudo-wild-type (pWt) with restored PSII function were used to study the effects of sulfur deprivation on H2-production activity. All of these mutants exhibited significant changes in the normal parameters associated with the H2-photoproduction process, such as a shorter aerobic phase, lower accumulation of starch, a prolonged anaerobic phase observed before the onset of H2-production, a shorter duration of H2-production, lower H2 yields compared to the pWt control, and slightly higher production of dark fermentation products such as acetate and formate. The more compromised the PSII photochemical activity, the more dramatic was the effect of sulfur deprivation on the H2-production process, which depends both on the presence of residual PSII activity and the amount of stored starch. PMID:17701084

  17. Biomass Productivities in Wild Type and Pigment Mutant of Cyclotella sp. (Diatom)

    SciTech Connect

    Huesemann, Michael H.; Hausmann, Tom S.; Bartha, Richard; Aksoy, M.; Weissman, Joseph C.; Benemann, John

    2008-07-03

    Microalgae are expected to play a significant role in greenhouse gas mitigation because they can utilize CO2 from powerplant flue gases directly while producing a variety of renewable carbon-neutral biofuels. In order for such a microalgal climate change mitigation strategy to become economically feasible, it will be necessary to significantly improve biomass productivities. One approach to achieve this objective is to reduce, via mutagenesis, the number of light harvesting pigments, which, according to theory, should significantly improve the light utilization efficiency, primarily by increasing the light intensity at which photosynthesis saturates (Is). Employing chemical (ethylmethylsulfonate, EMS) and UV mutagenesis of a wild type strain of the diatom Cyclotella, approximately 10,000 pigment mutants were generated, and two of the most promising ones (CM1 and CM1-1) were subjected to further testing in both laboratory cultures and outdoor ponds. Measurements of photosynthetic oxygen production rates as a function of light intensity (i.e., P-I curves) of samples taken from laboratory batch cultures during the exponential and linear growth phase indicated that the light intensity at which photosynthesis saturates (Is) was two to three times greater in the pigment mutant CM1-1 than in the wild type, i.e., 355-443 versus 116-169 μmole/m2∙sec, respectively. While theory, i.e., the Bush equation, predicts that such a significant gain in Is should increase light utilization efficiencies and thus biomass productivities, particularly at high light intensities, no improvements in biomass productivities were observed in either semi-continuous laboratory cultures or outdoor ponds. In fact, the maximum biomass productivity in semi-continuous laboratory culture was always greater in the wild type than in the mutant, namely 883 versus 725 mg/L∙d, respectively at low light intensity (200 μmole/m2∙sec) and 1229 versus 1043 mg/L∙d, respectively at high light intensity

  18. Analysis of Constructed E Gene Mutants of Mouse Hepatitis Virus Confirms a Pivotal Role for E Protein in Coronavirus Assembly

    PubMed Central

    Fischer, Françoise; Stegen, Carola F.; Masters, Paul S.; Samsonoff, William A.

    1998-01-01

    Expression studies have shown that the coronavirus small envelope protein E and the much more abundant membrane glycoprotein M are both necessary and sufficient for the assembly of virus-like particles in cells. As a step toward understanding the function of the mouse hepatitis virus (MHV) E protein, we carried out clustered charged-to-alanine mutagenesis on the E gene and incorporated the resulting mutations into the MHV genome by targeted recombination. Of the four possible clustered charged-to-alanine E gene mutants, one was apparently lethal and one had a wild-type phenotype. The two other mutants were partially temperature sensitive, forming small plaques at the nonpermissive temperature. Revertant analyses of these two mutants demonstrated that the created mutations were responsible for the temperature-sensitive phenotype of each and provided support for possible interactions among E protein monomers. Both temperature-sensitive mutants were also found to be markedly thermolabile when grown at the permissive temperature, suggesting that there was a flaw in their assembly. Most significantly, when virions of one of the mutants were examined by electron microscopy, they were found to have strikingly aberrant morphology in comparison to the wild type: most mutant virions had pinched and elongated shapes that were rarely seen among wild-type virions. These results demonstrate an important, probably essential, role for the E protein in coronavirus morphogenesis. PMID:9733825

  19. Identification and characterization of an anti-oxidative stress-associated mutant of Aspergillus fumigatus transformed by Agrobacterium tumefaciens

    PubMed Central

    FAN, ZHONGQI; YU, HUIMEI; GUO, QI; HE, DAN; XUE, BAIJI; XIE, XIANGLI; YOKOYAMA, KOJI; WANG, LI

    2016-01-01

    Aspergillus fumigatus is one of the most common opportunistic pathogenic fungi, surviving in various environmental conditions. Maintenance of the redox homeostasis of the fungus relies upon the well-organized regulation between reactive oxygen species generated by immune cells or its own organelles, and the activated anti-oxidative stress mechanism. To investigate such a mechanism, the present study obtained a number of randomly-inserted mutants of A. fumigatus, mediated by Agrobacterium tumefaciens. In addition, a high throughput hydrogen peroxide screening system was established to examine ~1,000 mutants. A total of 100 mutants exhibited changes in hydrogen peroxide sensitivity, among which a significant increase in sensitivity was observed in the AFM2658 mutant. Further investigations of the mutant were also performed, in which the sequence of this mutant was characterized using thermal asymmetric interlaced-polymerase chain reaction. This revealed that the insertion site was located on chromosome 2 afu1_92, and the 96 bp sequence was knocked out, which partially comprised a sequence localized between the integral membrane protein coding region and the helix-loop-helix transcription factor coding region. A decrease in the levels of anti-oxidative stress-associated mRNAs were observed, and an increase in reactive oxygen species were detected using fluorescence. The results of the present study demonstrated that this sequence may have a protective role in A. fumigatus in the presence of oxidative stress. PMID:26847000

  20. Transcriptome profiling identifies genes and pathways deregulated upon floxuridine treatment in colorectal cancer cells harboring GOF mutant p53.

    PubMed

    Datta, Arindam; Dey, Sanjib; Das, Pijush; Alam, Sk Kayum; Roychoudhury, Susanta

    2016-06-01

    Mutation in TP53 is a common genetic alteration in human cancers. Certain tumor associated p53 missense mutants acquire gain-of-function (GOF) properties and confer oncogenic phenotypes including enhanced chemoresistance. The colorectal cancers (CRC) harboring mutant p53 are generally aggressive in nature and difficult to treat. To identify a potential gene expression signature of GOF mutant p53-driven acquired chemoresistance in CRC, we performed transcriptome profiling of floxuridine (FUdR) treated SW480 cells expressing mutant p53(R273H) (GEO#: GSE77533). We obtained several genes differentially regulated between FUdR treated and untreated cells. Further, functional characterization and pathway analysis revealed significant enrichment of crucial biological processes and pathways upon FUdR treatment in SW480 cells. Our data suggest that in response to chemotherapeutics treatment, cancer cells with GOF mutant p53 can modulate key cellular pathways to withstand the cytotoxic effect of the drugs. The genes and pathways identified in the present study can be further validated and targeted for better chemotherapy response in colorectal cancer patients harboring mutant p53. PMID:27114909

  1. PIK3CA mutation uncouples tumor growth and Cyclin D1 regulation from MEK/ERK and mutant KRAS signaling

    PubMed Central

    Halilovic, Ensar; She, Qing-Bai; Ye, Qing; Pagliarini, Raymond; Sellers, William R.; Solit, David B.; Rosen, Neal

    2010-01-01

    Mutational activation of KRAS is a common event in human tumors. Identification of the key signaling pathways downstream of mutant KRAS is essential for our understanding of how to pharmacologically target these cancers in patients. We show that PD0325901, a small molecule MEK inhibitor, decreases MEK/ERK pathway signaling, and destabilizes Cyclin D1, resulting in significant anti-cancer activity in a subset of KRAS mutant tumors in vitro and in vivo. Mutational activation of PIK3CA, which commonly co-occurs with KRAS mutation, provides resistance to MEK inhibition through reactivation of AKT signaling. Genetic ablation of the mutant PIK3CA allele in MEK inhibitor-resistant cells restores MEK pathway sensitivity, and re-expression of mutant PIK3CA reinstates the resistance, highlighting the importance of this mutation in resistance to therapy in human cancers. In KRAS mutant tumors, PIK3CA mutation restores Cyclin D1 expression and G1/S cell cycle progression so that they are no longer dependent on KRAS and MEK/ERK signaling. Furthermore, the growth of KRAS mutant tumors with coexistent PIK3CA mutations in vivo is profoundly inhibited with combined pharmacologic inhibition of MEK and AKT. These data suggest that tumors with both KRAS and PI3K mutations are unlikely to respond to inhibition of the MEK pathway alone but will require effective inhibition of both MEK and PI3K/AKT pathway signaling. PMID:20699365

  2. Biocatalytic role of potato starch synthase III for α-glucan biosynthesis in Synechocystis sp. PCC6803 mutants.

    PubMed

    Yoo, Sang-Ho; Lee, Byung-Hoo; Li, Li; Perris, Shayani D N; Spalding, Martin H; Han, Sang Yun; Jane, Jay-lin

    2015-11-01

    A potato starch synthase III (PSSIII) was expressed in the Synechocystis mutants deficient in either glycogen synthase I (M1) or II (M2) to replenish α-(1,4) linkage synthesizing activity, resulting in new mutants, PM1 and PM2, respectively. These mutants were applied to study the role of exogenous plant starch synthase for starch/glycogen biosynthesis mechanism established in the cyanobacteria. The remaining glycogen synthase genes in PM1 and PM2 were further disrupted to make the mutants PM12 and PM21 which contained PSSIII as the sole glycogen/starch synthase. Among wild type and mutants, there were no significant differences in the amount of α-glucan produced. All the mutants harboring active PSSIII produced α-glucans with relatively much shorter and less longer α-1,4 chains than wild-type glycogen, which was exactly in accordance with the increase in glycogen branching enzyme activity. In fact, α-glucan structure of PM1 was very similar to those of PM12 and PM21, and PM2 had more intermediate chains than M2. This result suggests PSSIII may have distributive elongation property during α-glucan synthesis. In conclusion, the Synechocystis as an expression model system of plant enzymes can be applied to determine the role of starch synthesizing enzymes and their association during α-glucan synthesis. PMID:26358554

  3. Critical role of FANCC in JAK2 V617F mutant-induced resistance to DNA cross-linking drugs.

    PubMed

    Ueda, Fumihito; Sumi, Kazuya; Tago, Kenji; Kasahara, Tadashi; Funakoshi-Tago, Megumi

    2013-11-01

    A point mutation (V617F) of tyrosine kinase Janus kinase 2 (JAK2) is found in the majority of patients with myeloproliferative neoplasms (MPNs) and an aberrant signaling pathway induced by constitutively active JAK2 V617F mutant is a hallmark of MPNs. Cells transformed by JAK2 V617F mutant exhibited resistance to anti-cancer drugs such as cisplatin (CDDP), mitomycin C (MMC) and bleomycin (BLM). We first found that the expression of FANCC, a member of the Fanconi anemia (FA) proteins, was significantly induced by JAK2 V617F mutant through activation of signal transducers and activators of transcription 5 (STAT5). In addition, monoubiqitination and foci formation of FANCD2, which are critical for activation of the FA pathway, were increased in cells transformed by JAK2 V617F mutant, compared to cells expressing wild-type JAK2. Interestingly, knockdown of FANCC in cells expressing JAK2 V617F mutant induced not only the reduction of monoubiqitination and foci formation of FANCD2 but also the enhancement of sensitivity to DNA damage induced by CDDP and MMC but not BLM. Taken together, FANCC is most likely to be critical for resistance to DNA cross-linking drug-induced DNA damage in cells transformed by JAK2 V617F mutant. PMID:23838005

  4. Protein substrates for cGMP-dependent protein phosphorylation in cilia of wild type and atalanta mutants of Paramecium.

    PubMed

    Ann, K S; Nelson, D L

    1995-01-01

    In the ciliated protozoan Paramecium, swimming direction is regulated by voltage-gated Ca2+ channels in the ciliary membrane. In response to depolarizing stimuli, intraciliary Ca2+ rises, triggering reversal of the ciliary power stroke and backward swimming. One class of Ca(2+)-unresponsive behavioral mutants of Paramecium, atalanta mutants, cannot swim backward even though they have functional Ca2+ channels in their ciliary membrane. Several atalanta mutants were characterized with regard to several Ca(2+)-dependent activities, but no significant difference between wild type and the mutants was detected. However, one allelic group, atalanta A (initially characterized by Hinrichsen and Kung [1984: Genet. Res. Camb. 43:11-20]), showed a helical swimming path of opposite handedness from that of wild-type cells when detergent-permeabilized cells ("models") were reactivated with MgATP. When cGMP-dependent protein kinase purified from wild-type cells was added to atalanta A models, the handedness of the swimming path was reversed. Cyclic GMP stimulated in vitro phosphorylation of several proteins in isolated cilia, and the pattern of phosphoproteins was very similar for wild type and atalanta mutants, with one exception: a protein of 59 kDa was phosphorylated much less in the mutant ata A. When ciliary proteins were separated by gel electrophoresis and then phosphorylated "on blot" by purified cGMP-dependent protein kinase, phosphoprotein patterns were similar in wild type and ata mutants except that a 48 kDa protein (p48) from ata A3 was more heavily phosphorylated. This difference in p48 phosphorylation was also observed with cGMP-dependent protein kinase purified from ata A3 mutant cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7796456

  5. Pathogenicity of wild-type and temperature-sensitive mutants of herpes simplex virus type 2 in guinea pigs.

    PubMed Central

    Anderson, C A; August, M J; Hsiung, G D

    1980-01-01

    The pathogenicity of herpes simplex virus type 2 strain 186, the wild-type (WT) strain, and four temperature-sensitive (ts) mutants was studied after genital inoculation of female guinea pigs. Infection with the WT virus was generally severe, with extensive skin lesions in 89% and mortality in 37% of inoculated animals. Guinea pigs inoculated with ts mutants manifest remarkably mild disease, with lesions occurring in only 16% of the guinea pits and a mortality rate of 7%. WT virus was recovered from nerve and non-nerve tissues of all acutely infected animals and from the majority of latently infected animals (71%). Virus was isolated from nerve or genital tissues from only 13% of ts mutant-inoculated animals during acute infection and from 7% during latent infection. Three of the seven isolates from mutant-infected animals appeared to be WT virus. Identification of WT and ts mutant isolates was done by biological characterization in selective cell cultures at permissive (33 degrees C) and nonpermissive (38 degrees C) temperatures. One month after initial infection with WT virus, guinea pigs were challenged with the same virus and were completely resistant to overt clinical disease. Animals inoculated with ts mutants A1b and C2b had mild manifestations of disease after challenge with WT virus; however, the capacity of WT virus to establish latent infection was conserved. Although complement-required neutralizing antibodies were detectable after challenge in animals previously inoculated with mutant virus A1b, C2b, or D6b, there was no significant protection against subsequent infection with WT virus. No complement-required neutralizing antibodies were detected in F3b animals after challenge. The present study of WT and ts mutants of herpes simplex virus type 2 in the guinea pig model provides a means for better understanding the mechanisms of pathogenesis and latency after genital infection. Images Fig. 1 Fig. 1B Fig. 2 PMID:6254877

  6. An In Vivo Quantitative Comparison of Photoprotection in Arabidopsis Xanthophyll Mutants

    PubMed Central

    Ware, Maxwell A.; Dall’Osto, Luca; Ruban, Alexander V.

    2016-01-01

    Contribution of different LHCII antenna carotenoids to protective NPQ (pNPQ) were tested using a range of xanthophyll biosynthesis mutants of Arabidopsis: plants were either devoid of lutein (lut2), violaxanthin (npq2), or synthesized a single xanthophyll species, namely violaxanthin (aba4npq1lut2), zeaxanthin (npq2lut2), or lutein (chy1chy2lut5). A novel pulse amplitude modulated (PAM) fluorescence analysis procedure, that used a gradually increasing actinic light intensity, allowed the efficiency of pNPQ to be tested using the photochemical quenching (qP) parameter measured in the dark (qPd). Furthermore, the yield of photosystem II (ΦPSII) was calculated, and the light intensity which induces photoinhibition in 50% of leaves for each mutant was ascertained. Photoprotective capacities of each xanthophyll were quantified, taking into account chlorophyll a/b ratios and excitation pressure. Here, light tolerance, pNPQ capacity, and ΦPSII were highest in wild type plants. Of the carotenoid mutants, lut2 (lutein-deficient) plants had the highest light tolerance, and the joint the highest ΦPSII with violaxanthin only plants. We conclude that all studied mutants possess pNPQ and a more complete composition of xanthophylls in their natural binding sites is the most important factor governing photoprotection, rather than any one specific xanthophyll suggesting a strong structural effect of the molecules upon the LHCII antenna organization and discuss the results significance for future crop development. PMID:27446097

  7. Aberrant Subcellular Dynamics of Sigma-1 Receptor Mutants Underlying Neuromuscular Diseases.

    PubMed

    Wong, Adrian Y C; Hristova, Elitza; Ahlskog, Nina; Tasse, Louis-Alexandre; Ngsee, Johnny K; Chudalayandi, Prakash; Bergeron, Richard

    2016-09-01

    The sigma-1 receptor (σ-1R) is an endoplasmic reticulum resident chaperone protein involved in a plethora of cellular functions, and whose disruption has been implicated in a wide range of diseases. Genetic analysis has revealed two σ-1R mutants involved in neuromuscular disorders. A point mutation (E102Q) in the ligand-binding domain results in the juvenile form of amyotrophic lateral sclerosis (ALS16), and a 20 amino-acid deletion (Δ31-50) in the putative cytosolic domain leads to a form of distal hereditary motor neuropathy. We investigated the localization and functional properties of these mutants in cell lines using confocal imaging and electrophysiology. The σ-1R mutants exhibited a significant increase in mobility, aberrant localization, and enhanced block of the inwardly rectifying K(+) channel Kir2.1, compared with the wild-type σ-1R. Thus, these σ-1R mutants have different functional properties that could contribute to their disease phenotypes. PMID:27418673

  8. Altered Sporulation and Respiratory Patterns in Mutants of Bacillus subtilis Induced by Acridine Orange

    PubMed Central

    Bott, K. F.; Davidoff-Abelson, R.

    1966-01-01

    Bott, K. F. (The University of Chicago, Chicago, Ill.), and R. Davidoff-Abelson. Altered sporulation and respiratory patterns in mutants of Bacillus subtilis induced by acridine orange. J. Bacteriol. 92:229–240. 1966.—The addition of acridine orange to vegetative cultures of Bacillus subtilis induces the formation of sporulation mutants at a frequency of 20% or greater. These mutants are grouped into seven categories which reflect their different morphological properties. They are altered in their vegetative metabolism, as indicated by abnormal growth on synthetic media. Sporulation of these mutants is impaired at several levels, all of which are stable upon repeated subculturing. The initial stages of sporulation which require no increased metabolic activity (proteolytic enzyme activity and antibiotic production) are functional in all strains, but glucose dehydrogenase activity, an enzyme associated with early synthetic functions in spore synthesis, is significantly reduced. Reduced nicotinamide adenine dinucleotide oxidase is slightly depressed. It is suggested that acridine orange interacts with a cellular constituent controlling respiration and consequently prevents an increased metabolic activity that may be associated with normal spore synthesis. Images PMID:4957434

  9. Changes in photosynthesis and pigmentation in an agp deletion mutant of the cyanobacterium Synechocystis sp.

    PubMed

    Miao, Xiaoling; Wu, Qingyu; Wu, Guifang; Zhao, Nanming

    2003-03-01

    The agp gene encoding ADP-glucose pyrophosphorylase is involved in cyanobacterial glycogen synthesis. By in vitro DNA recombination technology, agp deletion mutant (agp-) of cyanobacterium Synechocystis sp. PCC 6803 was constructed. This mutation led to a complete absence of glycogen biosynthesis. As compared with WT (wild type), a 60% decrease in ratio of the c-phycocyanine/chlorophyll a and no significant change in the carotenoid/chlorophyll a were observed in agp- cells. The agp- mutant had 38% less photosynthetic capacity when grown in light over 600 micromol m(-2) s(-1). Under lower light intensity, the final biomass of the mutant strain was only 1.1 times of that of the WT strain under mixotrophic condition after 6 d culture. Under higher light intensity, however, the final biomass of the WT strain under mixotrophic conditions was 3 times that of the mutant strain after 6 d culture and 1.5 times under photoautotrophic conditions. The results indicate that there is a minimum requirement for glycogen synthesis for normal growth and development in cyanobacteria. PMID:12882559

  10. Reduced leu operon expression in a miaA mutant of Salmonella typhimurium.

    PubMed Central

    Blum, P H

    1988-01-01

    Salmonella typhimurium miaA mutants lacking the tRNA base modification cis-2-methylthioribosylzeatin (ms2io6A) were examined and found to be sensitive to a variety of chemical oxidants and unable to grow aerobically at 42 degrees C in a defined medium. Leucine supplementation suppressed both of these phenotypes, suggesting that leucine synthesis was defective. Intracellular levels of leucine decreased 40-fold in mutant strains after a shift from 30 to 42 degrees C during growth, and expression of a leu-lacZ transcriptional fusion ceased. Steady-state levels of leu mRNA were also significantly reduced during growth at elevated temperatures. Failure of miaA mutant leu-lacZ expression to be fully derepressed during L-leucine limitation at 30 degrees C and suppression of the miaA mutation by a mutation in the S. typhimurium leu attenuator suggests that translational control of the transcription termination mechanism regulating leu expression is defective. Since the S. typhimurium miaA mutation was also suppressed by the Escherichia coli leu operon in trans, phenotypic differences between E. coli and S. typhimurium miaA mutants may result from a difference between their respective leu operons. Images PMID:3141379

  11. Root graviresponsiveness and cellular differentiation in wild-type and a starchless mutant of Arabidopsis thaliana

    NASA Technical Reports Server (NTRS)

    Moore, R.

    1989-01-01

    Primary roots of a starchless mutant of Arabidopsis thaliana L. are strongly graviresponsive despite lacking amyloplasts in their columella cells. The ultrastructures of calyptrogen and peripheral cells in wild-type as compared to mutant seedlings are not significantly different. The largest difference in cellular differentiation in caps of mutant and wild-type roots is the relative volume of plastids in columella cells. Plastids occupy 12.3% of the volume of columella cells in wild-type seedlings, but only 3.69% of columella cells in mutant seedlings. These results indicate that: (1) amyloplasts and starch are not necessary for root graviresponsiveness; (2) the increase in relative volume of plastids that usually accompanies differentiation of columella cells is not necessary for root graviresponsiveness; and (3) the absence of starch and amyloplasts does not affect the structure of calyptrogen (i.e. meristematic) and secretory (i.e. peripheral) cells in root caps. These results are discussed relative to proposed models for root gravitropism.

  12. A few key residues determine the high redox potential shift in azurin mutants.

    PubMed

    Zanetti-Polzi, Laura; Bortolotti, Carlo A; Daidone, Isabella; Aschi, Massimiliano; Amadei, Andrea; Corni, Stefano

    2015-12-01

    The wide range of variability of the reduction potential (E(0)) of blue-copper proteins has been the subject of a large number of studies in the past several years. In particular, a series of azurin mutants have been recently rationally designed tuning E(0) over a very broad range (700 mV) without significantly altering the redox-active site [Marshall et al., Nature, 2009, 462, 113]. This clearly suggests that interactions outside the primary coordination sphere are relevant to determine E(0) in cupredoxins. However, the molecular determinants of the redox potential variability are still undisclosed. Here, by means of atomistic molecular dynamics simulations and hybrid quantum/classical calculations, the mechanisms that determine the E(0) shift of two azurin mutants with high potential shifts are unravelled. The reduction potentials of native azurin and of the mutants are calculated obtaining results in good agreement with the experiments. The analysis of the simulations reveals that only a small number of residues (including non-mutated ones) are relevant in determining the experimentally observed E(0) variation via site-specific, but diverse, mechanisms. These findings open the path to the rational design of new azurin mutants with different E(0). PMID:26381463

  13. Glucose-induced production of recombinant proteins in Hansenula polymorpha mutants deficient in catabolite repression.

    PubMed

    Krasovska, Olena S; Stasyk, Olena G; Nahorny, Viktor O; Stasyk, Oleh V; Granovski, Nikolai; Kordium, Vitaliy A; Vozianov, Oleksandr F; Sibirny, Andriy A

    2007-07-01

    The most commonly used expression platform for production of recombinant proteins in the methylotrophic yeast Hansenula polymorpha relies on the strong and strictly regulated promoter from the gene encoding peroxisomal enzyme alcohol (or methanol) oxidase (P(MOX)). Expression from P(MOX) is induced by methanol and is partially derepressed in glycerol or xylose medium, whereas in the presence of hexoses, disaccharides or ethanol, it is repressed. The need for methanol for maximal induction of gene expression in large-scale fermentation is a significant drawback, as this compound is toxic, flammable, supports a slow growth rate and requires extensive aeration. We isolated H. polymorpha mutants deficient in glucose repression of P(MOX) due to an impaired HpGCR1 gene, and other yet unidentified secondary mutations. The mutants exhibited pronounced defects in P(MOX) regulation only by hexoses and xylose, but not by disaccharides or ethanol. With one of these mutant strains as hosts, we developed a modified two-carbon source mode expression platform that utilizes convenient sugar substrates for growth (sucrose) and induction of recombinant protein expression (glucose or xylose). We demonstrate efficient regulatable by sugar carbon sources expression of three recombinant proteins: a secreted glucose oxidase from the fungus Aspergillus niger, a secreted mini pro-insulin, and an intracellular hepatitis B virus surface antigen in these mutant hosts. The modified expression platform preserves the favorable regulatable nature of P(MOX) without methanol, making a convenient alternative to the traditional system. PMID:17163508

  14. TORC1 Suppression Predicts Responsiveness to RAF and MEK Inhibition in BRAF-Mutant Melanoma

    PubMed Central

    Corcoran, Ryan B.; Rothenberg, Stephen Michael; Hata, Aaron N.; Faber, Anthony C.; Piris, Adriano; Nazarian, Rosalynn M.; Brown, Ronald D.; Godfrey, Jason T.; Winokur, Daniel; Walsh, John; Mino-Kenudson, Mari; Maheswaran, Shyamala; Settleman, Jeffrey; Wargo, Jennifer A.; Flaherty, Keith T.; Haber, Daniel A.; Engelman, Jeffrey A.

    2013-01-01

    RAF and MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase) inhibitors are effective in treating patients with BRAF-mutant melanoma. However, most responses are partial and short-lived, and many patients fail to respond at all. We found that suppression of TORC1 activity in response to RAF or MEK inhibitors, as measured by decreased phosphorylation of ribosomal protein S6 (P-S6), effectively predicted induction of cell death by the inhibitor in BRAF-mutant melanoma cell lines. In resistant melanomas, TORC1 activity was maintained after treatment with RAF or MEK inhibitors, in some cases despite robust suppression of mitogen-activated protein kinase (MAPK) signaling. In in vivo mouse models, suppression of TORC1 after MAPK inhibition was necessary for induction of apoptosis and tumor response. Finally, in paired biopsies obtained from patients with BRAF-mutant melanoma before treatment and after initiation of RAF inhibitor therapy, P-S6 suppression predicted significantly improved progression-free survival. Such a change in P-S6 could be readily monitored in real time by serial fine-needle aspiration biopsies, making quantitation of P-S6 a valuable biomarker to guide treatment in BRAF-mutant melanoma. PMID:23903755

  15. KRAS-Mutant Lung Cancers in the Era of Targeted Therapy.

    PubMed

    Naidoo, Jarushka; Drilon, Alexander

    2016-01-01

    KRAS-mutant lung cancers account for approximately 25% of non-small cell lung carcinomas, thus representing an enormous burden of cancer worldwide. KRAS mutations are clear drivers of tumor growth and are characterized by a complex biology involving the interaction between mutant KRAS, various growth factor pathways, and tumor suppressor genes. While KRAS mutations are classically associated with a significant smoking history, they are also identified in a substantial proportion of never-smokers. These mutations are found largely in lung adenocarcinomas with solid growth patterns and tumor-infiltrating lymphocytes. A variety of tools are available for diagnosis including Sanger sequencing, multiplex mutational hotspot profiling, and next-generation sequencing. The prognostic and predictive roles of KRAS status remain controversial. It has become increasingly clear, however, that KRAS mutations drive primary resistance to EGFR tyrosine kinase inhibition. Until recently, mutant KRAS was not thought of as a clinically-targetable driver in lung cancers. With the expansion of our knowledge regarding the biology of KRAS-mutant lung cancers and the role of MEK and PI3K/mTOR inhibition, the face of targeted therapeutics for this genomic subset of patients is slowly beginning to change. PMID:26667343

  16. Global carbon utilization profiles of wild-type, mutant, and transformant strains of Hypocrea jecorina.

    PubMed

    Druzhinina, Irina S; Schmoll, Monika; Seiboth, Bernhard; Kubicek, Christian P

    2006-03-01

    The ascomycete Hypocrea jecorina (Trichoderma reesei), an industrial producer of cellulases and hemicellulases, can efficiently degrade plant polysaccharides. However, the catabolic pathways for the resulting monomers and their relationship to enzyme induction are not well known. Here we used the Biolog Phenotype MicroArrays technique to evaluate the growth of H. jecorina on 95 carbon sources. For this purpose, we compared several wild-type isolates, mutants producing different amounts of cellulases, and strains transformed with a heterologous antibiotic resistance marker gene. The wild-type isolates and transformed strains had the highest variation in growth patterns on individual carbon sources. The cellulase mutants were relatively similar to their parental strains. Both in the mutant and in the transformed strains, the most significant changes occurred in utilization of xylitol, erythritol, D-sorbitol, D-ribose, D-galactose, L-arabinose, N-acetyl-D-glucosamine, maltotriose, and beta-methyl-glucoside. Increased production of cellulases was negatively correlated with the ability to grow on gamma-aminobutyrate, adonitol, and 2-ketogluconate; and positively correlated with that on d-sorbitol and saccharic acid. The reproducibility, relative simplicity, and high resolution (+/-10% of increase in mycelial density) of the phenotypic microarrays make them a useful tool for the characterization of mutant and transformed strains and for a global analysis of gene function. PMID:16517662

  17. Exoprotein Production Correlates with Morphotype Changes of Nonmotile Shewanella oneidensis Mutants

    PubMed Central

    Shi, Miaomiao; Wu, Lin; Xia, Yu; Chen, Haijiang; Luo, Qixia; Sun, Linlin

    2013-01-01

    We report a previously undescribed mechanism for the rugose morphotype in Shewanella oneidensis, a research model for investigating redox transformations of environmental contaminants. Bacteria may form smooth or rugose colonies on agar plates. In general, conversion from the smooth to rugose colony morphotype is attributed to increased production of exopolysaccharide (EPS). In this work, we discovered that aflagellate S. oneidensis mutants grew into rugose colonies, whereas those with nonfunctional flagellar filaments remained smooth. EPS production was not altered in either case, but mutants with the rugose morphotype showed significantly reduced exoprotein secretion. The idea that exoproteins at a reduced level correlate with rugosity gained support from smooth suppressor strains of an aflagellate rugose fliD (encoding the capping protein) mutant, which restored the exoprotein level to the levels of the wild-type and mutant strains with a smooth morphotype. Further analyses revealed that SO1072 (a putative GlcNAc-binding protein) was one of the highly upregulated exoproteins in these suppressor strains. Most intriguingly, this study identified a compensatory mechanism of SO1072 to flagellins possibly mediated by bis-(3′-5′)-cyclic dimeric GMP. PMID:23335418

  18. Conformational stability and warfarin-binding properties of human serum albumin studied by recombinant mutants.

    PubMed Central

    Watanabe, H; Kragh-Hansen, U; Tanase, S; Nakajou, K; Mitarai, M; Iwao, Y; Maruyama, T; Otagiri, M

    2001-01-01

    Correctly folded recombinant wild-type human serum albumin and the single-residue mutants K199A, W214A, R218H and H242Q were produced with the use of a yeast expression system. The changes in R218H resulted in a pronounced decrease in intrinsic fluorescence. Thermodynamic parameters for thermal denaturation of the present mutants and of five additional mutants have been determined, showing small increases in stability for two mutants (R218H and H242Q) and a larger decrease in stability for one (W214A). In the last of these, denaturation was a heterogeneous process starting at physiological temperature. The high-affinity binding constant for warfarin at pH 7.4 was determined by fluorescence spectroscopy: there was a significant increase in affinity for binding of warfarin to H242Q and K199A and a smaller decrease in affinity for W214A and R218H. The findings show that Trp-214 is not as essential for the high-affinity binding of warfarin as has previously been thought. PMID:11415459

  19. Antibiotic cross-resistance patterns of ambodryl and promazine resistant mutants.

    PubMed Central

    Ray, S.; Dastidar, S. G.; Chakrabarty, A. N.

    1980-01-01

    The antihistaminic drug ambodryl (Am) and the tranquillizer promazine (Pr), which had previously shown significant antimicrobial activities, were used to select bacterial mutants representing different levels of resistance to their action. These Am and Pr mutants were found to have simultaneously developed cross-resistance to penicillin (Pe), streptomycin (Sm), chloramphenicol (Cm), tetracycline (Tc) and kanamycin (Km). Certain first-step Am mutants of sensitive wild-type bacteria which were also sensitive to these antibiotics developed resistance to Pe and Sm as well as to Pr and the antihistamine benadryl (Bn). The next-step mutant of Am resistance not only doubled its resistance to these antibiotics and chemotherapeutics, but broadened its resistance-spectrum to cover Tc and Cm. These observations on cross-resistances seem to be best explained on the basis of a reduction in cell-membrane permeability acting in a nonspecific manner. The role of such cross-resistance in accounting for an overall increase in the minimum inhibitory concentrations (MIC) of many drugs against several groups of bacteria is evaluated. PMID:6108782

  20. TLR4 mutant mice are protected from renal fibrosis and chronic kidney disease progression

    PubMed Central

    Souza, Ana C P; Tsuji, Takayuki; Baranova, Irina N; Bocharov, Alexander V; Wilkins, Kenneth J; Street, Jonathan M; Alvarez-Prats, Alejandro; Hu, Xuzhen; Eggerman, Thomas; Yuen, Peter S T; Star, Robert A

    2015-01-01

    Chronic kidney disease (CKD) is associated with persistent low-grade inflammation and immunosuppression. In this study we tested the role of Toll-like receptor 4, the main receptor for endotoxin (LPS), in a mouse model of renal fibrosis and in a model of progressive CKD that better resembles the human disease. C3HeJ (TLR4 mutant) mice have a missense point mutation in the TLR4 gene, rendering the receptor nonfunctional. In a model of renal fibrosis after folic acid injection, TLR4 mutant mice developed less interstititial fibrosis in comparison to wild-type (WT) mice. Furthermore, 4 weeks after 5/6 nephrectomy with continuous low-dose angiotensin II infusion, C3HeOuJ (TLR4 WT) mice developed progressive CKD with albuminuria, increased serum levels of BUN and creatinine, glomerulosclerosis, and interstitial fibrosis, whereas TLR4 mutant mice were significantly protected from CKD progression. TLR4 WT mice also developed low-grade systemic inflammation, splenocyte apoptosis and increased expression of the immune inhibitory receptor PD-1 in the spleen, which were not observed in TLR4 mutant mice. In vitro, endotoxin (LPS) directly upregulated NLRP3 inflammasome expression in renal epithelial cells via TLR4. In summary, TLR4 contributes to renal fibrosis and CKD progression, at least in part, via inflammasome activation in renal epithelial cells, and may also participate in the dysregulated immune response that is associated with CKD. PMID:26416975

  1. Phospholipase D δ knock-out mutants are tolerant to severe drought stress

    PubMed Central

    Distéfano, Ayelen M; Valiñas, Matías A; Scuffi, Denise; Lamattina, Lorenzo; ten Have, Arjen; García-Mata, Carlos; Laxalt, Ana M

    2015-01-01

    Phospholipase D (PLD) is involved in different plant processes, ranging from responses to abiotic and biotic stress to plant development. Phospholipase Dδ (PLDδ) is activated in dehydration and salt stress, producing the lipid second messenger phosphatidic acid. In this work we show that pldδ Arabidopsis mutants were more tolerant to severe drought than wild-type plants. PLDδ has been shown to be required for ABA regulation of stomatal closure of isolated epidermal peels. However, there was no significant difference in stomatal conductance at the whole plant level between wild-type and pldδ mutants. Since PLD hydrolyses structural phospholipids, then we looked at membrane integrity. Ion leakage measurements showed that during dehydration of leaf discs pldδ mutant has less membrane degradation compared to the wild-type. We further analyzed the mutants and showed that pldδ have higher mRNA levels of RAB18 and RD29A compared to wild-type plants under normal growth conditions. Transient expression of AtPLDδ in Nicotiana benthamiana plants induced a wilting phenotype. These findings suggest that, in wt plants PLDδ disrupt membranes in severe drought stress and, in the absence of the protein (PLDδ knock-out) might drought-prime the plants, making them more tolerant to severe drought stress. The results are discussed in relation to PLDδ role in guard cell signaling and drought tolerance. PMID:26340512

  2. Plastid sedimentation kinetics in roots of wild-type and starch-deficient mutants of Arabidopsis

    NASA Technical Reports Server (NTRS)

    MacCleery, S. A.; Kiss, J. Z.

    1999-01-01

    Sedimentation and movement of plastids in columella cells of the root cap were measured in seedlings of wild-type, a reduced starch mutant, and a starchless mutant of Arabidopsis. To assay for sedimentation, we used both linear measurements and the change of angle from the cell center as indices in vertical and reoriented plants with the aid of computer-assisted image analysis. Seedlings were fixed at short periods after reorientation, and plastid sedimentation correlated with starch content in the three strains of Arabidopsis. Amyloplasts of wild-type seedlings showed the greatest sedimentation, whereas plastids of the starchless mutant showed no significant sedimentation in the vertically grown and reoriented seedlings. Because previous research has shown that a full complement of starch is needed for full gravitropic sensitivity, this study correlates increased sensitivity with plastid sedimentation. However, although plastid sedimentation contributed to gravisensitivity, it was not required, because the gravitropic starchless mutant had plastids that did not sediment. This is the first study, to our knowledge, to measure plastid sedimentation in Arabidopsis roots after reorientation of seedlings. Taken together, the results of this study are consistent with the classic plastid-based and protoplast-based models of graviperception and suggest that multiple systems of perception exist in plant cells.

  3. Comparative metabolic profiling of mce1 operon mutant vs wild-type Mycobacterium tuberculosis strains.

    PubMed

    Queiroz, Adriano; Medina-Cleghorn, Daniel; Marjanovic, Olivera; Nomura, Daniel K; Riley, Lee W

    2015-11-01

    Mycobacterium tuberculosis disrupted in a 13-gene operon (mce1) accumulates free mycolic acids (FM) in its cell wall and causes accelerated death in mice. Here, to more comprehensively analyze differences in their cell wall lipid composition, we used an untargeted metabolomics approach to compare the lipid profiles of wild-type and mce1 operon mutant strains. By liquid chromatography-mass spectrometry, we identified >400 distinct lipids significantly altered in the mce1 mutant compared to wild type. These lipids included decreased levels of saccharolipids and glycerophospholipids, and increased levels of alpha-, methoxy- and keto mycolic acids (MA), and hydroxyphthioceranic acid. The mutant showed reduced expression of mmpL8, mmpL10, stf0, pks2 and papA2 genes involved in transport and metabolism of lipids recognized to induce proinflammatory response; these lipids were found to be decreased in the mutant. In contrast, the transcripts of mmpL3, fasI, kasA, kasB, acpM and RV3451 involved in MA transport and metabolism increased; MA inhibits inflammatory response in macrophages. Since the mce1 operon is known to be regulated in intracellular M. tuberculosis, we speculate that the differences we observed in cell wall lipid metabolism and composition may affect host response to M. tuberculosis infection and determine the clinical outcome of such an infection. PMID:26319139

  4. Kinetic properties and thermal stabilities of mutant forms of mitochondrial aspartate aminotransferase.

    PubMed

    Azzariti, A; Vacca, R A; Giannattasio, S; Merafina, R S; Marra, E; Doonan, S

    1998-07-28

    Kinetic properties and thermal stabilities of the precursor form of mitochondrial aspartate aminotransferase, the mature form lacking 9 amino acids from the N-terminus, and forms of the mature protein in which cysteine-166 had been mutated to serine or alanine were compared with those of the mature enzyme. The precursor and the cysteine mutants showed moderately impaired catalytic properties consistent with decreased ability to undergo transition from the open to the closed conformation which is an integral part of the mechanism of action of the enzyme. The deletion mutant had a kcat only 2% of that of the mature enzyme but also much reduced Km values for both substrates. In addition it showed enhanced reactivity of cysteine-166 with 5,5'-dithiobis(2-nitrobenzoate), which is characteristic of the closed form of the enzyme, with no enhancement of reactivity in the presence of substrates. This is taken to show that the deletion mutant adopts a conformation that is significantly different from that of the mature enzyme particularly in respect of the small domain. The deletion mutant was found to be more resistant to thermal inactivation over a range of temperatures than were the other forms of the enzyme consistent with its having a more tightly packed small domain. PMID:9675237

  5. Mutant KRAS Conversion of Conventional T Cells into Regulatory T Cells.

    PubMed

    Zdanov, Stephanie; Mandapathil, Magis; Abu Eid, Rasha; Adamson-Fadeyi, Saudat; Wilson, Willie; Qian, Jiahua; Carnie, Andrea; Tarasova, Nadya; Mkrtichyan, Mikayel; Berzofsky, Jay A; Whiteside, Theresa L; Khleif, Samir N

    2016-04-01

    Constitutive activation of theKRASoncogene in human malignancies is associated with aggressive tumor growth and poor prognosis. Similar to other oncogenes,KRASacts in a cell-intrinsic manner to affect tumor growth or survival. However, we describe here a different, cell-extrinsic mechanism through which mutant KRAS contributes to tumor development. Tumor cells carrying mutated KRAS induced highly suppressive T cells, and silencing KRAS reversed this effect. Overexpression of the mutantKRAS(G12V)gene in wild-type KRAS tumor cells led to regulatory T-cell (Treg) induction. We also demonstrate that mutant KRAS induces the secretion of IL10 and transforming growth factor-β1 (both required for Treg induction) by tumor cells through the activation of the MEK-ERK-AP1 pathway. Finally, we report that inhibition of KRAS reduces the infiltration of Tregs in KRAS-driven lung tumorigenesis even before tumor formation. This cell-extrinsic mechanism allows tumor cells harboring a mutantKRASoncogene to escape immune recognition. Thus, an oncogene can promote tumor progression independent of its transforming activity by increasing the number and function of Tregs. This has a significant clinical potential, in which targeting KRAS and its downstream signaling pathways could be used as powerful immune modulators in cancer immunotherapy.Cancer Immunol Res; 4(4); 354-65. ©2016 AACR. PMID:26880715

  6. An In Vivo Quantitative Comparison of Photoprotection in Arabidopsis Xanthophyll Mutants.

    PubMed

    Ware, Maxwell A; Dall'Osto, Luca; Ruban, Alexander V

    2016-01-01

    Contribution of different LHCII antenna carotenoids to protective NPQ (pNPQ) were tested using a range of xanthophyll biosynthesis mutants of Arabidopsis: plants were either devoid of lutein (lut2), violaxanthin (npq2), or synthesized a single xanthophyll species, namely violaxanthin (aba4npq1lut2), zeaxanthin (npq2lut2), or lutein (chy1chy2lut5). A novel pulse amplitude modulated (PAM) fluorescence analysis procedure, that used a gradually increasing actinic light intensity, allowed the efficiency of pNPQ to be tested using the photochemical quenching (qP) parameter measured in the dark (qPd). Furthermore, the yield of photosystem II (ΦPSII) was calculated, and the light intensity which induces photoinhibition in 50% of leaves for each mutant was ascertained. Photoprotective capacities of each xanthophyll were quantified, taking into account chlorophyll a/b ratios and excitation pressure. Here, light tolerance, pNPQ capacity, and ΦPSII were highest in wild type plants. Of the carotenoid mutants, lut2 (lutein-deficient) plants had the highest light tolerance, and the joint the highest ΦPSII with violaxanthin only plants. We conclude that all studied mutants possess pNPQ and a more complete composition of xanthophylls in their natural binding sites is the most important factor governing photoprotection, rather than any one specific xanthophyll suggesting a strong structural effect of the molecules upon the LHCII antenna organization and discuss the results significance for future crop development. PMID:27446097

  7. Isolation and characterization of a mutant defective in triacylglycerol accumulation in nitrogen-starved Chlamydomonas reinhardtii.

    PubMed

    Hung, Chun-Hsien; Kanehara, Kazue; Nakamura, Yuki

    2016-09-01

    Triacylglycerol (TAG), a major source of biodiesel production, accumulates in nitrogen-starved Chlamydomonas reinhardtii. However, the metabolic pathway of starch-to-TAG conversion remains elusive because an enzyme that affects the starch degradation is unknown. Here, we isolated a new class of mutant bgal1, which expressed an overaccumulation of starch granules and defective photosynthetic growth. The bgal1 was a null mutant of a previously uncharacterized β-galactosidase-like gene (Cre02.g119700), which decreased total β-galactosidase activity 40% of the wild type. Upon nitrogen starvation, the bgal1 mutant showed decreased TAG accumulation mainly due to the reduced flux of de novo TAG biosynthesis evidenced by increased unsaturation of fatty acid composition in TAG and reduced TAG accumulation by additional supplementation of acetate to the culture media. Metabolomic analysis of the bgal1 mutant showed significantly reduced levels of metabolites following the hydrolysis of starch and substrates for TAG accumulation, whereas metabolites in TCA cycle were unaffected. Upon nitrogen starvation, while levels of glucose 6-phosphate, fructose 6-phosphate and acetyl-CoA remained lower, most of the other metabolites in glycolysis were increased but those in the TCA cycle were decreased, supporting TAG accumulation. We suggest that BGAL1 may be involved in the degradation of starch, which affects TAG accumulation in nitrogen-starved C. reinhardtii. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. PMID:27060488

  8. Phospholipase D δ knock-out mutants are tolerant to severe drought stress.

    PubMed

    Distéfano, Ayelen M; Valiñas, Matías A; Scuffi, Denise; Lamattina, Lorenzo; Ten Have, Arjen; García-Mata, Carlos; Laxalt, Ana M

    2015-01-01

    Phospholipase D (PLD) is involved in different plant processes, ranging from responses to abiotic and biotic stress to plant development. Phospholipase Dδ (PLDδ) is activated in dehydration and salt stress, producing the lipid second messenger phosphatidic acid. In this work we show that pldδ Arabidopsis mutants were more tolerant to severe drought than wild-type plants. PLDδ has been shown to be required for ABA regulation of stomatal closure of isolated epidermal peels. However, there was no significant difference in stomatal conductance at the whole plant level between wild-type and pldδ mutants. Since PLD hydrolyses structural phospholipids, then we looked at membrane integrity. Ion leakage measurements showed that during dehydration of leaf discs pldδ mutant has less membrane degradation compared to the wild-type. We further analyzed the mutants and showed that pldδ have higher mRNA levels of RAB18 and RD29A compared to wild-type plants under normal growth conditions. Transient expression of AtPLDδ in Nicotiana benthamiana plants induced a wilting phenotype. These findings suggest that, in wt plants PLDδ disrupt membranes in severe drought stress and, in the absence of the protein (PLDδ knock-out) might drought-prime the plants, making them more tolerant to severe drought stress. The results are discussed in relation to PLDδ role in guard cell signaling and drought tolerance. PMID:26340512

  9. Phenotypic classes of phenoloxidase-negative mutants of the lignin-degrading fungus Phanerochaete chrysosporium

    SciTech Connect

    Liwicki, R.; Paterson, A.; MacDonald, M.J.; Broda, P.

    1985-05-01

    This paper reports the isolation of phenoloxidase-negative mutants of the white-rot fungus Phanerochaete chrysosporium and the results of a survey of idiophasic functions among these mutants. The mutant strains were isolated from a medium containing o-anisidine after gamma irradiation of wild-type spores and fell into four classes, divided by the manner in which they mineralized /sup 14/C-lignin wheat lignocellulose. Examples are strain LMT7, which degraded lignin at a rate similar to that of the wild type; strain LMT26, in which degradation was enhanced; strain LMT16, whose degradation rate was apparently unaffected, although the onset of lignin attack was delayed compared with that in the wild type; and strain LMT24, which was unable to evolve significant amounts of /sup 14/CO/sub 2/ from the radiolabeled substrate. The mutants were not necessarily defective in other functions associated with idiophasic activities (intracellular cyclic AMP levels, sporulation, extracellular glucan production, veratryl alcohol synthesis). The authors conclude that phenoloxidase activity as detected by the o-anisidine plate test is not necessary for lignin degradation. In addition, mutations resulting in the loss of lignin-degrading ability were not necessarily pleiotropic with other idiophasic functions.

  10. Quantitative Analysis of Mutant Subclones in Chronic Myeloid Leukemia: Comparison of Different Methodological Approaches.

    PubMed

    Preuner, Sandra; Barna, Agnes; Frommlet, Florian; Czurda, Stefan; Konstantin, Byrgazov; Alikian, Mary; Machova Polakova, Katerina; Sacha, Tomasz; Richter, Johan; Lion, Thomas; Gabriel, Christian

    2016-01-01

    Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points. PMID:27136541

  11. Quantitative Analysis of Mutant Subclones in Chronic Myeloid Leukemia: Comparison of Different Methodological Approaches

    PubMed Central

    Preuner, Sandra; Barna, Agnes; Frommlet, Florian; Czurda, Stefan; Konstantin, Byrgazov; Alikian, Mary; Machova Polakova, Katerina; Sacha, Tomasz; Richter, Johan; Lion, Thomas; Gabriel, Christian

    2016-01-01

    Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points. PMID:27136541

  12. The kinetics of root gravitropism in PIN mutants suggest redundancy in the signal transduction pathway

    NASA Astrophysics Data System (ADS)

    Wolverton, Chris

    As nonmotile organisms, plants rely on differential growth responses to maximize exposure to the resources necessary for growth and reproduction. One of the primary environmental cues causing differential growth in roots is gravity, which is thought to be sensed predominately in the root cap. This gravity perception event is thought to be transduced into information in the form of an auxin gradient across the cap and propagating basipetally toward the elongation zone. The discovery of several families of auxin efflux and influx carriers has provided significant insight into the mechanisms of directional auxin transport, and the identification of mutants in the genes encoding these carriers provides the opportunity to test the roles of these transporters in plant gravitropism. In this study, we report the results of a systematic, high-resolution study of the kinetics of root gravitropism of mutants in the PIN family of auxin efflux carriers. Based on reported expression and localization patterns, we predicted mutations in PIN2, PIN3, PIN4, and PIN7 to cause the greatest reduction in root gravitropism. While pin2 mutants showed severe gravitropic deficiencies in roots as reported previously, several alleles of pin3, pin4 and pin7 remained strongly gravitropic. PIN3 has been localized to the central columella cells, the purported gravisensing cells in the root, and shown to rapidly relocate to the lower flank of the columella cells upon gravistimulation, suggesting an early role in auxin gradient formation. Mutant alleles of PIN3 showed an early delay in response, with just 7 deg of curvature in the first hour compared to approximately 15 deg h-1 in wild-type, but their rate of curvature recovered to near wild-type levels over the ensuing 3 h. Pin3 mutants also showed a slower overall growth rate (124 µm h-1 ), elongating at approximately half the rate of wild-type roots (240 µm h-1 ). PIN4 has been localized to the quiescent center in the root, where it presumably

  13. Preclinical efficacy of MEK inhibition in Nras-mutant AML

    PubMed Central

    Burgess, Michael R.; Hwang, Eugene; Firestone, Ari J.; Huang, Tannie; Xu, Jin; Zuber, Johannes; Bohin, Natacha; Wen, Tiffany; Kogan, Scott C.; Haigis, Kevin M.; Sampath, Deepak; Lowe, Scott; Shannon, Kevin

    2014-01-01

    Oncogenic NRAS mutations are highly prevalent in acute myeloid leukemia (AML). Genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis, but have limited independent prognostic significance. Using short hairpin RNA–mediated knockdown in human cell lines and primary mouse leukemias, we show that AML cells with NRAS/Nras mutations are dependent on continued oncogene expression in vitro and in vivo. Using the Mx1-Cre transgene to inactivate a conditional mutant Nras allele, we analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPCs) under normal and stressed conditions and found that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Treating recipient mice transplanted with primary NrasG12D AMLs with 2 potent allosteric mitogen-activated protein kinase kinase (MEK) inhibitors (PD0325901 or trametinib/GlaxoSmithKline 1120212) significantly prolonged survival and reduced proliferation but did not induce apoptosis, promote differentiation, or drive clonal evolution. The phosphatidylinositol 3-kinase inhibitor GDC-0941 was ineffective as a single agent and did not augment the activity of PD0325901. All mice ultimately succumbed to progressive leukemia. Together, these data validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors. PMID:25361812

  14. Dystonia and Cerebellar Degeneration in the Leaner Mouse Mutant

    PubMed Central

    Raike, Robert S.; Hess, Ellen J.; Jinnah, H.A.

    2015-01-01

    Cerebellar degeneration is traditionally associated with ataxia. Yet, there are examples of both ataxia and dystonia occurring in individuals with cerebellar degeneration. There is also substantial evidence suggesting that cerebellar dysfunction alone may cause dystonia. The types of cerebellar defects that may cause ataxia, dystonia, or both have not been delineated. In the current study, we explored the relationship between cerebellar degeneration and dystonia using the leaner mouse mutant. Leaner mice have severe dystonia that is associated with dysfunctional and degenerating cerebellar Purkinje cells. Whereas the density of Purkinje cells was not significantly reduced in 4 week-old leaner mice, approximately 50% of the neurons were lost by 34 weeks of age. On the other hand, the dystonia and associated functional disability became significantly less severe during this same interval. In other words, dystonia improved as Purkinje cells were lost, suggesting that dysfunctional Purkinje cells, rather than Purkinje cell loss, contribute to the dystonia. These results provide evidence that distorted cerebellar function may cause dystonia and support the concept that different types of cerebellar defects can have different functional consequences. PMID:25791619

  15. Natural Variation of Model Mutant Phenotypes in Ciona intestinalis

    PubMed Central

    Brown, Euan R.; Leccia, Nicola I.; Squarzoni, Paola; Tarallo, Raffaella; Alfano, Christian; Caputi, Luigi; D'Ambrosio, Palmira; Daniele, Paola; D'Aniello, Enrico; D'Aniello, Salvatore; Maiella, Sylvie; Miraglia, Valentina; Russo, Monia Teresa; Sorrenti, Gerarda; Branno, Margherita; Cariello, Lucio; Cirino, Paola; Locascio, Annamaria; Spagnuolo, Antonietta; Zanetti, Laura; Ristoratore, Filomena

    2008-01-01

    Background The study of ascidians (Chordata, Tunicata) has made a considerable contribution to our understanding of the origin and evolution of basal chordates. To provide further information to support forward genetics in Ciona intestinalis, we used a combination of natural variation and neutral population genetics as an approach for the systematic identification of new mutations. In addition to the significance of developmental variation for phenotype-driven studies, this approach can encompass important implications in evolutionary and population biology. Methodology/Principal Findings Here, we report a preliminary survey for naturally occurring mutations in three geographically interconnected populations of C. intestinalis. The influence of historical, geographical and environmental factors on the distribution of abnormal phenotypes was assessed by means of 12 microsatellites. We identified 37 possible mutant loci with stereotyped defects in embryonic development that segregate in a way typical of recessive alleles. Local populations were found to differ in genetic organization and frequency distribution of phenotypic classes. Conclusions/Significance Natural genetic polymorphism of C. intestinalis constitutes a valuable source of phenotypes for studying embryonic development in ascidians. Correlating genetic structure and the occurrence of abnormal phenotypes is a crucial focus for understanding the selective forces that shape natural finite populations, and may provide insights of great importance into the evolutionary mechanisms that generate animal diversity. PMID:18523552

  16. Methods of producing protoporphyrin IX and bacterial mutants therefor

    DOEpatents

    Zhou, Jizhong; Qiu, Dongru; He, Zhili; Xie, Ming

    2016-03-01

    The presently disclosed inventive concepts are directed in certain embodiments to a method of producing protoporphyrin IX by (1) cultivating a strain of Shewanella bacteria in a culture medium under conditions suitable for growth thereof, and (2) recovering the protoporphyrin IX from the culture medium. The strain of Shewanella bacteria comprises at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX. In certain embodiments of the method, the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or of shew_1140. In other embodiments, the presently disclosed inventive concepts are directed to mutant strains of Shewanella bacteria having at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX during cultivation of the bacteria. In certain embodiments the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or shew_1140.

  17. A computational study of λ-lac mutants

    NASA Astrophysics Data System (ADS)

    Werner, Maria; Aurell, Erik

    2009-12-01

    We present a comprehensive, computational study of the properties of bacteriophage λ mutants designed by Atsumi and Little (2006 Proc. Natl. Acad. Sci. 103 4558-63). These phages underwent a genetic reconstruction where Cro was replaced by a dimeric form of the Lac repressor. To clarify the theoretical characteristics of these mutants, we built a detailed thermodynamic model. The mutants all have a different genetic wiring than the wild-type λ. One group lacks regulation of PRM by the lytic protein. These mutants only exhibit the lysogenic equilibrium, with no transiently active PR. The other group lacks the negative feedback from CI. In this group, we identify a handful of bi-stable mutants, although the majority only exhibit the lysogenic equilibrium. The experimental identification of functional phages differs from our predictions. From a theoretical perspective, there is no reason why only 4 out of 900 mutants should be functional. The differences between theory and experiment can be explained in two ways. Either, the view of the λ phage as a bi-stable system needs to be revised, or the mutants have in fact not undergone a modular replacement, as intended by Atsumi and Little, but constitute instead a wider systemic change.

  18. Characterization of fig operon mutants of Francisella novicida U112

    PubMed Central

    Kiss, Katalin; Liu, Wei; Huntley, Jason F.; Norgard, Michael V.; Hansen, Eric J.

    2009-01-01

    Francisella species secrete a polycarboxylate siderophore that resembles rhizoferrin to acquire ferric iron. Several of the Francisella siderophore synthesis genes are contained in a Fur-regulated operon (designated fig or fsl) comprised of at least seven open reading frames (ORFs) including fur. Reverse transcriptase-PCR showed transcriptional linkage between figD and figE and between figE and figF. Mutations were constructed in four of these ORFs (figB, figC, figD, and figE) in F. novicida U112. All four of these new mutants and a F. novicida figA mutant grew at rates comparable to that of wild-type under iron-replete conditions but growth of all five mutants was stunted in iron-limiting media. When ferric rhizoferrin was added to the iron-limited media, growth of the figA, figB, figC, and figD mutants was restored to levels similar to those obtained in iron-replete media. However, this exogenously added siderophore could not rescue the figE mutant. When Chrome Azurol S assays were used to measure siderophore production, the figA, figB, and figC mutants were markedly deficient in their ability to synthesize siderophore whereas the figD and figE mutants produced siderophore at levels equivalent to the wild-type parent strain. PMID:18564336

  19. New Salmonella typhimurium mutants with altered outer membrane permeability.

    PubMed Central

    Sukupolvi, S; Vaara, M; Helander, I M; Viljanen, P; Mäkelä, P H

    1984-01-01

    We describe three new classes of Salmonella typhimurium mutants with increased sensitivity to hydrophobic agents. In contrast to many previously described mutants, the phage sensitivity pattern of these mutants did not give any indication of defective lipopolysaccharide. Furthermore, they had no detectable changes in their phospholipid or outer membrane protein composition, and their growth rate and cell morphology were normal. Class B mutants were nearly as sensitive to novobiocin, fusidic acid, erythromycin, rifampin, and clindamycin as are deep rough (heptoseless) mutants; in addition they were sensitive to methicillin, penicillin (to which heptoseless mutants are resistant), gentian violet, and anionic and cationic detergents. Class A and C mutants had less sensitive, but characteristic phenotypes. None of the three classes were sensitive to serum bactericidal action. The class B mutation mapped between map positions 7 and 11 on the S. typhimurium chromosome, and the class C mutation mapped between positions 5 and 7. The map position for the class A mutation remained undefined, but it was separate from the class B and C mutations and, like those, did not correspond to any gene loci known to participate in the synthesis of major outer membrane constituents. Images PMID:6378889

  20. Mapping Pathological Phenotypes in Reelin Mutant Mice

    PubMed Central

    Michetti, Caterina; Romano, Emilia; Altabella, Luisa; Caruso, Angela; Castelluccio, Paolo; Bedse, Gaurav; Gaetani, Silvana; Canese, Rossella; Laviola, Giovanni; Scattoni, Maria Luisa

    2014-01-01

    Autism Spectrum Disorders (ASD) are neurodevelopmental disorders with multifactorial origin characterized by social communication deficits and the presence of repetitive behaviors/interests. Several studies showed an association between the reelin gene mutation and increased risk of ASD and a reduced reelin expression in some brain regions of ASD subjects, suggesting a role for reelin deficiency in ASD etiology. Reelin is a large extracellular matrix glycoprotein playing important roles during development of the central nervous system. To deeply investigate the role of reelin dysfunction as vulnerability factor in ASD, we assessed the behavioral, neurochemical, and brain morphological features of reeler male mice. We recently reported a genotype-dependent deviation in the ultrasonic vocal repertoire and a general delay in motor development of reeler pups. We now report that adult male heterozygous (Het) reeler mice did not show social behavior and communication deficits during male–female social interactions. Wildtype and Het mice showed a typical light/dark locomotor activity profile, with a peak during the central interval of the dark phase. However, when faced with a mild stressful stimulus (a saline injection) only Het mice showed an over response to stress. In addition to the behavioral studies, we conducted high performance liquid chromatography and magnetic resonance imaging and spectroscopy to investigate whether reelin mutation influences brain monoamine and metabolites levels in regions involved in ASD. Low levels of dopamine in cortex and high levels of glutamate and taurine in hippocampus were detected in Het mice, in line with clinical data collected on ASD children. Altogether, our data detected subtle but relevant neurochemical abnormalities in reeler mice supporting this mutant line, particularly male subjects, as a valid experimental model to estimate the contribution played by reelin deficiency in the global ASD neurobehavioral phenotype. PMID

  1. The Burkholderia pseudomallei Δasd Mutant Exhibits Attenuated Intracellular Infectivity and Imparts Protection against Acute Inhalation Melioidosis in Mice ▿

    PubMed Central

    Norris, Michael H.; Propst, Katie L.; Kang, Yun; Dow, Steven W.; Schweizer, Herbert P.; Hoang, Tung T.

    2011-01-01

    Burkholderia pseudomallei, the cause of serious and life-threatening diseases in humans, is of national biodefense concern because of its potential use as a bioterrorism agent. This microbe is listed as a select agent by the CDC; therefore, development of vaccines is of significant importance. Here, we further investigated the growth characteristics of a recently created B. pseudomallei 1026b Δasd mutant in vitro, in a cell model, and in an animal model of infection. The mutant was typified by an inability to grow in the absence of exogenous diaminopimelate (DAP); upon single-copy complementation with a wild-type copy of the asd gene, growth was restored to wild-type levels. Further characterization of the B. pseudomallei Δasd mutant revealed a marked decrease in RAW264.7 murine macrophage cytotoxicity compared to the wild type and the complemented Δasd mutant. RAW264.7 cells infected by the Δasd mutant did not exhibit signs of cytopathology or multinucleated giant cell (MNGC) formation, which were observed in wild-type B. pseudomallei cell infections. The Δasd mutant was found to be avirulent in BALB/c mice, and mice vaccinated with the mutant were protected against acute inhalation melioidosis. Thus, the B. pseudomallei Δasd mutant may be a promising live attenuated vaccine strain and a biosafe strain for consideration of exclusion from the select agent list. PMID:21807903

  2. Characterization and responses to environmental cues of a photosynthetic antenna-deficient mutant of the filamentous cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Leganés, Francisco; Martínez-Granero, Francisco; Muñoz-Martín, M Ángeles; Marco, Eduardo; Jorge, Alberto; Carvajal, Laura; Vida, Teresa; González-Pleiter, Miguel; Fernández-Piñas, Francisca

    2014-07-01

    The cyanobacterial phycobilisome (PBS) is a giant pigment-protein complex which harvests light energy for photosynthesis and comprises two structures: a core and peripheral rods. Most studies on PBS structure and function are based on mutants of unicellular strains. In this report, we describe the phenotypic and genetic characterization of a transposon mutant of the filamentous Anabaena sp. strain PCC 7120, denoted LC1, which cannot synthesize the phycobiliprotein phycocyanin (PC), the main component of the rods; in this mutant, the transposon had inserted into the cpcB gene (orf alr0528) which putatively encodes PC-β chain. Mutant LC1 was able to synthesize phycoerythrocyanin (PEC), a phycobiliprotein (PBP) located at the terminal region of the rods; but in the absence of PC, PEC did not attach to the PBSs that only retained the allophycocyanin (APC) core; ferredoxin: NADP+-oxidoreductase (FNR) that is associated with the PBS in the wild type, was not found in isolated PBSs from LC1. The performance of the mutant exposed to different environmental conditions was evaluated. The mutant phenotype was successfully complemented by cloning and transfer of the wild type complete cpc operon to mutant LC1. Interestingly, LC1 compensated its mutation by significantly increasing the number of its core-PBS and the effective quantum yield of photosystem II (PSII) photochemistry; this feature suggests a more efficient energy conversion in the mutant which may be useful for biotechnological applications. PMID:24913049

  3. In Vitro selection of Neisseria gonorrhoeae mutants with elevated MIC values and increased resistance to cephalosporins.

    PubMed

    Johnson, Steven R; Grad, Yonatan; Ganakammal, Satishkumar Ranganathan; Burroughs, Mark; Frace, Mike; Lipsitch, Marc; Weil, Ryan; Trees, David

    2014-11-01

    Strains of Neisseria gonorrhoeae with mosaic penA genes bearing novel point mutations in penA have been isolated from ceftriaxone treatment failures. Such isolates exhibit significantly higher MIC values to third-generation cephalosporins. Here we report the in vitro isolation of two mutants with elevated MICs to cephalosporins. The first possesses a point mutation in the transpeptidase region of the mosaic penA gene, and the second contains an insertion mutation in pilQ. PMID:25199775

  4. Assessment of the Toxicity of CuO Nanoparticles by Using Saccharomyces cerevisiae Mutants with Multiple Genes Deleted

    PubMed Central

    Bao, Shaopan; Lu, Qicong; Dai, Heping; Zhang, Chao

    2015-01-01

    To develop applicable and susceptible models to evaluate the toxicity of nanoparticles, the antimicrobial effects of CuO nanoparticles (CuO-NPs) on various Saccharomyces cerevisiae (S. cerevisiae) strains (wild type, single-gene-deleted mutants, and multiple-gene-deleted mutants) were determined and compared. Further experiments were also conducted to analyze the mechanisms associated with toxicity using copper salt, bulk CuO (bCuO), carbon-shelled copper nanoparticles (C/Cu-NPs), and carbon nanoparticles (C-NPs) for comparisons. The results indicated that the growth inhibition rates of CuO-NPs for the wild-type and the single-gene-deleted strains were comparable, while for the multiple-gene deletion mutant, significantly higher toxicity was observed (P < 0.05). When the toxicity of the CuO-NPs to yeast cells was compared with the toxicities of copper salt and bCuO, we concluded that the toxicity of CuO-NPs should be attributed to soluble copper rather than to the nanoparticles. The striking difference in adverse effects of C-NPs and C/Cu-NPs with equivalent surface areas also proved this. A toxicity assay revealed that the multiple-gene-deleted mutant was significantly more sensitive to CuO-NPs than the wild type. Specifically, compared with the wild-type strain, copper was readily taken up by mutant strains when cell permeability genes were knocked out, and the mutants with deletions of genes regulated under oxidative stress (OS) were likely producing more reactive oxygen species (ROS). Hence, as mechanism-based gene inactivation could increase the susceptibility of yeast, the multiple-gene-deleted mutants should be improved model organisms to investigate the toxicity of nanoparticles. PMID:26386067

  5. Assessment of the toxicity of CuO nanoparticles by using Saccharomyces cerevisiae mutants with multiple genes deleted.

    PubMed

    Bao, Shaopan; Lu, Qicong; Fang, Tao; Dai, Heping; Zhang, Chao

    2015-12-01

    To develop applicable and susceptible models to evaluate the toxicity of nanoparticles, the antimicrobial effects of CuO nanoparticles (CuO-NPs) on various Saccharomyces cerevisiae (S. cerevisiae) strains (wild type, single-gene-deleted mutants, and multiple-gene-deleted mutants) were determined and compared. Further experiments were also conducted to analyze the mechanisms associated with toxicity using copper salt, bulk CuO (bCuO), carbon-shelled copper nanoparticles (C/Cu-NPs), and carbon nanoparticles (C-NPs) for comparisons. The results indicated that the growth inhibition rates of CuO-NPs for the wild-type and the single-gene-deleted strains were comparable, while for the multiple-gene deletion mutant, significantly higher toxicity was observed (P < 0.05). When the toxicity of the CuO-NPs to yeast cells was compared with the toxicities of copper salt and bCuO, we concluded that the toxicity of CuO-NPs should be attributed to soluble copper rather than to the nanoparticles. The striking difference in adverse effects of C-NPs and C/Cu-NPs with equivalent surface areas also proved this. A toxicity assay revealed that the multiple-gene-deleted mutant was significantly more sensitive to CuO-NPs than the wild type. Specifically, compared with the wild-type strain, copper was readily taken up by mutant strains when cell permeability genes were knocked out, and the mutants with deletions of genes regulated under oxidative stress (OS) were likely producing more reactive oxygen species (ROS). Hence, as mechanism-based gene inactivation could increase the susceptibility of yeast, the multiple-gene-deleted mutants should be improved model organisms to investigate the toxicity of nanoparticles. PMID:26386067

  6. Mutants of Saccharomycopsis lipolytica defective in lysine catabolism.

    PubMed Central

    Gaillardin, C; Fournier, P; Sylvestre, G; Heslot, H

    1976-01-01

    Wild-type strains of Saccharomycopsis lipolytica are able to use lysine as a carbon or a nitrogen source, but not as a unique source for both. Mutants were selected that could not use lysine either as a nitrogen or as a carbon source. Some of them, however, utilized N-6-acetyllysine or 5-aminovaleric acid. Many of the mutants appeared to be blocked in both utilizations, suggesting a unique pathway for lysine degradation (either as a carbon or as a nitrogen source). Genetic characterization of these mutants was achieved by complementation and recombination tests. PMID:1245461

  7. 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

  8. Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank

    PubMed Central

    Rathnaiah, Govardhan; Lamont, Elise A.; Harris, N. Beth; Fenton, Robert J.; Zinniel, Denise K.; Liu, Xiaofei; Sotos, Josh; Feng, Zhengyu; Livneh-Kol, Ayala; Shpigel, Nahum Y.; Czuprynski, Charles J.; Sreevatsan, Srinand; Barletta, Raúl G.

    2014-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated. PMID:25360421

  9. Extract from a mutant Rhodobacter sphaeroides as an enriched carotenoid source

    PubMed Central

    Wang, Chih-Chiang; Ding, Shangwu; Chiu, Kuo-Hsun; Liu, Wen-Sheng; Lin, Tai-Jung; Wen, Zhi-Hong

    2016-01-01

    Background The extract Lycogen™ from the phototrophic bacterium Rhodobacter sphaeroides (WL-APD911) has attracted significant attention because of its promising potential as a bioactive mixture, attributed in part to its anti-inflammatory properties and anti-oxidative activity. Objective This study aims to investigate the components of Lycogen™ and its anti-inflammatory properties and anti-oxidative activity. Design and results The mutant strain R. sphaeroides (WL-APD911) whose carotenoid 1,2-hydratase gene has been altered by chemical mutagenesis was used for the production of a new carotenoid. The strain was grown at 30°C on Luria–Bertani (LB) agar plates. After a 4-day culture period, the mutant strain displayed a 3.5-fold increase in carotenoid content, relative to the wild type. In the DPPH test, Lycogen™ showed more potent anti-oxidative activity than lycopene from the wild-type strain. Primary skin irritation test with hamsters showed no irritation response in hamster skins after 30 days of treatment with 0.2% Lycogen™. Chemical investigations of Lycogen™ using nuclear magnetic resonance (NMR) 1H, 13C, and COSY/DQCOSY spectra have identified spheroidenone and methoxyneurosporene. Quantitative analysis of these identified compounds based on spectral intensities indicates that spheroidenone and methoxyneurosporene are major components (approximately 1:1); very small quantities of other derivatives are also present in the sample. Conclusions In this study, we identified the major carotenoid compounds contained in Lycogen™, including spheroidenone and methoxyneurosporene by high-resolution NMR spectroscopy analysis. The carotenoid content of this mutant strain of R. sphaeroides was 3.5-fold higher than that in normal strain. Furthermore, Lycogen™ from the mutant strain is more potent than lycopene from the wild-type strain and does not cause irritation in hamster skins. These findings suggest that this mutant strain has the potential to be used

  10. Mutant p53 confers chemoresistance in non-small cell lung cancer by upregulating Nrf2

    PubMed Central

    Wang, Yao-Chen; He, Tsung-Ying; Lee, Ming-Ching; Yeh, Sauh-Der; Chen, Chih-Yi; Lee, Huei

    2015-01-01

    Nrf2 is a key transcription factor for genes coding for antioxidants, detoxification enzymes, and multiple drug resistance and it also confers resistance to anticancer drugs. Here, we hypothesized that mutant p53 could upregulate Nrf2 expression at the transcriptional level, thereby conferring cisplatin resistance in non-small cell lung cancer (NSCLC). Luciferase reporter assays and real-time PCR analysis indicated that the Nrf2 promoter activity and its mRNA levels were markedly suppressed by wild-type p53, but not by mutant p53. Chromatin immunoprecipitation (ChIP) further confirmed that wild-type p53 binds at the p53 putative binding site to block Sp1 binding to the Nrf2 promoter and consequently to suppress the Nrf2 promoter activity. The MTT assay indicated that an increase in Nrf2 expression by mutant p53 is responsible for cisplatin resistance. Among the Nrf2 downstream genes, Bcl-2 and Bcl-xL contribute more strongly to Nrf2-mediated cisplatin resistance when compared with heme oxygenase 1 (HO-1). Cox regression analysis showed that patients with high-Nrf2, high-Bcl-2, high-Bcl-xL mRNA tumors were more commonly occurred unfavorable response to cisplatin-based chemotherapy than their counterparts. The prognostic significance of Nrf2 mRNA levels on OS and RFS was also observed in patients who have received cisplatin-based chemotherapy, particularly in p53-mutant patients. Collectively, mutant p53 may confer cisplatin resistance via upregulation of Nrf2 expression, and Nrf2 mRNA level may predict chemotherapeutic response and outcomes in NSCLC. PMID:26497680

  11. Three classes of Escherichia coli mutants selected for aerobic expression of fumarate reductase.

    PubMed Central

    Iuchi, S; Kuritzkes, D R; Lin, E C

    1986-01-01

    Fumarate reductase (encoded by frd) and succinate dehydrogenase (encoded by sdh) of Escherichia coli are both known to catalyze the interconversion of fumarate and succinate. Fumarate reductase, however, is not inducible aerobically and therefore cannot participate in the dehydrogenation of succinate. Three classes of suppressor mutants, classified as frd oxygen-resistant [frd(Oxr)], constitutive [frd(Con)], and gene amplification [frd(Amp)] mutants, were selected from an sdh strain as pseudorevertants that regained the partial ability to grow aerobically on succinate. All contained increased aerobic levels of fumarate reductase activity. In frd(Oxr) mutants expression of the operon showed increased resistance to aerobic repression. Under anaerobic conditions expression of the operon became less dependent on the fnr+ gene product, a pleiotropic activator protein for genes encoding anaerobic respiratory enzymes. Exogenous fumarate, however, was still required for full induction, and repression by nitrate was undiminished. Thus, aerobic repression and anaerobic nitrate repression appear to involve separate mechanisms. In frd(Con) mutants expression of the operon became highly resistant to aerobic repression. Under anaerobic conditions expression of the operon no longer required the fnr+ gene product or exogenous fumarate and became immune to nitrate repression. In partial diploids bearing an frd(Oxr) or an frd(Con) allele and phi(frd+-lac) there was no mutual regulatory influence between the two genetic loci. Thus, the frd mutations act in cis and hence are probably in the promoter region. In frd(Amp) mutants the frd locus was amplified without significant alteration in the pattern of regulation. PMID:3536878

  12. Growth, seed development and genetic analysis in wild type and Def mutant of Pisum sativum L

    PubMed Central

    2011-01-01

    Background The def mutant pea (Pisum sativum L) showed non-abscission of seeds from the funicule. Here we present data on seed development and growth pattern and their relationship in predicting this particular trait in wild type and mutant lines as well as the inheritance pattern of the def allele in F2 and F3 populations. Findings Pod length and seed fresh weight increase with fruit maturity and this may affect the abscission event in pea seeds. However, the seed position in either the distal and proximal ends of the pod did not show any difference. The growth factors of seed fresh weight (FW), width of funicles (WFN), seed width (SW) and seed height (SH) were highly correlated and their relationships were determined in both wild type and def mutant peas. The coefficient of determination R2 values for the relationship between WFN and FW, SW and SH and their various interactions were higher for the def dwarf type. Stepwise multiple regression analysis showed that variation of WFN was associated with SH and SW. Pearson's chi square analysis revealed that the inheritance and segregation of the Def locus in 3:1 ratio was significant in two F2 populations. Structural analysis of the F3 population was used to confirm the inheritance status of the Def locus in F2 heterozygote plants. Conclusions This study investigated the inheritance of the presence or absence of the Def allele, controlling the presence of an abscission zone (AZ) or an abscission-less zone (ALZ) forming in wild type and mutant lines respectively. The single major gene (Def) controlling this phenotype was monogenic and def mutants were characterized and controlled by the homozygous recessive def allele that showed no palisade layers in the hilum region of the seed coat. PMID:22078070

  13. Transcriptomic analyses of maize ys1 and ys3 mutants reveal maize iron homeostasis.

    PubMed

    Nozoye, Tomoko; Nakanishi, Hiromi; Nishizawa, Naoko K

    2015-09-01

    To acquire iron (Fe), graminaceous plants secrete mugineic acid family phytosiderophores (MAs) (Takagi, 1976 [1]) through the MAs efflux transporter TOM1 (Nozoye et al., 2011 [2]) and take up Fe in the form of Fe(III)-MAs complexes through the Fe(III)-MAs transporter YS1 (Curie et al., 2001 [3]). Yellow stripe 1 (ys1) and ys3 are recessive mutants of maize (Zea mays L.) that result in symptoms typical of Fe deficiency, i.e., interveinal chlorosis of the leaves. The ys1 mutant is defective in the YS1 transporter and is therefore unable to take up Fe(III)-MAs complexes. While the ys3 mutant has been shown to be defective in MA release, the causative gene has not been identified. The objective of the present work was to identify the genes responsible for the ys1 and ys3 phenotypes, so as to extend our understanding of Fe homeostasis in maize by qRT-PCR. In agreement with previous reports, the expression level of YS1 was decreased in the ys1 mutant. Moreover, we identified that the expression level of a homolog of TOM1 in maize (ZmTOM1) was significantly decreased in the ys3 mutant. Here described the quality control and analysis that were performed on the dataset. The data is publicly available through the GEO database with accession number GSE44557. The interpretation and description of these data are included in a manuscript (Nozoye et al., 2013 [4]). PMID:26484234

  14. Silencing Mutant Ataxin-3 Rescues Motor Deficits and Neuropathology in Machado-Joseph Disease Transgenic Mice

    PubMed Central

    Onofre, Isabel; Albuquerque, David; Hirai, Hirokazu; Déglon, Nicole; de Almeida, Luís Pereira

    2013-01-01

    Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. This expansion translates into a polyglutamine tract that confers a toxic gain-of-function to the mutant protein – ataxin-3, leading to neurodegeneration in specific brain regions, with particular severity in the cerebellum. No treatment able to modify the disease progression is available. However, gene silencing by RNA interference has shown promising results. Therefore, in this study we investigated whether lentiviral-mediated allele-specific silencing of the mutant ataxin-3 gene, after disease onset, would rescue the motor behavior deficits and neuropathological features in a severely impaired transgenic mouse model of MJD. For this purpose, we injected lentiviral vectors encoding allele-specific silencing-sequences (shAtx3) into the cerebellum of diseased transgenic mice expressing the targeted C-variant of mutant ataxin-3 present in 70% of MJD patients. This variation permits to discriminate between the wild-type and mutant forms, maintaining the normal function of the wild-type allele and silencing only the mutant form. Quantitative analysis of rotarod performance, footprint and activity patterns revealed significant and robust alleviation of gait, balance (average 3-fold increase of rotarod test time), locomotor and exploratory activity impairments in shAtx3-injected mice, as compared to control ones injected with shGFP. An important improvement of neuropathology was also observed, regarding the number of intranuclear inclusions, calbindin and DARPP-32 immunoreactivity, fluorojade B and Golgi staining and molecular and granular layers thickness. These data demonstrate for the first time the efficacy of gene silencing in blocking the MJD-associated motor-behavior and neuropathological abnormalities after the onset of the disease, supporting the use of this strategy

  15. Thermostability of photosynthesis in two new chlorophyll b-less rice mutants.

    PubMed

    Lin, Zhifang; Peng, Changlian; Xu, Xinlan; Lin, Guizhu; Zhang, Jingliu

    2005-04-01

    Leaves of the two new chlorophyll b-less rice mutants VG28-1, VG30-5 and the wild type rice cv. Zhonghua 11 were subjected to temperatures 28, 36, 40, 44 and 48 degrees C in the dark for 30 min or gradually elevated temperature from 30 degrees C to 80 degrees C at 0.5 degrees C/min. The thermostability of photosynthetic apparatus was estimated by the changes in chlorophyll fluorescence parameters, photosynthetic rate and pigment content, chloroplast ultrastructure and tissue location of H2O2 accumulation. There were different patterns of F(o)-temperature curves between the Chl b-less mutants and the wild type plant, and the temperature of F(o) rising threshold was shifted 3 degrees C lower in the Chl b-less mutants (48 degrees C) than in the wild type (51 degrees C). At temperature up to about 45 degrees C, chloroplasts were swollen and thylakoid grana became misty accompanied with the complete loss of photosynthetic oxygen evolution in the two Chl b-less mutants, but chloroplast ultrastructure in the wild type showed no obvious alteration. After 55 degrees C exposure, the disordered thylakoid and significant H2O2 accumulation in leaves were found in the two Chl b-less mutants, whereas in the wild type plant, less H2O2 was accumulated and the swollen thylakoid still maintained a certain extent of stacking. A large extent of the changes in qP, NPQ and Fv/Fm was consistent with the Pn decreasing rate in the Chl b-less mutants during high temperature treatment as compared with the wild type. The results indicated that the Chl b-less mutants showed a tendency for higher thermosensitivity, and loss of Chl b in LHC II could lead to less thermostability of PSII structure and function. Heat damage to photosynthetic apparatus might be partially attributed to the internal oxidative stress produced at severely high temperature. PMID:15986886

  16. Deletion of Osr2 Partially Rescues Tooth Development in Runx2 Mutant Mice.

    PubMed

    Kwon, H J E; Park, E K; Jia, S; Liu, H; Lan, Y; Jiang, R

    2015-08-01

    Tooth organogenesis depends on genetically programmed sequential and reciprocal inductive interactions between the dental epithelium and neural crest-derived mesenchyme. Previous studies showed that the Msx1 and Runx2 transcription factors are required for activation of odontogenic signals, including Bmp4 and Fgf3, in the early tooth mesenchyme to drive tooth morphogenesis through the bud-to-cap transition and that Runx2 acts downstream of Msx1 to activate Fgf3 expression. Recent studies identified Osr2 as a repressor of tooth development and showed that inactivation of Osr2 rescued molar tooth morphogenesis in the Msx1(-/-) mutant mice as well as in mice with neural crest-specific inactivation of Bmp4. Here we show that Runx2 expression is expanded in the tooth bud mesenchyme in Osr2(-/-) mutant mouse embryos and is partially restored in the tooth mesenchyme in Msx1(-/-)Osr2(-/-) mutants in comparison with Msx1(-/-) and wild-type embryos. Whereas mandibular molar development arrested at the bud stage and maxillary molar development arrested at the bud-to-cap transition in Runx2(-/-) mutant mice, both mandibular and maxillary molar tooth germs progressed to the early bell stage, with rescued expression of Msx1 and Bmp4 in the dental papilla as well as expression of Bmp4, p21, and Shh in the primary enamel knot in the Osr2(-/-)Runx2(-/-) compound mutants. In contrast to the Msx1(-/-)Osr2(-/-) compound mutants, which exhibit nearly normal first molar morphogenesis, the Osr2(-/-)Runx2(-/-) compound mutant embryos failed to activate the expression of Fgf3 and Fgf10 in the dental papilla and exhibited significant deficit in cell proliferation in both the dental epithelium and mesenchyme in comparison with the control embryos. These data indicate that Runx2 synergizes with Msx1 to drive tooth morphogenesis through the bud-to-cap transition and that Runx2 controls continued tooth growth and morphogenesis beyond the cap stage through activation of Fgf3 and Fgf10 expression

  17. Proteomic analysis of Spirogyra varians mutant with high starch content and growth rate induced by gamma irradiation.

    PubMed

    Yoon, Minchul; Choi, Jong-il; Kim, Gwang Hoon; Kim, Dong-Ho; Park, Don-Hee

    2013-06-01

    This study was conducted to develop a high-efficiency strain of Spirogyra varians for the production of biomass by radiation breeding. The characteristics of wild-type and mutant S. varians were analyzed through phenomenological and proteomic observations. The results of our phenomenological observations of the S. varians mutant demonstrated increases in growth rate and content of chlorophyll a, b, and a + b; in particular, a significant threefold increase was observed in starch accumulation. Proteomic analysis to investigate the differences in expression between wild-type and mutant proteins identified 18 proteins with significantly different expressions. From the literature review, it was confirmed that the up-regulated proteins were mainly involved in photosynthesis, carbohydrate biosynthesis, and energy metabolism. These results suggest the possibility of algae development by radiation breeding for the production of biofuel. PMID:23370702

  18. Inhibition of cell wall turnover and autolysis by vancomycin in a highly vancomycin-resistant mutant of Staphylococcus aureus.

    PubMed Central

    Sieradzki, K; Tomasz, A

    1997-01-01

    A highly vancomycin-resistant mutant (MIC = 100 microg/ml) of Staphylococcus aureus, mutant VM, which was isolated in the laboratory by a step-pressure procedure, continued to grow and synthesize peptidoglycan in the presence of vancomycin (50 microg/ml) in the medium, but the antibiotic completely inhibited cell wall turnover and autolysis, resulting in the accumulation of cell wall material at the cell surface and inhibition of daughter cell separation. Cultures of mutant VM removed vancomycin from the growth medium through binding the antibiotic to the cell walls, from which the antibiotic could be quantitatively recovered in biologically active form. Vancomycin blocked the in vitro hydrolysis of cell walls by autolytic enzyme extracts, lysostaphin and mutanolysin. Analysis of UDP-linked peptidoglycan precursors showed no evidence for the presence of D-lactate-terminating muropeptides. While there was no significant difference in the composition of muropeptide units of mutant and parental cell walls, the peptidoglycan of VM had a significantly lower degree of cross-linkage. These observations and the results of vancomycin-binding studies suggest alterations in the structural organization of the mutant cell walls such that access of the vancomycin molecules to the sites of wall biosynthesis is blocked. PMID:9098053

  19. A rapid method to screen for cell-wall mutants using discriminant analysis of Fourier transform infrared spectra.

    PubMed

    Chen, L; Carpita, N C; Reiter, W D; Wilson, R H; Jeffries, C; McCann, M C

    1998-11-01

    We have developed a rapid method to screen large numbers of mutant plants for a broad range of cell wall phenotypes using Fourier transform infrared (FTIR) microspectroscopy of leaves. We established and validated a model that can discriminate between the leaves of wild-type and a previously defined set of cell-wall mutants of Arabidopsis. Exploratory principal component analysis indicated that mutants deficient in different cell-wall sugars can be distinguished from each other. Discrimination of cell-wall mutants from wild-type was independent of variability in starch content or additional unrelated mutations that might be present in a heavily mutagenised population. We then developed an analysis of FTIR spectra of leaves obtained from over 1000 mutagenised flax plants, and selected 59 plants whose spectral variation from wild-type was significantly out of the range of a wild-type population, determined by Mahalanobis distance. Cell wall sugars from the leaves of selected putative mutants were assayed by gas chromatography-mass spectrometry and 42 showed significant differences in neutral sugar composition. The FTIR spectra indicated that six of the remaining 17 plants have altered ester or protein content. We conclude that linear discriminant analysis of FTIR spectra is a robust method to identify a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification. PMID:9881159

  20. Inhibition of cell wall turnover and autolysis by vancomycin in a highly vancomycin-resistant mutant of Staphylococcus aureus.

    PubMed

    Sieradzki, K; Tomasz, A

    1997-04-01

    A highly vancomycin-resistant mutant (MIC = 100 microg/ml) of Staphylococcus aureus, mutant VM, which was isolated in the laboratory by a step-pressure procedure, continued to grow and synthesize peptidoglycan in the presence of vancomycin (50 microg/ml) in the medium, but the antibiotic completely inhibited cell wall turnover and autolysis, resulting in the accumulation of cell wall material at the cell surface and inhibition of daughter cell separation. Cultures of mutant VM removed vancomycin from the growth medium through binding the antibiotic to the cell walls, from which the antibiotic could be quantitatively recovered in biologically active form. Vancomycin blocked the in vitro hydrolysis of cell walls by autolytic enzyme extracts, lysostaphin and mutanolysin. Analysis of UDP-linked peptidoglycan precursors showed no evidence for the presence of D-lactate-terminating muropeptides. While there was no significant difference in the composition of muropeptide units of mutant and parental cell walls, the peptidoglycan of VM had a significantly lower degree of cross-linkage. These observations and the results of vancomycin-binding studies suggest alterations in the structural organization of the mutant cell walls such that access of the vancomycin molecules to the sites of wall biosynthesis is blocked. PMID:9098053

  1. Glutamate Racemase Mutants of Bacillus anthracis

    PubMed Central

    Oh, So-Young; Richter, Stefan G.; Missiakas, Dominique M.

    2015-01-01

    ABSTRACT d-Glutamate is an essential component of bacterial peptidoglycan and a building block of the poly-γ-d-glutamic acid (PDGA) capsule of Bacillus anthracis, the causative agent of anthrax. Earlier work suggested that two glutamate racemases, encoded by racE1 and racE2, are each essential for growth of B. anthracis, supplying d-glutamic acid for the synthesis of peptidoglycan and PDGA capsule. Earlier work could not explain, however, why two enzymes that catalyze the same reaction may be needed for bacterial growth. Here, we report that deletion of racE1 or racE2 did not prevent growth of B. anthracis Sterne (pXO1+ pXO2−), the noncapsulating vaccine strain, or of B. anthracis Ames (pXO1+ pXO2+), a fully virulent, capsulating isolate. While mutants with deletions in racE1 and racE2 were not viable, racE2 deletion delayed vegetative growth of B. anthracis following spore germination and caused aberrant cell shapes, phenotypes that were partially restored by exogenous d-glutamate. Deletion of racE1 or racE2 from B. anthracis Ames did not affect the production or stereochemical composition of the PDGA capsule. A model is presented whereby B. anthracis, similar to Bacillus subtilis, utilizes two functionally redundant racemase enzymes to synthesize d-glutamic acid for peptidoglycan synthesis. IMPORTANCE Glutamate racemases, enzymes that convert l-glutamate to d-glutamate, are targeted for antibiotic development. Glutamate racemase inhibitors may be useful for the treatment of bacterial infections such as anthrax, where the causative agent, B. anthracis, requires d-glutamate for the synthesis of peptidoglycan and poly-γ-d-glutamic acid (PDGA) capsule. Here we show that B. anthracis possesses two glutamate racemase genes that can be deleted without abolishing either bacterial growth or PDGA synthesis. These data indicate that drug candidates must inhibit both glutamate racemases, RacE1 and RacE2, in order to block B. anthracis growth and achieve therapeutic

  2. The Succinated Proteome of FH-Mutant Tumours.

    PubMed

    Yang, Ming; Ternette, Nicola; Su, Huizhong; Dabiri, Raliat; Kessler, Benedikt M; Adam, Julie; Teh, Bin Tean; Pollard, Patrick J

    2014-01-01

    Inherited mutations in the Krebs cycle enzyme fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity in HLRCC tumours causes accumulation of the Krebs cycle intermediate fumarate to high levels, which may act as an oncometabolite through various, but not necessarily mutually exclusive, mechanisms. One such mechanism, succination, is an irreversible non-enzymatic modification of cysteine residues by fumarate, to form S-(2-succino)cysteine (2SC). Previous studies have demonstrated that succination of proteins including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), kelch-like ECH-associated protein 1 (KEAP1) and mitochondrial aconitase (ACO2) can have profound effects on cellular metabolism. Furthermore, immunostaining for 2SC is a sensitive and specific biomarker for HLRCC tumours. Here, we performed a proteomic screen on an FH-mutant tumour and two HLRCC-derived cancer cell lines and identified 60 proteins where one or more cysteine residues were succinated; 10 of which were succinated at cysteine residues either predicted, or experimentally proven, to be functionally significant. Bioinformatic enrichment analyses identified most succinated targets to be involved in redox signaling. To our knowledge, this is the first proteomic-based succination screen performed in human tumours and cancer-derived cells and has identified novel 2SC targets that may be relevant to the pathogenesis of HLRCC. PMID:25105836

  3. The Succinated Proteome of FH-Mutant Tumours

    PubMed Central

    Yang, Ming; Ternette, Nicola; Su, Huizhong; Dabiri, Raliat; Kessler, Benedikt M.; Adam, Julie; Teh, Bin Tean; Pollard, Patrick J.

    2014-01-01

    Inherited mutations in the Krebs cycle enzyme fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity in HLRCC tumours causes accumulation of the Krebs cycle intermediate fumarate to high levels, which may act as an oncometabolite through various, but not necessarily mutually exclusive, mechanisms. One such mechanism, succination, is an irreversible non-enzymatic modification of cysteine residues by fumarate, to form S-(2-succino)cysteine (2SC). Previous studies have demonstrated that succination of proteins including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), kelch-like ECH-associated protein 1 (KEAP1) and mitochondrial aconitase (ACO2) can have profound effects on cellular metabolism. Furthermore, immunostaining for 2SC is a sensitive and specific biomarker for HLRCC tumours. Here, we performed a proteomic screen on an FH-mutant tumour and two HLRCC-derived cancer cell lines and identified 60 proteins where one or more cysteine residues were succinated; 10 of which were succinated at cysteine residues either predicted, or experimentally proven, to be functionally significant. Bioinformatic enrichment analyses identified most succinated targets to be involved in redox signaling. To our knowledge, this is the first proteomic-based succination screen performed in human tumours and cancer-derived cells and has identified novel 2SC targets that may be relevant to the pathogenesis of HLRCC. PMID:25105836

  4. Clear Plaque Mutants of Lactococcal Phage TP901-1

    PubMed Central

    Kot, Witold; Kilstrup, Mogens; Vogensen, Finn K.; Hammer, Karin

    2016-01-01

    We report a method for obtaining turbid plaques of the lactococcal bacteriophage TP901-1 and its derivative TP901-BC1034. We have further used the method to isolate clear plaque mutants of this phage. Analysis of 8 such mutants that were unable to lysogenize the host included whole genome resequencing. Four of the mutants had different mutations in structural genes with no relation to the genetic switch. However all 8 mutants had a mutation in the cI repressor gene region. Three of these were located in the promoter and Shine-Dalgarno sequences and five in the N-terminal part of the encoded CI protein involved in the DNA binding. The conclusion is that cI is the only gene involved in clear plaque formation i.e. the CI protein is the determining factor for the lysogenic pathway and its maintenance in the lactococcal phage TP901-1. PMID:27258092

  5. PATHOGENICITY AND IMMUNOGENICITY OF STREPTOMYCIN-DEPENDENT MUTANTS OF BRUCELLA

    PubMed Central

    Simon, Ellen M.; Berman, David T.

    1962-01-01

    Simon, Ellen M. (University of Wisconsin, Madison) and David T. Berman. Pathogenicity and immunogenicity of streptomycin-dependent mutants of Brucella. J. Bacteriol. 83:1347–1355. 1962.—Streptomycin-dependent (Sd) mutants of Brucella suis and B. abortus were avirulent for guinea pigs whether selected in the presence of streptomycin only or streptomycin and normal or immune serum. Administration of large quantities of streptomycin to guinea pigs increased the numbers of organisms which could be recovered, but did not cause the development of progressive infections. Vaccination with Sd mutants of B. abortus diminished the pathological response of guinea pigs infected with a large challenge dose of virulent B. abortus, but equal numbers of organisms were recovered from vaccinated animals and unvaccinated controls. Vaccination with Sd mutants of B. suis protected some guinea pigs from small challenge doses. Immunization by multiple injections or by one injection plus streptomycin was superior to a single inoculation of organisms. PMID:13913089

  6. An Arabidopsis mutant defective in the general phenylpropanoid pathway.

    PubMed Central

    Chapple, C C; Vogt, T; Ellis, B E; Somerville, C R

    1992-01-01

    Mutants of Arabidopsis deficient in a major leaf phenylpropanoid ester, 2-O-sinapoyl-L-malate, were identified by thin-layer chromatographic screening of methanolic leaf extracts from several thousand mutagenized plants. Mutations at a locus designated SIN1 also eliminate accumulation of the sinapic acid esters characteristic of seed tissues. Because of increased transparency to UV light, the sin1 mutants exhibit a characteristic red fluorescence under UV light, whereas wild-type plants have a blue-green appearance due to the fluorescence of sinapoyl malate in the upper epidermis. As determined by in vivo radiotracer feeding experiments, precursor supplementation studies, and enzymatic assays, the defect in the sin1 mutants appears to block the conversion of ferulate to 5-hydroxyferulate in the general phenylpropanoid pathway. As a result, the lignin of the mutant lacks the sinapic acid-derived components typical of wild-type lignin. PMID:1477555

  7. Nodule initiation elicited by noninfective mutants of Rhizobium phaseoli.

    PubMed

    Vandenbosch, K A; Noel, K D; Kaneko, Y; Newcomb, E H

    1985-06-01

    Rhizobium phaseoli CE106, CE110, and CE115, originally derived by transposon mutagenesis (Noel et al., J. Bacteriol. 158:149-155, 1984), induced the formation of uninfected root nodule-like swellings on bean (Phaseolus vulgaris). Bacteria densely colonized the root surface, and root hair curling and initiation of root cortical-cell divisions occurred normally in mutant-inoculated seedlings, although no infection threads formed. The nodules were ineffective, lacked leghemoglobin, and were anatomically distinct from normal nodules. Ultrastructural specialization for ureide synthesis, characteristic of legumes that form determinate nodules, was absent. Colony morphology of the mutant strains on agar plates was less mucoid than that of the wild type, and under some cultural conditions, the mutants did not react with Cellufluor, a fluorescent stain for beta-linked polysaccharide. These observations suggest that the genetic lesions in these mutants may be related to extracellular polysaccharide synthesis. PMID:3997785

  8. An assay for social interaction in Drosophila fragile X mutants

    PubMed Central

    Bolduc, Francois V.; Valente, Dan; Nguyen, Antoinette T.; Mitra, Partha P.; Tully, Tim

    2010-01-01

    We developed a novel assay to examine social interactions in Drosophila and, as a first attempt, apply it here at examining the behavior of Drosophila Fragile X Mental Retardation gene (dfmr1) mutants. Fragile X syndrome is the most common cause of single gene intellectual disability (ID) and is frequently associated with autism. Our results suggest that dfmr1 mutants are less active than wild-type flies and interact with each other less often. In addition, mutants for one allele of dfmr1, dfmr1B55, are more likely to come in close contact with a wild-type fly than another dfmr1B55 mutant. Our results raise the possibility of defective social expression with preserved receptive abilities. We further suggest that the assay may be applied in a general strategy of examining endophenoypes of complex human neurological disorders in Drosophila, and specifically in order to understand the genetic basis of social interaction defects linked with ID. PMID:20519966

  9. Resistant mechanism study of benzalkonium chloride selected Salmonella Typhimurium mutants.

    PubMed

    Guo, Wei; Cui, Shenghui; Xu, Xiao; Wang, Haoyan

    2014-02-01

    Benzalkonium chloride is one of the invaluable biocides that is extensively used in healthcare settings as well as in the food processing industry. After exposing wild-type Salmonella Typhimurium 14028s or its AcrAB inactivation mutant to gradually increasing levels of benzalkonium chloride, resistance mutants S-41, S-150, S-AB-23, S-AB-38, and S-AB-73 were selected and these mutants also showed a 2-64-fold stable minimum inhibitory concentration (MIC) increase to chloramphenicol, ciprofloxacin, nalidixic acid, and tetracycline. In S-41 and S-150, the expression of acrB was increased 2.7- and 7.6-fold, and ΔtolC or ΔacrAB mutants of S-41 and S-150 showed the same MICs to all tested antimicrobials as the equivalent Salmonella Typhimurium 14028s mutants. However, in S-AB-23, S-AB-38, and S-AB-73, the expression of acrF was increased 96-, 230-, and 267-fold, respectively, and ΔtolC or ΔacrEF mutants of S-AB-23, S-AB-38, and S-AB-73 showed the similar MICs to all tested antimicrobials as the ΔtolC mutant of Salmonella Typhimurium 14028s. Our data showed that constitutively over-expressed AcrAB working through TolC was the main resistance mechanism in ST14028s benzalkonium chloride resistance mutants. However, after AcrAB had been inactivated, benzalkonium chloride-resistant mutants could still be selected and constitutively over-expressed, AcrEF became the dominant efflux pump working through TolC and being responsible for the increasing antimicrobial resistance. These data indicated that different mechanisms existed for acrB and acrF constitutive over-expression. Since exposure to benzalkonium chloride may lead to Salmonella mutants with a decreased susceptibility to quinolones, which is currently one of the drugs of choice for the treatment of life-threatening salmonelosis, research into the pathogenesis and epidemiology of the benzalkonium chloride resistance mutants will be of increasing importance. PMID:23987991

  10. A low, adaptive dose of gamma-rays reduced the number and altered the spectrum of S1- mutants in human-hamster hybrid AL cells

    NASA Technical Reports Server (NTRS)

    Ueno, A. M.; Vannais, D. B.; Gustafson, D. L.; Wong, J. C.; Waldren, C. A.

    1996-01-01

    We examined the effects of a low, adaptive dose of 137Cs-gamma-irradiation (0.04 Gy) on the number and kinds of mutants induced in AL human-hamster hybrid cells by a later challenge dose of 4 Gy. The yield of S1- mutants was significantly less (by 53%) after exposure to both the adaptive and challenge doses compared to the challenge dose alone. The yield of hprt- mutants was similarly decreased. Incubation with cycloheximide (CX) or 3-aminobenzamide largely negated the decrease in mutant yield. The adaptive dose did not perturb the cell cycle, was not cytotoxic, and did not of itself increase the mutant yield above background. The adaptive dose did, however, alter the spectrum of S1- mutants from populations exposed only to the adaptive dose, as well as affecting the spectrum of S1- mutants generated by the challenge dose. The major change in both cases was a significant increase in the proportion of complex mutations compared to small mutations and simple deletions.

  11. [Riboflavin transport in cells of riboflavin-dependent yeast mutants].

    PubMed

    Sibirnyĭ, A A; Shavlovskiĭ, G M; Ksheminskaia, G P; Orlovskaia, A G

    1977-01-01

    Riboflavin was transported at a high rate into yeast cells of Pichia guilliermondii and Schwanniomyces occidentalis mutants capable of growth in a medium containing low concentrations of riboflavin, and having multiple susceptibility to some antibiotics and antimetabolites. Sucrose and sodium azide inhibited transport of riboflavin. Other riboflavin dependent mutants of Pichia guilliermondii, Pichia ohmeri, Torulopsis candida, and Saccharomyces cerevisiae, also growing in media containing low concentrations of riboflavin, were not capable of its active transport. PMID:329070

  12. Borrelia burgdorferi mutant lacking Osp: biological and immunological characterization.

    PubMed Central

    Sadziene, A; Thomas, D D; Barbour, A G

    1995-01-01

    All Borrelia burgdorferi sensu lato isolates characterized to date have one or a combination of several major outer surface proteins (Osps). Mutants of B. burgdorferi lacking Osps were selected with polyclonal or monoclonal antibodies at a frequency of 10(-6) to 10(-5). One mutant that lacked OspA, -B, -C, and -D was further characterized. It was distinguished from the OspA+B+ cells by its (i) autoaggregation and slower growth rate, (ii) decreased plating efficiency on solid medium, (iii) serum and complement sensitivity, and (iv) diminished capacity to adhere to human umbilical vein endothelial cells. The Osp-less mutant was unable to evoke a detectable immune response after intradermal live cell immunization even though mutant survived in mouse skin for the same duration as wild-type cells. Polyclonal mouse serum raised against Osp-less cells inhibited growth of the mutant but not of wild-type cells, an indication that other antigens are present on the surface of the Osp-less mutant. Two types of monoclonal antibodies (MAbs) with growth-inhibiting properties for mutant cells were identified. The first type bound to a 13-kDa surface protein of B. burgdorferi sensu stricto and of B. afzelii. The MIC of the Fab fragment of one MAb of this type was 0.2 micrograms/ml. The second type of MAb to the Osp-less mutant did not bind to B. burgdorferi components by Western blotting (immunoblotting) but did not bind to unfixed, viable cells in immunofluorescence and growth inhibition assays. These studies revealed possible functions Osp proteins in borrelias, specifically serum resistance, and indicated that in the absence of Osp proteins, other antigens are expressed or become accessible at the cell surface. PMID:7890424

  13. Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities.

    PubMed

    Kerr, Emma M; Gaude, Edoardo; Turrell, Frances K; Frezza, Christian; Martins, Carla P

    2016-03-01

    The RAS/MAPK (mitogen-activated protein kinase) signalling pathway is frequently deregulated in non-small-cell lung cancer, often through KRAS activating mutations. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations. We recently showed that advanced lung tumours from Kras(G12D/+);p53-null mice frequently exhibit Kras(G12D) allelic enrichment (Kras(G12D)/Kras(wild-type) > 1) (ref. 7), implying that mutant Kras copy gains are positively selected during progression. Here we show, through a comprehensive analysis of mutant Kras homozygous and heterozygous mouse embryonic fibroblasts and lung cancer cells, that these genotypes are phenotypically distinct. In particular, Kras(G12D/G12D) cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the tricarboxylic acid cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous non-small-cell lung cancer cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of Kras(G12D) copy gain), but not in the corresponding early tumours (Kras(G12D) heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprising two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated on the basis of their relative mutant allelic content. We also provide the first, to our knowledge, in vivo evidence of metabolic rewiring during lung cancer malignant progression. PMID:26909577

  14. Characterization of a multi-seeded (msd) mutant of sorghum that displays significant enhancement in seed number

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sorghum (Sorghum bicolor L. Moench) cultivars and germplasm display branched inflorescence or panicle, characterized by spikelets composed of a single sessile, fertile floret that develop into viable seed and one or two adjacent sterile pedicellate florets (Monoseeded [MSD] trait). Based on total nu...

  15. Neoceptor Concept Based on Molecular Complementarity in GPCRs: A Mutant Adenosine A3 Receptor with Selectively Enhanced Affinity for Amine-Modified Nucleosides

    PubMed Central

    Jacobson, Kenneth A.; Gao, Zhan-Guo; Chen, Aishe; Barak, Dov; Kim, Soon-Ai; Lee, Kyeong; Link, Andreas; Van Rompaey, Philippe; van Calenbergh, Serge; Liang, Bruce T.

    2012-01-01

    Adenosine A3 receptors are of interest in the treatment of cardiac ischemia, inflammation, and neurodegenerative diseases. In an effort to create a unique receptor mutant that would be activated by tailor-made synthetic ligands, we mutated the human A3 receptor at the site of a critical His residue in TM7, previously proposed to be involved in ligand recognition through interaction with the ribose moiety. The H272E mutant receptor displayed reduced affinity for most of the uncharged A3 receptor agonists and antagonists examined. For example, the nonselective agonist 1a was 19-fold less potent at the mutant receptor than at the wild-type receptor. The introduction of an amino group on the ribose moiety of adenosine resulted in either equipotency or enhanced binding affinity at the H272E mutant relative to wild-type A3 receptors, depending on the position of the amino group. 3′-Amino-3′-deoxyadenosine proved to be 7-fold more potent at the H272E mutant receptor than at the wild-type receptor, while the corresponding 2′- and 5′-amino analogues did not display significantly enhanced affinities. An 3′-amino-N6-iodobenzyl analogue showed only a small enhancement at the mutant (Ki = 320 nM) vs wild-type receptors. The 3′-amino group was intended for a direct electrostatic interaction with the negatively charged ribose-binding region of the mutant receptor, yet molecular modeling did not support this notion. This design approach is an example of engineering the structure of mutant receptors to recognize synthetic ligands for which they are selectively matched on the basis of molecular complementarity between the mutant receptor and the ligand. We have termed such engineered receptors “neoceptors”, since the ligand recognition profile of such mutant receptors need not correspond to the profile of the parent, native receptor. PMID:11708915

  16. Biofilm formation-defective mutants in Pseudomonas putida.

    PubMed

    López-Sánchez, Aroa; Leal-Morales, Antonio; Jiménez-Díaz, Lorena; Platero, Ana I; Bardallo-Pérez, Juan; Díaz-Romero, Alberto; Acemel, Rafael D; Illán, Juan M; Jiménez-López, Julia; Govantes, Fernando

    2016-07-01

    Out of 8000 candidates from a genetic screening for Pseudomonas putida KT2442 mutants showing defects in biofilm formation, 40 independent mutants with diminished levels of biofilm were analyzed. Most of these mutants carried insertions in genes of the lap cluster, whose products are responsible for synthesis, export and degradation of the adhesin LapA. All mutants in this class were strongly defective in biofilm formation. Mutants in the flagellar regulatory genes fleQ and flhF showed similar defects to that of the lap mutants. On the contrary, transposon insertions in the flagellar structural genes fliP and flgG, that also impair flagellar motility, had a modest defect in biofilm formation. A mutation in gacS, encoding the sensor element of the GacS/GacA two-component system, also had a moderate effect on biofilm formation. Additional insertions targeted genes involved in cell envelope function: PP3222, encoding the permease element of an ABC-type transporter and tolB, encoding the periplasmic component of the Tol-OprL system required for outer membrane stability. Our results underscore the central role of LapA, suggest cross-regulation between motility and adhesion functions and provide insights on the role of cell envelope trafficking and maintenance for biofilm development in P. putida. PMID:27190143

  17. Proteomic analysis of the flooding tolerance mechanism in mutant soybean.

    PubMed

    Komatsu, Setsuko; Nanjo, Yohei; Nishimura, Minoru

    2013-02-21

    Flooding stress of soybean is a serious problem because it reduces growth; however, flooding-tolerant cultivars have not been identified. To analyze the flooding tolerance mechanism of soybean, the flooding-tolerant mutant was isolated and analyzed using a proteomic technique. Flooding-tolerance tests were repeated five times using gamma-ray irradiated soybeans, whose root growth (M6 stage) was not suppressed even under flooding stress. Two-day-old wild-type and mutant plants were subjected to flooding stress for 2days, and proteins were identified using a gel-based proteomic technique. In wild-type under flooding stress, levels of proteins related to development, protein synthesis/degradation, secondary metabolism, and the cell wall changed; however, these proteins did not markedly differ in the mutant. In contrast, an increased number of fermentation-related proteins were identified in the mutant under flooding stress. The root tips of mutant plants were not affected by flooding stress, even though the wild-type plants had damaged root. Alcohol dehydrogenase activity in the mutant increased at an early stage of flooding stress compared with that of the wild-type. Taken together, these results suggest that activation of the fermentation system in the early stages of flooding may be an important factor for the acquisition of flooding tolerance in soybean. PMID:23313221

  18. The unfolded protein response selectively targets active smoothened mutants.

    PubMed

    Marada, Suresh; Stewart, Daniel P; Bodeen, William J; Han, Young-Goo; Ogden, Stacey K

    2013-06-01

    The Hedgehog signaling pathway, an essential regulator of developmental patterning, has been implicated in playing causative and survival roles in a range of human cancers. The signal-transducing component of the pathway, Smoothened, has revealed itself to be an efficacious therapeutic target in combating oncogenic signaling. However, therapeutic challenges remain in cases where tumors acquire resistance to Smoothened antagonists, and also in cases where signaling is driven by active Smoothened mutants that exhibit reduced sensitivity to these compounds. We previously demonstrated that active Smoothened mutants are subjected to prolonged endoplasmic reticulum (ER) retention, likely due to their mutations triggering conformation shifts that are detected by ER quality control. We attempted to exploit this biology and demonstrate that deregulated Hedgehog signaling driven by active Smoothened mutants is specifically attenuated by ER stressors that induce the unfolded protein response (UPR). Upon UPR induction, active Smoothened mutants are targeted by ER-associated degradation, resulting in attenuation of inappropriate pathway activity. Accordingly, we found that the UPR agonist thapsigargin attenuated mutant Smoothened-induced phenotypes in vivo in Drosophila melanogaster. Wild-type Smoothened and physiological Hedgehog patterning were not affected, suggesting that UPR modulation may provide a novel therapeutic window to be evaluated for targeting active Smoothened mutants in disease. PMID:23572559

  19. Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

    SciTech Connect

    Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.; Lamson, Jacob S.; He, Jennifer; Hoover, Cindi A.; Blow, Matthew J.; Bristow, James; Butland, Gareth; Arkin, Adam P.; Deutschbauer, Adam

    2015-05-12

    Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative D-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes

  20. Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

    DOE PAGESBeta

    Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.; Lamson, Jacob S.; He, Jennifer; Hoover, Cindi A.; Blow, Matthew J.; Bristow, James; Butland, Gareth; Arkin, Adam P.; et al

    2015-05-12

    Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with anymore » transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative D-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are

  1. Significance of duon mutations in cancer genomes.

    PubMed

    Yadav, Vinod Kumar; Smith, Kyle S; Flinders, Colin; Mumenthaler, Shannon M; De, Subhajyoti

    2016-01-01

    Functional mutations in coding regions not only affect the structure and function of the protein products, but may also modulate their expression in some cases. This class of mutations, recently dubbed "duon mutations" due to their dual roles, can potentially have major impacts on downstream pathways. However their significance in diseases such as cancer remain unclear. In a survey covering 4606 samples from 19 cancer types, and integrating allelic expression, overall mRNA expression, regulatory motif perturbation, and chromatin signatures in one composite index called REDACT score, we identified potential duon mutations. Several such mutations are detected in known cancer genes in multiple cancer types. For instance a potential duon mutation in TP53 is associated with increased expression of the mutant allelic gene copy, thereby possibly amplifying the functional effects on the downstream pathways. Another potential duon mutation in SF3B1 is associated with abnormal splicing and changes in angiogenesis and matrix degradation related pathways. Our findings emphasize the need to interrogate the mutations in coding regions beyond their obvious effects on protein structures. PMID:27272679

  2. Significance of duon mutations in cancer genomes

    PubMed Central

    Yadav, Vinod Kumar; Smith, Kyle S.; Flinders, Colin; Mumenthaler, Shannon M.; De, Subhajyoti

    2016-01-01

    Functional mutations in coding regions not only affect the structure and function of the protein products, but may also modulate their expression in some cases. This class of mutations, recently dubbed “duon mutations” due to their dual roles, can potentially have major impacts on downstream pathways. However their significance in diseases such as cancer remain unclear. In a survey covering 4606 samples from 19 cancer types, and integrating allelic expression, overall mRNA expression, regulatory motif perturbation, and chromatin signatures in one composite index called REDACT score, we identified potential duon mutations. Several such mutations are detected in known cancer genes in multiple cancer types. For instance a potential duon mutation in TP53 is associated with increased expression of the mutant allelic gene copy, thereby possibly amplifying the functional effects on the downstream pathways. Another potential duon mutation in SF3B1 is associated with abnormal splicing and changes in angiogenesis and matrix degradation related pathways. Our findings emphasize the need to interrogate the mutations in coding regions beyond their obvious effects on protein structures. PMID:27272679

  3. Significance of duon mutations in cancer genomes

    NASA Astrophysics Data System (ADS)

    Yadav, Vinod Kumar; Smith, Kyle S.; Flinders, Colin; Mumenthaler, Shannon M.; de, Subhajyoti

    2016-06-01

    Functional mutations in coding regions not only affect the structure and function of the protein products, but may also modulate their expression in some cases. This class of mutations, recently dubbed “duon mutations” due to their dual roles, can potentially have major impacts on downstream pathways. However their significance in diseases such as cancer remain unclear. In a survey covering 4606 samples from 19 cancer types, and integrating allelic expression, overall mRNA expression, regulatory motif perturbation, and chromatin signatures in one composite index called REDACT score, we identified potential duon mutations. Several such mutations are detected in known cancer genes in multiple cancer types. For instance a potential duon mutation in TP53 is associated with increased expression of the mutant allelic gene copy, thereby possibly amplifying the functional effects on the downstream pathways. Another potential duon mutation in SF3B1 is associated with abnormal splicing and changes in angiogenesis and matrix degradation related pathways. Our findings emphasize the need to interrogate the mutations in coding regions beyond their obvious effects on protein structures.

  4. An In silico Based Comparison of Drug Interactions in Wild and Mutant Human β-tubulin through Docking Studies

    PubMed Central

    Chellasamy, Selvaakumar; Mohammed, Sudheer M. M.

    2014-01-01

    Background Tubulin protein being the fundamental unit of microtubules is actively involved in cell division thus making them a potential anti-cancer drug target. In spite of many reported drugs against tubulin, few of them have started developing resistance in human β-tubulin due to amino acid substitutions. Methods In this study we generated three mutants (F270V, A364T and Q292E) using Modeller9v10 which were targeted with compounds from higher and lower plants along with marine isolates using iGEMDOCK2.0 to identify their residual interactions. Results The mutant F270V does not bring in any increase in the binding affinity in comparison with the taxol-wild type due to their conservative substitutions. However, it increases the volume of the active site. A364T mutant brings a better binding among few of the marine and higher plants isolates due to the substitution of the non-reactive methyl group with the polar residue. But this leads to reduced active site volume. Finally the mutant Q292E from epothilone binding site brings a remarkable change in drug binding in the mutants in comparison with the wild type due to the substitution of uncharged residue with the charged one. But as such there was no change in the volume of the active site observed in them. Conclusion Lower plants extracts were reported to exhibit better interactions with the taxol and epothilone binding sites. Whereas marine and higher plants isolates shows significant interactions only in the wild type instead of the mutants. In addition to this, the residual substitutions were also found to alter the conformations of the active sites in mutants PMID:24834310

  5. Functional analysis of N-linked glycosylation mutants of the measles virus fusion protein synthesized by recombinant vaccinia virus vectors.

    PubMed Central

    Alkhatib, G; Shen, S H; Briedis, D; Richardson, C; Massie, B; Weinberg, R; Smith, D; Taylor, J; Paoletti, E; Roder, J

    1994-01-01

    The role of N-linked glycosylation in the biological activity of the measles virus (MV) fusion (F) protein was analyzed by expressing glycosylation mutants with recombinant vaccinia virus vectors. There are three potential N-linked glycosylation sites located on the F2 subunit polypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant proteins was reduced by 50% relative to the wild-type level when glycosylation at either Asn-29 or Asn-61 was abolished. Despite the similar levels of cell surface expression, the Asn-29 and Asn-61 mutant proteins had different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not exhibit any significant cell fusion activity. Inactivation of the Asn-67 glycosylation site also reduced cell surface transport of mutant protein but had little effect on its ability to cause cell fusion. However, when the Asn-67 mutation was combined with mutations at either of the other two sites, cleavage-dependent activation, cell surface expression, and cell fusion activity were completely abolished. Our data show that the loss of N-linked oligosaccharides markedly impaired the proteolytic cleavage, stability, and biological activity of the MV F protein. The oligosaccharide side chains in MV F are thus essential for optimum conformation of the extracellular F2 subunit that is presumed to bind cellular membranes. Images PMID:8107215

  6. In silico study on multidrug resistance conferred by I223R/H275Y double mutant neuraminidase.

    PubMed

    Tan, Hua; Wei, Kun; Bao, Jiguang; Zhou, Xiaobo

    2013-11-01

    It was recently reported that an I223R/H275Y double mutant of neuraminidase (NA) creates a multidrug-resistant form of the pandemic influenza A (H1N1) virus. However, a comprehensive understanding of the molecular mechanisms is still lacking. We conducted a systematic in silico study to explore the structural basis underlying this multidrug resistance. By molecular docking analyses and molecular dynamics (MD) simulations, we compared various biochemical and biophysical properties of the wild type, the I223R single mutant and the I223R/H275Y double mutant NA with two inhibitors, zanamivir (ZMR) and oseltamivir (G39). The binding free energy of oseltamivir with all types of NA was substantially lower than its zanamivir counterpart. On the other hand, the binding free energy of each inhibitor with wild type NA was generally higher than that with mutant NAs. MD simulation outcomes exemplify distinct patterns for oseltamivir and zanamivir with all types of NA. In particular, the stronger resistance of the double mutant NA relative to the wild and single mutant types can be ascribed to the overall looser but locally more compact structure of the former. Specifically, as a whole the double mutant NA adapts to the larger gyration radius and greater distance between charged atom groups, which is contrary to the pattern in the local binding site region. The enhanced resistance of all types of NA to oseltamivir rather than zanamivir might be accounted for similarly. We expect these findings to provide significant insights into improving inhibitors for the multidrug-resistant neuraminidase of H1N1 influenza viruses. PMID:24056678

  7. Quantitative dynamics and increased variability of segmentation gene expression in the Drosophila Krüppel and knirps mutants.

    PubMed

    Surkova, Svetlana; Golubkova, Elena; Manu; Panok, Lena; Mamon, Lyudmila; Reinitz, John; Samsonova, Maria

    2013-04-01

    Here we characterize the response of the Drosophila segmentation system to mutations in two gap genes, Kr and kni, in the form of single or double homozygotes and single heterozygotes. Segmentation gene expression in these genotypes was quantitatively monitored with cellular resolution in space and 6.5 to 13min resolution in time. As is the case with wild type, we found that gene expression domains in the posterior portion of the embryo shift to the anterior over time. In certain cases, such as the gt posterior domain in Kr mutants, the shifts are significantly larger than is seen in wild type embryos. We also investigated the effects of Kr and kni on the variability of gene expression. Mutations often produce variable phenotypes, and it is well known that the cuticular phenotype of Kr mutants is variable. We sought to understand the molecular basis of this effect. We find that throughout cycle 14A the relative levels of eve and ftz expression in stripes 2 and 3 are variable among individual embryos. Moreover, in Kr and kni mutants, unlike wild type, the variability in positioning of the posterior Hb domain and eve stripe 7 is not decreased or filtered with time. The posterior Gt domain in Kr mutants is highly variable at early times, but this variability decreases when this domain shifts in the anterior direction to the position of the neighboring Kni domain. In contrast to these findings, positional variability throughout the embryo does not decrease over time in double Kr;kni mutants. In heterozygotes the early expression patterns of segmentation genes resemble patterns seen in homozygous mutants but by the onset of gastrulation they become similar to the wild type patterns. Finally, we note that gene expression levels are reduced in Kr and kni mutant embryos and have a tendency to decrease over time. This is a surprising result in view of the role that mutual repression is thought to play in the gap gene system. PMID:23333947

  8. Electrochemical and NMR spectroscopic studies of distal pocket mutants of nitrophorin 2: Stability, structure, and dynamics of axial ligand complexes

    PubMed Central

    Shokhireva, Tatjana Kh.; Berry, Robert E.; Uno, Elizabeth; Balfour, Celia A.; Zhang, Hongjun; Walker, F. Ann

    2003-01-01

    WT and leucine → valine distal pocket mutants of nitrophorin 2 (NP2) and their NO complexes have been investigated by spectroelectrochemistry. NO complexes of two of the mutants exhibit more positive reduction potential shifts than does the WT protein, thus indicating stabilization of the Fe(II)–NO state. This more positive reduction potential for NP2-L132V and the double mutant is consistent with the hypothesis that smaller valine residues may allow the heme to regain planarity instead of being significantly ruffled, as in WT NP2. Thus, ruffling may stabilize the Fe(III)–NO state, which is required for facile NO dissociation. NMR spectroscopic investigations show that the sterically demanding 2-methylimidazole ligand readily binds to all three distal pocket mutants to create low-spin Fe(III) complexes having axial ligands in nearly perpendicular planes; it also binds to the WT protein in the presence of higher concentrations of 2-methylimidazole, but yields a different ligand plane orientation than is present in any of the three distal pocket mutants. NOESY spectra of NP2–ImH mutants exhibit chemical exchange cross peaks, whereas WT NP2–ImH shows no chemical exchange. Chemical exchange in the case of the distal leucine → valine mutants is caused by ImH ligand orientational dynamics. The two angular orientations of the ImH ligand could be determined from the 1H chemical shifts of the heme methyls, and the rate of interconversion of the two forms could be estimated from the NOESY diagonal and cross peak intensities. Keq is 100 or larger and favors an orientation similar to that found for the WT NP2–ImH complex. PMID:12642672

  9. Gravitropism in a starchless mutant of Arabidopsis: implications for the starch-statolith theory of gravity sensing

    NASA Technical Reports Server (NTRS)

    Caspar, T.; Pickard, B. G.

    1989-01-01

    The starch-statolith theory of gravity reception has been tested with a mutant of Arabidopsis thaliana (L.) Heynh. which, lacking plastid phosphoglucomutase (EC 2.7.5.1) activity, does not synthesize starch. The hypocotyls and seedling roots of the mutant were examined by light and electron microscopy to confirm that they did not contain starch. In upright wild-type (WT) seedlings, starch-filled plastids in the starch sheath of the hypocotyl and in three of the five columellar layers of the root cap were piled on the cell floors, and sedimented to the ceilings when the plants were inverted. However, starchless plastids of the mutant were not significantly sedimented in these cells in either upright or inverted seedlings. Gravitropism of light-grown seedling roots was vigorous: e.g., 10 degrees curvature developed in mutants rotated on a clinostat following a 5 min induction at 1 g, compared with 14 degrees in the WT. Curvatures induced during intervals from 2.5 to 30 min were 70% as great in the mutant as the WT. Thus under these conditions the presence of starch and the sedimentation of plastids are unnecessary for reception of gravity by Arabidopsis roots. Gravitropism by hypocotyls of light-grown seedlings was less vigorous than that by roots, but the mutant hypocotyls exhibited an average of 70-80% as much curvature as the WT. Roots and hypocotyls of etiolated seedlings and flower stalks of mature plants were also gravitropic, although in these cases the mutant was generally less closely comparable to the WT. Thus, starch is also unnecessary for gravity reception in these tissues.

  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. Isolation and characterisation of transport-defective substrate-binding mutants of the tetracycline antiporter TetA(B).

    PubMed

    Wright, David J; Tate, Christopher G

    2015-10-01

    The tetracycline antiporter TetA(B) is a member of the Major Facilitator Superfamily which confers tetracycline resistance to cells by coupling the efflux of tetracycline to the influx of protons down their chemical potential gradient. Although it is a medically important transporter, its structure has yet to be determined. One possibility for why this has proven difficult is that the transporter may be conformationally heterogeneous in the purified state. To overcome this, we developed two strategies to rapidly identify TetA(B) mutants that were transport-defective and that could still bind tetracycline. Up to 9 amino acid residues could be deleted from the loop between transmembrane α-helices 6 and 7 with only a slight decrease in affinity of tetracycline binding as measured by isothermal titration calorimetry, although the mutant was transport-defective. Scanning mutagenesis where all the residues between 2 and 389 were mutated to either valine, alanine or glycine (VAG scan) identified 15 mutants that were significantly impaired in tetracycline transport. Of these mutants, 12 showed no evidence of tetracycline binding by isothermal titration calorimetry performed on the purified transporters. In contrast, the mutants G44V and G346V bound tetracycline 4-5 fold more weakly than TetA(B), with Kds of 28 μM and 36 μM, respectively, whereas the mutant R70G bound tetracycline 3-fold more strongly (Kd 2.1 μM). Systematic mutagenesis is thus an effective strategy for isolating transporter mutants that may be conformationally constrained and which represent attractive targets for crystallisation and structure determination. PMID:26143388

  12. Competitive Exclusion of Epiphytic Bacteria by Ice−Pseudomonas syringae Mutants

    PubMed Central

    Lindow, Steven E.

    1987-01-01

    The growth of ice nucleation-active and near-isogenic ice nucleation-deficient (Ice−) Pseudomonas syringae strains coexisting on leaf surfaces was examined to determine whether competition was sufficient to account for antagonism of phylloplane bacteria. The ice nucleation frequency spectra of 46 Ice−P. syringae mutants, obtained after mutagenesis with ethyl methanesulfonate, differed both quantitatively and qualitatively, but the mutants could be grouped into four distinct phenotypic classes. The numbers of ice nucleation-active bacteria and ice nuclei active at −5°C were reduced on plants colonized with Ice−P. syringae mutant strains before challenge inoculations with an Ice+P. syringae wild-type strain. Frost injury to plants pretreated with Ice−P. syringae strains was also reduced significantly compared with that to control plants and was correlated with the population size of the Ice+P. syringae strain and with the numbers of ice nuclei active at −5°C. An Ice−P. syringae strain colonized leaves, flowers, and young fruit of pears in field experiments and significantly reduced the colonization of these tissues by Ice+P. syringae strains and Erwinia amylovora as compared with untreated trees. PMID:16347468

  13. BET and BRAF inhibitors act synergistically against BRAF-mutant melanoma.

    PubMed

    Paoluzzi, Luca; Hanniford, Douglas; Sokolova, Elena; Osman, Iman; Darvishian, Farbod; Wang, Jinhua; Bradner, James E; Hernando, Eva

    2016-06-01

    Despite major advances in the treatment of metastatic melanoma, treatment failure is still inevitable in most cases. Manipulation of key epigenetic regulators, including inhibition of Bromodomain and extra-terminal domain (BET) family members impairs cell proliferation in vitro and tumor growth in vivo in different cancers, including melanoma. Here, we investigated the effect of combining the BET inhibitor JQ1 with the BRAF inhibitor Vemurafenib in in vitro and in vivo models of BRAF-mutant melanoma. We performed cytotoxicity and apoptosis assays, and a xenograft mouse model to determine the in vitro and in vivo efficacy of JQ1 in combination with Vemurafenib against BRAF-mutant melanoma cell lines. Further, to investigate the molecular mechanisms underlying the effects of combined treatment, we conducted antibody arrays of in vitro drug-treated cell lines and RNA sequencing of drug-treated xenograft tumors. The combination of JQ1 and Vemurafenib acted synergistically in BRAF-mutant cell lines, resulting in marked apoptosis in vitro, with upregulation of proapoptotic proteins. In vivo, combination treatment suppressed tumor growth and significantly improved survival compared to either drug alone. RNA sequencing of tumor tissues revealed almost four thousand genes that were uniquely modulated by the combination, with several anti-apoptotic genes significantly down-regulated. Collectively, our data provide a rationale for combined BET and BRAF inhibition as a novel strategy for the treatment of melanoma. PMID:27169980

  14. Evaluation of novel starch-deficient mutants of Chlorella sorokiniana for hyper-accumulation of lipids

    PubMed Central

    Vonlanthen, Sofie; Dauvillée, David; Purton, Saul

    2015-01-01

    When green algae are exposed to physiological stresses such as nutrient deprivation, growth is arrested and the cells channel fixed carbon instead into storage compounds, accumulating first starch granules and then lipid bodies containing triacylglycerides. In recent years there has been significant interest in the commercial exploitation of algal lipids as a sustainable source of biodiesel. Since starch and lipid biosynthesis involves the same C3 precursor pool, it has been proposed that mutations blocking starch accumulation should result in increased lipid yields, and indeed several studies have supported this. The fast-growing, thermotolerant alga Chlorella sorokiniana represents an attractive strain for industrial cultivation. We have therefore generated and characterized starch-deficient mutants of C. sorokiniana and determined whether lipid levels are increased in these strains under stress conditions. One mutant (ST68) is shown to lack isoamylase, whilst two others (ST3 and ST12) are defective in starch phosphorylase. However, we find no significant change in the accumulation or profile of fatty acids in these mutants compared to the wild-type, suggesting that a failure to accumulate starch per se is not sufficient for the hyper-accumulation of lipid, and that more subtle regulatory steps underlie the partitioning of carbon to the two storage products. PMID:26865991

  15. A histone mutant reproduces the phenotype caused by loss of histone-modifying factor Polycomb.

    PubMed

    Pengelly, Ana Raquel; Copur, Ömer; Jäckle, Herbert; Herzig, Alf; Müller, Jürg

    2013-02-01

    Although many metazoan enzymes that add or remove specific modifications on histone proteins are essential transcriptional regulators, the functional significance of posttranslational modifications on histone proteins is not well understood. Here, we show in Drosophila that a point mutation in lysine 27 of histone H3 (H3-K27) fails to repress transcription of genes that are normally repressed by Polycomb repressive complex 2 (PRC2), the methyltransferase that modifies H3-K27. Moreover, differentiated H3-K27 mutant cells show homeotic transformations like those seen in PRC2 mutant cells. Taken together, these analyses demonstrate that H3-K27 is the crucial physiological substrate that PRC2 modifies for Polycomb repression. PMID:23393264

  16. Analysis on the DNA Fingerprinting of Aspergillus Oryzae Mutant Induced by High Hydrostatic Pressure

    NASA Astrophysics Data System (ADS)

    Wang, Hua; Zhang, Jian; Yang, Fan; Wang, Kai; Shen, Si-Le; Liu, Bing-Bing; Zou, Bo; Zou, Guang-Tian

    2011-01-01

    The mutant strains of aspergillus oryzae (HP300a) are screened under 300 MPa for 20 min. Compared with the control strains, the screened mutant strains have unique properties such as genetic stability, rapid growth, lots of spores, and high protease activity. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) are used to analyze the DNA fingerprinting of HP300a and the control strains. There are 67.9% and 51.3% polymorphic bands obtained by these two markers, respectively, indicating significant genetic variations between HP300a and the control strains. In addition, comparison of HP300a and the control strains, the genetic distances of random sequence and simple sequence repeat of DNA are 0.51 and 0.34, respectively.

  17. DeltaznuADeltapurE Brucella abortus 2308 mutant as a live vaccine candidate.

    PubMed

    Yang, Xinghong; Thornburg, Theresa; Walters, Nancy; Pascual, David W

    2010-01-22

    To create a new, safe brucellosis live vaccine, a double mutant strain was constructed from Brucella abortus 2308. Using the DeltaznuA B. abortus 2308 mutant, a second mutation was introduced by deleting purE gene. The DeltaznuA DeltapurE B. abortus 2308 strain was less capable of surviving in macrophages. When evaluated in vivo, it was cleared within 8 weeks (wks) from mice, causing significantly less inflammation than spleens obtained from wild-type B. abortus 2308-infected mice. Furthermore, two doses of DeltaznuA DeltapurE B. abortus 2308 conferred 0.79 log protection, similar to S19 as did a single dose of DeltaznuA B. abortus 2308. Thus, this study shows the DeltaznuA DeltapurE B. abortus 2308 strain to be a potential livestock vaccine candidate. PMID:19914192

  18. Waved-1 mutant mice are hypersensitive to the locomotor actions of cocaine.

    PubMed

    Stanwood, Gregg D; Levitt, Pat

    2007-04-01

    Transforming growth factor-alpha (TGFalpha) is a well-known regulator of many developmental processes, and is expressed heavily in basal forebrain and striatal regions. When TGFalpha is reduced in Waved-1 (Wa-1) mutant mice, brain anatomy, biogenic amines, stress response, and behavior are normal prior to, but altered following puberty. As an initial screen for possible alterations in nigrostriatal and mesolimbic dopamine (DA) systems, we tested adult Wa-1 mutant mice in an open field, following acute injection with cocaine (15 mg/kg). Wa-1 mice exhibited significantly greater ambulatory distance, number of ambulatory episodes, and cocaine-induced motor stereotypies than do controls. These data indicate that adult Wa-1 mice are hypersensitive to the locomotor effects of cocaine and provide a new potential link between neurodevelopmental processes and adult psychostimulant responsiveness. PMID:17230552

  19. Prediction of Enzyme Mutant Activity Using Computational Mutagenesis and Incremental Transduction

    PubMed Central

    Basit, Nada; Wechsler, Harry

    2011-01-01

    Wet laboratory mutagenesis to determine enzyme activity changes is expensive and time consuming. This paper expands on standard one-shot learning by proposing an incremental transductive method (T2bRF) for the prediction of enzyme mutant activity during mutagenesis using Delaunay tessellation and 4-body statistical potentials for representation. Incremental learning is in tune with both eScience and actual experimentation, as it accounts for cumulative annotation effects of enzyme mutant activity over time. The experimental results reported, using cross-validation, show that overall the incremental transductive method proposed, using random forest as base classifier, yields better results compared to one-shot learning methods. T2bRF is shown to yield 90% on T4 and LAC (and 86% on HIV-1). This is significantly better than state-of-the-art competing methods, whose performance yield is at 80% or less using the same datasets. PMID:22007208

  20. Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency: Activity in Normal, Mutant, and Heterozygote-Cultured Human Skin Fibroblasts

    PubMed Central

    Fujimoto, Wilfred Y.; Seegmiller, J. Edwin

    1970-01-01

    Cultured skin fibroblasts from patients deficient for the enzyme hypoxanthine-guanine phosphoribosyltransferase (PRT) activity show very low but nevertheless significant levels of apparent PRT enzyme despite absence of detectable activity (<0.004% of normal) in erythrocytes of the same patients. In fibroblasts this mutant enzyme is more heat labile than the normal enzyme. These findings indicate that PRT deficiency in this disorder is not due to a deletion mutation of the PRT locus. Individual cultured skin fibroblasts from heterozygote females for PRT deficiency show normal, intermediate, or very low levels of PRT activity. The mosaicism demonstrated in the heterozygotes for this X-linked disorder accounts for the cells with normal and very low activities of PRT. Intermediate activity can best be explained by the phenomenon of metabolic cooperation presumably from the transfer of either PRT enzyme or messenger RNA, from normal to mutant cells. Images PMID:5267139

  1. Persistence of Escherichia coli O157:H7 and its mutants in soils.

    PubMed

    Ma, Jincai; Ibekwe, A Mark; Yi, Xuan; Wang, Haizhen; Yamazaki, Akihiro; Crowley, David E; Yang, Ching-Hong

    2011-01-01

    The persistence of Shiga toxin-producing E. coli O157:H7 in the environment poses a serious threat to public health. However, the role of Shiga toxins and other virulence factors in the survival of E. coli O157:H7 is poorly defined. The aim of this study was to determine if the virulence factors, stx₁, stx₂, stx₁₋₂, and eae in E. coli O157:H7 EDL933 play any significant role in the growth of this pathogen in rich media and in soils. Isogenic deletion mutants that were missing one of four virulence factors, stx₁, stx₂, stx₁₋₂, and eae in E. coli O157:H7 EDL933 were constructed, and their growth in rich media and survival in soils with distinct texture and chemistry were characterized. The survival data were successfully analyzed using Double Weibull model, and the modeling parameters of the mutant strains were not significantly different from those of the wild type. The calculated T(d) (time needed to reach the detection limit, 100 CFU/g soil) for loamy sand, sandy loam, and silty clay was 32, 80, and 110 days, respectively. It was also found that T(d) was positively correlated with soil structure (e.g. clay content), and soil chemistry (e.g. total nitrogen, total carbon, and water extractable organic carbon). The results of this study showed that the possession of Shiga toxins and intimin in E. coli O157:H7 might not play any important role in its survival in soils. The double deletion mutant of E. coli O157:H7 (stx₁⁻stx₂⁻) may be a good substitute to use for the investigation of transport, fate, and survival of E. coli O157:H7 in the environment where the use of pathogenic strains are prohibited by law since the mutants showed the same characteristics in both culture media and environmental samples. PMID:21826238

  2. Replication of HIV-1 deleted Nef mutants in chronically immune activated human T cells.

    PubMed

    Shapira-Nahor, Orit; Maayan, Shlomo; Peden, Keith W C; Rabinowitz, Ruth; Schlesinger, Michael; Alian, Akram; Panet, Amos

    2002-11-10

    Lymphocytes (PBMC) obtained from blood of HIV-sera negative Ethiopian immigrants (ETH) were highly susceptible to HIV-1 infection in vitro with no need for stimulation by mitogens. As the HIV nef gene product has been shown to enhance viral replication in stimulated primary lymphocytes, we investigated in this work the role of Nef in viral replication in the ETH cells. Lymphocytes obtained from ETH individuals supported high replication of wild-type HIV-1 and low but significant replication level of the two deleted Nef mutants (encode truncated Nef proteins consisting only of either the first 35 or the first 86 amino acids of Nef). In contrast, no replication was observed in nonactivated cells obtained from non-ETH individuals. After activation of the PBMC from ETH individuals with PHA, replication of both wild-type strains and the two deleted Nef mutant viruses further increased. The CD4(+) T cells of ETH individuals exhibited elevated levels of the surface activation markers CD45RO and HLA-DR, compared with T cells derived from non-ETH group. Likewise, expression of the chemokine receptors CCR5 and CXCR4 on these cells was higher in the ETH group than in the non-ETH group. Replication of HIV-1 wild-type and the isogenic-deleted Nef mutants was significantly correlated with the proportion of ETH cells expressing CD45RO and the chemokine receptors. This study suggests that HIV-1 may respond differently to several activation states characteristic of T cells. One activation state, defined by chronically activated lymphocytes from ETH individuals, is permissive to the wild-type HIV-1 and, to a lesser degree, to the Nef mutants. Further activation of these cells by exogenous stimuli enhances replication of the virus. Our results support the notion that Nef enhances the basal level of T cell activation and consequently, viral replication. PMID:12482665

  3. Functional Robustness of a Polycyclic Aromatic Hydrocarbon Metabolic Network Examined in a nidA Aromatic Ring-Hydroxylating Oxygenase Mutant of Mycobacterium vanbaalenii PYR-1

    PubMed Central

    Kim, Seong-Jae; Song, Jaekyeong; Kweon, Ohgew; Holland, Ricky D.; Kim, Dae-Wi; Kim, Jongnam; Yu, Li-Rong

    2012-01-01

    In this study, we obtained over 4,000 transposon mutants of Mycobacterium vanbaalenii PYR-1 and analyzed one of the mutants, 8F7, which appeared to lose its ability to degrade pyrene while still being able to degrade fluoranthene. This mutant was identified to be defective in nidA, encoding an aromatic ring-hydroxylating oxygenase (RHO), known to be involved in the initial oxidation step of pyrene degradation. When cultured with pyrene as a sole source of polycyclic aromatic hydrocarbon (PAH), high-pressure liquid chromatography analysis revealed that the nidA mutant showed a significant decrease in the rate of pyrene degradation compared to the wild-type PYR-1, although pyrene was still being degraded. However, when incubated with PAH mixtures including pyrene, phenanthrene, and fluoranthene, the pyrene degradation rate of the mutant was higher than that of the mutant previously incubated with pyrene as a sole source of PAH. There was no significant difference between wild-type PYR-1 and the mutant in the rates of phenanthrene and fluoranthene degradation. From the whole-cell proteome analysis of mutant 8F7 induced by pyrene, we identified expression of a number of RHO enzymes which are suspected to be responsible for pyrene degradation in the nidA mutant, which had no expression of NidA. Taken together, results in this study provide direct evidence for the in vivo functional role of nidA in pyrene degradation at the level of the ring-cleavage-process (RCP) functional module but also for the robustness of the PAH metabolic network (MN) to such a genetic perturbation. PMID:22407691

  4. Deciphering the Dynamics of Non-Covalent Interactions Affecting Thermal Stability of a Protein: Molecular Dynamics Study on Point Mutant of Thermus thermophilus Isopropylmalate Dehydrogenase.

    PubMed

    Sharma, Reetu; Sastry, G Narahari

    2015-01-01

    Thermus thermophilius isopropylmalate dehydrogenase catalyzes oxidative decarboxylation and dehydrogenation of isopropylmalate. Substitution of leucine to alanine at position 172 enhances the thermal stability among the known point mutants. Exploring the dynamic properties of non-covalent interactions such as saltbridges, hydrogen bonds and hydrophobic interactions to explain thermal stability of a protein is interesting in its own right. In this study dynamic changes in the non-covalent interactions are studied to decipher the deterministic features of thermal stability of a protein considering a case study of a point mutant in Thermus thermophilus isopropylmalate dehydrogenase. A total of four molecular dynamic simulations of 0.2 μs were carried out on wild type and mutant's functional dimers at 300 K and 337 K. Higher thermal stability of the mutant as compared to wild type is revealed by root mean square deviation, root mean square fluctuations and Cα-Cα distance with an increase in temperature from 300 K to 337 K. Most of the regions of wild type fluctuate higher than the corresponding regions of mutant with an increase in temperature. Cα-Cα distance analysis suggests that long distance networks are significantly affected in wild type as compared to the mutant. Short lived contacts are higher in wild type, while long lived contacts are lost at 337 K. The mutant forms less hydrogen bonds with water as compared to wild type at 337 K. In contrast to wild type, the mutant shows significant increase in unique saltbridges, hydrogen bonds and hydrophobic contacts at 337 K. The current study indicates that there is a strong inter-dependence of thermal stability on the way in which non-covalent interactions reorganize, and it is rewarding to explore this connection in single mutant studies. PMID:26657745

  5. Graviresponsiveness and abscisic-acid content of roots of carotenoid-deficient mutants of Zea mays L

    NASA Technical Reports Server (NTRS)

    Moore, R.; Smith, J. D.

    1985-01-01

    The abscisic-acid (ABA) content of roots of the carotenoid-deficient w-3, vp-5, and vp-7 mutants of Z. mays was analyzed using gas chromatography-mass spectrometry with an analysis sensitivity of 6 ng ABA g-1 fresh weight (FW). Roots of normal seedlings of the same lines were characterized by the following amounts of ABA (as ng ABA g-1 FW, +/- standard deviation): w-3, 279 +/- 43; vp-5, 237 +/- 26; vp-7, 338 +/- 61. We did not detect any ABA in roots of any of the mutants. Thus, the lack of carotenoids in these mutants correlated positively with the apparent absence of ABA. Primary roots of normal and mutant seedlings were positively gravitropic, with no significant differences in the curvatures of roots of normal as compared with mutant seedlings. These results indicate that ABA 1) is synthesized in maize roots via the carotenoid pathway, and 2) is not necessary for positive gravitropism by primary roots of Z. mays.

  6. Avian pathogenic Escherichia coli ΔtonB mutants are safe and protective live-attenuated vaccine candidates.

    PubMed

    Holden, Karen M; Browning, Glenn F; Noormohammadi, Amir H; Markham, Philip; Marenda, Marc S

    2014-10-10

    Avian pathogenic Escherichia coli (APEC) cause colibacillosis, a serious respiratory disease in poultry. Most APEC strains possess TonB-dependent outer membrane transporters for the siderophores salmochelin and aerobactin, which both contribute to their capacity to cause disease. To assess the potential of iron transport deficient mutants as vaccine candidates, the tonB gene was deleted in the APEC wild type strain E956 and a Δfur (ferric uptake repressor) mutant of E956. The growth of the ΔtonB and ΔtonB/Δfur mutants was impaired in iron-restricted conditions, but not in iron-replete media. Day old chicks were exposed to aerosols of the mutants to assess their efficacy as live attenuated vaccines. At day 18, the birds were challenged with aerosols of the virulent parent strain E956. Both mutants conferred protection against colibacillosis; weight gains and lesion scores were significantly different between the vaccinated groups and an unvaccinated challenged control group. Thus mutation of iron uptake systems can be used as a platform technology to generate protective live attenuated vaccines against extraintestinal E. coli infections, and potentially a range of Gram negative pathogens of importance in veterinary medicine. PMID:25205199

  7. Isolation of a spontaneous CHO amino acid transport mutant by a combination of tritium suicide and replica plating

    SciTech Connect

    Dantzig, A.H.; Slayman, C.W.; Adelberg, E.A.

    1982-07-01

    A spontaneous transport mutant of Chinese hamster ovary cells, CHY-1, was isolated by a combination of (/sup 3/H)proline suicide and replica plating. The mutant took up less tritium than the parent, resulting in a lower killing rate during storage. Transport by four separate amino acid transport systems (A, ASC, L, Ly+) was examined. The CHY-1 mutant exhibited normal uptake via the ASC, L, and Ly+ systems. By contrast, uptake of the most specific substrate of the A system, 2-(methylamino)-isobutyric acid, was significantly reduced at low, but not high, concentrations, due to a 3.5-fold increase in Km and a 1.5-fold increase in Vmax. Taken together, these data suggest that the CHY-1 mutation may be in the structural gene coding for the A transport protein. The tritium suicide procedure is discussed, and general equations are derived to predict the maximum storage time for the survival of one mutant cell and the optimum size of the cell population for maximum mutant enrichment.

  8. Waiting times for the appearance of cytotoxic T-lymphocyte escape mutants in chronic HIV-1 infection

    SciTech Connect

    Liu Yi . E-mail: yiliu197@u.washington.edu; Mullins, James I.; Mittler, John E.

    2006-03-30

    The failure of HIV-1 to escape at some cytotoxic T-lymphocyte (CTL) epitopes has generally been explained in terms of viral fitness costs or ineffective or attenuated CTL responses. Relatively little attention has been paid to the evolutionary time required for escape mutants to be detected. This time is significantly affected by selection, mutation rates, the presence of other advantageous mutations, and the effective population size of HIV-1 in vivo (typically estimated to be {approx}10{sup 3} in chronically infected patients, though one study has estimated it to be {approx}10{sup 5}). Here, we use a forward simulator with experimentally estimated HIV-1 parameters to show that these delays can be substantial. For an effective population size of 10{sup 3}, even highly advantageous mutants (s = 0.5) may not be detected for a couple of years in chronically infected patients, while moderately advantageous escape mutants (s = 0.1) may not be detected for up to 10 years. Even with an effective population size of 10{sup 5}, a moderately advantageous escape mutant (s = 0.1) may not be detected in the population within 2 years if it has to compete with other selectively advantageous mutants. Stochastic evolutionary forces, therefore, in addition to viral fitness costs and ineffective or attenuated CTL responses, must be taken into account when assessing the selection of CTL escape mutations.

  9. Effects of diet on synaptic vesicle release in dynactin complex mutants: a mechanism for improved vitality during motor disease

    PubMed Central

    Rawson, Joel M.; Kreko, Tabita; Davison, Holly; Mahoney, Rebekah; Bokov, Alex; Chang, Leo; Gelfond, Jon; Macleod, Greg T.; Eaton, Benjamin A.

    2012-01-01

    Summary Synaptic dysfunction is considered the primary substrate for the functional declines observed within the nervous system during age-related neurodegenerative disease. Dietary restriction (DR), which extends lifespan in numerous species, has been shown to have beneficial effects on many neurodegenerative disease models. Existing data sets suggest that the effects of DR during disease include the amelioration of synaptic dysfunction but evidence of the beneficial effects of diet on the synapse is lacking. Dynactin mutant flies have significant increases in mortality rates and exhibit progressive loss of motor function. Using a novel fly motor disease model, we demonstrate that mutant flies raised on a low calorie diet have enhanced motor function and improved survival compared to flies on a high calorie diet. Neurodegeneration in this model is characterized by an early impairment of neurotransmission that precedes the deterioration of neuromuscular junction (NMJ) morphology. In mutant flies, low calorie diet increases neurotransmission, but has little effect on morphology, supporting the hypothesis that enhanced neurotransmission contributes to the effects of diet on motor function. Importantly, the effects of diet on the synapse are not due to the reduction of mutant pathologies, but by the increased release of synaptic vesicles during activity. The generality of this effect is demonstrated by the observation that diet can also increase synaptic vesicle release at wild type NMJs. These studies reveal a novel presynaptic mechanism of diet that may contribute to the improved vigor observed in mutant flies raised on low calorie diet. PMID:22268717

  10. Biological features of an early-maturity mutant of sweet sorghum induced by carbon ions irradiation and its genetic polymorphism

    NASA Astrophysics Data System (ADS)

    Dong, Xicun; Li, Wenjian

    2012-08-01

    It is well known that heavy ions irradiation is characterized by a high linear energy transfer (LET) and relative biological effectiveness (RBE). These characters are believed to increase mutation frequency and mutation spectrum of plants or mammalian cells irradiated by heavy ions. Here we describe an early-maturity mutant of sweet sorghum induced by carbon ion irradiation. The growth period of this mutant was shortened by about 20 days compared to the wild type. The proline content of the mutant was increased by 11.05% while the malondialdehyde content was significantly lower than that of wild type. In addition, the RAPD analysis indicated that the percentage of polymorphism between the mutant KFJT-1 and the control KFJT-CK reached 5.26%. The gain of early-maturity might solve the problem in the northwest region of China where seeds of sweet sorghum cannot be mature because of early frost. The early-maturity mutant may be important for future space cultivation.

  11. Mutant Glycyl-tRNA Synthetase (Gars) Ameliorates SOD1G93A Motor Neuron Degeneration Phenotype but Has Little Affect on Loa Dynein Heavy Chain Mutant Mice

    PubMed Central

    Williams, Hazel P.; Chia, Ruth; Achilli, Francesca; Bryson, J. Barney; Greensmith, Linda; Fisher, Elizabeth M. C.

    2009-01-01

    Background In humans, mutations in the enzyme glycyl-tRNA synthetase (GARS) cause motor and sensory axon loss in the peripheral nervous system, and clinical phenotypes ranging from Charcot-Marie-Tooth neuropathy to a severe infantile form of spinal muscular atrophy. GARS is ubiquitously expressed and may have functions in addition to its canonical role in protein synthesis through catalyzing the addition of glycine to cognate tRNAs. Methodology/Principal Findings We have recently described a new mouse model with a point mutation in the Gars gene resulting in a cysteine to arginine change at residue 201. Heterozygous GarsC201R/+ mice have locomotor and sensory deficits. In an investigation of genetic mutations that lead to death of motor and sensory neurons, we have crossed the GarsC201R/+ mice to two other mutants: the TgSOD1G93A model of human amyotrophic lateral sclerosis and the Legs at odd angles mouse (Dync1h1Loa) which has a defect in the heavy chain of the dynein complex. We found the Dync1h1Loa/+;GarsC201R/+ double heterozygous mice are more impaired than either parent, and this is may be an additive effect of both mutations. Surprisingly, the GarsC201R mutation significantly delayed disease onset in the SOD1G93A;GarsC201R/+ double heterozygous mutant mice and increased lifespan by 29% on the genetic background investigated. Conclusions/Significance These findings raise intriguing possibilities for the study of pathogenetic mechanisms in all three mouse mutant strains. PMID:19593442

  12. La Crosse virus (LACV) Gc fusion peptide mutants have impaired growth and fusion phenotypes, but remain neurotoxic

    SciTech Connect

    Soldan, Samantha S.; Hollidge, Bradley S.; Wagner, Valentina; Weber, Friedemann; Gonzalez-Scarano, Francisco

    2010-09-01

    La Crosse virus is a leading cause of pediatric encephalitis in the Midwestern United States and an emerging pathogen in the American South. The LACV glycoprotein Gc plays a critical role in entry as the virus attachment protein. A 22 amino acid hydrophobic region within Gc (1066-1087) was recently identified as the LACV fusion peptide. To further define the role of Gc (1066-1087) in virus entry, fusion, and neuropathogenesis, a panel of recombinant LACV (rLACV) fusion peptide mutant viruses was generated. Replication of mutant rLACVs was significantly reduced. In addition, the fusion peptide mutants demonstrated decreased fusion phenotypes relative to LACV-WT. Interestingly, these viruses maintained their ability to cause neuronal loss in culture, suggesting that the fusion peptide of LACV Gc is a determinant of properties associated with neuroinvasion (growth to high titer in muscle cells and a robust fusion phenotype), but not necessarily of neurovirulence.

  13. Surveying the lipogenesis landscape in Yarrowia lipolytica through understanding the function of a Mga2p regulatory protein mutant.

    PubMed

    Liu, Leqian; Markham, Kelly; Blazeck, John; Zhou, Nijia; Leon, Dacia; Otoupal, Peter; Alper, Hal S

    2015-09-01

    Lipogenic organisms represent great starting points for metabolic engineering of oleochemical production. While previous engineering efforts were able to significantly improve lipid production in Yarrowia lipolytica, the lipogenesis landscape, especially with respect to regulatory elements, has not been fully explored. Through a comparative genomics and transcriptomics approach, we identified and validated a mutant mga2 protein that serves as a regulator of desaturase gene expression and potent lipogenesis factor. The resulting strain is enriched in unsaturated fatty acids. Comparing the underlying mechanism of this mutant to other previously engineered strains suggests that creating an imbalance between glycolysis and the TCA cycle can serve as a driving force for lipogenesis when combined with fatty acid catabolism overexpressions. Further comparative transcriptomics analysis revealed both distinct and convergent rewiring associated with these different genotypes. Finally, by combining metabolic engineering targets, it is possible to further engineer a strain containing the mutant mga2 gene to a lipid production titer of 25g/L. PMID:26219673

  14. Quantification of intrahost bottlenecks of West Nile virus in Culex pipiens mosquitoes using an artificial mutant swarm

    PubMed Central

    Ciota, Alexander T.; Ehrbar, Dylan J.; Van Slyke, Greta A.; Payne, Anne F.; Willsey, Graham G.; Viscio, Rachael E.; Kramer, Laura D.

    2012-01-01

    Mosquito-borne viruses are predominantly RNA viruses which exist within hosts as diverse mutant swarms. Defining the way in which stochastic forces within mosquito vectors shape these swarms is critical to advancing our understanding of the evolutionary and adaptive potential of these pathogens. There are multiple barriers within a mosquito which a viral swarm must traverse in order to ultimately be transmitted. Here, using artificial mutant swarms composed of neutral variants of West Nile virus (WNV), we tracked changes to swarm breadth over time and space in Culex pipiens mosquitoes. Our results demonstrate that all variants have the potential to survive intrahost bottlenecks, yet mean swarm breadth decreases during both midgut infection and transmission when starting populations contain higher levels of minority variants. In addition, WNV swarms are subject to temporal sweeps which act to significantly decrease intrahost diversity over time. Taken together, these data demonstrate the profound effects that stochastic forces can have in shaping arboviral mutant swarms. PMID:22326536

  15. Colletotrichum sublineolum genetic instability assessed by mutants resistant to chlorate.

    PubMed

    Cecília de Lima Fávaro, Léia; Luiz Araújo, Welington; Aparecida de Souza-Paccola, Ednéia; Lúcio Azevedo, João; Paccola-Meirelles, Luzia Doretto

    2007-01-01

    The fungus Colletotrichum sublineolum, causal agent of sorghum anthracnose, presents high variability, genetic instability and host specialization. The aims of the present work were to investigate the mechanisms involved in the genetic instability in this species. Mutants resistant to chlorate and unable to use nitrate (Nit mutants), were obtained spontaneously, isolated and characterized for complementation pattern, reversion frequency and RAPD profile. The results showed that chlorate-resistant mutants could be divided into six phenotypic classes that probably represented mutations in the structural nitrate reductase locus (nit1), in the structural nitrite reductase locus (nit6 and niiA of Neurospora and Aspergillus, respectively), in the specific regulator locus (nit3), in the main regulator locus (nit2), in loci that codified the cofactor containing molybdenum necessary for nitrate reductase activity (NitM), and one or more genes responsible for nitrate intake (crn). In addition, the genetic control of this metabolism in C. sublineolum seems to be similar to other fungi species such as Aspergillus, Neurospora and Fusarium. The high reversion frequency (10(-4) to 10(-5)) presented by nit1 mutants suggests that the instability in evaluated strains could be a result of transposable elements activity. The RAPD analysis enabled confirmation that the Nit mutants have a similar genetic background to original strain, and that polymorphism exists among wild-type strains, nit1 mutants and revertants of C. sublineolum. These are important aspects for the later direction of molecular analysis, where these mutants will be used as a tool to isolate the active transposable elements in the C. sublineolum genome. PMID:17158042

  16. Incomplete flagellar structures in nonflagellate mutants of Salmonella typhimurium.

    PubMed Central

    Suzuki, T; Iino, T; Horiguchi, T; Yamaguchi, S

    1978-01-01

    Incomplete flagellar structures were detected in osmotically shocked cells or membrane-associated fraction of many nonflagellate mutants of Salmonella typhimurium by electron microscopy. The predominant types of these structures in the mutants were cistron specific. The incomplete basal bodies were detected in flaFI, flaFIV, flaFVIII, and flaFIX mutants, the structure homologous to a basal body in flaFV mutants, the polyhook-basal body complex in flaR mutants, and the hook-basal body complex in flaL and flaU mutants. No structures homologous to flagellar bases or their parts were detected in the early-fla group nonflagellate mutants of flaAI, flaAII, flaAIII, flaB, flaC, flaD, flaE, flaFII, flaFIII, flaFVI, flaFVII, flaFX, flaK, and flaM. From these observations, a process of flagellar morphogenesis was postulated. The functions of the early-fla group are essential to the formation of S ring-M ring-rod complexes bound to the membrane. The completion of basal bodies requires succeeding functions of flaFI, flaFIV, flaFVIII, and flaFIX. Next, the formation of hooks attached to basal bodies proceeds by the function of flaFV and by flaR, which controls the hook length. Flagellar filaments appear at the tips of hooks because of the functions of flaL, flaU, and flagellin genes. Images PMID:342514

  17. Hydrocarbon assimilation and biosurfactant production in Pseudomonas aeruginosa mutants

    SciTech Connect

    Koch, A.K.; Fiechter, A.; Reiser, J. ); Kaeppeli, O. )

    1991-07-01

    The authors isolated transposon Tn5-GM-induced mutants of Pseudomonas aeruginosa PG201 that were unable to grow in minimal media containing hexadecane as a carbon source. Some of these mutants lacked extracellular rhamnolipids, as shown by measuring the surface and interfacial tensions of the cell culture supernatants. Furthermore, the concentrated culture media of the mutant strains were tested for the presence of rhamnolipids by thin-layer chromatography and for rhamnolipid activities, including hemolysis and growth inhibition of Bacillus subtilis. Mutant 65E12 was unable to produce extracellular rhamnolipids under any of the inhibition of Bacillus subtilis. Mutant 65E12 was unable to produce extracellular rhamnolipids under any of the conditions tested, lacked the capacity to take up {sup 14}C-labeled hexadecane, and did not grow in media containing individual alkanes with chain lengths ranging from C{sub 12} to C{sub 19}. However, growth on these alkanes and uptake of ({sup 14}C)hexadecane were restored when small amounts of purified rhamnolipids were added to the cultures. Mutant 59C7 was unable to grow in media containing hexadecane, nor was it able to take up ({sup 14}C)hexadecane uptake. The addition of small amounts of rhamnolipids restored on alkanes and ({sup 14}C)hexadecane uptake. In glucose-containing media, however, mutant 59C7 produced rhamnolipids at levels about twice as high as those of the wild-type strain. These results show that rhamnolipids play a major role in hexadecane uptake and utilization by P.aeruginosa.

  18. Altered lipid composition in Streptococcus pneumoniae cpoA mutants

    PubMed Central

    2014-01-01

    Background Penicillin-resistance in Streptococcus pneumoniae is mainly due to alterations in genes encoding the target enzymes for beta-lactams, the penicillin-binding proteins (PBPs). However, non-PBP genes are altered in beta-lactam-resistant laboratory mutants and confer decreased susceptibility to beta-lactam antibiotics. Two piperacillin resistant laboratory mutants of Streptococcus pneumoniae R6 contain mutations in the putative glycosyltransferase gene cpoA. The CpoA gene is part of an operon including another putative glycosyltransferase gene spr0982, both of which being homologous to glycolipid synthases present in other Gram-positive bacteria. Results We now show that the cpoA mutants as well as a cpoA deletion mutant are defective in the synthesis of galactosyl-glucosyl-diacylglycerol (GalGlcDAG) in vivo consistent with the in vitro function of CpoA as α-GalGlcDAG synthase as shown previously. In addition, the proportion of phosphatidylglycerol increased relative to cardiolipin in cpoA mutants. Moreover, cpoA mutants are more susceptible to acidic stress, have an increased requirement for Mg2+ at low pH, reveal a higher resistance to lysis inducing conditions and are hypersensitive to bacitracin. Conclusions The data show that deficiency of the major glycolipid GalGlcDAG causes a pleitotropic phenotype of cpoA mutant cells consistent with severe membrane alterations. We suggest that the cpoA mutations selected with piperacillin are directed against the lytic response induced by the beta-lactam antibiotic. PMID:24443834

  19. Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

    PubMed Central

    Hirata, Hiromi; Carta, Eloisa; Yamanaka, Iori; Harvey, Robert J.; Kuwada, John Y.

    2009-01-01

    Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Recently, in vivo analysis of glycinergic synaptic transmission has been pursued in zebrafish using molecular genetics. An ENU mutagenesis screen identified two behavioral mutants that are defective in glycinergic synaptic transmission. Zebrafish bandoneon (beo) mutants have a defect in glrbb, one of the duplicated glycine receptor (GlyR) β subunit genes. These mutants exhibit a loss of glycinergic synaptic transmission due to a lack of synaptic aggregation of GlyRs. Due to the consequent loss of reciprocal inhibition of motor circuits between the two sides of the spinal cord, motor neurons activate simultaneously on both sides resulting in bilateral contraction of axial muscles of beo mutants, eliciting the so-called ‘accordion’ phenotype. Similar defects in GlyR subunit genes have been observed in several mammals and are the basis for human hyperekplexia/startle disease. By contrast, zebrafish shocked (sho) mutants have a defect in slc6a9, encoding GlyT1, a glycine transporter that is expressed by astroglial cells surrounding the glycinergic synapse in the hindbrain and spinal cord. GlyT1 mediates rapid uptake of glycine from the synaptic cleft, terminating synaptic transmission. In zebrafish sho mutants, there appears to be elevated extracellular glycine resulting in persistent inhibition of postsynaptic neurons and subsequent reduced motility, causing the ‘twitch-once’ phenotype. We review current knowledge regarding zebrafish ‘accordion’ and ‘twitch-once’ mutants, including beo and sho, and report the identification of a new α2 subunit that revises the phylogeny of zebrafish GlyRs. PMID:20161699

  20. 31 CFR 561.328 - Reduce significantly, significantly reduced, and significant reduction.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false Reduce significantly, significantly reduced, and significant reduction. 561.328 Section 561.328 Money and Finance: Treasury Regulations... IRANIAN FINANCIAL SANCTIONS REGULATIONS General Definitions § 561.328 Reduce significantly,...

  1. 31 CFR 561.328 - Reduce significantly, significantly reduced, and significant reduction.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false Reduce significantly, significantly reduced, and significant reduction. 561.328 Section 561.328 Money and Finance: Treasury Regulations... IRANIAN FINANCIAL SANCTIONS REGULATIONS General Definitions § 561.328 Reduce significantly,...

  2. "Clinical" Significance: "Clinical" Significance and "Practical" Significance are NOT the Same Things

    ERIC Educational Resources Information Center

    Peterson, Lisa S.

    2008-01-01

    Clinical significance is an important concept in research, particularly in education and the social sciences. The present article first compares clinical significance to other measures of "significance" in statistics. The major methods used to determine clinical significance are explained and the strengths and weaknesses of clinical significance…

  3. Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase[W

    PubMed Central

    Catalanotti, Claudia; Dubini, Alexandra; Subramanian, Venkataramanan; Yang, Wenqiang; Magneschi, Leonardo; Mus, Florence; Seibert, Michael; Posewitz, Matthew C.; Grossman, Arthur R.

    2012-01-01

    Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism. PMID:22353371

  4. mTORC1 upregulation via ERK-dependent gene expression change confers intrinsic resistance to MEK inhibitors in oncogenic KRas-mutant cancer cells.

    PubMed

    Komatsu, N; Fujita, Y; Matsuda, M; Aoki, K

    2015-11-01

    Cancer cells harboring oncogenic BRaf mutants, but not oncogenic KRas mutants, are sensitive to MEK inhibitors (MEKi). The mechanism underlying the intrinsic resistance to MEKi in KRas-mutant cells is under intensive investigation. Here, we pursued this mechanism by live imaging of extracellular signal-regulated kinases (ERK) and mammalian target of rapamycin complex 1 (mTORC1) activities in oncogenic KRas or BRaf-mutant cancer cells. We established eight cancer cell lines expressing Förster resonance energy transfer (FRET) biosensors for ERK activity and S6K activity, which was used as a surrogate marker for mTORC1 activity. Under increasing concentrations of MEKi, ERK activity correlated linearly with the cell growth rate in BRaf-mutant cancer cells, but not KRas-mutant cancer cells. The administration of PI3K inhibitors resulted in a linear correlation between ERK activity and cell growth rate in KRas-mutant cancer cells. Intriguingly, mTORC1 activity was correlated linearly with the cell growth rate in both BRaf-mutant cancer cells and KRas-mutant cancer cells. These observations suggested that mTORC1 activity had a pivotal role in cell growth and that the mTORC1 activity was maintained primarily by the ERK pathway in BRaf-mutant cancer cells and by both the ERK and PI3K pathways in KRas-mutant cancer cells. FRET imaging revealed that MEKi inhibited mTORC1 activity with slow kinetics, implying transcriptional control of mTORC1 activity by ERK. In agreement with this observation, MEKi induced the expression of negative regulators of mTORC1, including TSC1, TSC2 and Deptor, which occurred more significantly in BRaf-mutant cells than in KRas-mutant cells. These findings suggested that the suppression of mTORC1 activity and induction of negative regulators of mTORC1 in cancer cells treated for at least 1 day could be used as surrogate markers for the MEKi sensitivity of cancer cells. PMID:25703330

  5. Differential mutant quantitation at the mouse lymphoma tk and CHO hgprt loci.

    PubMed

    Moore, M M; Harrington-Brock, K; Doerr, C L; Dearfield, K L

    1989-09-01

    Recent reports by several laboratories indicate that not all non-essential target loci are equally capable of detecting chromosomal mutations. The present study was undertaken to determine if both the tk locus in mouse lymphoma cells and the hgprt locus in Chinese hamster ovary (CHO) cells can be used to quantitate chromosomal mutations. Seven known mutagens for the tk locus were selected. These compounds were evaluated in the mouse lymphoma assay and in a suspension adapted CHO assay for their mutagenicity. In addition to the specific locus mutagenesis analysis, mouse lymphoma and CHO cells were evaluated for the frequency of gross chromosome aberrations. From these investigations, it appears that only those compounds [2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl] aminopropylamino)-acridine-dihydrochloride (ICR 170), ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS)] that induce significant numbers of large-colony thymidine kinase (TK) mutants also induce significant numbers of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) mutants. The four acrylates evaluated (methyl acrylate, ethyl acrylate, trimethylolpropane triacrylate and tetraethyleneglycol diacrylate) induced almost exclusively small-colony TK mutants and very few if any HGPRT mutants. Aberration analysis revealed that both the mouse lymphoma and CHO cells responded to the clastogenicity of the compounds (except for ICR 170 which was not positive in CHO cells) and that neither cell line was clearly more sensitive than the other to the clastogens tested. It is significant that the four acrylates give little or no evidence of genotoxicity when evaluated using selection for HGPRT-deficient mutants, yet are clearly clastogenic to the same cells in the same experiment. These results are consistent with the hypothesis that the hgprt locus may not be useful as a marker to evaluate the clastogenic component of a genotoxic compound. The present study adds to the increasing number of studies

  6. Cytokinin production by plant growth promoting rhizobacteria and selected mutants.

    PubMed

    García de Salamone, I E; Hynes, R K; Nelson, L M

    2001-05-01

    One of the proposed mechanisms by which rhizobacteria enhance plant growth is through the production of plant growth regulators. Five plant growth promoting rhizobacterial (PGPR) strains produced the cytokinin dihydrozeatin riboside (DHZR) in pure culture. Cytokinin production by Pseudomonas fluorescens G20-18, a rifampicin-resistant mutant (RIF), and two TnphoA-derived mutants (CNT1, CNT2), with reduced capacity to synthesize cytokinins, was further characterized in pure culture using immunoassay and thin layer chromatography. G20-18 produced higher amounts of three cytokinins, isopentenyl adenosine (IPA), trans-zeatin ribose (ZR), and DHZR than the three mutants during stationary phase. IPA was the major metabolite produced, but the proportion of ZR and DHZR accumulated by CNT1 and CNT2 increased with time. No differences were observed between strain G20-18 and the mutants in the amounts of indole acetic acid synthesized, nor were gibberellins detected in supernatants of any of the strains. Addition of 10(-5) M adenine increased cytokinin production in 96- and 168-h cultures of strain G20-18 by approximately 67%. G20-18 and the mutants CNT1 and CNT2 may be useful for determination of the role of cytokinin production in plant growth promotion by PGPR. PMID:11400730

  7. Biochemical and biological analysis of Mek1 phosphorylation site mutants.

    PubMed Central

    Huang, W; Kessler, D S; Erikson, R L

    1995-01-01

    Recently, we described the constitutive activation of Mek1 by mutation of its two serine phosphorylation sites. We have now characterized the biochemical properties of these Mek1 mutants and performed microinjection experiments to investigate the effect of an activated Mek on oocyte maturation. Single acidic substitution of either serine 218 or 222 activated Mek1 by 10-50 fold. The double acidic substitutions, [Asp218, Asp222] and [Asp218, Glu222], activated Mek1 over 6000-fold. The specific activity of the [Asp218, Asp222] and [Asp218, Glu222] Mek1 mutants, 29 nanomole phosphate per minute per milligram, is similar to that of wild-type Mek1 activated by Raf-1 in vitro. Although the mutants with double acidic substitutions could not be further activated by Raf-1, three of those with single acidic substitution were activated by Raf-1 to the specific activity of activated wild-type Mek1. Injection of the [Asp218, Asp222] Mek1 mutant into Xenopus oocytes activated both MAP kinase and histone H1 kinase and induced germinal vesicle breakdown, an effect that was only partially blocked by inhibition of protein synthesis. These data provide a measure of Mek's potential to influence cell functions and a quantitative basis to assess the biological effects of Mek1 mutants in a variety of circumstances. Images PMID:7612960

  8. Hepatitis B escape mutants in Scottish blood donors.

    PubMed

    Larralde, Osmany; Dow, Brian; Jarvis, Lisa; Davidson, Fiona; Petrik, Juraj

    2013-06-01

    Hepatitis B virus (HBV) remains as the viral infection with the highest risk of transmission by transfusion. This risk is associated with window period donations, occult HBV infection (OBI) and the emergence of escape mutants, which render blood donations false negative for hepatitis B surface antigen (HBsAg) serological testing. A retrospective study was conducted to gain insights into the molecular epidemiology of HBV escape mutants in Scottish blood donors. The criterion for selection was HBV positivity either by serology or nucleic acid testing (NAT). HBsAg detection was compared across several commercial immunoassays. The full length S gene from plasma samples was PCR amplified, cloned and expressed in HepG2 cells. Eight samples showed HBsAg discordant results, while 5 OBI samples were found. Four escape mutants, containing missense mutations in the S gene, are described here. These mutations impaired HBsAg detection both from HBV infected plasma samples and from recombinant proteins derived from its infected donors. Phylogenetic analysis showed that most of the mutants were clustered in the genotype D and were closely related to strains from Asia and the Middle East. We report here a proline substitution, outside the major hydrophilic region, that impaired HBsAg detection in vivo and in vitro, warning about the risk for the emergence of vaccine escape mutants with mutations outside the major neutralisation site. PMID:23274404

  9. Nitrate reduction mutants of Fusarium moniliforme (Gibberella fujikuroi)

    SciTech Connect

    Klittich, C.J.R.; Leslie, J.F.

    1988-03-01

    Twelve strains of Fusarium moniliforme were examined for their ability to sector spontaneously on toxic chlorate medium. All strains sectored frequently; 91% of over 1200 colonies examined formed chlorate-resistant, mutant sectors. Most of these mutants had lesions in the nitrate reduction pathway and were unable to utilize nitrate (nit mutants). nit mutations occurred in seven loci: a structural gene for nitrate reductase (nit1), a regulatory gene specific for the nitrate reduction pathway (nit3), and five genes controlling the production of a molybdenum-containing cofactors that is necessary for nitrate reductase activity (nit2, nit4, nit5, nit6, nit7). No mutations affecting nitrite reductase or a major nitrogen regulatory locus were found among over 1000 nit mutants. Mutations of nit1 were recovered most frequently (39-66%, depending on the strain) followed by nit3 mutations (23-42%). The frequency of isolation of each mutant type could be altered, however, by changing the source of nitrogen in the chlorate medium. The authors concluded that genetic control of nitrate reduction in F. moniliforme is similar to that in Aspergillus and Neurospora, but that the overall regulation of nitrogen metabolism may be different.

  10. Arabidopsis thaliana nucleosidase mutants provide new insights into nucleoside degradation

    PubMed Central

    Riegler, Heike; Geserick, Claudia; Zrenner, Rita

    2011-01-01

    A central step in nucleoside and nucleobase salvage pathways is the hydrolysis of nucleosides to their respective nucleobases. In plants this is solely accomplished by nucleosidases (EC 3.2.2.x). To elucidate the importance of nucleosidases for nucleoside degradation, general metabolism, and plant growth, thorough phenotypic and biochemical analyses were performed using Arabidopsis thaliana T-DNA insertion mutants lacking expression of the previously identified genes annotated as uridine ribohydrolases (URH1 and URH2). Comprehensive functional analyses of single and double mutants demonstrated that both isoforms are unimportant for seedling establishment and plant growth, while one participates in uridine degradation. Rather unexpectedly, nucleoside and nucleotide profiling and nucleosidase activity screening of soluble crude extracts revealed a deficiency of xanthosine and inosine hydrolysis in the single mutants, with substantial accumulation of xanthosine in one of them. Mixing of the two mutant extracts, and by in vitro activity reconstitution using a mixture of recombinant URH1 and URH2 proteins, both restored activity, thus providing biochemical evidence that at least these two isoforms are needed for inosine and xanthosine hydrolysis. This mutant study demonstrates the utility of in vivo systems for the examination of metabolic activities, with the discovery of the new substrate xanthosine and elucidation of a mechanism for expanding the nucleosidase substrate spectrum. PMID:21599668

  11. The Tennessee Mouse Genome Consortium: Identification of ocular mutants

    SciTech Connect

    Jablonski, Monica M.; Wang, Xiaofei; Lu, Lu; Miller, Darla R; Rinchik, Eugene M; Williams, Robert; Goldowitz, Daniel

    2005-06-01

    The Tennessee Mouse Genome Consortium (TMGC) is in its fifth year of a ethylnitrosourea (ENU)-based mutagenesis screen to detect recessive mutations that affect the eye and brain. Each pedigree is tested by various phenotyping domains including the eye, neurohistology, behavior, aging, ethanol, drug, social behavior, auditory, and epilepsy domains. The utilization of a highly efficient breeding protocol and coordination of various universities across Tennessee makes it possible for mice with ENU-induced mutations to be evaluated by nine distinct phenotyping domains within this large-scale project known as the TMGC. Our goal is to create mutant lines that model human diseases and disease syndromes and to make the mutant mice available to the scientific research community. Within the eye domain, mice are screened for anterior and posterior segment abnormalities using slit-lamp biomicroscopy, indirect ophthalmoscopy, fundus photography, eye weight, histology, and immunohistochemistry. As of January 2005, we have screened 958 pedigrees and 4800 mice, excluding those used in mapping studies. We have thus far identified seven pedigrees with primary ocular abnormalities. Six of the mutant pedigrees have retinal or subretinal aberrations, while the remaining pedigree presents with an abnormal eye size. Continued characterization of these mutant mice should in most cases lead to the identification of the mutated gene, as well as provide insight into the function of each gene. Mice from each of these pedigrees of mutant mice are available for distribution to researchers for independent study.

  12. Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase.

    PubMed Central

    Mat-Jan, F; Alam, K Y; Clark, D P

    1989-01-01

    Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion. PMID:2644194

  13. Regulatory Mutants at the his1 Locus of Yeast

    PubMed Central

    Lax, Carol; Fogel, Seymour; Cramer, Carole

    1979-01-01

    The his1 gene in Saccharomyces cerevisiae codes for phosphoribosyl transferase, an allosteric enzyme that catalyzes the initial step in histidine biosynthesis. Mutants that specifically alter the feedback regulatory function were isolated by selecting his1 prototrophic revertants that overproduce and excrete histidine. The prototrophs were obtained from diploids homoallelic for his1–7 and heterozygous for the flanking markers thr3 and arg6. Among six independently derived mutant isolates, three distinct levels of histidine excretion were detected. The mutants were shown to be second-site alterations mapping at the his1 locus by recovery of the original auoxtrophic parental alleles. The double mutants, HIS1–7e, are dominant with respect to catalytic function but recessive in regulatory function. When removed from this his1–7 background, the mutant regulatory site (HIS1–e) still confers prototrophy but not histidine excretion. To yield the excretion phenotype, the primary and altered secondary sites are required in cis array. Differences in histidine excretion levels correlate with resistance to the histidine analogue, triazoalanine. PMID:385447

  14. Mutant number distribution in an exponentially growing population

    NASA Astrophysics Data System (ADS)

    Keller, Peter; Antal, Tibor

    2015-01-01

    We present an explicit solution to a classic model of cell-population growth introduced by Luria and Delbrück (1943 Genetics 28 491-511) 70 years ago to study the emergence of mutations in bacterial populations. In this model a wild-type population is assumed to grow exponentially in a deterministic fashion. Proportional to the wild-type population size, mutants arrive randomly and initiate new sub-populations of mutants that grow stochastically according to a supercritical birth and death process. We give an exact expression for the generating function of the total number of mutants at a given wild-type population size. We present a simple expression for the probability of finding no mutants, and a recursion formula for the probability of finding a given number of mutants. In the ‘large population-small mutation’ limit we recover recent results of Kessler and Levine (2014 J. Stat. Phys. doi:10.1007/s10955-014-1143-3) for a fully stochastic version of the process.

  15. Analysis of a Bacillus subtilis Proteinase Mutant1

    PubMed Central

    Shoer, Ruth; Rappaport, Harry P.

    1972-01-01

    A Bacillus subtilis mutant having a phenotype manifesting reduced extracellular proteolytic activity was investigated. An extracellular protein was isolated and shown by fingerprint analysis to be a fragment of the wild-type enzyme. By using previously established molecular weights for the wild-type enzyme (2.9 × 104) and the two polypeptide chains derived from it (1.4 × 104 each), with the amino acid analysis and fingerprints of both wild-type and mutant proteins, a molecular weight of 1.57 × 104 was assigned to the mutant protein. 32P-diisopropylphosphate labeling of the mutant protein showed only 1 in 53 molecules to be functional. Thin-layer chromatography on Sephadex G-75 demonstrated that the active molecules were separable from the bulk of the isolated protein and had the same mobility as the wild-type enzyme. Fingerprints of tryptic digests of 32P-diisopropylphosphate-labeled wild-type and mutant proteins showed that the labeled peptides had identical characteristics. Images PMID:4621679

  16. Characterization of a mutant glucose isomerase from Thermoanaerobacterium saccharolyticum.

    PubMed

    Xu, Heng; Shen, Dong; Wu, Xue-Qiang; Liu, Zhi-Wei; Yang, Qi-He

    2014-10-01

    A series of site-directed mutant glucose isomerase at tryptophan 139 from Thermoanaerobacterium saccharolyticum strain B6A were purified to gel electrophoretic homogeneity, and the biochemical properties were determined. W139F mutation is the most efficient mutant derivative with a tenfold increase in its catalytic efficiency toward glucose compared with the native GI. With a maximal activity at 80 °C of 59.58 U/mg on glucose, this mutant derivative is the most active type ever reported. The enzyme activity was maximal at 90 °C and like other glucose isomerase, this mutant enzyme required Co(2+) or Mg(2+) for enzyme activity and thermal stability (stable for 20 h at 80 °C in the absence of substrate). Its optimum pH was around 7.0, and it had 86 % of its maximum activity at pH 6.0 incubated for 12 h at 60 °C. This enzyme was determined as thermostable and weak-acid stable. These findings indicated that the mutant GI W139F from T. saccharolyticum strain B6A is appropriate for use as a potential candidate for high-fructose corn syrup producing enzyme. PMID:25139657

  17. Photosensitivities of rhodopsin mutants with a displaced counterion.

    PubMed

    Tsutsui, Kei; Shichida, Yoshinori

    2010-11-30

    Visual pigments consist of a protein moiety opsin and an 11-cis-retinal chromophore that is covalently bound to the opsin via a Schiff base linkage. They have a high photosensitivity, which can be attributed to the high probability of photon absorption and the high photoisomerization quantum yield of the retinal chromophore. Both of these parameters are regulated by the opsin, though the precise mechanism is unknown. We previously found that counterion residue E113, which stabilizes the proton on the Schiff base, is involved in the efficient photoisomerization in vertebrate visual pigments. To test the positional effect of the counterion on the photon absorption and the photoisomerization, we measured the photosensitivities of a set of mutants of bovine rhodopsin in which the counterion was displaced to position 90, 94, 117, or 292. The molar extinction coefficient was reduced in many of the mutants, leading to reductions in the photosensitivity for monochromatic lights. However, the oscillator strength, the probability of photon absorption integrated over the entire wavenumber range of the absorption band, was relatively similar among the mutants and the wild type. In addition, the quantum yields of the mutants were not markedly different from that of the wild type. These results indicate that the counterion does not need to be located at position 113 for a high photosensitivity for natural light. Interestingly, all of the mutants exhibited greatly increased hydroxylamine sensitivity. This result suggests that the counterion in vertebrate visual pigments is optimally located for the stability of the Schiff base linkage. PMID:21038858

  18. Inhibition of DNA Methyltransferases Blocks Mutant Huntingtin-Induced Neurotoxicity

    PubMed Central

    Pan, Yanchun; Daito, Takuji; Sasaki, Yo; Chung, Yong Hee; Xing, Xiaoyun; Pondugula, Santhi; Swamidass, S. Joshua; Wang, Ting; Kim, Albert H.; Yano, Hiroko

    2016-01-01

    Although epigenetic abnormalities have been described in Huntington’s disease (HD), the causal epigenetic mechanisms driving neurodegeneration in HD cortex and striatum remain undefined. Using an epigenetic pathway-targeted drug screen, we report that inhibitors of DNA methyltransferases (DNMTs), decitabine and FdCyd, block mutant huntingtin (Htt)-induced toxicity in primary cortical and striatal neurons. In addition, knockdown of DNMT3A or DNMT1 protected neurons against mutant Htt-induced toxicity, together demonstrating a requirement for DNMTs in mutant Htt-triggered neuronal death and suggesting a neurodegenerative mechanism based on DNA methylation-mediated transcriptional repression. Inhibition of DNMTs in HD model primary cortical or striatal neurons restored the expression of several key genes, including Bdnf, an important neurotrophic factor implicated in HD. Accordingly, the Bdnf promoter exhibited aberrant cytosine methylation in mutant Htt-expressing cortical neurons. In vivo, pharmacological inhibition of DNMTs in HD mouse brains restored the mRNA levels of key striatal genes known to be downregulated in HD. Thus, disturbances in DNA methylation play a critical role in mutant Htt-induced neuronal dysfunction and death, raising the possibility that epigenetic strategies targeting abnormal DNA methylation may have therapeutic utility in HD. PMID:27516062

  19. Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse)

    PubMed Central

    Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R.; Marzban, Hassan

    2014-01-01

    The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18–19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22–23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation. PMID:24722417

  20. Purkinje cell compartmentation in the cerebellum of the lysosomal Acid phosphatase 2 mutant mouse (nax - naked-ataxia mutant mouse).

    PubMed

    Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R; Marzban, Hassan

    2014-01-01

    The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18-19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22-23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation. PMID:24722417

  1. α-Mangostin, a Natural Agent, Enhances the Response of NRAS Mutant Melanoma to Retinoic Acid.

    PubMed

    Xia, Yun; Chen, Jing; Gong, Chongwen; Chen, Hongxiang; Sun, Jiaming

    2016-01-01

    BACKGROUND The identification and use of novel compounds alone or in combination hold promise for the fight against NRAS mutant melanoma. MATERIAL AND METHODS We screened a kinase-specific inhibitor library through combining it with α-Mangostin in NRAS mutant melanoma cell line, and verified the enhancing effect of α-Mangostin through inhibition of the tumorigenesis pathway. RESULTS Within the kinase inhibitors, retinoic acid showed a significant synergistic effect with α-Mangostin. α-Mangostin also can reverse the drug resistance of retinoic acid in RARa siRNA-transduced sk-mel-2 cells. Colony assay, TUNEL staining, and the expressions of several apoptosis-related genes revealed that a-Mangostin enhanced the effect of retinoic acid-induced apoptosis. The combination treatment resulted in marked induction of ROS generation and inhibition of the AKT/S6 pathway. CONCLUSIONS These results indicate that the combination of these novel natural agents with retinoid acid may be clinically effective in NRAS mutant melanoma. PMID:27104669

  2. Detection of Cell Wall Chemical Variation in Zea Mays Mutants Using Near-Infrared Spectroscopy

    SciTech Connect

    Buyck, N.; Thomas, S.

    2001-01-01

    Corn stover is regarded as the prime candidate feedstock material for commercial biomass conversion in the United States. Variations in chemical composition of Zea mays cell walls can affect biomass conversion process yields and economics. Mutant lines were constructed by activating a Mu transposon system. The cell wall chemical composition of 48 mutant families was characterized using near-infrared (NIR) spectroscopy. NIR data were analyzed using a multivariate statistical analysis technique called Principal Component Analysis (PCA). PCA of the NIR data from 349 maize leaf samples reveals 57 individuals as outliers on one or more of six Principal Components (PCs) at the 95% confidence interval. Of these, 19 individuals from 16 families are outliers on either PC3 (9% of the variation) or PC6 (1% of the variation), the two PCs that contain information about cell wall polymers. Those individuals for which altered cell wall chemistry is confirmed with wet chemical analysis will then be subjected to fermentation analysis to determine whether or not biomass conversion process kinetics, yields and/or economics are significantly affected. Those mutants that provide indications for a decrease in process cost will be pursued further to identify the gene(s) responsible for the observed changes in cell wall composition and associated changes in process economics. These genes will eventually be incorporated into maize breeding programs directed at the development of a truly dual use crop.

  3. Blastocyst genotyping for quality control of mouse mutant archives: an ethical and economical approach.

    PubMed

    Scavizzi, Ferdinando; Ryder, Edward; Newman, Stuart; Raspa, Marcello; Gleeson, Diane; Wardle-Jones, Hannah; Montoliu, Lluis; Fernandez, Almudena; Dessain, Marie-Laure; Larrigaldie, Vanessa; Khorshidi, Zuzana; Vuolteenaho, Reetta; Soininen, Raija; André, Philippe; Jacquot, Sylvie; Hong, Yi; de Angelis, Martin Hrabe; Ramirez-Solis, Ramiro; Doe, Brendan

    2015-10-01

    With the advent of modern developmental biology and molecular genetics, the scientific community has generated thousands of newly genetically altered strains of laboratory mice with the aim of elucidating gene function. To this end, a large group of Institutions which form the International Mouse Phenotyping Consortium is generating and phenotyping a knockout mouse strain for each of the ~20,000 protein-coding genes using the mutant ES cell resource produced by the International Knockout Mouse Consortium. These strains are made available to the research community via public repositories, mostly as cryopreserved sperm or embryos. To ensure the quality of this frozen resource there is a requirement that for each strain the frozen sperm/embryos are proven able to produce viable mutant progeny, before the live animal resource is removed from cages. Given the current requirement to generate live pups to demonstrate their mutant genotype, this quality control check necessitates the use and generation of many animals and requires considerable time, cage space, technical and economic resources. Here, we describe a simple and efficient method of genotyping pre-implantation stage blastocysts with significant ethical and economic advantages especially beneficial for current and future large-scale mouse mutagenesis projects. PMID:26178246

  4. Generation and Standardized, Systemic Phenotypic Analysis of Pou3f3L423P Mutant Mice

    PubMed Central

    Kumar, Sudhir; Rathkolb, Birgit; Kemter, Elisabeth; Sabrautzki, Sibylle; Michel, Dian; Adler, Thure; Becker, Lore; Beckers, Johannes; Busch, Dirk H.; Garrett, Lillian; Hans, Wolfgang; Hölter, Sabine M.; Horsch, Marion; Klingenspor, Martin; Klopstock, Thomas; Rácz, Ildikó; Rozman, Jan; Vargas Panesso, Ingrid Liliana; Vernaleken, Alexandra; Zimmer, Andreas; Fuchs, Helmut; Gailus-Durner, Valérie; Hrabě de Angelis, Martin; Wolf, Eckhard; Aigner, Bernhard

    2016-01-01

    Increased levels of blood plasma urea were used as phenotypic parameter for establishing novel mouse models for kidney diseases on the genetic background of C3H inbred mice in the phenotype-driven Munich ENU mouse mutagenesis project. The phenotypically recessive mutant line HST011 was established and further analyzed. The causative mutation was detected in the POU domain, class 3 transcription factor 3 (Pou3f3) gene, which leads to the amino acid exchange Pou3f3L423P thereby affecting the conserved homeobox domain of the protein. Pou3f3 homozygous knockout mice are published and show perinatal death. Line Pou3f3L423P is a viable mouse model harboring a homozygous Pou3f3 mutation. Standardized, systemic phenotypic analysis of homozygous mutants was carried out in the German Mouse Clinic. Main phenotypic changes were low body weight and a state of low energy stores, kidney dysfunction and secondary effects thereof including low bone mineralization, multiple behavioral and neurological defects including locomotor, vestibular, auditory and nociceptive impairments, as well as multiple subtle changes in immunological parameters. Genome-wide transcriptome profiling analysis of kidney and brain of Pou3f3L423P homozygous mutants identified significantly regulated genes as compared to wild-type controls. PMID:27003440

  5. Deafness and permanently reduced potassium channel gene expression and function in hypothyroid Pit1dw mutants

    PubMed Central

    Mustapha, Mirna; Fang, Qing; Gong, Tzy-Wen; Dolan, David F.; Raphael, Yehoash; Camper, Sally A.; Duncan, R. Keith

    2012-01-01

    The absence of thyroid hormone (TH) during late gestation and early infancy can cause irreparable deafness in both humans and rodents. A variety of rodent models have been utilized in an effort to identify the underlying molecular mechanism. Here, we characterize a mouse model of secondary hypothyroidism, pituitary transcription factor 1 (Pit1dw), which has profound, congenital deafness that is rescued by oral TH replacement. These mutants have tectorial membrane abnormalities, including a prominent Hensen's stripe, elevated β-tectorin composition, and disrupted striated-sheet matrix. They lack distortion product otoacoustic emissions and cochlear microphonic responses, and exhibit reduced endocochlear potentials, suggesting defects in outer hair cell function and potassium recycling. Auditory system and hair cell physiology, histology and anatomy studies reveal novel defects of hormone deficiency related to deafness: (1) permanently impaired expression of KCNJ10 in the stria vascularis of Pit1dw mice, which likely contributes to the reduced endocochlear potential, (2) significant outer hair cell loss in the mutants, which may result from cellular stress induced by the lower KCNQ4 expression and current levels in Pit1dw mutant outer hair cells and (3) sensory and strial cell deterioration, which may have implications for thyroid hormone dysregulation in age related hearing impairment. In summary, we suggest that these defects in outer hair cell and strial cell function are important contributors to the hearing impairment in Pit1dw mice. PMID:19176829

  6. Mutant lamin A links prophase to a p53 independent senescence program

    PubMed Central

    Moiseeva, Olga; Lessard, Frédéric; Acevedo-Aquino, Mariana; Vernier, Mathieu; Tsantrizos, Youla S; Ferbeyre, Gerardo

    2015-01-01

    Expression of oncogenes or short telomeres can trigger an anticancer response known as cellular senescence activating the p53 and RB tumor suppressor pathways. This mechanism is switched off in most tumor cells by mutations in p53 and RB signaling pathways. Surprisingly, p53 disabled tumor cells could be forced into senescence by expression of a mutant allele of the nuclear envelope protein lamin A. The pro-senescence lamin A mutant contains a deletion in the sequence required for processing by the protease ZMPSTE24 leading to accumulation of farnesylated lamin A in the nuclear envelope. In addition, the serine at position 22, a target for CDK1-dependent phosphorylation, was mutated to alanine, preventing CDK1-catalyzed nuclear envelope disassembly. The accumulation of this mutant lamin A compromised prophase to prometaphase transition leading to invaginations of the nuclear lamina, nuclear fragmentation and impaired chromosome condensation. Cells exited this impaired mitosis without cytokinesis and re-replicated their DNA ultimately arresting in interphase as polyploid cells with features of cellular senescence including increased expression of inflammatory gene products and a significant reduction of tumorigenicity in vivo. PMID:26029982

  7. Production and Secretion of the Polysaccharide Biodispersan of Acinetobacter calcoaceticus A2 in Protein Secretion Mutants.

    PubMed

    Elkeles, A; Rosenberg, E; Ron, E Z

    1994-12-01

    Biodispersan is an extracellular anionic polysaccharide produced by Acinetobacter calcoaceticus A2 that changes the surface properties of limestone and acts both as a dispersant and as a grinding aid (E. Rosenberg, C. Rubinovitz, A. Gottlieb, S. Rosenhak, and E. Z. Ron, Appl. Environ. Microbiol. 54:317-322, 1988; E. Rosenberg, C. Rubinovitz, R. Legmann, and E. Z. Ron, Appl. Environ. Microbiol. 54:323-326, 1988; E. Rosenberg, Z. Schwartz, A. Tenenbaum, C. Rubinovitz, R. Legmann, and E. Z. Ron, J. Dispersion Sci. Technol. 10:241-250, 1989). Extracellular fluid also contains a high concentration of secreted proteins that create problems in the purification and application of biodispersan. In order to obtain preparations of biodispersan that contained smaller amounts of protein, we selected mutants of strain A2 that were defective in protein secretion. These mutants produced equal, or even higher, levels of total biodispersan compared with those of the parental strain. Moreover, although there was a significant drop in the concentration of extracellular proteins in the medium, the secretion of biodispersan was unaffected. These results suggest that secretion mutants are potentially useful for the production of extracellular polysaccharides. PMID:16349473

  8. Transcriptome analysis of all two-component regulatory system mutants of Escherichia coli K-12.

    PubMed

    Oshima, Taku; Aiba, Hirofumi; Masuda, Yasushi; Kanaya, Shigehiko; Sugiura, Masahito; Wanner, Barry L; Mori, Hirotada; Mizuno, Takeshi

    2002-10-01

    We have systematically examined the mRNA profiles of 36 two-component deletion mutants, which include all two-component regulatory systems of Escherichia coli, under a single growth condition. DNA microarray results revealed that the mutants belong to one of three groups based on their gene expression profiles in Luria-Bertani broth under aerobic conditions: (i) those with no or little change; (ii) those with significant changes; and (iii) those with drastic changes. Under these conditions, the anaeroresponsive ArcB/ArcA system, the osmoresponsive EnvZ/OmpR system and the response regulator UvrY showed the most drastic changes. Cellular functions such as flagellar synthesis and expression of the RpoS regulon were affected by multiple two-component systems. A high correlation coefficient of expression profile was found between several two-component mutants. Together, these results support the view that a network of functional interactions, such as cross-regulation, exists between different two-component systems. The compiled data are avail-able at our website (http://ecoli.aist-nara.ac.jp/xp_analysis/ 2_components). PMID:12366850

  9. α-Mangostin, a Natural Agent, Enhances the Response of NRAS Mutant Melanoma to Retinoic Acid

    PubMed Central

    Xia, Yun; Chen, Jing; Gong, Chongwen; Chen, Hongxiang; Sun, Jiaming

    2016-01-01

    Background The identification and use of novel compounds alone or in combination hold promise for the fight against NRAS mutant melanoma. Material/Methods We screened a kinase-specific inhibitor library through combining it with α-Mangostin in NRAS mutant melanoma cell line, and verified the enhancing effect of α-Mangostin through inhibition of the tumorigenesis pathway. Results Within the kinase inhibitors, retinoic acid showed a significant synergistic effect with α-Mangostin. α-Mangostin also can reverse the drug resistance of retinoic acid in RARa siRNA-transduced sk-mel-2 cells. Colony assay, TUNEL staining, and the expressions of several apoptosis-related genes revealed that α-Mangostin enhanced the effect of retinoic acid-induced apoptosis. The combination treatment resulted in marked induction of ROS generation and inhibition of the AKT/S6 pathway. Conclusions These results indicate that the combination of these novel natural agents with retinoid acid may be clinically effective in NRAS mutant melanoma. PMID:27104669

  10. Defective calmodulin-dependent rapid apical endocytosis in zebrafish sensory hair cell mutants.

    PubMed

    Seiler, C; Nicolson, T

    1999-11-15

    Vertebrate mechanosensory hair cells contain a narrow "pericuticular" zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1-43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function. PMID:10526320

  11. First TILLING Platform in Cucurbita pepo: A New Mutant Resource for Gene Function and Crop Improvement

    PubMed Central

    Vicente-Dólera, Nelly; Troadec, Christelle; Moya, Manuel; del Río-Celestino, Mercedes; Pomares-Viciana, Teresa; Bendahmane, Abdelhafid; Picó, Belén; Román, Belén; Gómez, Pedro

    2014-01-01

    Although the availability of genetic and genomic resources for Cucurbita pepo has increased significantly, functional genomic resources are still limited for this crop. In this direction, we have developed a high throughput reverse genetic tool: the first TILLING (Targeting Induced Local Lesions IN Genomes) resource for this species. Additionally, we have used this resource to demonstrate that the previous EMS mutant population we developed has the highest mutation density compared with other cucurbits mutant populations. The overall mutation density in this first C. pepo TILLING platform was estimated to be 1/133 Kb by screening five additional genes. In total, 58 mutations confirmed by sequencing were identified in the five targeted genes, thirteen of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was studied in a peroxidase gene, revealing that the phenotype of seedling homozygous for one of the isolated mutant alleles was albino. These results indicate that the TILLING approach in this species was successful at providing new mutations and can address the major challenge of linking sequence information to biological function and also the identification of novel variation for crop breeding. PMID:25386735

  12. Biofilm formation ability of Salmonella enterica serovar Typhimurium acrAB mutants.

    PubMed

    Schlisselberg, Dov B; Kler, Edna; Kisluk, Guy; Shachar, Dina; Yaron, Sima

    2015-10-01

    Recent studies offer contradictory findings about the role of multidrug efflux pumps in bacterial biofilm development. Thus, the aim of this study was to investigate the involvement of the AcrAB efflux pump in biofilm formation by investigating the ability of AcrB and AcrAB null mutants of Salmonella enterica serovar Typhimurium to produce biofilms. Three models were used to compare the ability of S. Typhimurium wild-type and its mutants to form biofilms: formation of biofilm on polystyrene surfaces; production of biofilm (mat model) on the air/liquid interface; and expression of curli and cellulose on Congo red-supplemented agar plates. All three investigated genotypes formed biofilms with similar characteristics. However, upon exposure to chloramphenicol, formation of biofilms on solid surfaces as well as the production of curli were either reduced or were delayed more significantly in both mutants, whilst there was no visible effect on pellicle formation. It can be concluded that when no selective pressure is applied, S. Typhimurium is able to produce biofilms even when the AcrAB efflux pumps are inactivated, implying that the use of efflux pump inhibitors to prevent biofilm formation is not a general solution and that combined treatments might be more efficient. Other factors that affect the ability to produce biofilms depending on efflux pump activity are yet to be identified. PMID:26260191

  13. Light-induced Enzyme Formation in a Chlorophyll-less Mutant of Euglena gracilis.

    PubMed

    Russell, G K; Draffan, A G

    1978-11-01

    A mutant strain, Y(9), of Euglena gracilis strain Z that is unable to produce protochlorophyll or chlorophyll has been isolated following treatment of wild type cells with nalidixic acid. Dark-grown cells of the mutant contain proplastids that show only limited ultrastructural development when placed in the light. Treatment of Y(9) cells with ultraviolet light brings about permanent cell bleaching with a target number similar to wild type Euglena, and with a slightly greater sensitivity to ultraviolet. Three enzymes of the reductive pentose phosphate cycle, fructose-1,6-diphosphate aldolase (class I), NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and 3-phosphoglycerate kinase, are detectable in dark-grown Y(9) cells at the low concentrations characteristic of dark-grown wild type cells, and increase substantially when these cells are exposed to light. The activity of ribulose-1,5-diphosphate carboxylase increases in the light to a lesser extent. Cytochrome 552, a carrier in the photosynthetic electron transport chain, is not present in light-grown cells of Y(9). The significance of this mutant for an understanding of the role of light in Euglena chloroplast development is discussed. PMID:16660582

  14. Genetics of Peripheral Vestibular Dysfunction: Lessons from Mutant Mouse Strains

    PubMed Central

    Jones, Sherri M.; Jones, Timothy A.

    2015-01-01

    Background A considerable amount of research has been published about genetic hearing impairment. Fifty to sixty percent of hearing loss is thought to have a genetic cause. Genes may also play a significant role in acquired hearing loss due to aging, noise exposure, or ototoxic medications. Between 1995 and 2012, over 100 causative genes have been identified for syndromic and nonsyndromic forms of hereditary hearing loss (see Hereditary Hearing Loss Homepage http://hereditaryhearingloss.org). Mouse models have been extremely valuable in facilitating the discovery of hearing loss genes, and in understanding inner ear pathology due to genetic mutations or elucidating fundamental mechanisms of inner ear development. Purpose Whereas much is being learned about hereditary hearing loss and the genetics of cochlear disorders, relatively little is known about the role genes may play in peripheral vestibular impairment. Here we review the literature with regard to genetics of vestibular dysfunction and discuss what we have learned from studies using mutant mouse models and direct measures of peripheral vestibular neural function. Results Several genes are considered that when mutated lead to varying degrees of inner ear vestibular dysfunction due to deficits in otoconia, stereocilia, hair cells, or neurons. Behavior often does not reveal the inner ear deficit. Many of the examples presented are also known to cause human disorders. Conclusions Knowledge regarding the roles of particular genes in the operation of the vestibular sensory apparatus is growing and it is clear that gene products co-expressed in the cochlea and vestibule may play different roles in the respective end organs. The discovery of new genes mediating critical inner ear vestibular function carries the promise of new strategies in diagnosing, treating and managing patients as well as predicting the course and level of morbidity in human vestibular disease. PMID:25032973

  15. Amdinocillin (Mecillinam) Resistance Mutations in Clinical Isolates and Laboratory-Selected Mutants of Escherichia coli

    PubMed Central

    Thulin, Elisabeth; Sundqvist, Martin

    2015-01-01

    Amdinocillin (mecillinam) is a β-lactam antibiotic that is used mainly for the treatment of uncomplicated urinary tract infections. The objectives of this study were to identify mutations that confer amdinocillin resistance on laboratory-isolated mutants and clinical isolates of Escherichia coli and to determine why amdinocillin resistance remains rare clinically even though resistance is easily selected in the laboratory. Under laboratory selection, frequencies of mutation to amdinocillin resistance varied from 8 × 10−8 to 2 × 10−5 per cell, depending on the concentration of amdinocillin used during selection. Several genes have been demonstrated to give amdinocillin resistance, but here eight novel genes previously unknown to be involved in amdinocillin resistance were identified. These genes encode functions involved in the respiratory chain, the ribosome, cysteine biosynthesis, tRNA synthesis, and pyrophosphate metabolism. The clinical isolates exhibited significantly greater fitness than the laboratory-isolated mutants and a different mutation spectrum. The cysB gene was mutated (inactivated) in all of the clinical isolates, in contrast to the laboratory-isolated mutants, where mainly other types of more costly mutations were found. Our results suggest that the frequency of mutation to amdinocillin resistance is high because of the large mutational target (at least 38 genes). However, the majority of these resistant mutants have a low growth rate, reducing the probability that they are stably maintained in the bladder. Inactivation of the cysB gene and a resulting loss of cysteine biosynthesis are the major mechanism of amdinocillin resistance in clinical isolates of E. coli. PMID:25583718

  16. Altered striatal function in a mutant mouse lacking D1A dopamine receptors.

    PubMed Central

    Drago, J; Gerfen, C R; Lachowicz, J E; Steiner, H; Hollon, T R; Love, P E; Ooi, G T