Cell line with endogenous EGFRvIII expression is a suitable model for research and drug development purposes.

PubMed

Stec, Wojciech J; Rosiak, Kamila; Siejka, Paulina; Peciak, Joanna; Popeda, Marta; Banaszczyk, Mateusz; Pawlowska, Roza; Treda, Cezary; Hulas-Bigoszewska, Krystyna; Piaskowski, Sylwester; Stoczynska-Fidelus, Ewelina; Rieske, Piotr

2016-05-31

Glioblastoma is the most common and malignant brain tumor, characterized by high cellular heterogeneity. About 50% of glioblastomas are positive for EGFR amplification, half of which express accompanying EGFR mutation, encoding truncated and constitutively active receptor termed EGFRvIII. Currently, no cell models suitable for development of EGFRvIII-targeting drugs exist, while the available ones lack the intratumoral heterogeneity or extrachromosomal nature of EGFRvIII.The reports regarding the biology of EGFRvIII expressed in the stable cell lines are often contradictory in observations and conclusions. In the present study, we use DK-MG cell line carrying endogenous non-modified EGFRvIII amplicons and derive a sub-line that is near depleted of amplicons, whilst remaining identical on the chromosomal level. By direct comparison of the two lines, we demonstrate positive effects of EGFRvIII on cell invasiveness and populational growth as a result of elevated cell survival but not proliferation rate. Investigation of the PI3K/Akt indicated no differences between the lines, whilst NFκB pathway was over-active in the line strongly expressing EGFRvIII, finding further supported by the effects of NFκB pathway specific inhibitors. Taken together, these results confirm the important role of EGFRvIII in intrinsic and extrinsic regulation of tumor behavior. Moreover, the proposed models are stable, making them suitable for research purposes as well as drug development process utilizing high throughput approach.

Changes in the EGFR amplification and EGFRvIII expression between paired primary and recurrent glioblastomas

PubMed Central

Gao, Ya; Kerkhof, Melissa; Kros, Johan M.; Gorlia, Thierry; van Zwieten, Kitty; Prince, Jory; van Duinen, Sjoerd; Sillevis Smitt, Peter A.; Taphoorn, Martin; French, Pim J.

2015-01-01

Abstract Background The efficacy of novel targeted therapies is often tested at the time of tumor recurrence. However, for glioblastoma (GBM) patients, surgical resections at recurrence are performed only in a minority of patients; therefore, molecular data are predominantly derived from the initial tumor. Molecular data of the initial tumor for patient selection into personalized medicine trials can therefore be used only when the specific genetic change is retained in the recurrent tumor. Methods In this study we determined whether EGFR amplification and expression of the most common mutation in GBMs (EGFRvIII) is retained at tumor recurrence. Because retention of genetic changes may be dependent on the initial treatment, we only used a cohort of GBM samples that were uniformly treated according to the current standard of care (ie, chemo-irradiation with temozolomide). Results Our data show that, in spite of some quantitative differences, the EGFR amplification status remains stable in the majority (84%) of tumors evaluated. EGFRvIII expression remained similar in 79% of GBMs. However, within the tumors expressing EGFRvIII at initial diagnosis, approximately one-half lose their EGFRvIII expression at tumor recurrence. Conclusions The relative stability of EGFR amplification indicates that molecular data obtained in the primary tumor can be used to predict the EGFR status of the recurrent tumor, but care should be taken in extrapolating EGFRvIII expression from the primary tumor, particularly when expressed at first diagnosis. PMID:25691693

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

PubMed

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

2015-09-15

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.

[Cell-ELA-based determination of binding affinity of DNA aptamer against U87-EGFRvIII cell].

PubMed

Tan, Yan; Liang, Huiyu; Wu, Xidong; Gao, Yubo; Zhang, Xingmei

2013-05-01

A15, a DNA aptamer with binding specificity for U87 glioma cells stably overexpressing the epidermal growth factor receptor variant III (U87-EGFRvIII), was generated by cell systematic evolution of ligands by exponential enrichment (cell-SELEX) using a random nucleotide library. Subsequently, we established a cell enzyme-linked assay (cell-ELA) to detect the affinity of A15 compared to an EGFR antibody. We used A15 as a detection probe and cultured U87-EGFRvIII cells as targets. Our data indicate that the equilibrium dissociation constants (K(d)) for A15 were below 100 nmol/L and had similar affinity compared to an EGFR antibody for U87-EGFRvIII. We demonstrated that the cell-ELA was a useful method to determine the equilibrium dissociation constants (K(d)) of aptamers generated by cell-SELEX.

Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma

PubMed Central

2013-01-01

Background Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells’ ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain. Methods A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model. Results Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in

Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma.

PubMed

Shen, Chan-Juan; Yang, Yu-Xiu; Han, Ethan Q; Cao, Na; Wang, Yun-Fei; Wang, Yi; Zhao, Ying-Ying; Zhao, Li-Ming; Cui, Jian; Gupta, Puja; Wong, Albert J; Han, Shuang-Yin

2013-05-09

Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells' ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain. A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model. Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in vivo growth of the EGFRvIII

Exploring the regulatory role of isocitrate dehydrogenase mutant protein on glioma stem cell proliferation.

PubMed

Lu, H-C; Ma, J; Zhuang, Z; Qiu, F; Cheng, H-L; Shi, J-X

2016-08-01

Glioma is the most lethal form of cancer that originates mostly from the brain and less frequently from the spine. Glioma is characterized by abnormal regulation of glial cell differentiation. The severity of the glioma was found to be relaxed in isocitrate dehydrogenase 1 (IDH1) mutant. The present study focused on histological discrimination and regulation of cancer stem cell between IDH1 mutant and in non-IDH1 mutant glioma tissue. Histology, immunohistochemistry and Western blotting techniques are used to analyze the glioma nature and variation in glioma stem cells that differ between IDH1 mutant and in non-IDH1 mutant glioma tissue. The aggressive form of non-IDH1 mutant glioma shows abnormal cellular histological variation with prominent larger nucleus along with abnormal clustering of cells. The longer survival form of IDH1 mutant glioma has a control over glioma stem cell proliferation. Immunohistochemistry with stem cell markers, CD133 and EGFRvIII are used to demonstrate that the IDH1 mutant glioma shows limited dependence on cancer stem cells and it shows marked apoptotic signals in TUNEL assay to regulate abnormal cells. The non-IDH1 mutant glioma failed to regulate misbehaving cells and it promotes cancer stem cell proliferation. Our finding supports that the IDH1 mutant glioma has a regulatory role in glioma stem cells and their survival.

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

PubMed

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

2014-01-01

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.

Isolation and characterization of a novel human scFv inhibiting EGFR vIII expressing cancers.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Dariushnejad, Hassan; Hosseini, Mohammad Kazem

2016-12-01

EGFRvIII, a mutant form of epidermal growth factor receptor is highly expressed in glioblastoma, carcinoma of the breast, ovary, and lung but not in normal cells. This tumor specific antigen has emerged as a promising candidate for antibody based therapy of several cancers. The aim of the present study was isolation and characterization of a human single chain antibody against EGFRvIII as a promising target for cancer therapy. For this, a synthetic peptide corresponding to EGFRvIII protein was used for screening the naive human scFv phage library. Selection was performed using a novel screening strategy for enrichment of rare specific clones. After five rounds of screening, six positive scFv clones against EGFRvIII were selected using monoclonal phage ELISA, among them, a clone with an amber mutation in VH CDR2 coding sequence showed higher reactivity. The mutation was corrected through site directed mutagenesis and then scFv fragment was expressed after subcloning into the bacterial expression vector. Expression in BL21 pLysS resulted in a highly soluble scFv appeared in soluble fraction of E. coli lysate. Bioinformatic in silico analysis between scFv and EGFRvIII sequences confirmed specific binding of desired scFv to EGFRvIII in CDR regions. The specific reactivity of the purified scFv with native EGFRvIII was confirmed by cell based ELISA and western blot. In conclusion, human anti- EGFRvIII scFv isolated from a scFv phage library displayed high reactivity with EGFRvIII. The scFv isolated in this study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFRvIII expressing cancers. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Production of a novel camel single-domain antibody specific for the type III mutant EGFR.

PubMed

Omidfar, K; Rasaee, M J; Modjtahedi, H; Forouzandeh, M; Taghikhani, M; Golmakani, N

2004-01-01

Camelids have a unique immune system capable of producing single-domain heavy-chain antibodies. The antigen-specific domain of these heavy-chain IgGs (VHH) are the smallest binding units produced by the immune system. In this study, we report the isolation and characterization of several binders against the epidermal growth factor receptor (EGFR) vIII retrieved from immune library of camels (Camelus bactrianus and Camelus dromedarius). The EGFRvIII is a ligand-independent, constitutively active, mutated form of the wild-type EGFR. The expression of EGFRvIII has been demonstrated in a wide range of human malignancies, including gliomas, and breast, prostate, ovarian and lung cancer. Camels were immunized with a synthetic peptide corresponding to a mutated sequence and tissue homogenates. Single-domain antibodies (VHH) were directly selected by panning a phage display library on successively decreasing amounts of synthetic peptide immobilized on magnetic beads. The anti-EGFRvIII camel single-domain antibodies selectively bound to the EGFRvIII peptide and reacted specifically with the immunoaffinity-purified antigen from a non-small cell lung cancer patient. These antibodies with affinities in the nanomolar range recognized the EGFRvIII peptide and affinity-purified mutated receptor. We concluded that using the phage display technique, antigen-specific VHH antibody fragments are readily accessible from the camelids. These antibodies may be good candidates for tumor-diagnostic and therapeutic applications. Copyright 2004 S. Karger AG, Basel.

Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors

PubMed Central

Ji, Hongbin; Zhao, Xiaojun; Yuza, Yuki; Shimamura, Takeshi; Li, Danan; Protopopov, Alexei; Jung, Boonim L.; McNamara, Kate; Xia, Huili; Glatt, Karen A.; Thomas, Roman K.; Sasaki, Hidefumi; Horner, James W.; Eck, Michael; Mitchell, Albert; Sun, Yangping; Al-Hashem, Ruqayyah; Bronson, Roderick T.; Rabindran, Sridhar K.; Discafani, Carolyn M.; Maher, Elizabeth; Shapiro, Geoffrey I.; Meyerson, Matthew; Wong, Kwok-Kin

2006-01-01

The tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) have shown anti-tumor activity in the treatment of non-small cell lung cancer (NSCLC). Dramatic and durable responses have occurred in NSCLC tumors with mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). In contrast, these inhibitors have shown limited efficacy in glioblastoma, where a distinct EGFR mutation, the variant III (vIII) in-frame deletion of exons 2–7, is commonly found. In this study, we determined that EGFRvIII mutation was present in 5% (3/56) of analyzed human lung squamous cell carcinoma (SCC) but was not present in human lung adenocarcinoma (0/123). We analyzed the role of the EGFRvIII mutation in lung tumorigenesis and its response to tyrosine kinase inhibition. Tissue-specific expression of EGFRvIII in the murine lung led to the development of NSCLC. Most importantly, these lung tumors depend on EGFRvIII expression for maintenance. Treatment with an irreversible EGFR inhibitor, HKI-272, dramatically reduced the size of these EGFRvIII-driven murine tumors in 1 week. Similarly, Ba/F3 cells transformed with the EGFRvIII mutant were relatively resistant to gefitinib and erlotinib in vitro but proved sensitive to HKI-272. These findings suggest a therapeutic strategy for cancers harboring the EGFRvIII mutation. PMID:16672372

Recombinant expression of extracellular domain of mutant Epidermal Growth Factor Receptor in prokaryotic and baculovirus expression systems.

PubMed

Vettath, Sunitha Kodengil; Shivashankar, Gaganashree; Menon, Krishnakumar N; Vijayachandran, Lakshmi S

2018-04-15

Epidermal Growth Factor Receptor variant III (EGFRvIII) is a tumor specific antigen detected in various tumors including gliomas, breast cancer, lung cancer, head and neck squamous cell carcinoma (HNSCC). Screening of EGFRvIII targeting drug molecules can be accelerated by developing drug screening platforms using recombinantly expressed protein. Choice of expression system is one of the major factors deciding the success of recombinant expression of a protein. In our study, we have tried to express and purify the extracellular domain (ECD) of this highly unstable protein using bacterial and baculovirus expression systems to select the expression system suited for our purpose. Even though the protein was successfully expressed in prokaryotic system, purification could be done only under denaturing conditions. But in the baculovirus expression system, the protein was expressed in soluble form and could be purified under native conditions, with single step of purification. Based on our results, we conclude that insect cells are better choice over E. coli cells for expressing EGFRvIII ECD in soluble form. This study provides insights for other researchers involved in expression of similar unstable membrane proteins, on selecting the best expression system and challenges involved. Copyright © 2018 Elsevier B.V. All rights reserved.

Quantitative proteomic analysis reveals effects of epidermal growth factor receptor (EGFR) on invasion-promoting proteins secreted by glioblastoma cells.

PubMed

Sangar, Vineet; Funk, Cory C; Kusebauch, Ulrike; Campbell, David S; Moritz, Robert L; Price, Nathan D

2014-10-01

Glioblastoma multiforme is a highly invasive and aggressive brain tumor with an invariably poor prognosis. The overexpression of epidermal growth factor receptor (EGFR) is a primary influencer of invasion and proliferation in tumor cells and the constitutively active EGFRvIII mutant, found in 30-65% of Glioblastoma multiforme, confers more aggressive invasion. To better understand how EGFR contributes to tumor aggressiveness, we investigated the effect of EGFR on the secreted levels of 65 rationally selected proteins involved in invasion. We employed selected reaction monitoring targeted mass spectrometry using stable isotope labeled internal peptide standards to quantity proteins in the secretome from five GBM (U87) isogenic cell lines in which EGFR, EGFRvIII, and/or PTEN were expressed. Our results show that cell lines with EGFR overexpression and constitutive EGFRvIII expression differ remarkably in the expression profiles for both secreted and intracellular signaling proteins, and alterations in EGFR signaling result in reproducible changes in concentrations of secreted proteins. Furthermore, the EGFRvIII-expressing mutant cell line secretes the majority of the selected invasion-promoting proteins at higher levels than other cell lines tested. Additionally, the intracellular and extracellular protein measurements indicate elevated oxidative stress in the EGFRvIII-expressing cell line. In conclusion, the results of our study demonstrate that EGFR signaling has a significant effect on the levels of secreted invasion-promoting proteins, likely contributing to the aggressiveness of Glioblastoma multiforme. Further characterization of these proteins may provide candidates for new therapeutic strategies and targets as well as biomarkers for this aggressive disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

TTSS2-deficient hha mutant of Salmonella Typhimurium exhibits significant systemic attenuation in immunocompromised hosts

PubMed Central

Vishwakarma, Vikalp; Pati, Niladri Bhusan; Ray, Shilpa; Das, Susmita; Suar, Mrutyunjay

2014-01-01

Non-typhoidal Salmonella (NTS) infections are emerging as leading problem worldwide and the variations in host immune status append to the concern of NTS. Salmonella enterica serovar Typhimurium is one of the causative agents of NTS infections and has been extensively studied. The inactivation of Salmonella pathogenicity island 2 (SPI2) encoded type-III secretion system 2 (TTSS2) has been reported rendering the strain incapable for systemic dissemination to host sites and has also been proposed as live-attenuated vaccine. However, infections from TTSS2-deficient Salmonella have also been reported. In this study, mutant strain MT15 was developed by inactivation of the hemolysin expression modulating protein (hha) in TTSS2-deficient S. Typhimurium background. The MT15 strain showed significant level of attenuation in immune-deprived murine colitis model when tested in iNos−/−, IL10−/−, and CD40L−/− mice groups in C57BL/6 background. Further, the mutation in hha does not implicate any defect in bacterial colonization to the host gut. The long-term infection of developed mutant strain conferred protective immune responses to suitably immunized streptomycin pre-treated C57BL/6 mice. The immunization enhanced the CD4+ and CD8+ cell types involved in bacterial clearance. The serum IgG and luminal secretory IgA (sIgA) was also found to be elevated after the due course of infection. Additionally, the immunized C57BL/6 mice were protected from the subsequent lethal infection of Salmonella Typhimurium. Collectively, these findings implicate the involvement of hemolysin expression modulating protein (Hha) in establishment of bacterial infection. In light of the observed attenuation of the developed mutant strain, this study proposes the possible significance of SPI2-deficient hha mutant as an alternative live-attenuated vaccine strain for use against lethal Salmonella infections. PMID:24401482

New anti-cancer chemicals Ertredin and its derivatives, regulate oxidative phosphorylation and glycolysis and suppress sphere formation in vitro and tumor growth in EGFRvIII-transformed cells.

PubMed

Atsumi, Sonoko; Nosaka, Chisato; Adachi, Hayamitsu; Kimura, Tomoyuki; Kobayashi, Yoshihiko; Takada, Hisashi; Watanabe, Takumi; Ohba, Shun-Ichi; Inoue, Hiroyuki; Kawada, Manabu; Shibasaki, Masakatsu; Shibuya, Masabumi

2016-07-19

EGFRvIII is a mutant form of the epidermal growth factor receptor gene (EGFR) that lacks exons 2-7. The resulting protein does not bind to ligands and is constitutively activated. The expression of EGFRvIII is likely confined to various types of cancer, particularly glioblastomas. Although an anti-EGFRvIII vaccine is of great interest, low-molecular-weight substances are needed to obtain better therapeutic efficacy. Thus, the purpose of this study is to identify low molecular weight substances that can suppress EGFRvIII-dependent transformation. We constructed a new throughput screening system and searched for substances that decreased cell survival of NIH3T3/EGFRvIII spheres under 3-dimensional (3D)-culture conditions, but retained normal NIH3T3 cell growth under 2D-culture conditions. In vivo activity was examined using a mouse transplantation model, and derivatives were chemically synthesized. Functional characterization of the candidate molecules was investigated using an EGFR kinase assay, immunoprecipitation, western blotting, microarray analysis, quantitative polymerase chain reaction analysis, and measurement of lactate and ATP synthesis. In the course of screening 30,000 substances, a reagent, "Ertredin" was found to inhibit anchorage-independent 3D growth of sphere-forming cells transfected with EGFRvIII cDNA. Ertredin also inhibited sphere formation in cells expressing wild-type EGFR in the presence of EGF. However, it did not affect anchorage-dependent 2D growth of parental NIH3T3 cells. The 3D-growth-inhibitory activity of some derivatives, including those with new structures, was similar to Ertredin. Furthermore, we demonstrated that Ertredin suppressed tumor growth in an allograft transplantation mouse model injected with EGFRvIII- or wild-type EGFR-expressing cells; a clear toxicity to host animals was not observed. Functional characterization of Ertredin in cells expressing EGFRvIII indicated that it stimulated EGFRvIII ubiquitination, suppressed

Enhanced transpiration rate in the high pigment 1 tomato mutant and its physiological significance.

PubMed

Carvalho, R F; Aidar, S T; Azevedo, R A; Dodd, I C; Peres, L E P

2011-05-01

Tomato high pigment (hp) mutants represent an interesting horticultural resource due to their enhanced accumulation of carotenoids, flavonoids and vitamin C. Since hp mutants are known for their exaggerated light responses, the molecules accumulated are likely to be antioxidants, recruited to deal with light and others stresses. Further phenotypes displayed by hp mutations are reduced growth and an apparent disturbance in water loss. Here, we examined the impact of the hp1 mutation and its near isogenic line cv Micro-Tom (MT) on stomatal conductance (gs), transpiration (E), CO(2) assimilation (A) and water use efficiency (WUE). Detached hp1 leaves lost water more rapidly than control leaves, but this behaviour was reversed by exogenous abscisic acid (ABA), indicating the ability of hp1 to respond to this hormone. Although attached hp1 leaves had enhanced gs, E and A compared to control leaves, genotypic differences were lost when water was withheld. Both instantaneous leaf-level WUE and long-term whole plant WUE did not differ between hp1 and MT. Our results indicate a link between exaggerated light response and water loss in hp1, which has important implications for the use of this mutant in both basic and horticultural research. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

No Significant Increase in the Δ4- and Δ7-Dafachronic Acid Concentration in the Long-Lived glp-1 Mutant, nor in the Mutants Defective in Dauer Formation.

PubMed

Li, Tie-Mei; Liu, Weilong; Lu, Shan; Zhang, Yan-Ping; Jia, Le-Mei; Chen, Jie; Li, Xiangke; Lei, Xiaoguang; Dong, Meng-Qiu

2015-05-12

The steroid hormone dafachronic acid (DA) regulates dauer formation and lifespan in Caenorhabditis elegans by binding to the nuclear receptor DAF-12. However, little is known about how DA concentrations change under various physiologic conditions and about how DA/DAF-12 signaling interacts with other signaling pathways that also regulate dauer formation and lifespan. Using a sensitive bioanalytical method, we quantified the endogenous DA concentrations in a long-lived germline-less glp-1 mutant and in the Dauer formation-defective (Daf-d) mutants daf-12, daf-16, daf-5, and daf-3. We found that the DA concentration in the glp-1 mutant was similar to that in the wild type (WT). This result is contrary to the long-held belief that germline loss-induced longevity involves increased DA production and suggests instead that this type of longevity involves an enhanced response to DA. We also found evidence suggesting that increased DA sensitivity underlies lifespan extension triggered by exogenous DA. At the L2/L3 stage, the DA concentration in a daf-12 null mutant decreased to 22% of the WT level. This finding is consistent with the previously proposed positive feedback regulation between DAF-12 and DA production. Surprisingly, the DA concentrations in the daf-16, daf-5, and daf-3 mutants were only 19-34% of the WT level at the L2/L3 stage, slightly greater than those in the Dauer formation-constitutive (Daf-c) mutants at the pre-dauer stage (4-15% of the WT L2 control). Our experimental evidence suggested that the positive feedback between DA and DAF-12 was partially induced in the three Daf-d mutants. Copyright © 2015 Li et al.

Sharing mutants and experimental information prepublication using FgMutantDb (https://scabusa.org/FgMutantDb).

PubMed

Baldwin, Thomas T; Basenko, Evelina; Harb, Omar; Brown, Neil A; Urban, Martin; Hammond-Kosack, Kim E; Bregitzer, Phil P

2018-06-01

There is no comprehensive storage for generated mutants of Fusarium graminearum or data associated with these mutants. Instead, researchers relied on several independent and non-integrated databases. FgMutantDb was designed as a simple spreadsheet that is accessible globally on the web that will function as a centralized source of information on F. graminearum mutants. FgMutantDb aids in the maintenance and sharing of mutants within a research community. It will serve also as a platform for disseminating prepublication results as well as negative results that often go unreported. Additionally, the highly curated information on mutants in FgMutantDb will be shared with other databases (FungiDB, Ensembl, PhytoPath, and PHI-base) through updating reports. Here we describe the creation and potential usefulness of FgMutantDb to the F. graminearum research community, and provide a tutorial on its use. This type of database could be easily emulated for other fungal species. Published by Elsevier Inc.

EGFRvIII/integrin β3 interaction in hypoxic and vitronectinenriching microenvironment promote GBM progression and metastasis.

PubMed

Liu, Zhaoyu; Han, Lei; Dong, Yucui; Tan, Yanli; Li, Yongsheng; Zhao, Manli; Xie, Hui; Ju, Huanyu; Wang, He; Zhao, Yu; Zheng, Qifan; Wang, Qixue; Su, Jun; Fang, Chuan; Fu, Songbin; Jiang, Tao; Liu, Jiaren; Li, Xia; Kang, Chunsheng; Ren, Huan

2016-01-26

Glioblastoma (GBM) is one of the most lethal brain tumors with a short survival time. EGFR amplification and mutation is the most significant genetic signature in GBM. About half of the GBMs with EGFR amplification express a constitutively autophosphorylated variant of EGFR, known as EGFRvIII. Our in vitro data demonstrated further enhanced EGFRvIII activity and tumor cell invasion in the tumor microenvironment of hypoxia plus extracellular matrix (ECM) vitronectin, in which EGFRvIII and integrin β3 tended to form complexes. The treatment with ITGB3 siRNA or the integrin antagonist cilengetide preferentially interrupted the EGFRvIII/integrin β3 complex, effectively reduced tumor cell invasion and activation of downstream signaling effectors. Cilengitide is recently failed in Phase III CENTRIC trial in unselected patients with GBM. However, we found that cilengitide demonstrated efficacious tumor regression via inhibition of tumor growth and angiogenesis in EGFRvIII orthotopic xenografts. Bioinformatics analysis emphasized key roles of integrin β3, hypoxia and vitronectin and their strong correlations with EGFRvIII expression in malignant glioma patient samples in vivo. In conclusion, we demonstrate that EGFRvIII/integrin β3 complexes promote GBM progression and metastasis in the environment of hypoxia and vitronectin-enrichment, and cilengitide may serve as a promising therapeutics for EGFRvIII-positive GBMs.

[Construction and prokaryotic expression of recombinant gene EGFRvIII HBcAg and immunogenicity analysis of the fusion protein].

PubMed

Duan, Xiao-yi; Wang, Jian-sheng; Guo, You-min; Han, Jun-li; Wang, Quan-ying; Yang, Guang-xiao

2007-01-01

To construct recombinant prokaryotic expression plasmid pET28a(+)/c-PEP-3-c and evaluate the immunogenicity of the fusion protein. cDNA fragment encoding PEP-3 was obtained from pGEM-T Easy/PEP-3 and inserted into recombinant plasmid pGEMEX/HBcAg. Then it was subcloned in prokaryotic expression vector and transformed into E.coli BL21(DE3). The fusion protein was expressed by inducing IPTG and purified by Ni(2+)-NTA affinity chromatography. BALB/c mice were immunized with fusion protein and the antibody titre was determined by indirect ELISA. The recombinant gene was confirmed to be correct by restriction enzyme digestion and DNA sequencing. After prokaryotic expression, fusion protein existed in sediment and accounted for 56% of all bacterial lysate. The purified product accounted for 92% of all protein and its concentration was 8 g/L. The antibody titre in blood serum reached 1:16 000 after the fourth immunization and reached 1:2.56x10(5) after the sixth immunization. The titre of anti-PEP-3 antibody reached 1:1.28x10(5) and the titre of anti-HBcAg antibody was less than 1:4x10(3). Fusion gene PEP-3-HBcAg is highly expressed in E.coli BL21. The expressed fusion protein can induce neutralizing antibody with high titer and specificity, which lays a foundation for the study of genetically engineering vaccine for malignant tumors with the high expression of EGFRvIII.

Chaperone-mediated autophagy degrades mutant p53

PubMed Central

Vakifahmetoglu-Norberg, Helin; Kim, Minsu; Xia, Hong-guang; Iwanicki, Marcin P.; Ofengeim, Dimitry; Coloff, Jonathan L.; Pan, Lifeng; Ince, Tan A.; Kroemer, Guido; Brugge, Joan S.; Yuan, Junying

2013-01-01

Missense mutations in the gene TP53, which encodes p53, one of the most important tumor suppressors, are common in human cancers. Accumulated mutant p53 proteins are known to actively contribute to tumor development and metastasis. Thus, promoting the removal of mutant p53 proteins in cancer cells may have therapeutic significance. Here we investigated the mechanisms that govern the turnover of mutant p53 in nonproliferating tumor cells using a combination of pharmacological and genetic approaches. We show that suppression of macroautophagy by multiple means promotes the degradation of mutant p53 through chaperone-mediated autophagy in a lysosome-dependent fashion. In addition, depletion of mutant p53 expression due to macroautophagy inhibition sensitizes the death of dormant cancer cells under nonproliferating conditions. Taken together, our results delineate a novel strategy for killing tumor cells that depend on mutant p53 expression by the activation of chaperone-mediated autophagy and potential pharmacological means to reduce the levels of accumulated mutant p53 without the restriction of mutant p53 conformation in quiescent tumor cells. PMID:23913924

Aggressive behavior of the white-eye mutant crickets, Gryllus bimaculatus.

PubMed

Sakura, Midori; Watanabe, T; Aonuma, H

2012-01-01

Aggressive behavior of white-eye mutant crickets was investigated and compared with that of wild-type crickets. In the dark, wild-type pairs performed long-lasting fights with significantly higher aggressive levels compared to those in the light. In contrast, fights between two white-eye mutants were not significantly different with those between two wild-type crickets both in duration and the aggressive levels. Ethograms of aggressive behavior showed that the mutants could show typical sequentially escalating fight with the same behavioral categories as the wild-type crickets. These results indicate that the white-eye mutants are able to express normal aggressive behavior.

Identification of EGFRvIII-derived CTL epitopes restricted by HLA A0201 for dendritic cell based immunotherapy of gliomas.

PubMed

Wu, An-hua; Xiao, Jing; Anker, Lars; Hall, Walter A; Gregerson, Dale S; Cavenee, Webster K; Chen, Wei; Low, Walter C

2006-01-01

The type III variant of the epidermal growth factor receptor (EGFRvIII) mutation is present in 20-25% of patients with glioblastoma multiforme (GBM). EGFRvIII is not expressed in normal tissue and is therefore a suitable candidate antigen for dendritic cell (DC) based immunotherapy of GBM. To identify the antigenic epitope(s) that may serve as targets for EGFRvIII-specific cytotoxic T lymphocytes (CTLs), the peptide sequence of EGFRvIII was screened with two software programs to predict candidate epitopes restricted by the major histocompatibility complex class I subtype HLA-A0201, which is the predominant subtype in most ethnic groups. Three predicted peptides were constructed and loaded to mature human DCs generated from peripheral blood monocytes. Autologous CD8+ T cells were stimulated in vitro with the EGFRvIII peptide-pulsed DCs. One of the three peptides was found to induce EGFRvIII-specific CTLs as demonstrated by IFN-gamma production and cytotoxicity against HLA-A0201+ EGFRvIII transfected U87 glioma cells. These results suggest that vaccination with EGFRvIII peptide-pulsed DCs or adoptive transfer of in vitro elicited EGFRvIII-specific CTLs by EGFRvIII peptide-pulsed DCs are potential approaches to the treatment of glioma patients.

Significance of coronavirus mutants in feces and diseased tissues of cats suffering from feline infectious peritonitis.

PubMed

Pedersen, Niels C; Liu, Hongwei; Dodd, Kimberly A; Pesavento, Patricia A

2009-09-01

The internal FECV→FIPV mutation theory and three of its correlates were tested in four sibs/half-sib kittens, a healthy contact cat, and in four unrelated cats that died of FIP at geographically disparate regions. Coronavirus from feces and extraintestinal FIP lesions from the same cat were always >99% related in accessory and structural gene sequences. SNPs and deletions causing a truncation of the 3c gene product were found in almost all isolates from the diseased tissues of the eight cats suffering from FIP, whereas most, but not all fecal isolates from these same cats had intact 3c genes. Other accessory and structural genes appeared normal in both fecal and lesional viruses. Deliterious mutations in the 3c gene were unique to each cat, indicating that they did not originate in one cat and were subsequently passed horizontally to the others. Compartmentalization of the parental and mutant forms was not absolute; virus of lesional type was sometimes found in feces of affected cats and virus identical to fecal type was occasionally identified in diseased tissues. Although 3c gene mutants in this study were not horizontally transmitted, the parental fecal virus was readily transmitted by contact from a cat that died of FIP to its housemate. There was a high rate of mutability in all structural and accessory genes both within and between cats, leading to minor genetic variants. More than one variant could be identified in both diseased tissues and feces of the same cat. Laboratory cats inoculated with a mixture of two closely related variants from the same FIP cat developed disease from one or the other variant, but not both. Significant genetic drift existed between isolates from geographically distinct regions of the Western US.

Significance of Coronavirus Mutants in Feces and Diseased Tissues of Cats Suffering from Feline Infectious Peritonitis

PubMed Central

Pedersen, Niels C.; Liu, Hongwei; Dodd, Kimberly A.; Pesavento, Patricia A.

2009-01-01

The internal FECV→FIPV mutation theory and three of its correlates were tested in four sibs/half-sib kittens, a healthy contact cat, and in four unrelated cats that died of FIP at geographically disparate regions. Coronavirus from feces and extraintestinal FIP lesions from the same cat were always >99% related in accessory and structural gene sequences. SNPs and deletions causing a truncation of the 3c gene product were found in almost all isolates from the diseased tissues of the eight cats suffering from FIP, whereas most, but not all fecal isolates from these same cats had intact 3c genes. Other accessory and structural genes appeared normal in both fecal and lesional viruses. Deliterious mutations in the 3c gene were unique to each cat, indicating that they did not originate in one cat and were subsequently passed horizontally to the others. Compartmentalization of the parental and mutant forms was not absolute; virus of lesional type was sometimes found in feces of affected cats and virus identical to fecal type was occasionally identified in diseased tissues. Although 3c gene mutants in this study were not horizontally transmitted, the parental fecal virus was readily transmitted by contact from a cat that died of FIP to its housemate. There was a high rate of mutability in all structural and accessory genes both within and between cats, leading to minor genetic variants. More than one variant could be identified in both diseased tissues and feces of the same cat. Laboratory cats inoculated with a mixture of two closely related variants from the same FIP cat developed disease from one or the other variant, but not both. Significant genetic drift existed between isolates from geographically distinct regions of the Western US. PMID:21994544

Sharing mutants and experimental information prepublication using FgMutantDB

USDA-ARS?s Scientific Manuscript database

There has been no central location for storing generated mutants of Fusarium graminearum or for data associated with these mutants. Instead researchers relied on several independent, non-integrated databases. FgMutantDB was designed as a simple spreadsheet that is accessible globally on the web th...

Isolation and characterization of acid-sensitive mutants of Pediococcus acidilactici.

PubMed

Kurdi, Peter; Smitinont, Thitapha; Valyasevi, Ruud

2009-02-01

Acid-sensitive mutants of Pediococcus acidilactici BCC 9545, a starter culture of the Thai fermented pork sausage nham, were isolated as spontaneous neomycin resistant mutants. The mutants generally produced less acid and acidified the culture media less than the parent strain in a 72 h culturing period. Interestingly, the ATPase activities of the mutants did not differ considerably from that of the parent strain in acidic conditions. It was also found that the internal pH values of the mutant strains were somewhat lower in neutral environment, while at pH 5.0 their internal pHs were significantly lower compared to the parent's. Inhibiting the H(+)-ATPase activities in energized cells by N,N'-dicyclohexyl carbodiimide also revealed that protons were leaking from the mutants at neutral pH, which increased under acidic conditions. In contrast, the parent strain exhibited a smaller proton leak and only under acidic conditions. The membrane fatty acid analysis of the mutants indicated that under acidic conditions the mutants had a significantly smaller major unsaturated/saturated fatty acids ratio ((C(18:1)+C(18:3n6))/(C(16:0)+C(18:0))) compared to the parent strain's membrane. Taken together, these observations suggest there is a reasonable possibility that the membrane fatty acid profile differences in the mutants resulted in their acid-sensitivity.

GBM heterogeneity as a function of variable epidermal growth factor receptor variant III activity.

PubMed

Lindberg, Olle R; McKinney, Andrew; Engler, Jane R; Koshkakaryan, Gayane; Gong, Henry; Robinson, Aaron E; Ewald, Andrew J; Huillard, Emmanuelle; David James, C; Molinaro, Annette M; Shieh, Joseph T; Phillips, Joanna J

2016-11-29

Abnormal activation of the epidermal growth factor receptor (EGFR) due to a deletion of exons 2-7 of EGFR (EGFRvIII) is a common alteration in glioblastoma (GBM). While this alteration can drive gliomagenesis, tumors harboring EGFRvIII are heterogeneous. To investigate the role for EGFRvIII activation in tumor phenotype we used a neural progenitor cell-based murine model of GBM driven by EGFR signaling and generated tumor progenitor cells with high and low EGFRvIII activation, pEGFRHi and pEGFRLo. In vivo, ex vivo, and in vitro studies suggested a direct association between EGFRvIII activity and increased tumor cell proliferation, decreased tumor cell adhesion to the extracellular matrix, and altered progenitor cell phenotype. Time-lapse confocal imaging of tumor cells in brain slice cultures demonstrated blood vessel co-option by tumor cells and highlighted differences in invasive pattern. Inhibition of EGFR signaling in pEGFRHi promoted cell differentiation and increased cell-matrix adhesion. Conversely, increased EGFRvIII activation in pEGFRLo reduced cell-matrix adhesion. Our study using a murine model for GBM driven by a single genetic driver, suggests differences in EGFR activation contribute to tumor heterogeneity and aggressiveness.

Value of bilirubin oxidase and its mutants in the diagnosis of hyperbilirubinemia.

PubMed

Zhang, Lei; Zhang, Xiao; Luo, Zhi-Ying

2005-11-01

To elucidate the significance of the coordination amino acid residues in bilirubin oxidase (BO) and their kinetic characteristics, and evaluate whether BO mutants may serve as better diagnostic agent for hyperbilirubinemia. The BO mutants I402G and C457S were obtained by site-directed mutagenesis and confirmed by amino acid sequence analysis. Ru-incorporated C457S mutant was obtained by direct incubation of ruthenium compounds with the mutant. The electron paramagnetic resonance (EPR) spectra of the recombinant BO and the mutants were investigated, and the enzyme kinetics of the recombinant BO and I402G mutant were measured with bilirubin as the substrate at 25 degrees C. The BO mutants were expressed and purified successfully. The mutant I402G showed low enzyme activity, and had C457S virtually no enzyme activity. Nevertheless Ru-incorporation conferred higher enzyme activity to C457S mutant. The enzyme kinetic investigations revealed that the kinetic parameter k(cat) of the recombinant BO and I402G mutant was 235.8 min(-1) and 6.9 min(-1), respectively, suggesting higher enzyme activity of the recombinant BO. The coordinating amino acids have important significance in maintaining the integrity of active centers and enzyme activities of recombinant BO and its mutants. The enzyme activities of the mutants I402G and C457S are much lower than those of recombinant BO, therefore they are not appropriate for diagnostic purpose. Ru-incorporation facilitates the formation of a new intact active center in C457S mutant, which therefore acquires enzyme activity.

Human IP10-scFv and DC-induced CTL synergistically inhibit the growth of glioma in a xenograft model.

PubMed

Wang, Xuan; Zhang, Fang-Cheng; Zhao, Hong-Yang; Lu, Xiao-Ling; Sun, Yun; Xiong, Zhi-Yong; Jiang, Xiao-Bing

2014-08-01

The epidermal growth factor receptor (EGFR) mutant of EGFRvIII is highly expressed in glioma cells, and the EGFRvIII-specific dendritic cell (DC)-induced tumor antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) may hold promise in cancer immunotherapy. Interferon (IFN)-γ-inducible protein (IP)-10 (IP-10) is a potent inhibitor of angiogenesis and can recruit CXCR3(+) T cells, including CD8(+) T cells, which are important for the control of tumor growth. In this study, we assessed if the combination of IP10-EGFRvIIIscFv with DC-induced CTLs would improve the therapeutic antitumor efficacy. IP10-scFv was generated by linking the human IP-10 gene with the DNA fragment for anti-EGFRvIIIscFv with a (Gly4Ser)3 flexible linker, purified by affinity chromatography, and characterized for its anti-EGFRvIII immunoreactivity and chemotactic activity. DCs were isolated from human peripheral blood monocyte cells and pulsed with EGFRvIII-peptide, then co-cultured with autologous CD8(+) T cells. BALB/c-nu mice were inoculated with human glioma U87-EGFRvIII cells in the brain and treated intracranially with IP10-scFv and/or intravenously with DC-induced CTLs for evaluating the therapeutic effect. Treatment with both IP10-scFv and EGFRvIII peptide-pulsed, DC-induced CTL synergistically inhibited the growth of glioma and prolonged the survival of tumor-bearing mice, which was accompanied by the inhibition of tumor angiogenesis and enhancement of cytotoxicity, thereby increasing the numbers of brain-infiltrating lymphocytes (BILs) and prolonging the residence time of CTLs in the tumor.

Dual targeting of glioblastoma with chimeric antigen receptor-engineered natural killer cells overcomes heterogeneity of target antigen expression and enhances antitumor activity and survival.

PubMed

Genßler, Sabrina; Burger, Michael C; Zhang, Congcong; Oelsner, Sarah; Mildenberger, Iris; Wagner, Marlies; Steinbach, Joachim P; Wels, Winfried S

2016-04-01

Epidermal growth factor receptor (EGFR) and its mutant form EGFRvIII are overexpressed in a large proportion of glioblastomas (GBM). Immunotherapy with an EGFRvIII-specific vaccine has shown efficacy against GBM in clinical studies. However, immune escape by antigen-loss variants and lack of control of EGFR wild-type positive clones limit the usefulness of this approach. Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells may represent an alternative immunotherapeutic strategy. For targeting to GBM, we generated variants of the clinically applicable human NK cell line NK-92 that express CARs carrying a composite CD28-CD3ζ domain for signaling, and scFv antibody fragments for cell binding either recognizing EGFR, EGFRvIII, or an epitope common to both antigens. In vitro analysis revealed high and specific cytotoxicity of EGFR-targeted NK-92 against established and primary human GBM cells, which was dependent on EGFR expression and CAR signaling. EGFRvIII-targeted NK-92 only lysed EGFRvIII-positive GBM cells, while dual-specific NK cells expressing a cetuximab-based CAR were active against both types of tumor cells. In immunodeficient mice carrying intracranial GBM xenografts either expressing EGFR, EGFRvIII or both receptors, local treatment with dual-specific NK cells was superior to treatment with the corresponding monospecific CAR NK cells. This resulted in a marked extension of survival without inducing rapid immune escape as observed upon therapy with monospecific effectors. Our results demonstrate that dual targeting of CAR NK cells reduces the risk of immune escape and suggest that EGFR/EGFRvIII-targeted dual-specific CAR NK cells may have potential for adoptive immunotherapy of glioblastoma.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gu Ning; Laboratory of Neurochemistry, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto; Adachi, Tetsuya

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 2more » 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.« less

Accelerated bang recovery in Drosophila genderblind mutants.

PubMed

Featherstone, David E; Yanoga, Fatoumata; Grosjean, Yael

2008-07-01

Cystine-glutamate transporters import cystine into cells for glutathione synthesis and protection from oxidative stress, but also export significant amounts of glutamate. Increasing evidence suggests that 'ambient extracellular glutamate' secreted by cystine-glutamate transporters in the nervous system modulates glutamatergic synapse strength and behavior. To date, the only cystine-glutamate transporter mutants examined behaviorally are Drosophila genderblind mutants. These animals contain loss-of-function mutations in the 'genderblind' gene, which encodes an xCT subunit essential for cystine-glutamate transporter function. Genderblind was named based on a mutant courtship phenotype: male genderblind mutants are attracted to normally aversive male pheromones and thus court and attempt to copulate with both male and female partners equally. However, genderblind protein is expressed in many parts of the fly brain and thus might be expected to also regulate other behaviors, including behaviors not related to male courtship or chemosensation. Here, we show that genderblind mutants display faster recovery and increased negative geotaxis after strong mechanical stimuli (e.g., they climb faster and farther after vial banging). This phenotype is displayed by both males and females, consistent with strong genderblind expression in both sexes.

The H159A mutant of yeast enolase 1 has significant activity.

PubMed

Brewer, J M; Holland, M J; Lebioda, L

2000-10-05

The function of His159 in the enolase mechanism is disputed. Recently, Vinarov and Nowak (Biochemistry (1999) 38, 12138-12149) prepared the H159A mutant of yeast enolase 1 and expressed this in Escherichia coli. They reported minimal (ca. 0.01% of the native value) activity, though the protein appeared to be correctly folded, according to its CD spectrum, tryptophan fluorescence, and binding of metal ion and substrate. We prepared H159A enolase using a multicopy plasmid and expressed the enzyme in yeast. Our preparations of H159A enolase have 0.2-0.4% of the native activity under standard assay conditions and are further activated by Mg(2+) concentrations above 1 mM to 1-1.5% of the native activity. Native enolase 1 (and enolase 2) are inhibited by such Mg(2+) concentrations. It is possible that His159 is necessary for correct folding of the enzyme and that expression in E. coli leads to largely misfolded protein. Copyright 2000 Academic Press.

Dendritic cells pulsed with a tumor-specific peptide induce long-lasting immunity and are effective against murine intracerebral melanoma.

PubMed

Heimberger, Amy B; Archer, Gary E; Crotty, Laura E; McLendon, Roger E; Friedman, Allan H; Friedman, Henry S; Bigner, Darell D; Sampson, John H

2002-01-01

Dendritic cells (DCs) are specialized cells of the immune system that are capable of generating potent immune responses that are active even within the "immunologically privileged" central nervous system. However, immune responses generated by DCs have also been demonstrated to produce clinically significant autoimmunity. Targeting the epidermal growth factor receptor variant III (EGFRvIII), which is a mutation specific to tumor tissue, could eliminate this risk. The purpose of this study was to demonstrate that DC-based immunizations directed solely against this tumor-specific antigen, which is commonly found on tumors that originate within or metastasize to the brain, could be efficacious. C3H mice were vaccinated with DCs mixed with a keyhole limpet hemocyanin conjugate of the tumor-specific peptide, PEP-3, which spans the EGFRvIII mutation, or the random-sequence peptide, PEP-1, and were intracerebrally challenged with a syngeneic melanoma expressing a murine homologue of EGFRvIII. Systemic immunization with DCs mixed with PEP-3-keyhole limpet hemocyanin generated antigen-specific immunity. Among mice challenged with intracerebral tumors, this resulted in an approximately 600% increase in the median survival time (>300 d, P < 0.0016), relative to control values. Sixty-three percent of mice treated with DCs mixed with the tumor-specific peptide survived in the long term and 100% survived rechallenge with tumor, indicating that antitumor immunological memory was also induced. In a murine melanoma model, immunization with DCs mixed with tumor-specific peptide results in an antigen-specific immunological response that recognizes the EGFRvIII mutation, has potent antitumor efficacy against intracerebral tumors that express EGFRvIII, and results in long-lasting antitumor immunity.

Retinoblastoma protein (pRB) was significantly phosphorylated through a Ras-to-MAPK pathway in mutant K-ras stably transfected human adrenocortical cells.

PubMed

Chen, Y-F; Chiu, H-H; Wu, C-H; Wang, J-Y; Chen, F-M; Tzou, W-H; Shin, S-J; Lin, S-R

2003-10-01

Our previous studies have shown that the cell proliferation rate, mRNA levels of p450scc, p450c17, and 3betaHSD, and secretion of cortisol were significantly increased in human adrenocortical cells stably transfected with mutated K-ras expression plasmid "pK568MRSV" after being inducted with IPTG. In addition, the increased level was a time-dependent manner. However, the levels of p450, p450scc, p450c17, 3betaHSD, cortisol, and cell proliferation rate were inhibited by a MEK phospholation inhibitor, PD098059. The above results prove that mutated K-ras oncogene is able to regulate tumorigenesis and steroidogenesis through a Ras-RAF-MEK-MAPK signal transduction pathway. The aim of this study was to investigate regulated factors in this pathway and also examine whether the other signal transduction pathways or other moles involved in tumorigenesis or steroidogenesis. In the first year, we analyzed gene profiles of mutant K-ras-transfected adrenocortical cells by DNA microarray to determine the gene expression related to cell cycle, signal transduction, apoptosis, tumorigenesis, steroidogenesis, and other expressed sequence tag. After being affected by the K-ras mutant, gene expression was significantly increased in some upregulated genes. Human zinc-finger protein 22 increased by 28.5 times, Osteopontin increased by 5.8 times, LIM domain Kinase 2 (LIMK2) increased by 3.3 times, Homo sapiens dual-specificity tyrosine-(Y)-phosphorylation regulated Kinase 2 (DYRK2) increased by 2.2 times, and human syntaxin 3 increased by two times. On the other hand, significant decreases in gene expression were also observed in some downregulated genes. Retinoblastoma binding protein 1 (RBBP1) decreased by four times, Homo sapiens craniofacial development protein 1 (CFDP1) decreased by 2.4 times, DAP Kinase-related apoptosis-inducing protein Kinase 1 (DRAK1) decreased by 2.3 times, SKI-interacting protein (SKIP) decreased by 2.2 times, and human poly(A)-Binding protein (PABP) decreased

Selective targeting of KRAS-Mutant cells by miR-126 through repression of multiple genes essential for the survival of KRAS-Mutant cells

PubMed Central

Hara, Toshifumi; Jones, Matthew F.; Subramanian, Murugan; Li, Xiao Ling; Ou, Oliver; Zhu, Yuelin; Yang, Yuan; Wakefield, Lalage M.; Hussain, S. Perwez; Gaedcke, Jochen; Ried, Thomas; Luo, Ji; Caplen, Natasha J.; Lal, Ashish

2014-01-01

MicroRNAs (miRNAs) regulate the expression of hundreds of genes. However, identifying the critical targets within a miRNA-regulated gene network is challenging. One approach is to identify miRNAs that exert a context-dependent effect, followed by expression profiling to determine how specific targets contribute to this selective effect. In this study, we performed miRNA mimic screens in isogenic KRAS-Wild-type (WT) and KRAS-Mutant colorectal cancer (CRC) cell lines to identify miRNAs selectively targeting KRAS-Mutant cells. One of the miRNAs we identified as a selective inhibitor of the survival of multiple KRAS-Mutant CRC lines was miR-126. In KRAS-Mutant cells, miR-126 over-expression increased the G1 compartment, inhibited clonogenicity and tumorigenicity, while exerting no effect on KRAS-WT cells. Unexpectedly, the miR-126-regulated transcriptome of KRAS-WT and KRAS-Mutant cells showed no significant differences. However, by analyzing the overlap between miR-126 targets with the synthetic lethal genes identified by RNAi in KRAS-Mutant cells, we identified and validated a subset of miR-126-regulated genes selectively required for the survival and clonogenicity of KRAS-Mutant cells. Our strategy therefore identified critical target genes within the miR-126-regulated gene network. We propose that the selective effect of miR-126 on KRAS-Mutant cells could be utilized for the development of targeted therapy for KRAS mutant tumors. PMID:25245095

Towards an informative mutant phenotype for every bacterial gene

DOE PAGES

Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly 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

Increased Steady-State Mutant Huntingtin mRNA in Huntington's Disease Brain.

PubMed

Liu, Wanzhao; Chaurette, Joanna; Pfister, Edith L; Kennington, Lori A; Chase, Kathryn O; Bullock, Jocelyn; Vonsattel, Jean Paul G; Faull, Richard L M; Macdonald, Douglas; DiFiglia, Marian; Zamore, Phillip D; Aronin, Neil

2013-01-01

Huntington's disease is caused by expansion of CAG trinucleotide repeats in the first exon of the huntingtin gene, which is essential for both development and neurogenesis. Huntington's disease is autosomal dominant. The normal allele contains 6 to 35 CAG triplets (average, 18) and the mutant, disease-causing allele contains >36 CAG triplets (average, 42). We examined 279 postmortem brain samples, including 148 HD and 131 non-HD controls. A total of 108 samples from 87 HD patients that are heterozygous at SNP rs362307, with a normal allele (18 to 27 CAG repeats) and a mutant allele (39 to 73 CAG repeats) were used to measure relative abundance of mutant and wild-type huntingtin mRNA. We used allele-specific, quantitative RT-PCR based on SNP heterozygosity to estimate the relative amount of mutant versus normal huntingtin mRNA in postmortem brain samples from patients with Huntington's disease. In the cortex and striatum, the amount of mRNA from the mutant allele exceeds that from the normal allele in 75% of patients. In the cerebellum, no significant difference between the two alleles was evident. Brain tissues from non-HD controls show no significant difference between two alleles of huntingtin mRNAs. Allelic differences were more pronounced at early neuropathological grades (grades 1 and 2) than at late grades (grades 3 and 4). More mutant HTT than normal could arise from increased transcription of mutant HTT allele, or decreased clearance of mutant HTT mRNA, or both. An implication is that equimolar silencing of both alleles would increase the mutant HTT to normal HTT ratio.

Novel, improved grading system(s) for IDH-mutant astrocytic gliomas.

PubMed

Shirahata, Mitsuaki; Ono, Takahiro; Stichel, Damian; Schrimpf, Daniel; Reuss, David E; Sahm, Felix; Koelsche, Christian; Wefers, Annika; Reinhardt, Annekathrin; Huang, Kristin; Sievers, Philipp; Shimizu, Hiroaki; Nanjo, Hiroshi; Kobayashi, Yusuke; Miyake, Yohei; Suzuki, Tomonari; Adachi, Jun-Ichi; Mishima, Kazuhiko; Sasaki, Atsushi; Nishikawa, Ryo; Bewerunge-Hudler, Melanie; Ryzhova, Marina; Absalyamova, Oksana; Golanov, Andrey; Sinn, Peter; Platten, Michael; Jungk, Christine; Winkler, Frank; Wick, Antje; Hänggi, Daniel; Unterberg, Andreas; Pfister, Stefan M; Jones, David T W; van den Bent, Martin; Hegi, Monika; French, Pim; Baumert, Brigitta G; Stupp, Roger; Gorlia, Thierry; Weller, Michael; Capper, David; Korshunov, Andrey; Herold-Mende, Christel; Wick, Wolfgang; Louis, David N; von Deimling, Andreas

2018-04-23

According to the 2016 World Health Organization Classification of Tumors of the Central Nervous System (2016 CNS WHO), IDH-mutant astrocytic gliomas comprised WHO grade II diffuse astrocytoma, IDH-mutant (AII IDHmut ), WHO grade III anaplastic astrocytoma, IDH-mutant (AAIII IDHmut ), and WHO grade IV glioblastoma, IDH-mutant (GBM IDHmut ). Notably, IDH gene status has been made the major criterion for classification while the manner of grading has remained unchanged: it is based on histological criteria that arose from studies which antedated knowledge of the importance of IDH status in diffuse astrocytic tumor prognostic assessment. Several studies have now demonstrated that the anticipated differences in survival between the newly defined AII IDHmut and AAIII IDHmut have lost their significance. In contrast, GBM IDHmut still exhibits a significantly worse outcome than its lower grade IDH-mutant counterparts. To address the problem of establishing prognostically significant grading for IDH-mutant astrocytic gliomas in the IDH era, we undertook a comprehensive study that included assessment of histological and genetic approaches to prognosis in these tumors. A discovery cohort of 211 IDH-mutant astrocytic gliomas with an extended observation was subjected to histological review, image analysis, and DNA methylation studies. Tumor group-specific methylation profiles and copy number variation (CNV) profiles were established for all gliomas. Algorithms for automated CNV analysis were developed. All tumors exhibiting 1p/19q codeletion were excluded from the series. We developed algorithms for grading, based on molecular, morphological and clinical data. Performance of these algorithms was compared with that of WHO grading. Three independent cohorts of 108, 154 and 224 IDH-mutant astrocytic gliomas were used to validate this approach. In the discovery cohort several molecular and clinical parameters were of prognostic relevance. Most relevant for overall survival (OS

Expression of CALR mutants causes mpl-dependent thrombocytosis in zebrafish.

PubMed

Lim, K-H; Chang, Y-C; Chiang, Y-H; Lin, H-C; Chang, C-Y; Lin, C-S; Huang, L; Wang, W-T; Gon-Shen Chen, C; Chou, W-C; Kuo, Y-Y

2016-10-07

CALR mutations are identified in about 30% of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs) including essential thrombocythemia (ET) and primary myelofibrosis. Although the molecular pathogenesis of CALR mutations leading to MPNs has been studied using in vitro cell lines models, how mutant CALR may affect developmental hematopoiesis remains unknown. Here we took advantage of the zebrafish model to examine the effects of mutant CALR on early hematopoiesis and model human CALR-mutated MPNs. We identified three zebrafish genes orthologous to human CALR, referred to as calr, calr3a and calr3b. The expression of CALR-del52 and CALR-ins5 mutants caused an increase in the hematopoietic stem/progenitor cells followed by thrombocytosis without affecting normal angiogenesis. The expression of CALR mutants also perturbed early developmental hematopoiesis in zebrafish. Importantly, morpholino knockdown of mpl but not epor or csf3r could significantly attenuate the effects of mutant CALR. Furthermore, the expression of mutant CALR caused jak-stat signaling activation in zebrafish that could be blocked by JAK inhibitors (ruxolitinib and fedratinib). These findings showed that mutant CALR activates jak-stat signaling through an mpl-dependent mechanism to mediate pathogenic thrombopoiesis in zebrafish, and illustrated that the signaling machinery related to mutant CALR tumorigenesis are conserved between human and zebrafish.

Formulation of a mmaA4 Gene Deletion Mutant of Mycobacterium bovis BCG in Cationic Liposomes Significantly Enhances Protection against Tuberculosis

PubMed Central

Derrick, Steven C.; Dao, Dee; Yang, Amy; Kolibab, Kris; Jacobs, William R.; Morris, Sheldon L.

2012-01-01

A new vaccination strategy is urgently needed for improved control of the global tuberculosis (TB) epidemic. Using a mouse aerosol Mycobacterium tuberculosis challenge model, we investigated the protective efficacy of a mmaA4 gene deletion mutant of Mycobacterium bovis BCG (ΔmmaA4BCG) formulated in dimethyl dioctadecyl ammonium bromide (DDA) – D(+) trehalose 6,6 dibenenate (TDB) (DDA/TDB) adjuvant. In previous studies, deletion of the mmaA4 gene was shown to reduce the suppression of IL-12 production often seen after mycobacterial infections. While the non-adjuvanted ΔmmaA4BCG strain did not protect mice substantially better than conventional BCG against a tuberculous challenge in four protection experiments, the protective responses induced by the ΔmmaA4BCG vaccine formulated in DDA/TDB adjuvant was consistently increased relative to nonadjuvanted BCG controls. Furthermore, the ΔmmaA4BCG-DDA/TDB vaccine induced significantly higher frequencies of multifunctional (MFT) CD4 T cells expressing both IFNγ and TNFα (double positive) or IFNγ, TNFα and IL-2 (triple positive) than CD4 T cells derived from mice vaccinated with BCG. These MFT cells were characterized by having higher IFNγ and TNFα median fluorescence intensity (MFI) values than monofunctional CD4 T cells. Interestingly, both BCG/adjuvant and ΔmmaA4BCG/adjuvant formulations induced significantly higher frequencies of CD4 T cells expressing TNFα and IL-2 than nonadjuvanted BCG or ΔmmaA4BCG vaccines indicating that BCG/adjuvant mixtures may be more effective at inducing central memory T cells. Importantly, when either conventional BCG or the mutant were formulated in adjuvant and administered to SCID mice or immunocompromised mice depleted of IFNγ, significantly lower vaccine-derived mycobacterial CFU were detected relative to immunodeficient mice injected with non-adjuvanted BCG. Overall, these data suggest that immunization with the ΔmmaA4BCG/adjuvant formulation may be an effective, safe

Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis.

PubMed

Novak, K D; Peterson, M D; Reedy, M C; Titus, M A

1995-12-01

The functional relationship between three Dictyostelium myosin Is, myoA, myoB, and myoC, has been examined through the creation of double mutants. Two double mutants, myoA-/B- and myoB-/C-, exhibit similar conditional defects in fluid-phase pinocytosis. Double mutants grown in suspension culture are significantly impaired in their ability to take in nutrients from the medium, whereas they are almost indistinguishable from wild-type and single mutant strains when grown on a surface. The double mutants are also found to internalize gp126, a 116-kD membrane protein, at a slower rate than either the wild-type or single mutant cells. Ultrastructural analysis reveals that both double mutants possess numerous small vesicles, in contrast to the wild-type or myosin I single mutants that exhibit several large, clear vacuoles. The alterations in fluid and membrane internalization in the suspension-grown double mutants, coupled with the altered vesicular profile, suggest that these cells may be compromised during the early stages of pinocytosis, a process that has been proposed to occur via actin-based cytoskeletal rearrangements. Scanning electron microscopy and rhodamine-phalloidin staining indicates that the myosin I double mutants appear to extend a larger number of actin-filled structures, such as filopodia and crowns, than wild-type cells. Rhodamine-phalloidin staining of the F-actin cytoskeleton of these suspension-grown cells also reveals that the double mutant cells are delayed in the rearrangement of cortical actin-rich structures upon adhesion to a substrate. We propose that myoA, myoB, and myoC play roles in controlling F-actin filled membrane projections that are required for pinosome internalization in suspension.

Genetic interactions of the unfinished flower development (ufd) mutant support a significant role of the tomato UFD gene in regulating floral organogenesis.

PubMed

Poyatos-Pertíñez, Sandra; Quinet, Muriel; Ortíz-Atienza, Ana; Bretones, Sandra; Yuste-Lisbona, Fernando J; Lozano, Rafael

2016-09-01

Genetic interactions of UFD gene support its specific function during reproductive development of tomato; in this process, UFD could play a pivotal role between inflorescence architecture and flower initiation genes. Tomato (Solanum lycopersicum L.) is a major vegetable crop that also constitutes a model species for the study of plant developmental processes. To gain insight into the control of flowering and floral development, a novel tomato mutant, unfinished flower development (ufd), whose inflorescence and flowers were unable to complete their normal development was characterized using double mutant and gene expression analyses. Genetic interactions of ufd with mutations affecting inflorescence fate (uniflora, jointless and single flower truss) were additive and resulted in double mutants displaying the inflorescence structure of the non-ufd parental mutant and the flower phenotype of the ufd mutant. In addition, ufd mutation promotes an earlier inflorescence meristem termination. Taken together, both results indicated that UFD is not involved in the maintenance of inflorescence meristem identity, although it could participate in the regulatory system that modulates the rate of meristem maturation. Regarding the floral meristem identity, the falsiflora mutation was epistatic to the ufd mutation even though FALSIFLORA was upregulated in ufd inflorescences. In terms of floral organ identity, the ufd mutation was epistatic to macrocalyx, and MACROCALYX expression was differently regulated depending on the inflorescence developmental stage. These results suggest that the UFD gene may play a pivotal role between the genes required for flowering initiation and inflorescence development (such as UNIFLORA, FALSIFLORA, JOINTLESS and SINGLE FLOWER TRUSS) and those required for further floral organ development such as the floral organ identity genes.

PDGFRA-mutant syndrome.

PubMed

Ricci, Riccardo; Martini, Maurizio; Cenci, Tonia; Carbone, Arnaldo; Lanza, Paola; Biondi, Alberto; Rindi, Guido; Cassano, Alessandra; Larghi, Alberto; Persiani, Roberto; Larocca, Luigi M

2015-07-01

Germline PDGFRA mutations cause multiple heterogeneous gastrointestinal mesenchymal tumors. In its familial form this disease, which was formerly termed intestinal neurofibromatosis/neurofibromatosis 3b (INF/NF3b), has been included among familial gastrointestinal stromal tumors (GISTs) because of its genotype, described when GIST was the only known PDGFRA-mutant gastrointestinal tumor. Shortly afterwards, however, inflammatory fibroid polyps also revealed PDGFRA mutations. Subsequently, gastrointestinal CD34+ 'fibrous tumors' of uncertain classification were described in a germline PDGFRA-mutant context. Our aim was to characterize the syndrome produced by germline PDGFRA mutations and establish diagnostic criteria and management strategies for this hitherto puzzling disease. We studied a kindred displaying multiple gastrointestinal mesenchymal tumors, comparing it with published families/individuals with possible analogous conditions. We identified a novel inherited PDGFRA mutation (P653L), constituting the third reported example of familial PDGFRA mutation. In adult mutants we detected inflammatory fibroid polyps, gastric GISTs and gastrointestinal fibrous tumors of uncertain nosology. We demonstrate that the syndrome formerly defined as INF/NF3b (exemplified by the family reported herein) is simplistically considered a form of familial GIST, because inflammatory fibroid polyps often prevail. Fibrous tumors appear variants of inflammatory fibroid polyps. 'INF/NF3b' and 'familial GIST' are misleading terms which we propose changing to 'PDGFRA-mutant syndrome'. In this condition, unlike KIT-dependent familial GIST syndromes, if present, GISTs are stomach-restricted and diffuse Cajal cell hyperplasia is not observed. This restriction of GISTs to the stomach in PDGFRA-mutant syndrome: (i) focuses oncological concern on gastric masses, as inflammatory fibroid polyps are benign; (ii) supports a selective role of gastric environment for PDGFRA mutations to elicit GISTs

Aptamer-conjugated PEGylated quantum dots targeting epidermal growth factor receptor variant III for fluorescence imaging of glioma.

PubMed

Tang, Jiaze; Huang, Ning; Zhang, Xiang; Zhou, Tao; Tan, Ying; Pi, Jiangli; Pi, Li; Cheng, Si; Zheng, Huzhi; Cheng, Yuan

2017-01-01

The extent of resection is a significant prognostic factor in glioma patients. However, the maximum safe resection level is difficult to determine due to the inherent infiltrative character of tumors. Recently, fluorescence-guided surgery has emerged as a new technique that allows safe resection of glioma. In this study, we constructed a new kind of quantum dot (QD)-labeled aptamer (QD-Apt) nanoprobe by conjugating aptamer 32 (A32) to the QDs surface, which can specially bind to the tumors. A32 is a single-stranded DNA capable of binding to the epidermal growth factor receptor variant III (EGFRvIII) specially distributed on the surface of glioma cells. To detect the expression of EGFRvIII in human brain tissues, 120 specimens, including 110 glioma tissues and 10 normal brain tissues, were examined by immunohistochemistry, and the results showed that the rate of positive expression of EGFRvIII in the glioma tissues was 41.82%, and 0.00% in normal brain tissues. Besides, the physiochemical properties of QD-Apt nanoparticles (NPs) were thoroughly characterized. Biocompatibility of the NPs was evaluated, and the results suggested that the QD-Apt was nontoxic in vivo and vitro. Furthermore, the use of the QD-Apt in labeling glioma cell lines and human brain glioma tissues, and target gliomas in situ was also investigated. We found that not only could QD-Apt specially bind to the U87-EGFRvIII glioma cells but also bind to human glioma tissues in vitro. Fluorescence imaging in vivo with orthotopic glioma model mice bearing U87-EGFRvIII showed that QD-Apt could penetrate the blood-brain barrier and then selectively accumulate in the tumors through binding to EGFRvIII, and consequently, generate a strong fluorescence, which contributed to the margins of gliomas that were visualized clearly, and thus, help the surgeons realize the maximum safe resection of glioma. In addition, QD-Apt can also be applied in preoperative diagnosis and postoperative examination of glioma

Aptamer-conjugated PEGylated quantum dots targeting epidermal growth factor receptor variant III for fluorescence imaging of glioma

PubMed Central

Tang, Jiaze; Huang, Ning; Zhang, Xiang; Zhou, Tao; Tan, Ying; Pi, Jiangli; Pi, Li; Cheng, Si; Zheng, Huzhi; Cheng, Yuan

2017-01-01

The extent of resection is a significant prognostic factor in glioma patients. However, the maximum safe resection level is difficult to determine due to the inherent infiltrative character of tumors. Recently, fluorescence-guided surgery has emerged as a new technique that allows safe resection of glioma. In this study, we constructed a new kind of quantum dot (QD)-labeled aptamer (QD-Apt) nanoprobe by conjugating aptamer 32 (A32) to the QDs surface, which can specially bind to the tumors. A32 is a single-stranded DNA capable of binding to the epidermal growth factor receptor variant III (EGFRvIII) specially distributed on the surface of glioma cells. To detect the expression of EGFRvIII in human brain tissues, 120 specimens, including 110 glioma tissues and 10 normal brain tissues, were examined by immunohistochemistry, and the results showed that the rate of positive expression of EGFRvIII in the glioma tissues was 41.82%, and 0.00% in normal brain tissues. Besides, the physiochemical properties of QD-Apt nanoparticles (NPs) were thoroughly characterized. Biocompatibility of the NPs was evaluated, and the results suggested that the QD-Apt was nontoxic in vivo and vitro. Furthermore, the use of the QD-Apt in labeling glioma cell lines and human brain glioma tissues, and target gliomas in situ was also investigated. We found that not only could QD-Apt specially bind to the U87-EGFRvIII glioma cells but also bind to human glioma tissues in vitro. Fluorescence imaging in vivo with orthotopic glioma model mice bearing U87-EGFRvIII showed that QD-Apt could penetrate the blood–brain barrier and then selectively accumulate in the tumors through binding to EGFRvIII, and consequently, generate a strong fluorescence, which contributed to the margins of gliomas that were visualized clearly, and thus, help the surgeons realize the maximum safe resection of glioma. In addition, QD-Apt can also be applied in preoperative diagnosis and postoperative examination of glioma

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

PubMed Central

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

2016-01-01

Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. 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, and STM3734 are needed for Salmonella to 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

Active site-directed double mutants of dihydrofolate reductase.

PubMed

Ercikan-Abali, E A; Mineishi, S; Tong, Y; Nakahara, S; Waltham, M C; Banerjee, D; Chen, W; Sadelain, M; Bertino, J R

1996-09-15

Variants of dihydrofolate reductase (DHFR), which confer resistance to antifolates, are used as dominant selectable markers in vitro and in vivo and may be useful in the context of gene therapy. To identify improved mutant human DHFRs with increased catalytic efficiency and decreased binding to methotrexate, we constructed by site-directed mutagenesis four variants with substitutions at both Leu22 and Phe31 (i.e., Phe22-Ser31, Tyr22-Ser31, Phe22-Gly31, and Tyr22-Gly31). Antifolate resistance has been observed previously when individual changes are made at these active-site residues. Substrate and antifolate binding properties of these "double" mutants revealed that each have greatly diminished affinity for antifolates (> 10,000-fold) yet only slightly reduced substrate affinity. Comparison of in vitro measured properties with those of single-residue variants indicates that double mutants are indeed significantly superior. This was verified for one of the double mutants that provided high-level methotrexate resistance following retrovirus-mediated gene transfer in NIH3T3 cells.

Physiology and pathogenicity of cpdB deleted mutant of avian pathogenic Escherichia coli.

PubMed

Liu, Huifang; Chen, Liping; Si, Wei; Wang, Chunlai; Zhu, Fangna; Li, Guangxing; Liu, Siguo

2017-04-01

Avian colibacillosis is one of the most common infectious diseases caused partially or entirely by avian pathogenic Escherichia coli (APEC) in birds. In addition to spontaneous infection, APEC can also cause secondary infections that result in greater severity of illness and greater losses to the poultry industry. In order to assess the role of 2', 3'-cyclic phosphodiesterase (cpdB) in APEC on disease physiology and pathogenicity, an avian pathogenic Escherichia coli-34 (APEC-34) cpdB mutant was obtained using the Red system. The cpdB mutant grew at a slower rate than the natural strain APEC-34. Scanning electron microscopy (SEM) indicated that the bacteria of the cpdB mutant were significantly longer than the bacteria observed in the natural strain (P<0.01), and that the width of the cpdB mutant was significantly smaller than its natural counterpart (P<0.01). In order to evaluate the role of cpdB in APEC in the colonization of internal organs (lung, liver and spleen) in poultry, seven-day-old SPF chicks were infected with 10 9 CFU/chick of the cpdB mutant or the natural strain. No colonizations of cpdB mutants were observed in the internal organs 10days following the infection, though numerous natural strains were observed at 20days following infection. Additionally, the relative expression of division protein ftsZ, outer membrane protein A ompA, ferric uptake regulator fur and tryptophanase tnaA genes in the mutant strain were all significantly lower than in the natural strain (P<0.05 or P<0.01). These results suggested that cpdB is involved in the long-term colonization of APEC in the internal organs of the test subjects. The deletion of the cpdB gene also significantly affected the APEC growth and morphology. Copyright © 2016. Published by Elsevier Ltd.

Identification and Characterization of an Arabidopsis thaliana Mutant lbt With High Tolerance to Boron Deficiency

PubMed Central

Huai, Zexun; Peng, Lishun; Wang, Sheliang; Zhao, Hua; Shi, Lei; Xu, Fangsen

2018-01-01

Boron (B) is an essential micronutrient of plants. In the present study, we characterized an Arabidopsis mutant lbt with significant low-boron tolerance that was identified based on our previous mapping of QTL for B efficiency in Arabidopsis. Multiple nutrient-deficiency analyses point out that lbt mutant is insensitive to only B-limitation stress. Compared with wild-type Col-0, the fresh weight, leaf area, root length and root elongation rate of lbt mutant were significantly improved under B deficiency during vegetative growth. lbt mutant also showed the improvements in plant height, branches and inflorescences compared with Col-0 during the reproductive stage under B limitation. Ultrastructure analysis of the leaves showed that starch accumulation in lbt mutant was significantly diminished compared with Col-0. Furthermore, there were no significant differences in the expression of transporter-related genes and B concentrations between Col-0 and lbt mutant under both normal B and low-B conditions. These results suggest that lbt mutant has a lower B demand than Col-0. Genetic analysis suggests that the low-B tolerant phenotype of lbt mutant is under the control of a monogenic recessive gene. Based on the high-density SNP linkage genetic map, only one QTL for low-B tolerance was mapped on chromosome 4 between 10.4 and 14.8 Mb. No any reported B-relative genes exist in the QTL interval, suggesting that a gene with unknown function controls the tolerance of lbt to B limitation. Taken together, lbt is a low-B tolerant mutant that does not depend on the uptake or transport of B and is controlled by a monogenic recessive gene mapped on chromosome 4, and cloning and functional analysis of LBT gene are expected to reveal novel mechanisms for plant resistance to B deficiency.

Working-for-Food Behaviors: A Preclinical Study in Prader-Willi Mutant Mice.

PubMed

Lassi, Glenda; Maggi, Silvia; Balzani, Edoardo; Cosentini, Ilaria; Garcia-Garcia, Celina; Tucci, Valter

2016-11-01

Abnormal feeding behavior is one of the main symptoms of Prader-Willi syndrome (PWS). By studying a PWS mouse mutant line, which carries a paternally inherited deletion of the small nucleolar RNA 116 (Snord116), we observed significant changes in working-for-food behavioral responses at various timescales. In particular, we report that PWS mutant mice show a significant delay compared to wild-type littermate controls in responding to both hour-scale and seconds-to-minutes-scale time intervals. This timing shift in mutant mice is associated with better performance in the working-for-food task, and results in better decision making in these mutant mice. The results of our study reveal a novel aspect of the organization of feeding behavior, and advance the understanding of the interplay between the metabolic functions and cognitive mechanisms of PWS. Copyright © 2016 by the Genetics Society of America.

Phosphorodiamidate morpholino oligomers suppress mutant huntingtin expression and attenuate neurotoxicity

PubMed Central

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.

2014-01-01

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 HdhQ7/Q150 knock-in HD mice. These data demonstrate the potential of PMOs as an approach to suppressing the expression of mutant HTT. PMID:25035419

Creation, characterization and utilization of Cryptococcus neoformans mutants sensitive to micafungin.

PubMed

Toh-E, Akio; Ohkusu, Misako; Shimizu, Kiminori; Yamaguchi, Masashi; Ishiwada, Naruhiko; Watanabe, Akira; Kamei, Katsuhiko

2017-12-01

We constructed deletion mutants of Cryptococcus neoformans var neoformans (serotype D) genes encoding late ergosterol biosynthetic pathway enzymes and found that the mutations enhanced susceptibility to various drugs including micafungin, one of the echinocandins, to which wild-type Cryptococcus strains show no susceptibility. Furthermore, through isolation of a mutant resistant to micafungin from a micafungin-sensitive erg mutant and genetic analysis of it, we found that the responsible mutation occurred in the hotspot 2 of FKS1 encoding β-1, 3-glucan synthase, indicating that micafungin inhibited the growth of the erg mutant via inhibiting Fks1 activity. Addition of ergosterol to the culture of the erg mutants recovered the resistance to micafungin, suggesting that the presence of ergosterol in membrane inhibits the accession of micafungin to its target. We found that a loss of one of genes encoding subunits of v-ATPase, VPH1, made Cryptococcus cells sensitive to micafungin. Our observation that the erg2 vph1 double mutant was more sensitive to micafungin than either single mutant suggests that these two genes act differently in becoming resistant to micafungin. The erg mutants allowed us to study the physiological significance of β-1, 3-glucan synthesis in C. neoformans; the inhibition of β-1, 3-glucan synthesis induced cell death and changes in cellular morphology. By observing the erg mutant cells recovering from the growth inhibition imposed by micafungin, we recognized β-1, 3-glucan synthesis would suppress filamentous growth in C. neoformans.

Mitochondrial dysfunction precedes neurodegeneration in mahogunin (Mgrn1) mutant mice

PubMed Central

Sun, Kaihua; Johnson, Brian S.; Gunn, Teresa M.

2007-01-01

Oxidative stress, ubiquitination defects and mitochondrial dysfunction are commonly associated with neurodegeneration. Mice lacking mahogunin ring finger-1 (MGRN1) or attractin (ATRN) develop age-dependent spongiform neurodegeneration through an unknown mechanism. It has been suggested that they act in a common pathway. As MGRN1 is an E3 ubiquitin ligase, proteomic analysis of Mgrn1 mutant and control brains was performed to explore the hypothesis that loss of MGRN1 causes neurodegeneration via accumulation of its substrates. Many mitochondrial proteins were reduced in Mgrn1 mutants. Subsequent assays confirmed significantly reduced mitochondrial complex IV expression and activity as well as increased oxidative stress in mutant brains. Mitochondrial dysfunction was obvious many months before onset of vacuolation, implicating this as a causative factor. Compatible with the hypothesis that ATRN and MGRN1 act in the same pathway, mitochondrial dysfunction and increased oxidative stress were also observed in the brains of Atrn mutants. Our results suggest that the study of Mgrn1 and Atrn mutant mice will provide insight into a causative molecular mechanism common to many neurodegenerative disorders. PMID:17720281

Characterization of Staphylococcus aureus mutants expressing reduced susceptibility to common house-cleaners

PubMed Central

Davis, A.O.; O’Leary, J.O.; Muthaiyan, A.; Langevin, M.J.; Delgado, A.; Abalos, A.T.; Fajardo, A.R.; Marek, J.; Wilkinson, B.J.; Gustafson, J.E.

2013-01-01

Aims To characterize mutants of Staphylococcus aureus expressing reduced susceptibility to house cleaners (HC), assess the impact of the alternative sigma factor SigB on HC susceptibility, and determine the MIC of clinical methicillin-resistant S. aureus (MRSA) to a HC. Methods and Results Susceptibility to HC, HC components, H2O2, vancomycin and oxacillin and physiological parameters were determined for HC-reduced susceptibility (HCRS) mutants, parent strain COL and COLsigB::kan. HCRS mutants selected with three HC expressed reduced susceptibility to multiple HC, HC components, H2O2 and vancomycin. Two unique HCRS mutants also lost the methicillin resistance determinant. In addition, all HCRS mutants exhibited better growth at two temperatures, and one HCRS mutant expressed reduced carotenoid production. COLsigB::kan demonstrated increased susceptibility to all HC and many HC components. sigB operon mutations were not detected in one HCRS mutant background. Of 76 clinical MRSA, 20 exhibited reduced susceptibility to a HC. Conclusions HCRS mutants demonstrate altered susceptibility to multiple antimicrobials. While sigB is required for full HC resistance, one HCRS mechanism does not involve sigB operon mutations. Clinical MRSA expressing reduced susceptibility to a common HC were detected. Significance and Impact of the Study This study suggests that HCRS mutants are not protected against, nor selected by, practical HC concentrations. PMID:15659191

[Construction and characterization of a gspL mutant of avian pathogenic Escherichia coli].

PubMed

Fan, Guobo; Han, Yue; Zhang, Yuxi; Han, Xiangan; Wang, Shaohui; Bai, Hao; Meng, Qingmei; Qi, Kezong; Ding, Chan; Yu, Shengqing

2015-01-04

To study the role of gspL gene in avian pathogenic Escherichia coli. The gspL mutant of Avian pathogenic Escherichia coli (APEC) was constructed by homologous recombination assay. The growth characteristics, the ability of adhesion and invasion to DF1 cells, the virulence genes transcription level and median lethal dose (LD50) were analyzed between the gspL mutant strain and the wild strain. Compared with the wild strain, the mutant strain had no significant difference in the growth status. However, its ability of adhesion and invasion was significantly lower. The transcription of genes pfs, fyuA, iss and vat increased obviously, the tsh decreased and the transcription level of luxS, ibeA, stx2f and ompA had no significant change. LD50 showed that the gspL mutant strain had 12-fold increase in virulence. The deletion of gspL gene could abate the ability of adhesion and invasion, regulate and control some virulence gene transcription level, enhance the virulence of APEC. The results show that the gspL gene play roles in pathogenicity of APEC.

Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3

PubMed Central

Meng, He; Zhang, Xiaojie; Yu, Genggeng; Lee, Soo Jung; Chen, Y. Eugene; Prudovsky, Igor; Wang, Michael M.

2012-01-01

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the best understood cause of dominantly inherited stroke and results from NOTCH3 mutations that lead to NOTCH3 protein accumulation and selective arterial smooth muscle degeneration. Previous studies show that NOTCH3 protein forms multimers. Here, we investigate protein interactions between NOTCH3 and other vascular Notch isoforms and characterize the effects of elevated NOTCH3 on smooth muscle gene regulation. We demonstrate that NOTCH3 forms heterodimers with NOTCH1, NOTCH3, and NOTCH4. R90C and C49Y mutant NOTCH3 form complexes which are more resistant to detergents than wild type NOTCH3 complexes. Using quantitative NOTCH3-luciferase clearance assays, we found significant inhibition of mutant NOTCH3 clearance. In coculture assays of NOTCH function, overexpressed wild type and mutant NOTCH3 significantly repressed NOTCH-regulated smooth muscle transcripts and potently impaired the activity of three independent smooth muscle promoters. Wildtype and R90C recombinant NOTCH3 proteins applied to cell cultures also blocked canonical Notch fuction. We conclude that CADASIL mutants of NOTCH3 complex with NOTCH1, 3, and 4, slow NOTCH3 clearance, and that overexpressed wild type and mutant NOTCH3 protein interfere with key NOTCH-mediated functions in smooth muscle cells. PMID:23028706

Pretreatment Dynamic Susceptibility Contrast MRI Perfusion in Glioblastoma: Prediction of EGFR Gene Amplification.

PubMed

Gupta, A; Young, R J; Shah, A D; Schweitzer, A D; Graber, J J; Shi, W; Zhang, Z; Huse, J; Omuro, A M P

2015-06-01

Molecular and genetic testing is becoming increasingly relevant in GBM. We sought to determine whether dynamic susceptibility contrast (DSC) magnetic resonance imaging (MRI) perfusion imaging could predict EGFR-defined subtypes of GBM. We retrospectively identified 106 consecutive glioblastoma (GBM) patients with known EGFR gene amplification, and a subset of 65 patients who also had known EGFRvIII gene mutation status. All patients underwent T2* DSC MRI perfusion. DSC perfusion maps and T2* signal intensity time curves were evaluated, and the following measures of tumor perfusion were recorded: (1) maximum relative cerebral blood volume (rCBV), (2) relative peak height (rPH), and (3) percent signal recovery (PSR). The imaging metrics were correlated to EGFR gene amplification and EGFRvIII mutation status using univariate analyses. EGFR amplification was present in 44 (41.5 %) subjects and absent in 62 (58.5 %). Among the 65 subjects who had undergone EGFRvIII mutation transcript analysis, 18 subjects (27.7 %) tested positive for the EGFRvIII mutation, whereas 47 (72.3 %) did not. Higher median rCBV (3.31 versus 2.62, p = 0.01) and lower PSR (0.70 versus 0.78, p = 0.03) were associated with high levels of EGFR amplification. Higher median rPH (3.68 versus 2.76, p = 0.03) was associated with EGFRvIII mutation. DSC MRI perfusion may have a role in identifying patients with EGFR gene amplification and EGFRvIII gene mutation status, potential targets for individualized treatment protocols. Our results raise the need for further investigation for imaging biomarkers of genetically unique GBM subtypes.

Defeat mutant KRAS with synthetic lethality

PubMed Central

Pang, Xiufeng; Liu, Mingyao

2017-01-01

ABSTRACT Ras proteins are considered as the founding members of a large superfamily of small GTPases that control fundamental cellular functions. Mutationally activated RAS genes were discovered in human cancer cells more than 3 decades ago, but intensive efforts on Ras structure, biochemistry, function and signaling continue even now. Because mutant Ras proteins are inherently difficult to inhibit and have yet been therapeutically conquered, it was designated as “the Everest of oncogenes” in the cancer genome landscape, further promoting a “renaissance” in RAS research. Different paths to directly or indirectly targeting mutant Ras signaling are currently under investigation in the hope of finding an efficacious regimen. Inhibitors directly binding to KRASG12C to block its downstream signaling have been revealed, supporting the notion of Ras' druggability. An alternative indirect approach by targeting synthetic lethal interactors of mutant RAS is underway. We recently employed a synthetic lethal drug screen plus a combinatorial strategy using a panel of clinical agents and discovered that KRAS-mutant cancers were fragile to the combined inhibition of polo-like kinase 1 (Plk1) and RhoA/Rho kinase (ROCK). The combined regimen of BI-2536 (a Plk1 inhibitor) and fasudil (a ROCK inhibitor) promoted a significant inhibition of patient-derived lung cancer xenografts and prolonged the survival of LSL-KRASG12D mice. In this commentary, we will summarize the state-of-the art for the direction of synthetic lethality, and also speculate on the future development of this approach. PMID:27463838

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

Invert biopanning: A novel method for efficient and rapid isolation of scFvs by phage display technology.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Veisi, Kamal; Tanomand, Asghar; Akbari, Bahman

2016-11-01

Phage display is a prominent screening technique for development of novel high affinity antibodies against almost any antigen. However, removing false positive clones in screening process remains a challenge. The aim of this study was to develop an efficient and rapid method for isolation of high affinity scFvs by removing NSBs without losing rare specific clones. Therefore, a novel two rounds strategy called invert biopanning was developed for isolating high affinity scFvs against EGFRvIII antigen from human scFv library. The efficiency of invert biopanning method (procedure III) was analyzed by comparing with results of conventional biopanning methods (procedures I and II). According to the results of polyclonal ELISA, the second round of procedure III displayed highest binding affinity against EGFRvIII peptide accompanied by lowest NSB comparing to other two procedures. Several positive clones were identified among output phages of procedure III by monoclonal phage ELISA which displayed high affinity to EGFRvIII antigen. In conclusion, results of our study indicate that invert biopanning is an efficient method for avoiding NSBs and conservation of rare specific clones during screening of a scFv phage library. Novel anti EGFRvIII scFv isolated could be a promising candidate for potential use in treatment of EGFRvIII expressing cancers. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Leptin gene promoter DNA methylation in WNIN obese mutant rats

PubMed Central

2014-01-01

Background Obesity has become an epidemic in worldwide population. Leptin gene defect could be one of the causes for obesity. Two mutant obese rats WNIN/Ob and WNIN/GROb, isolated at National Centre for Laboratory Animal Sciences (NCLAS), Hyderabad, India, were found to be leptin resistant. The present study aims to understand the regulatory mechanisms underlying the resistance by promoter DNA methylation of leptin gene in these mutant obese rats. Methods Male obese mutant homozygous, carrier and heterozygous rats of WNIN/Ob and WNIN/GROb strain of 6 months old were studied to check the leptin gene expression (RT-PCR) and promoter DNA methylation (MassARRAY Compact system, SEQUENOM) of leptin gene by invivo and insilico approach. Results Homozygous WNIN/Ob and WNIN/GROb showed significantly higher leptin gene expression compared to carrier and lean counterparts. Leptin gene promoter DNA sequence region was analyzed ranging from transcription start site (TSS) to-550 bp length and found four CpGs in this sequence among them only three CpG loci (-309, -481, -502) were methylated in these WNIN mutant rat phenotypes. Conclusion The increased percentage of methylation in WNIN mutant lean and carrier phenotypes is positively correlated with transcription levels. Thus genetic variation may have effect on methylation percentages and subsequently on the regulation of leptin gene expression which may lead to obesity in these obese mutant rat strains. PMID:24495350

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.

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

Interactions of Nitrate and CO2 Enrichment on Growth, Carbohydrates, and Rubisco in Arabidopsis Starch Mutants. Significance of Starch and Hexose1

PubMed Central

Sun, Jindong; Gibson, Kelly M.; Kiirats, Olavi; Okita, Thomas W.; Edwards, Gerald E.

2002-01-01

Wild-type (wt) Arabidopsis plants, the starch-deficient mutant TL46, and the near-starchless mutant TL25 were grown in hydroponics under two levels of nitrate, 0.2 versus 6 mm, and two levels of CO2, 35 versus 100 Pa. Growth (fresh weight and leaf area basis) was highest in wt plants, lower in TL46, and much lower in TL25 plants under a given treatment. It is surprising that the inability to synthesize starch restricted leaf area development under both low N (NL) and high N (NH). For each genotype, the order of greatest growth among the four treatments was high CO2/NH > low CO2/NH, > high CO2/NL, which was similar to low CO2/NL. Under high CO2/NL, wt and TL46 plants retained considerable starch in leaves at the end of the night period, and TL25 accumulated large amounts of soluble sugars, indicative of N-limited restraints on utilization of photosynthates. The lowest ribulose-1,5-bisphosphate carboxylase/oxygenase per leaf area was in plants grown under high CO2/NL. When N supply is limited, the increase in soluble sugars, particularly in the starch mutants, apparently accentuates the feedback and down-regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase, resulting in greater reduction of growth. With an adequate supply of N, growth is limited in the starch mutants due to insufficient carbohydrate reserves during the dark period. A combination of limited N and a limited capacity to synthesize starch, which restrict the capacity to use photosynthate, and high CO2, which increases the potential to produce photosynthate, provides conditions for strong down-regulation of photosynthesis. PMID:12428022

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.

Control of glioblastoma tumorigenesis by feed-forward cytokine signaling

PubMed Central

Jahani-Asl, Arezu; Yin, Hang; Soleimani, Vahab D; Haque, Takrima; Luchman, H Artee; Chang, Natasha C; Sincennes, Marie-Claude; Puram, Sidharth V; Scott, Andrew M; Lorimer, Ian A J; Perkins, Theodore J; Ligon, Keith L; Weiss, Samuel; Rudnicki, Michael A; Bonni, Azad

2016-01-01

EGFRvIII-STAT3 signaling is important in glioblastoma pathogenesis. Here, we identified the cytokine receptor OSMR as a direct target gene of the transcription factor STAT3 in mouse astrocytes and human brain tumor stem cells (BTSCs). We found that OSMR functioned as an essential co-receptor for EGFRvIII. OSMR formed a physical complex with EGFRvIII, and depletion of OSMR impaired EGFRvIII-STAT3 signaling. Conversely, pharmacological inhibition of EGFRvIII phosphorylation inhibited the EGFRvIII-OSMR interaction and activation of STAT3. EGFRvIII-OSMR signaling in tumors operated constitutively, whereas EGFR-OSMR signaling in nontumor cells was synergistically activated by the ligands EGF and OSM. Finally, knockdown of OSMR strongly suppressed cell proliferation and tumor growth of mouse glioblastoma cells and human BTSC xenografts in mice, and prolonged the lifespan of those mice. Our findings identify OSMR as a critical regulator of glioblastoma tumor growth that orchestrates a feed-forward signaling mechanism with EGFRvIII and STAT3 to drive tumorigenesis. PMID:27110918

Remote intracranial recurrence of IDH mutant gliomas is associated with TP53 mutations and an 8q gain

PubMed Central

Nakae, Shunsuke; Kato, Takema; Murayama, Kazuhiro; Sasaki, Hikaru; Abe, Masato; Kumon, Masanobu; Kumai, Tadashi; Yamashiro, Kei; Inamasu, Joji; Hasegawa, Mitsuhiro; Kurahashi, Hiroki; Hirose, Yuichi

2017-01-01

Most IDH mutant gliomas harbor either 1p/19q co-deletions or TP53 mutation; 1p/19q co-deleted tumors have significantly better prognoses than tumors harboring TP53 mutations. To investigate the clinical factors that contribute to differences in tumor progression of IDH mutant gliomas, we classified recurrent tumor patterns based on MRI and correlated these patterns with their genomic characterization. Accordingly, in IDH mutant gliomas (N = 66), 1p/19 co-deleted gliomas only recurred locally, whereas TP53 mutant gliomas recurred both locally and in remote intracranial regions. In addition, diffuse tensor imaging suggested that remote intracranial recurrence in the astrocytomas, IDH-mutant with TP53 mutations may occur along major fiber bundles. Remotely recurrent tumors resulted in a higher mortality and significantly harbored an 8q gain; astrocytomas with an 8q gain resulted in significantly shorter overall survival than those without an 8q gain. OncoScan® arrays and next-generation sequencing revealed specific 8q regions (i.e., between 8q22 and 8q24) show a high copy number. In conclusion, only tumors with TP53 mutations showed patterns of remote recurrence in IDH mutant gliomas. Furthermore, an 8q gain was significantly associated with remote intracranial recurrence and can be considered a poor prognostic factor in astrocytomas, IDH-mutant. PMID:29156679

Superior triacylglycerol (TAG) accumulation in starchless mutants of Scenedesmus obliquus: (I) mutant generation and characterization

PubMed Central

2014-01-01

Background Microalgae are a promising platform for producing neutral lipids, to be used in the application for biofuels or commodities in the feed and food industry. A very promising candidate is the oleaginous green microalga Scenedesmus obliquus, because it accumulates up to 45% w/w triacylglycerol (TAG) under nitrogen starvation. Under these conditions, starch is accumulated as well. Starch can amount up to 38% w/w under nitrogen starvation, which is a substantial part of the total carbon captured. When aiming for optimized TAG production, blocking the formation of starch could potentially increase carbon allocation towards TAG. In an attempt to increase TAG content, productivity and yield, starchless mutants of this high potential strain were generated using UV mutagenesis. Previous studies in Chlamydomonas reinhardtii have shown that blocking the starch synthesis yields higher TAG contents, although these TAG contents do not surpass those of oleaginous microalgae yet. So far no starchless mutants in oleaginous green microalgae have been isolated that result in higher TAG productivities. Results Five starchless mutants have been isolated successfully from over 3,500 mutants. The effect of the mutation on biomass and total fatty acid (TFA) and TAG productivity under nitrogen-replete and nitrogen-depleted conditions was studied. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion, reached a TAG content of 49.4% (% of dry weight) and had no substantial change in biomass productivity compared to the wild type. Conclusions The improved S. obliquus TAG production strains are the first starchless mutants in an oleaginous green microalga that show enhanced TAG content under

Photosynthesis, Light Use Efficiency, and Yield of Reduced-Chlorophyll Soybean Mutants in Field Conditions.

PubMed

Slattery, Rebecca A; VanLoocke, Andy; Bernacchi, Carl J; Zhu, Xin-Guang; Ort, Donald R

2017-01-01

Reducing chlorophyll (chl) content may improve the conversion efficiency of absorbed photosynthetically active radiation into biomass and therefore yield in dense monoculture crops by improving light penetration and distribution within the canopy. The effects of reduced chl on leaf and canopy photosynthesis and photosynthetic efficiency were studied in two reportedly robust reduced-chl soybean mutants, Y11y11 and y9y9 , in comparison to the wild-type (WT) "Clark" cultivar. Both mutants were characterized during the 2012 growing season whereas only the Y11y11 mutant was characterized during the 2013 growing season. Chl deficiency led to greater rates of leaf-level photosynthesis per absorbed photon early in the growing season when mutant chl content was ∼35% of the WT, but there was no effect on photosynthesis later in the season when mutant leaf chl approached 50% of the WT. Transient benefits of reduced chl at the leaf level did not translate to improvements in canopy-level processes. Reduced pigmentation in these mutants was linked to lower water use efficiency, which may have dampened any photosynthetic benefits of reduced chl, especially since both growing seasons experienced significant drought conditions. These results, while not confirming our hypothesis or an earlier published study in which the Y11y11 mutant significantly outyielded the WT, do demonstrate that soybean significantly overinvests in chl. Despite a >50% chl reduction, there was little negative impact on biomass accumulation or yield, and the small negative effects present were likely due to pleiotropic effects of the mutation. This outcome points to an opportunity to reinvest nitrogen and energy resources that would otherwise be used in pigment-proteins into increasing biochemical photosynthetic capacity, thereby improving canopy photosynthesis and biomass production.

Photosynthesis, Light Use Efficiency, and Yield of Reduced-Chlorophyll Soybean Mutants in Field Conditions

PubMed Central

Slattery, Rebecca A.; VanLoocke, Andy; Bernacchi, Carl J.; Zhu, Xin-Guang; Ort, Donald R.

2017-01-01

Reducing chlorophyll (chl) content may improve the conversion efficiency of absorbed photosynthetically active radiation into biomass and therefore yield in dense monoculture crops by improving light penetration and distribution within the canopy. The effects of reduced chl on leaf and canopy photosynthesis and photosynthetic efficiency were studied in two reportedly robust reduced-chl soybean mutants, Y11y11 and y9y9, in comparison to the wild-type (WT) “Clark” cultivar. Both mutants were characterized during the 2012 growing season whereas only the Y11y11 mutant was characterized during the 2013 growing season. Chl deficiency led to greater rates of leaf-level photosynthesis per absorbed photon early in the growing season when mutant chl content was ∼35% of the WT, but there was no effect on photosynthesis later in the season when mutant leaf chl approached 50% of the WT. Transient benefits of reduced chl at the leaf level did not translate to improvements in canopy-level processes. Reduced pigmentation in these mutants was linked to lower water use efficiency, which may have dampened any photosynthetic benefits of reduced chl, especially since both growing seasons experienced significant drought conditions. These results, while not confirming our hypothesis or an earlier published study in which the Y11y11 mutant significantly outyielded the WT, do demonstrate that soybean significantly overinvests in chl. Despite a >50% chl reduction, there was little negative impact on biomass accumulation or yield, and the small negative effects present were likely due to pleiotropic effects of the mutation. This outcome points to an opportunity to reinvest nitrogen and energy resources that would otherwise be used in pigment-proteins into increasing biochemical photosynthetic capacity, thereby improving canopy photosynthesis and biomass production. PMID:28458677

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…

Endocochlear potential generation is associated with intercellular communication in the stria vascularis: structural analysis in the viable dominant spotting mouse mutant.

PubMed

Carlisle, L; Steel, K; Forge, A

1990-11-01

Deafness in the viable dominant spotting mouse mutant is due to a primary defect of the stria vascularis which results in absence of the positive endocochlear potential in scala media. Endocochlear potentials were measured and the structure of stria vascularis of mutants with potentials close to zero was compared with that in normal littermate controls by use of morphometric methods. The stria vascularis was significantly thinner in mutants. Marginal cells were not significantly different from controls in terms of volume density or intramembrane particle density but the network density of tight junctions was significantly reduced in the mutants. A virtual absence of gap junctions between basal cells and marginal or intermediate cells was observed, but intramembrane particle density and junctional complexes between adjacent basal cells were not different from controls. The volume density of basal cells was significantly greater in mutants. Intermediate cells accounted for a significantly smaller volume density of the stria vascularis in mutants and had a lower density of intramembrane particles than controls. Melanocytes were not identified in the stria vascularis of mutants. These results suggest that communication between marginal, intermediate and basal cells might be important to the normal function of the stria vascularis.

Gravitropism in lateral roots of Arabidopsis pgm-1 mutants is indistinguishable from that of wild-type

PubMed Central

Bai, Hanwen

2011-01-01

The majority of understanding of root gravity responses comes from the study of primary roots, even though lateral roots make a far greater contribution to root system architecture. The focus of this report is the analysis of gravitropic responses in lateral roots of wild-type background and pgm-1 mutants. Despite the significant reduction in gravitropic response of primary roots of pgm-1 mutants, the lateral roots of this mutant demonstrate wild-type rates of gravitropism, suggesting a significant difference in gravity signal transduction between primary and lateral roots. PMID:21921698

Gravitropism in lateral roots of Arabidopsis pgm-1 mutants is indistinguishable from that of wild-type.

PubMed

Bai, Hanwen; Wolverton, Chris

2011-10-01

The majority of understanding of root gravity responses comes from the study of primary roots, even though lateral roots make a far greater contribution to root system architecture. The focus of this report is the analysis of gravitropic responses in lateral roots of wild-type background and pgm-1 mutants. Despite the significant reduction in gravitropic response of primary roots of pgm-1 mutants, the lateral roots of this mutant demonstrate wild-type rates of gravitropism, suggesting a significant difference in gravity signal transduction between primary and lateral roots.

Brassinosteroid-Insensitive Dwarf Mutants of Arabidopsis Accumulate Brassinosteroids1

PubMed Central

Noguchi, Takahiro; Fujioka, Shozo; Choe, Sunghwa; Takatsuto, Suguru; Yoshida, Shigeo; Yuan, Heng; Feldmann, Kenneth A.; Tax, Frans E.

1999-01-01

Seven dwarf mutants resembling brassinosteroid (BR)-biosynthetic dwarfs were isolated that did not respond significantly to the application of exogenous BRs. Genetic and molecular analyses revealed that these were novel alleles of BRI1 (Brassinosteroid-Insensitive 1), which encodes a receptor kinase that may act as a receptor for BRs or be involved in downstream signaling. The results of morphological and molecular analyses indicated that these represent a range of alleles from weak to null. The endogenous BRs were examined from 5-week-old plants of a null allele (bri1-4) and two weak alleles (bri1-5 and bri1-6). Previous analysis of endogenous BRs in several BR-biosynthetic dwarf mutants revealed that active BRs are deficient in these mutants. However, bri1-4 plants accumulated very high levels of brassinolide, castasterone, and typhasterol (57-, 128-, and 33-fold higher, respectively, than those of wild-type plants). Weaker alleles (bri1-5 and bri1-6) also accumulated considerable levels of brassinolide, castasterone, and typhasterol, but less than the null allele (bri1-4). The levels of 6-deoxoBRs in bri1 mutants were comparable to that of wild type. The accumulation of biologically active BRs may result from the inability to utilize these active BRs, the inability to regulate BR biosynthesis in bri1 mutants, or both. Therefore, BRI1 is required for the homeostasis of endogenous BR levels. PMID:10557222

Emerged HA and NA Mutants of the Pandemic Influenza H1N1 Viruses with Increasing Epidemiological Significance in Taipei and Kaohsiung, Taiwan, 2009–10

PubMed Central

Kao, Chuan-Liang; Chan, Ta-Chien; Tsai, Chu-Han; Chu, Kuan-Ying; Chuang, Shu-Fang; Lee, Chang-Chun; Li, Zheng-Rong Tiger; Wu, Ko-Wen; Chang, Luan-Yin; Shen, Yea-Huei; Huang, Li-Min; Lee, Ping-Ing; Yang, ChingLai; Compans, Richard; Rouse, Barry T.; King, Chwan-Chuen

2012-01-01

The 2009 influenza pandemic provided an opportunity to observe dynamic changes of the hemagglutinin (HA) and neuraminidase (NA) of pH1N1 strains that spread in two metropolitan areas -Taipei and Kaohsiung. We observed cumulative increases of amino acid substitutions of both HA and NA that were higher in the post–peak than in the pre-peak period of the epidemic. About 14.94% and 3.44% of 174 isolates had one and two amino acids changes, respective, in the four antigenic sites. One unique adaptive mutation of HA2 (E374K) was first detected three weeks before the epidemic peak. This mutation evolved through the epidemic, and finally emerged as the major circulated strain, with significantly higher frequency in the post-peak period than in the pre-peak (64.65% vs 9.28%, p<0.0001). E374K persisted until ten months post-nationwide vaccination without further antigenic changes (e.g. prior to the highest selective pressure). In public health measures, the epidemic peaked at seven weeks after oseltamivir treatment was initiated. The emerging E374K mutants spread before the first peak of school class suspension, extended their survival in high-density population areas before vaccination, dominated in the second wave of class suspension, and were fixed as herd immunity developed. The tempo-spatial spreading of E374K mutants was more concentrated during the post–peak (p = 0.000004) in seven districts with higher spatial clusters (p<0.001). This is the first study examining viral changes during the naïve phase of a pandemic of influenza through integrated virological/serological/clinical surveillance, tempo-spatial analysis, and intervention policies. The vaccination increased the percentage of E374K mutants (22.86% vs 72.34%, p<0.001) and significantly elevated the frequency of mutations in Sa antigenic site (2.36% vs 23.40%, p<0.001). Future pre-vaccination public health efforts should monitor amino acids of HA and NA of pandemic influenza viruses isolated at

Mutant power: using mutant allele collections for yeast functional genomics.

PubMed

Norman, Kaitlyn L; Kumar, Anuj

2016-03-01

The budding yeast has long served as a model eukaryote for the functional genomic analysis of highly conserved signaling pathways, cellular processes and mechanisms underlying human disease. The collection of reagents available for genomics in yeast is extensive, encompassing a growing diversity of mutant collections beyond gene deletion sets in the standard wild-type S288C genetic background. We review here three main types of mutant allele collections: transposon mutagen collections, essential gene collections and overexpression libraries. Each collection provides unique and identifiable alleles that can be utilized in genome-wide, high-throughput studies. These genomic reagents are particularly informative in identifying synthetic phenotypes and functions associated with essential genes, including those modeled most effectively in complex genetic backgrounds. Several examples of genomic studies in filamentous/pseudohyphal backgrounds are provided here to illustrate this point. Additionally, the limitations of each approach are examined. Collectively, these mutant allele collections in Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans promise insights toward an advanced understanding of eukaryotic molecular and cellular biology. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

Misfolded rhodopsin mutants display variable aggregation properties.

PubMed

Gragg, Megan; Park, Paul S-H

2018-06-08

The largest class of rhodopsin mutations causing autosomal dominant retinitis pigmentosa (adRP) is mutations that lead to misfolding and aggregation of the receptor. The misfolding mutants have been characterized biochemically, and categorized as either partial or complete misfolding mutants. This classification is incomplete and does not provide sufficient information to fully understand the disease pathogenesis and evaluate therapeutic strategies. A Förster resonance energy transfer (FRET) method was utilized to directly assess the aggregation properties of misfolding rhodopsin mutants within the cell. Partial (P23H and P267L) and complete (G188R, H211P, and P267R) misfolding mutants were characterized to reveal variability in aggregation properties. The complete misfolding mutants all behaved similarly, forming aggregates when expressed alone, minimally interacting with the wild-type receptor when coexpressed, and were unresponsive to treatment with the pharmacological chaperone 9-cis retinal. In contrast, variability was observed between the partial misfolding mutants. In the opsin form, the P23H mutant behaved similarly as the complete misfolding mutants. In contrast, the opsin form of the P267L mutant existed as both aggregates and oligomers when expressed alone and formed mostly oligomers with the wild-type receptor when coexpressed. The partial misfolding mutants both reacted similarly to the pharmacological chaperone 9-cis retinal, displaying improved folding and oligomerization when expressed alone but aggregating with wild-type receptor when coexpressed. The observed differences in aggregation properties and effect of 9-cis retinal predict different outcomes in disease pathophysiology and suggest that retinoid-based chaperones will be ineffective or even detrimental. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

Neurobehavioral Mutants Identified in an ENU Mutagenesis Project

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, Melloni N.; Dunning, Jonathan P; Wiley, Ronald G

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 responsivenessmore » 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.« less

Isolation and characterization of OmpC porin mutants with altered pore properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Misra, R.; Benson, S.A.

1988-02-01

The LamB protien is normally required for the uptake of maltodextrins. Starting with a LamB/sup -/ OmpF/sup -/ strain, we have isolated mutants that will grow on maltodextrins. The mutation conferring the Dex/sup +/ phenotype in the majority of these mutants has been mapped to the ompC locus. These mutants, unlike LamB/sup -/ OmpF/sup -/ strains, grew on maltotriose and maltotetraose, but not on maltopentaose, and showed a significantly higher rate of (/sup 14/C) maltose uptake than the parent strain did. In addition, these mutants showed increased sensitivity to certain ..beta..-lactam antibiotics and sodium dodecyl sulfate, but did not exhibitmore » an increase in sensitivity to other antibiotics and detergents. The nucleotide sequence of these mutants has been determined. In all cases, residue 74 (arginine) of the mature OmpC protein was affected. The results suggest that this region of the OmpC protein is involved in the pore domain and that the alterations lead to an increased pore size.« less

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. Copyright © 2014. Published by

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

Polarity-defective mutants of Aspergillus nidulans.

PubMed

Osherov, N; Mathew, J; May, G S

2000-12-01

We have identified two polarity-defective (pod) mutants in Aspergillus nidulans from a collection of heat-sensitive lethal mutants. At restrictive temperature, these mutants are capable of nuclear division but are unable to establish polar hyphal growth. We cloned the two pod genes by complementation of their heat-sensitive lethal phenotypes. The libraries used to clone the pod genes are under the control of the bidirectional niaD and niiA promoters. Complementation of the pod mutants is dependent on growth on inducing medium. We show that rescue of the heat-sensitive phenotype on inducing media is independent of the orientation of the gene relative to the niaD or niiA promoters, demonstrating that the intergenic region between the niaD and the niiA genes functions as an orientation-independent enhancer and repressor that is capable of functioning over long distances. The products of the podG and the podH genes were identified as homologues of the alpha subunit of yeast mitochondrial phenylalanyl--tRNA synthetase and transcription factor IIF interacting component of the CTD phosphatase. Neither of these gene products would have been predicted to produce a pod mutant phenotype based on studies of cellular polarity mutants in other organisms. The implications of these results are discussed. Copyright 2000 Academic Press.

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.

Metabolic pathway profiling of mitochondrial respiratory chain mutants in C. elegans

PubMed Central

MJ, Falk; Z, Zhang; Rosenjack; Nissim; E, Daikhin; Nissim; MM, Sedensky; M, Yudkoff; PG, Morgan

2008-01-01

C. elegans affords a model of primary mitochondrial dysfunction that provides insight into cellular adaptations which accompany mutations in nuclear gene that encode mitochondrial proteins. To this end, we characterized genome-wide expression profiles of C. elegans strains with mutations in nuclear-encoded subunits of respiratory chain complexes. Our goal was to detect concordant changes among clusters of genes that comprise defined metabolic pathways. Results indicate that respiratory chain mutants significantly upregulate a variety of basic cellular metabolic pathways involved in carbohydrate, amino acid, and fatty acid metabolism, as well as cellular defense pathways such as the metabolism of P450 and glutathione. To further confirm and extend expression analysis findings, quantitation of whole worm free amino acid levels was performed in C. elegans mitochondrial mutants for subunits of complexes I, II, and III. Significant differences were seen for 13 of 16 amino acid levels in complex I mutants compared with controls, as well as overarching similarities among profiles of complex I, II, and III mutants compared with controls. The specific pattern of amino acid alterations observed provides novel evidence to suggest that an increase in glutamate-linked transamination reactions caused by the failure of NAD+ dependent oxidation of ketoacids occurs in primary mitochondrial respiratory chain mutants. Recognition of consistent alterations among patterns of nuclear gene expression for multiple biochemical pathways and in quantitative amino acid profiles in a translational genetic model of mitochondrial dysfunction allows insight into the complex pathogenesis underlying primary mitochondrial disease. Such knowledge may enable the development of a metabolomic profiling diagnostic tool applicable to human mitochondrial disease. PMID:18178500

A METABOLIC SIGNATURE FOR LONG-LIFE IN THE C. ELEGANS MIT MUTANTS

PubMed Central

Butler, Jeffrey A.; Mishur, Robert J.; Bhaskaran, Shylesh; Rea, Shane L.

2012-01-01

SUMMARY Mit mutations that disrupt function of the mitochondrial electron transport chain can, inexplicably, prolong Caenorhabditis elegans lifespan. In this study we use a metabolomics approach to identify an ensemble of mitochondrial-derived α-ketoacids and α-hydroxyacids that are produced by long-lived Mit mutants but not by other long-lived mutants or by short-lived mitochondrial mutants. We show that accumulation of these compounds is dependent upon concerted inhibition of three α-ketoacid dehydrogenases that share dihydrolipoamide dehydrogenase (DLD) as a common subunit, a protein previously linked in humans with increased risk of Alzheimer’s disease. When the expression of DLD in wild type animals was reduced using RNA interference we observed an unprecedented effect on lifespan - as RNAi dosage was increased lifespan was significantly shortened but, at higher doses, it was significantly lengthened, suggesting DLD plays a unique role in modulating length of life. Our findings provide novel insight into the origin of the Mit phenotype. PMID:23173729

Lack of chemically induced mutation in repair-deficient mutants of yeast.

PubMed

Prakash, L

1974-12-01

Two genes, rad6 and rad9, that confer radiation sensitivity in the yeast Saccharomyces cerevisiae also greatly reduce the frequency of chemically-induced reversions of a tester mutant cyc1-131, which is a chain initiation mutant in the structural gene determining iso-1-cytochrome c. Mutations induced by ethyl methanesulfonate (EMS), diethyl sulfate (DES), methyl methanesulfonate (MMS), dimethyl sulfate (DMS), nitroquinoline oxide (NQO), nitrosoguanidine (NTG), nitrogen mustard (HN2), beta-propiolactone, and tritiated uridine, as well as mutations induced by ultraviolet light (UV) and ionizing radiation were greatly diminished in strains homozygous for either the rad6 or rad9 gene. Nitrous acid and nitrosoimidazolidone (NIL), on the other hand, were highly mutagenic in these repair-deficient mutants, and at low doses, these mutagens acted with about the same efficiency as in the normal RAD strain. At high doses of either nitrous acid or NIL, however, reversion frequencies were significantly reduced in the two rad mutants compared to normal strains. Although both rad mutants are immutable to about the same extent, the rad9 strains tend to be less sensitive to the lethal effect of chemical mutagens than rad6 strains. It is concluded that yeast requires a functional repair system for mutation induction by chemical agents.

Escherichia coli mutants impaired in maltodextrin transport.

PubMed

Wandersman, C; Schwartz, M; Ferenci, T

1979-10-01

Wild-type Escherichia coli K-12 was found to grow equally well on maltose and on maltodextrins containing up to seven glucose residues. Three classes of mutants unable to grow on maltodextrins, but still able to grow on maltose, were investigated in detail. The first class, already known, was composed of phage lambda-resistant mutants, which lack the outer membrane protein coded by gene lamB. These mutants grow on maltose and maltotriose but not at all on maltotetraose and longer maltodextrins which cannot cross the outer membrane. A second class of mutants were affected in malE, the structural gene of the periplasmic maltose binding protein. The maltose binding proteins isolated from the new mutants were altered in their substrate binding properties, but not in a way that could account for the mutant phenotypes. Rather, the results of growth experiments and transport studies suggest that these malE mutants are impaired in their ability to transport maltodextrins across the outer membrane. This implies that the maltose binding protein (in wild-type strains) cooperates with the lambda receptor in permeation through the outer membrane. The last class of mutants described in this paper were affected in malG, or perhaps in an as yet undetected gene close to malG. They were defective in the transfer of maltodextrins from the periplasmic space to the cytoplasm but only slightly affected in the transport of maltose.

Imaginal Disc Abnormalities in Lethal Mutants of Drosophila

PubMed Central

Shearn, Allen; Rice, Thomas; Garen, Alan; Gehring, Walter

1971-01-01

Late lethal mutants of Drosophila melanogaster, dying after the larval stage of development, were isolated. The homozygous mutant larvae were examined for abnormal imaginal disc morphology, and the discs were injected into normal larval hosts to test their capacities to differentiate into adult structures. In about half of the mutants analyzed, disc abnormalities were found. Included among the abnormalities were missing discs, small discs incapable of differentiating, morphologically normal discs with limited capacities for differentiation, and discs with homeotic transformations. In some mutants all discs were affected, and in others only certain discs. The most extreme abnormal phenotype is a class of “discless” mutants. The viability of these mutant larvae indicates that the discs are essential only for the development of an adult and not of a larva. The late lethals are therefore a major source of mutants for studying the genetic control of disc formation. Images PMID:5002822

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.

Construction and functional analysis of Trichoderma harzianum mutants that modulate maize resistance to the pathogen Curvularia lunata.

PubMed

Fan, Lili; Fu, Kehe; Yu, Chuanjin; Ma, Jia; Li, Yaqian; Chen, Jie

2014-01-01

Agrobacterium tumefaciens-mediated transformation (ATMT) was used to generate an insertional mutant library of the mycelial fungus Trichoderma harzianum. From a total of 450 mutants, six mutants that showed significant influence on maize resistance to C. lunata were analyzed in detail. Maize coated with these mutants was more susceptible to C. lunata compared with those coated with a wild-type (WT) strain. Similar to other fungal ATMT libraries, all six mutants were single copy integrations, which occurred preferentially in noncoding regions (except two mutants) and were frequently accompanied by the loss of border sequences. Two mutants (T66 and T312) that were linked to resistance were characterized further. Maize seeds coated with T66 and T312 were more susceptible to C. lunata than those treated with WT. Moreover, the mutants affected the resistance of maize to C. lunata by enhancing jasmonate-responsive gene expression. T66 and T312 induced maize resistance to C. lunata infection through a jasmonic acid-dependent pathway.

Identification and quantification of flavonoids in yellow grain mutant of rice (Oryza sativa L.).

PubMed

Kim, Backki; Woo, Sunmin; Kim, Mi-Jung; Kwon, Soon-Wook; Lee, Joohyun; Sung, Sang Hyun; Koh, Hee-Jong

2018-02-15

Flavonoids are naturally occurring phenolic compounds with potential health-promoting activities. Although anthocyanins and phenolic acids in coloured rice have been investigated, few studies have focused on flavonoids. Herein, we analysed flavonoids in a yellow grain rice mutant using UHPLC-DAD-ESI-Q-TOF-MS, and identified 19 flavonoids by comparing retention times and accurate mass measurements. Among them, six flavonoids, isoorientin, isoorientin 2″-O-glucoside, vitexin 2″-O-glucoside, isovitexin, isoscoparin 2″-O-glucoside and isoscoparin, were isolated and fully identified from the yellow grain rice mutant, and the levels were significantly higher than wild-type, with isoorientin particularly abundant in mutant embryo. Significant differences in total phenolic compounds and antioxidant activity were observed in mutant rice by DPPH, FRAP and TEAC assays. The results suggest that the representative six flavonoids may play an important role in colouration and antioxidant activity of embryo and endosperm tissue. The findings provide insight into flavonoid biosynthesis and the possibility of improving functionality in rice. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Characterization and classification of zebrafish brain morphology mutants

PubMed Central

Lowery, Laura Anne; De Rienzo, Gianluca; Gutzman, Jennifer H.; Sive, Hazel

2010-01-01

The mechanisms by which the vertebrate brain achieves its three-dimensional structure are clearly complex, requiring the functions of many genes. Using the zebrafish as a model, we have begun to define genes required for brain morphogenesis, including brain ventricle formation, by studying 16 mutants previously identified as having embryonic brain morphology defects. We report the phenotypic characterization of these mutants at several time-points, using brain ventricle dye injection, imaging, and immunohistochemistry with neuronal markers. Most of these mutants display early phenotypes, affecting initial brain shaping, while others show later phenotypes, affecting brain ventricle expansion. In the early phenotype group, we further define four phenotypic classes and corresponding functions required for brain morphogenesis. Although we did not use known genotypes for this classification, basing it solely on phenotypes, many mutants with defects in functionally related genes clustered in a single class. In particular, class 1 mutants show midline separation defects, corresponding to epithelial junction defects; class 2 mutants show reduced brain ventricle size; class 3 mutants show midbrain-hindbrain abnormalities, corresponding to basement membrane defects; and class 4 mutants show absence of ventricle lumen inflation, corresponding to defective ion pumping. Later brain ventricle expansion requires the extracellular matrix, cardiovascular circulation, and transcription/splicing-dependent events. We suggest that these mutants define processes likely to be used during brain morphogenesis throughout the vertebrates. PMID:19051268

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

Biomass productivities in wild type and pigment mutant of Cyclotella sp. (Diatom).

PubMed

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

2009-06-01

Microalgae are expected to play a significant role in greenhouse gas mitigation because they can utilize CO(2) from power plant 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 (I(s)). Employing chemical (ethylmethylsulfonate) 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 (I(s)) was two to three times greater in the pigment mutant CM1-1 than in the wild type, i.e., 355-443 versus 116-169 mumol/m(2) s, respectively. While theory, i.e., the Bush equation, predicts that such a significant gain in I(s) 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 day, respectively, at low light intensity (200 micromol/m(2) s) and 1,229 versus 1,043 mg/L day, respectively, at high light

Molecular characterization of baculovirus Bombyx mori nucleopolyhedrovirus polyhedron mutants.

PubMed

Katsuma, S; Noguchi, Y; Shimada, T; Nagata, M; Kobayashi, M; Maeda, S

1999-01-01

Four newly isolated and two previously isolated polyhedron mutants of Bombyx mori nucleopolyhedrovirus (BmNPV) were studied. Two polyhedron deficient mutants, #126 and #136, produced small uncrystallized particles of polyhedrin in the nuclei and cytoplasm of infected cells. Mutant #211 produced a large number of variably sized polyhedra in the nucleus and #220 produced a few large cuboidal polyhedra in the nucleus. Mutant #24 and #128 were previously isolated BmNPV mutants. Mutant #24 could not produce polyhedrin mRNA and polyhedra produced by mutant #128 lacked oral infectivity. Nucleotide sequence analysis indicated that five mutants (#126, #136, #211, #220 and #128) had amino acid substitutions in polyhedrin and mutant #24 had a point mutation only in the promoter region of the polyhedrin gene. Cotransfection experiments showed that the altered phenotypes were due to the mutations found in the polyhedrin gene regions. In mutants #126 and #136, amino acid sequences of the nuclear localization signal of polyhedrin were identical to those of wild-type BmNPV, suggesting that this sequence was necessary but not sufficient for nuclear localization of polyhedrin. Electron microscopic observation revealed that fewer occluded virions were contained in polyhedra of #128 and #220.

Cercosporin-deficient mutants by plasmid tagging in the asexual fungus Cercospora nicotianae.

PubMed

Chung, K-R; Ehrenshaft, M; Wetzel, D K; Daub, M E

2003-11-01

We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.

Saccharomyces cerevisiae mutants with enhanced induced mutation and altered mitotic gene conversion.

PubMed

Ivanov, E L; Kovaltzova, S V; Korolev, V G

1989-08-01

We have developed a method to isolate yeast (Saccharomyces cerevisiae) mutants with enhanced induced mutagenesis based on nitrous acid-induced reversion of the ade2-42 allele. Six mutants have been isolated and designated him (high induced mutagenesis), and 4 of them were studied in more detail. The him mutants displayed enhanced reversion of the ade2-42 allele, either spontaneous or induced by nitrous acid, UV light, and the base analog 6-N-hydroxylaminopurine, but not by gamma-irradiation. It is worth noting that the him mutants turned out not to be sensitive to the lethal effects of the mutagens used. The enhancement in mutation induced by nitrous acid, UV light, and 6-N-hydroxylaminopurine has been confirmed in a forward-mutation assay (induction of mutations in the ADE1, ADE2 genes). The latter agent revealed the most apparent differences between the him mutants and the wild-type strain and was, therefore, chosen for the genetic analysis of mutants, him mutations analyzed behaved as a single Mendelian trait; complementation tests indicated 3 complementation groups (HIM1, HIM2, and HIM3), each containing 1 mutant allele. Uracil-DNA glycosylase activity was determined in crude cell extracts, and no significant differences between the wild-type and him strains were detected. Spontaneous mitotic gene conversion at the ADE2 locus is altered in him1 strains, either increased or decreased, depending on the particular heteroallelic combination. Genetic evidence strongly suggests him mutations to be involved in a process of mismatch correction of molecular heteroduplexes.

Molecular synergy underlies the co-occurrence patterns and phenotype of NPM1-mutant acute myeloid leukemia

PubMed Central

Dovey, Oliver M.; Cooper, Jonathan L.; Mupo, Annalisa; Grove, Carolyn S.; Lynn, Claire; Conte, Nathalie; Andrews, Robert M.; Pacharne, Suruchi; Tzelepis, Konstantinos; Vijayabaskar, M. S.; Green, Paul; Rad, Roland; Arends, Mark; Wright, Penny; Yusa, Kosuke; Bradley, Allan; Varela, Ignacio

2017-01-01

NPM1 mutations define the commonest subgroup of acute myeloid leukemia (AML) and frequently co-occur with FLT3 internal tandem duplications (ITD) or, less commonly, NRAS or KRAS mutations. Co-occurrence of mutant NPM1 with FLT3-ITD carries a significantly worse prognosis than NPM1-RAS combinations. To understand the molecular basis of these observations, we compare the effects of the 2 combinations on hematopoiesis and leukemogenesis in knock-in mice. Early effects of these mutations on hematopoiesis show that compound Npm1cA/+;NrasG12D/+ or Npm1cA;Flt3ITD share a number of features: Hox gene overexpression, enhanced self-renewal, expansion of hematopoietic progenitors, and myeloid differentiation bias. However, Npm1cA;Flt3ITD mutants displayed significantly higher peripheral leukocyte counts, early depletion of common lymphoid progenitors, and a monocytic bias in comparison with the granulocytic bias in Npm1cA/+;NrasG12D/+ mutants. Underlying this was a striking molecular synergy manifested as a dramatically altered gene expression profile in Npm1cA;Flt3ITD, but not Npm1cA/+;NrasG12D/+, progenitors compared with wild-type. Both double-mutant models developed high-penetrance AML, although latency was significantly longer with Npm1cA/+;NrasG12D/+. During AML evolution, both models acquired additional copies of the mutant Flt3 or Nras alleles, but only Npm1cA/+;NrasG12D/+ mice showed acquisition of other human AML mutations, including IDH1 R132Q. We also find, using primary Cas9-expressing AMLs, that Hoxa genes and selected interactors or downstream targets are required for survival of both types of double-mutant AML. Our results show that molecular complementarity underlies the higher frequency and significantly worse prognosis associated with NPM1c/FLT3-ITD vs NPM1/NRAS-G12D-mutant AML and functionally confirm the role of HOXA genes in NPM1c-driven AML. PMID:28835438

Increased sensitivity to radiochemotherapy in IDH1 mutant glioblastoma as demonstrated by serial quantitative MR volumetry

PubMed Central

Tran, Anh N.; Lai, Albert; Li, Sichen; Pope, Whitney B.; Teixeira, Stephanie; Harris, Robert J.; Woodworth, Davis C.; Nghiemphu, Phioanh L.; Cloughesy, Timothy F.; Ellingson, Benjamin M.

2014-01-01

Background Isocitrate dehydrogenase 1 (IDH1) mutations have been linked to favorable outcomes in patients with glioblastoma multiforme (GBM). Recent in vitro experiments suggest that IDH1 mutation sensitizes tumors to radiation damage. We hypothesized that radiographic treatment response would be significantly different between IDH1 mutant versus wild-type GBMs after radiotherapy (RT) and concurrent temozolomide (TMZ). Methods A total of 39 newly diagnosed GBM patients with known IDH1 mutational status (10 IDH1 mutants), who followed standard therapy and had regular post-contrast T1W (T1+C) and T2W/ fluid-attenuated inversion recovery (FLAIR) images in the 6-month period after starting RT, were enrolled. The volume of contrast-enhancing and FLAIR hyperintensity were calculated from each scan. Linear and polynomial regression techniques were used to estimate the rate of change and temporal patterns in tumor volumes. Results IDH1 mutant GBMs demonstrated a favorable response to RT/TMZ in the study period, as demonstrated by 10 of 10 mutants showing radiographic response (decreasing VT1+C), compared with 13 of 29 wild-types (P < .001). During the study period, VT1+C and VFLAIR changed at −3.6% per week and +0.6% per week in IDH1 mutant tumors, respectively, as compared with +0.8% per week and +5.2% per week in IDH1 wild-type tumors (P = .0076 and P = .0118, respectively). Amongst the radiographic responders, IDH1 mutant GBMs still demonstrated significant progression-free and overall survival benefit. Aggregated tumor kinetics by group showed significant lower rate in IDH1 mutant GBMs in specific periods: >105 days for VFLAIR and 95–120 and >150 days for VT1+C from starting RT/TMZ. Conclusions The current study supports the hypothesis that IDH1 mutant GBMs are more sensitive to radiochemotherapy than IDH1 wild-type GBMs. PMID:24305712

Mutant IDH1 is required for IDH1 mutated tumor cell growth

PubMed Central

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

2012-01-01

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 IDH1R132C 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

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

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.

Diaminopurine-Resistant Mutants of Cultured, Diploid Human Fibroblasts

PubMed Central

Rappaport, Harriet; DeMars, Robert

1973-01-01

Clones of cells resistant to 2,6-diaminopurine were detected in skin fibroblast cultures derived from 13 of 21 normal humans of both sexes from 17 unrelated families. Almost all of the cultures that yielded mutants were chosen for further study from among a total of 83 surveyed because they displayed a slight resistance to low concentrations of diaminopurine. The incidences of mutant colonies ranged between about 10-5 and 10-4 per cell surviving prior mutagenic treatment with MNNG. The incidences of spontaneous mutants were about 10-7 to 10-5 in three unrelated cultures. Most independent mutants had distinctly reduced activity of adenine phosphoribosyltransferase but some had apparently normal amounts of activity. Two mutants from unrelated boys had little or no detectable enzyme activity and were unable to effectively use exogenous adenine for growth when purine biosynthesis was blocked with azaserine. Most mutants could utilize exogenous adenine, just as most azaguanine-resistant fibroblast mutants can utilize exogenous hypoxanthine, even when their hypoxanthine-guanine phosphoribosyltransferase activity is reduced. Diverse genetic changes conferred diaminopurine resistance but their specific natures are still undefined. Gross numerical or structural chromosome abnormalities were not observed in the mutants examined so far. Since at least one gene responsible for adenine phosphoribosyltransferase activity is on autosome No. 16 our results suggest that at least some of the cultures yielding mutants were heterozygous and that alleles conferring diaminopurine resistance may be frequent enough to comprise a polymorphism. PMID:4358687

Dictyostelium discoideum mutants with conditional defects in phagocytosis

PubMed Central

1994-01-01

We have isolated and characterized Dictyostelium discoideum mutants with conditional defects in phagocytosis. Under suspension conditions, the mutants exhibited dramatic reductions in the uptake of bacteria and polystyrene latex beads. The initial binding of these ligands was unaffected, however, indicating that the defect was not in a plasma membrane receptor: Because of the phagocytosis defect, the mutants were unable to grow when cultured in suspensions of heat-killed bacteria. The mutants exhibited normal capacities for fluid phase endocytosis and grew as rapidly as parental (AX4) cells in axenic medium. Both the defects in phagocytosis and growth on bacteria were corrected when the mutant Dictyostelium cells were cultured on solid substrates. Reversion and genetic complementation analysis suggested that the mutant phenotypes were caused by single gene defects. While the precise site of action of the mutations was not established, the mutations are likely to affect an early signaling event because the binding of bacteria to mutant cells in suspension was unable to trigger the localized polymerization of actin filaments required for ingestion; other aspects of actin function appeared normal. This class of conditional phagocytosis mutant should prove to be useful for the expression cloning of the affected gene(s). PMID:7519624

An annotated database of Arabidopsis mutants of acyl lipid metabolism

DOE PAGES

McGlew, Kathleen; Shaw, Vincent; Zhang, Meng; ...

2014-12-10

Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism ( fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypesmore » of mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. Mutants for at least 30 % of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. Furthermore, the database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research.« less

Downregulation of aldose reductase is responsible for developmental abnormalities of the silkworm purple quail-like mutant (q-lp).

PubMed

Wang, Pingyang; Bi, Simin; Wei, Weiyang; Qiu, Zhiyong; Xia, Dingguo; Shen, Xingjia; Zhao, Qiaoling

2018-05-03

Aldose reductase (AR) is a rate-limiting enzyme in the polyol pathway and is also the key enzyme involved in diabetic complications. The silkworm purple quail-like mutant (q-l p ) exhibits pigmented dots on its epidermis. The q-l p mutant also shows developmental abnormalities and decreased vitality. In this study, fat bodies from the q-l p mutant and the wildtype 932VR strain were subjected to two-dimensional gel electrophoresis (2-DE) analysis, and the Bombyx mori AR (BmAR) protein was found to be significantly downregulated in the q-l p mutant. The expression of BmAR at the mRNA level was also significantly downregulated, as verified through quantitative reverse transcription PCR (qRT-PCR). Knockdown of the expression of BmAR via RNAi resulted in a reduction of silkworm weight. The sorbitol level in q-l p was significantly lower than in the wildtype. These results suggested that the BmAR gene is closely related to the development of the q-l p mutant. Investigation of the cause of BmAR downregulation in the q-l p mutant could contribute to revealing the function of AR in insects and offers a new method of identifying AR inhibitors for the treatment of diabetic complications. Copyright © 2017. Published by Elsevier B.V.

A metabolic signature for long life in the Caenorhabditis elegans Mit mutants.

PubMed

Butler, Jeffrey A; Mishur, Robert J; Bhaskaran, Shylesh; Rea, Shane L

2013-02-01

Mit mutations that disrupt function of the mitochondrial electron transport chain can, inexplicably, prolong Caenorhabditis elegans lifespan. In this study we use a metabolomics approach to identify an ensemble of mitochondrial-derived α-ketoacids and α-hydroxyacids that are produced by long-lived Mit mutants but not by other long-lived mutants or by short-lived mitochondrial mutants. We show that accumulation of these compounds is dependent on concerted inhibition of three α-ketoacid dehydrogenases that share dihydrolipoamide dehydrogenase (DLD) as a common subunit, a protein previously linked in humans with increased risk of Alzheimer's disease. When the expression of DLD in wild-type animals was reduced using RNA interference we observed an unprecedented effect on lifespan - as RNAi dosage was increased lifespan was significantly shortened, but, at higher doses, it was significantly lengthened, suggesting that DLD plays a unique role in modulating length of life. Our findings provide novel insight into the origin of the Mit phenotype. © 2012 The Authors Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

Isolation and Characterization of Mms-Sensitive Mutants of SACCHAROMYCES CEREVISIAE

PubMed Central

Prakash, Louise; Prakash, Satya

1977-01-01

We have isolated mutants sensitive to methyl methanesulfonate (MMS) in Saccharomyces cerevisiae. Alleles of rad1, rad4, rad6, rad52, rad55 and rad57 were found among these mms mutants. Twenty-nine of the mms mutants which complement the existing radiation-sensitive (rad and rev ) mutants belong to 22 new complementation groups. Mutants from five complementation groups are sensitive only to MMS. Mutants of 11 complementation groups are sensitive to UV or X rays in addition to MMS, mutants of six complementation groups are sensitive to all three agents. The cross-sensitivities of these mms mutants to UV and X rays are discussed in terms of their possible involvement in DNA repair. Sporulation is reduced or absent in homozygous diploids of mms mutants from nine complementation groups. PMID:195865

Comparison of arabidopsis stomatal density mutants indicates variation in water stress responses and potential epistatic effects

Treesearch

Shaneka S. Lawson; Paula M. Pijut; Charles H. Michler

2014-01-01

Recent physiological analysis of Arabidopsis stomatal density (SD) mutants indicated that SD was not the major factor controlling aboveground biomass accumulation. Despite the general theory that plants with fewer stomata have limited biomass acquisition capabilities, epf1 and several other Arabidopsis mutants varied significantly in leaf fresh...

Bending patterns of chlamydomonas flagella: III. A radial spoke head deficient mutant and a central pair deficient mutant.

PubMed

Brokaw, C J; Luck, D J

1985-01-01

Flash photomicrography at frequencies up to 300 Hz and computer-assisted image analysis have been used to obtain parameters describing the flagellar bending patterns of mutants of Chlamydomonas reinhardtii. All strains contained the uni1 mutation, to facilitate photography. The radial spoke head deficient mutant pf17, and the central pair deficient mutant, pf15, in combination with suppressor mutations that restore motility without restoring the ultrastructural or biochemical deficiencies, both generate forward mode bending patterns with increased shear amplitude and decreased asymmetry relative to the "wild-type" uni1 flagella described previously. In the reverse beating mode, the suppressed pf17 mutants generate reverse bending patterns with large shear amplitudes. Reverse beating of the suppressed pf15 mutants is rare. There is a reciprocal relationship between increased shear amplitude and decreased beat frequency, so that the velocity of sliding between flagellar microtubules is not increased by an increase in shear amplitude. The suppressor mutations alone cause decreased frequency and sliding velocity in both forward and reverse mode beating, with little change in shear amplitude or symmetry.

Modulation of mutant Huntingtin aggregates and toxicity by human myeloid leukemia factors.

PubMed

Banerjee, Manisha; Datta, Moumita; Bhattacharyya, Nitai P

2017-01-01

Increased poly glutamine (polyQ) stretch at N-terminal of Huntingtin (HTT) causes Huntington's disease. HTT interacts with large number of proteins, although the preference for such interactions with wild type or mutated HTT protein remains largely unknown. HYPK, an intrinsically unstructured protein chaperone and interactor of mutant HTT was found to interact with myeloid leukemia factor 1 (MLF1) and 2 (MLF2). To identify the role of these two proteins in mutant HTT mediated aggregate formation and toxicity in a cell model, both the proteins were found to preferentially interact with the mutated N-terminal HTT. They significantly reduced the number of cells containing mutant HTT aggregates and subsequent apoptosis in Neuro2A cells. Additionally, in FRAP assay, mobile fraction of mutant HTT aggregates was increased in the presence of MLF1 or MLF2. Further, MLF1 could release transcription factors like p53, CBP and CREB from mutant HTT aggregates. Moreover, in HeLa cell co-expressing mutant HTT exon1 and full length MLF1, p53 was released from the aggregates, leading to the recovery of the expression of the GADD45A transcript, a p53 regulated gene. Taking together, these results showed that MLF1 and MLF2 modulated the formation of aggregates and induction of apoptosis as well as the expressions of genes indirectly. Copyright © 2016 Elsevier Ltd. All rights reserved.

Loss of circadian clock accelerates aging in neurodegeneration-prone mutants.

PubMed

Krishnan, Natraj; Rakshit, Kuntol; Chow, Eileen S; Wentzell, Jill S; Kretzschmar, Doris; Giebultowicz, Jadwiga M

2012-03-01

Circadian clocks generate rhythms in molecular, cellular, physiological, and behavioral processes. Recent studies suggest that disruption of the clock mechanism accelerates organismal senescence and age-related pathologies in mammals. Impaired circadian rhythms are observed in many neurological diseases; however, it is not clear whether loss of rhythms is the cause or result of neurodegeneration, or both. To address this important question, we examined the effects of circadian disruption in Drosophila melanogaster mutants that display clock-unrelated neurodegenerative phenotypes. We combined a null mutation in the clock gene period (per(01)) that abolishes circadian rhythms, with a hypomorphic mutation in the carbonyl reductase gene sniffer (sni(1)), which displays oxidative stress induced neurodegeneration. We report that disruption of circadian rhythms in sni(1) mutants significantly reduces their lifespan compared to single mutants. Shortened lifespan in double mutants was coupled with accelerated neuronal degeneration evidenced by vacuolization in the adult brain. In addition, per(01)sni(1) flies showed drastically impaired vertical mobility and increased accumulation of carbonylated proteins compared to age-matched single mutant flies. Loss of per function does not affect sni mRNA expression, suggesting that these genes act via independent pathways producing additive effects. Finally, we show that per(01) mutation accelerates the onset of brain pathologies when combined with neurodegeneration-prone mutation in another gene, swiss cheese (sws(1)), which does not operate through the oxidative stress pathway. Taken together, our data suggest that the period gene may be causally involved in neuroprotective pathways in aging Drosophila. Copyright © 2011 Elsevier Inc. All rights reserved.

Loss of circadian clock accelerates aging in neurodegeneration-prone mutants

PubMed Central

Krishnan, Natraj; Rakshit, Kuntol; Chow, Eileen S.; Wentzell, Jill S.; Kretzschmar, Doris; Giebultowicz, Jadwiga M.

2012-01-01

Circadian clocks generate rhythms in molecular, cellular, physiological, and behavioral processes. Recent studies suggest that disruption of the clock mechanism accelerates organismal senescence and age-related pathologies in mammals. Impaired circadian rhythms are observed in many neurological diseases; however, it is not clear whether loss of rhythms is the cause or result of neurodegeneration, or both. To address this important question, we examined the effects of circadian disruption in Drosophila melanogaster mutants that display clock-unrelated neurodegenerative phenotypes. We combined a null mutation in the clock gene period (per01) that abolishes circadian rhythms, with a hypomorphic mutation in the carbonyl reductase gene sniffer (sni1), which displays oxidative stress induced neurodegeneration. We report that disruption of circadian rhythms in sni1 mutants significantly reduces their lifespan compared to single mutants. Shortened lifespan in double mutants was coupled with accelerated neuronal degeneration evidenced by vacuolization in the adult brain. In addition, per01 sni1 flies showed drastically impaired vertical mobility and increased accumulation of carbonylated proteins compared to age-matched single mutant flies. Loss of per function does not affect sni mRNA expression, suggesting that these genes act via independent pathways producing additive effects. Finally, we show that per01 mutation accelerates the onset of brain pathologies when combined with neurodegeneration-prone mutation in another gene, swiss cheese (sws1), which does not operate through the oxidative stress pathway. Taken together, our data suggest that the period gene may be causally involved in neuroprotective pathways in aging Drosophila. PMID:22227001

Characterization of a Weak Allele of Zebrafish cloche Mutant

PubMed Central

Ma, Ning; Huang, Zhibin; Chen, Xiaohui; He, Fei; Wang, Kun; Liu, Wei; Zhao, Linfeng; Xu, Xiangmin; Liao, Wangjun; Ruan, Hua; Luo, Shenqiu; Zhang, Wenqing

2011-01-01

Hematopoiesis is a complicated and dynamic process about which the molecular mechanisms remain poorly understood. Danio rerio (zebrafish) is an excellent vertebrate system for studying hematopoiesis and developmental mechanisms. In the previous study, we isolated and identified a cloche 172 (clo 172) mutant, a novel allele compared to the original cloche (clo) mutant, through using complementation test and initial mapping. Here, according to whole mount in-situ hybridization, we report that the endothelial cells in clo 172 mutant embryos, although initially developed, failed to form the functional vascular system eventually. In addition, further characterization indicates that the clo 172 mutant exhibited weaker defects instead of completely lost in primitive erythroid cells and definitive hematopoietic cells compared with the clo s5 mutant. In contrast, primitive myeloid cells were totally lost in clo 172 mutant. Furthermore, these reappeared definitive myeloid cells were demonstrated to initiate from the remaining hematopoietic stem cells (HSCs) in clo 172 mutant, confirmed by the dramatic decrease of lyc in clo 172 runx1w84x double mutant. Collectively, the clo 172 mutant is a weak allele compared to the clo s5 mutant, therefore providing a model for studying the early development of hematopoietic and vascular system, as well as an opportunity to further understand the function of the cloche gene. PMID:22132109

Reduced heart size and increased myocardial fuel substrate oxidation in ACC2 mutant mice

PubMed Central

Essop, M. Faadiel; Camp, Heidi S.; Choi, Cheol Soo; Sharma, Saumya; Fryer, Ryan M.; Reinhart, Glenn A.; Guthrie, Patrick H.; Bentebibel, Assia; Gu, Zeiwei; Shulman, Gerald I.; Taegtmeyer, Heinrich; Wakil, Salih J.; Abu-Elheiga, Lutfi

2008-01-01

The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC2) is a key regulator of mitochondrial fatty acid (FA) uptake via carnitine palmitoyltransferase 1 (CPT1). To test the hypothesis that oxidative metabolism is upregulated in hearts from animals lacking ACC2 (employing a transgenic Acc2-mutant mouse), we assessed cardiac function in vivo and determined rates of myocardial substrate oxidation ex vivo. When examined by echocardiography, there was no difference in systolic function, but left ventricular mass of the Acc2-mutant (MUT) mouse was significantly reduced (∼25%) compared with wild-types (WT). Reduced activation of the mammalian target of rapamycin (mTOR) and its downstream target p70S6K was found in MUT hearts. Exogenous oxidation rates of oleate were increased ∼22%, and, unexpectedly, exogenous glucose oxidation rates were also increased in MUT hearts. Using a hyperinsulinemic-euglycemic clamp, we found that glucose uptake in MUT hearts was increased by ∼83%. Myocardial triglyceride levels were significantly reduced in MUT vs. WT while glycogen content was the same. In parallel, transcript levels of PPARα and its target genes, pyruvate dehydrogenase kinase-4 (PDK-4), malonyl-CoA decarboxylase (MCD), and mCPT1, were downregulated in MUT mice. In summary, we report that 1) Acc2-mutant hearts exhibit a marked preference for the oxidation of both glucose and FAs coupled with greater utilization of endogenous fuel substrates (triglycerides), 2) attenuated mTOR signaling may result in reduced heart sizes observed in Acc2-mutant mice, and 3) Acc2-mutant hearts displayed normal functional parameters despite a significant decrease in size. PMID:18487439

Proteostasis and ageing: insights from long-lived mutant mice.

PubMed

Sands, William A; Page, Melissa M; Selman, Colin

2017-10-15

The global increase in life expectancy is creating significant medical, social and economic challenges to current and future generations. Consequently, there is a need to identify the fundamental mechanisms underlying the ageing process. This knowledge should help develop realistic interventions capable of combatting age-related disease, and thus improving late-life health and vitality. While several mechanisms have been proposed as conserved lifespan determinants, the loss of proteostasis - where proteostasis is defined here as the maintenance of the proteome - appears highly relevant to both ageing and disease. Several studies have shown that multiple proteostatic mechanisms, including the endoplasmic reticulum (ER)-induced unfolded protein response (UPR), the ubiquitin-proteasome system (UPS) and autophagy, appear indispensable for longevity in many long-lived invertebrate mutants. Similarly, interspecific comparisons suggest that proteostasis may be an important lifespan determinant in vertebrates. Over the last 20 years a number of long-lived mouse mutants have been described, many of which carry single-gene mutations within the growth-hormone, insulin/IGF-1 or mTOR signalling pathways. However, we still do not know how these mutations act mechanistically to increase lifespan and healthspan, and accordingly whether mechanistic commonality occurs between different mutants. Recent evidence supports the premise that the successful maintenance of the proteome during ageing may be linked to the increased lifespan and healthspan of long-lived mouse mutants. © 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Analyses of tomato fruit brightness mutants uncover both cutin-deficient and cutin-abundant mutants and a new hypomorphic allele of GDSL lipase.

PubMed

Petit, Johann; Bres, Cécile; Just, Daniel; Garcia, Virginie; Mauxion, Jean-Philippe; Marion, Didier; Bakan, Bénédicte; Joubès, Jérôme; Domergue, Frédéric; Rothan, Christophe

2014-02-01

The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, postharvest shelf-life, and brightness. To identify tomato mutants with modified cuticle composition and architecture and to further decipher the relationships between fruit brightness and cuticle in tomato, we screened an ethyl methanesulfonate mutant collection in the miniature tomato cultivar Micro-Tom for mutants with altered fruit brightness. Our screen resulted in the isolation of 16 glossy and 8 dull mutants displaying changes in the amount and/or composition of wax and cutin, cuticle thickness, and surface aspect of the fruit as characterized by optical and environmental scanning electron microscopy. The main conclusions on the relationships between fruit brightness and cuticle features were as follows: (1) screening for fruit brightness is an effective way to identify tomato cuticle mutants; (2) fruit brightness is independent from wax load variations; (3) glossy mutants show either reduced or increased cutin load; and (4) dull mutants display alterations in epidermal cell number and shape. Cuticle composition analyses further allowed the identification of groups of mutants displaying remarkable cuticle changes, such as mutants with increased dicarboxylic acids in cutin. Using genetic mapping of a strong cutin-deficient mutation, we discovered a novel hypomorphic allele of GDSL lipase carrying a splice junction mutation, thus highlighting the potential of tomato brightness mutants for advancing our understanding of cuticle formation in plants.

Identification and suppression of epidermal growth factor receptor variant III signaling in fibroblast-like synoviocytes from aggressive rheumatoid arthritis by the mimotope.

PubMed

Niu, Jianying; Li, Changhong; Jin, Yinji; Xing, Rui; Sun, Lin; Yu, Ruohan; Jian, Leilei; Liu, Xiangyuan; Yang, Lin

2018-06-01

Epidermal growth factor receptor (EGFR) signaling has been reported to play a vital role in the pathogenesis of rheumatoid arthritis (RA). In current study, we sought to observe whether the active immunization induced by the mimotope could recognize EGFR, inhibit their signaling and disrupt the pathogenic behavior of fibroblast-like synoviocytes (FLS) from RA patients. We prepared a linked EGFR mimotope and performed series of experiments to detect whether the mimotope could induce the desired immune responses. To our surprises, we detected the expression of EGFR variant III (EGFRvIII), but not EGFR in the synovial tissues and FLS from patients with aggressive RA by the linked EGFR mimotope-induced antibodies (LEMIA). Meanwhile, LEMIA could inhibit the signaling caused by the autophosphorylation of EGFRvIII in the FLS. The proliferation, migration, invasion and anti-apoptosis capabilities of the EGFRvIII-expressed FLS were disrupted by LEMIA. These results suggest that EGFRvIII signaling may participate in the malignant behaviors of FLS from aggressive RA. Meanwhile, the linked EGFR mimotope could be used to detect the expression of EGFRvIII and developed to be a potential therapy agent against the aggressive FLS. Copyright © 2018 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Bacterio-opsin mutants of Halobacterium halobium

PubMed Central

Betlach, Mary; Pfeifer, Felicitas; Friedman, James; Boyer, Herbert W.

1983-01-01

The bacterio-opsin (bop) gene of Halobacterium halobium R1 has been cloned with about 40 kilobases of flanking genomic sequence. The 40-kilobase segment is derived from the (G+C)-rich fraction of the chromosome and is not homologous to the major (pHH1) or minor endogenous covalently closed circular DNA species of H. halobium. A 5.1-kilobase Pst I fragment containing the bop gene was subcloned in pBR322 and a partial restriction map was determined. Defined restriction fragments of this clone were used as probes to analyze the defects associated with the bop gene in 12 bacterio-opsin mutants. Eleven out of 12 of the mutants examined had inserts ranging from 350 to 3,000 base pairs either in the bop gene or up to 1,400 base pairs upstream. The positions of the inserts were localized to four regions in the 5.1-kilobase genomic fragment: within the gene (one mutant), in a region that overlaps the 5′ end of the gene (seven mutants), and in two different upstream regions (three mutants). Two revertants of the mutant with the most distal insert had an additional insert in the same region. The polar effects of these inserts are discussed in terms of inactivation of a regulatory gene or disruption of part of a coordinately expressed operon. Given the defined nature of the bop mRNA—i.e., it has a 5′ leader sequence of three ribonucleotides—these observations indicate that the bop mRNA might be processed from a large mRNA transcript. Images PMID:16593291

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.

Structure based discovery of clomifene as a potent inhibitor of cancer-associated mutant IDH1

PubMed Central

Luan, Shanshan; Li, Dan; Chen, Renqi; Zhang, Qian; Chen, Lixia; Huang, Jiangeng; Li, Hua

2017-01-01

Isocitrate dehydrogenase (IDH) plays an indispensable role in the tricarboxylic acid cycle, and IDH mutations are present in nearly 75% of glioma and 20% of acute myeloid leukemia. One IDH1R132H inhibitor (clomifene citrate) was found by virtual screening method, which can selectively suppress mutant enzyme activities in vitro and in vivo with a dose-dependent manner. The molecular docking indicated that clomifene occupied the allosteric site of the mutant IDH1. Enzymatic kinetics also demonstrated that clomifene inhibited mutant enzyme in a non-competitive manner. Moreover, knockdown of mutant IDH1 in HT1080 cells decreased the sensitivity to clomifene. In vivo studies indicated that clomifene significantly suppressed the tumor growth of HT1080-bearing CB-17/Icr-scid mice with oral administration of 100 mg/kg and 50 mg/kg per day. In short, our findings highlight clomifene may have clinical potential in tumor therapies as a safe and effective inhibitor of mutant IDH1. PMID:28498812

[Construction and stress tolerance of trehalase mutant in Saccharomyces cerevisiae].

PubMed

Lv, Ye; Xiao, Dongguang; He, Dongqin; Guo, Xuewu

2008-10-01

Accumulation of trehalose is critical in improving the stress tolerance of Saccharomyces cerevisiae. Two enzymes are capable of hydrolyzing trehalose: a neutral trehalase (NTH1) and an acidic trehalase (ATH1). We constructed trehalase disruption mutants to provide a basis for future commercial application. To retain the accumulation of trehalose in yeast cell, we constructed diploid homozygous neutral trehalase mutants (Deltanth1), acid trehalase mutants (Deltaath1) and double mutants (Deltaath1Deltanth1) by using gene disruption. We tested mutants'trehalose content and their tolerance to freezing, heat, high-sugar and ethanol concentrations. These trehalase disruption mutants were further confirmed by PCR amplification and southern blot. All mutant strains accumulated higher levels of cellular trehalose and grew to a higher cell density than the isogenic parent strain. In addition, the levels of trehalose in these mutants correlated with increased tolerance to freezing, heat, high-sugar and ethanol concentration. The improved tolerance of trehalase mutants may make them useful in commercial applications, including baking and brewing protein.

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

Computing border bases using mutant strategies

NASA Astrophysics Data System (ADS)

Ullah, E.; Abbas Khan, S.

2014-01-01

Border bases, a generalization of Gröbner bases, have actively been addressed during recent years due to their applicability to industrial problems. In cryptography and coding theory a useful application of border based is to solve zero-dimensional systems of polynomial equations over finite fields, which motivates us for developing optimizations of the algorithms that compute border bases. In 2006, Kehrein and Kreuzer formulated the Border Basis Algorithm (BBA), an algorithm which allows the computation of border bases that relate to a degree compatible term ordering. In 2007, J. Ding et al. introduced mutant strategies bases on finding special lower degree polynomials in the ideal. The mutant strategies aim to distinguish special lower degree polynomials (mutants) from the other polynomials and give them priority in the process of generating new polynomials in the ideal. In this paper we develop hybrid algorithms that use the ideas of J. Ding et al. involving the concept of mutants to optimize the Border Basis Algorithm for solving systems of polynomial equations over finite fields. In particular, we recall a version of the Border Basis Algorithm which is actually called the Improved Border Basis Algorithm and propose two hybrid algorithms, called MBBA and IMBBA. The new mutants variants provide us space efficiency as well as time efficiency. The efficiency of these newly developed hybrid algorithms is discussed using standard cryptographic examples.

Study the Expression of ompf Gene in Esherichia coli Mutants.

PubMed

Jaktaji, R Pourahmad; Heidari, F

2013-09-01

The outer membrane porin proteins are the major factors in controlling the permeability of cell membrane. OmpF is an example of porin proteins in Esherichia coli. In normal growth condition a large amount of this protein is synthesised, but under stress condition, such as the presence of antibiotics in environment its expression is decreased inhibiting the entrance of antibiotics into cell. The expression of ompF is inhibited by antisense RNA transcribed from micF. In normal condition the expression of micF is low, but in the presence of antibiotics its expression is increased and causes multiple resistances to irrelevant antibiotics. The aims of this research were to study first, the intactness of micF and then quantify the expression of ompF in ciprofloxacin and tetracycline resistant mutants of E. coli. For this purpose the 5' end of micF was amplified and then sequenced. None of these mutants except one and its clone has a mutation in this gene. Then the relative expression of ompF in these mutants was quantified by real time PCR. There was no significant difference between ompF transcription of mutants and wild type strain. Based on this study and previous study it is concluded that low to intermediate levels of resistance to ciprofloxacin and tetracycline does not decrease ompF transcription.

Study the Expression of ompf Gene in Esherichia coli Mutants

PubMed Central

Jaktaji, R. Pourahmad; Heidari, F.

2013-01-01

The outer membrane porin proteins are the major factors in controlling the permeability of cell membrane. OmpF is an example of porin proteins in Esherichia coli. In normal growth condition a large amount of this protein is synthesised, but under stress condition, such as the presence of antibiotics in environment its expression is decreased inhibiting the entrance of antibiotics into cell. The expression of ompF is inhibited by antisense RNA transcribed from micF. In normal condition the expression of micF is low, but in the presence of antibiotics its expression is increased and causes multiple resistances to irrelevant antibiotics. The aims of this research were to study first, the intactness of micF and then quantify the expression of ompF in ciprofloxacin and tetracycline resistant mutants of E. coli. For this purpose the 5’ end of micF was amplified and then sequenced. None of these mutants except one and its clone has a mutation in this gene. Then the relative expression of ompF in these mutants was quantified by real time PCR. There was no significant difference between ompF transcription of mutants and wild type strain. Based on this study and previous study it is concluded that low to intermediate levels of resistance to ciprofloxacin and tetracycline does not decrease ompF transcription. PMID:24403654

An attenuated quadruple gene mutant of Mycobacterium tuberculosis imparts protection against tuberculosis in guinea pigs

PubMed Central

Chauhan, Priyanka

2018-01-01

ABSTRACT Previously we had developed a triple gene mutant of Mycobacterium tuberculosis (MtbΔmms) harboring disruption in three genes, namely mptpA, mptpB and sapM. Though vaccination with MtbΔmms strain induced protection in the lungs of guinea pigs, the mutant strain failed to control the hematogenous spread of the challenge strain to the spleen. Additionally, inoculation with MtbΔmms resulted in some pathological damage to the spleens in the early phase of infection. In order to generate a strain that overcomes the pathology caused by MtbΔmms in spleen of guinea pigs and controls dissemination of the challenge strain, MtbΔmms was genetically modified by disrupting bioA gene to generate MtbΔmmsb strain. Further, in vivo attenuation of MtbΔmmsb was evaluated and its protective efficacy was assessed against virulent M. tuberculosis challenge in guinea pigs. MtbΔmmsb mutant strain was highly attenuated for growth and virulence in guinea pigs. Vaccination with MtbΔmmsb mutant generated significant protection in comparison to sham-immunized animals at 4 and 12 weeks post-infection in lungs and spleen of infected animals. However, the protection imparted by MtbΔmmsb was significantly less in comparison to BCG immunized animals. This study indicates the importance of attenuated multiple gene deletion mutants of M. tuberculosis for generating protection against tuberculosis. PMID:29242198

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

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

Isolation and characterisation of a dwarf rice mutant exhibiting defective gibberellins biosynthesis.

PubMed

Ji, S H; Gururani, M A; Lee, J W; Ahn, B-O; Chun, S-C

2014-03-01

We have isolated a severe dwarf mutant derived from a Ds (Dissociation) insertion mutant rice (Oryza sativa var. japonica c.v. Dongjin). This severe dwarf phenotype, has short and dark green leaves, reduced shoot growth early in the seedling stage, and later severe dwarfism with failure to initiate flowering. When treated with bioactive GA3 , mutants are restored to the normal wild-type phenotype. Reverse transcription PCR analyses of 22 candidate genes related to the gibberellin (GA) biosynthesis pathway revealed that among 22 candidate genes tested, a dwarf mutant transcript was not expressed only in one OsKS2 gene. Genetic analysis revealed that the severe dwarf phenotype was controlled by recessive mutation of a single nuclear gene. The putative OsKS2 gene was a chromosome 4-located ent-kaurene synthase (KS), encoding the enzyme that catalyses an early step of the GA biosynthesis pathway. Sequence analysis revealed that osks2 carried a 1-bp deletion in the ORF region of OsKS2, which led to a loss-of-function mutation. The expression pattern of OsKS2 in wild-type cv Dongjin, showed that it is expressed in all organs, most prominently in the stem and floral organs. Morphological characteristics of the dwarf mutant showed dramatic modifications in internal structure and external morphology. We propose that dwarfism in this mutant is caused by a point mutation in OsKS2, which plays a significant role in growth and development of higher plants. Further investigation on OsKS2 and other OsKS-like proteins is underway and may yield better understanding of the putative role of OsKS in severe dwarf mutants. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

Isolation and characterization of gallium resistant Pseudomonas aeruginosa mutants.

PubMed

García-Contreras, Rodolfo; Lira-Silva, Elizabeth; Jasso-Chávez, Ricardo; Hernández-González, Ismael L; Maeda, Toshinari; Hashimoto, Takahiro; Boogerd, Fred C; Sheng, Lili; Wood, Thomas K; Moreno-Sánchez, Rafael

2013-12-01

Pseudomonas aeruginosa PA14 cells resistant to the novel antimicrobial gallium nitrate (Ga) were developed using transposon mutagenesis and by selecting spontaneous mutants. The mutants showing the highest growth in the presence of Ga were selected for further characterization. These mutants showed 4- to 12-fold higher Ga minimal inhibitory growth concentrations and a greater than 8-fold increase in the minimum biofilm eliminating Ga concentration. Both types of mutants produced Ga resistant biofilms whereas the formation of wild-type biofilms was strongly inhibited by Ga. The gene interrupted in the transposon mutant was hitA, which encodes a periplasmic iron binding protein that delivers Fe³⁺ to the HitB iron permease; complementation of the mutant with the hitA gene restored the Ga sensitivity. This hitA mutant showed a 14-fold decrease in Ga internalization versus the wild-type strain, indicating that the HitAB system is also involved in the Ga uptake. Ga uptake in the spontaneous mutant was also lower, although no mutations were found in the hitAB genes. Instead, this mutant harbored 64 non-silent mutations in several genes including those of the phenazine pyocyanin biosynthesis. The spontaneous mutant produced 2-fold higher pyocyanin basal levels than the wild-type; the addition of this phenazine to wild-type cultures protected them from the Ga bacteriostatic effect. The present data indicate that mutations affecting Ga transport and probably pyocyanin biosynthesis enable cells to develop resistance to Ga. Copyright © 2013 Elsevier GmbH. All rights reserved.

Prominent dominant negative effect of a mutant Fas molecule lacking death domain on cell-mediated induction of apoptosis.

PubMed

Yokota, Aya; Takeuchi, Emiko; Iizuka, Misao; Ikegami, Yuko; Takayama, Hajime; Shinohara, Nobukata

2005-01-01

Using a panel of transfectant B lymphoma cells expressing varying amounts of the mutant Fas together with the endogenous wild type Fas, semi-quantitative studies on the dominant negative effect of a murine mutant Fas molecule lacking death domain were carried out. In anti-Fas antibody-mediated induction of apoptosis, the mutant molecules exerted significant dominant-negative effect only when their expression level was comparable to or higher than that of wild type molecules, or when exposed to low amounts of the antibody. The inhibitory effect was accompanied by the failure in DISC formation in spite of Fas aggregation. When they were subjected to T cell-mediated Fas-based induction of apoptosis, however, the dominant negative effect was prominent such that the expression of even a small amount of the mutant molecules resulted in significant inhibition. Such a strong inhibitory effect explains the dominant phenotype of this type of mutant Fas molecules in ALPS heterozygous patients and also implies that the physiological effectors for Fas in vivo are cells, i.e., FasL-expressing activated T cells.

Circulation of Pneumocystis dihydropteroate synthase mutants in France.

PubMed

Le Gal, Solène; Damiani, Céline; Perrot, Maëla; Rouillé, Amélie; Virmaux, Michèle; Quinio, Dorothée; Moalic, Elodie; Saliou, Philippe; Berthou, Christian; Le Meur, Yann; Totet, Anne; Nevez, Gilles

2012-10-01

Data on the prevalence of Pneumocystis jirovecii (P. jirovecii) dihydropteroate synthase (DHPS) mutants in France are still limited. In this study, mutant prevalence in the Brest region (western France) was determined. Archival pulmonary specimens from 85 patients infected with P. jirovecii and admitted to our institution (University Hospital, Brest) from October 2007 to February 2010 were retrospectively typed at the DHPS locus using a polymerase chain reaction-restriction fragment length polymorphism assay. Type identification was successful in 66 of 85 patients. Sixty-four patients were infected with a wild type, whereas mutants were found in 2 patients (2/66, 3%). Medical chart analysis revealed that these 2 patients usually lived in Paris. Another patient usually lived on the French Riviera, whereas 63 patients were from the city of Brest. Thus, the corrected prevalence of mutants in patients who effectively lived in our geographic area was 0% (0/63). Taking into account that i) Paris is characterized by a high prevalence of mutants from 18.5% to 40%, ii) infection diagnoses were performed in the 2 Parisians during their vacation <30 days, iii) infection incubation is assumed to last about 2 months, the results provide evidence of mutant circulation from Paris to Brest through infected vacationers. The study shows that the usual city of patient residence, rather than the city of infection diagnosis, is a predictor of mutants and that P. jirovecii infections involving mutants do not represent a public health issue in western France. Copyright © 2012 Elsevier Inc. All rights reserved.

Leishmania infantum Lipophosphoglycan-Deficient Mutants: A Tool to Study Host Cell-Parasite Interplay

PubMed Central

Lázaro-Souza, Milena; Matte, Christine; Lima, Jonilson B.; Arango Duque, Guillermo; Quintela-Carvalho, Graziele; de Carvalho Vivarini, Áislan; Moura-Pontes, Sara; Figueira, Cláudio P.; Jesus-Santos, Flávio H.; Gazos Lopes, Ulisses; Farias, Leonardo P.; Araújo-Santos, Théo; Descoteaux, Albert; Borges, Valéria M.

2018-01-01

Lipophosphoglycan (LPG) is the major surface glycoconjugate of metacyclic Leishmania promastigotes and is associated with virulence in various species of this parasite. Here, we generated a LPG-deficient mutant of Leishmania infantum, the foremost etiologic agent of visceral leishmaniasis in Brazil. The L. infantum LPG-deficient mutant (Δlpg1) was obtained by homologous recombination and complemented via episomal expression of LPG1 (Δlpg1 + LPG1). Deletion of LPG1 had no observable effect on parasite morphology or on the presence of subcellular organelles, such as lipid droplets. While both wild-type and add-back parasites reached late phase in axenic cultures, the growth of Δlpg1 parasites was delayed. Additionally, the deletion of LPG1 impaired the outcome of infection in murine bone marrow-derived macrophages. Although no significant differences were observed in parasite load after 4 h of infection, survival of Δlpg1 parasites was significantly reduced at 72 h post-infection. Interestingly, L. infantum LPG-deficient mutants induced a strong NF-κB-dependent activation of the inducible nitric oxide synthase (iNOS) promoter compared to wild type and Δlpg1 + LPG1 parasites. In conclusion, the L. infantum Δlpg1 mutant constitutes a powerful tool to investigate the role(s) played by LPG in host cell-parasite interactions. PMID:29675001

Transcriptome sequencing and metabolic pathways of astaxanthin accumulated in Haematococcus pluvialis mutant under 15% CO2.

PubMed

Cheng, Jun; Li, Ke; Zhu, Yanxia; Yang, Weijuan; Zhou, Junhu; Cen, Kefa

2017-03-01

Transcriptome sequencing and annotation was performed on Haematococcus pluvialis mutant red cells induced with high light under 15% CO 2 to demonstrate why astaxanthin yield of the mutant was 1.7 times higher than that of a wild strain. It was found that 56% of 1947 differentially expressed genes were upregulated in mutant cells. Most significant differences were found in unigenes related to photosynthesis, carotenoid biosynthesis and fatty acid biosynthesis pathways. The pyruvate kinase increased by 3.5-fold in mutant cells. Thus, more pyruvate, which was beneficial to carotenoids and fatty acid biosynthesis, was generated. Phytoene synthase, zeta-carotene desaturase, lycopene beta-cyclase involved in β-carotene biosynthesis in mutant cells were upregulated by 10.4-, 4.4-, and 5.8-fold, respectively. Beta-carotene 3-hydroxylase catalyzing conversion of β-carotene into astaxanthin was upregulated by 18.4-fold. The fatty acid biosynthesis was promoted because of the upregulation of acetyl-CoA synthetase and acetyl-CoA carboxylase, thus increasing astaxanthin esterification and accumulation in mutant cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Native Mutant Huntingtin in Human Brain

PubMed Central

Sapp, Ellen; Valencia, Antonio; Li, Xueyi; Aronin, Neil; Kegel, Kimberly B.; Vonsattel, Jean-Paul; Young, Anne B.; Wexler, Nancy; DiFiglia, Marian

2012-01-01

Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575–850 kDa in control brain and at 650–885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1–17)) and increased when lysates were treated with denaturants (SDS, 8 m urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670–880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43–50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 m urea + DTT. Native full-length mutant htt in embryonic HD140Q/140Q mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer. PMID:22375012

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. © 2015 The Society for Applied Microbiology.

Blinded histopathological characterisation of POLE exonuclease domain-mutant endometrial cancers: sheep in wolf's clothing.

PubMed

Van Gool, Inge C; Ubachs, Jef E H; Stelloo, Ellen; de Kroon, Cor D; Goeman, Jelle J; Smit, Vincent T H B M; Creutzberg, Carien L; Bosse, Tjalling

2018-01-01

POLE exonuclease domain mutations identify a subset of endometrial cancer (EC) patients with an excellent prognosis. The use of this biomarker has been suggested to refine adjuvant treatment decisions, but the necessary sequencing is not widely performed and is relatively expensive. Therefore, we aimed to identify histopathological and immunohistochemical characteristics to aid in the detection of POLE-mutant ECs. Fifty-one POLE-mutant endometrioid, 67 POLE-wild-type endometrioid and 15 POLE-wild-type serous ECs were included (total N = 133). An expert gynaecopathologist, blinded to molecular features, evaluated each case (two or more slides) for 16 morphological characteristics. Immunohistochemistry was performed for p53, p16, MLH1, MSH2, MSH6, and PMS2. POLE-mutant ECs were characterised by a prominent immune infiltrate: 80% showed peritumoral lymphocytes and 59% showed tumour-infiltrating lymphocytes, as compared with 43% and 28% of POLE-wild-type endometrioid ECs, and 27% and 13% of their serous counterparts (P < 0.01, all comparisons). Of POLE-mutant ECs, 33% contained tumour giant cells; this proportion was significantly higher than that in POLE-wild-type endometrioid ECs (10%; P = 0.003), but not significantly different from that in serous ECs (53%). Serous-like features were as often (focally) present in POLE-mutant as in POLE-wild-type endometrioid ECs (6-24%, depending on the feature). The majority of POLE-mutant ECs showed wild-type p53 (86%), negative/focal p16 (82%) and normal mismatch repair protein expression (90%). A simple combination of morphological and immunohistochemical characteristics (tumour type, grade, peritumoral lymphocytes, MLH1, and p53 expression) can assist in prescreening for POLE exonuclease domain mutations in EC, increasing the probability of a mutation being detected from 7% to 33%. This facilitates the use of this important prognostic biomarker in routine pathology. © 2017 John Wiley & Sons Ltd.

Misfolded opsin mutants display elevated β -sheet structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, Lisa M.; Gragg, Megan; Kim, Tae Gyun

Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Also, both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate themore » aggregation of misfolded opsin mutants. In conclusion, the effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself.« less

Misfolded opsin mutants display elevated β -sheet structure

DOE PAGES

Miller, Lisa M.; Gragg, Megan; Kim, Tae Gyun; ...

2015-09-07

Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Also, both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate themore » aggregation of misfolded opsin mutants. In conclusion, the effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself.« less

Whole-Genome Sequencing of Sordaria macrospora Mutants Identifies Developmental Genes.

PubMed

Nowrousian, Minou; Teichert, Ines; Masloff, Sandra; Kück, Ulrich

2012-02-01

The study of mutants to elucidate gene functions has a long and successful history; however, to discover causative mutations in mutants that were generated by random mutagenesis often takes years of laboratory work and requires previously generated genetic and/or physical markers, or resources like DNA libraries for complementation. Here, we present an alternative method to identify defective genes in developmental mutants of the filamentous fungus Sordaria macrospora through Illumina/Solexa whole-genome sequencing. We sequenced pooled DNA from progeny of crosses of three mutants and the wild type and were able to pinpoint the causative mutations in the mutant strains through bioinformatics analysis. One mutant is a spore color mutant, and the mutated gene encodes a melanin biosynthesis enzyme. The causative mutation is a G to A change in the first base of an intron, leading to a splice defect. The second mutant carries an allelic mutation in the pro41 gene encoding a protein essential for sexual development. In the mutant, we detected a complex pattern of deletion/rearrangements at the pro41 locus. In the third mutant, a point mutation in the stop codon of a transcription factor-encoding gene leads to the production of immature fruiting bodies. For all mutants, transformation with a wild type-copy of the affected gene restored the wild-type phenotype. Our data demonstrate that whole-genome sequencing of mutant strains is a rapid method to identify developmental genes in an organism that can be genetically crossed and where a reference genome sequence is available, even without prior mapping information.

Whole-Genome Sequencing of Sordaria macrospora Mutants Identifies Developmental Genes

PubMed Central

Nowrousian, Minou; Teichert, Ines; Masloff, Sandra; Kück, Ulrich

2012-01-01

The study of mutants to elucidate gene functions has a long and successful history; however, to discover causative mutations in mutants that were generated by random mutagenesis often takes years of laboratory work and requires previously generated genetic and/or physical markers, or resources like DNA libraries for complementation. Here, we present an alternative method to identify defective genes in developmental mutants of the filamentous fungus Sordaria macrospora through Illumina/Solexa whole-genome sequencing. We sequenced pooled DNA from progeny of crosses of three mutants and the wild type and were able to pinpoint the causative mutations in the mutant strains through bioinformatics analysis. One mutant is a spore color mutant, and the mutated gene encodes a melanin biosynthesis enzyme. The causative mutation is a G to A change in the first base of an intron, leading to a splice defect. The second mutant carries an allelic mutation in the pro41 gene encoding a protein essential for sexual development. In the mutant, we detected a complex pattern of deletion/rearrangements at the pro41 locus. In the third mutant, a point mutation in the stop codon of a transcription factor-encoding gene leads to the production of immature fruiting bodies. For all mutants, transformation with a wild type-copy of the affected gene restored the wild-type phenotype. Our data demonstrate that whole-genome sequencing of mutant strains is a rapid method to identify developmental genes in an organism that can be genetically crossed and where a reference genome sequence is available, even without prior mapping information. PMID:22384404

NRAS-mutant melanoma: current challenges and future prospect

PubMed Central

Muñoz-Couselo, Eva; Adelantado, Ester Zamora; Ortiz, Carolina; García, Jesús Soberino; Perez-Garcia, José

2017-01-01

Melanoma is one of the most common cutaneous cancers worldwide. Activating mutations in RAS oncogenes are found in a third of all human cancers and NRAS mutations are found in 15%–20% of melanomas. The NRAS-mutant subset of melanoma is more aggressive and associated with poorer outcomes, compared to non-NRAS-mutant melanoma. Although immune checkpoint inhibitors and targeted therapies for BRAF-mutant melanoma are transforming the treatment of metastatic melanoma, the ideal treatment for NRAS-mutant melanoma remains unknown. Despite promising preclinical data, current therapies for NRAS-mutant melanoma remain limited, showing a modest increase in progression-free survival but without any benefit in overall survival. Combining MEK inhibitors with agents inhibiting cell cycling and the PI3K–AKT pathway appears to provide additional benefit; in particular, a strategy of MEK inhibition and CDK4/6 inhibition is likely to be a viable treatment option in the future. Patients whose tumors had NRAS mutations had better response to immunotherapy and better outcomes than patients whose tumors had other genetic subtypes, suggesting that immune therapies – especially immune checkpoint inhibitors – may be particularly effective as treatment options for NRAS-mutant melanoma. Improved understanding of NRAS-mutant melanoma will be essential to develop new treatment strategies for this subset of patients with melanoma. PMID:28860801

Comparison of Zebrafish tmem88a mutant and morpholino knockdown phenotypes

PubMed Central

Place, Elsie S.; Smith, James C.

2017-01-01

Tmem88a is a transmembrane protein that is thought to be a negative regulator of the Wnt signalling pathway. Several groups have used antisense morpholino oligonucleotides in an effort to characterise the role of tmem88a in zebrafish cardiovascular development, but they have not obtained consistent results. Here, we generate an 8 bp deletion in the coding region of the tmem88a locus using TALENs, and we have gone on to establish a viable homozygous tmem88aΔ8 mutant line. Although tmem88aΔ8 mutants have reduced expression of some key haematopoietic genes, differentiation of erythrocytes and neutrophils is unaffected, contradicting our previous study using antisense morpholino oligonucleotides. We find that expression of the tmem88a paralogue tmem88b is not significantly changed in tmem88aΔ8 mutants and injection of the tmem88a splice-blocking morpholino oligonucleotide into tmem88aΔ8 mutants recapitulates the reduction of erythrocytes observed in morphants using o-Dianisidine. This suggests that there is a partial, but inessential, requirement for tmem88a during haematopoiesis and that morpholino injection exacerbates this phenotype in tmem88a morpholino knockdown embryos. PMID:28192479

Isolation, characterization, and complementation of a motility mutant of Spiroplasma citri.

PubMed Central

Jacob, C; Nouzières, F; Duret, S; Bové, J M; Renaudin, J

1997-01-01

The helical mollicute Spiroplasma citri, when growing on low-agar medium, forms fuzzy colonies with occasional surrounding satellite colonies due to the ability of the spiroplasmal cells to move through the agar matrix. In liquid medium, these helical organisms flex, twist, and rotate rapidly. By using Tn4001 insertion mutagenesis, a motility mutant was isolated on the basis of its nondiffuse, sharp-edged colonies. Dark-field microscopy observations revealed that the organism flexed at a low frequency and had lost the ability to rotate about the helix axis. In this mutant, the transposon was shown to be inserted into an open reading frame encoding a putative polypeptide of 409 amino acids for which no significant homology with known proteins was found. The corresponding gene, named scm1, was recovered from the wild-type strain and introduced into the motility mutant by using the S. citri oriC plasmid pBOT1 as the vector. The appearance of fuzzy colonies and the observation that spiroplasma cells displayed rotatory and flexional movements showed the motile phenotype to be restored in the spiroplasmal transformants. The functional complementation of the motility mutant proves the scm1 gene product to be involved in the motility mechanism of S. citri. PMID:9244268

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.

Genetics of Ustilago violacea. I. Carotenoid mutants and carotenogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garber, E.D.; Baird, M.L.; Chapman, D.J.

1975-12-01

Wild-type strains of Ustilago violacea produce pink colonies on laboratory medium and yield white, orange, pumpkin, and yellow colonies after uv mutagenesis. The wild-type strains contain neurosporene and lycopene; one orange mutant, $gamma$-carotene; and one yellow mutant, $beta$-carotene. One white mutant had no detectable carotenoids. Diploid colonies heterozygous for wild type and orange, pumpkin, yellow, or white are phenotypically wild type. Diploid colonies heterozygous for yellow and orange are also phenotypically wild type. Diploid colonies heterozygous for white and orange; white and yellow; and white, yellow, and orange are phenotypically light orange, light yellow, and orange- yellow, respectively. The whitemore » mutants give a circular complementation map; the color mutants fit a linear complementation map. We propose a multienzyme of four identical dehydrogenases and one or two identical cyclases for carotenogenesis in this species. The white and color mutants represent structural mutations altering the conformation of the dehydrogenase or cyclase, respectively. Furthermore, cyclases may or may not aggregate in association with the dehydrogenase aggregate to form the multienzyme aggregate responsible for the color mutants. (auth)« less

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.

Efficacy of BET bromodomain inhibition in Kras-mutant non-small cell lung cancer

PubMed Central

Shimamura, Takeshi; Chen, Zhao; Soucheray, Margaret; Carretero, Julian; Kikuchi, Eiki; Tchaicha, Jeremy H.; Gao, Yandi; Cheng, Katherine A.; Cohoon, Travis J.; Qi, Jun; Akbay, Esra; Kimmelman, Alec C.; Kung, Andrew L.; Bradner, James E.; Wong, Kwok-Kin

2013-01-01

Purpose Amplification of MYC is one of the most common genetic alterations in lung cancer, contributing to a myriad of phenotypes associated with growth, invasion and drug resistance. Murine genetics has established both the centrality of somatic alterations of Kras in lung cancer, as well as the dependency of mutant Kras tumors on MYC function. Unfortunately, drug-like small-molecule inhibitors of KRAS and MYC have yet to be realized. The recent discovery, in hematologic malignancies, that BET bromodomain inhibition impairs MYC expression and MYC transcriptional function established the rationale of targeting KRAS-driven NSCLC with BET inhibition. Experimental Design We performed functional assays to evaluate the effects of JQ1 in genetically defined NSCLC cells lines harboring KRAS and/or LKB1 mutations. Furthermore, we evaluated JQ1 in transgenic mouse lung cancer models expressing mutant kras or concurrent mutant kras and lkb1. Effects of bromodomain inhibition on transcriptional pathways were explored and validated by expression analysis. Results While JQ1 is broadly active in NSCLC cells, activity of JQ1 in mutant KRAS NSCLC is abrogated by concurrent alteration or genetic knock-down of LKB1. In sensitive NSCLC models, JQ1 treatment results in the coordinate downregulation of the MYC-dependent transcriptional program. We found that JQ1 treatment produces significant tumor regression in mutant kras mice. As predicted, tumors from mutant kras and lkb1 mice did not respond to JQ1. Conclusion Bromodomain inhibition comprises a promising therapeutic strategy for KRAS mutant NSCLC with wild-type LKB1, via inhibition of MYC function. Clinical studies of BET bromodomain inhibitors in aggressive NSCLC will be actively pursued. PMID:24045185

CHO-cell mutant with a defect in cytokinesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson, L.H.; Lindl, P.A.

1976-01-01

In a selection procedure designed to enrich for temperature-sensitive mutant cells blocked in mitosis a CHO-cell mutant was isolated which has a defect in cytokinesis as the basis of its temperature-sensitive phenotype. Cultures of the mutant had an abnormally high percentage (ie, 34 percent) of polyploid cells at the permissive temperature of 34/sup 0/C and showed further increased frequencies of polyploidy as well as many multinucleated cells at 38.5/sup 0/ and 39.5/sup 0/. When the mutant cells were synchronized in metaphase by Colcemid arrest and then placed into fresh medium at nonpermissive temperature, they did not divide although the completionmore » of mitosis appeared cytologically normal. Ultrastructural examination by electron microscopy of such synchronized cells at telophase revealed no specific defects in cellular components other than failure of development of a normal midbody. The sensitivity of the mutant to cytochalasin B and to Colcemid was the same as for wild-type cells. This mutation behaved as recessive in tetraploid cell hybrids constructed by fusing the mutant with a CHO strain which was wild-type with respect to temperature sensitivity.« less

When the good go bad: Mutant NPM1 in acute myeloid leukemia.

PubMed

Kunchala, Preethi; Kuravi, Sudhakiranmayi; Jensen, Roy; McGuirk, Joseph; Balusu, Ramesh

2018-05-01

Nucleophosmin 1 (NPM1) is a nucleolar phosphoprotein that performs diverse biological functions including molecular chaperoning, ribosome biogenesis, DNA repair, and genome stability. Acute myeloid leukemia (AML) is a heterogeneous disease, more than half of the AML cases exhibit normal karyotype (NK). Approximately 50-60 percent of patients with NK-AML carry NPM1 mutations which are characterized by cytoplasmic dislocation of the NPM1 protein. In AML, mutant NPM1 (NPM1c+) acts in a dominant negative fashion and also blocks the differentiation of myeloid cells through gain-of-function for the AML phenotype. Currently, there is limited knowledge on the gain-of-function mechanism of mutant NPM1. Here, we review the known mechanisms of mutant NPM1 in the pathogenesis of AML. We describe genetic abnormalities, the clinical significance of exon-12 mutations in the NPM1 gene, and chromosomal translocations including the recently discovered NPM1-TYK2, and NPM1-HAUS1. Also, we outline the possible therapeutic interventions for the treatment of AML by targeting NPM1. Overall, the review will summarize present knowledge on mutant NPM1 origin, pathogenesis, and therapy in AML. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

EGFRvIII-specific chimeric antigen receptor T cells migrate to and kill tumor deposits infiltrating the brain parenchyma in an invasive xenograft model of glioblastoma.

PubMed

Miao, Hongsheng; Choi, Bryan D; Suryadevara, Carter M; Sanchez-Perez, Luis; Yang, Shicheng; De Leon, Gabriel; Sayour, Elias J; McLendon, Roger; Herndon, James E; Healy, Patrick; Archer, Gary E; Bigner, Darell D; Johnson, Laura A; Sampson, John H

2014-01-01

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and is uniformly lethal. T-cell-based immunotherapy offers a promising platform for treatment given its potential to specifically target tumor tissue while sparing the normal brain. However, the diffuse and infiltrative nature of these tumors in the brain parenchyma may pose an exceptional hurdle to successful immunotherapy in patients. Areas of invasive tumor are thought to reside behind an intact blood brain barrier, isolating them from effective immunosurveillance and thereby predisposing the development of "immunologically silent" tumor peninsulas. Therefore, it remains unclear if adoptively transferred T cells can migrate to and mediate regression in areas of invasive GBM. One barrier has been the lack of a preclinical mouse model that accurately recapitulates the growth patterns of human GBM in vivo. Here, we demonstrate that D-270 MG xenografts exhibit the classical features of GBM and produce the diffuse and invasive tumors seen in patients. Using this model, we designed experiments to assess whether T cells expressing third-generation chimeric antigen receptors (CARs) targeting the tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, would localize to and treat invasive intracerebral GBM. EGFRvIII-targeted CAR (EGFRvIII+ CAR) T cells demonstrated in vitro EGFRvIII antigen-specific recognition and reactivity to the D-270 MG cell line, which naturally expresses EGFRvIII. Moreover, when administered systemically, EGFRvIII+ CAR T cells localized to areas of invasive tumor, suppressed tumor growth, and enhanced survival of mice with established intracranial D-270 MG tumors. Together, these data demonstrate that systemically administered T cells are capable of migrating to the invasive edges of GBM to mediate antitumor efficacy and tumor regression.

Protein expression profiling of the drosophila fragile X mutant brain reveals up-regulation of monoamine synthesis.

PubMed

Zhang, Yong Q; Friedman, David B; Wang, Zhe; Woodruff, Elvin; Pan, Luyuan; O'donnell, Janis; Broadie, Kendal

2005-03-01

Fragile X syndrome is the most common form of inherited mental retardation, associated with both cognitive and behavioral anomalies. The disease is caused by silencing of the fragile X mental retardation 1 (fmr1) gene, which encodes the mRNA-binding, translational regulator FMRP. Previously we established a disease model through mutation of Drosophila fmr1 (dfmr1) and showed that loss of dFMRP causes defects in neuronal structure, function, and behavioral output similar to the human disease state. To uncover molecular targets of dFMRP in the brain, we use here a proteomic approach involving two-dimensional difference gel electrophoresis analyses followed by mass spectrometry identification of proteins with significantly altered expression in dfmr1 null mutants. We then focus on two misregulated enzymes, phenylalanine hydroxylase (Henna) and GTP cyclohydrolase (Punch), both of which mediate in concert the synthetic pathways of two key monoamine neuromodulators, dopamine and serotonin. Brain enzymatic assays show a nearly 2-fold elevation of Punch activity in dfmr1 null mutants. Consistently brain neurochemical assays show that both dopamine and serotonin are significantly increased in dfmr1 null mutants. At a cellular level, dfmr1 null mutant neurons display a highly significant elevation of the dense core vesicles that package these monoamine neuromodulators for secretion. Taken together, these data indicate that dFMRP normally down-regulates the monoamine pathway, which is consequently up-regulated in the mutant condition. Elevated brain levels of dopamine and serotonin provide a plausible mechanistic explanation for aspects of cognitive and behavioral deficits in human patients.

Chemotaxis-defective mutants of the nematode Caenorhabditis elegans.

PubMed

Dusenbery, D B; Sheridan, R E; Russell, R L

1975-06-01

The technique of countercurrent separation has been used to isolate 17 independent chemotaxis-defective mutants of the nematode Caenorhabditis elegans. The mutants, selected to be relatively insensitive to the normally attractive salt NaCl, show varying degrees of residual sensitivity; some are actually weakly repelled by NaCl. The mutants are due to single gene defects, are autosomal and recessive, and identify at least five complementation groups.

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

Variations in seed protein content of cotton (Gossypium hirsutum L.) mutant lines by in vivo and in vitro mutagenesis.

PubMed

Muthusamy, Annamalai; Jayabalan, Narayanasamy

2013-01-01

The present work describes the influence of gamma irradiation (GR), ethyl methane sulphonate (EMS) and sodium azide (SA) treatment on yield and protein content of selected mutant lines of cotton. Seeds of MCU 5 and MCU 11 were exposed to gamma rays (GR), ethyl methane sulphonate (EMS) and sodium azide (SA). Lower dose of gamma irradiation (100-500 Gy), 10-50 mM EMS and SA at lower concentration effectively influences in improving the yield and protein content. Significant increase in yield (258.9 g plant(-1)) and protein content (18.63 mg g(-1) d. wt.) as compared to parental lines was noted in M2 generations. During the subsequent field trials, number of mutant lines varied morphologically in terms of yield as well as biochemical characters such as protein. The selected mutant lines were bred true to their characters in M3 and M4 generations. The significant increase in protein content and profiles of the mutant lines with range of 10.21-18.63 mg g(-1). The SDS-PAGE analysis of mutant lines revealed 9 distinct bands of different intensities with range of 26-81 kDa. The difference in intensity of bands was more (41, 50 and 58 kDa) in the mutant lines obtained from in vitro mutation than in vivo mutation. Significance of such stimulation in protein content correlated with yielding ability of the mutant lines of cotton in terms of seed weight per plant. The results confirm that in cotton it is possible to enhance the both yield and biochemical characters by in vivo and in vitro mutagenic treatments.

Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors

NASA Astrophysics Data System (ADS)

Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.

1990-12-01

Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

Mutant matrix metalloproteinase-9 reduces postoperative peritoneal adhesions in rats.

PubMed

Atta, Hussein; El-Rehany, Mahmoud; Roeb, Elke; Abdel-Ghany, Hend; Ramzy, Maggie; Gaber, Shereen

2016-02-01

Postoperative peritoneal adhesions continue to be a major source of morbidity and occasional mortality. Studies have shown that matrix metalloproteinase-9 (MMP-9) levels are decreased postoperatively which may limits matrix degradation and participate in the development of peritoneal adhesions. In this proof-of-principle study, we evaluated the effect of gene therapy with catalytically inactive mutant MMP-9 on postoperative peritoneal adhesions in rats. Adenovirus encoding mutant MMP-9 (Ad-mMMP-9) or saline was instilled in the peritoneal cavity after cecal and parietal peritoneal injury in rats. Expression of mutant MMP-9 transcript was verified by sequencing. Adenovirus E4 gene expression, adhesion scores, MMP-9, tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and transforming growth factor-β1 (TGF-β1) expression were evaluated at sacrifice one week after treatment. Both mutant MMP-9 transcripts and adenovirus E4 gene were expressed in Ad-mMMP-9 treated adhesions. Adhesions severity decreased significantly (p = 0.036) in the Ad-mMMP-9-treated compared with saline-treated adhesions. Expression of MMP-9 mRNA and protein were elevated (p = 0.001 and p = 0.029, respectively) in the Ad-mMMP-9-treated adhesions compared with saline-treated adhesions. While tPA levels were increased (p = 0.02) in Ad-mMMP-9 treated adhesions compared with saline-treated adhesions, TGF-β1 and PAI-1 levels were decreased (p = 0.017 and p = 0.042, respectively). No difference in mortality were found between groups (p = 0.64). Mutant MMP-9 gene therapy effectively transduced peritoneal adhesions resulting in reduction of severity of primary peritoneal adhesions. Copyright © 2016 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.

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

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.

Mutant p53 proteins counteract autophagic mechanism sensitizing cancer cells to mTOR inhibition.

PubMed

Cordani, Marco; Oppici, Elisa; Dando, Ilaria; Butturini, Elena; Dalla Pozza, Elisa; Nadal-Serrano, Mercedes; Oliver, Jordi; Roca, Pilar; Mariotto, Sofia; Cellini, Barbara; Blandino, Giovanni; Palmieri, Marta; Di Agostino, Silvia; Donadelli, Massimo

2016-08-01

Mutations in TP53 gene play a pivotal role in tumorigenesis and cancer development. Here, we report that gain-of-function mutant p53 proteins inhibit the autophagic pathway favoring antiapoptotic effects as well as proliferation of pancreas and breast cancer cells. We found that mutant p53 significantly counteracts the formation of autophagic vesicles and their fusion with lysosomes throughout the repression of some key autophagy-related proteins and enzymes as BECN1 (and P-BECN1), DRAM1, ATG12, SESN1/2 and P-AMPK with the concomitant stimulation of mTOR signaling. As a paradigm of this mechanism, we show that atg12 gene repression was mediated by the recruitment of the p50 NF-κB/mutant p53 protein complex onto the atg12 promoter. Either mutant p53 or p50 NF-κB depletion downregulates atg12 gene expression. We further correlated the low expression levels of autophagic genes (atg12, becn1, sesn1, and dram1) with a reduced relapse free survival (RFS) and distant metastasis free survival (DMFS) of breast cancer patients carrying TP53 gene mutations conferring a prognostic value to this mutant p53-and autophagy-related signature. Interestingly, the mutant p53-driven mTOR stimulation sensitized cancer cells to the treatment with the mTOR inhibitor everolimus. All these results reveal a novel mechanism through which mutant p53 proteins promote cancer cell proliferation with the concomitant inhibition of autophagy. Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Registration of two allelic erect leaf mutants of sorghum

USDA-ARS?s Scientific Manuscript database

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...

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

Biochemical Analysis of Two Single Mutants that Give Rise to a Polymorphic G6PD A-Double Mutant

PubMed Central

Ramírez-Nava, Edson Jiovany; González-Valdez, Abigail; Vanoye-Carlo, America; Hernández-Ochoa, Beatriz; Sierra-Palacios, Edgar; Hernández-Pineda, Jessica; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto; Oria-Hernández, Jesús; Reyes-Vivas, Horacio; Marcial-Quino, Jaime

2017-01-01

Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme that plays a crucial role in the regulation of cellular energy and redox balance. Mutations in the gene encoding G6PD cause the most common enzymopathy that drives hereditary nonspherocytic hemolytic anemia. To gain insights into the effects of mutations in G6PD enzyme efficiency, we have investigated the biochemical, kinetic, and structural changes of three clinical G6PD variants, the single mutations G6PD A+ (Asn126AspD) and G6PD Nefza (Leu323Pro), and the double mutant G6PD A− (Asn126Asp + Leu323Pro). The mutants showed lower residual activity (≤50% of WT G6PD) and displayed important kinetic changes. Although all Class III mutants were located in different regions of the three-dimensional structure of the enzyme and were not close to the active site, these mutants had a deleterious effect over catalytic activity and structural stability. The results indicated that the G6PD Nefza mutation was mainly responsible for the functional and structural alterations observed in the double mutant G6PD A−. Moreover, our study suggests that the G6PD Nefza and G6PD A− mutations affect enzyme functions in a similar fashion to those reported for Class I mutations. PMID:29072585

Isolation of New Gravitropic Mutants under Hypergravity Conditions.

PubMed

Mori, Akiko; Toyota, Masatsugu; Shimada, Masayoshi; Mekata, Mika; Kurata, Tetsuya; Tasaka, Masao; Morita, Miyo T

2016-01-01

Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upward. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes). In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using next-generation sequencing (NGS) and single nucleotide polymorphism (SNP)-based markers. Using the endodermal-amyloplast less 1 ( eal1 ) mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g) restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene ( enhancer of eal1 ) mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis .

Isolation of ntrA-like mutants of Azotobacter vinelandii.

PubMed Central

Santero, E; Luque, F; Medina, J R; Tortolero, M

1986-01-01

A number of chlorate-resistant mutants of Azotobacter vinelandii affected in a general control of nitrogen metabolism were isolated. These mutants could not utilize dinitrogen, nitrate, or nitrite as a nitrogen source. The reason for this inability is that they were simultaneously deficient in nitrogenase and nitrate and nitrite reductase activities. They were complemented by a cosmid carrying a DNA fragment of A. vinelandii able to complement ntrA mutants of Escherichia coli, so they seemed to be ntrA-like mutants. PMID:3009406

Isolation and preliminary characterization of temperature-sensitive mutants of influenza virus.

PubMed

Sugiura, A; Tobita, K; Kilbourne, E D

1972-10-01

Isolation of temperature-sensitive (ts) mutants was attempted from the WSN strain of influenza A virus which was grown and assayed in MDBK cells. After growth of wild-type virus in the presence of 5-fluorouracil, 15 ts mutants were selected for which the ratio of plaquing efficiency at 39.5 C to that at 33 C was 10(-3) or less. In pairwise crosses of ts mutants, recombination and complementation were either very efficient or undetectable. It is suggested, therefore, that the viral genome consists of physically discrete units and recombination occurs as an exchange of these units. All 15 mutants have been assigned with certainty into five recombination groups. Three mutants are suspected to be double mutants. Any two complementing mutants always recombined with each other, and noncomplementing mutants did not recombine. In physiological tests, mutants showed diverse patterns of functional defects at the nonpermissive temperature. However, it was not always possible to correlate these physiological defects with the results of genetic characterization.

Analysis of AtCry1 and Mutants

NASA Astrophysics Data System (ADS)

Burdick, Derek; Purvis, Adam; Ahmad, Margaret; Link, Justin J.; Engle, Dorothy

Cryptochrome is an incredibly versatile protein that influences numerous biological processes such as plant growth, bird migration, and sleep cycles. Due to the versatility of this protein, understanding the mechanism would allow for advances in numerous fields such as crop growth, animal behavior, and sleep disorders. It is known that cryptochrome requires blue light to function, but the exact processes in the regulation of biological activity are still not fully understood. It is believed that the c-terminal domain of the protein undergoes a conformational change when exposed to blue light which allows for biological function. Three different non-functioning mutants were tested during this study to gain insight on the mechanism of cryptochrome. Absorbance spectra showed a difference between two of the mutants and the wild type with one mutant showing little difference. Immunoprecipitation experiments were also conducted to identify the different c-terminal responses of the mutants. By studying non functioning mutants of this protein, the mechanism of the protein can be further characterized. This two-month research experience in Paris allowed us to experience international and interdisciplinary collaborations in science and immerse in a different culture. The Borcer Fund for Student Research, Xavier University, Cincinnati, OH, and John Hauck Foundation.

Thermosensitivity of a barosensitive Saccharomyces cerevisiae mutant obtained by UV mutagenesis

NASA Astrophysics Data System (ADS)

Shigematsu, Toru; Nomura, Kazuki; Nasuhara, Yusuke; Ikarashi, Kenta; Nagai, Gen; Hirayama, Masao; Hayashi, Mayumi; Ueno, Shigeaki; Fujii, Tomoyuki

2010-12-01

Using UV mutagenesis, a high pressure (HP)-sensitive (barosensitive) mutant of Saccharomyces cerevisiae was obtained. The mutant strain a924E1 showed a significant loss of viability at HP levels of 175 to 250 MPa at 20 °C compared with the parent strain. This strain also showed a significant loss of viability following heat treatment at 50-58 °C at 0.1 MPa. These results showed that the mutation caused a significant thermosensitivity as well as barosensitivity. The activation volume and activation energy values for the inactivation of strain a924E1 were equivalent to those of the parent strain. This suggested that the mechanism for the HP and thermal inactivation reaction of strain a924E1 was basically the same as that of the parent strain. Strain a924E1 showed no deficiency in growth and fermentation ability as well as auxotrophic property. Although the identification of the genetic sites of mutation introduced is underway, these phenotypes are favorable for the application of HP treatment and heat-assisted HP treatment on fermentation control.

Computational Design of a Thermostable Mutant of Cocaine Esterase via Molecular Dynamics Simulations

PubMed Central

Huang, Xiaoqin; Gao, Daquan; Zhan, Chang-Guo

2015-01-01

Cocaine esterase (CocE) has been known as the most efficient native enzyme for metabolizing the naturally occurring cocaine. A major obstacle to the clinical application of CocE is the thermoinstability of native CocE with a half-life of only ~11 min at physiological temperature (37°C). It is highly desirable to develop a thermostable mutant of CocE for therapeutic treatment of cocaine overdose and addiction. To establish a structure-thermostability relationship, we carried out molecular dynamics (MD) simulations at 400 K on wild-type CocE and previously known thermostable mutants, demonstrating that the thermostability of the active form of the enzyme correlates with the fluctuation (characterized as the RMSD and RMSF of atomic positions) of the catalytic residues (Y44, S117, Y118, H287, and D259) in the simulated enzyme. In light of the structure-thermostability correlation, further computational modeling including MD simulations at 400 K predicted that the active site structure of the L169K mutant should be more thermostable. The prediction has been confirmed by wet experimental tests showing that the active form of the L169K mutant had a half-life of 570 min at 37°C, which is significantly longer than those of the wild-type and previously known thermostable mutants. The encouraging outcome suggests that the high-temperature MD simulations and the structure-thermostability may be considered as a valuable tool for computational design of thermostable mutants of an enzyme. PMID:21373712

Computational design of a thermostable mutant of cocaine esterase via molecular dynamics simulations.

PubMed

Huang, Xiaoqin; Gao, Daquan; Zhan, Chang-Guo

2011-06-07

Cocaine esterase (CocE) has been known as the most efficient native enzyme for metabolizing naturally occurring cocaine. A major obstacle to the clinical application of CocE is the thermoinstability of native CocE with a half-life of only ∼11 min at physiological temperature (37 °C). It is highly desirable to develop a thermostable mutant of CocE for therapeutic treatment of cocaine overdose and addiction. To establish a structure-thermostability relationship, we carried out molecular dynamics (MD) simulations at 400 K on wild-type CocE and previously known thermostable mutants, demonstrating that the thermostability of the active form of the enzyme correlates with the fluctuation (characterized as the root-mean square deviation and root-mean square fluctuation of atomic positions) of the catalytic residues (Y44, S117, Y118, H287, and D259) in the simulated enzyme. In light of the structure-thermostability correlation, further computational modelling including MD simulations at 400 K predicted that the active site structure of the L169K mutant should be more thermostable. The prediction has been confirmed by wet experimental tests showing that the active form of the L169K mutant had a half-life of 570 min at 37 °C, which is significantly longer than those of the wild-type and previously known thermostable mutants. The encouraging outcome suggests that the high-temperature MD simulations and the structure-thermostability relationship may be considered as a valuable tool for the computational design of thermostable mutants of an enzyme.

PIK3CA mutant tumors depend on oxoglutarate dehydrogenase | Office of Cancer Genomics

Cancer.gov

Oncogenic PIK3CA mutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found that PIK3CA mutant cancer cells require PIK3CA but also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH).

Mechanical properties of elytra from Tribolium castaneum wild-type and body color mutant strains.

PubMed

Lomakin, Joseph; Arakane, Yasuyuki; Kramer, Karl J; Beeman, Richard W; Kanost, Michael R; Gehrke, Stevin H

2010-12-01

Cuticle tanning in insects involves simultaneous cuticular pigmentation and hardening or sclerotization. The dynamic mechanical properties of the highly modified and cuticle-rich forewings (elytra) from Tribolium castaneum (red flour beetle) wild-type and body color mutant strains were investigated to relate body coloration and elytral mechanical properties. There was no statistically significant variation in the storage modulus E' among the elytra from jet, cola, sooty and black mutants or between the mutants and the wild-type GA-1 strain: E' averaged 5.1 ± 0.6 GPa regardless of body color. E' is a power law function of oscillation frequency for all types. The power law exponent, n, averaged 0.032 ± 0.001 for elytra from all genotypes except black; this small value indicated that the elytra are cross-linked. Black elytra, however, displayed a significantly larger n of 0.047 ± 0.004 and an increased loss tangent (tan δ), suggesting that metabolic differences in the black mutant strain result in elytra that are less cross-linked and more pigmented than the other types. These results are consistent with the hypothesis that black elytra have a β-alanine-deficient and dopamine-abundant metabolism, leading to greater melanin (black pigment) production, probably at the expense of cross-linking of cuticular proteins mediated by N-β-alanyldopamine quinone. Copyright © 2010 Elsevier Ltd. All rights reserved.

Listeria monocytogenes mutants with altered growth phenotypes at refrigeration temperature and high salt concentrations.

PubMed

Burall, Laurel S; Laksanalamai, Pongpan; Datta, Atin R

2012-02-01

Listeria monocytogenes can survive and grow in refrigerated temperatures and high-salt environments. In an effort to better understand the associated mechanisms, a library of ∼ 5,200 transposon mutants of LS411, a food isolate from the Jalisco cheese outbreak, were screened for their ability to grow in brain heart infusion (BHI) broth at 5°C or in the presence of 7% NaCl and two mutants with altered growth profiles were identified. The LS522 mutant has a transposon insertion between secA2 and iap and showed a significant reduction in growth in BHI broth at 5°C and in the presence of 7% NaCl. Reverse transcriptase quantitative PCR (RT-qPCR) revealed a substantial reduction in the expression of iap. Additionally, a hypothetical gene (met), containing a putative S-adenosylmethionine-dependent methyltransferase domain, downstream of iap had downregulated expression. In-frame deletion mutants of iap and met were created in LS411. The LS560 (LS411 Δiap) mutant showed reduced growth at 5°C and in the presence of 7% salt, confirming its role in cold and salt growth attenuation. Surprisingly, the LS655 (LS411 Δmet) mutant showed slightly increased growth during refrigeration, though no alteration was seen in salt growth relative to the wild-type strain. The LS527 mutant, containing an insertion 36 bp upstream of the gbu operon, showed reduced expression of the gbu transcript by RT-qPCR and also showed growth reduction at 5°C and in the presence of 7% salt. This attenuation was severely exacerbated when the mutant was grown under the combined stresses. Analysis of the gbu operon deletion mutant showed decreased growth in 7% salt and refrigeration, supporting the previously characterized role for this gene in cold and salt adaptation. These studies indicate the potential for an intricate relationship between environmental stress regulation and virulence in L. monocytogenes.

Listeria monocytogenes Mutants with Altered Growth Phenotypes at Refrigeration Temperature and High Salt Concentrations

PubMed Central

Burall, Laurel S.; Laksanalamai, Pongpan

2012-01-01

Listeria monocytogenes can survive and grow in refrigerated temperatures and high-salt environments. In an effort to better understand the associated mechanisms, a library of ∼ 5,200 transposon mutants of LS411, a food isolate from the Jalisco cheese outbreak, were screened for their ability to grow in brain heart infusion (BHI) broth at 5°C or in the presence of 7% NaCl and two mutants with altered growth profiles were identified. The LS522 mutant has a transposon insertion between secA2 and iap and showed a significant reduction in growth in BHI broth at 5°C and in the presence of 7% NaCl. Reverse transcriptase quantitative PCR (RT-qPCR) revealed a substantial reduction in the expression of iap. Additionally, a hypothetical gene (met), containing a putative S-adenosylmethionine-dependent methyltransferase domain, downstream of iap had downregulated expression. In-frame deletion mutants of iap and met were created in LS411. The LS560 (LS411 Δiap) mutant showed reduced growth at 5°C and in the presence of 7% salt, confirming its role in cold and salt growth attenuation. Surprisingly, the LS655 (LS411 Δmet) mutant showed slightly increased growth during refrigeration, though no alteration was seen in salt growth relative to the wild-type strain. The LS527 mutant, containing an insertion 36 bp upstream of the gbu operon, showed reduced expression of the gbu transcript by RT-qPCR and also showed growth reduction at 5°C and in the presence of 7% salt. This attenuation was severely exacerbated when the mutant was grown under the combined stresses. Analysis of the gbu operon deletion mutant showed decreased growth in 7% salt and refrigeration, supporting the previously characterized role for this gene in cold and salt adaptation. These studies indicate the potential for an intricate relationship between environmental stress regulation and virulence in L. monocytogenes. PMID:22179239

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.

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.

Cortex content of asporogenous mutants of Bacillus subtilis.

PubMed Central

Imae, Y; Strominger, J L

1976-01-01

A method for the measurement of muramic lactam, which is specifically located in the cortical peptidoglycan of bacterial spores, was developed as a quantitative assay method for spore cortex content. During sporulation of Bacillus subtilis 168, muramic lactam (i.e., spore cortex) began to appear at state IV of sporulation and continued to increase over most of the late stages of sporulation. Spore cortex contents of various spo mutants of B. subitils were surveyed. Cortex was not detected in mutants in which sporulation was blocked earlier than stage II sporulation. Spores of spo IV mutant had about 40% of the cortex content of the wild-type spores. One spo III mutant had a low amount of cortex, but four others had none. PMID:1262319

Enterocin A Mutants Identified by Saturation Mutagenesis Enhance Potency Towards Vancomycin-Resistant Enterococci

PubMed Central

McClintock, Maria K.; Kaznessis, Yiannis N.; Hackel, Benjamin J.

2016-01-01

Vancomycin-resistant Enterococci infections are a significant clinical problem. One proposed solution is to use probiotics, such as lactic acid bacteria, to produce antimicrobial peptides at the site of infection. Enterocin A, a class 2a bacteriocin, exhibits inhibitory activity against E. faecium and E. faecalis, which account for 86% of vancomycin-resistant Enterococci infections. In this study, we aimed to engineer enterocin A mutants with enhanced potency within a lactic acid bacterial production system. Peptide mutants resulting from saturation mutagenesis at sites A24 and T27 were efficiently screened in a 96-well plate assay for inhibition of pathogen growth. Several mutants exhibit increased potency relative to wild-type enterocin A in both liquid- and solid-medium growth assays. In particular, A24P and T27G exhibit enhanced inhibition of multiple strains of E. faecium and E. faecalis, including clinically isolated vancomycin-resistant strains. A24P and T27G enhance killing of E. faecium 8 by 13±3- and 18±4-fold, respectively. The engineered enterocin A/lactic acid bacteria systems offer significant potential to combat antibiotic-resistant infections. PMID:26191783

Enterocin A mutants identified by saturation mutagenesis enhance potency towards vancomycin-resistant Enterococci.

PubMed

McClintock, Maria K; Kaznessis, Yiannis N; Hackel, Benjamin J

2016-02-01

Vancomycin-resistant Enterococci infections are a significant clinical problem. One proposed solution is to use probiotics, such as lactic acid bacteria, to produce antimicrobial peptides at the site of infection. Enterocin A, a class 2a bacteriocin, exhibits inhibitory activity against E. faecium and E. faecalis, which account for 86% of vancomycin-resistant Enterococci infections. In this study, we aimed to engineer enterocin A mutants with enhanced potency within a lactic acid bacterial production system. Peptide mutants resulting from saturation mutagenesis at sites A24 and T27 were efficiently screened in a 96-well plate assay for inhibition of pathogen growth. Several mutants exhibit increased potency relative to wild-type enterocin A in both liquid- and solid-medium growth assays. In particular, A24P and T27G exhibit enhanced inhibition of multiple strains of E. faecium and E. faecalis, including clinically isolated vancomycin-resistant strains. A24P and T27G enhance killing of E. faecium 8 by 13 ± 3- and 18 ± 4-fold, respectively. The engineered enterocin A/lactic acid bacteria systems offer significant potential to combat antibiotic-resistant infections. © 2015 Wiley Periodicals, Inc.

Mutant DD genotype of NFKB1 gene is associated with the susceptibility and severity of coronary artery disease.

PubMed

Luo, Jun-Yi; Li, Xiao-Mei; Zhou, Yun; Zhao, Qiang; Chen, Bang-Dang; Liu, Fen; Chen, Xiao-Cui; Zheng, Hong; Ma, Yi-Tong; Gao, Xiao-Ming; Yang, Yi-Ning

2017-02-01

Nuclear factor κappa B (NF-κB) is an important transcription factor in the development and progression of coronary artery disease (CAD). Recent evidence suggests that -94 ATTG ins/del mutant in the promoter of NFKB1 gene is an essential functional mutant. The present study demonstrated the frequencies of the del/del (DD) genotype and del (D) allele were significantly higher in CAD patients than in controls. CAD patients carrying mutant DD genotype had worse stenosis of diseased coronary arteries compared to those carrying ins/ins (II) or ins/del (ID) genotype. Plasma levels of endothelial nitric oxide synthase (eNOS) were lower, while inflammatory cytokine incnterlukin-6 (IL-6) was higher in CAD patients with DD genotype than those with II or ID genotype (both P<0.05). In vitro study showed that mutant human umbilical vein endothelial cells (DD genotype HUVECs) were more susceptible to H 2 O 2 -induced apoptosis, which was accompanied with a decreased Bcl-2 expression. Further, mutant HUVECs had lower eNOS but higher IL-6 mRNA levels and decreased phosphorylation of eNOS under H 2 O 2 -stimulation (both P<0.05). Compared to wild type cells (II genotype), significantly downregulated protein expression of total NF-κB p50 subunit were observed in mutant HUVECs with or without oxidative stress, and a lower expression of unclear p50 was associated with a decreased p50 nuclear translocation in mutant HUVECs versus wild type cells under H 2 O 2 -stimulation (both P<0.05). In conclusion, mutant DD genotype of NFKB1 gene is associated with the risk and severity of CAD. Dwonregulation of NF-κB p50 subunit leads to exacerbated endothelial dysfunction and apoptosis and enhanced inflammatory response that is the potential underlying mechanism. Copyright © 2017 Elsevier Ltd. All rights reserved.

UV-induced lethal sectoring and pure mutant clones in yeast.

PubMed

Hannan, M A; Duck, P; Nasim, A

1976-08-01

The induction of lethal sectoring and pure mutant clones by ultraviolet light has been studied in a homogeneous G1 population of Saccharomyces cerevisiae grown in a normal growth medium. At the lowest UV dose of 250 ergs, which corresponds to a shoulder in the survival curve, all mutants appeared as pure clones. At higher doses the frequency of mosaic mutants progressively increased. These results indicate a relationship between the highest frequency of complete mutants and the maximum repair activity. In addition, the frequency of lethal sectoring at all doses tested was too low to account for the origin of pure mutant clones.

Grain Filling Characteristics and Their Relations with Endogenous Hormones in Large- and Small-Grain Mutants of Rice.

PubMed

Zhang, Weiyang; Cao, Zhuanqin; Zhou, Qun; Chen, Jing; Xu, Gengwen; Gu, Junfei; Liu, Lijun; Wang, Zhiqin; Yang, Jianchang; Zhang, Hao

2016-01-01

This study determined if the variation in grain filling parameters between two different spikelet types of rice (Oryza sativa L.) is regulated by the hormonal levels in the grains. Two rice mutants, namely, a large-grain mutant (AZU-M) and a small-grain mutant (ZF802-M), and their respective wild types (AZU-WT and ZF802-WT) were grown in the field. The endosperm cell division rate, filling rate, and hormonal levels: zeatin + zeatin riboside (Z+ZR), indo-3-acetic acid (IAA), polyamines (PAs), and abscisic acid (ABA) were determined. The results showed that there was no significant difference between the filling and endosperm cell division rates. These rates were synchronous between the superior and inferior spikelets for both mutants. However, the abovementioned parameters were significantly different between the two spikelet types for the two wild types. The superior spikelets filled faster and their filling rate was higher compared to the inferior ones. Changes in the concentrations of plant hormones were consistent with the observed endosperm cell division rate and the filling rate for both types of spikelets of mutant and wild type plants. Regression analysis showed a significant positive correlation between cell division and filling rates with the concentrations of the investigated hormones. Exogenous chemical application verified the role of ABA, IAA, and PAs in grain filling. The results indicate that poor filling of inferior spikelets in rice occurs primarily due to the reduced hormone concentrations therein, leading to lower division rate of endosperm cells, fewer endosperm cells, slower filling rate, and smaller grain weight.

Grain Filling Characteristics and Their Relations with Endogenous Hormones in Large- and Small-Grain Mutants of Rice

PubMed Central

Zhang, Weiyang; Cao, Zhuanqin; Zhou, Qun; Chen, Jing; Xu, Gengwen; Gu, Junfei; Liu, Lijun; Wang, Zhiqin; Yang, Jianchang; Zhang, Hao

2016-01-01

This study determined if the variation in grain filling parameters between two different spikelet types of rice (Oryza sativa L.) is regulated by the hormonal levels in the grains. Two rice mutants, namely, a large-grain mutant (AZU-M) and a small-grain mutant (ZF802-M), and their respective wild types (AZU-WT and ZF802-WT) were grown in the field. The endosperm cell division rate, filling rate, and hormonal levels: zeatin + zeatin riboside (Z+ZR), indo-3-acetic acid (IAA), polyamines (PAs), and abscisic acid (ABA) were determined. The results showed that there was no significant difference between the filling and endosperm cell division rates. These rates were synchronous between the superior and inferior spikelets for both mutants. However, the abovementioned parameters were significantly different between the two spikelet types for the two wild types. The superior spikelets filled faster and their filling rate was higher compared to the inferior ones. Changes in the concentrations of plant hormones were consistent with the observed endosperm cell division rate and the filling rate for both types of spikelets of mutant and wild type plants. Regression analysis showed a significant positive correlation between cell division and filling rates with the concentrations of the investigated hormones. Exogenous chemical application verified the role of ABA, IAA, and PAs in grain filling. The results indicate that poor filling of inferior spikelets in rice occurs primarily due to the reduced hormone concentrations therein, leading to lower division rate of endosperm cells, fewer endosperm cells, slower filling rate, and smaller grain weight. PMID:27780273

Abnormal lignin in a loblolly pine mutant.

PubMed

Ralph, J; MacKay, J J; Hatfield, R D; O'Malley, D M; Whetten, R W; Sederoff, R R

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.

Analyses of Tomato Fruit Brightness Mutants Uncover Both Cutin-Deficient and Cutin-Abundant Mutants and a New Hypomorphic Allele of GDSL Lipase[C][W][OPEN

PubMed Central

Petit, Johann; Bres, Cécile; Just, Daniel; Garcia, Virginie; Mauxion, Jean-Philippe; Marion, Didier; Bakan, Bénédicte; Joubès, Jérôme; Domergue, Frédéric; Rothan, Christophe

2014-01-01

The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, postharvest shelf-life, and brightness. To identify tomato mutants with modified cuticle composition and architecture and to further decipher the relationships between fruit brightness and cuticle in tomato, we screened an ethyl methanesulfonate mutant collection in the miniature tomato cultivar Micro-Tom for mutants with altered fruit brightness. Our screen resulted in the isolation of 16 glossy and 8 dull mutants displaying changes in the amount and/or composition of wax and cutin, cuticle thickness, and surface aspect of the fruit as characterized by optical and environmental scanning electron microscopy. The main conclusions on the relationships between fruit brightness and cuticle features were as follows: (1) screening for fruit brightness is an effective way to identify tomato cuticle mutants; (2) fruit brightness is independent from wax load variations; (3) glossy mutants show either reduced or increased cutin load; and (4) dull mutants display alterations in epidermal cell number and shape. Cuticle composition analyses further allowed the identification of groups of mutants displaying remarkable cuticle changes, such as mutants with increased dicarboxylic acids in cutin. Using genetic mapping of a strong cutin-deficient mutation, we discovered a novel hypomorphic allele of GDSL lipase carrying a splice junction mutation, thus highlighting the potential of tomato brightness mutants for advancing our understanding of cuticle formation in plants. PMID:24357602

Characteristics of invasion-reduced hilA gene mutant of Salmonella Enteritidis in vitro and in vivo.

PubMed

Lv, Shuang; Si, Wei; Yu, Shenye; Li, Zhaoli; Wang, Xiumei; Chen, Liping; Zhang, Wanjiang; Liu, Siguo

2015-08-01

Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) is a facultative intracellular pathogen that causes huge losses in poultry industry and also food poisoning in humans due to its being a food-borne pathogen. Functions of Invasion-related genes need to be explored, as invasion is a key step for Salmonella infection. In this study, a transposon mutant library of Salmonella Enteritidis isolate SM6 was constructed and screened for the invasion-related genes via incubation with Caco-2 cells. Three stably attenuated mutants were identified for significantly reduced invasion with insertions all in hilA (hyperinvasive locus A) gene. We constructed and evaluated the hilA deletion mutant in vivo and in vitro. SM6△hilA showed significantly reduced ability to invade Caco-2 cells and decreased pathogenicity in chicks. However, the bacterial load and pathological damage in the cecum were significantly higher than those in the SM6 in vivo. Present results provide new evidences for pathogenicity research on Salmonella Enteritidis. Copyright © 2015 Elsevier B.V. All rights reserved.

Isolation of New Gravitropic Mutants under Hypergravity Conditions

PubMed Central

Mori, Akiko; Toyota, Masatsugu; Shimada, Masayoshi; Mekata, Mika; Kurata, Tetsuya; Tasaka, Masao; Morita, Miyo T.

2016-01-01

Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upward. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes). In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using next-generation sequencing (NGS) and single nucleotide polymorphism (SNP)-based markers. Using the endodermal-amyloplast less 1 (eal1) mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g) restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene (enhancer of eal1) mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis. PMID:27746791

Polyamines and Flower Development in the Male Sterile Stamenless-2 Mutant of Tomato (Lycopersicon esculentum Mill.) 1

PubMed Central

Rastogi, Rajeev; Sawhney, Vipen K.

1990-01-01

The floral organs of the male sterile stamenless-2 (sl-2/sl-2) mutant of tomato (Lycopersicon esculentum Mill.) contain significantly higher level of polyamines than those of the normal (R Rastogi, VK Sawhney [1990] Plant Physiol 93: 439-445). The effects of putrescine, spermidine and spermine, and three different inhibitors of polyamine biosynthesis on the in vitro development of floral buds of the normal and sl-2/sl-2 mutant were studied. The polyamines were inhibitory to the in vitro growth and development of both the normal and mutant floral buds and they induced abnormal stamen development in normal flowers. The inhibitors of polyamine biosynthesis also inhibited the growth and development of floral organs of the two genotypes, but the normal flowers showed greater sensitivity than the mutant. The inhibitors also promoted the formation of normal-looking pollen in stamens of some mutant flowers. The effect of the inhibitors on polyamine levels was not determined. The polyamine-induced abnormal stamen development in the normal, and the inhibitor-induced production of normal-looking pollen in mutant flowers support the suggestion that the elevated polyamine levels contribute to abnormal stamen development in the sl-2/sl-2 mutant of tomato. Images Figure 3 Figure 5 PMID:16667486

Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum.

PubMed

Woodcock, M Ryan; Vaughn-Wolfe, Jennifer; Elias, Alexandra; Kump, D Kevin; Kendall, Katharina Denise; Timoshevskaya, Nataliya; Timoshevskiy, Vladimir; Perry, Dustin W; Smith, Jeramiah J; Spiewak, Jessica E; Parichy, David M; Voss, S Randal

2017-01-31

The molecular genetic toolkit of the Mexican axolotl, a classic model organism, has matured to the point where it is now possible to identify genes for mutant phenotypes. We used a positional cloning-candidate gene approach to identify molecular bases for two historic axolotl pigment phenotypes: white and albino. White (d/d) mutants have defects in pigment cell morphogenesis and differentiation, whereas albino (a/a) mutants lack melanin. We identified in white mutants a transcriptional defect in endothelin 3 (edn3), encoding a peptide factor that promotes pigment cell migration and differentiation in other vertebrates. Transgenic restoration of Edn3 expression rescued the homozygous white mutant phenotype. We mapped the albino locus to tyrosinase (tyr) and identified polymorphisms shared between the albino allele (tyr a ) and tyr alleles in a Minnesota population of tiger salamanders from which the albino trait was introgressed. tyr a has a 142 bp deletion and similar engineered alleles recapitulated the albino phenotype. Finally, we show that historical introgression of tyr a significantly altered genomic composition of the laboratory axolotl, yielding a distinct, hybrid strain of ambystomatid salamander. Our results demonstrate the feasibility of identifying genes for traits in the laboratory Mexican axolotl.

EGFR mutant allelic-specific imbalance assessment in routine samples of non-small cell lung cancer.

PubMed

Malapelle, Umberto; Vatrano, Simona; Russo, Stefania; Bellevicine, Claudio; de Luca, Caterina; Sgariglia, Roberta; Rocco, Danilo; de Pietro, Livia; Riccardi, Fernando; Gobbini, Elisa; Righi, Luisella; Troncone, Giancarlo

2015-09-01

In non-small cell lung cancer (NSCLC), the epidermal growth factor receptor (EGFR) gene may undergo both mutations and copy number gains. EGFR mutant allele-specific imbalance (MASI) occurs when the ratio of mutant-to-wild-type alleles increases significantly. In this study, by using a previously validated microfluidic-chip-based technology, EGFR-MASI occurred in 25/67 mutant cases (37%), being more frequently associated with EGFR exon 19 deletions (p=0.033). In a subset of 49 treated patients, we assessed whether MASI is a modifier of anti-EGFR treatment benefit. The difference in progression-free survival and overall survival between EGFR-MASI-positive and EGFR-MASI-negative groups of patients did not show a statistical significance. In conclusion, EGFR-MASI is a significant event in NSCLC, specifically associated with EGFR exon 19 deletions. However, EGFR-MASI does not seem to play a role in predicting the response to first-generation EGFR small molecules inhibitors. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

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.

Targeting ESR1-Mutant Breast Cancer

DTIC Science & Technology

2015-09-01

AWARD NUMBER: W81XWH-14-1-0359 TITLE: Targeting ESR1 -Mutant Breast Cancer PRINCIPAL INVESTIGATOR: Dr. Sarat Chandarlapaty CONTRACTING...31 Aug 2015 4. TITLE AND SUBTITLE Targeting ESR1 -Mutant Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0359 5c. PROGRAM ELEMENT...Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The hypothesis of this proposal is that LBD mutations in ESR1 promote resistance to

Targeting ESR1-Mutant Breast Cancer

DTIC Science & Technology

2015-09-01

Award Number: W81XWH-14-1-0360 TITLE: Targeting ESR1 -Mutant Breast Cancer PRINCIPAL INVESTIGATOR: Geoffrey L. Greene, Ph.D. CONTRACTING...ADDRESS. 1. REPORT DATE September 2015 2. REPORT TYPE Annual 3. DATES COVERED 1 Sep 2014 - 31 Aug 2015 4. TITLE AND SUBTITLE Targeting ESR1 -Mutant...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The hypothesis of this proposal is that LBD mutations in ESR1 promote resistance to current FDA

Eicosapentaenoic acid prevents arterial calcification in klotho mutant mice.

PubMed

Nakamura, Kazufumi; Miura, Daiji; Saito, Yukihiro; Yunoki, Kei; Koyama, Yasushi; Satoh, Minoru; Kondo, Megumi; Osawa, Kazuhiro; Hatipoglu, Omer F; Miyoshi, Toru; Yoshida, Masashi; Morita, Hiroshi; Ito, Hiroshi

2017-01-01

The klotho gene was identified as an "aging-suppressor" gene that accelerates arterial calcification when disrupted. Serum and vascular klotho levels are reduced in patients with chronic kidney disease, and the reduced levels are associated with arterial calcification. Intake of eicosapentaenoic acid (EPA), an n-3 fatty acid, reduces the risk of fatal coronary artery disease. However, the effects of EPA on arterial calcification have not been fully elucidated. The aim of this study was to determine the effect of EPA on arterial calcification in klotho mutant mice. Four-week-old klotho mutant mice and wild-type (WT) mice were given a diet containing 5% EPA (EPA food, klotho and WT: n = 12, each) or not containing EPA (control food, klotho and WT: n = 12, each) for 4 weeks. Calcium volume scores of thoracic and abdominal aortas assessed by computed tomography were significantly elevated in klotho mice after 4 weeks of control food, but they were not elevated in klotho mice after EPA food or in WT mice. Serum levels of EPA and resolvin E1, an active metabolite of EPA, in EPA food-fed mice were significantly increased compared to those in control food-fed mice. An oxidative stress PCR array followed by quantitative PCR revealed that NADPH oxidase-4 (NOX4), an enzyme that generates superoxide, gene expression was up-regulated in arterial smooth muscle cells (SMCs) of klotho mice. Activity of NOX was also significantly higher in SMCs of klotho mice than in those of WT mice. EPA decreased expression levels of the NOX4 gene and NOX activity. GPR120, a receptor of n-3 fatty acids, gene knockdown by siRNA canceled effects of EPA on NOX4 gene expression and NOX activity in arterial SMCs of klotho mice. EPA prevents arterial calcification together with reduction of NOX gene expression and activity via GPR120 in klotho mutant mice.

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.

A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis

PubMed Central

1989-01-01

A severin deficient mutant of Dictyostelium discoideum has been isolated by the use of colony immunoblotting after chemical mutagenesis. In homogenates of wild-type cells, severin is easily detected as a very active F-actin fragmenting protein. Tests for severin in the mutant, HG1132, included viscometry for the assay of F- actin fragmentation in fractions from DEAE-cellulose columns, labeling of blots with monoclonal and polyclonal antibodies, and immunofluorescent-labeling of cryosections. Severin could not be detected in the mutant using these methods. The mutation in HG1132 is recessive and has been mapped to linkage group VII. The mutant failed to produce the normal severin mRNA, but small amounts of a transcript that was approximately 100 bases larger than the wild-type mRNA were detected in the mutant throughout all stages of development. On the DNA level a new Mbo II restriction site was found in the mutant within the coding region of the severin gene. The severin deficient mutant cells grew at an approximately normal rate, aggregated and formed fruiting bodies with viable spores. By the use of an image processing system, speed of cell movement, turning rates, and precision of chemotactic orientation in a stable gradient of cyclic AMP were quantitated, and no significant differences between wild-type and mutant cells were found. Thus, under the culture conditions used, severin proved to be neither essential for growth of D. discoideum nor for any cell function that is important for aggregation or later development. PMID:2537840

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

Hush puppy: a new mouse mutant with pinna, ossicle, and inner ear defects.

PubMed

Pau, Henry; Fuchs, Helmut; de Angelis, Martin Hrabé; Steel, Karen P

2005-01-01

Deafness can be associated with abnormalities of the pinna, ossicles, and cochlea. The authors studied a newly generated mouse mutant with pinna defects and asked whether these defects are associated with peripheral auditory or facial skeletal abnormalities, or both. Furthermore, the authors investigated where the mutation responsible for these defects was located in the mouse genome. The hearing of hush puppy mutants was assessed by Preyer reflex and electrophysiological measurement. The morphological features of their middle and inner ears were investigated by microdissection, paint-filling of the labyrinth, and scanning electron microscopy. Skeletal staining of skulls was performed to assess the craniofacial dimensions. Genome scanning was performed using microsatellite markers to localize the mutation to a chromosomal region. Some hush puppy mutants showed early onset of hearing impairment. They had small, bat-like pinnae and normal malleus but abnormal incus and stapes. Some mutants had asymmetrical defects and showed reduced penetrance of the ear abnormalities. Paint-filling of newborns' inner ears revealed no morphological abnormality, although half of the mice studied were expected to carry the mutation. Reduced numbers of outer hair cells were demonstrated in mutants' cochlea on scanning electron microscopy. Skeletal staining showed that the mutants have significantly shorter snouts and mandibles. Genome scan revealed that the mutation lies on chromosome 8 between markers D8Mit58 and D8Mit289. The study results indicate developmental problems of the first and second branchial arches and otocyst as a result of a single gene mutation. Similar defects are found in humans, and hush puppy provides a mouse model for investigation of such defects.

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. Copyright © 2012 Elsevier B.V. All rights reserved.

Cell-to-cell stimulation of movement in nonmotile mutants of Myxococcus

PubMed Central

Hodgkin, Jonathan; Kaiser, Dale

1977-01-01

A large number of nonmotile mutants of the gliding bacterium Myxococcus xanthus have been isolated and partly characterized. About [unk] of these mutants are conditional mutants of a novel kind: mutant cells become transiently motile after contact with nonmutant cells or with cells of a different mutant type. These “stimulatable” mutants fall into five phenotypic classes (types B, C, D, E, and F). Most mutants are nonstimulatable (type A) and never become motile, but type A cells (and wild-type cells) can stimulate cells of any of the other five types. Stimulatable mutants of different types are capable of stimulating each other. For example, in a mixture of B and C cells, both become motile. Linkage analysis using a generalized transducing phage has shown that each of types B, C, D, E, and F corresponds to a single distinct genetic locus. Type A mutants, by contrast, belong to at least 17 different loci. Stimulation depends on close apposition of interacting cells, because stimulation does not occur when contact between cells is prevented. It is possible that the stimulatable mutants are defective in components of the gliding mechanism that can be exchanged between cells. Alternatively, they may be defective in a system of cell communication controlling the coordinated cell movements observed in Myxococcus. Images PMID:16592422

Identification of Gold Nanoparticle-Resistant Mutants of Saccharomyces cerevisiae Suggests a Role for Respiratory Metabolism in Mediating Toxicity

PubMed Central

Smith, Mark R.; Boenzli, Matthew G.; Hindagolla, Vihangi; Ding, Jun; Miller, John M.; Hutchison, James E.; Greenwood, Jeffrey A.; Abeliovich, Hagai

2013-01-01

Positively charged gold nanoparticles (0.8-nm core diameter) reduced yeast survival, but not growth, at a concentration of 10 to 100 μg/ml. Among 17 resistant deletion mutants isolated in a genome-wide screen, highly significant enrichment was observed for respiration-deficient mutants lacking genes encoding proteins associated with the mitochondrion. PMID:23144132

Autolytic defective mutant of Streptococcus faecalis.

PubMed Central

Cornett, J B; Redman, B E; Shockman, G D

1978-01-01

Properties of a variant of Streptococcus faecalis ATCC 9790 with defective cellular autolysis are described. The mutant strain was selected as a survivor from a mutagenized cell population simultaneously challenged with two antibiotics which inhibit cell wall biosynthesis, penicillin G and cycloserine. Compared to the parental strain, the mutant strain exhibited: (i) a thermosensitive pattern of cellular autolysis; (ii) an autolytic enzyme activity that had only a slightly increased thermolability when tested in solution in the absence of wall substrate; and (iii) an isolated autolysin that had hydrolytic activity on isolated S. faecalis wall substrate indistinguishable from that of the parental strain, but that was inactive when tested on walls of Micrococcus lysodeikticus as a substrate. These data indicate an alteration in the substrate specificity of the autolytic enzyme of the mutant which appears to result from the synthesis of an altered form of autolytic enzyme. PMID:415045

Vaccination of guinea pigs using mce operon mutants of Mycobacterium tuberculosis

PubMed Central

Obregón-Henao, Andrés; Shanley, Crystal; Bianco, María Verónica; Cataldi, Angel A; Basaraba, Randall J; Orme, Ian M; Bigi, Fabiana

2011-01-01

The limited efficacy of the BCG vaccine for tuberculosis, coupled with emerging information suggesting that it is poorly protective against newly emerging strains of Mycobacterium tuberculosis such as the W-Beijing isolates, makes it paramount to search for more potent alternatives. One such class of candidates is attenuated mutants derived from M. tuberculosis itself. We demonstrate here, in an initial short term assay, that mutants derived from disruption of the mce genes of the bacillus were highly protective in guinea pigs exposed by low dose aerosol infection with the virulent W-Beijing isolate SA161. This protection was demonstrated by a significant reduction in the numbers of bacilli harvested from the lungs, and dramatic improvements in lung histopathology. PMID:21515327

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

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.

Safety, Protective Immunity, and DIVA Capability of a Rough Mutant Salmonella Pullorum Vaccine Candidate in Broilers.

PubMed

Guo, Rongxian; Jiao, Yang; Li, Zhuoyang; Zhu, Shanshan; Fei, Xiao; Geng, Shizhong; Pan, Zhiming; Chen, Xiang; Li, Qiuchun; Jiao, Xinan

2017-01-01

Salmonella enterica subsp. enterica serovar Gallinarum biovar Pullorum ( Salmonella Pullorum) is highly adapted to chickens causing an acute systemic disease that results in high mortality. Vaccination represents one approach for promoting animal health, food safety and reducing environmental persistence in Salmonella control. An important consideration is that Salmonella vaccination in poultry should not interfere with the salmonellosis monitoring program. This is the basis of the DIVA (Differentiation of Infected and Vaccinated Animals) program. In order to achieve this goal, waaL mutant was developed on a spiC mutant that was developed previously. The safety, efficacy, and DIVA features of this vaccine candidate ( Salmonella Pullorum Δ spiC Δ waaL ) were evaluated in broilers. Our results show that the truncated LPS in the vaccine strain has a differentiating use as both a bacteriological marker (rough phenotype) and also as a serological marker facilitating the differentiation between infected and vaccinated chickens. The rough mutant showed adequate safety being avirulent in the host chicks and showed increased sensitivity to environmental stresses. Single intramuscular immunization of day-old broiler chicks with the mutant confers ideal protection against lethal wild type challenge by significantly stimulating both humoral and cellular immune responses as well as reducing the colonization of the challenge strain. Significantly lower mean pathology scores were observed in the vaccination group compared to the control group. Additionally, the mutant strain generated cross-protection against challenge with the wild type Salmonella Gallinarum thereby improving survival and with the wild type Salmonella Enteritidis thereby reducing colonization. These results suggest that the double-mutant strain may be a safe, effective, and cross-protective vaccine against Salmonella infection in chicks while conforming to the requirements of the DIVA program.

Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient.

PubMed Central

Howden, R; Goldsbrough, P B; Andersen, C R; Cobbett, C S

1995-01-01

An allelic series of cad1, cadmium-sensitive mutants of Arabidopsis thaliana, was isolated. These mutants were sensitive to cadmium to different extents and were deficient in their ability to form cadmium-peptide complexes as detected by gel-filtration chromatography. Each mutant was deficient in its ability to accumulate phytochelatins (PCs) as detected by high-performance liquid chromatography and the amount of PCs accumulated by each mutant correlated with its degree of sensitivity to cadmium. The mutants had wild-type levels of glutathione, the substrate for PC biosynthesis, and in vitro assays demonstrated that each of the mutants was deficient in PC synthase activity. These results demonstrate conclusively the importance of PCs for cadmium tolerance in plants. PMID:7770517

Isolation and Characterization of Sex-Linked Female-Sterile Mutants in DROSOPHILA MELANOGASTER

PubMed Central

Gans, Madeleine; Audit, Claudie; Masson, Michele

1975-01-01

The purpose of the experiments described was to identify X chromosome genes functioning mainly or exclusively during oogenesis. Two mutagenesis experiments were carried out with ethyl methane sulfonate. Following treatment inducing 60% lethals, 9% of the treated X chromosomes carried a female sterility mutation which did not otherwise seriously affect viability. Among —95 isolated mutants, 19 were heat-sensitive and 5 cold-sensitive. The mutants have been classified as follows: I (16 mutants; 12 complementation groups): the females laid few or no eggs; the defect concerned either ovulation or oogenesis. II (37 mutants; 18 complementation groups): the female laid morphologically abnormal eggs, often with increased membrane permeability. III A (13 mutants; at least 8 complementation groups): the homozygous females were sterile if mated to mutant males; their progeny (homo- and hemizygous) died at a late embryonic stage (11 mutants), at the larval stage (1 mutant) or at the pupal stage (1 mutant). However fertility was partly restored by breeding to wild-type males as shown by survival of some heterozygous descendants. III B (29 mutants; 22 complementation groups): the fertility of the females was not restored by breeding to a wild-type male. Most of the eggs of 13 of the mutants died at a late stage of embryogenesis. The eggs of the others ceased development earlier or, perhaps, remained unfertilized. The distribution of the number of mutants per complementation group led to an estimation of a total of about 150 X-linked genes involved in female fertility. The females of three mutants, heat-sensitive and totally sterile at 29°, produced at a lower temperature descendants morphologically abnormal or deprived of germ cells. Three other mutants not described in detail showed a reduction in female fertility with many descendants lacking germ cells. A desirable mutant which was not recovered was one with normal fertile females producing descendants which, regardless of

Lenalidomide enhances the function of chimeric antigen receptor T cells against the epidermal growth factor receptor variant III by enhancing immune synapses.

PubMed

Kuramitsu, S; Ohno, M; Ohka, F; Shiina, S; Yamamichi, A; Kato, A; Tanahashi, K; Motomura, K; Kondo, G; Kurimoto, M; Senga, T; Wakabayashi, T; Natsume, A

2015-10-01

The epidermal growth factor receptor variant III (EGFRvIII) is exclusively expressed on the cell surface in ~50% of glioblastoma multiforme (GBM). This variant strongly and persistently activates the phosphatidylinositol 3-kinase-Akt signaling pathway in a ligand-independent manner resulting in enhanced tumorigenicity, cellular motility and resistance to chemoradiotherapy. Our group generated a recombinant single-chain variable fragment (scFv) antibody specific to the EGFRvIII, referred to as 3C10-scFv. In the current study, we constructed a lentiviral vector transducing the chimeric antigen receptor (CAR) that consisted of 3C10-scFv, CD3ζ, CD28 and 4-1BB (3C10-CAR). The 3C10-CAR-transduced peripheral blood mononuclear cells (PBMCs) and CD3(+) T cells specifically lysed the glioma cells that express EGFRvIII. Moreover, we demonstrated that CAR CD3(+) T cells migrated to the intracranial xenograft of GBM in the mice treated with 3C10-CAR PBMCs. An important and novel finding of our study was that a thalidomide derivative lenalidomide induced 3C10-CAR PBMC proliferation and enhanced the persistent antitumor effect of the cells in vivo. Lenalidomide also exhibited enhanced immunological synapses between the effector cells and the target cells as determined by CD11a and F-actin polymerization. Collectively, lentiviral-mediated transduction of CAR effectors targeting the EGFRvIII showed specific efficacy, and lenalidomide even intensified CAR cell therapy by enhanced formation of immunological synapses.

Engineering Chimeric Antigen Receptor T cells to Treat Glioblastoma.

PubMed

Choi, Bryan D; O'Rourke, Donald M; Maus, Marcela V

2017-08-01

Immunotherapy has emerged as a promising strategy for glioblastoma (GBM), a disease that remains universally fatal despite currently available standard-of-care. Adoptive T cell therapy has been shown to produce potent antitumor immunity while obviating the need for traditional antigen presentation and primary immune responses. Chimeric antigen receptors (CARs) are specialized molecules that can be expressed on the surface of T cells allowing for redirected cytotoxicity against tumor antigens of interest. To date, the application of CAR T cells for GBM has been relatively limited, in large part due to a dearth of well-described tumor specific antigens that are both homogenously and frequently expressed. A mutated version of the epidermal growth factor receptor, EGFRvIII, is a constitutively activated tyrosine kinase that is expressed on the surface of GBM and other common neoplasms, but completely absent from all normal tissues. We have recently generated CAR T cells directed against EGFRvIII and reported results from a Phase I clinical trial investigating this platform in patients with EGFRvIII-expressing GBM. Our study showed that despite conventional notions of central nervous system "immune-privilege," EGFRvIII CAR T cells trafficked to intracerebral tumors, leading to successful targeting and eradication of this antigen in the brain. Here, we review our experience with EGFRvIII CAR T cells and highlight important considerations for the clinical translation of this therapy in patients with GBM.

Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice

PubMed Central

Filippov, Andrey A.; Sergueev, Kirill V.; He, Yunxiu; Huang, Xiao-Zhe; Gnade, Bryan T.; Mueller, Allen J.; Fernandez-Prada, Carmen M.; Nikolich, Mikeljon P.

2011-01-01

Background Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. Methodology/Principal Findings The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD50 and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. Conclusions/Significance We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis. PMID:21980477

Evaluation of Brown Midrib Sorghum Mutants as a Potential Biomass Feedstock for 2,3-Butanediol Biosynthesis.

PubMed

Guragain, Yadhu N; Srinivasa Rao, P; Vara Prasad, P V; Vadlani, Praveen V

2017-11-01

Three sorghum backgrounds [Atlas, Early Hegari (EH), and Kansas Collier (KC)] and two bmr mutants (bmr6 and bmr12) of each line were evaluated and compared for grain and biomass yield, biomass composition, and 2,3-butanediol production from biomass. The data showed that the bmr6 mutation in EH background led to a significant decrease in stover yield and increase in grain yield, whereas the stover yield was increased by 64% without affecting grain yield in KC background. The bmr mutants had 10 to 25% and 2 to 9% less lignin and structural carbohydrate contents, respectively, and 24 to 93% more non-structural sugars than their parents in all sorghum lines, except EH bmr12. The total fermentable sugars released were 22 to 36% more in bmr mutants than in parents for Atlas and KC, but not for EH. The bmr6 mutation in KC background produced the most promising feedstock, among the evaluated bmr mutants, for 2,3-butanediol production without affecting grain yield, followed by KC bmr12 and Atlas bmr6, but the bmr mutation had an adverse effect in EH background. This indicated that the genetic background of the parent line and type of bmr mutation significantly affect the biomass quality as a feedstock for biochemical production.

Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer

DTIC Science & Technology

2015-09-01

AWARD NUMBER: W81XWH-14-1-0177 TITLE: Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer PRINCIPAL INVESTIGATOR: Katerina Politi...CONTRACT NUMBER Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer 5b. GRANT NUMBER W81XWH-14-1-0177 5c. PROGRAM ELEMENT NUMBER 6...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Phenotypic changes have been observed in EGFR mutant lung cancers that become resistant to targeted

Immunogenicity and protection induced by a Mycobacterium tuberculosis sigE mutant in a BALB/c mouse model of progressive pulmonary tuberculosis.

PubMed

Hernandez Pando, Rogelio; Aguilar, Leon Diana; Smith, Issar; Manganelli, Riccardo

2010-07-01

Tuberculosis is still one of the main challenges to human global health, leading to about two million deaths every year. One of the reasons for its success is the lack of efficacy of the widely used vaccine Mycobacterium bovis BCG. In this article, we analyze the potential use of an attenuated mutant of Mycobacterium tuberculosis H37Rv lacking the sigma factor sigma(E) as a live vaccine. We have demonstrated that BALB/c mice infected by the intratracheal route with this mutant strain showed significantly higher survival rates and less tissue damage than animals infected with the parental or complemented mutant strain. Although animals infected with the sigE mutant had low bacillary loads, their lungs showed significantly higher production of the protective factors gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), inducible nitric oxide synthase (iNOS), and beta-defensins than those of animals infected with the parental or complemented mutant strain. Moreover, we demonstrate that the sigE mutant, when inoculated subcutaneously, was more attenuated than BCG in immunodeficient nude mice, thus representing a good candidate for a novel attenuated live vaccine strain. Finally, when we used the sigE mutant as a subcutaneous vaccine, it was able to induce a higher level of protection than did BCG with both H37Rv and a highly virulent strain of M. tuberculosis (Beijing code 9501000). Taken together, our findings suggest that the sigE mutant is a very promising strain for the development of a new vaccine against tuberculosis.

A potential functional association between mutant BMPR2 and primary ovarian insufficiency.

PubMed

Patiño, Liliana Catherine; Silgado, Daniel; Laissue, Paul

2017-06-01

Primary ovarian insufficiency (POI) affects ~1% of women in the general population. Despite numerous attempts at identifying POI genetic aetiology, coding mutations in only a few genes have been functionally related to POI pathogenesis. It has been suggested that mutant BMPR2 might contribute towards the phenotype. Several BMP15 (a BMPR2 ligand) coding mutations in human species have been related to POI pathogenesis. The BMPR2 p.Ser987Phe mutation, previously identified in a woman with POI, might therefore lead to cellular dysfunction contributing to the phenotype. To explore such an assumption, the present study assessed potential pathogenic subcellular localization/aggregation patterns associated with the p.Ser987Phe mutant form of BMPR2 in a relevant model for studying ovarian function. A significant increase in protein-like aggregation patterns was identified at the endoplasmic reticulum (ER) which permitted us to establish, for the first time, a potential functional association between mutant BMPR2 and POI aetiology. Since BMPR2 mutant forms were previously related to idiopathic pulmonary arterial hypertension, BMPR2 mutations may be related to an as-yet-to-be described syndromic form of POI involving pulmonary dysfunction. Additional assays are necessary to confirm that BMPR2 abnormal subcellular patterns are composed by aggregates. POI: primary ovarian insufficiency; ER: endoplasmic reticulum; NGS: next generation sequencing.

Phenotypic characterization of a photomorphogenic mutant.

PubMed

Fankhauser, Christian; Casal, Jorge J

2004-09-01

Light is arguably the most important abiotic factor controlling plant growth and development throughout their life cycle. Plants have evolved sophisticated light-sensing mechanisms to monitor fluctuations in light quality, intensity, direction and periodicity (day length). In Arabidopsis, three families of photoreceptors have been identified by molecular genetic studies. The UV-A/blue light receptors cryptochromes and the red/far-red receptors phytochromes control an overlapping set of responses including photoperiodic flowering induction and de-etiolation. Phototropins are the primary photoreceptors for a set of specific responses to UV-A/blue light such as phototropism, chloroplast movement and stomatal opening. Mutants affecting a photoreceptor have a characteristic phenotype. It is therefore possible to determine the specific developmental responses and the photoreceptor pathway(s) affected in a mutant by performing an appropriate set of photobiological and genetic experiments. In this paper, we outline the principal and easiest experiments that can be performed to obtain a first indication about the nature of the photobiological defect in a given mutant.

Personalized treatment of EGFR mutant and ALK-positive patients in NSCLC.

PubMed

Somasundaram, Aswin; Socinski, Mark A; Burns, Timothy F

2014-12-01

The epidermal growth factor receptor (EGFR) is mutated in 15% of adenocarcinomas of the lung. In addition, the anaplastic lymphoma kinase (ALK) is altered in 8% of adenocarcinomas of the lung. Treatment of EGFR mutant and ALK translocation-positive tumors in NSCLC with tyrosine kinase inhibitors (TKI) results in a dramatic therapeutic response and has revolutionized therapy. Unfortunately, resistance to TKIs invariably develops. Many promising new therapies are under investigation to overcome the resistance. We analyzed the current primary literature and recent national meetings to evaluate the clinical characteristics and therapeutic implications of relevant treatments for EGFR mutant and ALK-positive NSCLC in the first-line, acquired resistance, and adjuvant settings. Treatment with EGFR TKIs in the first-line setting of EGFR mutant NSCLC results in a significant clinical benefit. Several promising third generation EGFR TKIs are being evaluated in Phase II and III trials in the acquired resistance setting. Crizotinib is superior to chemotherapy in the first-line setting for ALK-positive NSCLC. Ceritinib is effective and approved for ALK-positive NSCLC in the acquired resistance setting. Continued investigation is needed to develop novel therapies to overcome acquired resistance to TKIs.

Modeling the competition between antenna size mutant and wild type microalgae in outdoor mass culture.

PubMed

de Mooij, Tim; Schediwy, Kira; Wijffels, René H; Janssen, Marcel

2016-12-20

Under high light conditions, microalgae are oversaturated with light which significantly reduces the light use efficiency. Microalgae with a reduced pigment content, antenna size mutants, have been proposed as a potential solution to increase the light use efficiency. The goal of this study was to investigate the competition between antenna size mutants and wild type microalgae in mass cultures. Using a kinetic model and literature-derived experimental data from wild type Chlorella sorokiniana, the productivity and competition of wild type cells and antenna size mutants were simulated. Cultivation was simulated in an outdoor microalgal raceway pond production system which was assumed to be limited by light only. Light conditions were based on a Mediterranean location (Tunisia) and a more temperate location (the Netherlands). Several wild type contamination levels were simulated in each mutant culture separately to predict the effect on the productivity over the cultivation time of a hypothetical summer season of 100days. The simulations demonstrate a good potential of antenna size reduction to increase the biomass productivity of microalgal cultures. However, it was also found that after a contamination with wild type cells the mutant cultures will be rapidly overgrown resulting in productivity loss. Copyright © 2016 Elsevier B.V. All rights reserved.

Synthesis of bacteriophage phiC DNA in dna mutants of Esherichia coli.

PubMed

Kodaira, K I; Taketo, A

1978-06-01

Host dna functions involved in the replication of microvirid phage phiC DNA were investigated in vivo. Although growth of this phage was markedly inhibited even at 35-37 degrees C even in dna+ host, conversion of the infecting single-stranded DNA into the double-stranded parental replicative form (stage I synthesis) occurred normally at 43 degrees C in dna+, dnaA, dnaB, dnaC(D), and dnaE cells. In dnaG mutant, the stage I synthesis was severely inhibited at 43 degrees C but not at 30 degrees C. The stage I replication of phiC DNA was clearly thermosensitive in dnaZ cells incubated in nutrient broth. In Tris-casamino acids-glucose medium, however, the dnaZ mutant sufficiently supported synthesis of the parental replicative form. At 43 degrees C, synthesis of the progeny replicative form DNA (stage II replication) was significantly inhibited even in dna+ cells and was nearly completely blocked in dnaB or dnaC(D) mutant. At 37 degrees C, the stage II replication proceeded normally in dna+ bacteria.

shl, a New set of Arabidopsis mutants with exaggerated developmental responses to available red, far-red, and blue light.

PubMed

Pepper, A E; Seong-Kim, M; Hebst, S M; Ivey, K N; Kwak, S J; Broyles, D E

2001-09-01

The interaction of light perception with development is the subject of intensive genetic analysis in the model plant Arabidopsis. We performed genetic screens in low white light-a threshold condition in which photomorphogenetic signaling pathways are only partially active-for ethyl methane sulfonate-generated mutants with altered developmental phenotypes. Recessive mutants with exaggerated developmental responses were obtained in eight complementation groups designated shl for seedlings hyperresponsive to light. shl1, shl2, shl5, and shl3 shl4 (double mutant) seedlings showed limited or no phenotypic effects in darkness, but showed significantly enhanced inhibition of hypocotyl elongation in low-white, red, far-red, blue, and green light across a range of fluences. These results reflect developmental hyper-responsiveness to signals generated by both phytochrome and cryptochrome photoreceptors. The shl11 mutant retained significant phenotypic effects on hypocotyl length in both the phyA mutant and phyB mutant backgrounds but may be dependent on CRY1 for phenotypic expression in blue light. The shl2 phenotype was partially dependent on PHYB, PHYA, and CRY1 in red, far-red, and blue light, respectively. shl2 and, in particular, shl1 were partially dependent on HY5 activity for their light-hyperresponsive phenotypes. The SHL genes act (genetically) as light-dependent negative regulators of photomorphogenesis, possibly in a downstream signaling or developmental pathway that is shared by CRY1, PHYA, and PHYB and other photoreceptors (CRY2, PHYC, PHYD, and PHYE).

Determination quercetin content, antioxidant and antimicrobial activity of genotype mutant Samosir shallots irradiated by gamma rays

NASA Astrophysics Data System (ADS)

Sinuraya, M.; Hanafiah, D. S.; Romulo, A.; Barus, A.

2018-02-01

The aim of the research was to study the variation in antioxidant and antimicrobial activity as well as the total quercetin content of the fifth generation genotypes mutant Samosir shallot irradiated by gamma rays. The studies conducted included the assessment of quercetin content, antioxidant and antimicrobial activity in shallot bulbs after long-term storage (6 months in the room temperature). Quercetin content of 20 selected genotype mutants of irradiated shallot bulbs along with untreated populations were calculated using quercetin (QU) as a standard. Antioxidant activities of 8 genotype mutant were determined using DPPH. Antimicrobial activity of bulb extracts were tested against six bacteria including Staphylococcus aurous, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae and oneyeastCandida albicans. The results showed that population of genotype mutants irradiated with dosage 2Gy, 4 Gy, 5 Gy and 6 Gy have higher quercetin content than control samples. None of the genotype mutants exhibited antibacterial inhibitory against all microorganism tested except for the sample number 2 and 6 (bulbs generated from the plants irradiated by gamma rays with dosage at 2 Gy and 6 Gy). There was also none of the genotypes observed exhibited significant antioxidant efficacy.

Pharmacokinetic properties of radiolabeled mutant Interleukin-2v: a PET imaging study

PubMed Central

Hartimath, Siddesh V.; Manuelli, Valeria; Zijlma, Rolf; Signore, Alberto; Nayak, Tapan K.; Freimoser-Grundschober, Anne; Klein, Christian; Dierckx, Rudi A.J.O.; de Vries, Erik F.J.

2018-01-01

Interleukin-2 (IL2) is a cytokine that can stimulate cytotoxic immune cells to attack infected and malignant cells. Unfortunately, IL2 can also cause serious immune-related toxicity. Recently, a mutant of IL2 (IL2v) with abolished CD25 binding, increased plasma half-life and less toxicity was engineered. Unlike wild-type IL2 (wt-IL2), mutant IL2v does not bind to the α-subunit (CD25) of the high affinity IL2αβγ receptor, but only to its β and γ subunit. Here, we investigated the biological properties of IL2v and compared with the wt-IL2 using fluorine-18 and PET. [18F]FB-IL2v binds specifically to IL2 receptors (IL2R) on activated human peripheral blood monocytes (hPBMCs) and is cleared mainly by the kidneys (Balb/c mice). [18F]FB-IL2v PET studies in SCID mice injected with hPBMCs revealed high uptake in the implant (0.85 ± 0.15 SUV), which was significantly reduced after pretreatment with wt-IL2 or mutant IL2v (SUV 0.26 ± 0.1 and 0.46 ± 0.1, p < 0.01). Compartment modeling and Logan graphical analysis in wistar rats inoculated with hPBMCs indicated that the binding of [18F]FB-IL2v to IL2R was reversible. The volume of distribution (VT) and the non-displaceable binding potential (BPnd) of mutant [18F]FB-IL2v in the implant were approximately 3 times lower than those of wild-type [18F]FB-IL2 (p < 0.01). Pretreatment with wt-IL2 significantly reduced the VT and BPnd of mutant [18F]FB-IL2v in the implant (p < 0.001). This demonstrates that wild-type [18F]FB-IL2 binds stronger to IL2R and has faster kinetics than [18F]FB-IL2v, which makes it less suitable as a therapeutic drug. [18F]FB-IL2v, on the other hand, seems to have better properties for use as a therapeutic drug. PMID:29467958

Pharmacokinetic properties of radiolabeled mutant Interleukin-2v: a PET imaging study.

PubMed

Hartimath, Siddesh V; Manuelli, Valeria; Zijlma, Rolf; Signore, Alberto; Nayak, Tapan K; Freimoser-Grundschober, Anne; Klein, Christian; Dierckx, Rudi A J O; de Vries, Erik F J

2018-01-23

Interleukin-2 (IL2) is a cytokine that can stimulate cytotoxic immune cells to attack infected and malignant cells. Unfortunately, IL2 can also cause serious immune-related toxicity. Recently, a mutant of IL2 (IL2v) with abolished CD25 binding, increased plasma half-life and less toxicity was engineered. Unlike wild-type IL2 (wt-IL2), mutant IL2v does not bind to the α-subunit (CD25) of the high affinity IL2αβγ receptor, but only to its β and γ subunit. Here, we investigated the biological properties of IL2v and compared with the wt-IL2 using fluorine-18 and PET. [ 18 F]FB-IL2v binds specifically to IL2 receptors (IL2R) on activated human peripheral blood monocytes (hPBMCs) and is cleared mainly by the kidneys (Balb/c mice). [ 18 F]FB-IL2v PET studies in SCID mice injected with hPBMCs revealed high uptake in the implant (0.85 ± 0.15 SUV), which was significantly reduced after pretreatment with wt-IL2 or mutant IL2v (SUV 0.26 ± 0.1 and 0.46 ± 0.1, p < 0.01). Compartment modeling and Logan graphical analysis in wistar rats inoculated with hPBMCs indicated that the binding of [ 18 F]FB-IL2v to IL2R was reversible. The volume of distribution (V T ) and the non-displaceable binding potential (BP nd ) of mutant [ 18 F]FB-IL2v in the implant were approximately 3 times lower than those of wild-type [ 18 F]FB-IL2 ( p < 0.01). Pretreatment with wt-IL2 significantly reduced the V T and BPnd of mutant [ 18 F]FB-IL2v in the implant ( p < 0.001). This demonstrates that wild-type [ 18 F]FB-IL2 binds stronger to IL2R and has faster kinetics than [18F]FB-IL2v, which makes it less suitable as a therapeutic drug. [ 18 F]FB-IL2v, on the other hand, seems to have better properties for use as a therapeutic drug.

The Phenotype performance of M3 red rice mutant (Oryza sativa L.)

NASA Astrophysics Data System (ADS)

Kasim, N.; Sjahril, R.; Riadi, M.; Arbie, F.

2018-05-01

Local rice genotype generally has colour, flavour and scent more preferred by consumers, yet unfortunately it has long-lived planting period and low production. Therefore, the plant breeding practices in rice needs to be implemented for better rice varieties which are superior in terms of both quality and quantity. Our findings describe the growth character performance and the production of red rice mutant from M3 generation. This study was conducted in the Agriculture Faculty wetlands, Hasanuddin University, Makassar, by using ANOVA test with some red rice mutant genotypes i.e. 7 genotypes mutants (G1, G2, G3, G4, G5, G6 and G7) and controls/parent-plants (not the mutant). Results show that there were difference in growth performance and production of red rice mutant. Each parameter observed on each genotype had different results. Mutants produced best response in tillers production were G4 mutant with the tillers grain weight at 99.2 g, whereas by the results of the analysis of rank, mutants showed the best overall response were found in G6 mutants.

An efficient screen for peroxisome-deficient mutants of Pichia pastoris.

PubMed Central

Liu, H; Tan, X; Veenhuis, M; McCollum, D; Cregg, J M

1992-01-01

We describe a rapid and efficient screen for peroxisome-deficient (per) mutants in the yeast Pichia pastoris. The screen relies on the unusual ability of P. pastoris to grow on two carbon sources, methanol and oleic acid, both of which absolutely require peroxisomes to be metabolized. A collection of 280 methanol utilization-defective (Mut-) P. pastoris mutants was isolated, organized into 46 complementation groups, and tested for those that were also oleate-utilization defective (Out-) but still capable of growth on ethanol and glucose. Mutants in 10 groups met this phenotypic description, and 8 of these were observed by electron microscopy to be peroxisome deficient (Per-). In each per mutant, Mut-, Out-, and Per- phenotypes were tightly linked and therefore were most likely due to a mutation at a single locus. Subcellular fractionation experiments indicated that the peroxisomal marker enzyme catalase was mislocalized to the cytosol in both methanol- and oleate-induced cultures of the mutants. In contrast, alcohol oxidase, a peroxisomal methanol utilization pathway enzyme, was virtually absent from per mutant cells. The relative ease of per mutant isolation in P. pastoris, in conjunction with well-developed procedures for its molecular and genetic manipulation, makes this organism an attractive system for studies on peroxisome biogenesis. Images PMID:1629154

Identification of Proteus mirabilis Mutants with Increased Sensitivity to Antimicrobial Peptides

PubMed Central

McCoy, Andrea J.; Liu, Hongjian; Falla, Timothy J.; Gunn, John S.

2001-01-01

Antimicrobial peptides (APs) are important components of the innate defenses of animals, plants, and microorganisms. However, some bacterial pathogens are resistant to the action of APs. For example, Proteus mirabilis is highly resistant to the action of APs, such as polymyxin B (PM), protegrin, and the synthetic protegrin analog IB-367. To better understand this resistance, a transposon mutagenesis approach was used to generate P. mirabilis mutants sensitive to APs. Four unique PM-sensitive mutants of P. mirabilis were identified (these mutants were >2 to >128 times more sensitive than the wild type). Two of these mutants were also sensitive to IB-367 (16 and 128 times more sensitive than the wild type). Lipopolysaccharide (LPS) profiles of the PM- and protegrin-sensitive mutants demonstrated marked differences in both the lipid A and O-antigen regions, while the PM-sensitive mutants appeared to have alterations of either lipid A or O antigen. Matrix-assisted laser desorption ionization–time of flight mass spectrometry analysis of the wild-type and PM-sensitive mutant lipid A showed species with one or two aminoarabinose groups, while lipid A from the PM- and protegrin-sensitive mutants was devoid of aminoarabinose. When the mutants were streaked on an agar-containing medium, the swarming motility of the PM- and protegrin-sensitive mutants was completely inhibited and the swarming motility of the mutants sensitive to only PM was markedly decreased. DNA sequence analysis of the mutagenized loci revealed similarities to an O-acetyltransferase (PM and protegrin sensitive) and ATP synthase and sap loci (PM sensitive). These data further support the role of LPS modifications as an elaborate mechanism in the resistance of certain bacterial species to APs and suggest that LPS surface charge alterations may play a role in P. mirabilis swarming motility. PMID:11408219

Characterization of spontaneous mutants of Magnaporthe grisea expressing stable resistance to the Qo-inhibiting fungicide azoxystrobin.

PubMed

Avila-Adame, Cruz; Köller, Wolfram

2003-03-01

The class of Qo-inhibiting fungicides (QoIs) act as respiration inhibitors by binding to the Qo center of cytochrome b. The longevity of these fungicides has been challenged by the selection of fungal sub-populations resisting high doses of QoI fungicides, with a G143A amino acid exchange in the cytochrome b target site identified as the most common cause of resistance. In contrast, the mechanism of alternative respiration, as another mechanism of fungal QoI resistance, has thus far not been affiliated with practical resistance. In the present study, azoxystrobin-resistant mutants of Magnaporthe grisea were generated and characterized. Emergence of these spontaneous mutants was facilitated when resting melanized mycelia were allowed to escape full inhibition by azoxystrobin. This escape was related to the intactness of alternative respiration, indicating that residual expression of this rescue mechanism was involved in the spontaneous emergence of target-site mutants. The two mutants characterized resisted high doses of the QoI, azoxystrobin, with resistance factors exceeding 1,000. Two different mutations of the cytochrome b gene were identified as exchanges of guanine, leading to a G143A or a G143S amino acid exchange. Resistance of both target-site mutants remained stable during four consecutive disease cycles in the absence of azoxystrobin. Several parameters tested to measure fitness penalties inherent to the mutational changes revealed that the G143A mutant was not compromised. In contrast, the conidia production of the G143S mutant was significantly lower under both saprophytic and pathogenic conditions of reproduction.

Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin.

PubMed

Ramkumar, Srinivasagan; Fujii, Noriko; Fujii, Norihiko; Thankappan, Bency; Sakaue, Hiroaki; Ingu, Kim; Natarajaseenivasan, Kalimuthusamy; Anbarasu, Kumarasamy

2014-01-01

To study the comparative structural and functional changes between wild-type (wt) and N-terminal congenital cataract causing αA-crystallin mutants (R12C, R21L, R49C, and R54C) upon exposure to different dosages of gamma rays. Alpha A crystallin N-terminal mutants were created with the site-directed mutagenesis method. The recombinantly overexpressed and purified wt and mutant proteins were used for further studies. A (60)Co source was used to generate gamma rays to irradiate wild and mutant proteins at dosages of 0.5, 1.0, and 2.0 kGy. The biophysical property of the gamma irradiated (GI) and non-gamma irradiated (NGI) αA-crystallin wt and N-terminal mutants were determined. Oligomeric size was determined by size exclusion high-performance liquid chromatography (HPLC), the secondary structure with circular dichroism (CD) spectrometry, conformation of proteins with surface hydrophobicity, and the functional characterization were determined regarding chaperone activity using the alcohol dehydrogenase (ADH) aggregation assay. αA-crystallin N-terminal mutants formed high molecular weight (HMW) cross-linked products as well as aggregates when exposed to GI compared to the NGI wt counterparts. Furthermore, all mutants exhibited changed β-sheet and random coil structure. The GI mutants demonstrated decreased surface hydrophobicity when compared to αA-crystallin wt at 0, 1.0, and 1.5 kGy; however, at 2.0 kGy a drastic increase in hydrophobicity was observed only in the mutant R54C, not the wt. In contrast, chaperone activity toward ADH was gradually elevated at the minimum level in all GI mutants, and significant elevation was observed in the R12C mutant. Our findings suggest that the N-terminal mutants of αA-crystallin are structurally and functionally more sensitive to GI when compared to their NGI counterparts and wt. Protein oxidation as a result of gamma irradiation drives the protein to cross-link and aggregate culminating in cataract formation.

Detection of a gravitropism phenotype in glutamate receptor-like 3.3 mutants of Arabidopsis thaliana using machine vision and computation.

PubMed

Miller, Nathan D; Durham Brooks, Tessa L; Assadi, Amir H; Spalding, Edgar P

2010-10-01

Gene disruption frequently produces no phenotype in the model plant Arabidopsis thaliana, complicating studies of gene function. Functional redundancy between gene family members is one common explanation but inadequate detection methods could also be responsible. Here, newly developed methods for automated capture and processing of time series of images, followed by computational analysis employing modified linear discriminant analysis (LDA) and wavelet-based differentiation, were employed in a study of mutants lacking the Glutamate Receptor-Like 3.3 gene. Root gravitropism was selected as the process to study with high spatiotemporal resolution because the ligand-gated Ca(2+)-permeable channel encoded by GLR3.3 may contribute to the ion fluxes associated with gravity signal transduction in roots. Time series of root tip angles were collected from wild type and two different glr3.3 mutants across a grid of seed-size and seedling-age conditions previously found to be important to gravitropism. Statistical tests of average responses detected no significant difference between populations, but LDA separated both mutant alleles from the wild type. After projecting the data onto LDA solution vectors, glr3.3 mutants displayed greater population variance than the wild type in all four conditions. In three conditions the projection means also differed significantly between mutant and wild type. Wavelet analysis of the raw response curves showed that the LDA-detected phenotypes related to an early deceleration and subsequent slower-bending phase in glr3.3 mutants. These statistically significant, heritable, computation-based phenotypes generated insight into functions of GLR3.3 in gravitropism. The methods could be generally applicable to the study of phenotypes and therefore gene function.

Detection of a Gravitropism Phenotype in glutamate receptor-like 3.3 Mutants of Arabidopsis thaliana Using Machine Vision and Computation

PubMed Central

Miller, Nathan D.; Durham Brooks, Tessa L.; Assadi, Amir H.; Spalding, Edgar P.

2010-01-01

Gene disruption frequently produces no phenotype in the model plant Arabidopsis thaliana, complicating studies of gene function. Functional redundancy between gene family members is one common explanation but inadequate detection methods could also be responsible. Here, newly developed methods for automated capture and processing of time series of images, followed by computational analysis employing modified linear discriminant analysis (LDA) and wavelet-based differentiation, were employed in a study of mutants lacking the Glutamate Receptor-Like 3.3 gene. Root gravitropism was selected as the process to study with high spatiotemporal resolution because the ligand-gated Ca2+-permeable channel encoded by GLR3.3 may contribute to the ion fluxes associated with gravity signal transduction in roots. Time series of root tip angles were collected from wild type and two different glr3.3 mutants across a grid of seed-size and seedling-age conditions previously found to be important to gravitropism. Statistical tests of average responses detected no significant difference between populations, but LDA separated both mutant alleles from the wild type. After projecting the data onto LDA solution vectors, glr3.3 mutants displayed greater population variance than the wild type in all four conditions. In three conditions the projection means also differed significantly between mutant and wild type. Wavelet analysis of the raw response curves showed that the LDA-detected phenotypes related to an early deceleration and subsequent slower-bending phase in glr3.3 mutants. These statistically significant, heritable, computation-based phenotypes generated insight into functions of GLR3.3 in gravitropism. The methods could be generally applicable to the study of phenotypes and therefore gene function. PMID:20647506

Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank.

PubMed

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.

A cadmium-sensitive, glutathione-deficient mutant of Arabidopsis thaliana.

PubMed Central

Howden, R; Andersen, C R; Goldsbrough, P B; Cobbett, C S

1995-01-01

The roots of the cadmium-sensitive mutant of Arabidopsis thaliana, cad1-1, become brown in the presence of cadmium. A new cadmium-sensitive mutant affected at a second locus, cad2, has been identified using this phenotype. Genetic analysis has grown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus. Assays of cadmium accumulation by intact plants indicated that the mutant is deficient in its ability to sequester cadmium. Undifferentiated callus tissue was also cadmium sensitive, suggesting that the mutant phenotype is expressed at the cellular level. The level of cadmium-binding complexes formed in vivo was decreased compared with the wild type and accumulation of phytochelatins was about 10% of that in the wild type. The level of glutathione, the substrate for phytochelatin biosynthesis, in tissues of the mutant was decreased to about 15 to 30% of that in the wild type. Thus, the deficiency in phytochelatin biosynthesis can be explained by a deficiency in glutathione. PMID:7770518

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

Applications of Protein Thermodynamic Database for Understanding Protein Mutant Stability and Designing Stable Mutants.

PubMed

Gromiha, M Michael; Anoosha, P; Huang, Liang-Tsung

2016-01-01

Protein stability is the free energy difference between unfolded and folded states of a protein, which lies in the range of 5-25 kcal/mol. Experimentally, protein stability is measured with circular dichroism, differential scanning calorimetry, and fluorescence spectroscopy using thermal and denaturant denaturation methods. These experimental data have been accumulated in the form of a database, ProTherm, thermodynamic database for proteins and mutants. It also contains sequence and structure information of a protein, experimental methods and conditions, and literature information. Different features such as search, display, and sorting options and visualization tools have been incorporated in the database. ProTherm is a valuable resource for understanding/predicting the stability of proteins and it can be accessed at http://www.abren.net/protherm/ . ProTherm has been effectively used to examine the relationship among thermodynamics, structure, and function of proteins. We describe the recent progress on the development of methods for understanding/predicting protein stability, such as (1) general trends on mutational effects on stability, (2) relationship between the stability of protein mutants and amino acid properties, (3) applications of protein three-dimensional structures for predicting their stability upon point mutations, (4) prediction of protein stability upon single mutations from amino acid sequence, and (5) prediction methods for addressing double mutants. A list of online resources for predicting has also been provided.

Inhibition of p53 Mutant Peptide Aggregation In Vitro by Cationic Osmolyte Acetylcholine Chloride.

PubMed

Chen, Zhaolin; Kanapathipillai, Mathumai

2017-01-01

Mutations of tumor suppressor protein p53 are present in almost about 50% of all cancers. It has been reported that the p53 mutations cause aggregation and subsequent loss of p53 function, leading to cancer progression. Here in this study we focus on the inhibitory effects of cationic osmolyte molecules acetylcholine chloride, and choline on an aggregation prone 10 amino acid p53 mutant peptide WRPILTIITL, and the corresponding wildtype peptide RRPILTIITL in vitro. The characterization tools used for this study include Thioflavin- T (ThT) induced fluorescence, transmission electron microscopy (TEM), congo red binding, turbidity, dynamic light scattering (DLS), and cell viability assays. The results show that acetylcholine chloride in micromolar concentrations significantly inhibit p53 mutant peptide aggregation in vitro, and could be promising candidate for p53 mutant/ misfolded protein aggregation inhibition. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Rice mutants deficient in ω-3 fatty acid desaturase (FAD8) fail to acclimate to cold temperatures.

PubMed

Tovuu, Altanzaya; Zulfugarov, Ismayil S; Wu, Guangxi; Kang, In Soon; Kim, Choongrak; Moon, Byoung Yong; An, Gynheung; Lee, Choon-Hwan

2016-12-01

To investigate the role of ω-3 fatty acid (FA) desaturase (FAD8) during cold acclimation in higher plants, we characterized three independent T-DNA insertional knock-out mutants of OsFAD8 from rice (Oryza sativa L.). At room temperature (28 °C), osfad8 plants exhibited significant alterations in fatty acid (FA) unsaturation for all four investigated plastidic lipid classes. During a 5-d acclimation period at 4 °C, further changes in FA unsaturation in both wild-type (WT) and mutant plants varied according to the type of lipid. We also monitored the fluidity of the thylakoid membrane using a threshold temperature to represent the change in fluorescence. The values were altered significantly by both FAD8 mutation and cold acclimation, suggesting that factors other than FAD8 are involved in C18 FA unsaturation and fluctuations in membrane fluidity. Similarly, significant changes were noted for both the mutant and WT samples in terms of their FA compositions as well as activities related to photosystem (PS) I, PSII, and photoprotection. This included the development of non-photochemical quenching and increased zeaxanthin accumulation. Despite the relatively small changes in FA composition during cold acclimation, cold-inducible FAD8 knock-out mutants displayed strong differences in photoprotective activities and a further drop in membrane fluidity. The mutants were more sensitive than WT to short-term low-temperature stress that resulted in increased production of reactive oxygen species after 5 d of chilling. Taken together, our findings suggest that FA unsaturation by OsFAD8 is crucial for the acclimation of higher plants to low-temperature stress. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Nonbehavioral Selection for Pawns, Mutants of PARAMECIUM AURELIA with Decreased Excitability

PubMed Central

Schein, Stanley J.

1976-01-01

The reversal response in Paramecium aurelia is mediated by calcium which carries the inward current during excitation. Electrophysiological studies indicate that strontium and barium can also carry the inward current. Exposure to high concentrations of barium rapidly paralyzes and later kills wild-type paramecia. Following mutagenesis with nitrosoguanidine, seven mutants which continued to swim in the `high-barium' solution were selected. All of the mutants show decreased reversal behavior, with phenotypes ranging from extremely non-reversing (`extreme' pawns) to nearly wild-type reversal behavior (`partial' pawns). The mutations fall into three complementation groups, identical to the pwA, pwB, and pwC genes of Kung et al. (1975). All of the pwA and pwB mutants withstand longer exposure to barium, the pwB mutants surviving longer than the pwA mutants. Among mutants of each gene, survival is correlated with loss of reversal behavior. Double mutants (A–B, A–C, B–C), identified in the exautogamous progeny of crosses between `partial' mutants, exhibited a more extreme non-reversing phenotype than either of their single-mutant (`partial' pawn) parents.———Inability to reverse could be expected from an alteration in the calcium-activated reversal mechanism or in excitation. A normal calcium-activated structure was demonstrated in all pawns by chlorpromazine treatment. In a separate report (Schein, Bennett and Katz 1976) the results of electrophysiological investigations directly demonstrate decreased excitability in all of the mutants, a decrease due to an altered calcium activation. The studies of the genetics, the survival in barium and the electro-physiology of the pawns demonstrate that the pwA and pwB genes have different effects on calcium activation. PMID:1001878

Low ergosterol content in yeast adh1 mutant enhances chitin maldistribution and sensitivity to paraquat-induced oxidative stress.

PubMed

Marisco, G; Saito, S T; Ganda, I S; Brendel, M; Pungartnik, C

2011-05-01

Alcohol dehydrogenases catalyse the reversible oxidation of alcohols to aldehydes or ketones, with concomitant reduction of NAD(+) or NADP(+) . Adh1p is responsible for the reduction of acetaldehyde to ethanol, while Adh2p catalyses the reverse reaction, the oxidation of ethanol to acetaldehyde. Lack of Adh1p shifts the cellular redox balance towards excess NADH/NADPH and acetaldehyde, while absence of Adh2p does the opposite. Yeast mutant adh1Δ had a slow growth rate, whereas adh2Δ grew like the isogenic wild-type (WT) during prediauxic shift fermentative metabolism. After 48 h WT and mutants reached the same number of viable cells. When exponentially growing (LOG) cells were exposed to calcofluor white, only mutant adh1Δ displayed an irregular deposition of chitin. Quantitative analyses of both LOG and stationary-phase cells showed that adh1Δ mutant contained significantly less ergosterol than cells of WT and adh2Δ mutant, whereas the erg3Δ mutant contained extremely low ergosterol pools. Both adh1Δ and adh2Δ mutants showed higher-than-WT resistance to heat shock and to H(2) O(2) but had WT resistance when exposed to ultraviolet (UV) light and the DNA cross-linking agent diepoxyoctane, indicating normal DNA repair capacity. Mutant adh1Δ was specifically sensitive to acetaldehyde and to membrane peroxidizing paraquat. Our results link the pleiotropic phenotype of adh1Δ mutants to low pools of ergosterol and to reductive stress, and introduce the two new phenotypes, resistance to heat shock and to H(2) O(2) , for the adh2Δ mutant, most probably related to increased ROS production in mitochondria, which leads to the induction of oxidative stress protection. Copyright © 2011 John Wiley & Sons, Ltd.

Pharmacological chaperones facilitate the post-ER transport of recombinant N370S mutant β-glucocerebrosidase in plant cells: Evidence that N370S is a folding mutant

PubMed Central

Babajani, Gholamreza; Tropak, Michael B.; Mahuran, Don J.; Kermode, Allison R.

2012-01-01

Gaucher disease is a prevalent lysosomal storage disease in which affected individuals inherit mutations in the gene (GBA1) encoding lysosomal acid β-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45). One of the most prevalent disease-causing mutations in humans is a N370S missense mutation in the GCase protein. As part of a larger endeavor to study the fate of mutant human proteins expressed in plant cells, the N370S mutant protein along with the wild-type- (WT)-GCase, both equipped with a signal peptide, were synthesized in transgenic tobacco BY2 cells, which do not possess lysosomes. The enzymatic activity of plant-recombinant N370S GCase lines was significantly lower (by 81–95%) than that of the WT-GCase lines. In contrast to the WT-GCase protein, which was efficiently secreted from tobacco BY2 cells, and detected in large amounts in the culture medium, only a small proportion of the N370S GCase was secreted. Pharmacological chaperones such as N-(n-nonyl) deoxynojirimycin and ambroxol increased the steady-state mutant protein levels both inside the plant cells and in the culture medium. These findings contradict the assertion that small molecule chaperones increase N370S GCase activity (as assayed in treated patient cell lysates) by stabilizing the enzyme in the lysosome, and suggest that the mutant protein is impaired in its ability to obtain its functional folded conformation, which is a requirement for exiting the lumen of the ER. PMID:22592100

Analysis of Photosynthetic Antenna Function in a Mutant of Arabidopsis thaliana (L.) Lacking trans-Hexadecenoic Acid 1

PubMed Central

McCourt, Peter; Browse, John; Watson, Jan; Arntzen, Charles J.; Somerville, Chris R.

1985-01-01

Several lines of evidence support the proposal that the unusual chloroplast-specific lipid acyl group Δ3,trans-hexadecenoic acid (trans-C16:1) stimulates the formation or maintenance of the oligomeric form of the light-harvesting chlorophyll a/b complex (LHCP). To assess the functional significance of this apparent association we have analyzed LHCP structure and function in a mutant of Arabidopsis thaliana (L.) which lacks trans-C16:1 by electrophoretic analysis of the protein-chlorophyll complexes and by measurements of chlorophyll fluorescence under a variety of conditions. By these criteria the putative oligomeric form of LHCP appears to be slightly more labile to detergent-mediated dissociation in the mutant. The oligomeric PSI chlorophyll-protein complex, associated with PSI, was also more labile to detergent-mediated dissociation in the mutant, suggesting a previously unsuspected association of trans-C16:1 with the PSI complex. However, no significant effect of the mutation on the efficiency of energy transfer from LHCP to the photochemical reaction centers was observed under any of the various conditions imposed. Also, the stability of the chlorophyll-protein complexes to temperature-induced dissociation was unaffected in the mutant. The role of trans-C16:1 is very subtle or is only conditionally expressed. Images Fig. 1 PMID:16664340

The PEP-3-KLH (CDX-110) vaccine in glioblastoma multiforme patients

PubMed Central

Heimberger, Amy B.; Sampson, John H

2009-01-01

Conventional therapies for glioblastoma multiforme (GBM) fail to target tumor cells exclusively resulting in non-specific toxicity. Immune targeting of tumor-specific mutations may allow for more precise eradication of neoplastic cells. The epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific mutation that is widely expressed on GBM and other neoplasms and its expression enhances tumorigenicity. This in-frame deletion mutation splits a codon resulting in a novel glycine at the fusion junction producing a tumor-specific epitope target for cellular or humoral immunotherapy. We have previously shown that vaccination with a peptide that spans the EGFRvIII fusion junction (PEPvIII-KLH/CDX-110) is an efficacious immunotherapy in syngeneic murine models. In this review, we summarize our results in GBM patients targeting this mutation in multiple, multi-institutional Phase II immunotherapy trials. These trials demonstrated that a selected population of GBM patients who received the vaccines targeting EGFRvIII had an unexpectedly long survival time. Further therapeutic strategies and potential pitfalls using this approach are discussed. PMID:19591631

The PEPvIII-KLH (CDX-110) vaccine in glioblastoma multiforme patients.

PubMed

Heimberger, Amy B; Sampson, John H

2009-08-01

Conventional therapies for glioblastoma multiforme (GBM) fail to target tumor cells exclusively, resulting in non-specific toxicity. Immune targeting of tumor-specific mutations may allow for more precise eradication of neoplastic cells. EGFR variant III (EGFRvIII) is a tumor-specific mutation that is widely expressed in GBM and other neoplasms and its expression enhances tumorigenicity. This in-frame deletion mutation splits a codon, resulting in a novel glycine at the fusion junction producing a tumor-specific epitope target for cellular or humoral immunotherapy. We have previously shown that vaccination with a peptide that spans the EGFRvIII fusion junction (PEPvIII-KLH/CDX-110) is an efficacious immunotherapy in syngeneic murine models. In this review, we summarize our results in GBM patients targeting this mutation in multiple, multi-institutional Phase II immunotherapy trials. These trials demonstrated that a selected population of GBM patients who received vaccines targeting EGFRvIII had an unexpectedly long survival time. Further therapeutic strategies and potential pitfalls of using this approach are discussed.

Mutant Huntingtin Causes a Selective Decrease in the Expression of Synaptic Vesicle Protein 2C.

PubMed

Peng, Chaohua; Zhu, Gaochun; Liu, Xiangqian; Li, He

2018-04-30

Huntington's disease (HD) is a neurodegenerative disease caused by a polyglutamine expansion in the huntingtin (Htt) protein. Mutant Htt causes synaptic transmission dysfunctions by interfering in the expression of synaptic proteins, leading to early HD symptoms. Synaptic vesicle proteins 2 (SV2s), a family of synaptic vesicle proteins including 3 members, SV2A, SV2B, and SV2C, plays important roles in synaptic physiology. Here, we investigated whether the expression of SV2s is affected by mutant Htt in the brains of HD transgenic (TG) mice and Neuro2a mouse neuroblastoma cells (N2a cells) expressing mutant Htt. Western blot analysis showed that the protein levels of SV2A and SV2B were not significantly changed in the brains of HD TG mice expressing mutant Htt with 82 glutamine repeats. However, in the TG mouse brain there was a dramatic decrease in the protein level of SV2C, which has a restricted distribution pattern in regions particularly vulnerable in HD. Immunostaining revealed that the immunoreactivity of SV2C was progressively weakened in the basal ganglia and hippocampus of TG mice. RT-PCR demonstrated that the mRNA level of SV2C progressively declined in the TG mouse brain without detectable changes in the mRNA levels of SV2A and SV2B, indicating that mutant Htt selectively inhibits the transcriptional expression of SV2C. Furthermore, we found that only SV2C expression was progressively inhibited in N2a cells expressing a mutant Htt containing 120 glutamine repeats. These findings suggest that the synaptic dysfunction in HD results from the mutant Htt-mediated inhibition of SV2C transcriptional expression. These data also imply that the restricted distribution and decreased expression of SV2C contribute to the brain region-selective pathology of HD.

Polysaccharide production by a reduced pigmentation mutant of Aureobasidium pullulans NYS-1.

PubMed

West, T P; Strohfus, B

2001-08-01

To isolate a reduced pigmentation mutant of Aureobasidium pullulans NYS-1 and characterize its cellular pigmentation plus its polysaccharide and biomass production relative to carbon source. Cellular pigmentation, polysaccharide levels and biomass production by the isolated mutant NYSRP-1 were analysed relative to carbon source. Cellular pigmentation of the mutant was lower than its parent strain using either carbon source. The mutant elaborated higher polysaccharide levels on sucrose than on corn syrup. The pullulan content of the polysaccharide synthesized and biomass production by the mutant rose as the carbon source concentration was increased. It is feasible to isolate a reduced pigmentation mutant from strain NYS-1 that exhibits elevated polysaccharide production using corn syrup as a carbon source. The mutant provides an advantage for commercial pullulan production because of its reduced pigmentation and enhanced polysaccharide synthesis.

Evaluation of nigrostriatal dopaminergic function in adult +/+ and +/- BDNF mutant mice.

PubMed

Dluzen, D E; Gao, X; Story, G M; Anderson, L I; Kucera, J; Walro, J M

2001-07-01

Deletion of a single copy of the BDNF gene has been shown to affect the nigrostriatal dopaminergic system of young adult BDNF mice. In the present report we evaluated various indices of nigrostriatal dopaminergic function between 9-month-old wild-type (+/+) and heterozygous (+/-) BDNF mutant mice. Performance in a sensorimotor beam walking task was significantly decreased in +/- mice as indicated by increased times required to traverse both a wide (21 mm) and narrow (6 mm) beam. No differences in spontaneous locomotor behavior were observed between the +/+ and +/- mice. Amphetamine-stimulated (5 mg/kg) locomotor behavior was increased to a greater degree in the +/- mice, with the number of movements performed by these mice being significantly greater than their +/+ controls. Corpus striatal dopamine concentrations were significantly greater in the +/- BDNF mice. The absence of any significant differences for dopamine concentrations within the hypothalamus and olfactory bulb of these mice, as well as an absence of any difference in striatal norepinephrine concentrations, suggested a relative specificity of these effects to the corpus striatum. Both the +/- and +/+ mice showed similar reductions in striatal dopamine concentrations in response to a neurotoxic regimen of methamphetamine (20 mg/kg). Collectively these data show increased levels of striatal dopamine concentrations associated with altered behavioral responses involving the nigrostriatal dopaminergic system within the heterozygous BDNF mutant mice. Copyright 2001 Academic Press.

Isolation and Characterization of Sex-Linked Female-Sterile Mutants in DROSOPHILA MELANOGASTER with Special Attention to Eggshell Mutants

PubMed Central

Komitopoulou, Katia; Gans, Madeleine; Margaritis, Lukas H.; Kafatos, Fotis C.; Masson, Michele

1983-01-01

To study genes that function mainly or exclusively during oogenesis, we have isolated and analyzed female-sterile mutations, with special emphasis on those that affect eggshell formation. Following treatment that induced 61 to 66% lethals, 8.1% of the 1071 X chromosomes tested carried recessive female sterility mutations (87 isolates), and 8.0% carried partial female-sterile mutations (86 isolates), respectively. In addition, three dominant female steriles were recovered. Some of the mutants had very low fecundity, and others laid morphologically normal eggs that failed to develop. A third category included 29 mutants that laid eggs with morphological abnormalities: 26 were female steriles, two were partial female steriles and one was fertile. Mutants of this third category were characterized in some detail and compared with 40 previously isolated mutants that laid similarly abnormal eggs. Approximately 28–31 complementation groups with morphological abnormalities were detected, some of which were large allelic series (11, 9, 7, 6 and 5 alleles). Twenty-four groups were mapped genetically or cytogenetically, and 21 were partially characterized by ultrastructural and biochemical procedures. Of the latter, one group showed clear deficiency of yolk proteins, and nine showed prominent ultrastructural defects in the chorion (at least eight accompanied by deficiencies in characterized chorion proteins). At least six groups with clear-cut effects were found at loci not previously identified with known chorion structural genes. PMID:17246182

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

Intragenic Mapping of Chemically Induced ad-7 Mutants of Schizosaccharomyces pombe

PubMed Central

Loprieno, Nicola

1967-01-01

Thirty adenine-requiring ad-7 mutants of Schizosaccharomyces pombe, induced by ethylmethanesulfonate, methyl-methanesulfonate, and hydroxylamine and exhibiting low spontaneous reversion frequencies, were located by intragenic recombination analysis. Their identification as ad-7 mutants was assessed in relation to two previously mapped ad-7 mutants. Each mutant was found to occupy a distinct mutational site; the smallest recombination fraction observed between the two closest mutational sites was of the order of 0.5 × 10−6. PMID:6051345

Repair of Ultraviolet Radiation Damage in Sensitive Mutants of Micrococcus radiodurans

PubMed Central

Moseley, B. E. B.

1969-01-01

Various aspects of the repair of ultraviolet (UV) radiation-induced damage were compared in wild-type Micrococcus radiodurans and two UV-sensitive mutants. Unlike the wild type, the mutants are more sensitive to radiation at 265 nm than at 280 nm. The delay in deoxyribonucleic acid (DNA) synthesis following exposure to UV is about seven times as long in the mutants as in the wild type. All three strains excise UV-induced pyrimidine dimers from their DNA, although the rate at which cytosine-thymine dimers are excised is slower in the mutants. The three strains also mend the single-strand breaks that appear in the irradiated DNA as a result of dimer excision, although the process is less efficient in the mutants. It is suggested that the increased sensitivity of the mutants to UV radiation may be caused by a partial defect in the second step of dimer excision. PMID:5773016

Isolation and characterization of xylitol-assimilating mutants of recombinant Saccharomyces cerevisiae.

PubMed

Tani, Tatsunori; Taguchi, Hisataka; Fujimori, Kazuhiro E; Sahara, Takehiko; Ohgiya, Satoru; Kamagata, Yoichi; Akamatsu, Takashi

2016-10-01

To clarify the mechanisms of xylitol utilization, three xylitol-assimilating mutants were isolated from recombinant Saccharomyces cerevisiae strains showing highly efficient xylose-utilization. The nucleotide sequences of the mutant genomes were analyzed and compared with those of the wild-type strains and the mutation sites were identified. gal80 mutations were common to all the mutants, and recessive to the wild-type allele. Hence we constructed a gal80Δ mutant and confirmed that the gal80Δ mutant showed a xylitol-assimilation phenotype. When the constructed gal80Δ mutant was crossed with the three isolated mutants, all diploid hybrids showed xylitol assimilation, indicating that the mutations were all located in the GAL80. We analyzed the role of the galactose permease Gal2, controlled by the regulatory protein Gal80, in assimilating xylitol. A gal2Δ gal80Δ double mutant did not show xylitol assimilation, whereas expression of GAL2 under the control of the TDH3 promoter in the GAL80 strain did result in assimilation. These data indicate that Gal2 was needed for xylitol assimilation in the wild-type strain. When the gal80 mutant with an initial cell concentration of A660 = 20 was used for batch fermentation in a complex medium containing 20 g/L xylose or 20 g/L xylitol at pH 5.0 and 30°C under oxygen limitation, the gal80 mutant consumed 100% of the xylose within 12 h, but <30% of the xylitol within 100 h, indicating that xylose reductase is required for xylitol consumption in oxygen-limited conditions. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities

PubMed Central

Kerr, Emma; Gaude, Edoardo; Turrell, Frances; Frezza, Christian; Martins, Carla P

2016-01-01

Summary The RAS/MAPK-signalling pathway is frequently deregulated in non-small cell lung cancer (NSCLC), often through KRAS activating mutations1-3. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations4-7. We recently showed that advanced lung tumours from KrasG12D/+;p53-null mice frequently exhibit KrasG12D allelic enrichment (KrasG12D/Kraswild-type>1)7, implying that mutant Kras copy gains are positively selected during progression. Through a comprehensive analysis of mutant Kras homozygous and heterozygous MEFs and lung cancer cells we now show that these genotypes are phenotypically distinct. In particular, KrasG12D/G12D cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the TCA cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous NSCLC cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of KrasG12D copy gain), but not in the corresponding early tumours (KrasG12D 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 comprised of two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated based on their relative mutant allelic content. We also provide the first in vivo evidence of metabolic rewiring during lung cancer malignant progression. PMID:26909577

Increased Tau Phosphorylation and Tau Truncation, and Decreased Synaptophysin Levels in Mutant BRI2/Tau Transgenic Mice

PubMed Central

Garringer, Holly J.; Murrell, Jill; Sammeta, Neeraja; Gnezda, Anita; Ghetti, Bernardino; Vidal, Ruben

2013-01-01

Familial Danish dementia (FDD) is an autosomal dominant neurodegenerative disease caused by a 10-nucleotide duplication-insertion in the BRI2 gene. FDD is clinically characterized by loss of vision, hearing impairment, cerebellar ataxia and dementia. The main neuropathologic findings in FDD are the deposition of Danish amyloid (ADan) and the presence of neurofibrillary tangles (NFTs). Here we investigated tau accumulation and truncation in double transgenic (Tg-FDD-Tau) mice generated by crossing transgenic mice expressing human Danish mutant BRI2 (Tg-FDD) with mice expressing human 4-repeat mutant Tau-P301S (Tg-Tau). Compared to Tg-Tau mice, we observed a significant enhancement of tau deposition in Tg-FDD-Tau mice. In addition, a significant increase in tau cleaved at aspartic acid (Asp) 421 was observed in Tg-FDD-Tau mice. Tg-FDD-Tau mice also showed a significant decrease in synaptophysin levels, occurring before widespread deposition of fibrillar ADan and tau can be observed. Thus, the presence of soluble ADan/mutant BRI2 can lead to significant changes in tau metabolism and synaptic dysfunction. Our data provide new in vivo insights into the pathogenesis of FDD and the pathogenic pathway(s) by which amyloidogenic peptides, regardless of their primary amino acid sequence, can cause neurodegeneration. PMID:23418567

Increased tau phosphorylation and tau truncation, and decreased synaptophysin levels in mutant BRI2/tau transgenic mice.

PubMed

Garringer, Holly J; Murrell, Jill; Sammeta, Neeraja; Gnezda, Anita; Ghetti, Bernardino; Vidal, Ruben

2013-01-01

Familial Danish dementia (FDD) is an autosomal dominant neurodegenerative disease caused by a 10-nucleotide duplication-insertion in the BRI(2) gene. FDD is clinically characterized by loss of vision, hearing impairment, cerebellar ataxia and dementia. The main neuropathologic findings in FDD are the deposition of Danish amyloid (ADan) and the presence of neurofibrillary tangles (NFTs). Here we investigated tau accumulation and truncation in double transgenic (Tg-FDD-Tau) mice generated by crossing transgenic mice expressing human Danish mutant BRI(2) (Tg-FDD) with mice expressing human 4-repeat mutant Tau-P301S (Tg-Tau). Compared to Tg-Tau mice, we observed a significant enhancement of tau deposition in Tg-FDD-Tau mice. In addition, a significant increase in tau cleaved at aspartic acid (Asp) 421 was observed in Tg-FDD-Tau mice. Tg-FDD-Tau mice also showed a significant decrease in synaptophysin levels, occurring before widespread deposition of fibrillar ADan and tau can be observed. Thus, the presence of soluble ADan/mutant BRI(2) can lead to significant changes in tau metabolism and synaptic dysfunction. Our data provide new in vivo insights into the pathogenesis of FDD and the pathogenic pathway(s) by which amyloidogenic peptides, regardless of their primary amino acid sequence, can cause neurodegeneration.

Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants

PubMed Central

Noguera, Martín E.; Vazquez, Diego S.; Ferrer-Sueta, Gerardo; Agudelo, William A.; Howard, Eduardo; Rasia, Rodolfo M.; Manta, Bruno; Cousido-Siah, Alexandra; Mitschler, André; Podjarny, Alberto; Santos, Javier

2017-01-01

Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity. PMID:28181556

Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants

NASA Astrophysics Data System (ADS)

Noguera, Martín E.; Vazquez, Diego S.; Ferrer-Sueta, Gerardo; Agudelo, William A.; Howard, Eduardo; Rasia, Rodolfo M.; Manta, Bruno; Cousido-Siah, Alexandra; Mitschler, André; Podjarny, Alberto; Santos, Javier

2017-02-01

Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity.

Isolation of a spontaneous CHO amino acid transport mutant by a combination of tritium suicide and replica plating

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dantzig, A.H.; Slayman, C.W.; Adelberg, E.A.

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 increasemore » 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.« less

Checkpoint Blockade Cancer Immunotherapy Targets Tumour-Specific Mutant Antigens

PubMed Central

Gubin, Matthew M.; Zhang, Xiuli; Schuster, Heiko; Caron, Etienne; Ward, Jeffrey P.; Noguchi, Takuro; Ivanova, Yulia; Hundal, Jasreet; Arthur, Cora D.; Krebber, Willem-Jan; Mulder, Gwenn E.; Toebes, Mireille; Vesely, Matthew D.; Lam, Samuel S.K.; Korman, Alan J.; Allison, James P.; Freeman, Gordon J.; Sharpe, Arlene H.; Pearce, Erika L.; Schumacher, Ton N.; Aebersold, Ruedi; Rammensee, Hans-Georg; Melief, Cornelis J. M.; Mardis, Elaine R.; Gillanders, William E.; Artyomov, Maxim N.; Schreiber, Robert D.

2014-01-01

The immune system plays key roles in determining the fate of developing cancers by not only functioning as a tumour promoter facilitating cellular transformation, promoting tumour growth and sculpting tumour cell immunogenicity1–6, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion1,2,7. Yet clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer induced immunosuppression. In many individuals, immunosuppression is mediated by Cytotoxic T-Lymphocyte Associated Antigen-4 (CTLA-4) and Programmed Death-1 (PD-1), two immunomodulatory receptors expressed on T cells8,9. Monoclonal antibody (mAb) based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits—including durable responses—to patients with different malignancies10–13. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Herein, we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T cell rejection antigens following αPD-1 and/or αCTLA-4 therapy of mice bearing progressively growing sarcomas and show that therapeutic synthetic long peptide (SLP) vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Whereas, mutant tumour antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with αPD-1- and/or αCTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens (TSMA) are not only important targets of checkpoint blockade therapy but also can be

Acquired Substrate Preference for GAB1 Protein Bestows Transforming Activity to ERBB2 Kinase Lung Cancer Mutants

PubMed Central

Fan, Ying-Xin; Wong, Lily; Marino, Michael P.; Ou, Wu; Shen, Yi; Wu, Wen Jin; Wong, Kwok-Kin; Reiser, Jakob; Johnson, Gibbes R.

2013-01-01

Activating mutations in the αC-β4 loop of the ERBB2 kinase domain, such as ERBB2YVMA and ERBB2G776VC, have been identified in human lung cancers and found to drive tumor formation. Here we observe that the docking protein GAB1 is hyper-phosphorylated in carcinomas from transgenic mice and in cell lines expressing these ERBB2 cancer mutants. Using dominant negative GAB1 mutants lacking canonical tyrosine residues for SHP2 and PI3K interactions or lentiviral shRNA that targets GAB1, we demonstrate that GAB1 phosphorylation is required for ERBB2 mutant-induced cell signaling, cell transformation, and tumorigenesis. An enzyme kinetic analysis comparing ERBB2YVMA to wild type using physiologically relevant peptide substrates reveals that ERBB2YVMA kinase adopts a striking preference for GAB1 phosphorylation sites as evidenced by ∼150-fold increases in the specificity constants (kcat/Km) for several GAB1 peptides, and this change in substrate selectivity was predominantly attributed to the peptide binding affinities as reflected by the apparent Km values. Furthermore, we demonstrate that ERBB2YVMA phosphorylates GAB1 protein ∼70-fold faster than wild type ERBB2 in vitro. Notably, the mutation does not significantly alter the Km for ATP or sensitivity to lapatinib, suggesting that, unlike EGFR lung cancer mutants, the ATP binding cleft of the kinase is not significantly changed. Taken together, our results indicate that the acquired substrate preference for GAB1 is critical for the ERBB2 mutant-induced oncogenesis. PMID:23612964

A Dominant Loss-of-Function GJA1 (Cx43) Mutant Impairs Parturition in the Mouse1

PubMed Central

Tong, Dan; Lu, Xuerong; Wang, Hong-Xing; Plante, Isabelle; Lui, Ed; Laird, Dale W.; Bai, Donglin; Kidder, Gerald M.

2009-01-01

Expression of GJA1 (commonly known as connexin43 or Cx43), a major myometrial gap junction protein, is upregulated before the onset of delivery, suggesting an essential role for Cx43-mediated gap junctional intercellular communication (GJIC) in normal uterine contraction during parturition. To determine how a disease-linked Cx43 mutation affects myometrial function, we studied a mutant mouse model carrying an autosomal dominant mutation (Gja1Jrt) in the gene encoding Cx43 that displays features of the human genetic disease oculodentodigital dysplasia. We found that Cx43 level, specifically the phosphorylated species of the protein, is significantly reduced in the myometrium of the mutant mice (Gja1Jrt/+), as revealed by Western blotting and immunostaining. Patch-clamp electrophysiological measurements demonstrated that coupling between myometrial smooth muscle cells is reduced to <15% of wild-type, indicating that the mutant protein acts dominantly on its wild-type counterpart. The phosphorylated species of Cx43 in the mutant myometrium failed to increase prior to parturition as well as in response to exogenous estrogen. Correspondingly, in vitro experiments with uterine strips revealed weaker contraction of the mutant myometrium and reduced responsiveness to oxytocin, providing an explanation for the prolonged gestation and presence of suffocated fetuses in the uteri that were observed in some of the mutant mice. We conclude that the Gja1Jrt mutation has a dominant-negative effect on Cx43 function in the myometrium, severely reducing GJIC, leading to impaired parturition. PMID:19176884

The temperature-sensitive mutants of Toxoplasma gondii and ocular toxoplasmosis.

PubMed

Lu, Fangli; Huang, Shiguang; Kasper, Lloyd H

2009-01-22

The risk of blindness caused by ocular toxoplasmosis supports efforts to improve our understanding for control of this disease. In this study, the involvement of CD8(+), CD4(+), B cell, and IL-10 gene in the immune response of primary ocular infection with the temperature-sensitive mutant (ts-4) of the RH Toxoplasma gondii strain, and in the protective immunity of ocular ts-4 vaccination and challenge with RH strain was investigated in murine models utilizing inbred C57BL/6 mice-deficient in CD4(+), CD8(+), B cells (microMT), or IL-10 gene. Compared to naive mice, all WT and mutant mice had different degree of ocular pathological changes after ts-4 ocular infection, in which both CD8 KO and IL-10 KO mice showed the most severe ocular lesions. Immunized by ts-4 intracameral (i.c.) inoculation, all mutant mice had partially decreased vaccine-induced resistance associated with increased ocular parasite burdens after RH strain challenge. A significant increase of the percentages of B cells and CD8(+) T cells in the draining lymph nodes were observed in WT and IL-10 KO mice after either infection or challenge. The levels of specific anti-toxoplasma IgG in both eye fluid and serum from all the mice were significantly increased after ts-4 i.c. immunization, except microMT mice. These results suggest that the avirulent ts-4 of T. gondii inoculated intracamerally can induce both ocular pathology and ocular protective immunity; CD4(+), CD8(+), B cell, and IL-10 gene are all necessary to the vaccine-induced resistance to ocular challenge by virulent RH strain, in which CD8(+) T cells are the most important component.

Germination-defective mutant of Neurospora crassa that responds to siderophores

NASA Technical Reports Server (NTRS)

Charlang, G.; Williams, N. P.

1977-01-01

A conditionally germination-defective mutant of Neurospora crassa has been found to be partially curable by ferricrocin and other siderophores. The mutant conidia rapidly lose their membrane-bound siderophores when suspended in buffer or growth media. Germination is consequently delayed unless large numbers of conidia are present (positive population effect). This indicates that the mutant has a membrane defect involving the siderophore attachment site.

Disoxaril mutants of Coxsackievirus B1: phenotypic characteristics and analysis of the target VP1 gene.

PubMed

Nikolova, Ivanka; Galabov, Angel S; Petkova, Rumena; Chakarov, Stoyan; Atanasov, Boris

2011-01-01

Disoxaril inhibits enterovirus replication by binding to the hydrophobic pocket within the VP1 coat protein, thus stabilizing the virion and blocking its uncoating. Disoxaril-resistant (RES) mutants of the Coxsackievirus B1 (CVB1/RES) were derived from the wild disoxaril-sensitive (SOF) strain (CVB1/SOF) using a selection approach. A disoxaril-dependent (DEP) mutant (CVB1/DEP) was obtained following nine consecutive passages of the disoxaril-resistant mutant in the presence of disoxaril. Phenotypic characteristics of the disoxaril mutants were investigated. A timing-of-addition study of the CVB1/DEP replication demonstrated that in the absence of disoxaril the virus particle assembly stopped. VP1 RNA sequences of disoxaril mutants were compared with the existing Gen Bank CVB1 reference structure. The amino acid sequence of a large VP1 196-258 peptide (disoxaril-binding region) of CVB1/RES was significantly different from that of the CVB1/SOF. Crucially important changes in CVB1/RES were two point mutations, M213H and F237L, both in the ligand-binding pocket. The sequence analysis of the CVB1/DEP showed some reversion to CVB1/SOF. The amino acid sequences of the three VP1 proteins are presented.

Effect of Mutant p53 Proteins on Glycolysis and Mitochondrial Metabolism.

PubMed

Eriksson, Matilda; Ambroise, Gorbatchev; Ouchida, Amanda Tomie; Lima Queiroz, Andre; Smith, Dominique; Gimenez-Cassina, Alfredo; Iwanicki, Marcin P; Muller, Patricia A; Norberg, Erik; Vakifahmetoglu-Norberg, Helin

2017-12-15

TP53 is one of the most commonly mutated genes in human cancers. Unlike other tumor suppressors that are frequently deleted or acquire loss-of-function mutations, the majority of TP53 mutations in tumors are missense substitutions, which lead to the expression of full-length mutant proteins that accumulate in cancer cells and may confer unique gain-of-function (GOF) activities to promote tumorigenic events. Recently, mutant p53 proteins have been shown to mediate metabolic changes as a novel GOF to promote tumor development. There is a strong rationale that the GOF activities, including alterations in cellular metabolism, might vary between the different p53 mutants. Accordingly, the effect of different mutant p53 proteins on cancer cell metabolism is largely unknown. In this study, we have metabolically profiled several individual frequently occurring p53 mutants in cancers, focusing on glycolytic and mitochondrial oxidative phosphorylation pathways. Our investigation highlights the diversity of different p53 mutants in terms of their effect on metabolism, which might provide a foundation for the development of more effective targeted pharmacological approaches toward variants of mutant p53. Copyright © 2017 American Society for Microbiology.

Isolation and characterisation of transport-defective substrate-binding mutants of the tetracycline antiporter TetA(B)

PubMed Central

Wright, David J.; Tate, Christopher G.

2015-01-01

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

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.

Ozone-Sensitive Arabidopsis Mutants with Deficiencies in Photorespiratory Enzymes.

PubMed

Saji, Shoko; Bathula, Srinivas; Kubo, Akihiro; Tamaoki, Masanori; Aono, Mitsuko; Sano, Tomoharu; Tobe, Kazuo; Timm, Stefan; Bauwe, Hermann; Nakajima, Nobuyoshi; Saji, Hikaru

2017-05-01

An ozone-sensitive mutant was isolated from T-DNA-tagged lines of Arabidopsis thaliana. The T-DNA was inserted at a locus on chromosome 3, where two genes encoding glycolate oxidases, GOX1 and GOX2, peroxisomal enzymes involved in photorespiration, reside contiguously. The amounts of the mutant's foliar transcripts for these genes were reduced, and glycolate oxidase activity was approximately 60% of that of the wild-type plants. No difference in growth and appearance was observed between the mutant and the wild-type plants under normal conditions with ambient air under a light intensity of 100 µmol photons m-2 s-1. However, signs of severe damage, such as chlorosis and ion leakage from the tissue, rapidly appeared in mutant leaves in response to ozone treatment at a concentration of 0.2 µl l-1 under a higher light intensity of 350 µmol photons m-2 s-1 that caused no such symptoms in the wild-type plant. The mutant also exhibited sensitivity to sulfur dioxide and long-term high-intensity light. Arabidopsis mutants with deficiencies in other photorespiratory enzymes such as glutamate:glyoxylate aminotransferase and hydroxypyruvate reductase also exhibited ozone sensitivities. Therefore, photorespiration appears to be involved in protection against photooxidative stress caused by ozone and other abiotic factors under high-intensity light. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Isolation of peroxisome-deficient mutants of Saccharomyces cerevisiae.

PubMed Central

Erdmann, R; Veenhuis, M; Mertens, D; Kunau, W H

1989-01-01

Two mutants of Saccharomyces cerevisiae affected in peroxisomal assembly (pas mutants) have been isolated and characterized. Each strain contains a single mutation that results in (i) the inability to grow on oleic acid, (ii) accumulation of peroxisomal matrix enzymes in the cytosol, and (iii) absence of detectable peroxisomes at the ultrastructural level. These lesions (pas1-1 and pas2) are shown to be nonallelic and recessive. Crossing of pas1-1 and pas2 strains resulted in diploid cells that had regained the ability to grow on oleic acid as sole carbon source and to form peroxisomes. These pas mutants may provide useful tools for future studies on the molecular mechanisms involved in peroxisomal assembly. Images PMID:2568633

The Arabidopsis mutant cev1 links cell wall signaling to jasmonate and ethylene responses.

PubMed

Ellis, Christine; Karafyllidis, Ioannis; Wasternack, Claus; Turner, John G

2002-07-01

Biotic and abiotic stresses stimulate the synthesis of jasmonates and ethylene, which, in turn, induce the expression of genes involved in stress response and enhance defense responses. The cev1 mutant has constitutive expression of stress response genes and has enhanced resistance to fungal pathogens. Here, we show that cev1 plants have increased production of jasmonate and ethylene and that its phenotype is suppressed by mutations that interrupt jasmonate and ethylene signaling. Genetic mapping, complementation analysis, and sequence analysis revealed that CEV1 is the cellulose synthase CeSA3. CEV1 was expressed predominantly in root tissues, and cev1 roots contained less cellulose than wild-type roots. Significantly, the cev1 mutant phenotype could be reproduced by treating wild-type plants with cellulose biosynthesis inhibitors, and the cellulose synthase mutant rsw1 also had constitutive expression of VSP. We propose that the cell wall can signal stress responses in plants.

The Arabidopsis Mutant cev1 Links Cell Wall Signaling to Jasmonate and Ethylene Responses

PubMed Central

Ellis, Christine; Karafyllidis, Ioannis; Wasternack, Claus; Turner, John G.

2002-01-01

Biotic and abiotic stresses stimulate the synthesis of jasmonates and ethylene, which, in turn, induce the expression of genes involved in stress response and enhance defense responses. The cev1 mutant has constitutive expression of stress response genes and has enhanced resistance to fungal pathogens. Here, we show that cev1 plants have increased production of jasmonate and ethylene and that its phenotype is suppressed by mutations that interrupt jasmonate and ethylene signaling. Genetic mapping, complementation analysis, and sequence analysis revealed that CEV1 is the cellulose synthase CeSA3. CEV1 was expressed predominantly in root tissues, and cev1 roots contained less cellulose than wild-type roots. Significantly, the cev1 mutant phenotype could be reproduced by treating wild-type plants with cellulose biosynthesis inhibitors, and the cellulose synthase mutant rsw1 also had constitutive expression of VSP. We propose that the cell wall can signal stress responses in plants. PMID:12119374

Electrical phenotypes of calcium transport mutant strains of a filamentous fungus, Neurospora crassa.

PubMed

Hamam, Ahmed; Lew, Roger R

2012-05-01

We characterized the electrical phenotypes of mutants with mutations in genes encoding calcium transporters-a mechanosensitive channel homolog (MscS), a Ca(2+)/H(+) exchange protein (cax), and Ca(2+)-ATPases (nca-1, nca-2, nca-3)-as well as those of double mutants (the nca-2 cax, nca-2 nca-3, and nca-3 cax mutants). The electrical characterization used dual impalements to obtain cable-corrected current-voltage measurements. Only two types of mutants (the MscS mutant; the nca-2 mutant and nca-2-containing double mutants) exhibited lower resting potentials. For the nca-2 mutant, on the basis of unchanged conductance and cyanide-induced depolarization of the potential, the cause is attenuated H(+)-ATPase activity. The growth of the nca-2 mutant-containing strains was inhibited by elevated extracellular Ca(2+) levels, indicative of lesions in Ca(2+) homeostasis. However, the net Ca(2+) effluxes of the nca-2 mutant, measured noninvasively with a self-referencing Ca(2+)-selective microelectrode, were similar to those of the wild type. All of the mutants exhibited osmosensitivity similar to that of the wild type (the turgor of the nca-2 mutant was also similar to that of the wild type), suggesting that Ca(2+) signaling does not play a role in osmoregulation. The hyphal tip morphology and tip-localized mitochondria of the nca-2 mutant were similar to those of the wild type, even when the external [Ca(2+)] was elevated. Thus, although Ca(2+) homeostasis is perturbed in the nca-2 mutant (B. J. Bowman et al., Eukaryot. Cell 10:654-661, 2011), the phenotype does not extend to tip growth or to osmoregulation but is revealed by lower H(+)-ATPase activity.

Electrical Phenotypes of Calcium Transport Mutant Strains of a Filamentous Fungus, Neurospora crassa

PubMed Central

Hamam, Ahmed

2012-01-01

We characterized the electrical phenotypes of mutants with mutations in genes encoding calcium transporters—a mechanosensitive channel homolog (MscS), a Ca2+/H+ exchange protein (cax), and Ca2+-ATPases (nca-1, nca-2, nca-3)—as well as those of double mutants (the nca-2 cax, nca-2 nca-3, and nca-3 cax mutants). The electrical characterization used dual impalements to obtain cable-corrected current-voltage measurements. Only two types of mutants (the MscS mutant; the nca-2 mutant and nca-2-containing double mutants) exhibited lower resting potentials. For the nca-2 mutant, on the basis of unchanged conductance and cyanide-induced depolarization of the potential, the cause is attenuated H+-ATPase activity. The growth of the nca-2 mutant-containing strains was inhibited by elevated extracellular Ca2+ levels, indicative of lesions in Ca2+ homeostasis. However, the net Ca2+ effluxes of the nca-2 mutant, measured noninvasively with a self-referencing Ca2+-selective microelectrode, were similar to those of the wild type. All of the mutants exhibited osmosensitivity similar to that of the wild type (the turgor of the nca-2 mutant was also similar to that of the wild type), suggesting that Ca2+ signaling does not play a role in osmoregulation. The hyphal tip morphology and tip-localized mitochondria of the nca-2 mutant were similar to those of the wild type, even when the external [Ca2+] was elevated. Thus, although Ca2+ homeostasis is perturbed in the nca-2 mutant (B. J. Bowman et al., Eukaryot. Cell 10:654–661, 2011), the phenotype does not extend to tip growth or to osmoregulation but is revealed by lower H+-ATPase activity. PMID:22408225

Improved Medium for Selecting Nitrate-Nonutilizing (nit) Mutants of Verticillium dahliae.

PubMed

Korolev, N; Katan, T

1997-10-01

ABSTRACT Nitrate-nonutilizing (nit) mutants are commonly used to determine vegetative compatibility between isolates of Verticillium dahliae by complementation (heterokaryon) testing. These mutants emerge spontaneously as chlorate-resistant sectors growing out of partially restricted, wild-type colonies on chlorate-amended media. The commonly used chlorate media are based on minimal medium (MMC) or cornmeal agar (CMC), amended with potassium chlorate. nit mutants recovered on these media constituted 10 to 36%(on MMC) and 25 to 45%(on CMC) of the apparently resistant sectors. An improved water agar chlorate medium (WAC) is described that is more effective for selecting chlorate-resistant nit mutants. WAC medium consists of agar (2%), glucose (0.02%), and potassium chlorate (2 to 5%). On WAC, growth of most V. dahliae isolates was strongly inhibited, and 66 to 100%(average >80%) of the chlorate-resistant sectors formed were nit mutants. Most mutants were characterized as nit1, and about 6% as NitM.

A dinoflagellate mutant with higher frequency of multiple fission.

PubMed

Lam, C M; Chong, C; Wong, J T

2001-01-01

The dinoflagellate Crypthecodinium cohnii Biecheler propagates by both binary and multiple fission. By a newly developed mutagenesis protocol based on using ethyl methanesulfonate and a cell size screening method, a cell cycle mutant, mf2, was isolated with giant cells which predominantly divide by multiple fission. The average cell size of the mutant mf2 is larger than the control C. cohnii. Cell cycle synchronization experiments suggest that mutant mf2, when compared with the control strain, has a prolonged G1 phase with a corresponding delay of the G2 + M phase.

Structurally altered capsular polysaccharides produced by mutant bacteria

NASA Technical Reports Server (NTRS)

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

Impaired discrimination learning in interneuronal NMDAR-GluN2B mutant mice.

PubMed

Brigman, Jonathan L; Daut, Rachel A; Saksida, Lisa; Bussey, Timothy J; Nakazawa, Kazu; Holmes, Andrew

2015-06-17

Previous studies have established a role for N-methyl-D-aspartate receptor (NMDAR) containing the GluN2B subunit in efficient learning behavior on a variety of tasks. Recent findings have suggested that NMDAR on GABAergic interneurons may underlie the modulation of striatal function necessary to balance efficient action with cortical excitatory input. Here we investigated how loss of GluN2B-containing NMDAR on GABAergic interneurons altered corticostriatal-mediated associative learning. Mutant mice (floxed-GluN2B×Ppp1r2-Cre) were generated to produce loss of GluN2B on forebrain interneurons and phenotyped on a touchscreen-based pairwise visual learning paradigm. We found that the mutants showed normal performance during Pavlovian and instrumental pretraining, but were significantly impaired on a discrimination learning task. Detailed analysis of the microstructure of discrimination performance revealed reduced win→stay behavior in the mutants. These results further support the role of NMDAR, and GluN2B in particular, on modulation of striatal function necessary for efficient choice behavior and suggest that NMDAR on interneurons may play a critical role in associative learning.

Nitrogen fixation system of tungsten-resistant mutants of Azotobacter vinelandii.

PubMed Central

Riddle, G D; Simonson, J G; Hales, B J; Braymer, H D

1982-01-01

Mutants of Azotobacter vinelandii ATCC 12837 were isolated which could fix N2 in the presence of high tungsten concentrations. The most studied of these mutants (WD2) grew well in N-free modified Burk broth containing 10 mM W, whereas the wild type would not grow in this medium. WD2 would also grow in Burk N-free broth at about the same rate as the wild type. WD2 in broth containing W exhibited 22% of the whole cell acetylene reduction activity of the wild type in broth containing Mo and showed a lowered affinity for acetylene. Two-dimensional gel electrophoresis experiments showed that N2-fixing cells of WD2 from broth containing W or Mo did not produce significant amounts of component I of native nitrogenase protein. Electron spin resonance spectra of whole cells and cell-free extracts of WD2 from broth containing W lacked any trace of the g = 3.6 resonance associated with FeMoCo. Images PMID:6956567

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.

[Correlation of chromosome 1p and 19q status and expression of R132H mutant IDH1 protein in oligodendroglial tumors].

PubMed

Yao, Kun; Duan, Zejun; Hu, Zeliang; Bian, Yu; Qi, Xueling

2014-10-01

To correlate the presence of chromosome 1p/19q deletion with the expression of R132H mutant IDH1 status in oligodendroglial tumors, and to explore molecular markers for predicting chemosensitivity of oligodendroglial tumors. The study included 75 oligodendroglial tumors (38 oligodendrogliomas and 37 oligoastrocytomas). Immunohistochemistry was used to detect the expression of R132H mutant IDH1 protein, and fluorescence in situ hybridization (FISH) was employed to detect 1p/19q deletion. Deletion of chromosome 1p and/or 19q was detected in 37 cases (37/75, 49.3%), among which co-deletion of 1p and 19q was seen in 34 cases (closely correlated, P < 0.01). Oligodendrogliomas WHOIIhad a slightly higher deletion rate than oligodendrogliomas WHO III, although without statistical significance. Oligodendrogliomas WHO IIand WHO III had a significantly higher deletion rate of chromosome 1p/19q than oligoastrocytomas WHO II and WHO III (P < 0.05). While combined loss of 1p/19q was always detected in oligodendrogliomas when FISH was positive, isolated 1p or 19q deletion was only found in oligoastrocytomas. The expression of R132H mutant IDH1 was detected in 51 of 75 cases (68.0%), in which oligodendrogliomas had a higher positive rate than oligoastrocytomas. Statistical analysis demonstrated a significant correlation between the expression of R132H mutant IDH1 protein and the presence of combined 1p/19q deletion in oligodendrogliomas (P < 0.05). A significant correlation was observed between the expression of R132H mutant protein and 1p/19q LOH.Expression of 132H mutant IDH1 protein is the potential biomarker for predicating the presence of 1p/19q deletion in oligodendrogliomas.

Prediction of protein mutant stability using classification and regression tool.

PubMed

Huang, Liang-Tsung; Saraboji, K; Ho, Shinn-Ying; Hwang, Shiow-Fen; Ponnuswamy, M N; Gromiha, M Michael

2007-02-01

Prediction of protein stability upon amino acid substitutions is an important problem in molecular biology and the solving of which would help for designing stable mutants. In this work, we have analyzed the stability of protein mutants using two different datasets of 1396 and 2204 mutants obtained from ProTherm database, respectively for free energy change due to thermal (DeltaDeltaG) and denaturant denaturations (DeltaDeltaG(H(2)O)). We have used a set of 48 physical, chemical energetic and conformational properties of amino acid residues and computed the difference of amino acid properties for each mutant in both sets of data. These differences in amino acid properties have been related to protein stability (DeltaDeltaG and DeltaDeltaG(H(2)O)) and are used to train with classification and regression tool for predicting the stability of protein mutants. Further, we have tested the method with 4 fold, 5 fold and 10 fold cross validation procedures. We found that the physical properties, shape and flexibility are important determinants of protein stability. The classification of mutants based on secondary structure (helix, strand, turn and coil) and solvent accessibility (buried, partially buried, partially exposed and exposed) distinguished the stabilizing/destabilizing mutants at an average accuracy of 81% and 80%, respectively for DeltaDeltaG and DeltaDeltaG(H(2)O). The correlation between the experimental and predicted stability change is 0.61 for DeltaDeltaG and 0.44 for DeltaDeltaG(H(2)O). Further, the free energy change due to the replacement of amino acid residue has been predicted within an average error of 1.08 kcal/mol and 1.37 kcal/mol for thermal and chemical denaturation, respectively. The relative importance of secondary structure and solvent accessibility, and the influence of the dataset on prediction of protein mutant stability have been discussed.

Purkinje cells from RyR2 mutant mice are highly arrhythmogenic but responsive to targeted therapy.

PubMed

Kang, Guoxin; Giovannone, Steven F; Liu, Nian; Liu, Fang-Yu; Zhang, Jie; Priori, Silvia G; Fishman, Glenn I

2010-08-20

The Purkinje fiber network has been proposed as the source of arrhythmogenic Ca(2+) release events in catecholaminergic polymorphic ventricular tachycardia (CPVT), yet evidence supporting this mechanism at the cellular level is lacking. We sought to determine the frequency and severity of spontaneous Ca(2+) release events and the response to the antiarrhythmic agent flecainide in Purkinje cells and ventricular myocytes from RyR2(R4496C/+) CPVT mutant mice and littermate controls. We crossed RyR2(R4496C/+) knock-in mice with the newly described Cntn2-EGFP BAC transgenic mice, which express a fluorescent reporter gene in cells of the cardiac conduction system, including the distal Purkinje fiber network. Isolated ventricular myocytes (EGFP(-)) and Purkinje cells (EGFP(+)) from wild-type hearts and mutant hearts were distinguished by epifluorescence and intracellular Ca(2+) dynamics recorded by microfluorimetry. Both wild-type and RyR2(R4496C/+) mutant Purkinje cells displayed significantly slower kinetics of activation and relaxation compared to ventricular myocytes of the same genotype, and tau(decay) in the mutant Purkinje cells was significantly slower than that observed in wild-type Purkinje cells. Of the 4 groups studied, RyR2(R4496C/+) mutant Purkinje cells were also most likely to develop spontaneous Ca(2+) release events, and the number of events per cell was also significantly greater. Furthermore, with isoproterenol treatment, although all 4 groups showed increases in the frequency of arrhythmogenic Ca(2+(i)) events, the RyR2(R4496C/+) Purkinje cells responded with the most profound abnormalities in intracellular Ca(2+) handling, including a significant increase in the frequency of unstimulated Ca(2+(i)) events and the development of alternans, as well as isolated and sustained runs of triggered beats. Both Purkinje cells and ventricular myocytes from wild-type mice showed suppression of spontaneous Ca(2+) release events with flecainide, whereas in RyR2(R

Corticostriatal circuit defects in Hoxb8 mutant mice

PubMed Central

Nagarajan, Naveen; Jones, Bryan W.; West, Peter J.; Marc, Robert; Capecchi, Mario R.

2018-01-01

Hoxb8 mutant mice exhibit compulsive grooming and hair removal dysfunction similar to humans with the OCD-spectrum disorder, trichotillomania. Since, in the mouse brain, the only detectable cells that label with Hoxb8 cell lineage appear to be microglia, we suggested that defective microglia cause the neuropsychiatric disorder. Does the Hoxb8 mutation in microglia lead to neural circuit dysfunctions? We demonstrate that Hoxb8 mutants contain corticostriatal circuit defects. Golgi staining, ultra-structural, and electrophysiological studies of mutants reveal excess dendritic spines, pre- and post-synaptic structural defects, long-term potentiation and miniature postsynaptic current defects. Hoxb8 mutants also exhibit hyperanxiety and social behavioral deficits similar to mice with neuronal mutations in Sapap3, Slitrk5 and Shank3, reported models of OCD and autism spectrum disorders (ASD’s). Long-term treatment of Hoxb8 mutants with fluoxetine, a serotonin reuptake inhibitor (SSRI), reduces excessive grooming, hyperanxiety and social behavioral impairments. These studies provide linkage between the neuronal defects induced by defective Hoxb8-microglia, and neuronal dysfunctions directly generated by mutations in synaptic components that result in mice that display similar pathological grooming, hyperanxiety and social impairment deficits. Our results shed light on Hoxb8 microglia driven circuit-specific defects and therapeutic approaches that will become essential to developing novel therapies for neuropsychiatric diseases such as OCD and ASD’s with Hoxb8-microglia being the central target. PMID:28948967

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.

Characterization of Sugar Insensitive (sis) Mutants of Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gibson, Susan I.

Despite the fact that soluble sugar levels have been postulated to play an important role in the control of a wide variety of plant metabolic and developmental pathways, the mechanisms by which plants respond to soluble sugar levels remain poorly understood. Plant responses to soluble sugar levels are also important in bioenergy production, as plant sugar responses are believed to help regulate both carbon fixation and carbon partitioning. For example, accumulation of soluble sugars, such as sucrose and glucose, in source tissues leads to feedback inhibition of photosynthesis, thereby decreasing rates of carbon fixation. Soluble sugar levels can also affectmore » sink strengths, affecting the rates of accumulation of carbon-based compounds into both particular molecular forms (e.g. carbohydrates versus lipids versus proteins) and particular plant organs and tissues. Mutants of Arabidopsis that are defective in the ability to respond to soluble sugar levels were isolated and used as tools to identify some of the factors involved in plant sugar response. These sugar insensitive (sis) mutants were isolated by screening mutagenized seeds for those that were able to germinate and develop relatively normal shoot systems on media containing 0.3 M glucose or 0.3 M sucrose. At these sugar concentrations, wild-type Arabidopsis germinate and produce substantial root systems, but show little to no shoot development. Twenty-eight sis mutants were isolated during the course of four independent mutant screens. Based on a preliminary characterization of all of these mutants, sis3 and sis6 were chosen for further study. Both of these mutations appear to lie in previously uncharacterized loci. Unlike many other sugar-response mutants, sis3 mutants exhibit a wild-type or near wild-type response in all phytohormone-response assays conducted to date. The sis6-1 mutation is unusual in that it appears to be due to overexpression of a gene, rather than representing a loss of function

Rapid screening astaxanthin-hyperproducing Haematococcus pluvialis mutants through near-infrared spectroscopy.

PubMed

Liu, J H; Song, L; Huang, Q

2016-02-01

The unicellular freshwater green microalga Haematococcus pluvialis is the richest source of natural astaxanthin. Since accumulation of astaxanthin differs significantly among various algal strains at different stages, it is therefore critical to develop an effective high-throughput assay for rapid screening astaxanthin-hyperproducing strains. In the present study, near-infrared spectroscopy (NIRS) in combination with biochemical assay was employed for evaluation of the wide-type H. Pluvialis strains. The partial least squares (PLS) models of total biomass, astaxanthin content and astaxanthin expressed as a percentage of dry weight (DW) were developed with the R(2) values as 0·959, 0·982 and 0·952, the prediction correlation factor (r) values as 0·979, 0·988 and 0·966, and the residual predictive deviation (RPD) values as 4·88, 6·22 and 3·86, respectively. Furthermore, the PLS models were employed to evaluate H. pluvialis mutants, with the r values as 0·973, 0·983 and 0·976, and the RPD values as 3·45, 7·59 and 4·07, respectively. This work thus demonstrates that NIRS is an easy, fast and non-invasive approach that can be applied in high-throughput screening of astaxanthin-hyperproducing algal mutants. Haematococcus pluvialis has potential application for its ability to accumulate natural antioxidant astaxanthin. In this study, we initiated the application of near-infrared spectroscopy (NIRS) in the analysis of total biomass and astaxanthin content of different mutant strains, demonstrating that NIRS can be very useful in the screening of axataxanthin-hyperproducing mutant strains. © 2015 The Society for Applied Microbiology.

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

PubMed

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

2009-11-01

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 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 Phox and Bem1 (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 as 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.

Vaccination of rainbow trout against infectious hematopoietic necrosis (IHN) by using attenuated mutants selected by neutralizing monoclonal antibodies

USGS Publications Warehouse

Roberti, K.A.; Rohovec, J.S.; Winton, J.R.

1998-01-01

A neutralizing monoclonal antibody against infectious hematopoietic necrosis virus (IHNV) was used to select neutralization-resistant mutants from isolates of virus obtained from adult steelhead Oncorhynchus mykiss returning to the Round Butte Hatchery (RB mutants) on the Deschutes River in Oregon, USA, and from rainbow trout (nonanadromous O. mykiss) at a commercial hatchery in the Hagerman Valley of Idaho, USA (193-110 mutants). Two of the mutants, RB-1 and 193-110-4, were significantly (P 0.05) in protection among fish exposed to the RB-1 vaccine strain at a dose of 1 x 105 TCID50/mL for periods of either 1, 12, or 24 h, held for 14 d, and then challenged with the wild-type RB isolate, although the 1-h exposure seemed to be somewhat less effective. Fish were vaccinated with the RB-1 strain at 1 x 103-1 x 105 TCID50/mL for 24 h then challenged after 1, 7, 14, or 21 d with the wild-type RB isolate. No significant (P > 0.1) protection was observed at 1 d postvaccination, but the relative percent survival increased progressively at each subsequent challenge period, becoming statistically significant by day 7 (P < 0.001) and beyond. These results suggested that resistance to challenge with wild-type virus resulted from development of IHNV-specific immunity and not from viral interference or interferon induction, and they reinforce the potential of an attenuated vaccine to control this important disease.

Understanding the Basis of Drug Resistance of the Mutants of αβ-Tubulin Dimer via Molecular Dynamics Simulations

PubMed Central

Natarajan, Kathiresan; Senapati, Sanjib

2012-01-01

The vital role of tubulin dimer in cell division makes it an attractive drug target. Drugs that target tubulin showed significant clinical success in treating various cancers. However, the efficacy of these drugs is attenuated by the emergence of tubulin mutants that are unsusceptible to several classes of tubulin binding drugs. The molecular basis of drug resistance of the tubulin mutants is yet to be unraveled. Here, we employ molecular dynamics simulations, protein-ligand docking, and MMPB(GB)SA analyses to examine the binding of anticancer drugs, taxol and epothilone to the reported point mutants of tubulin - T274I, R282Q, and Q292E. Results suggest that the mutations significantly alter the tubulin structure and dynamics, thereby weaken the interactions and binding of the drugs, primarily by modifying the M loop conformation and enlarging the pocket volume. Interestingly, these mutations also affect the tubulin distal sites that are associated with microtubule building processes. PMID:22879949

Transcriptome analysis of the epidermis of the purple quail-like (q-lp) mutant of silkworm, Bombyx mori.

PubMed

Wang, Pingyang; Qiu, Zhiyong; Xia, Dingguo; Tang, Shunming; Shen, Xingjia; Zhao, Qiaoling

2017-01-01

A new purple quail-like (q-lp) mutant found from the plain silkworm strain 932VR has pigment dots on the epidermis similar to the pigment mutant quail (q). In addition, q-lp mutant larvae are inactive, consume little and grow slowly, with a high death rate and other developmental abnormalities. Pigmentation of the silkworm epidermis consists of melanin, ommochrome and pteridine. Silkworm development is regulated by ecdysone and juvenile hormone. In this study, we performed RNA-Seq on the epidermis of the q-lp mutant in the 4th instar during molting, with 932VR serving as the control. The results showed 515 differentially expressed genes, of which 234 were upregulated and 281 downregulated in q-lp. BLASTGO analysis indicated that the downregulated genes mainly encode protein-binding proteins, membrane components, oxidation/reduction enzymes, and proteolytic enzymes, whereas the upregulated genes largely encode cuticle structural constituents, membrane components, transport related proteins, and protein-binding proteins. Quantitative reverse transcription PCR was used to verify the accuracy of the RNA-Seq data, focusing on key genes for biosynthesis of the three pigments and chitin as well as genes encoding cuticular proteins and several related nuclear receptors, which are thought to play key roles in the q-lp mutant. We drew three conclusions based on the results: 1) melanin, ommochrome and pteridine pigments are all increased in the q-lp mutant; 2) more cuticle proteins are expressed in q-lp than in 932VR, and the number of upregulated cuticular genes is significantly greater than downregulated genes; 3) the downstream pathway regulated by ecdysone is blocked in the q-lp mutant. Our research findings lay the foundation for further research on the developmental changes responsible for the q-lp mutant.

Diversity and stability study on rice mutants induced in space environment.

PubMed

Lu, Wei-Hong; Wang, Xin-Zhu; Zheng, Qi; Guan, Shuang-Hong; Xin, Ping; Sun, Ye-Qing

2008-03-01

To further study the characteristics of changes on the molecular level of rice mutants induced in space environment, we analyzed proteins in leaves and seeds of four rice mutants (two high-tillering and two low-tillering) in the 8(th) and 9(th) generations after a 15-day spaceflight, and compared with their ground controls by two-dimentional polyacrylamide gel electrophoresis and reverse phase liquid chromatography (RPLC). In addition, the albumin, globulin, prolamine, glutelin, and amylose of the mutant seeds were analyzed by RPLC and ultra-violet spectrometry. The results showed that the low-abundance proteins of leaves in the peak tillering stage are more likely to be induced compared with their corresponding controls. The albumin, globulin, and prolamine of the mutant seeds revealed changes when compared with their controls, and the characteristics of changes in different mutants were stably inherited in the 8(th) and 9(th) generations, suggesting that they can be used as bio markers to identity the mutants induced by spaceflight. Moreover, two proteins (SSP9111 and SSP6302) were found to be expressed with high intensity (two-fold change) in different mutants, which were both correlated with photosystem according to mass spectrometry and database searching.

Gamma ray-induced small plaque mutants of western equine encephalitis virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simizu, B.; Yamazaki, S.; Suzuki, K.

1973-12-01

Small plaque mutants of Western equine encephalitis virus were obtained from the surviving fractions of wild-type virus which was irradiated with gamma rays. The frequency with which small plaque mutants appeared in the surviving fraction increased with the radiation dose. These mutants were not more resistant to radiation than wild-type virus. The growth rate of a mutant, S127, was lower than that of wild-type. Clonally purified mutant virions presented two peaks in a velocity sedimentation profile; peak 1 corresponded to the peak of wild type and peak 2 moved faster than peak 1. Virions of both peaks were infectious andmore » consistently formed small plaques in chicken embryo cells. Virions reisolated from either peak and grown in chicken embryo cells also revealed two peaks in sedimentation analysis. In the electron microscope examination peak 2 proved to consist of giant form particles, each of which contained more than one nucleoid surrounded with a common envelope. Despite this remarkable morphological difference, densities of the wild-type and S127 mutant virions were similar in cesium chloride gradients. The RNAs and proteins of mutant virions could not be distinguished from those of wild types on the basis of size or change. (auth)« less

Accumulation of 10 Fluoroquinolones by Wild-Type or Efflux Mutant Streptococcus pneumoniae

PubMed Central

Piddock, Laura J. V.; Johnson, M. M.

2002-01-01

A method for measuring fluoroquinolone accumulation by Streptococcus pneumoniae was rigorously examined. The accumulation of ciprofloxacin, clinafloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, sitafloxacin, and trovafloxacin in the presence and absence of either carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) or reserpine was determined for two wild-type fluoroquinolone-susceptible capsulated S. pneumoniae strains (M3 and M4) and the noncapsulated strain R6. Two efflux mutants, R6N (which overexpresses PmrA) and a mutant of M4, M22 (no expression of PmrA), were also examined. Essentially, the fluoroquinolones fell into two groups. (i) One group consisting of ciprofloxacin, grepafloxacin, and norfloxacin accumulated to 72 to 92 ng/mg (dry weight) of cells in all strains. (ii) The remainder of the agents accumulated to 3 to 30 ng/mg (dry weight) of cells. With a decrease in hydrophobicity, there was a decrease in the concentration accumulated. With an increase in the molecular weight of the free form of each agent, there was also a decrease in the concentration accumulated. The strains differed in their responses to reserpine and CCCP. For the three fluoroquinolone-susceptible strains, only reserpine had a significant effect upon accumulation of moxifloxacin and clinafloxacin by M3 and showed no effect for the other agents and strains. For M3 and M4, CCCP enhanced the concentration of ciprofloxacin and norfloxacin accumulated, whereas for R6, the effect was only statistically significant for ofloxacin. Efflux mutant M22 accumulated less ciprofloxacin, gatifloxacin, and ofloxacin than M4 did. M22 accumulated more norfloxacin than M4 did. Reserpine and CCCP had variable effects as for the other strains. Differences in the accumulation of fluoroquinolones by R6 and R6N were highly dependent upon growth phase, and only for norfloxacin was there a significant difference between two strains. PMID:11850266

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Catalanotti, C.; Dubini, A.; Subramanian, V.

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 doublemore » 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.« less

Selection and Characterization of Dunaliella salina Mutants Defective in Haloadaptation 1

PubMed Central

Chitlaru, Edith; Pick, Uri

1989-01-01

A technique for selection of Dunaliella mutants defective in their capacity to recover from osmotic shocks has been developed. The selection is based on physical separation of mutants on density gradients. This technique takes advantage of the fact that Dunaliella cells, when exposed to osmotic shocks, initially change volume and density due to water gain or loss and subsequently recover their volume and density by readjusting their intracellular glycerol. Eight mutants that do not recover their original density following hyperosmotic shocks have been isolated. The mutants grow similar to wild type cells in 1 molar NaCl, and recover like the wild type from hypotonic shocks but are defective in recovering from hypertonic shocks. A partial characterization of one of the mutants is described. Images Figure 1 PMID:16667101

Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

DOE PAGES

Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.; ...

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

Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.

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

EMMA—mouse mutant resources for the international scientific community

PubMed Central

Wilkinson, Phil; Sengerova, Jitka; Matteoni, Raffaele; Chen, Chao-Kung; Soulat, Gaetan; Ureta-Vidal, Abel; Fessele, Sabine; Hagn, Michael; Massimi, Marzia; Pickford, Karen; Butler, Richard H.; Marschall, Susan; Mallon, Ann-Marie; Pickard, Amanda; Raspa, Marcello; Scavizzi, Ferdinando; Fray, Martin; Larrigaldie, Vanessa; Leyritz, Johan; Birney, Ewan; Tocchini-Valentini, Glauco P.; Brown, Steve; Herault, Yann; Montoliu, Lluis; de Angelis, Martin Hrabé; Smedley, Damian

2010-01-01

The laboratory mouse is the premier animal model for studying human disease and thousands of mutants have been identified or produced, most recently through gene-specific mutagenesis approaches. High throughput strategies by the International Knockout Mouse Consortium (IKMC) are producing mutants for all protein coding genes. Generating a knock-out line involves huge monetary and time costs so capture of both the data describing each mutant alongside archiving of the line for distribution to future researchers is critical. The European Mouse Mutant Archive (EMMA) is a leading international network infrastructure for archiving and worldwide provision of mouse mutant strains. It operates in collaboration with the other members of the Federation of International Mouse Resources (FIMRe), EMMA being the European component. Additionally EMMA is one of four repositories involved in the IKMC, and therefore the current figure of 1700 archived lines will rise markedly. The EMMA database gathers and curates extensive data on each line and presents it through a user-friendly website. A BioMart interface allows advanced searching including integrated querying with other resources e.g. Ensembl. Other resources are able to display EMMA data by accessing our Distributed Annotation System server. EMMA database access is publicly available at http://www.emmanet.org. PMID:19783817

Respiratory-deficient mutants of the unicellular green alga Chlamydomonas: a review.

PubMed

Salinas, Thalia; Larosa, Véronique; Cardol, Pierre; Maréchal-Drouard, Laurence; Remacle, Claire

2014-05-01

Genetic manipulation of the unicellular green alga Chlamydomonas reinhardtii is straightforward. Nuclear genes can be interrupted by insertional mutagenesis or targeted by RNA interference whereas random or site-directed mutagenesis allows the introduction of mutations in the mitochondrial genome. This, combined with a screen that easily allows discriminating respiratory-deficient mutants, makes Chlamydomonas a model system of choice to study mitochondria biology in photosynthetic organisms. Since the first description of Chlamydomonas respiratory-deficient mutants in 1977 by random mutagenesis, many other mutants affected in mitochondrial components have been characterized. These respiratory-deficient mutants increased our knowledge on function and assembly of the respiratory enzyme complexes. More recently some of these mutants allowed the study of mitochondrial gene expression processes poorly understood in Chlamydomonas. In this review, we update the data concerning the respiratory components with a special focus on the assembly factors identified on other organisms. In addition, we make an inventory of different mitochondrial respiratory mutants that are inactivated either on mitochondrial or nuclear genes. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

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

Altered respiratory responses to hypoxia in mutant mice deficient in neuronal nitric oxide synthase

PubMed Central

Kline, David D; Yang, Tianen; Huang, Paul L; Prabhakar, Nanduri R

1998-01-01

The role of endogenous nitric oxide (NO) generated by neuronal nitric oxide synthase (NOS-1) in the control of respiration during hypoxia and hypercapnia was assessed using mutant mice deficient in NOS-1. Experiments were performed on awake and anaesthetized mutant and wild-type control mice. Respiratory responses to varying levels of inspired oxygen (100, 21 and 12 % O2) and carbon dioxide (3 and 5 % CO2 balanced oxygen) were analysed. In awake animals, respiration was monitored by body plethysmograph along with oxygen consumption (V̇O2), CO2 production (V̇CO2) and body temperature. In anaesthetized, spontaneously breathing mice, integrated efferent phrenic nerve activity was monitored as an index of neural respiration along with arterial blood pressure and blood gases. Cyclic 3′,5′-guanosine monophosphate (cGMP) levels in the brainstem were analysed by radioimmunoassay as an index of nitric oxide generation. Unanaesthetized mutant mice exhibited greater respiratory responses during 21 and 12 % O2 than the wild-type controls. Respiratory responses were associated with significant decreases in oxygen consumption in both groups of mice, and the magnitude of change was greater in mutant than wild-type mice. Changes in CO2 production and body temperature, however, were comparable between both groups of mice. Similar augmentation of respiratory responses during hypoxia was also observed in anaesthetized mutant mice. In addition, five of the fourteen mutant mice displayed periodic oscillations in respiration (brief episodes of increases in respiratory rate and tidal phrenic nerve activity) while breathing 21 and 12 % O2, but not during 100 % O2. The time interval between the episodes decreased by reducing inspired oxygen from 21 to 12 % O2. Changes in arterial blood pressure and arterial blood gases were comparable at any given level of inspired oxygen between both groups of mice, indicating that changes in these variables do not account for the differences in the

An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

PubMed Central

Qualls, David A.; Crosby, Keith; Brown, Hilda; Borchelt, David R.

2013-01-01

Background By mechanisms yet to be discerned, the co-expression of high levels of wild-type human superoxide dismutase 1 (hSOD1) with variants of hSOD1 encoding mutations linked familial amyotrophic lateral sclerosis (fALS) hastens the onset of motor neuron degeneration in transgenic mice. Although it is known that spinal cords of paralyzed mice accumulate detergent insoluble forms of WT hSOD1 along with mutant hSOD1, it has been difficult to determine whether there is co-deposition of the proteins in inclusion structures. Methodology/Principal Findings In the present study, we use cell culture models of mutant SOD1 aggregation, focusing on the A4V, G37R, and G85R variants, to examine interactions between WT-hSOD1 and misfolded mutant SOD1. In these studies, we fuse WT and mutant proteins to either yellow or red fluorescent protein so that the two proteins can be distinguished within inclusions structures. Conclusions/Significance Although the interpretation of the data is not entirely straightforward because we have strong evidence that the nature of the fused fluorophores affects the organization of the inclusions that form, our data are most consistent with the idea that normal dimeric WT-hSOD1 does not readily interact with misfolded forms of mutant hSOD1. We also demonstrate the monomerization of WT-hSOD1 by experimental mutation does induce the protein to aggregate, although such monomerization may enable interactions with misfolded mutant SOD1. Our data suggest that WT-hSOD1 is not prone to become intimately associated with misfolded mutant hSOD1 within intracellular inclusions that can be generated in cultured cells. PMID:24391857

Andrographolide induces degradation of mutant p53 via activation of Hsp70.

PubMed

Sato, Hirofumi; Hiraki, Masatsugu; Namba, Takushi; Egawa, Noriyuki; Baba, Koichi; Tanaka, Tomokazu; Noshiro, Hirokazu

2018-05-22

The tumor suppressor gene p53 encodes a transcription factor that regulates various cellular functions, including DNA repair, apoptosis and cell cycle progression. Approximately half of all human cancers carry mutations in p53 that lead to loss of tumor suppressor function or gain of functions that promote the cancer phenotype. Thus, targeting mutant p53 as an anticancer therapy has attracted considerable attention. In the current study, a small-molecule screen identified andrographlide (ANDRO) as a mutant p53 suppressor. The effects of ANDRO, a small molecule isolated from the Chinese herb Andrographis paniculata, on tumor cells carrying wild-type or mutant p53 were examined. ANDRO suppressed expression of mutant p53, induced expression of the cyclin-dependent kinase inhibitor p21 and pro-apoptotic proteins genes, and inhibited the growth of cancer cells harboring mutant p53. ANDRO also induced expression of the heat-shock protein (Hsp70) and increased binding between Hsp70 and mutant p53 protein, thus promoting proteasomal degradation of p53. These results provide novel insights into the mechanisms regulating the function of mutant p53 and suggest that activation of Hsp70 may be a new strategy for the treatment of cancers harboring mutant p53.

Analysis of Induced Pluripotent Stem Cells from a BRCA1 Mutant Family

PubMed Central

Soyombo, Abigail A.; Wu, Yipin; Kolski, Lauren; Rios, Jonathan J.; Rakheja, Dinesh; Chen, Alice; Kehler, James; Hampel, Heather; Coughran, Alanna; Ross, Theodora S.

2013-01-01

Summary Understanding BRCA1 mutant cancers is hampered by difficulties in obtaining primary cells from patients. We therefore generated and characterized 24 induced pluripotent stem cell (iPSC) lines from fibroblasts of eight individuals from a BRCA1 5382insC mutant family. All BRCA1 5382insC heterozygous fibroblasts, iPSCs, and teratomas maintained equivalent expression of both wild-type and mutant BRCA1 transcripts. Although no difference in differentiation capacity was observed between BRCA1 wild-type and mutant iPSCs, there was elevated protein kinase C-theta (PKC-theta) in BRCA1 mutant iPSCs. Cancer cell lines with BRCA1 mutations and hormone-receptor-negative breast cancers also displayed elevated PKC-theta. Genome sequencing of the 24 iPSC lines showed a similar frequency of reprogramming-associated de novo mutations in BRCA1 mutant and wild-type iPSCs. These data indicate that iPSC lines can be derived from BRCA1 mutant fibroblasts to study the effects of the mutation on gene expression and genome stability. PMID:24319668

Methylammonium-resistant mutants of Nicotiana plumbaginifolia are affected in nitrate transport.

PubMed

Godon, C; Krapp, A; Leydecker, M T; Daniel-Vedele, F; Caboche, M

1996-02-25

This work reports the isolation and preliminary characterization of Nicotiana plumbaginifolia mutants resistant to methylammonium. Nicotiana plumbaginifolia plants cannot grow on low levels of nitrate in the presence of methylammonium. Methylammonium is not used as a nitrogen source, although it can be efficiently taken up by Nicotiana plumbaginifolia cells and converted into methylglutamine, an analog of glutamine. Glutamine is known to repress the expression of the enzymes that mediate the first two steps in the nitrate assimilatory pathway, nitrate reductase (NR) and nitrite reductase (NiR). Methylammonium has therefore been used, in combination with low concentrations of nitrate, as a selective agent in order to screen for mutants in which the nitrate pathway is de-repressed. Eleven semi-dominant mutants, all belonging to the same complementation group, were identified. The mutant showing the highest resistance to methylammonium was not affected either in the utilization of ammonium, accumulation of methylammonium or in glutamine synthase activity. A series of experiments showed that utilization of nitrite by the wild-type and the mutant was comparable, in the presence or the absence of methylammonium, thus suggesting that the mutation specifically affected nitrate transport or reduction. Although NR mRNA levels were less repressed by methylammonium treatment of the wild-type than the mutant, NR activities of the mutant remained comparable with or without methylammonium, leading to the hypothesis that modified expression of NR is probably not responsible for resistance to methylammonium. Methylammonium inhibited nitrate uptake in the wild-type but had only a limited effect in the mutant. The implications of these results are discussed.

A Metagenome-Wide Association Study and Arrayed Mutant Library Confirm Acetobacter Lipopolysaccharide Genes Are Necessary for Association with Drosophila melanogaster.

PubMed

White, K Makay; Matthews, Melinda K; Hughes, Rachel C; Sommer, Andrew J; Griffitts, Joel S; Newell, Peter D; Chaston, John M

2018-03-28

A metagenome wide association (MGWA) study of bacterial host association determinants in Drosophila predicted that LPS biosynthesis genes are significantly associated with host colonization. We were unable to create site-directed mutants for each of the predicted genes in Acetobacter , so we created an arrayed transposon insertion library using Acetobacter fabarum DsW_054 isolated from Drosophila Creation of the A. fabarum DsW_054 gene knock-out library was performed by combinatorial mapping and Illumina sequencing of random transposon insertion mutants. Transposon insertion locations for 6,418 mutants were successfully mapped, including hits within 63% of annotated genes in the A. fabarum DsW_054 genome. For 45/45 members of the library, insertion sites were verified by arbitrary PCR and Sanger sequencing. Mutants with insertions in four different LPS biosynthesis genes were selected from the library to validate the MGWA predictions. Insertion mutations in two genes biosynthetically upstream of Lipid-A formation, lpxC and lpxB , show significant differences in host association, whereas mutations in two genes encoding LPS biosynthesis functions downstream of Lipid-A biosynthesis had no effect. These results suggest an impact of bacterial cell surface molecules on the bacterial capacity for host association. Also, the transposon insertion mutant library will be a useful resource for ongoing research on the genetic basis for Acetobacter traits. Copyright © 2018 White et al.

Differentially expressed genes in the ovary of the sixth day of pupal "Ming" lethal egg mutant of silkworm, Bombyx mori.

PubMed

Gao, Peng; Chen, An-Li; Zhao, Qiao-Ling; Shen, Xing-Jia; Qiu, Zhi-Yong; Xia, Ding-Guo; Tang, Shun-Ming; Zhang, Guo-Zheng

2013-09-15

The "Ming" lethal egg mutant (l-em) is a vitelline membrane mutant in silkworm, Bombyx mori. The eggs laid by the l-em mutant lose water, ultimately causing death within an hour. Previous studies have shown that the deletion of BmEP80 is responsible for the l-em mutation in silkworm, B. mori. In the current study, digital gene expression (DGE) was performed to investigate the difference of gene expression in ovaries between wild type and l-em mutant on the sixth day of the pupal stage to obtain a global view of gene expression profiles using the ovaries of three l-em mutants and three wild types. The results showed a total of 3,463,495 and 3,607,936 clean tags in the wild type and the l-em mutant libraries, respectively. Compared with those of wild type, 239 differentially expressed genes were detected in the l-em mutant, wherein 181 genes are up-regulated and 58 genes are down-regulated in the mutant strain. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results showed that no pathway was significantly enriched and three pathways are tightly related to protein synthesis among the five leading pathways. Moreover, the expression profiles of eight important differentially expressed genes related to oogenesis changed. These results provide a comprehensive gene expression analysis of oogenesis and vitellogenesis in B. mori which facilitates understanding of both the specific molecular mechanism of the 1-em mutant and Lepidopteran oogenesis in general. Copyright © 2013 Elsevier B.V. All rights reserved.

The Arabidopsis mutant, fy-1, has an ABA-insensitive germination phenotype

PubMed Central

Jiang, Shiling; Kumar, Santosh; Eu, Young-Jae; Jami, Sravan Kumar; Stasolla, Claudio; Hill, Robert D.

2012-01-01

Arabidopsis FY, a homologue of the yeast RNA 3' processing factor Pfs2p, regulates the autonomous floral transition pathway through its interaction with FCA, an RNA binding protein. It is demonstrated here that FY also influences seed dormancy. Freshly-harvested seed of the Arabidopsis fy-1 mutant germinated readily in the absence of stratification or after-ripening. Furthermore, the fy-1 mutant showed less ABA sensitivity compared with the wild type, Ler, under identical conditions. Freshly-harvested seed of fy-1 had significantly higher ABA levels than Ler, even though Ler was dormant and fy-1 germinated readily. The PPLPP domains of FY, which are required for flowering control, were not essential for the ABA-influenced repression of germination. FLC expression analysis in seeds of different genotypes suggested that the effect of FY on dormancy may not be elicited through FLC. No significant differences in CYP707A1, CYP707A2, NCED9, ABI3, and ABI4 were observed between freshly-harvested Ler and fy-1 imbibed for 48 h. GA3ox1 and GA3ox2 rapidly increased over the 48 h imbibition period for fy-1, with no significant increases in these transcripts for Ler. ABI5 levels were significantly lower in fy-1 over the 48 h imbibition period. The results suggest that FY is involved in the development of dormancy and ABA sensitivity in Arabidopsis seed. PMID:22282534

The Arabidopsis mutant, fy-1, has an ABA-insensitive germination phenotype.

PubMed

Jiang, Shiling; Kumar, Santosh; Eu, Young-Jae; Jami, Sravan Kumar; Stasolla, Claudio; Hill, Robert D

2012-04-01

Arabidopsis FY, a homologue of the yeast RNA 3' processing factor Pfs2p, regulates the autonomous floral transition pathway through its interaction with FCA, an RNA binding protein. It is demonstrated here that FY also influences seed dormancy. Freshly-harvested seed of the Arabidopsis fy-1 mutant germinated readily in the absence of stratification or after-ripening. Furthermore, the fy-1 mutant showed less ABA sensitivity compared with the wild type, Ler, under identical conditions. Freshly-harvested seed of fy-1 had significantly higher ABA levels than Ler, even though Ler was dormant and fy-1 germinated readily. The PPLPP domains of FY, which are required for flowering control, were not essential for the ABA-influenced repression of germination. FLC expression analysis in seeds of different genotypes suggested that the effect of FY on dormancy may not be elicited through FLC. No significant differences in CYP707A1, CYP707A2, NCED9, ABI3, and ABI4 were observed between freshly-harvested Ler and fy-1 imbibed for 48 h. GA3ox1 and GA3ox2 rapidly increased over the 48 h imbibition period for fy-1, with no significant increases in these transcripts for Ler. ABI5 levels were significantly lower in fy-1 over the 48 h imbibition period. The results suggest that FY is involved in the development of dormancy and ABA sensitivity in Arabidopsis seed.

Comparative production of 6-aminopenicillanic acid by different E. coli strains and their acridine orange (AO) induced mutants.

PubMed

Arshad, Rubina; Farooq, Shafqat; Ali, Syed Shahid

2007-11-01

The present study was conducted to see the difference in production of 6-APA I) between wild strains of E. coli collected from local environment and their acridine orange (AO) induced mutants and ii) between mutants and E. coli strains (ATCC 11105 and ATCC 9637) of American Type Culture Collection (ATCC) used commercially for enzymatic production of 6-APA. The optimum conditions for bioconversion were standardized and 6-APA was obtained in crystalline form. Relative PGA activity of local and foreign E. coli strains varied significantly with the highest being 12.7 in mutant strain (BDCS-N-M36) and the lowest 4.3 mg 6-APA h(-1) mg(-1) wet cells in foreign strain (ATCC 11105). The enzyme activity exhibited by mutant strain (BDCS-N-M36) was also two folds higher compared to that in wild parent BDCS-N-W50 (6.3 mg 6-APA h(-1) mg(-1) wet cells). The overall production of 6-APA and conversion ratios ranged between 0.25-0.41 g of 6-APA per 0.5 g of penicillin G and 51-83%, respectively. Maximum conversion ratio (83%) was achieved by using crude cells of mutant strain (BDCS-N-M36) which is the highest value ever reported by crude cells on a shake-flask scale whereas reported 6-APA production by immobilized cells is 60-90% in batch and continuous systems. Results are being discussed with reference to importance of local bacterial strains and their significance for industrially important enzymes.

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

High-resolution analysis of locomotor activity rhythms in disconnected, a visual-system mutant of Drosophila melanogaster.

PubMed

Dowse, H B; Dushay, M S; Hall, J C; Ringo, J M

1989-07-01

Free-running locomotor activity and eclosion rhythms of Drosophila melanogaster, mutant at the disconnected (disco) locus, are substantially different from the wild-type phenotype. Initial periodogram analysis revealed little or no rhythmicity (Dushay et al., 1989). We have reanalyzed the locomotor activity data using high-resolution signal analysis (maximum-entropy spectral analysis, or MESA). These analyses, corroborated by autocorrelograms, uncovered significant residual circadian rhythmicity and strong ultradian rhythms in most of the animals tested. In this regard the disco mutants are much like flies expressing mutant alleles of the period gene, as well as wild-type flies reared throughout life in constant darkness. We hypothesize that light normally triggers the coupling of multiple ultradian oscillators into a functional circadian clock and that this process is disrupted in disco flies as a result of the neural lesion.

Mutant MiRP1 subunits modulate HERG K+ channel gating: a mechanism for pro-arrhythmia in long QT syndrome type 6

PubMed Central

Lu, Yu; Mahaut-Smith, Martyn P; Huang, Christopher L-H; Vandenberg, Jamie I

2003-01-01

Mutations in KCNE2, which encodes the minK-related protein 1 (MiRP1), are associated with an increased risk of arrhythmias; however, the underlying mechanisms are unknown. MiRP1 is thought to associate with many K+ channel α-subunits, including HERG K+ channels, which have a major role in suppressing arrhythmias initiated by premature beats. In this study we have investigated in chinese hamster ovary (CHO) cells at 37 °C the effects of co-expressing HERG K+ channels with either wild-type (WT) MiRP1 or one of three mutant MiRP1 subunits, T8A, Q9E and M54T. The most significant effects of MiRP1 subunits on HERG channels were a more negative steady-state activation for HERG + T8A MiRP1 and a more positive steady-state activation for HERG + M54T MiRP1 compared to either HERG + WT MiRP1 or HERG alone. All three mutants caused a significant slowing of deactivation at depolarised potentials. T8A MiRP1 also caused an acceleration of inactivation and recovery from inactivation compared to HERG + WT MiRP1. During ventricular action potential clamp experiments there was a significant decrease in current in the early phases of the action potential for HERG + WT MiRP1 channels compared to HERG alone. This effect was not as prominent for the mutant MiRP1 subunits. During premature action potential clamp protocols, the T8A and Q9E mutants, but not the M54T mutant, resulted in significantly larger current spikes during closely coupled premature beats, compared to HERG + WT MiRP1. At longer coupling intervals, all three mutants resulted in larger current spikes than HERG alone or HERG + WT MiRP1 channels. It is therefore possible that augmentation of HERG currents in the early diastolic period may be pro-arrhythmic. PMID:12923204

Isolation and Characterization of Escherichia coli tolC Mutants Defective in Secreting Enzymatically Active Alpha-Hemolysin

PubMed Central

Vakharia, Hema; German, Greg J.; Misra, Rajeev

2001-01-01

This study describes the isolation and characterization of a unique class of TolC mutants that, under steady-state growth conditions, secreted normal levels of largely inactive alpha-hemolysin. Unlike the reduced activity in the culture supernatants, the cell-associated hemolytic activity in these mutants was identical to that in the parental strain, thus reflecting a normal intracellular toxin activation event. Treatment of the secreted toxin with guanidine hydrochloride significantly restored cytolytic activity, suggesting that the diminished activity may have been due to the aggregation or misfolding of the toxin molecules. Consistent with this notion, sedimentation and filtration analyses showed that alpha-hemolysin secreted from the mutant strain has a mass greater than that secreted from the parental strain. Experiments designed to monitor the time course of alpha-hemolysin release showed delayed appearance of toxin in the culture supernatant of the mutant strain, thus indicating a possible defect in alpha-hemolysin translocation or release. Eight different TolC substitutions displaying this toxin secretion defect were scattered throughout the protein, of which six localized in the periplasmically exposed α-helical domain, while the remaining two mapped within the outer membrane-embedded β-barrel domain of TolC. A plausible model for the secretion of inactive alpha-hemolysin in these TolC mutants is discussed in the context of the recently determined three-dimensional structure of TolC. PMID:11698380

Replication of transformation-defective mutants of the Prague strain of Rous sarcoma virus and isolation of a td mutant from duck-adapted PR-RSV-C.

PubMed

Geryk, J; Mazo, A; Svoboda, J; Hlozánek, I

1980-01-01

The replication of transformation-defective mutants of the Prague strain of Rous sarcoma virus subgroup C was studied using roller cultures. Under such conditions, 10(5)--10(6) infectous units of virus per 0.2 ml were produced, as revealed in both the reverse transcriptase and 16Q complementation tests. A new td daPR-RSV-C mutant was isolated from duck-adapted PR-RSV-C. This mutant replicated in roller cultures with equal efficiency as the original td PR-RSV-C. It was verified that td daPR-RSV-C does not transform chicken fibroblasts, is not oncogenic for 3-week-old chickens and has subgroup C host-range specificity. Both td mutants replicate in duck cells and reach the same titres.

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. Copyright © 2012 Elsevier B.V. All rights reserved.

Reversion of autocrine transformation by a dominant negative platelet-derived growth factor mutant.

PubMed

Vassbotn, F S; Andersson, M; Westermark, B; Heldin, C H; Ostman, A

1993-07-01

A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions.

Production and characterization of streptomycin dependent mutants of Pasteurella multocida from bovine haemorrhagic septicaemia.

PubMed Central

de Alwis, M C; Carter, G R; Chengappa, M M

1980-01-01

A large number of streptomycin dependent mutants were produced from bovine haemorrhagic septicaemia strains of Pasteurella multocida. The mutants required a minimum concentration of 25-50 microgram/mL streptomycin for growth and tolerated a concentration of 200 mg/mL. These mutants were avirulent to mice, when inoculated alone, but some mutants killed mice when inoculated with streptomycin. Biochemically all mutants were uniform and similar to the wild type. Most mutants were stable, but a few produced streptomycin independent revertants. The rate of reversion varied with each mutant. Most revertants were highly virulent for mice, some totally avirulant and a few relatively avirulent. PMID:6778598

Elevated Cell Wall Serine in Pleiotropic Staphylococcal Mutants

PubMed Central

Korman, Ruth Z.

1966-01-01

Korman, Ruth Z. (Cornell University, Ithaca, N.Y.). Elevated cell wall serine in pleiotropic staphylococcal mutants. J. Bacteriol. 92:762–768. 1966.—Physically purified cell walls were prepared from two staphylococcal strains and from pleiotropic variants derived from them. The quantitative amino acid and amino sugar content of these walls is reported. The pleiotypes, which are identified culturally by their failure to elaborate coagulase, their resistance to bacteriophage, and their sensitivity to mannitol, have altered molar ratios of amino acids and amino sugars in their cell walls. In comparison with lysine content, the serine content of the mutant wall is elevated and the glycine content is reduced. The glucosamine content is reduced also. It is postulated that the pleiotropic mutants possess an altered cell wall biosynthetic pathway. Images PMID:5922547

Isolation and characterization of low-sulphur-tolerant mutants of Arabidopsis.

PubMed

Wu, Yu; Zhao, Qing; Gao, Lei; Yu, Xiao-Min; Fang, Ping; Oliver, David J; Xiang, Cheng-Bin

2010-07-01

Sulphur is an essential element for plant growth and development as well as for defence against biotic and abiotic stresses. Increasing sulphate utilization efficiency (SUE) is an important issue for crop improvement. Little is known about the genetic determinants of sulphate utilization efficiency. No gain-of-function mutants with improved SUE have been reported to date. Here the isolation and characterization of two low-sulphur-tolerant mutants, sue3 and sue4 are reported using a high-throughput genetic screen where a 'sulphur-free' solid medium was devised to give the selection pressure necessary to suppress the growth of the wild-type seedlings. Both mutants showed improved tolerance to low sulphur conditions and well-developed root systems. The mutant phenotype of both sue3 and sue4 was specific to sulphate deficiency and the mutants displayed enhanced tolerance to heavy metal and oxidative stress. Genetic analysis revealed that sue3 was caused by a single recessive nuclear mutation while sue4 was caused by a single dominant nuclear mutation. The recessive locus in sue3 is the previously identified VirE2-interacting Protein 1. The dominant locus in sue4 is a function-unknown locus activated by the four enhancers on the T-DNA. The function of SUE3 and SUE4 in low sulphur tolerance was confirmed either by multiple mutant alleles or by recapitulation analysis. Taken together, our results demonstrate that this genetic screen is a reasonable approach to isolate Arabidopsis mutants with improved low sulphur tolerance and potentially with enhanced sulphate utilization efficiency. The two loci identified in sue3 and sue4 should assist in understanding the molecular mechanisms of low sulphur tolerance.

Agrobacterium tumefaciens mutants affected in attachment to plant cells.

PubMed Central

Douglas, C J; Halperin, W; Nester, E W

1982-01-01

An analysis of Agrobacterium tumefaciens mutants with Tn5 insertions in chromosomal DNA showed that the chromosome of A. tumefaciens codes for a specific ability of this bacterium to attach to plant cells. This ability is associated with tumorigenesis by A. tumefaciens, the ability of avirulent A. tumefaciens to inhibit tumorigenesis, and the ability to adsorb certain phages. A second class of chromosomal mutations affects tumorigenesis without altering the ability to attach to plant cells. The attachment of A. tumefaciens to plant cells was assayed by mixing radiolabeled bacteria with suspensions of tobacco tissue culture cells or freshly isolated Zinnia leaf mesophyll cells. Under the conditions of this assay, an avirulent Ti plasmid-cured strain attached to the same extent as the same strain containing pTiB6806. Six of eight avirulent mutants with Tn5 insertions in chromosomal DNA showed defective attachment, whereas two retained wild-type attachment ability. In contrast to the strains showing wild-type attachment, the attachment-defective mutants failed to inhibit tumorigenesis when inoculated onto Jerusalem artichoke slices before inoculation of a virulent strain and also showed a loss of sensitivity to two Agrobacterium phages. The loss of phage sensitivity appeared to be due to a loss of ability to adsorb the phages. Staining with Calcofluor indicated that the mutants retained the ability to synthesize cellulose fibrils, which have been implicated in the attachment process. Southern filter hybridizations demonstrated that each mutant contained a single Tn5 insertion, and genetic linkage between the Tn5 insertion in one mutant and the attachment phenotype has also been demonstrated. Images PMID:6292165

Heterozygous inactivation of tsc2 enhances tumorigenesis in p53 mutant zebrafish

PubMed Central

Kim, Seok-Hyung; Kowalski, Marie L.; Carson, Robert P.; Bridges, L. Richard; Ess, Kevin C.

2013-01-01

SUMMARY Tuberous sclerosis complex (TSC) is a multi-organ disorder caused by mutations of the TSC1 or TSC2 genes. A key function of these genes is to inhibit mTORC1 (mechanistic target of rapamycin complex 1) kinase signaling. Cells deficient for TSC1 or TSC2 have increased mTORC1 signaling and give rise to benign tumors, although, as a rule, true malignancies are rarely seen. In contrast, other disorders with increased mTOR signaling typically have overt malignancies. A better understanding of genetic mechanisms that govern the transformation of benign cells to malignant ones is crucial to understand cancer pathogenesis. We generated a zebrafish model of TSC and cancer progression by placing a heterozygous mutation of the tsc2 gene in a p53 mutant background. Unlike tsc2 heterozygous mutant zebrafish, which never exhibited cancers, compound tsc2;p53 mutants had malignant tumors in multiple organs. Tumorigenesis was enhanced compared with p53 mutant zebrafish. p53 mutants also had increased mTORC1 signaling that was further enhanced in tsc2;p53 compound mutants. We found increased expression of Hif1-α, Hif2-α and Vegf-c in tsc2;p53 compound mutant zebrafish compared with p53 mutant zebrafish. Expression of these proteins probably underlies the increased angiogenesis seen in compound mutant zebrafish compared with p53 mutants and might further drive cancer progression. Treatment of p53 and compound mutant zebrafish with the mTORC1 inhibitor rapamycin caused rapid shrinkage of tumor size and decreased caliber of tumor-associated blood vessels. This is the first report using an animal model to show interactions between tsc2, mTORC1 and p53 during tumorigenesis. These results might explain why individuals with TSC rarely have malignant tumors, but also suggest that cancer arising in individuals without TSC might be influenced by the status of TSC1 and/or TSC2 mutations and be potentially treatable with mTORC1 inhibitors. PMID:23580196

Phenotypic Restoration by Molybdate of Nitrate Reductase Activity in chlD Mutants of Escherichia coli

PubMed Central

Glaser, J. H.; DeMoss, J. A.

1971-01-01

ChlD mutants of Escherichia coli are pleiotropic, lacking formate-nitrate reductase activity as well as formate-hydrogenlyase activity. Whole-chain formate-nitrate reductase activity, assayed with formate as the electron donor and measuring the amount of nitrite produced, was restored to wild-type levels in the mutants by addition of 10−4m molybdate to the growth medium. Under these conditions, the activity of each of the components of the membrane-bound nitrate reductase chain increased after molybdate supplementation. In the absence of nitrate, the activities of the formate-hydrogenlyase system were also restored by molybdate. Strains deleted for the chlD gene responded in a similar way to molybdate supplementation. The concentration of molybdenum in the chlD mutant cells did not differ significantly from that in the wild-type cells at either low or high concentrations of molybdate in the medium. However, the distribution of molybdenum between the soluble protein and membrane fractions differed significantly from wild type. We conclude that the chlD gene product cannot be a structural component of the formate-hydrogenlyase pathway or the formate-nitrate reductase pathway, but that it must have an indirect role in processing molybdate to a form necessary for both electron transport systems. PMID:4942767

Mitochondrial Calcium Dysregulation Contributes to Dendrite Degeneration Mediated by PD/LBD-Associated LRRK2 Mutants.

PubMed

Verma, Manish; Callio, Jason; Otero, P Anthony; Sekler, Israel; Wills, Zachary P; Chu, Charleen T

2017-11-15

Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to development of late-onset familial Parkinson's disease (PD), with clinical features of motor and cognitive dysfunction indistinguishable from sporadic PD. Calcium dysregulation plays an important role in PD pathogenesis, but the mechanisms of neurodegeneration remain unclear. Recent reports indicate enhanced excitatory neurotransmission in cortical neurons expressing mutant LRRK2, which occurs before the well-characterized phenotype of dendritic shortening. As mitochondria play a major role in the rapid buffering of cytosolic calcium, we hypothesized that altered mitochondrial calcium handling contributes to dendritic retraction elicited by the LRRK2-G2019S and -R1441C mutations. In primary mouse cortical neurons, we observed increased depolarization-induced mitochondrial calcium uptake. We found that expression of mutant LRRK2 elicited transcriptional upregulation of the mitochondrial calcium uniporter (MCU) and the mitochondrial calcium uptake 1 protein (MICU1) with no change in levels of the mitochondrial calcium antiporter NCLX. Elevated MCU and MICU1 were also observed in LRRK2-mutated patient fibroblasts, along with increased mitochondrial calcium uptake, and in postmortem brains of sporadic PD/PDD patients of both sexes. Transcriptional upregulation of MCU and MICU1 was caused by activation of the ERK1/2 (MAPK3/1) pathway. Inhibiting ERK1/2 conferred protection against mutant LRRK2-induced neurite shortening. Pharmacological inhibitors or RNAi knockdown of MCU attenuated mitochondrial calcium uptake and dendritic/neuritic shortening elicited by mutant LRRK2, whereas expression of a constitutively active mutant of NCLX that enhances calcium export from mitochondria was neuroprotective. These data suggest that an increased susceptibility to mitochondrial calcium dysregulation contributes to dendritic injury in mutant LRRK2 pathogenesis. SIGNIFICANCE STATEMENT Cognitive dysfunction and dementia are

Spontaneous Gac Mutants of Pseudomonas Biological Control Strains: Cheaters or Mutualists? ▿

PubMed Central

Driscoll, William W.; Pepper, John W.; Pierson, Leland S.; Pierson, Elizabeth A.

2011-01-01

Bacteria rely on a range of extracellular metabolites to suppress competitors, gain access to resources, and exploit plant or animal hosts. The GacS/GacA two-component regulatory system positively controls the expression of many of these beneficial external products in pseudomonad bacteria. Natural populations often contain variants with defective Gac systems that do not produce most external products. These mutants benefit from a decreased metabolic load but do not appear to displace the wild type in nature. How could natural selection maintain the wild type in the presence of a mutant with enhanced growth? One hypothesis is that Gac mutants are “cheaters” that do not contribute to the public good, favored within groups but selected against between groups, as groups containing more mutants lose access to ecologically important external products. An alternative hypothesis is that Gac mutants have a mutualistic interaction with the wild type, so that each variant benefits by the presence of the other. In the biocontrol bacterium Pseudomonas chlororaphis strain 30-84, Gac mutants do not produce phenazines, which suppress competitor growth and are critical for biofilm formation. Here, we test the predictions of these alternative hypotheses by quantifying interactions between the wild type and the phenazine- and biofilm-deficient Gac mutant within growing biofilms. We find evidence that the wild type and Gac mutants interact mutualistically in the biofilm context, whereas a phenazine-defective structural mutant does not. Our results suggest that the persistence of alternative Gac phenotypes may be due to the stabilizing role of local mutualistic interactions. PMID:21873476

Possible association between mutant frequency in peripheral lymphocytes and domestic radon concentrations.

PubMed

Bridges, B A; Cole, J; Arlett, C F; Green, M H; Waugh, A P; Beare, D; Henshaw, D L; Last, R D

1991-05-18

To investigate whether previously found geographical correlations between leukaemia incidence and exposure to radon are reflected in a detectable mutagenic effect on individuals, the frequency of mutations in the hypoxanthine guanine phosphoribosyl transferase gene (hprt) in peripheral blood T lymphocytes was measured in subjects with known domestic radon concentrations. These concentrations were measured in December, 1989, in houses in Street, Somerset, UK, by passive alpha-track radon detectors. 20 non-smoking subjects aged 36-55 years were selected from the patient list at the local health centre on the basis of the radon concentrations in their homes--the range selected varied by a factor of ten. Blood samples for preparation of T lymphocytes were taken in July, 1990. There was a significant association between the log mutant frequency and radon concentration (t = 3.47, p less than 0.01). A second analysis of a further set of radon measurements (October, 1990, to January, 1991), in both living rooms and bedrooms, and repeated mutant frequency determinations also showed a significant relation, which remained significant even after exclusion of the highest frequency and adjustment for subject's age and cloning efficiency. These data must be regarded as preliminary and further more extensive studies should be done to determine whether the observed association is causal.

Mutant Enrichment in the Colonial Alga, EUDORINA ELEGANS

PubMed Central

Toby, A. L.; Kemp, C. L.

1975-01-01

An enrichment procedure has been developed that results in at least a 200x increase in mutation frequency in the colonial alga, Eudorina elegans. A period of nitrogen starvation followed by treatment with 8-azaguanine results in the death of wild-type cells and the maintenance of mutants. N'-nitro-N-nitro-soguanidine-induced acetate, p-aminobenzoic acid and reduced nitrogen requiring mutants have been isolated by this procedure. PMID:1205128

Defining the requirements for the pathogenic interaction between mutant calreticulin and MPL in MPN

PubMed Central

Elf, Shannon; Abdelfattah, Nouran S.; Baral, April J.; Beeson, Danielle; Rivera, Jeanne F.; Ko, Amy; Florescu, Natalie; Birrane, Gabriel; Chen, Edwin

2018-01-01

Mutations in calreticulin (CALR) are phenotypic drivers in the pathogenesis of myeloproliferative neoplasms. Mechanistic studies have demonstrated that mutant CALR binds to the thrombopoietin receptor MPL, and that the positive electrostatic charge of the mutant CALR C terminus is required for mutant CALR-mediated activation of JAK-STAT signaling. Here we demonstrate that although binding between mutant CALR and MPL is required for mutant CALR to transform hematopoietic cells; binding alone is insufficient for cytokine independent growth. We further show that the threshold of positive charge in the mutant CALR C terminus influences both binding of mutant CALR to MPL and activation of MPL signaling. We find that mutant CALR binds to the extracellular domain of MPL and that 3 tyrosine residues within the intracellular domain of MPL are required to activate signaling. With respect to mutant CALR function, we show that its lectin-dependent function is required for binding to MPL and for cytokine independent growth, whereas its chaperone and polypeptide-binding functionalities are dispensable. Together, our findings provide additional insights into the mechanism of the pathogenic mutant CALR-MPL interaction in myeloproliferative neoplasms. PMID:29288169

Defining the requirements for the pathogenic interaction between mutant calreticulin and MPL in MPN.

PubMed

Elf, Shannon; Abdelfattah, Nouran S; Baral, April J; Beeson, Danielle; Rivera, Jeanne F; Ko, Amy; Florescu, Natalie; Birrane, Gabriel; Chen, Edwin; Mullally, Ann

2018-02-15

Mutations in calreticulin ( CALR ) are phenotypic drivers in the pathogenesis of myeloproliferative neoplasms. Mechanistic studies have demonstrated that mutant CALR binds to the thrombopoietin receptor MPL, and that the positive electrostatic charge of the mutant CALR C terminus is required for mutant CALR-mediated activation of JAK-STAT signaling. Here we demonstrate that although binding between mutant CALR and MPL is required for mutant CALR to transform hematopoietic cells; binding alone is insufficient for cytokine independent growth. We further show that the threshold of positive charge in the mutant CALR C terminus influences both binding of mutant CALR to MPL and activation of MPL signaling. We find that mutant CALR binds to the extracellular domain of MPL and that 3 tyrosine residues within the intracellular domain of MPL are required to activate signaling. With respect to mutant CALR function, we show that its lectin-dependent function is required for binding to MPL and for cytokine independent growth, whereas its chaperone and polypeptide-binding functionalities are dispensable. Together, our findings provide additional insights into the mechanism of the pathogenic mutant CALR-MPL interaction in myeloproliferative neoplasms. © 2018 by The American Society of Hematology.

Arabidopsis Brassinosteroid-Insensitive dwarf12 Mutants Are Semidominant and Defective in a Glycogen Synthase Kinase 3β-Like Kinase1

PubMed Central

Choe, Sunghwa; Schmitz, Robert J.; Fujioka, Shozo; Takatsuto, Suguru; Lee, Mi-Ok; Yoshida, Shigeo; Feldmann, Kenneth A.; Tax, Frans E.

2002-01-01

Mutants defective in the biosynthesis or signaling of brassinosteroids (BRs), plant steroid hormones, display dwarfism. Loss-of-function mutants for the gene encoding the plasma membrane-located BR receptor BRI1 are resistant to exogenous application of BRs, and characterization of this protein has contributed significantly to the understanding of BR signaling. We have isolated two new BR-insensitive mutants (dwarf12-1D and dwf12-2D) after screening Arabidopsis ethyl methanesulfonate mutant populations. dwf12 mutants displayed the characteristic morphology of previously reported BR dwarfs including short stature, short round leaves, infertility, and abnormal de-etiolation. In addition, dwf12 mutants exhibited several unique phenotypes, including severe downward curling of the leaves. Genetic analysis indicates that the two mutations are semidominant in that heterozygous plants show a semidwarf phenotype whose height is intermediate between wild-type and homozygous mutant plants. Unlike BR biosynthetic mutants, dwf12 plants were not rescued by high doses of exogenously applied BRs. Like bri1 mutants, dwf12 plants accumulated castasterone and brassinolide, 43- and 15-fold higher, respectively, providing further evidence that DWF12 is a component of the BR signaling pathway that includes BRI1. Map-based cloning of the DWF12 gene revealed that DWF12 belongs to a member of the glycogen synthase kinase 3β family. Unlike human glycogen synthase kinase 3β, DWF12 lacks the conserved serine-9 residue in the auto-inhibitory N terminus. In addition, dwf12-1D and dwf12-2D encode changes in consecutive glutamate residues in a highly conserved TREE domain. Together with previous reports that both bin2 and ucu1 mutants contain mutations in this TREE domain, this provides evidence that the TREE domain is of critical importance for proper function of DWF12/BIN2/UCU1 in BR signal transduction pathways. PMID:12428015

φX-174 Bacteriophage Structural Mutants Which Affect Deoxyribonucleic Acid Synthesis

PubMed Central

Siegel, Jeff E. D.; Hayashi, Masaki

1969-01-01

Seven cistrons in φX-174 were identified and one in particular was studied intensively: cistron A, which is assigned a protein in the mature phage. Amber mutants in this cistron synthesize a new deoxyribonucleic acid (DNA) form in addition to circular phage DNA upon infection of the restrictive host. This DNA is linear, non-infectious, and single-stranded; it is formed from the phage strand of replicative form φX-174 DNA. These mutants produce two different defective particles in the restrictive host. One particle contains circular phage DNA but is not infectious; the other contains the new DNA form and is similar to the 70S particles found in wild-type phage lysates. The mutant A gene product acts independently of normal A protein upon mixed infection of the restrictive host with an A mutant and a mutant from any other cistron or wild type. PMID:5823229

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. Copyright 1999 John Wiley & Sons, Inc.

Restoration of mutant bestrophin-1 expression, localisation and function in a polarised epithelial cell model

PubMed Central

Briant, Kit; Streit, Anne-Kathrin; Thomson, Steven; Koay, Yee Hui

2016-01-01

ABSTRACT Autosomal recessive bestrophinopathy (ARB) is a retinopathy caused by mutations in the bestrophin-1 protein, which is thought to function as a Ca2+-gated Cl− channel in the basolateral surface of the retinal pigment epithelium (RPE). Using a stably transfected polarised epithelial cell model, we show that four ARB mutant bestrophin-1 proteins were mislocalised and subjected to proteasomal degradation. In contrast to the wild-type bestrophin-1, each of the four mutant proteins also failed to conduct Cl− ions in transiently transfected cells as determined by whole-cell patch clamp. We demonstrate that a combination of two clinically approved drugs, bortezomib and 4-phenylbutyrate (4PBA), successfully restored the expression and localisation of all four ARB mutant bestrophin-1 proteins. Importantly, the Cl− conductance function of each of the mutant bestrophin-1 proteins was fully restored to that of wild-type bestrophin-1 by treatment of cells with 4PBA alone. The functional rescue achieved with 4PBA is significant because it suggests that this drug, which is already approved for long-term use in infants and adults, might represent a promising therapy for the treatment of ARB and other bestrophinopathies resulting from missense mutations in BEST1. PMID:27519691

Restoration of mutant bestrophin-1 expression, localisation and function in a polarised epithelial cell model.

PubMed

Uggenti, Carolina; Briant, Kit; Streit, Anne-Kathrin; Thomson, Steven; Koay, Yee Hui; Baines, Richard A; Swanton, Eileithyia; Manson, Forbes D

2016-11-01

Autosomal recessive bestrophinopathy (ARB) is a retinopathy caused by mutations in the bestrophin-1 protein, which is thought to function as a Ca 2+ -gated Cl - channel in the basolateral surface of the retinal pigment epithelium (RPE). Using a stably transfected polarised epithelial cell model, we show that four ARB mutant bestrophin-1 proteins were mislocalised and subjected to proteasomal degradation. In contrast to the wild-type bestrophin-1, each of the four mutant proteins also failed to conduct Cl - ions in transiently transfected cells as determined by whole-cell patch clamp. We demonstrate that a combination of two clinically approved drugs, bortezomib and 4-phenylbutyrate (4PBA), successfully restored the expression and localisation of all four ARB mutant bestrophin-1 proteins. Importantly, the Cl - conductance function of each of the mutant bestrophin-1 proteins was fully restored to that of wild-type bestrophin-1 by treatment of cells with 4PBA alone. The functional rescue achieved with 4PBA is significant because it suggests that this drug, which is already approved for long-term use in infants and adults, might represent a promising therapy for the treatment of ARB and other bestrophinopathies resulting from missense mutations in BEST1. © 2016. Published by The Company of Biologists Ltd.

A Caveolin Dominant Negative Mutant Associates with Lipid Bodies and Induces Intracellular Cholesterol Imbalance

PubMed Central

Pol, Albert; Luetterforst, Robert; Lindsay, Margaret; Heino, Sanna; Ikonen, Elina; Parton, Robert G.

2001-01-01

Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3DGV, specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1–3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cavDGV to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts. PMID:11238460

Secondary Metabolism and Interspecific Competition Affect Accumulation of Spontaneous Mutants in the GacS-GacA Regulatory System in Pseudomonas protegens

PubMed Central

Yan, Qing; Lopes, Lucas D.; Shaffer, Brenda T.; Kidarsa, Teresa A.; Vining, Oliver; Philmus, Benjamin; Song, Chunxu; Stockwell, Virginia O.; Raaijmakers, Jos M.; McPhail, Kerry L.; Andreote, Fernando D.; Chang, Jeff H.

2018-01-01

ABSTRACT Secondary metabolites are synthesized by many microorganisms and provide a fitness benefit in the presence of competitors and predators. Secondary metabolism also can be costly, as it shunts energy and intermediates from primary metabolism. In Pseudomonas spp., secondary metabolism is controlled by the GacS-GacA global regulatory system. Intriguingly, spontaneous mutations in gacS or gacA (Gac− mutants) are commonly observed in laboratory cultures. Here we investigated the role of secondary metabolism in the accumulation of Gac− mutants in Pseudomonas protegens strain Pf-5. Our results showed that secondary metabolism, specifically biosynthesis of the antimicrobial compound pyoluteorin, contributes significantly to the accumulation of Gac− mutants. Pyoluteorin biosynthesis, which poses a metabolic burden on the producer cells, but not pyoluteorin itself, leads to the accumulation of the spontaneous mutants. Interspecific competition also influenced the accumulation of the Gac− mutants: a reduced proportion of Gac− mutants accumulated when P. protegens Pf-5 was cocultured with Bacillus subtilis than in pure cultures of strain Pf-5. Overall, our study associated a fitness trade-off with secondary metabolism, with metabolic costs versus competitive benefits of production influencing the evolution of P. protegens, assessed by the accumulation of Gac− mutants. PMID:29339425

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.

Two-Pore Channels: Lessons from Mutant Mouse Models

PubMed Central

Ruas, Margarida; Galione, Antony; Parrington, John

2016-01-01

Recent interest in two-pore channels (TPCs) has resulted in a variety of studies dealing with the functional role and mechanism of action of these endo-lysosomal proteins in diverse physiological processes. With the availability of mouse lines harbouring mutant alleles for Tpcnl and/or Tpcn2 genes, several studies have made use of them to validate, consolidate and discover new roles for these channels not only at the cellular level but, importantly, also at the level of the whole organism. The different mutant mouse lines that have been used were derived from distinct genetic manipulation strategies, with the aim of knocking out expression of TPC proteins. However, the expression of different residual TPC sequences predicted to occur in these mutant mouse lines, together with the varied degree to which the effects on Tpcn expression have been studied, makes it important to assess the true knockout status of some of the lines. In this review we summarize these Tpcn mutant mouse lines with regard to their predicted effect on Tpcn expression and the extent to which they have been characterized. Additionally, we discuss how results derived from studies using these Tpcn mutant mouse lines have consolidated previously proposed roles for TPCs, such as mediators of NAADP signalling, endo-lysosomal functions, and pancreatic β cell physiology. We will also review how they have been instrumental in the assignment of new physiological roles for these cation channels in processes such as membrane electrical excitability, neoangiogenesis, viral infection and brown adipose tissue and heart function, revealing, in some cases, a specific contribution of a particular TPC isoform. PMID:27330869

Isolation and characterization of low-sulphur-tolerant mutants of Arabidopsis

PubMed Central

Wu, Yu; Zhao, Qing; Gao, Lei; Yu, Xiao-Min; Fang, Ping; Oliver, David J.; Xiang, Cheng-Bin

2010-01-01

Sulphur is an essential element for plant growth and development as well as for defence against biotic and abiotic stresses. Increasing sulphate utilization efficiency (SUE) is an important issue for crop improvement. Little is known about the genetic determinants of sulphate utilization efficiency. No gain-of-function mutants with improved SUE have been reported to date. Here the isolation and characterization of two low-sulphur-tolerant mutants, sue3 and sue4 are reported using a high-throughput genetic screen where a ‘sulphur-free’ solid medium was devised to give the selection pressure necessary to suppress the growth of the wild-type seedlings. Both mutants showed improved tolerance to low sulphur conditions and well-developed root systems. The mutant phenotype of both sue3 and sue4 was specific to sulphate deficiency and the mutants displayed enhanced tolerance to heavy metal and oxidative stress. Genetic analysis revealed that sue3 was caused by a single recessive nuclear mutation while sue4 was caused by a single dominant nuclear mutation. The recessive locus in sue3 is the previously identified VirE2-interacting Protein 1. The dominant locus in sue4 is a function-unknown locus activated by the four enhancers on the T-DNA. The function of SUE3 and SUE4 in low sulphur tolerance was confirmed either by multiple mutant alleles or by recapitulation analysis. Taken together, our results demonstrate that this genetic screen is a reasonable approach to isolate Arabidopsis mutants with improved low sulphur tolerance and potentially with enhanced sulphate utilization efficiency. The two loci identified in sue3 and sue4 should assist in understanding the molecular mechanisms of low sulphur tolerance. PMID:20547563

Development and characterisation of highly antibiotic resistant Bartonella bacilliformis mutants

PubMed Central

Gomes, Cláudia; Martínez-Puchol, Sandra; Ruiz-Roldán, Lidia; Pons, Maria J.; del Valle Mendoza, Juana; Ruiz, Joaquim

2016-01-01

The objective was to develop and characterise in vitro Bartonella bacilliformis antibiotic resistant mutants. Three B. bacilliformis strains were plated 35 or 40 times with azithromycin, chloramphenicol, ciprofloxacin or rifampicin discs. Resistance-stability was assessed performing 5 serial passages without antibiotic pressure. MICs were determined with/without Phe-Arg-β-Napthylamide and artesunate. Target alterations were screened in the 23S rRNA, rplD, rplV, gyrA, gyrB, parC, parE and rpoB genes. Chloramphenicol and ciprofloxacin resistance were the most difficult and easiest (>37.3 and 10.6 passages) to be selected, respectively. All mutants but one selected with chloramphenicol achieved high resistance levels. All rifampicin, one azithromycin and one ciprofloxacin mutants did not totally revert when cultured without antibiotic pressure. Azithromycin resistance was related to L4 substitutions Gln-66 → Lys or Gly-70 → Arg; L4 deletion Δ62–65 (Lys-Met-Tyr-Lys) or L22 insertion 83::Val-Ser-Glu-Ala-His-Val-Gly-Lys-Ser; in two chloramphenicol-resistant mutants the 23S rRNA mutation G2372A was detected. GyrA Ala-91 → Val and Asp-95 → Gly and GyrB Glu474 → Lys were detected in ciprofloxacin-resistant mutants. RpoB substitutions Gln-527 → Arg, His-540 → Tyr and Ser-545 → Phe plus Ser-588 → Tyr were detected in rifampicin-resistant mutants. In 5 mutants the effect of efflux pumps on resistance was observed. Antibiotic resistance was mainly related to target mutations and overexpression of efflux pumps, which might underlie microbiological failures during treatments. PMID:27667026

Tomato TILLING Technology: Development of a Reverse Genetics Tool for the Efficient Isolation of Mutants from Micro-Tom Mutant Libraries

PubMed Central

Okabe, Yoshihiro; Asamizu, Erika; Saito, Takeshi; Matsukura, Chiaki; Ariizumi, Tohru; Brès, Cécile; Rothan, Christophe; Mizoguchi, Tsuyoshi; Ezura, Hiroshi

2011-01-01

To accelerate functional genomic research in tomato, we developed a Micro-Tom TILLING (Targeting Induced Local Lesions In Genomes) platform. DNA pools were constructed from 3,052 ethyl methanesulfonate (EMS) mutant lines treated with 0.5 or 1.0% EMS. The mutation frequency was calculated by screening 10 genes. The 0.5% EMS population had a mild mutation frequency of one mutation per 1,710 kb, whereas the 1.0% EMS population had a frequency of one mutation per 737 kb, a frequency suitable for producing an allelic series of mutations in the target genes. The overall mutation frequency was one mutation per 1,237 kb, which affected an average of three alleles per kilobase screened. To assess whether a Micro-Tom TILLING platform could be used for efficient mutant isolation, six ethylene receptor genes in tomato (SlETR1–SlETR6) were screened. Two allelic mutants of SlETR1 (Sletr1-1 and Sletr1-2) that resulted in reduced ethylene responses were identified, indicating that our Micro-Tom TILLING platform provides a powerful tool for the rapid detection of mutations in an EMS mutant library. This work provides a practical and publicly accessible tool for the study of fruit biology and for obtaining novel genetic material that can be used to improve important agronomic traits in tomato. PMID:21965606

Tomato TILLING technology: development of a reverse genetics tool for the efficient isolation of mutants from Micro-Tom mutant libraries.

PubMed

Okabe, Yoshihiro; Asamizu, Erika; Saito, Takeshi; Matsukura, Chiaki; Ariizumi, Tohru; Brès, Cécile; Rothan, Christophe; Mizoguchi, Tsuyoshi; Ezura, Hiroshi

2011-11-01

To accelerate functional genomic research in tomato, we developed a Micro-Tom TILLING (Targeting Induced Local Lesions In Genomes) platform. DNA pools were constructed from 3,052 ethyl methanesulfonate (EMS) mutant lines treated with 0.5 or 1.0% EMS. The mutation frequency was calculated by screening 10 genes. The 0.5% EMS population had a mild mutation frequency of one mutation per 1,710 kb, whereas the 1.0% EMS population had a frequency of one mutation per 737 kb, a frequency suitable for producing an allelic series of mutations in the target genes. The overall mutation frequency was one mutation per 1,237 kb, which affected an average of three alleles per kilobase screened. To assess whether a Micro-Tom TILLING platform could be used for efficient mutant isolation, six ethylene receptor genes in tomato (SlETR1-SlETR6) were screened. Two allelic mutants of SlETR1 (Sletr1-1 and Sletr1-2) that resulted in reduced ethylene responses were identified, indicating that our Micro-Tom TILLING platform provides a powerful tool for the rapid detection of mutations in an EMS mutant library. This work provides a practical and publicly accessible tool for the study of fruit biology and for obtaining novel genetic material that can be used to improve important agronomic traits in tomato.

Quinolone-resistant gyrase mutants demonstrate decreased susceptibility to triclosan.

PubMed

Webber, Mark A; Buckner, Michelle M C; Redgrave, Liam S; Ifill, Gyles; Mitchenall, Lesley A; Webb, Carly; Iddles, Robyn; Maxwell, Anthony; Piddock, Laura J V

2017-10-01

Cross-resistance between antibiotics and biocides is a potentially important driver of MDR. A relationship between susceptibility of Salmonella to quinolones and triclosan has been observed. This study aimed to: (i) investigate the mechanism underpinning this; (ii) determine whether the phenotype is conserved in Escherichia coli; and (iii) evaluate the potential for triclosan to select for quinolone resistance. WT E. coli, Salmonella enterica serovar Typhimurium and gyrA mutants were used. These were characterized by determining antimicrobial susceptibility, DNA gyrase activity and sensitivity to inhibition. Expression of stress response pathways (SOS, RpoS, RpoN and RpoH) was measured, as was the fitness of mutants. The potential for triclosan to select for quinolone resistance was determined. All gyrase mutants showed increased triclosan MICs and altered supercoiling activity. There was no evidence for direct interaction between triclosan and gyrase. Identical substitutions in GyrA had different impacts on supercoiling in the two species. For both, there was a correlation between altered supercoiling and expression of stress responses. This was more marked in E. coli, where an Asp87Gly GyrA mutant demonstrated greatly increased fitness in the presence of triclosan. Exposure of parental strains to low concentrations of triclosan did not select for quinolone resistance. Our data suggest gyrA mutants are less susceptible to triclosan due to up-regulation of stress responses. The impact of gyrA mutation differs between E. coli and Salmonella. The impacts of gyrA mutation beyond quinolone resistance have implications for the fitness and selection of gyrA mutants in the presence of non-quinolone antimicrobials. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Yarrowia lipolytica morphological mutant enables lasting in situ immobilization in bioreactor.

PubMed

Vandermies, Marie; Kar, Tambi; Carly, Frédéric; Nicaud, Jean-Marc; Delvigne, Frank; Fickers, Patrick

2018-04-26

In the present study, we have isolated and characterized a Yarrowia lipolytica morphological mutant growing exclusively in the pseudohyphal morphology. The gene responsible for this phenotype, YALI0E06519g, was identified as homologous to the mitosis regulation gene HSL1 from Saccharomyces cerevisiae. Taking advantage of its morphology, we achieved the immobilization of the Δhsl1 mutant on the metallic structured packing of immobilized-cell bioreactors. We obtained significant cell retention and growth on the support during shake flask and bioreactor experiments without an attachment step prior to the culture. The system of medium aspersion on the packing ensured oxygen availability in the absence of agitation and minimized the potential release of cells in the culture medium. Additionally, the metallic packing proved its facility of cleaning and sterilization after fermentation. This combined use of morphological mutation and bioreactor design is a promising strategy to develop continuous processes for the production of recombinant protein and metabolites using Y. lipolytica. Graphical Abstract.

Integrative Genomic Analysis of Cholangiocarcinoma Identifies Distinct IDH-Mutant Molecular Profiles.

PubMed

Farshidfar, Farshad; Zheng, Siyuan; Gingras, Marie-Claude; Newton, Yulia; Shih, Juliann; Robertson, A Gordon; Hinoue, Toshinori; Hoadley, Katherine A; Gibb, Ewan A; Roszik, Jason; Covington, Kyle R; Wu, Chia-Chin; Shinbrot, Eve; Stransky, Nicolas; Hegde, Apurva; Yang, Ju Dong; Reznik, Ed; Sadeghi, Sara; Pedamallu, Chandra Sekhar; Ojesina, Akinyemi I; Hess, Julian M; Auman, J Todd; Rhie, Suhn K; Bowlby, Reanne; Borad, Mitesh J; Zhu, Andrew X; Stuart, Josh M; Sander, Chris; Akbani, Rehan; Cherniack, Andrew D; Deshpande, Vikram; Mounajjed, Taofic; Foo, Wai Chin; Torbenson, Michael S; Kleiner, David E; Laird, Peter W; Wheeler, David A; McRee, Autumn J; Bathe, Oliver F; Andersen, Jesper B; Bardeesy, Nabeel; Roberts, Lewis R; Kwong, Lawrence N

2017-03-14

Cholangiocarcinoma (CCA) is an aggressive malignancy of the bile ducts, with poor prognosis and limited treatment options. Here, we describe the integrated analysis of somatic mutations, RNA expression, copy number, and DNA methylation by The Cancer Genome Atlas of a set of predominantly intrahepatic CCA cases and propose a molecular classification scheme. We identified an IDH mutant-enriched subtype with distinct molecular features including low expression of chromatin modifiers, elevated expression of mitochondrial genes, and increased mitochondrial DNA copy number. Leveraging the multi-platform data, we observed that ARID1A exhibited DNA hypermethylation and decreased expression in the IDH mutant subtype. More broadly, we found that IDH mutations are associated with an expanded histological spectrum of liver tumors with molecular features that stratify with CCA. Our studies reveal insights into the molecular pathogenesis and heterogeneity of cholangiocarcinoma and provide classification information of potential therapeutic significance. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Neutral lipid accumulation at elevated temperature in conditional mutants of two microalgae species.

PubMed

Yao, Shuo; Brandt, Anders; Egsgaard, Helge; Gjermansen, Claes

2012-12-01

Triacylglycerols, an energy storage compound in microalgae, are known to be accumulated after nitrogen starvation of microalgae cells. Microalgae could be of importance for future biodiesel production due to their fast growth rate and high oil content. In collections of temperature sensitive mutants of Chlamydomonas reinhardtii and Chlorella vulgaris, nine out of fourty-one mutants in C. reinhardtii and eleven out of fifty-three mutants in C. vulgaris contained increased amounts of neutral lipids, predominantly as triacylglycerols. Upon temperature induced cell-cycle arrest, these mutants showed enlarged cellular volume compared with the wild type. The C. reinhardtii mutants were analyzed further and one type of mutants displayed a shift in lipid composition from polar membrane lipids to neutral lipids after a temperature up-shift, while the second type of mutants accumulated more total lipid per cell, predominantly as neutral lipids as compared with the wild type. Three C. reinhardtii mutants were analyzed further and found to be arrested after DNA synthesis but prior to cell division in the cell cycle. These mutants will be useful in order to further understand neutral lipid accumulation in microalgae and suggest possibilities for biodiesel production by specific induction of lipid accumulation in miroalgal cultures by cell-cycle inhibition. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Let-7 Sensitizes KRAS Mutant Tumor Cells to Chemotherapy

PubMed Central

Dai, Xin; Jiang, Ying; Tan, Chalet

2015-01-01

KRAS is the most commonly mutated oncogene in human cancers and is associated with poor prognosis and drug resistance. Let-7 is a family of tumor suppressor microRNAs that are frequently suppressed in solid tumors, where KRAS mutations are highly prevalent. In this study, we investigated the potential use of let-7 as a chemosensitizer. We found that let-7b repletion selectively sensitized KRAS mutant tumor cells to the cytotoxicity of paclitaxel and gemcitabine. Transfection of let-7b mimic downregulated the expression of mutant but not wild-type KRAS. Combination of let-7b mimic with paclitaxel or gemcitabine diminished MEK/ERK and PI3K/AKT signaling concurrently, triggered the onset of apoptosis, and reverted the epithelial-mesenchymal transition in KRAS mutant tumor cells. In addition, let-7b repletion downregulated the expression of β-tubulin III and ribonucleotide reductase subunit M2, two proteins known to mediate tumor resistance to paclitaxel and gemcitabine, respectively. Let-7 may represent a new class of chemosensitizer for the treatment of KRAS mutant tumors. PMID:25946136

Enhanced cellulase producing mutants developed from heterokaryotic Aspergillus strain.

PubMed

Kaur, Baljit; Oberoi, H S; Chadha, B S

2014-03-01

A heterokaryon 28, derived through protoplast fusion between Aspergillus nidulans and Aspergillus tubingensis (Dal8), was subjected cyclic mutagenesis followed by selection on increasing levels of 2-deoxy glucose (2-DG) as selection marker. The derived deregulated cellulase hyper producing mutant '64', when compared to fusant 28, produced 9.83, 7.8, 3.2, 4.2 and 19.74 folds higher endoglucanase, β-glucosidase, cellobiohydrolase, FPase and xylanase, respectively, under shake cultures. The sequence analysis of PCR amplified β-glucosidase gene from wild and mutant showed nucleotide deletion/substitution. The mutants showed highly catalytic efficient β-glucosidase as evident from low Km and high Vmax values. The expression profiling through zymogram analysis also indicated towards over-expression of cellulases. The up/down regulated expressed proteins observed through SDS-PAGE were identified by Peptide mass fingerprinting The cellulase produced by mutants in conjunction with cellulase free xylanase derived from Thermomyces lanuginosus was used for efficient utilization of alkali treated rice straw for obtaining xylo-oligosaccharides and ethanol. Copyright © 2014 Elsevier Ltd. All rights reserved.

Early transcriptional responses of internalization defective Brucella abortus mutants in professional phagocytes, RAW 264.7.

PubMed

Cha, Seung Bin; Lee, Won Jung; Shin, Min Kyoung; Jung, Myung Hwan; Shin, Seung Won; Yoo, An Na; Kim, Jong Wan; Yoo, Han Sang

2013-06-27

Brucella abortus is an intracellular zoonotic pathogen which causes undulant fever, endocarditis, arthritis and osteomyelitis in human and abortion and infertility in cattle. This bacterium is able to invade and replicate in host macrophage instead of getting removed by this defense mechanism. Therefore, understanding the interaction between virulence of the bacteria and the host cell is important to control brucellosis. Previously, we generated internalization defective mutants and analyzed the envelope proteins. The present study was undertaken to evaluate the changes in early transcriptional responses between wild type and internalization defective mutants infected mouse macrophage, RAW 264.7. Both of the wild type and mutant infected macrophages showed increased expression levels in proinflammatory cytokines, chemokines, apoptosis and G-protein coupled receptors (Gpr84, Gpr109a and Adora2b) while the genes related with small GTPase which mediate intracellular trafficking was decreased. Moreover, cytohesin 1 interacting protein (Cytip) and genes related to ubiquitination (Arrdc3 and Fbxo21) were down-regulated, suggesting the survival strategy of this bacterium. However, we could not detect any significant changes in the mutant infected groups compared to the wild type infected group. In summary, it was very difficult to clarify the alterations in host cellular transcription in response to infection with internalization defective mutants. However, we found several novel gene changes related to the GPCR system, ubiquitin-proteosome system, and growth arrest and DNA damages in response to B. abortus infection. These findings may contribute to a better understanding of the molecular mechanisms underlying host-pathogen interactions and need to be studied further.

Physiochemical properties and kinetics of glucoamylase produced from deoxy-d-glucose resistant mutant of Aspergillus niger for soluble starch hydrolysis.

PubMed

Riaz, Muhammad; Rashid, Muhammad Hamid; Sawyer, Lindsay; Akhtar, Saeed; Javed, Muhammad Rizwan; Nadeem, Habibullah; Wear, Martin

2012-01-01

Glucoamylases (GAs) from a wild and a deoxy-d-glucose-resistant mutant of a locally isolated Aspergillus niger were purified to apparent homogeneity. The subunit molecular mass estimated by SDS-PAGE was 93 kDa for both strains, while the molecular masses determined by MALDI-TOF for wild and mutant GAs were 72.876 and 72.063 kDa, respectively. The monomeric nature of the enzymes was confirmed through activity staining. Significant improvement was observed in the kinetic properties of the mutant GA relative to the wild type enzyme. Kinetic constants of starch hydrolysis for A. niger parent and mutant GAs calculated on the basis of molecular masses determined through MALDI-TOF were as follows: k cat = 343 and 727 s -1 , K m = 0.25 and 0.16 mg mL -1 , k cat / K m (specificity constant) = 1374 and 4510 mg mL -1 s -1 , respectively. Thermodynamic parameters for soluble starch hydrolysis also suggested that mutant GA was more efficient compared to the parent enzyme.

Structures of oncogenic, suppressor and rescued p53 core-domain variants: mechanisms of mutant p53 rescue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wallentine, Brad D.; Wang, Ying; Tretyachenko-Ladokhina, Vira

2013-10-01

X-ray crystallographic structures of four p53 core-domain variants were determined in order to gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53. To gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53, X-ray crystallographic structures of four p53 core-domain variants were determined. These include an oncogenic mutant, V157F, two single-site suppressor mutants, N235K and N239Y, and the rescued cancer mutant V157F/N235K/N239Y. The V157F mutationmore » substitutes a smaller hydrophobic valine with a larger hydrophobic phenylalanine within strand S4 of the hydrophobic core. The structure of this cancer mutant shows no gross structural changes in the overall fold of the p53 core domain, only minor rearrangements of side chains within the hydrophobic core of the protein. Based on biochemical analysis, these small local perturbations induce instability in the protein, increasing the free energy by 3.6 kcal mol{sup −1} (15.1 kJ mol{sup −1}). Further biochemical evidence shows that each suppressor mutation, N235K or N239Y, acts individually to restore thermodynamic stability to V157F and that both together are more effective than either alone. All rescued mutants were found to have wild-type DNA-binding activity when assessed at a permissive temperature, thus pointing to thermodynamic stability as the critical underlying variable. Interestingly, thermodynamic analysis shows that while N239Y demonstrates stabilization of the wild-type p53 core domain, N235K does not. These observations suggest distinct structural mechanisms of rescue. A new salt bridge between Lys235 and Glu198, found in both the N235K and rescued cancer mutant structures, suggests a rescue mechanism that relies on stabilizing the �

Benomyl-resistant mutant strain of Trichoderma sp. with increased mycoparasitic activity.

PubMed

Olejníková, P; Ondrusová, Z; Krystofová, S; Hudecová, D

2010-01-01

Application of UV radiation to the strain Trichoderma sp. T-bt (isolated from lignite) resulted in the T-brm mutant which was resistant to the systemic fungicide benomyl. The tub2 gene sequence in the T-brm mutant differed from the parent as well as the collection strain (replacing tyrosine with histidine in the TUB2 protein). Under in vitro conditions this mutant exhibited a higher mycoparasitic activity toward phytopathogenic fungi.

Defined single-gene and multi-gene deletion mutant collections in Salmonella enterica sv Typhimurium.

PubMed

Porwollik, Steffen; Santiviago, Carlos A; Cheng, Pui; Long, Fred; Desai, Prerak; Fredlund, Jennifer; Srikumar, Shabarinath; Silva, Cecilia A; Chu, Weiping; Chen, Xin; Canals, Rocío; Reynolds, M Megan; Bogomolnaya, Lydia; Shields, Christine; Cui, Ping; Guo, Jinbai; Zheng, Yi; Endicott-Yazdani, Tiana; Yang, Hee-Jeong; Maple, Aimee; Ragoza, Yury; Blondel, Carlos J; Valenzuela, Camila; Andrews-Polymenis, Helene; McClelland, Michael

2014-01-01

We constructed two collections of targeted single gene deletion (SGD) mutants and two collections of targeted multi-gene deletion (MGD) mutants in Salmonella enterica sv Typhimurium 14028s. The SGD mutant collections contain (1), 3517 mutants in which a single gene is replaced by a cassette containing a kanamycin resistance (KanR) gene oriented in the sense direction (SGD-K), and (2), 3376 mutants with a chloramphenicol resistance gene (CamR) oriented in the antisense direction (SGD-C). A combined total of 3773 individual genes were deleted across these SGD collections. The MGD collections contain mutants bearing deletions of contiguous regions of three or more genes and include (3), 198 mutants spanning 2543 genes replaced by a KanR cassette (MGD-K), and (4), 251 mutants spanning 2799 genes replaced by a CamR cassette (MGD-C). Overall, 3476 genes were deleted in at least one MGD collection. The collections with different antibiotic markers permit construction of all viable combinations of mutants in the same background. Together, the libraries allow hierarchical screening of MGDs for different phenotypic followed by screening of SGDs within the target MGD regions. The mutants of these collections are stored at BEI Resources (www.beiresources.org) and publicly available.

Persistence of Escherichia coli O157:H7 and Its Mutants in Soils

PubMed Central

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, stx 1, stx 2, stx 1–2, 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, stx 1, stx 2, stx 1–2, 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 Td (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 Td 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 (stx 1 − stx 2 −) 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

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

Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chandra, P. Manish; Brannigan, James A., E-mail: jab@ysbl.york.ac.uk; Prabhune, Asmita

The production, crystallization and characterization of three inactive mutants of penicillin V acylase from B. sphaericus in their respective precursor and processed forms are reported. The space groups are different for the native enzyme and the mutants. The crystallization of three catalytically inactive mutants of penicillin V acylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants willmore » provide three-dimensional structures of precursor PVA forms, plus open a route to the study of enzyme–substrate complexes for this industrially important enzyme.« less

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.

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.

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…

Growth and sporulation of a pyrimidine spore color mutant of Sordaria fimicola.

PubMed

el-Ani, A S

1967-04-07

A nonautonomous spore color mutant of Sordaria fimicola is a pyrimidine auxotroph that produces hyaline nonviable ascospores. Uracil, uridine, and cytidine are more effective growth factors than cytosine and thymine and, in high concentrations, render the mutant self-fertile by inducing the ascospores to resume development and maturation. Crosses with the unlinked arginine non-autonomus spore color mutant st-59 yielded the double mutant st-59 pyr that requires both arginine and a pyrimidine for growth, which indicates a lack of suppression of the pyrimidine requirement by the arginine locus.

Prognostic Significance of POLE Proofreading Mutations in Endometrial Cancer

PubMed Central

Church, David N.; Stelloo, Ellen; Nout, Remi A.; Valtcheva, Nadejda; Depreeuw, Jeroen; ter Haar, Natalja; Noske, Aurelia; Amant, Frederic; Wild, Peter J.; Lambrechts, Diether; Jürgenliemk-Schulz, Ina M.; Jobsen, Jan J.; Smit, Vincent T. H. B. M.; Creutzberg, Carien L.; Bosse, Tjalling

2015-01-01

Background: Current risk stratification in endometrial cancer (EC) results in frequent over- and underuse of adjuvant therapy, and may be improved by novel biomarkers. We examined whether POLE proofreading mutations, recently reported in about 7% of ECs, predict prognosis. Methods: We performed targeted POLE sequencing in ECs from the PORTEC-1 and -2 trials (n = 788), and analyzed clinical outcome according to POLE status. We combined these results with those from three additional series (n = 628) by meta-analysis to generate multivariable-adjusted, pooled hazard ratios (HRs) for recurrence-free survival (RFS) and cancer-specific survival (CSS) of POLE-mutant ECs. All statistical tests were two-sided. Results: POLE mutations were detected in 48 of 788 (6.1%) ECs from PORTEC-1 and-2 and were associated with high tumor grade (P < .001). Women with POLE-mutant ECs had fewer recurrences (6.2% vs 14.1%) and EC deaths (2.3% vs 9.7%), though, in the total PORTEC cohort, differences in RFS and CSS were not statistically significant (multivariable-adjusted HR = 0.43, 95% CI = 0.13 to 1.37, P = .15; HR = 0.19, 95% CI = 0.03 to 1.44, P = .11 respectively). However, of 109 grade 3 tumors, 0 of 15 POLE-mutant ECs recurred, compared with 29 of 94 (30.9%) POLE wild-type cancers; reflected in statistically significantly greater RFS (multivariable-adjusted HR = 0.11, 95% CI = 0.001 to 0.84, P = .03). In the additional series, there were no EC-related events in any of 33 POLE-mutant ECs, resulting in a multivariable-adjusted, pooled HR of 0.33 for RFS (95% CI = 0.12 to 0.91, P = .03) and 0.26 for CSS (95% CI = 0.06 to 1.08, P = .06). Conclusion: POLE proofreading mutations predict favorable EC prognosis, independently of other clinicopathological variables, with the greatest effect seen in high-grade tumors. This novel biomarker may help to reduce overtreatment in EC. PMID:25505230

Characterization of a novel gravitropic mutant of morning glory, weeping2

NASA Astrophysics Data System (ADS)

Kitazawa, Daisuke; Miyazawa, Yutaka; Fujii, Nobuharu; Nitasaka, Eiji; Takahashi, Hideyuki

2008-09-01

In higher plants, gravity is a major environmental cue that governs growth orientation, a phenomenon termed gravitropism. It has been suggested that gravity also affects other aspects of morphogenesis, such as circumnutation and winding movements. Previously, we showed that these aspects of plant growth morphology require amyloplast sedimentation inside gravisensing endodermal cells. However, the molecular mechanism of the graviresponse and its relationship to circumnutation and winding remains obscure. Here, we have characterized a novel shoot gravitropic mutant of morning glory, weeping2 ( we2). In the we2 mutant, the gravitropic response of the stem was absent, and hypocotyls exhibited a severely reduced gravitropic response, whereas roots showed normal gravitropism. In agreement with our previous studies, we found that we2 mutant has defects in shoot circumnutation and winding. Histological analysis showed that we2 mutant forms abnormal endodermal cells. We identified a mutation in the morning glory homolog of SHORT-ROOT ( PnSHR1) that was genetically linked to the agravitropic phenotype of we2 mutant, and which may underlie the abnormal differentiation of endodermal cells in this plant. These results suggest that the phenotype of we2 mutant is due to a mutation of PnSHR1, and that PnSHR1 regulates gravimorphogenesis, including circumnutation and winding movements, in morning glory.

A combinatorial strategy for treating KRAS mutant lung cancer

PubMed Central

Manchado, Eusebio; Weissmueller, Susann; Morris, John P.; Chen, Chi-Chao; Wullenkord, Ramona; Lujambio, Amaia; de Stanchina, Elisa; Poirier, John T.; Gainor, Justin F.; Corcoran, Ryan B.; Engelman, Jeffrey A.; Rudin, Charles M.; Rosen, Neal; Lowe, Scott W.

2016-01-01

Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proven difficult to inhibit, and the effectiveness of agents that target key KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, an FDA-approved MEK inhibitor that acts downstream of KRAS to suppress signaling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA (shRNA) screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signaling rebound and adaptive drug resistance. As a consequence, genetic or pharmacologic inhibition of FGFR1 in combination with trametinib enhances tumor cell death in vitro and in vivo. This compensatory response shows distinct specificities – it is dominated by FGFR1 in KRAS mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patient tumors treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer. PMID:27338794

The Tennessee Mouse Genome Consortium: Identification of ocular mutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jablonski, Monica M.; Wang, Xiaofei; Lu, Lu

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 andmore » 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.« less

The tillering phenotype of the rice plastid terminal oxidase (PTOX) loss-of-function mutant is associated with strigolactone deficiency.

PubMed

Tamiru, Muluneh; Abe, Akira; Utsushi, Hiroe; Yoshida, Kakoto; Takagi, Hiroki; Fujisaki, Koki; Undan, Jerwin R; Rakshit, Sujay; Takaichi, Shinichi; Jikumaru, Yusuke; Yokota, Takao; Terry, Matthew J; Terauchi, Ryohei

2014-04-01

The significance of plastid terminal oxidase (PTOX) in phytoene desaturation and chloroplast function has been demonstrated using PTOX-deficient mutants, particularly in Arabidopsis. However, studies on its role in monocots are lacking. Here, we report cloning and characterization of the rice (Oryza sativa) PTOX1 gene. Using Ecotype Targeting Induced Local Lesions IN Genomes (EcoTILLING) and TILLING as forward genetic tools, we identified the causative mutation of an EMS mutant characterized by excessive tillering, semi-dwarfism and leaf variegation that corresponded to the PTOX1 gene. The tillering and semi-dwarf phenotypes of the ptox1 mutant are similar to phenotypes of known strigolactone (SL)-related rice mutants, and both phenotypic traits could be rescued by application of the synthetic SL GR24. The ptox1 mutant accumulated phytoene in white leaf sectors with a corresponding deficiency in β-carotene, consistent with the expected function of PTOX1 in promoting phytoene desaturase activity. There was also no accumulation of the carotenoid-derived SL ent-2'-epi-5-deoxystrigol in root exudates. Elevated concentrations of auxin were detected in the mutant, supporting previous observations that SL interaction with auxin is important in shoot branching control. Our results demonstrate that PTOX1 is required for both carotenoid and SL synthesis resulting in SL-deficient phenotypes in rice. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Tissue-Specific Profiling Reveals Transcriptome Alterations in Arabidopsis Mutants Lacking Morphological Phenotypes[C][W

PubMed Central

Simon, Marissa; Bruex, Angela; Kainkaryam, Raghunandan M.; Zheng, Xiaohua; Huang, Ling; Woolf, Peter J.; Schiefelbein, John

2013-01-01

Traditional genetic analysis relies on mutants with observable phenotypes. Mutants lacking visible abnormalities may nevertheless exhibit molecular differences useful for defining gene function. To examine this, we analyzed tissue-specific transcript profiles from Arabidopsis thaliana transcription factor gene mutants with known roles in root epidermis development, but lacking a single-gene mutant phenotype due to genetic redundancy. We discovered substantial transcriptional changes in each mutant, preferentially affecting root epidermal genes in a manner consistent with the known double mutant effects. Furthermore, comparing transcript profiles of single and double mutants, we observed remarkable variation in the sensitivity of target genes to the loss of one or both paralogous genes, including preferential effects on specific branches of the epidermal gene network, likely reflecting the pathways of paralog subfunctionalization during evolution. In addition, we analyzed the root epidermal transcriptome of the transparent testa glabra2 mutant to clarify its role in the network. These findings provide insight into the molecular basis of genetic redundancy and duplicate gene diversification at the level of a specific gene regulatory network, and they demonstrate the usefulness of tissue-specific transcript profiling to define gene function in mutants lacking informative visible changes in phenotype. PMID:24014549

Lipopolysaccharide and Aldoheptose Biosynthesis in Transketolase Mutants of Salmonella typhimurium

PubMed Central

Eidels, L.; Osborn, M. J.

1971-01-01

Genetic and biochemical evidence that sedoheptulose-7-phosphate is an obligatory precursor of the L-glycero-D-mannoheptose residues of the lipopolysaccharide of Salmonella was obtained by isolation and characterization of transketolase-negative mutants of Salmonella typhimurium. These mutants, which are defective in synthesis of sedoheptulose-7-phosphate, were found to produce an incomplete heptose-deficient lipopolysaccharide, and were also sensitive to bile salts, a characteristic property of heptose-deficient mutants. Phenotypic repair of the defect in lipopolysaccharide synthesis was obtained by addition of exogenous sedoheptulose-7-phosphate to growing cultures of the mutant strains. Characterization of revertants isolated either as transketolase-positive or heptose-positive provided further evidence that the heptose deficiency resulted from mutation at the transketolase locus. On the basis of these findings a possible pathway for conversion of sedoheptulose-7-phosphate to L-glycero-D-mannoheptose is proposed. PMID:4942911

[Biosorption ability of mutants of Rhodotorula mucilaginosa UCM Y-1776].

PubMed

Mamieieva, O H; Kasatkina, T P; Lavrinchuk, V Ia

2007-01-01

Twenty stable mutants with various coloration intensity have been allocated in carotene-synthesizing natural strain Rhodotorula mucilaginosa UCM Y-1776 (wild type) after nitrosoguanidine action. Two brightly orange mutants 4L and 11 and one non-pigmented mutant 2 were chosen for the further researches. The ultraviolet was inefficient as a mutagen. Resistance to high concentration of copper ions (up to 200 mg/g), high sorption ability (Qmax = 9.1 mmol/g) was characteristic of R. mucilaginosa UCM Y-1776. Concentration of copper ions 50 mg/l was toxic for mutants 4L, 11 and 2, which sorption ability was lower in comparison with carotene pigmented R. mucilaginosa UCM Y-1776. It was shown, for the first time that there was a direct dependence between the presence of carotenoid pigments, resistance to high concentration of copper ions and sorption ability for yeast R. mucilaginosa UCM Y-1776.

A detailed study of gerJ mutants of Bacillus subtilis.

PubMed

Warburg, R J; Buchanan, C E; Parent, K; Halvorson, H O

1986-08-01

A total of nine gerJ mutants have now been isolated in Bacillus subtilis. All are defective in their spore germination properties, being blocked at an intermediate (phase grey) stage. The dormant spores are sensitive to heating at 90 degrees C and two of the mutants (generated by transposon insertion) produce spores sensitive at 80 degrees C. The spores of these two more extreme mutants had a visibly defective cortex when studied by electron microscopy, as did some of the other mutants. During sporulation, the acquisition of spore resistance properties and the appearance of the sporulation-specific penicillin-binding protein PBP5* were delayed. A strain probably carrying a lacZ fusion to the gerJ promoter demonstrated increased expression between t2 and t4. We propose that the gerJ locus is involved in the control of one or more sporulation-specific genes.

Altered sexual and social behaviors in trp2 mutant mice

PubMed Central

Leypold, Bradley G.; Yu, C. Ron; Leinders-Zufall, Trese; Kim, Michelle M.; Zufall, Frank; Axel, Richard

2002-01-01

We have used gene targeting to generate mice with a homozygous deficiency in trp2, a cation channel expressed in the vomeronasal organ (VNO). Trp2 mutant animals reveal a striking reduction in the electrophysiological response to pheromones in the VNO, suggesting that trp2 plays a central role in mediating the pheromone response. These mutants therefore afford the opportunity to examine the role of the VNO in the generation of innate sexual and social behaviors in mice. Trp2 mutant males and nursing females are docile and fail to initiate aggressive attacks on intruder males. Male–female sexual behavior appears normal, but trp2 mutant males also vigorously mount other males. These results suggest that the cation channel trp2 is required in the VNO to detect male-specific pheromones that elicit aggressive behaviors and dictate the choice of sexual partners. PMID:11972034

LEE-encoded regulator (Ler) mutants elicit serotype-specific protection, but not cross protection, against attaching and effacing E. coli strains.

PubMed

Zhu, C; Feng, S; Yang, Z; Davis, K; Rios, H; Kaper, J B; Boedeker, E C

2007-02-26

We previously showed that single dose orogastric immunization with an attenuated regulatory Lee-encoded regulator (ler) mutant of the rabbit enteropathogenic Escherichia coli (REPEC) strain E22 (O103:H2) protected rabbits from fatal infection with the highly virulent parent strain. In the current study we assessed the degree of homologous (serotype-specific) and heterologous (cross-serotype) protection induced by immunization with REPEC ler mutant strains of differing serotypes, or with a prototype strain RDEC-1 (O15:H-) which expresses a full array of ler up-regulated proteins. We constructed an additional ler mutant using RDEC-1 thus, permitting immunization with a ler mutant of either serotype, O15 or O103, followed by challenge with a virulent REPEC strain of the same or different serotypes. Consistent with our previous data, the current study demonstrated that rabbits immunized with a RDEC-1 ler mutant were protected from challenge with virulent RDEC-H19A (RDEC-1 transduced with Shiga toxin-producing phage H19A) of the same serotype. Rabbits immunized with RDEC-1 or E22 derivative ler mutants demonstrated significant increase in serum antibody titers to the respective whole bacterial cells expressing O antigen but not to the LEE-encoded proteins. However, immunization with the ler mutants of either E22 or RDEC-1 failed to protect rabbits from infections with virulent organisms belonging to different serotypes. In contrast, rabbits immunized with the prototype RDEC-1 were cross protected against challenge with the heterologous E22 strain as shown by normal weight gain, and the absence of clinical signs of disease or characteristic attaching and effacing (A/E) lesions. Immunization with RDEC-1 induced significantly elevated serum IgG titers to LEE-encoded proteins. We thus, demonstrated homologous protection induced by the REPEC ler mutants and heterologous protection by RDEC-1. The observed correlation between elevated immune responses to the LEE

Survival, growth, and localization of epiphytic fitness mutants of pseudomonas syringae on leaves

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beattie, G.A.; Lindow, S.E.

Among 82 epiphytic fitness mutants of a Pseudomonas syringae pv. syringae strain that were characterized in a previous study, 4 mutants were particularly intolerant of the stresses associated with dry leaf surfaces. These four mutants each exhibited distinctive behaviors when inoculated into and into plant leaves. For example, while non showed measurable growth on dry potato leaf surfaces, they grew to different population sizes in the intercellular space of bean leaves and on dry bean leaf surfaces, and one mutant appeared incapable of growth in both environments although it grew well on moist bean leaves. The presence of the parentalmore » strain did not influence the survival of the mutants immediately following exposure of leaves to dry, high-light incubation conditions, suggesting that the reduced survival of the mutants did not result from an inability to produce extracellular factors in planta. On moist bean leaves that were colonized by either a mutant or the wild type, the proportion of the total epiphytic population that was located in sizes protected from a surface sterilant was smaller for the mutants than for the wild type, indicating that the mutants were reduced in their ability to locate, multiply in, and/or survive in such protected sites. This reduced ability was only one of possible several factors contributing to the reduced epiphytic fitness of each mutant. Their reduced fitness was not specific to the host plant bean, since they also exhibited reduced fitness on the nonhost plant potato; the functions altered in these strains are thus of interest for their contribution to the general fitness of bacterial epiphytes. 52 refs., 6 figs., 1 tab.« less

Improved Properties of Baker's Yeast Mutants Resistant to 2-Deoxy-d-Glucose

PubMed Central

Rincón, Ana M.; Codón, Antonio C.; Castrejón, Francisco; Benítez, Tahía

2001-01-01

We isolated spontaneous mutants from Saccharomyces cerevisiae (baker's yeast V1) that were resistant to 2-deoxy-d-glucose and had improved fermentative capacity on sweet doughs. Three mutants could grow at the same rate as the wild type in minimal SD medium (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 2% glucose) and had stable elevated levels of maltase and/or invertase under repression conditions but lower levels in maltose-supplemented media. Two of the mutants also had high levels of phosphatase active on 2-deoxy-d-glucose-6-phosphate. Dough fermentation (CO2 liberation) by two of the mutants was faster and/or produced higher final volumes than that by the wild type, both under laboratory and industrial conditions, when the doughs were supplemented with glucose or sucrose. However, the three mutants were slower when fermenting plain doughs. Fermented sweet bakery products obtained with these mutants were of better quality than those produced by the wild type, with regard to their texture and their organoleptic properties. PMID:11526034

2-deoxyglucose as a selective agent for derepressed mutants of Pichia stipitis

Treesearch

Hassan K. Sreenath; Thomas W. Jeffries

1998-01-01

The glucose analog 2-deoxyglucose (2-DOG) has been used to obtain mutants derepressed for pentose metabolism. Some researchers have used 2-DOG alone whereas others have used it in the presence of a glucoserepressible carbon source. We examined both methods and screened mutant strains for improved use of xylose in the presence of glucose. Pichia stipitis mutants...

Phenotypic Characterization of a copA Mutant of Neisseria gonorrhoeae Identifies a Link between Copper and Nitrosative Stress

PubMed Central

Djoko, Karrera Y.; Franiek, Jessica A.; Edwards, Jennifer L.; Falsetta, Megan L.; Kidd, Stephen P.; Potter, Adam J.; Chen, Nathan H.; Apicella, Michael A.; Jennings, Michael P.

2012-01-01

NGO0579 is annotated copA in the Neisseria gonorrhoeae chromosome, suggesting that it encodes a cation-transporting ATPase specific for copper ions. Compared to wild-type cells, a copA mutant was more sensitive to killing by copper ions but not to other transition metals. The mutant also accumulated a greater amount of copper, consistent with the predicted role of CopA as a copper efflux pump. The copA mutant showed a reduced ability to invade and survive within human cervical epithelial cells, although its ability to form a biofilm on the surface of these cells was not significantly different from that of the wild type. In the presence of copper, the copA mutant exhibited increased sensitivity to killing by nitrite or nitric oxide. Therefore, we concluded that copper ion efflux catalyzed by CopA is linked to the nitrosative stress defense system of Neisseria gonorrhoeae. These observations suggest that copper may exert its effects as an antibacterial agent in the innate immune system via an interaction with reactive nitrogen species. PMID:22184419

Probucol ameliorates renal and metabolic sequelae of primary CoQ deficiency in Pdss2 mutant mice.

PubMed

Falk, Marni J; Polyak, Erzsebet; Zhang, Zhe; Peng, Min; King, Rhonda; Maltzman, Jonathan S; Okwuego, Ezinne; Horyn, Oksana; Nakamaru-Ogiso, Eiko; Ostrovsky, Julian; Xie, Letian X; Chen, Jia Yan; Marbois, Beth; Nissim, Itzhak; Clarke, Catherine F; Gasser, David L

2011-07-01

Therapy of mitochondrial respiratory chain diseases is complicated by limited understanding of cellular mechanisms that cause the widely variable clinical findings. Here, we show that focal segmental glomerulopathy-like kidney disease in Pdss2 mutant animals with primary coenzyme Q (CoQ) deficiency is significantly ameliorated by oral treatment with probucol (1% w/w). Preventative effects in missense mutant mice are similar whether fed probucol from weaning or for 3 weeks prior to typical nephritis onset. Furthermore, treating symptomatic animals for 2 weeks with probucol significantly reduces albuminuria. Probucol has a more pronounced health benefit than high-dose CoQ(10) supplementation and uniquely restores CoQ(9) content in mutant kidney. Probucol substantially mitigates transcriptional alterations across many intermediary metabolic domains, including peroxisome proliferator-activated receptor (PPAR) pathway signaling. Probucol's beneficial effects on the renal and metabolic manifestations of Pdss2 disease occur despite modest induction of oxidant stress and appear independent of its hypolipidemic effects. Rather, decreased CoQ(9) content and altered PPAR pathway signaling appear, respectively, to orchestrate the glomerular and global metabolic consequences of primary CoQ deficiency, which are both preventable and treatable with oral probucol therapy. Copyright © 2011 EMBO Molecular Medicine.

Metaphase to Anaphase (mat) Transition–Defective Mutants inCaenorhabditis elegans

PubMed Central

Golden, Andy; Sadler, Penny L.; Wallenfang, Matthew R.; Schumacher, Jill M.; Hamill, Danielle R.; Bates, Gayle; Bowerman, Bruce; Seydoux, Geraldine; Shakes, Diane C.

2000-01-01

The metaphase to anaphase transition is a critical stage of the eukaryotic cell cycle, and, thus, it is highly regulated. Errors during this transition can lead to chromosome segregation defects and death of the organism. In genetic screens for temperature-sensitive maternal effect embryonic lethal (Mel) mutants, we have identified 32 mutants in the nematode Caenorhabditis elegans in which fertilized embryos arrest as one-cell embryos. In these mutant embryos, the oocyte chromosomes arrest in metaphase of meiosis I without transitioning to anaphase or producing polar bodies. An additional block in M phase exit is evidenced by the failure to form pronuclei and the persistence of phosphohistone H3 and MPM-2 antibody staining. Spermatocyte meiosis is also perturbed; primary spermatocytes arrest in metaphase of meiosis I and fail to produce secondary spermatocytes. Analogous mitotic defects cause M phase delays in mitotic germline proliferation. We have named this class of mutants “mat” for metaphase to anaphase transition defective. These mutants, representing six different complementation groups, all map near genes that encode subunits of the anaphase promoting complex or cyclosome, and, here, we show that one of the genes, emb-27, encodes the C. elegans CDC16 ortholog. PMID:11134076

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. Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

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.

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

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

2016-04-29

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.

[The isolation and characteristics of mutants of the Saccharopolyspora erythraea strain resistant to thiostrepton].

PubMed

Nastasiak, I N; Fedorenko, V A; Danilenko, V N

1997-01-01

The formation of thiostreptone resistant spontaneous and nitrosoguanidine-induced mutants in the erythromycin-producing organism Saccharopolyspora erythraea was investigated. The investigated collection of the mutants was heterogeneous by the level of the thiostreptone resistance (2.5 to 20 micrograms/ml). The thiostreptone resistance mutations had a pleiotropic effect: 17 per cent of the mutants was characterized by the growth thermosensitivity and 26 and 5.8 per cent of the mutants were characterized by loss of the ability to form melanine and aerial mycelium respectively. Such phenotypes were most frequent in the mutants resistant to low concentrations of thiostreptone (2 to 5 micrograms/ml). The absolute majority of the isolated thiostreptone resistant mutants was unstable and formed both the antibiotic resistant and the antibiotic sensitive clones. The greatest portion of the strains with high antibiotic activity (20 per cent) was detected among the S. erythraea spontaneous mutants on the medium with 2.5 micrograms/ ml of thiostreptone. It was shown that the instability of the high antibiotic activity in the mutants was associated with loss of the thiostreptone resistance property.

Mutant DnaAs of Escherichia coli that are refractory to negative control

PubMed Central

Chodavarapu, Sundari; Felczak, Magdalena M.; Simmons, Lyle A.; Murillo, Alec; Kaguni, Jon M.

2013-01-01

DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (β clamp), and both DnaAcos and H202Y resist inhibition by the Hda-β clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-β clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initiation in Escherichia coli. PMID:23990329

Mutant DnaAs of Escherichia coli that are refractory to negative control.

PubMed

Chodavarapu, Sundari; Felczak, Magdalena M; Simmons, Lyle A; Murillo, Alec; Kaguni, Jon M

2013-12-01

DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (β clamp), and both DnaAcos and H202Y resist inhibition by the Hda-β clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-β clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initiation in Escherichia coli.

Severity of mutant phenotype in a series of chlorophyll-deficient wheat mutants depends on light intensity and the severity of the block in chlorophyll synthesis.

PubMed

Falbel, T G; Meehl, J B; Staehelin, L A

1996-10-01

Analyses of a series of allelic chlorina mutants of wheat (Triticum aestivum L.), which have partial blocks in chlorophyll (Chl) synthesis and, therefore, a limited Chl supply, reinforce the principle that Chl is required for the stable accumulation of Chl-binding proteins and that only reaction centers accumulate when the supply of Chl is severely limited. Depending on the rate of Chl accumulation (determined by the severity of the mutation) and on the rate of turnover of Chl and its precursors (determined by the environment in which the plant is grown), the mutants each reach an equilibrium of Chl synthesis and degradation. Together these mutants generate a spectrum of phenotypes. Under the harshest conditions (high illumination), plants with moderate blocks in Chl synthesis have membranes with very little Chl and Chl-proteins and membrane stacks resembling the thylakoids of the lethal xantha mutants of barely grown at low to medium light intensities (which have more severe blocks). In contrast, when grown under low-light conditions the same plants with moderate blocks have thylakoids resembling those of the wild type. The wide range of phenotypes of Chl b-deficient mutants has historically produced more confusion than enlightenment, but incomparable growth conditions can now explain the discrepancies reported in the literature.

Severity of mutant phenotype in a series of chlorophyll-deficient wheat mutants depends on light intensity and the severity of the block in chlorophyll synthesis.

PubMed Central

Falbel, T G; Meehl, J B; Staehelin, L A

1996-01-01

Analyses of a series of allelic chlorina mutants of wheat (Triticum aestivum L.), which have partial blocks in chlorophyll (Chl) synthesis and, therefore, a limited Chl supply, reinforce the principle that Chl is required for the stable accumulation of Chl-binding proteins and that only reaction centers accumulate when the supply of Chl is severely limited. Depending on the rate of Chl accumulation (determined by the severity of the mutation) and on the rate of turnover of Chl and its precursors (determined by the environment in which the plant is grown), the mutants each reach an equilibrium of Chl synthesis and degradation. Together these mutants generate a spectrum of phenotypes. Under the harshest conditions (high illumination), plants with moderate blocks in Chl synthesis have membranes with very little Chl and Chl-proteins and membrane stacks resembling the thylakoids of the lethal xantha mutants of barely grown at low to medium light intensities (which have more severe blocks). In contrast, when grown under low-light conditions the same plants with moderate blocks have thylakoids resembling those of the wild type. The wide range of phenotypes of Chl b-deficient mutants has historically produced more confusion than enlightenment, but incomparable growth conditions can now explain the discrepancies reported in the literature. PMID:8883392

Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS.

PubMed

Yao, Zhan; Yaeger, Rona; Rodrik-Outmezguine, Vanessa S; Tao, Anthony; Torres, Neilawattie M; Chang, Matthew T; Drosten, Matthias; Zhao, Huiyong; Cecchi, Fabiola; Hembrough, Todd; Michels, Judith; Baumert, Hervé; Miles, Linde; Campbell, Naomi M; de Stanchina, Elisa; Solit, David B; Barbacid, Mariano; Taylor, Barry S; Rosen, Neal

2017-08-10

Approximately 200 BRAF mutant alleles have been identified in human tumours. Activating BRAF mutants cause feedback inhibition of GTP-bound RAS, are RAS-independent and signal either as active monomers (class 1) or constitutively active dimers (class 2). Here we characterize a third class of BRAF mutants-those that have impaired kinase activity or are kinase-dead. These mutants are sensitive to ERK-mediated feedback and their activation of signalling is RAS-dependent. The mutants bind more tightly than wild-type BRAF to RAS-GTP, and their binding to and activation of wild-type CRAF is enhanced, leading to increased ERK signalling. The model suggests that dysregulation of signalling by these mutants in tumours requires coexistent mechanisms for maintaining RAS activation despite ERK-dependent feedback. Consistent with this hypothesis, melanomas with these class 3 BRAF mutations also harbour RAS mutations or NF1 deletions. By contrast, in lung and colorectal cancers with class 3 BRAF mutants, RAS is typically activated by receptor tyrosine kinase signalling. These tumours are sensitive to the inhibition of RAS activation by inhibitors of receptor tyrosine kinases. We have thus defined three distinct functional classes of BRAF mutants in human tumours. The mutants activate ERK signalling by different mechanisms that dictate their sensitivity to therapeutic inhibitors of the pathway.

Leaf hydraulic conductance varies with vein anatomy across Arabidopsis thaliana wild-type and leaf vein mutants.

PubMed

Caringella, Marissa A; Bongers, Franca J; Sack, Lawren

2015-12-01

Leaf venation is diverse across plant species and has practical applications from paleobotany to modern agriculture. However, the impact of vein traits on plant performance has not yet been tested in a model system such as Arabidopsis thaliana. Previous studies analysed cotyledons of A. thaliana vein mutants and identified visible differences in their vein systems from the wild type (WT). We measured leaf hydraulic conductance (Kleaf ), vein traits, and xylem and mesophyll anatomy for A. thaliana WT (Col-0) and four vein mutants (dot3-111 and dot3-134, and cvp1-3 and cvp2-1). Mutant true leaves did not possess the qualitative venation anomalies previously shown in the cotyledons, but varied quantitatively in vein traits and leaf anatomy across genotypes. The WT had significantly higher mean Kleaf . Across all genotypes, there was a strong correlation of Kleaf with traits related to hydraulic conductance across the bundle sheath, as influenced by the number and radial diameter of bundle sheath cells and vein length per area. These findings support the hypothesis that vein traits influence Kleaf , indicating the usefulness of this mutant system for testing theory that was primarily established comparatively across species, and supports a strong role for the bundle sheath in influencing Kleaf . © 2015 John Wiley & Sons Ltd.

Mutants of feline immunodeficiency virus resistant to 2',3'-dideoxy-2',3'-didehydrothymidine.

PubMed Central

Zhu, Y Q; Remington, K M; North, T W

1996-01-01

We selected mutants of feline immunodeficiency virus (FIV) that are resistant to 2',3'-dideoxy-2',3'-didehydrothymidine (d4T). Two mutants were selected in cultured cells with a stepwise increase in d4T concentration, resulting in mutants able to replicate in 100 microM d4T. These mutants were three- to sixfold more resistant to d4T than wild-type FIV. They were also cross-resistant to 3'-azido-3'-deoxythymidine (AZT), 3'-fluoro-2',3'-dideoxythymidine, 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and 9-(2-phosphonylmethoxyethyl)adenine, and they were highly resistant to phosphonoformic acid (PFA). Plaque-purified mutants were isolated from each of the mutant populations. The mutant phenotype was stable, because both of the plaque-purified mutants remained d4T resistant even after three passages in the absence of d4T. One of the plaque-purified mutants, designated D4R-3c, was further characterized. Compared with wild-type reverse transcriptase (RT), RT purified from D4R-3c was 3-fold resistant to inhibition by the 5'-triphosphate of d4T, 10-fold resistant to inhibition by the 5'-triphosphate of AZT, and 6-fold resistant to PFA. D4R-3c had a single point mutation in the RT-encoding region of the pol gene at position 2474, resulting in a Val to Ile mutation at codon 47 of the FIV RT. The role of this mutation in d4T resistance was confirmed by site-directed mutagenesis. PMID:8878567

Targeted Mutants of Cochliobolus carbonum Lacking the Two Major Extracellular Polygalacturonases

PubMed Central

Scott-Craig, John S.; Cheng, Yi-Qiang; Cervone, Felice; De Lorenzo, Giulia; Pitkin, John W.; Walton, Jonathan D.

1998-01-01

The filamentous fungus Cochliobolus carbonum produces endo-α1,4-polygalacturonase (endoPG), exo-α1,4-polygalacturonase (exoPG), and pectin methylesterase when grown in culture on pectin. Residual activity in a pgn1 mutant (lacking endoPG) was due to exoPG activity, and the responsible protein has now been purified. After chemical deglycosylation, the molecular mass of the purified protein decreased from greater than 60 to 45 kDa. The gene that encodes exoPG, PGX1, was isolated with PCR primers based on peptide sequences from the protein. The product of PGX1, Pgx1p, has a predicted molecular mass of 48 kDa, 12 potential N-glycosylation sites, and 61% amino acid identity to an exoPG from the saprophytic fungus Aspergillus tubingensis. Strains of C. carbonum mutated in PGX1 were constructed by targeted gene disruption and by gene replacement. Growth of pgx1 mutant strains on pectin was reduced by ca. 20%, and they were still pathogenic on maize. A double pgn1/pgx1 mutant strain was constructed by crossing. The double mutant grew as well as the pgx1 single mutant on pectin and was still pathogenic despite having less than 1% of total wild-type PG activity. Double mutants retained a small amount of PG activity with the same cation-exchange retention time as Pgn1p and also pectin methylesterase and a PG activity associated with the mycelium. Continued growth of the pgn1/pgx1 mutant on pectin could be due to one or more of these residual activities. PMID:9546185

Targeting Autophagy Sensitizes BRAF-Mutant Thyroid Cancer to Vemurafenib

PubMed Central

Wang, Weibin; Kang, Helen; Zhao, Yinu; Min, Irene; Wyrwas, Brian; Moore, Maureen; Teng, Lisong; Zarnegar, Rasa; Jiang, Xuejun

2017-01-01

Context: The RAF inhibitor vemurafenib has provided a major advance for the treatment of patients with BRAF-mutant metastatic melanoma. However, BRAF-mutant thyroid cancer is relatively resistant to vemurafenib, and the reason for this disparity remains unclear. Anticancer therapy–induced autophagy can trigger adaptive drug resistance in a variety of cancer types and treatments. To date, role of autophagy during BRAF inhibition in thyroid cancer remains unknown. Objective: In this study, we investigate if autophagy is activated in vemurafenib-treated BRAF-mutant thyroid cancer cells, and whether autophagy inhibition improves or impairs the treatment efficacy of vemurafenib. Design: Autophagy level was determined by western blot assay and transmission electron microscopy. The combined effects of autophagy inhibitor and vemurafenib were assessed in terms of cell viability in vitro and tumor growth rate in vivo. Whether the endoplasmic reticulum (ER) stress was in response to vemurafenib-induced autophagy was also analyzed. Results: Vemurafenib induced a high level of autophagy in BRAF-mutant thyroid cancer cells. Inhibition of autophagy by either a pharmacological inhibitor or interfering RNA knockdown of essential autophagy genes augmented vemurafenib-induced cell death. Vemurafenib-induced autophagy was independent of MAPK signaling pathway and was mediated through the ER stress response. Finally, administration of vemurafenib with the autophagy inhibitor hydroxychloroquine promoted more pronounced tumor suppression in vivo. Conclusions: Our data demonstrate that vemurafenib induces ER stress response–mediated autophagy in thyroid cancer and autophagy inhibition may be a beneficial strategy to sensitize BRAF-mutant thyroid cancer to vemurafenib. PMID:27754804

Targeting Autophagy Sensitizes BRAF-Mutant Thyroid Cancer to Vemurafenib.

PubMed

Wang, Weibin; Kang, Helen; Zhao, Yinu; Min, Irene; Wyrwas, Brian; Moore, Maureen; Teng, Lisong; Zarnegar, Rasa; Jiang, Xuejun; Fahey, Thomas J

2017-02-01

The RAF inhibitor vemurafenib has provided a major advance for the treatment of patients with BRAF-mutant metastatic melanoma. However, BRAF-mutant thyroid cancer is relatively resistant to vemurafenib, and the reason for this disparity remains unclear. Anticancer therapy-induced autophagy can trigger adaptive drug resistance in a variety of cancer types and treatments. To date, role of autophagy during BRAF inhibition in thyroid cancer remains unknown. In this study, we investigate if autophagy is activated in vemurafenib-treated BRAF-mutant thyroid cancer cells, and whether autophagy inhibition improves or impairs the treatment efficacy of vemurafenib. Autophagy level was determined by western blot assay and transmission electron microscopy. The combined effects of autophagy inhibitor and vemurafenib were assessed in terms of cell viability in vitro and tumor growth rate in vivo. Whether the endoplasmic reticulum (ER) stress was in response to vemurafenib-induced autophagy was also analyzed. Vemurafenib induced a high level of autophagy in BRAF-mutant thyroid cancer cells. Inhibition of autophagy by either a pharmacological inhibitor or interfering RNA knockdown of essential autophagy genes augmented vemurafenib-induced cell death. Vemurafenib-induced autophagy was independent of MAPK signaling pathway and was mediated through the ER stress response. Finally, administration of vemurafenib with the autophagy inhibitor hydroxychloroquine promoted more pronounced tumor suppression in vivo. Our data demonstrate that vemurafenib induces ER stress response-mediated autophagy in thyroid cancer and autophagy inhibition may be a beneficial strategy to sensitize BRAF-mutant thyroid cancer to vemurafenib. Copyright © 2017 by the Endocrine Society

Early transcriptional responses of internalization defective Brucella abortus mutants in professional phagocytes, RAW 264.7

PubMed Central

2013-01-01

Background Brucella abortus is an intracellular zoonotic pathogen which causes undulant fever, endocarditis, arthritis and osteomyelitis in human and abortion and infertility in cattle. This bacterium is able to invade and replicate in host macrophage instead of getting removed by this defense mechanism. Therefore, understanding the interaction between virulence of the bacteria and the host cell is important to control brucellosis. Previously, we generated internalization defective mutants and analyzed the envelope proteins. The present study was undertaken to evaluate the changes in early transcriptional responses between wild type and internalization defective mutants infected mouse macrophage, RAW 264.7. Results Both of the wild type and mutant infected macrophages showed increased expression levels in proinflammatory cytokines, chemokines, apoptosis and G-protein coupled receptors (Gpr84, Gpr109a and Adora2b) while the genes related with small GTPase which mediate intracellular trafficking was decreased. Moreover, cytohesin 1 interacting protein (Cytip) and genes related to ubiquitination (Arrdc3 and Fbxo21) were down-regulated, suggesting the survival strategy of this bacterium. However, we could not detect any significant changes in the mutant infected groups compared to the wild type infected group. Conclusions In summary, it was very difficult to clarify the alterations in host cellular transcription in response to infection with internalization defective mutants. However, we found several novel gene changes related to the GPCR system, ubiquitin-proteosome system, and growth arrest and DNA damages in response to B. abortus infection. These findings may contribute to a better understanding of the molecular mechanisms underlying host-pathogen interactions and need to be studied further. PMID:23802650

Characterization of sarcoplasmic reticulum Ca{sup 2+} ATPase nucleotide binding domain mutants using NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Myint, Wazo; Gong, Qingguo; Ahn, Jinwoo

2011-02-04

Research highlights: {yields} Structural consequence by substitution mutations on the isolated SERCA-nucleotide binding (SERCA-N) domain was studied. {yields} The study fills a gap between the previous clinical, physiological, and biochemical data and the molecular basis of SERCA-N. {yields} The E412G mutation, known to be seen in patients with Darier's disease, was found to maintain the active conformation but exhibit reduced protein stability. -- Abstract: Sarcoplasmic reticulum Ca{sup 2+} ATPase (SERCA) is essential for muscle function by transporting Ca{sup 2+} from the cytosol into the sarcoplasmic reticulum through ATP hydrolysis. In this report, the effects of substitution mutations on the isolatedmore » SERCA-nucleotide binding domain (SERCA-N) were studied using NMR. {sup 15}N-{sup 1}H HSQC spectra of substitution mutants at the nucleotide binding site, T441A, R560V, and C561A, showed chemical shift changes, primarily in residues adjacent to the mutation sites, indicating only local effects. Further, the patterns of chemical shift changes upon AMP-PNP binding to these mutants were similar to that of the wild type SERCA-N (WT). In contrast to these nucleotide binding site mutants, a mutant found in patients with Darier's disease, E412G, showed small but significant chemical shift changes throughout the protein and rapid precipitation. However, the AMP-PNP dissociation constant ({approx}2.5 mM) was similar to that of WT ({approx}3.8 mM). These results indicate that the E412G mutant retains its catalytic activity but most likely reduces its stability. Our findings provide molecular insight into previous clinical, physiological, and biochemical observations.« less

Inactivation of JAK2/STAT3 Signaling Axis and Downregulation of M1 mAChR Cause Cognitive Impairment in klotho Mutant Mice, a Genetic Model of Aging

PubMed Central

Park, Seok-Joo; Shin, Eun-Joo; Min, Sun Seek; An, Jihua; Li, Zhengyi; Hee Chung, Yoon; Hoon Jeong, Ji; Bach, Jae-Hyung; Nah, Seung-Yeol; Kim, Won-Ki; Jang, Choon-Gon; Kim, Yong-Sun; Nabeshima, Yo-ichi; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2013-01-01

We previously reported cognitive dysfunction in klotho mutant mice. In the present study, we further examined novel mechanisms involved in cognitive impairment in these mice. Significantly decreased janus kinase 2 (JAK2) and signal transducer and activator of transcription3 (STAT3) phosphorylation were observed in the hippocampus of klotho mutant mice. A selective decrease in protein expression and binding density of the M1 muscarinic cholinergic receptor (M1 mAChR) was observed in these mice. Cholinergic parameters (ie, acetylcholine (ACh), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE)) and NMDAR-dependent long-term potentiation (LTP) were significantly impaired in klotho mutant mice. McN-A-343 (McN), an M1 mAChR agonist, significantly attenuated these impairments. AG490 (AG), a JAK2 inhibitor, counteracted the attenuating effects of McN, although AG did not significantly alter the McN-induced effect on AChE. Furthermore, AG significantly inhibited the attenuating effects of McN on decreased NMDAR-dependent LTP, protein kinase C βII, p-ERK, p-CREB, BDNF, and p-JAK2/p-STAT3-expression in klotho mutant mice. In addition, k252a, a BDNF receptor tyrosine kinase B (TrkB) inhibitor, significantly counteracted McN effects on decreased ChAT, ACh, and M1 mAChR and p-JAK2/p-STAT3 expression. McN-induced effects on cognitive impairment in klotho mutant mice were consistently counteracted by either AG or k252a. Our results suggest that inactivation of the JAK2/STAT3 signaling axis and M1 mAChR downregulation play a critical role in cognitive impairment observed in klotho mutant mice. PMID:23389690

Characterization of a bi-pistil mutant in Medicago truncatula Gaertn

USDA-ARS?s Scientific Manuscript database

We propose the name bi-pistil, bip, for a floral organ mutant observed in transgenic Medicago truncatula plants. The mutant has two separate stigmas borne on two separate styles that emerge from a single superior carpel primordium. The bip plant was crossed to a previously reported male sterile mtap...

Serum response factor: positive and negative regulation of an epithelial gene expression network in the destrin mutant cornea

PubMed Central

Kawakami-Schulz, Sharolyn V.; Verdoni, Angela M.; Sattler, Shannon G.; Jessen, Erik; Kao, Winston W.-Y.; Ikeda, Akihiro

2014-01-01

Increased angiogenesis, inflammation, and proliferation are hallmarks of diseased tissues, and in vivo models of these disease phenotypes can provide insight into disease pathology. Dstncorn1 mice, deficient for the actin depolymerizing factor destrin (DSTN), display an increase of serum response factor (SRF) that results in epithelial hyperproliferation, inflammation, and neovascularization in the cornea. Previous work demonstrated that conditional ablation of Srf from the corneal epithelium of Dstncorn1 mice returns the cornea to a wild-type (WT) like state. This result implicated SRF as a major regulator of genes that contributes to abnormal phenotypes in Dstncorn1 cornea. The purpose of this study is to identify gene networks that are affected by increased expression of Srf in the Dstncorn1 cornea. Microarray analysis led to characterization of gene expression changes that occur when conditional knockout of Srf rescues mutant phenotypes in the cornea of Dstncorn1 mice. Comparison of gene expression values from WT, Dstncorn1 mutant, and Dstncorn1 rescued cornea identified >400 differentially expressed genes that are downstream from SRF. Srf ablation had a significant effect on genes associated with epithelial cell-cell junctions and regulation of actin dynamics. The majority of genes affected by SRF are downregulated in the Dstncorn1 mutant cornea, suggesting that increased SRF negatively affects transcription of SRF gene targets. ChIP-seq analysis on Dstncorn1 mutant and WT tissue revealed that, despite being present in higher abundance, SRF binding is significantly decreased in the Dstncorn1 mutant cornea. This study uses a unique model combining genetic and genomic approaches to identify genes that are regulated by SRF. These findings expand current understanding of the role of SRF in both normal and abnormal tissue homeostasis. PMID:24550211

Reversion of autocrine transformation by a dominant negative platelet-derived growth factor mutant.

PubMed Central

Vassbotn, F S; Andersson, M; Westermark, B; Heldin, C H; Ostman, A

1993-01-01

A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions. Images PMID:8321214

Mutant Analysis Reveals Allosteric Regulation of ClpB Disaggregase

PubMed Central

Franke, Kamila B.; Bukau, Bernd; Mogk, Axel

2017-01-01

The members of the hexameric AAA+ disaggregase of E. coli and S. cerevisiae, ClpB, and Hsp104, cooperate with the Hsp70 chaperone system in the solubilization of aggregated proteins. Aggregate solubilization relies on a substrate threading activity of ClpB/Hsp104 fueled by ATP hydrolysis in both ATPase rings (AAA-1, AAA-2). ClpB/Hsp104 ATPase activity is controlled by the M-domains, which associate to the AAA-1 ring to downregulate ATP hydrolysis. Keeping M-domains displaced from the AAA-1 ring by association with Hsp70 increases ATPase activity due to enhanced communication between protomers. This communication involves conserved arginine fingers. The control of ClpB/Hsp104 activity is crucial, as hyperactive mutants with permanently dissociated M-domains exhibit cellular toxicity. Here, we analyzed AAA-1 inter-ring communication in relation to the M-domain mediated ATPase regulation, by subjecting a conserved residue of the AAA-1 domain subunit interface of ClpB (A328) to mutational analysis. While all A328X mutants have reduced disaggregation activities, their ATPase activities strongly differed. ClpB-A328I/L mutants have reduced ATPase activity and when combined with the hyperactive ClpB-K476C M-domain mutation, suppress cellular toxicity. This underlines that ClpB ATPase activation by M-domain dissociation relies on increased subunit communication. The ClpB-A328V mutant in contrast has very high ATPase activity and exhibits cellular toxicity on its own, qualifying it as novel hyperactive ClpB mutant. ClpB-A328V hyperactivity is however, different from that of M-domain mutants as M-domains stay associated with the AAA-1 ring. The high ATPase activity of ClpB-A328V primarily relies on the AAA-2 ring and correlates with distinct conformational changes in the AAA-2 catalytic site. These findings characterize the subunit interface residue A328 as crucial regulatory element to control ATP hydrolysis in both AAA rings. PMID:28275610

Stability of Iowa mutant and wild type Aβ-peptide aggregates

NASA Astrophysics Data System (ADS)

Alred, Erik J.; Scheele, Emily G.; Berhanu, Workalemahu M.; Hansmann, Ulrich H. E.

2014-11-01

Recent experiments indicate a connection between the structure of amyloid aggregates and their cytotoxicity as related to neurodegenerative diseases. Of particular interest is the Iowa Mutant, which causes early-onset of Alzheimer's disease. While wild-type Amyloid β-peptides form only parallel beta-sheet aggregates, the mutant also forms meta-stable antiparallel beta sheets. Since these structural variations may cause the difference in the pathological effects of the two Aβ-peptides, we have studied in silico the relative stability of the wild type and Iowa mutant in both parallel and antiparallel forms. We compare regular molecular dynamics simulations with such where the viscosity of the samples is reduced, which, we show, leads to higher sampling efficiency. By analyzing and comparing these four sets of all-atom molecular dynamics simulations, we probe the role of the various factors that could lead to the structural differences. Our analysis indicates that the parallel forms of both wild type and Iowa mutant aggregates are stable, while the antiparallel aggregates are meta-stable for the Iowa mutant and not stable for the wild type. The differences result from the direct alignment of hydrophobic interactions in the in-register parallel oligomers, making them more stable than the antiparallel aggregates. The slightly higher thermodynamic stability of the Iowa mutant fibril-like oligomers in its parallel organization over that in antiparallel form is supported by previous experimental measurements showing slow inter-conversion of antiparallel aggregates into parallel ones. Knowledge of the mechanism that selects between parallel and antiparallel conformations and determines their relative stability may open new avenues for the development of therapies targeting familial forms of early-onset Alzheimer's disease.

Rapid degeneration of rod photoreceptors expressing self-association-deficient arrestin-1 mutant

PubMed Central

Song, Xiufeng; Seo, Jungwon; Baameur, Faiza; Vishnivetskiy, Sergey A.; Chen, Qiuyan; Kook, Seunghyi; Kim, Miyeon; Brooks, Evan K.; Altenbach, Christian; Hong, Yuan; Hanson, Susan M.; Palazzo, Maria C.; Chen, Jeannie; Hubbell, Wayne L.; Gurevich, Eugenia V.; Gurevich, Vsevolod V.

2013-01-01

Arrestin-1 binds light-activated phosphorhodopsin and ensures timely signal shutoff. We show that high transgenic expression of an arrestin-1 mutant with enhanced rhodopsin binding and impaired oligomerization causes apoptotic rod death in mice. Dark rearing does not prevent mutant-induced cell death, ruling out the role of arrestin complexes with light-activated rhodopsin. Similar expression of WT arrestin-1 that robustly oligomerizes, which leads to only modest increase in the monomer concentration, does not affect rod survival. Moreover, WT arrestin-1 co-expressed with the mutant delays retinal degeneration. Thus, arrestin-1 mutant directly affects cell survival via binding partner(s) other than light-activated rhodopsin. Due to impaired self-association of the mutant its high expression dramatically increases the concentration of the monomer. The data suggest that monomeric arrestin-1 is cytotoxic and WT arrestin-1 protects rods by forming mixed oligomers with the mutant and/or competing with it for the binding to non-receptor partners. Thus, arrestin-1 self-association likely serves to keep low concentration of the toxic monomer. The reduction of the concentration of harmful monomer is an earlier unappreciated biological function of protein oligomerization. PMID:24012956

Rapid degeneration of rod photoreceptors expressing self-association-deficient arrestin-1 mutant.

PubMed

Song, Xiufeng; Seo, Jungwon; Baameur, Faiza; Vishnivetskiy, Sergey A; Chen, Qiuyan; Kook, Seunghyi; Kim, Miyeon; Brooks, Evan K; Altenbach, Christian; Hong, Yuan; Hanson, Susan M; Palazzo, Maria C; Chen, Jeannie; Hubbell, Wayne L; Gurevich, Eugenia V; Gurevich, Vsevolod V

2013-12-01

Arrestin-1 binds light-activated phosphorhodopsin and ensures timely signal shutoff. We show that high transgenic expression of an arrestin-1 mutant with enhanced rhodopsin binding and impaired oligomerization causes apoptotic rod death in mice. Dark rearing does not prevent mutant-induced cell death, ruling out the role of arrestin complexes with light-activated rhodopsin. Similar expression of WT arrestin-1 that robustly oligomerizes, which leads to only modest increase in the monomer concentration, does not affect rod survival. Moreover, WT arrestin-1 co-expressed with the mutant delays retinal degeneration. Thus, arrestin-1 mutant directly affects cell survival via binding partner(s) other than light-activated rhodopsin. Due to impaired self-association of the mutant its high expression dramatically increases the concentration of the monomer. The data suggest that monomeric arrestin-1 is cytotoxic and WT arrestin-1 protects rods by forming mixed oligomers with the mutant and/or competing with it for the binding to non-receptor partners. Thus, arrestin-1 self-association likely serves to keep low concentration of the toxic monomer. The reduction of the concentration of harmful monomer is an earlier unappreciated biological function of protein oligomerization. © 2013.

Suppression of mutants aberrant in light intensity responses of complementary chromatic adaptation.

PubMed Central

Casey, E S; Kehoe, D M; Grossman, A R

1997-01-01

Complementary chromatic adaptation is a process in which cyanobacteria alter the pigment protein (phycocyanin and phycoerythrin) composition of their light-harvesting complexes, the phycobilisomes, to help optimize the absorbance of prevalent wavelengths of light in the environment. Several classes of mutants that display aberrant complementary chromatic adaptation have been isolated. One of the mutant classes, designated "blue" or FdB, accumulates high levels of the blue chromoprotein phycocyanin in low-intensity green light, a condition that normally suppresses phycocyanin synthesis. We demonstrate here that the synthesis of the phycocyanin protein and mRNA in the FdB mutants can be suppressed by increasing the intensity of green light. Hence, these mutants have a decreased sensitivity to green light with respect to suppression of phycocyanin synthesis. Although we were unable to complement the blue mutants, we did isolate genes that could suppress the mutant phenotype. These genes, which have been identified previously, encode a histidine kinase sensor and response regulator protein that play key roles in controlling complementary chromatic adaptation. These findings are discussed with respect to the mechanism by which light quality and quantity control the biosynthesis of the phycobilisome. PMID:9226271

Mutant fatty acid desaturase and methods for directed mutagenesis

DOEpatents

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

2008-01-29

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

Identification of a novel Lymantria dispar nucleopolyhedrovirus mutant that exhibits abnormal polyhedron formation and virion occlusion.

PubMed

Slavicek, J M; Mercer, M J; Pohlman, D; Kelly, M E; Bischoff, D S

1998-07-01

In previous studies on the formation of Lymantria dispar nuclear polyhedrosis virus (LdMNPV) few polyhedra (FP) mutants, several polyhedron formation mutants (PFM) were identified that appeared to be unique. These viral mutants are being characterized to investigate the processes of polyhedron formation and virion occlusion. LdMNPV isolate PFM-1 is one of these mutants, and is described in this report. Genetic techniques were used to determine if isolate PFM-1 contained a mutation in the polyhedrin or 25K FP gene. Wild-type viruses were recovered after coinfection of Ld652Y cells with isolate PFM-1 and a FP mutant, and with isolates PFM-1 and PFM-C (isolate PFM-C contains a mutation in the polyhedrin gene). These viruses were analyzed by genomic restriction endonuclease digestion and found to be chimeras of the original PFMs used in the coinfections. Marker rescue studies mapped the mutation in isolate PFM-1 to a genomic region that does not include the polyhedrin or 25K FP genes. Isolate PFM-1 produced approximately 14-fold fewer polyhedra than LdMNPV isolate A21-MPV, an isolate that produces wild-type levels of polyhedra, and approximately 2-fold more polyhedra compared to the FP isolate 122-2. Polyhedra generated by isolate PFM-1 were normal in size and shape but contained very few viral nucleocapsids. The same amount of budded virus (BV) was released from cells infected with isolates PFM-1 and A21-MPV. In contrast, isolate 122-2 yielded significantly more BV than isolates PFM-1 and A21-MPV.