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

International Nuclear Information System (INIS)

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

1982-01-01

A spontaneous transport mutant of Chinese hamster ovary cells, CHY-1, was isolated by a combination of [ 3 H]proline suicide and replica plating. The mutant took up less tritium than the parent, resulting in a lower killing rate during storage. Transport by four separate amino acid transport systems (A, ASC, L, Ly+) was examined. The CHY-1 mutant exhibited normal uptake via the ASC, L, and Ly+ systems. By contrast, uptake of the most specific substrate of the A system, 2-(methylamino)-isobutyric acid, was significantly reduced at low, but not high, concentrations, due to a 3.5-fold increase in Km and a 1.5-fold increase in Vmax. Taken together, these data suggest that the CHY-1 mutation may be in the structural gene coding for the A transport protein. The tritium suicide procedure is discussed, and general equations are derived to predict the maximum storage time for the survival of one mutant cell and the optimum size of the cell population for maximum mutant enrichment

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

Science.gov (United States)

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.

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

Science.gov (United States)

Ng, Jason Liang Pin; Hassan, Samira; Truong, Thy T.; Hocart, Charles H.; Laffont, Carole; Frugier, Florian; Mathesius, Ulrike

2015-01-01

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation. PMID:26253705

Impaired renal secretion of substrates for the multidrug resistance protein 2 in mutant transport-deficient (TR-) rats.

NARCIS (Netherlands)

Masereeuw, R.; Notenboom, S.; Smeets, P.H.E.; Wouterse, A.C.; Russel, F.G.M.

2003-01-01

Previous studies with mutant transport-deficient rats (TR(-)), in which the multidrug resistance protein 2 (Mrp2) is lacking, have emphasized the importance of this transport protein in the biliary excretion of a wide variety of glutathione conjugates, glucuronides, and other organic anions. Mrp2 is

The Arabidopsis cax1 mutant exhibits impaired ion homeostasis, development, and hormonal responses and reveals interplay among vacuolar transporters.

Science.gov (United States)

Cheng, Ning-Hui; Pittman, Jon K; Barkla, Bronwyn J; Shigaki, Toshiro; Hirschi, Kendal D

2003-02-01

The Arabidopsis Ca(2+)/H(+) transporter CAX1 (Cation Exchanger1) may be an important regulator of intracellular Ca(2+) levels. Here, we describe the preliminary localization of CAX1 to the tonoplast and the molecular and biochemical characterization of cax1 mutants. We show that these mutants exhibit a 50% reduction in tonoplast Ca(2+)/H(+) antiport activity, a 40% reduction in tonoplast V-type H(+)-translocating ATPase activity, a 36% increase in tonoplast Ca(2+)-ATPase activity, and increased expression of the putative vacuolar Ca(2+)/H(+) antiporters CAX3 and CAX4. Enhanced growth was displayed by the cax1 lines under Mn(2+) and Mg(2+) stress conditions. The mutants exhibited altered plant development, perturbed hormone sensitivities, and altered expression of an auxin-regulated promoter-reporter gene fusion. We propose that CAX1 regulates myriad plant processes and discuss the observed phenotypes with regard to the compensatory alterations in other transporters.

The Arabidopsis cax1 Mutant Exhibits Impaired Ion Homeostasis, Development, and Hormonal Responses and Reveals Interplay among Vacuolar Transporters

Science.gov (United States)

Cheng, Ning-Hui; Pittman, Jon K.; Barkla, Bronwyn J.; Shigaki, Toshiro; Hirschi, Kendal D.

2003-01-01

The Arabidopsis Ca2+/H+ transporter CAX1 (Cation Exchanger1) may be an important regulator of intracellular Ca2+ levels. Here, we describe the preliminary localization of CAX1 to the tonoplast and the molecular and biochemical characterization of cax1 mutants. We show that these mutants exhibit a 50% reduction in tonoplast Ca2+/H+ antiport activity, a 40% reduction in tonoplast V-type H+-translocating ATPase activity, a 36% increase in tonoplast Ca2+-ATPase activity, and increased expression of the putative vacuolar Ca2+/H+ antiporters CAX3 and CAX4. Enhanced growth was displayed by the cax1 lines under Mn2+ and Mg2+ stress conditions. The mutants exhibited altered plant development, perturbed hormone sensitivities, and altered expression of an auxin-regulated promoter-reporter gene fusion. We propose that CAX1 regulates myriad plant processes and discuss the observed phenotypes with regard to the compensatory alterations in other transporters. PMID:12566577

Functional Rescue of a Misfolded Drosophila melanogaster Dopamine Transporter Mutant Associated with a Sleepless Phenotype by Pharmacological Chaperones.

Science.gov (United States)

Kasture, Ameya; El-Kasaby, Ali; Szöllősi, Daniel; Asjad, H M Mazhar; Grimm, Alexandra; Stockner, Thomas; Hummel, Thomas; Freissmuth, Michael; Sucic, Sonja

2016-09-30

Folding-defective mutants of the human dopamine transporter (DAT) cause a syndrome of infantile dystonia/parkinsonism. Here, we provide a proof-of-principle that the folding deficit is amenable to correction in vivo by two means, the cognate DAT ligand noribogaine and the HSP70 inhibitor, pifithrin-μ. We examined the Drosophila melanogaster (d) mutant dDAT-G108Q, which leads to a sleepless phenotype in flies harboring this mutation. Molecular dynamics simulations suggested an unstable structure of dDAT-G108Q consistent with a folding defect. This conjecture was verified; heterologously expressed dDAT-G108Q and the human (h) equivalent hDAT-G140Q were retained in the endoplasmic reticulum in a complex with endogenous folding sensors (calnexin and HSP70-1A). Incubation of the cells with noribogaine (a DAT ligand selective for the inward-facing state) and/or pifithrin-μ (an HSP70 inhibitor) restored folding of, and hence dopamine transport by, dDAT-G108Q and hDAT-G140Q. The mutated versions of DAT were confined to the cell bodies of the dopaminergic neurons in the fly brain and failed to reach the axonal compartments. Axonal delivery was restored, and sleep time was increased to normal length (from 300 to 1000 min/day) if the dDAT-G108Q-expressing flies were treated with noribogaine and/or pifithrin-μ. Rescuing misfolded versions of DAT by pharmacochaperoning is of therapeutic interest; it may provide opportunities to remedy disorders arising from folding-defective mutants of human DAT and of other related SLC6 transporters. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The transcriptional response of Lactobacillus sanfranciscensis DSM 20451T and its tcyB mutant lacking a functional cystine transporter to diamide stress.

Science.gov (United States)

Stetina, Mandy; Behr, Jürgen; Vogel, Rudi F

2014-07-01

As a result of its strong adaptation to wheat and rye sourdoughs, Lactobacillus sanfranciscensis has the smallest genome within the genus Lactobacillus. The concomitant absence of some important antioxidative enzymes and the inability to synthesize glutathione suggest a role of cystine transport in maintenance of an intracellular thiol balance. Diamide [synonym 1,1'-azobis(N,N-dimethylformamide)] disturbs intracellular and membrane thiol levels in oxidizing protein thiols depending on its initial concentration. In this study, RNA sequencing was used to reveal the transcriptional response of L. sanfranciscensis DSM 20451(T) (wild type [WT]) and its ΔtcyB mutant with a nonfunctional cystine transporter after thiol stress caused by diamide. Along with the different expression of genes involved in amino acid starvation, pyrimidine synthesis, and energy production, our results show that thiol stress in the wild type can be compensated through activation of diverse chaperones and proteases whereas the ΔtcyB mutant shifts its metabolism in the direction of survival. Only a small set of genes are significantly differentially expressed between the wild type and the mutant. In the WT, mainly genes which are associated with a heat shock response are upregulated whereas glutamine import and synthesis genes are downregulated. In the ΔtcyB mutant, the whole opp operon was more highly expressed, as well as a protein which probably includes enzymes for methionine transport. The two proteins encoded by spxA and nrdH, which are involved in direct or indirect oxidative stress responses, are also upregulated in the mutant. This work emphasizes that even in the absence of definitive antioxidative enzymes, bacteria with a small genome and a high frequency of gene inactivation and elimination use small molecules such as the cysteine/cystine couple to overcome potential cell damage resulting from oxidative stress. Copyright © 2014, American Society for Microbiology. All Rights

Structural Characterization of Heme Environmental Mutants of CgHmuT that Shuttles Heme Molecules to Heme Transporters

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Norifumi Muraki

2016-05-01

Full Text Available Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow.

ICK is essential for cell type-specific ciliogenesis and the regulation of ciliary transport.

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Chaya, Taro; Omori, Yoshihiro; Kuwahara, Ryusuke; Furukawa, Takahisa

2014-06-02

Cilia and flagella are formed and maintained by intraflagellar transport (IFT) and play important roles in sensing and moving across species. At the distal tip of the cilia/flagella, IFT complexes turn around to switch from anterograde to retrograde transport; however, the underlying regulatory mechanism is unclear. Here, we identified ICK localization at the tip of cilia as a regulator of ciliary transport. In ICK-deficient mice, we found ciliary defects in neuronal progenitor cells with Hedgehog signal defects. ICK-deficient cells formed cilia with mislocalized Hedgehog signaling components. Loss of ICK caused the accumulation of IFT-A, IFT-B, and BBSome components at the ciliary tips. In contrast, overexpression of ICK induced the strong accumulation of IFT-B, but not IFT-A or BBSome components at ciliary tips. In addition, ICK directly phosphorylated Kif3a, while inhibition of this Kif3a phosphorylation affected ciliary formation. Our results suggest that ICK is a Kif3a kinase and essential for proper ciliogenesis in development by regulating ciliary transport at the tip of cilia. © 2014 The Authors.

The Meckel syndrome- associated protein MKS1 functionally interacts with components of the BBSome and IFT complexes to mediate ciliary trafficking and hedgehog signaling

Science.gov (United States)

Barrington, Chloe L.; Katsanis, Nicholas

2017-01-01

The importance of primary cilia in human health is underscored by the link between ciliary dysfunction and a group of primarily recessive genetic disorders with overlapping clinical features, now known as ciliopathies. Many of the proteins encoded by ciliopathy-associated genes are components of a handful of multi-protein complexes important for the transport of cargo to the basal body and/or into the cilium. A key question is whether different complexes cooperate in cilia formation, and whether they participate in cilium assembly in conjunction with intraflagellar transport (IFT) proteins. To examine how ciliopathy protein complexes might function together, we have analyzed double mutants of an allele of the Meckel syndrome (MKS) complex protein MKS1 and the BBSome protein BBS4. We find that Mks1; Bbs4 double mutant mouse embryos exhibit exacerbated defects in Hedgehog (Hh) dependent patterning compared to either single mutant, and die by E14.5. Cells from double mutant embryos exhibit a defect in the trafficking of ARL13B, a ciliary membrane protein, resulting in disrupted ciliary structure and signaling. We also examined the relationship between the MKS complex and IFT proteins by analyzing double mutant between Mks1 and a hypomorphic allele of the IFTB component Ift172. Despite each single mutant surviving until around birth, Mks1; Ift172avc1 double mutants die at mid-gestation, and exhibit a dramatic failure of cilia formation. We also find that Mks1 interacts genetically with an allele of Dync2h1, the IFT retrograde motor. Thus, we have demonstrated that the MKS transition zone complex cooperates with the BBSome to mediate trafficking of specific trans-membrane receptors to the cilium. Moreover, the genetic interaction of Mks1 with components of IFT machinery suggests that the transition zone complex facilitates IFT to promote cilium assembly and structure. PMID:28291807

The Meckel syndrome- associated protein MKS1 functionally interacts with components of the BBSome and IFT complexes to mediate ciliary trafficking and hedgehog signaling.

Directory of Open Access Journals (Sweden)

Sarah C Goetz

Full Text Available The importance of primary cilia in human health is underscored by the link between ciliary dysfunction and a group of primarily recessive genetic disorders with overlapping clinical features, now known as ciliopathies. Many of the proteins encoded by ciliopathy-associated genes are components of a handful of multi-protein complexes important for the transport of cargo to the basal body and/or into the cilium. A key question is whether different complexes cooperate in cilia formation, and whether they participate in cilium assembly in conjunction with intraflagellar transport (IFT proteins. To examine how ciliopathy protein complexes might function together, we have analyzed double mutants of an allele of the Meckel syndrome (MKS complex protein MKS1 and the BBSome protein BBS4. We find that Mks1; Bbs4 double mutant mouse embryos exhibit exacerbated defects in Hedgehog (Hh dependent patterning compared to either single mutant, and die by E14.5. Cells from double mutant embryos exhibit a defect in the trafficking of ARL13B, a ciliary membrane protein, resulting in disrupted ciliary structure and signaling. We also examined the relationship between the MKS complex and IFT proteins by analyzing double mutant between Mks1 and a hypomorphic allele of the IFTB component Ift172. Despite each single mutant surviving until around birth, Mks1; Ift172avc1 double mutants die at mid-gestation, and exhibit a dramatic failure of cilia formation. We also find that Mks1 interacts genetically with an allele of Dync2h1, the IFT retrograde motor. Thus, we have demonstrated that the MKS transition zone complex cooperates with the BBSome to mediate trafficking of specific trans-membrane receptors to the cilium. Moreover, the genetic interaction of Mks1 with components of IFT machinery suggests that the transition zone complex facilitates IFT to promote cilium assembly and structure.

nitrosoguanidine-induced cadmium resistant mutants of Aspergillus

Indian Academy of Sciences (India)

Unknown

nitrosoguanidine-induced cadmium resistant mutants of. Aspergillus niger. SAMAR ... gens and UV irradiation to study transportation of cad- mium ion through cell ..... Rowley W S 1993 Yeast bZib proteins mediate pleiotropic drug and metal ...

The intraflagellar transport machinery in ciliary signaling

DEFF Research Database (Denmark)

Mourão, André; Christensen, Søren Tvorup; Lorentzen, Esben

2016-01-01

Cilia and flagella on eukaryotic cells are slender microtubule-based projections surrounded by a membrane with a unique lipid and protein composition. It is now appreciated that cilia in addition to their established roles in motility also constitute hubs for cellular signaling by sensing external...

Using mass spectrometry for identification of ABC transporters from Xanthomonas citri and mutants expressed in different growth conditions

International Nuclear Information System (INIS)

Faria, J.N.; Balan, A.; Paes Leme, A.F.

2012-01-01

Full text: Xanthomonas citri is a phytopathogenic bacterium that infects citrus plants causing significant losses for the economy. In our group, we have focused on the identification and characterization of ABC transport proteins of this bacterium, in order to determinate their function for growth in vitro and in vivo, during infection. ABC transporters represent one of the largest families of proteins, which transport since small molecules as ions up to oligopeptides and sugars. In prokaryotic cells many works have reported the ABC transport function in pathogenesis, resistance, biofilm formation, infectivity and DNA repair, but until our knowledge, there is no data related to these transporters and X. citri. So, In order to determinate which transporters are expressed in X. citri, we started a proteomic analysis based on mono and bi-dimensional gels associated to mass spectrometry analyses. After growing X. citri and two different mutants deleted for ssuA and nitA genes in LB and minimum media, cellular extracts were obtained and used for preparation of mono and bi-dimensional gels. Seven bands covering the expected mass of ABC transporter components (20 kDa to 50 kDa) in SDS-PAGE were cut off the gel, treated with trypsin and submitted to the MS for protein identification. The results of 2D gels were good enough and will serve as a standard for development of similar experiments in large scale. (author)

Differential auxin transport and accumulation in the stem base lead to profuse adventitious root primordia formation in the aerial roots (aer) mutant of tomato (Solanum lycopersicum L.).

Science.gov (United States)

Mignolli, F; Mariotti, L; Picciarelli, P; Vidoz, M L

2017-06-01

The aerial roots (aer) mutant of tomato is characterized by a profuse and precocious formation of adventitious root primordia along the stem. We demonstrated that auxin is involved in the aer phenotype but ruled out higher auxin sensitivity of mutant plants. Interestingly, polar auxin transport was altered in aer, as young seedlings showed a reduced response to an auxin transport inhibitor and higher expression of auxin export carriers SlPIN1 and SlPIN3. An abrupt reduction in transcripts of auxin efflux and influx genes in older aer hypocotyls caused a marked deceleration of auxin transport in more mature tissues. Indeed, in 20days old aer plants, the transport of labeled IAA was faster in apices than in hypocotyls, displaying an opposite trend in comparison to a wild type. In addition, auxin transport facilitators (SlPIN1, SlPIN4, SlLAX5) were more expressed in aer apices than in hypocotyls, suggesting that auxin moves faster from the upper to the lower part of the stem. Consequently, a significantly higher level of free and conjugated IAA was found at the base of aer stems with respect to their apices. This auxin accumulation is likely the cause of the aer phenotype. Copyright © 2017 Elsevier GmbH. All rights reserved.

Neurotransmitter Transporter-Like: a male germline-specific SLC6 transporter required for Drosophila spermiogenesis.

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Nabanita Chatterjee

2011-01-01

Full Text Available The SLC6 class of membrane transporters, known primarily as neurotransmitter transporters, is increasingly appreciated for its roles in nutritional uptake of amino acids and other developmentally specific functions. A Drosophila SLC6 gene, Neurotransmitter transporter-like (Ntl, is expressed only in the male germline. Mobilization of a transposon inserted near the 3' end of the Ntl coding region yields male-sterile mutants defining a single complementation group. Germline transformation with Ntl cDNAs under control of male germline-specific control elements restores Ntl/Ntl homozygotes to normal fertility, indicating that Ntl is required only in the germ cells. In mutant males, sperm morphogenesis appears normal, with elongated, individualized and coiled spermiogenic cysts accumulating at the base of the testes. However, no sperm are transferred to the seminal vesicle. The level of polyglycylation of Ntl mutant sperm tubulin appears to be significantly lower than that of wild type controls. Glycine transporters are the most closely related SLC6 transporters to Ntl, suggesting that Ntl functions as a glycine transporter in developing sperm, where augmentation of the cytosolic pool of glycine may be required for the polyglycylation of the massive amounts of tubulin in the fly's giant sperm. The male-sterile phenotype of Ntl mutants may provide a powerful genetic system for studying the function of an SLC6 transporter family in a model organism.

Isolation of parafluorophenylalanine-resistant mutants from HeLa cell cultures

International Nuclear Information System (INIS)

Yim, L.K.; Stuart, W.D.

1983-01-01

This report describes a method to isolate temperature-conditional phenylalanine transport mutants from the transformed human cell line HeLa. Using ultraviolet light as a mutagenic agent and DL-parafluorophenylalanine (PFPA), a poisonous analogue of L-phenylalanine, as a selective agent, mutagenized cells were selected for survival in the presence of PFPA at a temperature of 39 degrees C. Survivors of the mutagenesis and selection procedures were removed from the culture dishes by trypsin and cloned at a temperature of 35 degrees C. Seven of these lines isolated demonstrated continued resistance to PFPA at 39 degrees C. These lines were tested for uptake of L-phenylalanine at an external concentration of 100 microM and for continued resistance to PFPA at two concentrations. Cells were tested at 35 and at 39 degrees C. The data were compared to those obtained for the parental HeLa cell line under identical conditions. The seven mutant cell lines demonstrated varying resistances to PFPA and varying levels of accumulation of L-phenylalanine when tested at 35 and 39 degrees C. Three mutant lines were additionally tested for L-phenylalanine tRNA charging levels and for transport of L-arginine. The lines had parental cell levels of tRNA charging and L-arginine transport which suggest that the induced genetic defect affects a specific L-phenylalanine transport system

Testing the time-of-flight model for flagellar length sensing.

Science.gov (United States)

Ishikawa, Hiroaki; Marshall, Wallace F

2017-11-07

Cilia and flagella are microtubule-based organelles that protrude from the surface of most cells, are important to the sensing of extracellular signals, and make a driving force for fluid flow. Maintenance of flagellar length requires an active transport process known as intraflagellar transport (IFT). Recent studies reveal that the amount of IFT injection negatively correlates with the length of flagella. These observations suggest that a length-dependent feedback regulates IFT. However, it is unknown how cells recognize the length of flagella and control IFT. Several theoretical models try to explain this feedback system. We focused on one of the models, the "time-of-flight" model, which measures the length of flagella on the basis of the travel time of IFT protein in the flagellar compartment. We tested the time-of-flight model using Chlamydomonas dynein mutant cells, which show slower retrograde transport speed. The amount of IFT injection in dynein mutant cells was higher than that in control cells. This observation does not support the prediction of the time-of-flight model and suggests that Chlamydomonas uses another length-control feedback system rather than that described by the time-of-flight model. © 2017 Ishikawa and Marshall. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Tyrosine Phosphorylation of the Human Serotonin Transporter: A Role in the Transporter Stability and Function

Science.gov (United States)

Annamalai, Balasubramaniam; Mannangatti, Padmanabhan; Arapulisamy, Obulakshmi; Shippenberg, Toni S.; Jayanthi, Lankupalle D.

2012-01-01

The serotonin (5-HT) transporter (SERT) regulates serotoninergic neurotransmission by clearing 5-HT released into the synaptic space. Phosphorylation of SERT on serine and threonine mediates SERT regulation. Whether tyrosine phosphorylation regulates SERT is unknown. Here, we tested the hypothesis that tyrosine-phosphorylation of SERT regulates 5-HT transport. In support of this, alkali-resistant 32P-labeled SERT was found in rat platelets, and Src-tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4,d]pyrimidine (PP2) decreased platelet SERT function and expression. In human placental trophoblast cells expressing SERT, PP2 reduced transporter function, expression, and stability. Although siRNA silencing of Src expression decreased SERT function and expression, coexpression of Src resulted in PP2-sensitive increases in SERT function and expression. PP2 treatment markedly decreased SERT protein stability. Compared with WT-SERT, SERT tyrosine mutants Y47F and Y142F exhibited reduced 5-HT transport despite their higher total and cell surface expression levels. Moreover, Src-coexpression increased total and cell surface expression of Y47F and Y142F SERT mutants without affecting their 5-HT transport capacity. It is noteworthy that Y47F and Y142F mutants exhibited higher protein stability compared with WT-SERT. However, similar to WT-SERT, PP2 treatment decreased the stability of Y47F and Y142F mutants. Furthermore, compared with WT-SERT, Y47F and Y142F mutants exhibited lower basal tyrosine phosphorylation and no further enhancement of tyrosine phosphorylation in response to Src coexpression. These results provide the first evidence that SERT tyrosine phosphorylation supports transporter protein stability and 5HT transport. PMID:21992875

Genes that act downstream of sensory neurons to influence longevity, dauer formation, and pathogen responses in Caenorhabditis elegans.

Directory of Open Access Journals (Sweden)

Marta M Gaglia

Full Text Available The sensory systems of multicellular organisms are designed to provide information about the environment and thus elicit appropriate changes in physiology and behavior. In the nematode Caenorhabditis elegans, sensory neurons affect the decision to arrest during development in a diapause state, the dauer larva, and modulate the lifespan of the animals in adulthood. However, the mechanisms underlying these effects are incompletely understood. Using whole-genome microarray analysis, we identified transcripts whose levels are altered by mutations in the intraflagellar transport protein daf-10, which result in impaired development and function of many sensory neurons in C. elegans. In agreement with existing genetic data, the expression of genes regulated by the transcription factor DAF-16/FOXO was affected by daf-10 mutations. In addition, we found altered expression of transcriptional targets of the DAF-12/nuclear hormone receptor in the daf-10 mutants and showed that this pathway influences specifically the dauer formation phenotype of these animals. Unexpectedly, pathogen-responsive genes were repressed in daf-10 mutant animals, and these sensory mutants exhibited altered susceptibility to and behavioral avoidance of bacterial pathogens. Moreover, we found that a solute transporter gene mct-1/2, which was induced by daf-10 mutations, was necessary and sufficient for longevity. Thus, sensory input seems to influence an extensive transcriptional network that modulates basic biological processes in C. elegans. This situation is reminiscent of the complex regulation of physiology by the mammalian hypothalamus, which also receives innervations from sensory systems, most notably the visual and olfactory systems.

Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae.

Science.gov (United States)

Nijland, Jeroen G; Shin, Hyun Yong; Boender, Leonie G M; de Waal, Paul P; Klaassen, Paul; Driessen, Arnold J M

2017-06-01

Engineering Saccharomyces cerevisiae for the utilization of pentose sugars is an important goal for the production of second-generation bioethanol and biochemicals. However, S. cerevisiae lacks specific pentose transporters, and in the presence of glucose, pentoses enter the cell inefficiently via endogenous hexose transporters (HXTs). By means of in vivo engineering, we have developed a quadruple hexokinase deletion mutant of S. cerevisiae that evolved into a strain that efficiently utilizes d-xylose in the presence of high d-glucose concentrations. A genome sequence analysis revealed a mutation (Y353C) in the general corepressor CYC8 , or SSN6 , which was found to be responsible for the phenotype when introduced individually in the nonevolved strain. A transcriptome analysis revealed altered expression of 95 genes in total, including genes involved in (i) hexose transport, (ii) maltose metabolism, (iii) cell wall function (mannoprotein family), and (iv) unknown functions (seripauperin multigene family). Of the 18 known HXTs, genes for 9 were upregulated, especially the low or nonexpressed HXT10 , HXT13 , HXT15 , and HXT16 Mutant cells showed increased uptake rates of d-xylose in the presence of d-glucose, as well as elevated maximum rates of metabolism ( V max ) for both d-glucose and d-xylose transport. The data suggest that the increased expression of multiple hexose transporters renders d-xylose metabolism less sensitive to d-glucose inhibition due to an elevated transport rate of d-xylose into the cell. IMPORTANCE The yeast Saccharomyces cerevisiae is used for second-generation bioethanol formation. However, growth on xylose is limited by pentose transport through the endogenous hexose transporters (HXTs), as uptake is outcompeted by the preferred substrate, glucose. Mutant strains were obtained with improved growth characteristics on xylose in the presence of glucose, and the mutations mapped to the regulator Cyc8. The inactivation of Cyc8 caused increased

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

Science.gov (United States)

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.

Impaired phloem loading in zmsweet13a,b,c sucrose transporter triple knock-out mutants in Zea mays.

Science.gov (United States)

Bezrutczyk, Margaret; Hartwig, Thomas; Horschman, Marc; Char, Si Nian; Yang, Jinliang; Yang, Bing; Frommer, Wolf B; Sosso, Davide

2018-04-01

Crop yield depends on efficient allocation of sucrose from leaves to seeds. In Arabidopsis, phloem loading is mediated by a combination of SWEET sucrose effluxers and subsequent uptake by SUT1/SUC2 sucrose/H + symporters. ZmSUT1 is essential for carbon allocation in maize, but the relative contribution to apoplasmic phloem loading and retrieval of sucrose leaking from the translocation path is not known. Here we analysed the contribution of SWEETs to phloem loading in maize. We identified three leaf-expressed SWEET sucrose transporters as key components of apoplasmic phloem loading in Zea mays L. ZmSWEET13 paralogues (a, b, c) are among the most highly expressed genes in the leaf vasculature. Genome-edited triple knock-out mutants were severely stunted. Photosynthesis of mutants was impaired and leaves accumulated high levels of soluble sugars and starch. RNA-seq revealed profound transcriptional deregulation of genes associated with photosynthesis and carbohydrate metabolism. Genome-wide association study (GWAS) analyses may indicate that variability in ZmSWEET13s correlates with agronomical traits, especifically flowering time and leaf angle. This work provides support for cooperation of three ZmSWEET13s with ZmSUT1 in phloem loading in Z. mays. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

A cataract-causing connexin 50 mutant is mislocalized to the ER due to loss of the fourth transmembrane domain and cytoplasmic domain.

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Somaraju Chalasani, Madhavi Latha; Muppirala, Madhavi; G Ponnam, Surya Prakash; Kannabiran, Chitra; Swarup, Ghanshyam

2013-01-01

Mutations in the eye lens gap junction protein connexin 50 cause cataract. Earlier we identified a frameshift mutant of connexin 50 (c.670insA; p.Thr203AsnfsX47) in a family with autosomal recessive cataract. The mutant protein is smaller and contains 46 aberrant amino acids at the C-terminus after amino acid 202. Here, we have analysed this frameshift mutant and observed that it localized to the endoplasmic reticulum (ER) but not in the plasma membrane. Moreover, overexpression of the mutant resulted in disintegration of the ER-Golgi intermediate compartment (ERGIC), reduction in the level of ERGIC-53 protein and breakdown of the Golgi in many cells. Overexpression of the frameshift mutant partially inhibited the transport of wild type connexin 50 to the plasma membrane. A deletion mutant lacking the aberrant sequence showed predominant localization in the ER and inhibited anterograde protein transport suggesting, therefore, that the aberrant sequence is not responsible for improper localization of the frameshift mutant. Further deletion analysis showed that the fourth transmembrane domain and a membrane proximal region (231-294 amino acids) of the cytoplasmic domain are needed for transport from the ER and localization to the plasma membrane. Our results show that a frameshift mutant of connexin 50 mislocalizes to the ER and causes disintegration of the ERGIC and Golgi. We have also identified a sequence of connexin 50 crucial for transport from the ER and localization to the plasma membrane.

Mutants induced in winter rye (Secale cereale L.): Short straw-mutant No. 2714 and late-senescence mutant

Energy Technology Data Exchange (ETDEWEB)

Muszynski, S; Darlewska, M [Department of Plant Breeding and Seed Science, Warsaw Agricultural University, Warsaw (Poland)

1990-01-01

Full text: Mutants were induced by treating dormant seeds with ionizing radiation (fast neutrons) or chemicals (N-nitroso-N-ethyl urea or sodium azide). Among several mutants obtained, of special value is the short-straw mutant No. 2714 and a late senescent mutant. (author)

Regulation of auxin transport during gravitropism

Science.gov (United States)

Rashotte, A.; Brady, S.; Kirpalani, N.; Buer, C.; Muday, G.

Plants respond to changes in the gravity vector by differential growth across the gravity-stimulated organ. The plant hormone auxin, which is normally basipetally transported, changes in direction and auxin redistribution has been suggested to drive this differential growth or gravitropism. The mechanisms by which auxin transport directionality changes in response to a change in gravity vector are largely unknown. Using the model plant, Arabidopsis thaliana, we have been exploring several regulatory mechanisms that may control auxin transport. Mutations that alter protein phosphorylation suggest that auxin transport in arabidopsis roots may be controlled via phosphorylation and this signal may facilitate gravitropic bending. The protein kinase mutant pinoid (pid9) has reduced auxin transport; whereas the protein phosphatase mutant, rcn1, has elevated transport, suggesting reciprocal regulation of auxin transport by reversible protein phosphorylation. In both of these mutants, the auxin transport defects are accompanied by gravitropic defects, linking phosphorylation signaling to gravity-induced changes in auxin transport. Additionally, auxin transport may be regulated during gravity response by changes in an endogenous auxin efflux inhibitor. Flavonoids, such as quercetin and kaempferol, have been implicated in regulation of auxin transport in vivo and in vitro. Mutants that make no flavonoids have reduced root gravitropic bending. Furthermore, changes in auxin-induced gene expression and flavonoid accumulation patterns have been observed during gravity stimulation. Current studies are examining whether there are spatial and temporal changes in flavonoid accumulation that precede gravitropic bending and whether the absence of these changes are the cause of the altered gravity response in plants with mutations that block flavonoid synthesis. These results support the idea that auxin transport may be regulated during gravity response by several mechanisms including

Phosphorus Partitioning of Soybean Lines Containing Different Mutant Alleles of Two Soybean Seed-Specific Adenosine Triphosphate-Binding Cassette Phytic Acid Transporter Paralogs

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Jason D. Gillman

2013-03-01

Full Text Available Seed phytate is a repository of P and minerals in soybean [ (L. Merr.] seeds that limits P and mineral bioavailability for monogastric animals (e.g., humans, swine [], and poultry [especially chicken, ] due to insufficient digestive tract phytase activity. We previously identified epistatic recessive mutations affecting two paralogous adenosine triphosphate-binding cassette phytic acid transporter genes (one a nonsense mutation in and the other a missense mutation in as the molecular genetic basis in the ethyl methanesulfonate (EMS-induced mutant low phytate soybean line M153. An additional mutant low phytate line, M766, contained one single nucleotide polymorphism within the ninth intron of the locus as well as a nonsense mutation in . The objectives of this research were to clarify the genetics underlying the low phytate phenotype in line M766 and to determine P partitioning in new combinations of mutant alleles from M766 and M153. Inheritance of nonsense alleles affecting both ( genes (one from M153 and one from M766 led to the production of viable seeds that contained transgressive reductions in total seed phytate and significantly higher levels of inorganic phosphate than has been reported for nontransgenic soybean material and will allow efficient molecular selection of soybeans with even greater reductions of phytate for improved quality soybean meal.

Defective glycinergic synaptic transmission in zebrafish motility mutants

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Hiromi Hirata

2010-01-01

Full Text Available Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Recently, in vivo analysis of glycinergic synaptic transmission has been pursued in zebrafish using molecular genetics. An ENU mutagenesis screen identified two behavioral mutants that are defective in glycinergic synaptic transmission. Zebrafish bandoneon (beo mutants have a defect in glrbb, one of the duplicated glycine receptor (GlyR β subunit genes. These mutants exhibit a loss of glycinergic synaptic transmission due to a lack of synaptic aggregation of GlyRs. Due to the consequent loss of reciprocal inhibition of motor circuits between the two sides of the spinal cord, motor neurons activate simultaneously on both sides resulting in bilateral contraction of axial muscles of beo mutants, eliciting the so-called ‘accordion’ phenotype. Similar defects in GlyR subunit genes have been observed in several mammals and are the basis for human hyperekplexia/startle disease. By contrast, zebrafish shocked (sho mutants have a defect in slc6a9, encoding GlyT1, a glycine transporter that is expressed by astroglial cells surrounding the glycinergic synapse in the hindbrain and spinal cord. GlyT1 mediates rapid uptake of glycine from the synaptic cleft, terminating synaptic transmission. In zebrafish sho mutants, there appears to be elevated extracellular glycine resulting in persistent inhibition of postsynaptic neurons and subsequent reduced motility, causing the ‘twitch once’ phenotype. We review current knowledge regarding zebrafish ‘accordion’ and ‘twitch once’ mutants, including beo and sho, and report the identification of a new α2 subunit that revises the phylogeny of zebrafish GlyRs.

Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

Science.gov (United States)

Hirata, Hiromi; Carta, Eloisa; Yamanaka, Iori; Harvey, Robert J.; Kuwada, John Y.

2009-01-01

Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Recently, in vivo analysis of glycinergic synaptic transmission has been pursued in zebrafish using molecular genetics. An ENU mutagenesis screen identified two behavioral mutants that are defective in glycinergic synaptic transmission. Zebrafish bandoneon (beo) mutants have a defect in glrbb, one of the duplicated glycine receptor (GlyR) β subunit genes. These mutants exhibit a loss of glycinergic synaptic transmission due to a lack of synaptic aggregation of GlyRs. Due to the consequent loss of reciprocal inhibition of motor circuits between the two sides of the spinal cord, motor neurons activate simultaneously on both sides resulting in bilateral contraction of axial muscles of beo mutants, eliciting the so-called ‘accordion’ phenotype. Similar defects in GlyR subunit genes have been observed in several mammals and are the basis for human hyperekplexia/startle disease. By contrast, zebrafish shocked (sho) mutants have a defect in slc6a9, encoding GlyT1, a glycine transporter that is expressed by astroglial cells surrounding the glycinergic synapse in the hindbrain and spinal cord. GlyT1 mediates rapid uptake of glycine from the synaptic cleft, terminating synaptic transmission. In zebrafish sho mutants, there appears to be elevated extracellular glycine resulting in persistent inhibition of postsynaptic neurons and subsequent reduced motility, causing the ‘twitch-once’ phenotype. We review current knowledge regarding zebrafish ‘accordion’ and ‘twitch-once’ mutants, including beo and sho, and report the identification of a new α2 subunit that revises the phylogeny of zebrafish GlyRs. PMID:20161699

A Neisseria meningitidis fbpABC mutant is incapable of using nonheme iron for growth.

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Khun, H H; Kirby, S D; Lee, B C

1998-05-01

The neisserial fbpABC locus has been proposed to act as an iron-specific ABC transporter system. To confirm this assigned function, we constructed an fbpABC mutant in Neisseria meningitidis by insertional inactivation of fbpABC with a selectable antibiotic marker. The mutant was unable to use iron supplied from human transferrin, human lactoferrin, or iron chelates. However, the use of iron from heme and human hemoglobin was unimpaired. These results support the obligatory participation of fbpABC in neisserial periplasmic iron transport and do not indicate a role for this genetic locus in the heme iron pathway.

Bridging the gap between chemistry, physiology, and evolution: Quantifying the functionality of sperm whale myoglobin mutants

DEFF Research Database (Denmark)

Dasmeh, Pouria; Kepp, Kasper Planeta

2011-01-01

This work merges a large set of previously reported thermochemical data for myoglobin (Mb) mutants with a physiological model of O2-transport and -storage. The model allows a quantification of the functional proficiency of myoglobin (Mb) mutants under various physiological conditions, i.e. O2-con...

The capacity of Listeria monocytogenes mutants with in-frame deletions in putative ATP-binding cassette transporters to form biofilms and comparison with the wild type

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Marina Ceruso

2014-02-01

Full Text Available Listeria monocytogenes (Lm is a food-borne pathogen responsible for human listeriosis, an invasive infection with high mortality rates. Lm has developed efficient strategies for survival under stress conditions such as starvation and wide variations in temperature, pH, and osmolarity. Therefore, Lm can survive in food under multiple stress conditions. Detailed studies to determine the mode of action of this pathogen for survival under stress conditions are important to control Lm in food. It has been shown that genes encoding for ATP-binding cassette (ABC transporters are induced in Lm in food, in particular under stress conditions. Previous studies showed that these genes are involved in sensitivity to nisin, acids, and salt. The aim of this study was to determine the involvement of some ABC transporters in biofilm formation. Therefore, deletion mutants of ABC transporter genes (LMOf2365_1875 and LMOf2365_1877 were created in Lm F2365, and then were compared to the wild type for their capacity to form biofilms. Lm strain F2365 was chosen as reference since the genome is fully sequenced and furthermore this strain is particularly involved in food-borne outbreaks of listeriosis. Our results showed that DLMOf2365_1875 had an increased capacity to form biofilms compared to the wild type, indicating that LMOf2365_1875 negatively regulates biofilm formation. A deeper knowledge on the ability to form biofilms in these mutants may help in the development of intervention strategies to control Lm in food and in the environment.

Human liver cell trafficking mutants: characterization and whole exome sequencing.

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Fei Yuan

Full Text Available The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α''. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.

Mutational scanning of the human serotonin transporter reveals fast translocating serotonin transporter mutants

DEFF Research Database (Denmark)

Kristensen, Anders S; Larsen, Mads B; Johnsen, Laust B

2004-01-01

The serotonin transporter (SERT) belongs to a family of sodium-chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from the extracellular space. SERT represents a major pharmacological target in the treatment of several clinical conditions, including depressi...

Rate and Regulation of Copper Transport by Human Copper Transporter 1 (hCTR1)*

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Maryon, Edward B.; Molloy, Shannon A.; Ivy, Kristin; Yu, Huijun; Kaplan, Jack H.

2013-01-01

Human copper transporter 1 (hCTR1) is a homotrimer of a 190-amino acid monomer having three transmembrane domains believed to form a pore for copper permeation through the plasma membrane. The hCTR1-mediated copper transport mechanism is not well understood, nor has any measurement been made of the rate at which copper ions are transported by hCTR1. In this study, we estimated the rate of copper transport by the hCTR1 trimer in cultured cells using 64Cu uptake assays and quantification of plasma membrane hCTR1. For endogenous hCTR1, we estimated a turnover number of about 10 ions/trimer/s. When overexpressed in HEK293 cells, a second transmembrane domain mutant of hCTR1 (H139R) had a 3-fold higher Km value and a 4-fold higher turnover number than WT. Truncations of the intracellular C-terminal tail and an AAA substitution of the putative metal-binding HCH C-terminal tripeptide (thought to be required for transport) also exhibited elevated transport rates and Km values when compared with WT hCTR1. Unlike WT hCTR1, H139R and the C-terminal mutants did not undergo regulatory endocytosis in elevated copper. hCTR1 mutants combining methionine substitutions that block transport (M150L,M154L) on the extracellular side of the pore and the high transport H139R or AAA intracellular side mutations exhibited the blocked transport of M150L,M154L, confirming that Cu+ first interacts with the methionines during permeation. Our results show that hCTR1 elements on the intracellular side of the hCTR1 pore, including the carboxyl tail, are not essential for permeation, but serve to regulate the rate of copper entry. PMID:23658018

SUCROSE TRANSPORTER 5 supplies Arabidopsis embryos with biotin and affects triacylglycerol accumulation

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Pommerrenig, Benjamin; Popko, Jennifer; Heilmann, Mareike; Schulmeister, Sylwia; Dietel, Katharina; Schmitt, Bianca; Stadler, Ruth; Feussner, Ivo; Sauer, Norbert

2013-01-01

The Arabidopsis SUC5 protein represents a classical sucrose/H+ symporter. Functional analyses previously revealed that SUC5 also transports biotin, an essential co-factor for fatty acid synthesis. However, evidence for a dual role in transport of the structurally unrelated compounds sucrose and biotin in plants was lacking. Here we show that SUC5 localizes to the plasma membrane, and that the SUC5 gene is expressed in developing embryos, confirming the role of the SUC5 protein as substrate carrier across apoplastic barriers in seeds. We show that transport of biotin but not of sucrose across these barriers is impaired in suc5 mutant embryos. In addition, we show that SUC5 is essential for the delivery of biotin into the embryo of biotin biosynthesis-defective mutants (bio1 and bio2). We compared embryo and seedling development as well as triacylglycerol accumulation and fatty acid composition in seeds of single mutants (suc5, bio1 or bio2), double mutants (suc5 bio1 and suc5 bio2) and wild-type plants. Although suc5 mutants were like the wild-type, bio1 and bio2 mutants showed developmental defects and reduced triacylglycerol contents. In suc5 bio1 and suc5 bio2 double mutants, developmental defects were severely increased and the triacylglycerol content was reduced to a greater extent in comparison to the single mutants. Supplementation with externally applied biotin helped to reduce symptoms in both single and double mutants, but the efficacy of supplementation was significantly lower in double than in single mutants, showing that transport of biotin into the embryo is lower in the absence of SUC5. PMID:23031218

Brucella abortus mutants lacking ATP-binding cassette transporter proteins are highly attenuated in virulence and confer protective immunity against virulent B. abortus challenge in BALB/c mice.

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Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Park, Bo-Kyoung; Hahn, Tae-Wook

2016-06-01

Brucella abortus RB51 is an attenuated vaccine strain that has been most frequently used for bovine brucellosis. Although it is known to provide good protection in cattle, it still has some drawbacks including resistance to rifampicin, residual virulence and pathogenicity in humans. Thus, there has been a continuous interest on new safe and effective bovine vaccine candidates. In the present study, we have constructed unmarked mutants by deleting singly cydD and cydC genes, which encode ATP-binding cassette transporter proteins, from the chromosome of the virulent Brucella abortus isolate from Korean cow (referred to as IVK15). Both IVK15ΔcydD and ΔcydC mutants showed increased sensitivity to metal ions, hydrogen peroxide and acidic pH, which are mimic to intracellular environment during host infection. Additionally, the mutants exhibited a significant growth defect in RAW264.7 cells and greatly attenuated in mice. Vaccination of mice with either IVK15ΔcydC or IVK15ΔcydD mutant could elicit an anti-Brucella specific immunoglobulin G (IgG) and IgG subclass responses as well as enhance the secretion of interferon-gamma, and provided better protection against challenge with B. abortus strain 2308 than with the commercial B. abortus strain RB51 vaccine. Collectively, these results suggest that both IVK15ΔcydC and IVK15ΔcydD mutants could be an attenuated vaccine candidate against B. abortus. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterization of Brucella abortus mutant strain Δ22915, a potential vaccine candidate.

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Bao, Yanqing; Tian, Mingxing; Li, Peng; Liu, Jiameng; Ding, Chan; Yu, Shengqing

2017-04-04

Brucellosis, caused by Brucella spp., is an important zoonosis worldwide. Vaccination is an effective strategy for protection against Brucella infection in livestock in developing countries and in wildlife in developed countries. However, current vaccine strains including S19 and RB51 are pathogenic to humans and pregnant animals, limiting their use. In this study, we constructed the Brucella abortus (B. abortus) S2308 mutant strain Δ22915, in which the putative lytic transglycosylase gene BAB_RS22915 was deleted. The biological properties of mutant strain Δ22915 were characterized and protection of mice against virulent S2308 challenge was evaluated. The mutant strain Δ22915 showed reduced survival within RAW264.7 cells and survival in vivo in mice. In addition, the mutant strain Δ22915 failed to escape fusion with lysosomes within host cells, and caused no observable pathological damage. RNA-seq analysis indicated that four genes associated with amino acid/nucleotide transport and metabolism were significantly upregulated in mutant strain Δ22915. Furthermore, inoculation of ∆22915 at 10 5 colony forming units induced effective host immune responses and long-term protection of BALB/c mice. Therefore, mutant strain ∆22915 could be used as a novel vaccine candidate in the future to protect animals against B. abortus infection.

ABC transporters in Arthropods: genomic comparison and role in insecticide transport and resistance

NARCIS (Netherlands)

Dermauw, W.; Van Leeuwen, T.

2014-01-01

About a 100 years ago, the Drosophila white mutant marked the birth of Drosophila genetics. The white gene turned out to encode the first well studied ABC transporter in arthropods. The ABC gene family is now recognized as one of the largest transporter families in all kingdoms of life. The majority

The accessibility in the external part of the TM5 of the glutamate transporter EAAT1 is conformationally sensitive during the transport cycle.

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Xiuping Zhang

Full Text Available BACKGROUND: Excitatory amino acid transporter 1 (EAAT1 is a glutamate transporter which is a key element in the termination of the synaptic actions of glutamate. It serves to keep the extracellular glutamate concentration below neurotoxic level. However the functional significance and the change of accessibility of residues in transmembrane domain (TM 5 of the EAAT1 are not clear yet. METHODOLOGY/PRINCIPAL FINDINGS: We used cysteine mutagenesis with treatments with membrane-impermeable sulfhydryl reagent MTSET [(2-trimethylammonium methanethiosulfonate] to investigate the change of accessibility of TM5. Cysteine mutants were introduced from position 291 to 300 of the cysteine-less version of EAAT1. We checked the activity and kinetic parameters of the mutants before and after treatments with MTSET, furthermore we analyzed the effect of the substrate and blocker on the inhibition of the cysteine mutants by MTSET. Inhibition of transport by MTSET was observed in the mutants L296C, I297C and G299C, while the activity of K300C got higher after exposure to MTSET. V(max of L296C and G299C got lower while that of K300C got higher after treated by MTSET. The L296C, G299C, K300C single cysteine mutants showed a conformationally sensitive reactivity pattern. The sensitivity of L296C to MTSET was potentiated by glutamate and TBOA,but the sensitivity of G299C to MTSET was potentiated only by TBOA. CONCLUSIONS/SIGNIFICANCE: All these facts suggest that the accessibility of some positions of the external part of the TM5 is conformationally sensitive during the transport cycle. Our results indicate that some residues of TM5 take part in the transport pathway during the transport cycle.

A mutant of a mutant of a mutant of a ...: Irradiation of progressive radiation-induced mutants in a mutation-breeding programme with Chrysanthenum morifolium RAM

International Nuclear Information System (INIS)

Broertjes, C.; Koene, P.; Veen, J.W.H. van.

1980-01-01

Radiation-induced sports in Chrysanthemum morifolium RAM. have been reported for several years. It has become an everyday practice to produce flower-colour mutants from outstanding cross-breeding products, even before they are distributed for the commercial production of cut flowers. One of the most successful and recent examples is that of cv. Horim, of which hundreds of mutants were produced by successive use of radiation-induced mutants in the mutation-breeding programme. Over about 4 years a variety of flower-colour mutants was obtained, not only largely including the outstanding characteristics of the original cultivar but sometimes even with an appreciable improvement in quality and yield. It is expected that the latter types, the Miros group, will soon completely supersede the spontaneous or raditation-induced Horim sports and mutants and take over the leading position of the Horim group in the production of all-year-round (AYR) cut-flowers. (orig.)

Structural defects in cilia of the choroid plexus, subfornical organ and ventricular ependyma are associated with ventriculomegaly

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Swiderski Ruth E

2012-10-01

Full Text Available Abstract Background Hydrocephalus is a heterogeneous disorder with multiple etiologies that are not yet fully understood. Animal models have implicated dysfunctional cilia of the ependyma and choroid plexus in the development of the disorder. In this report, we sought to determine the origin of the ventriculomegaly in four Bardet Biedl syndrome (BBS mutant mouse strains as models of a ciliopathy. Methods Evans Blue dye was injected into the lateral ventricle of wild- type and BBS mutant mice to determine whether obstruction of intra- or extra-ventricular CSF flow contributed to ventriculomegaly. Transmission electron microscopy (TEM was used to examine the ultrastructure of the choroid plexus, subfornical organ (SFO, subcommisural organ (SCO, and ventricular ependyma to evaluate their ultrastructure and the morphology of their primary and motile cilia. Results and discussion No obstruction of intra- or extra-ventricular CSF flow was observed, implying a communicating form of hydrocephalus in BBS mutant mice. TEM analyses of the mutants showed no evidence of choroidal papillomas or breakdown of the blood:CSF barrier. In contrast, structural defects were observed in a subpopulation of cilia lining the choroid plexus, SFO, and ventricular ependyma. These included disruptions of the microtubular structure of the axoneme and the presence of electron-dense vesicular-like material along the ciliary shaft and at the tips of cilia. Conclusions Abnormalities in cilia structure and function have the potential to influence ciliary intraflagellar transport (IFT, cilia maintenance, protein trafficking, and regulation of CSF production. Ciliary structural defects are the only consistent pathological features associated with CSF-related structures in BBS mutant mice. These defects are observed from an early age, and may contribute to the underlying pathophysiology of ventriculomegaly.

Primary cilia regulate Shh activity in the control of molar tooth number

Czech Academy of Sciences Publication Activity Database

Ohazama, A.; Haycraft, C. J.; Seppala, M.; Ghafoor, S.; Cobourne, M.; Martinelli, D. C.; Fan, CH. M.; Peterková, Renata; Lesot, H.; Yoder, B. K.; Sharpe, P. T.

2009-01-01

Roč. 136, č. 6 (2009), s. 897-903 ISSN 0950-1991 R&D Projects: GA ČR GA304/07/0223; GA MŠk OC B23.002 Institutional research plan: CEZ:AV0Z50390512 Keywords : intraflagellar transport * supernumerary tooth * tooth development Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.194, year: 2009

A divergent calponin homology (NN–CH) domain defines a novel family

DEFF Research Database (Denmark)

Schou, Kenneth Bødtker; Andersen, Jens S.; Pedersen, Lotte Bang

2014-01-01

and NUF2 share evolutionary ancestry with a novel protein family in mammals comprising, besides NDC80/HEC1 and NUF2, three Intraflagellar Transport (IFT) complex B subunits (IFT81, IFT57, CLUAP1) as well as six proteins with poorly defined function (FAM98A-C, CCDC22, CCDC93 and C14orf166). We show...

Impaired pH homeostasis in Arabidopsis lacking the vacuolar dicarboxylate transporter and analysis of carboxylic acid transport across the tonoplast.

Science.gov (United States)

Hurth, Marco Alois; Suh, Su Jeoung; Kretzschmar, Tobias; Geis, Tina; Bregante, Monica; Gambale, Franco; Martinoia, Enrico; Neuhaus, H Ekkehard

2005-03-01

Arabidopsis (Arabidopsis thaliana) mutants lacking the tonoplastic malate transporter AttDT (A. thaliana tonoplast dicarboxylate transporter) and wild-type plants showed no phenotypic differences when grown under standard conditions. To identify putative metabolic changes in AttDT knock-out plants, we provoked a metabolic scenario connected to an increased consumption of dicarboxylates. Acidification of leaf discs stimulated dicarboxylate consumption and led to extremely low levels of dicarboxylates in mutants. To investigate whether reduced dicarboxylate concentrations in mutant leaf cells and, hence, reduced capacity to produce OH(-) to overcome acidification might affect metabolism, we measured photosynthetic oxygen evolution under conditions where the cytosol is acidified. AttDT::tDNA protoplasts showed a much stronger inhibition of oxygen evolution at low pH values when compared to wild-type protoplasts. Apparently citrate, which is present in higher amounts in knock-out plants, is not able to replace dicarboxylates to overcome acidification. To raise more information on the cellular level, we performed localization studies of carboxylates. Although the total pool of carboxylates in mutant vacuoles was nearly unaltered, these organelles contained a lower proportion of malate and fumarate and a higher proportion of citrate when compared to wild-type vacuoles. These alterations concur with the observation that radioactively labeled malate and citrate are transported into Arabidopsis vacuoles by different carriers. In addition, wild-type vacuoles and corresponding organelles from AttDT::tDNA mutants exhibited similar malate channel activities. In conclusion, these results show that Arabidopsis vacuoles contain at least two transporters and a channel for dicarboxylates and citrate and that the activity of AttDT is critical for regulation of pH homeostasis.

An evolved xylose transporter from Zymomonas mobilis enhances sugar transport in Escherichia coli

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Zhang Jingqing

2009-12-01

Full Text Available Abstract Background Xylose is a second most abundant sugar component of lignocellulose besides glucose. Efficient fermentation of xylose is important for the economics of biomass-based biorefineries. However, sugar mixtures are sequentially consumed in xylose co-fermentation with glucose due to carbon catabolite repression (CCR in microorganisms. As xylose transmembrance transport is one of the steps repressed by CCR, it is therefore of interest to develop a transporter that is less sensitive to the glucose inhibition or CCR. Results The glucose facilitator protein Glf transporter from Zymomonas mobilis, also an efficient transporter for xylose, was chosen as the target transporter for engineering to eliminate glucose inhibition on xylose uptake. The evolution of Glf transporter was carried out with a mixture of glucose and xylose in E. coli. Error-prone PCR and random deletion were employed respectively in two rounds of evolution. Aided by a high-throughput screening assay using xylose analog p-nitrophenyl-β-D-xylopyranoside (pNPX in 96-well plates, a best mutant 2-RD5 was obtained that contains several mutations, and a deletion of 134 residues (about 28% of total residues, or three fewer transmembrane sections (TMSs. It showed a 10.8-fold improvement in terms of pNPX transport activity in the presence of glucose. The fermentation performance results showed that this mutant improved xylose consumption by 42% with M9 minimal medium containing 20 g L-1 xylose only, while with the mixture sugar of xylose and glucose, 28% more glucose was consumed, but no obvious co-utilization of xylose was observed. Further glucose fed-batch experiments suggested that the intracellular metabolism of xylose was repressed by glucose. Conclusions Through random mutagenesis and partial deletion coupled with high-throughput screening, a mutant of the Glf transporter (2-RD5 was obtained that relieved the inhibition of xylose transport by glucose. The fermentation

The Golgin GMAP210/TRIP11 anchors IFT20 to the Golgi complex.

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John A Follit

2008-12-01

Full Text Available Eukaryotic cells often use proteins localized to the ciliary membrane to monitor the extracellular environment. The mechanism by which proteins are sorted, specifically to this subdomain of the plasma membrane, is almost completely unknown. Previously, we showed that the IFT20 subunit of the intraflagellar transport particle is localized to the Golgi complex, in addition to the cilium and centrosome, and hypothesized that the Golgi pool of IFT20 plays a role in sorting proteins to the ciliary membrane. Here, we show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP210/Trip11. Mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction, ventricular septal defects of the heart, omphalocele, and lung hypoplasia. Cells lacking GMAP210 have normal Golgi structure, but IFT20 is no longer localized to this organelle. GMAP210 is not absolutely required for ciliary assembly, but cilia on GMAP210 mutant cells are shorter than normal and have reduced amounts of the membrane protein polycystin-2 localized to them. This work suggests that GMAP210 and IFT20 function together at the Golgi in the sorting or transport of proteins destined for the ciliary membrane.

Impaired pH Homeostasis in Arabidopsis Lacking the Vacuolar Dicarboxylate Transporter and Analysis of Carboxylic Acid Transport across the Tonoplast1

Science.gov (United States)

Hurth, Marco Alois; Suh, Su Jeoung; Kretzschmar, Tobias; Geis, Tina; Bregante, Monica; Gambale, Franco; Martinoia, Enrico; Neuhaus, H. Ekkehard

2005-01-01

Arabidopsis (Arabidopsis thaliana) mutants lacking the tonoplastic malate transporter AttDT (A. thaliana tonoplast dicarboxylate transporter) and wild-type plants showed no phenotypic differences when grown under standard conditions. To identify putative metabolic changes in AttDT knock-out plants, we provoked a metabolic scenario connected to an increased consumption of dicarboxylates. Acidification of leaf discs stimulated dicarboxylate consumption and led to extremely low levels of dicarboxylates in mutants. To investigate whether reduced dicarboxylate concentrations in mutant leaf cells and, hence, reduced capacity to produce OH− to overcome acidification might affect metabolism, we measured photosynthetic oxygen evolution under conditions where the cytosol is acidified. AttDT::tDNA protoplasts showed a much stronger inhibition of oxygen evolution at low pH values when compared to wild-type protoplasts. Apparently citrate, which is present in higher amounts in knock-out plants, is not able to replace dicarboxylates to overcome acidification. To raise more information on the cellular level, we performed localization studies of carboxylates. Although the total pool of carboxylates in mutant vacuoles was nearly unaltered, these organelles contained a lower proportion of malate and fumarate and a higher proportion of citrate when compared to wild-type vacuoles. These alterations concur with the observation that radioactively labeled malate and citrate are transported into Arabidopsis vacuoles by different carriers. In addition, wild-type vacuoles and corresponding organelles from AttDT::tDNA mutants exhibited similar malate channel activities. In conclusion, these results show that Arabidopsis vacuoles contain at least two transporters and a channel for dicarboxylates and citrate and that the activity of AttDT is critical for regulation of pH homeostasis. PMID:15728336

Long-lived mitochondrial (Mit) mutants of Caenorhabditis elegans utilize a novel metabolism.

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Butler, Jeffrey A; Ventura, Natascia; Johnson, Thomas E; Rea, Shane L

2010-12-01

The Caenorhabditis elegans mitochondrial (Mit) mutants have disrupted mitochondrial electron transport chain (ETC) functionality, yet, surprisingly, they are long lived. We have previously proposed that Mit mutants supplement their energy needs by exploiting alternate energy production pathways normally used by wild-type animals only when exposed to hypoxic conditions. We have also proposed that longevity in the Mit mutants arises as a property of their new metabolic state. If longevity does arise as a function of metabolic state, we would expect to find a common metabolic signature among these animals. To test these predictions, we established a novel approach monitoring the C. elegans exometabolism as a surrogate marker for internal metabolic events. Using HPLC-ultraviolet-based metabolomics and multivariate analyses, we show that long-lived clk-1(qm30) and isp-1(qm150) Mit mutants have a common metabolic profile that is distinct from that of aerobically cultured wild-type animals and, unexpectedly, wild-type animals cultured under severe oxygen deprivation. Moreover, we show that 2 short-lived mitochondrial ETC mutants, mev-1(kn1) and ucr-2.3(pk732), also share a common metabolic signature that is unique. We show that removal of soluble fumarate reductase unexpectedly increases health span in several genetically defined Mit mutants, identifying at least 1 alternate energy production pathway, malate dismutation, that is operative in these animals. Our study suggests long-lived, genetically specified Mit mutants employ a novel metabolism and that life span may well arise as a function of metabolic state.

Potent inhibition of HIV-1 replication by a Tat mutant.

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Luke W Meredith

Full Text Available Herein we describe a mutant of the two-exon HIV-1 Tat protein, termed Nullbasic, that potently inhibits multiple steps of the HIV-1 replication cycle. Nullbasic was created by replacing the entire arginine-rich basic domain of wild type Tat with glycine/alanine residues. Like similarly mutated one-exon Tat mutants, Nullbasic exhibited transdominant negative effects on Tat-dependent transactivation. However, unlike previously reported mutants, we discovered that Nullbasic also strongly suppressed the expression of unspliced and singly-spliced viral mRNA, an activity likely caused by redistribution and thus functional inhibition of HIV-1 Rev. Furthermore, HIV-1 virion particles produced by cells expressing Nullbasic had severely reduced infectivity, a defect attributable to a reduced ability of the virions to undergo reverse transcription. Combination of these inhibitory effects on transactivation, Rev-dependent mRNA transport and reverse transcription meant that permissive cells constitutively expressing Nullbasic were highly resistant to a spreading infection by HIV-1. Nullbasic and its activities thus provide potential insights into the development of potent antiviral therapeutics that target multiple stages of HIV-1 infection.

Comparative metabolic profiling of mce1 operon mutant vs wild-type Mycobacterium tuberculosis strains.

Science.gov (United States)

Queiroz, Adriano; Medina-Cleghorn, Daniel; Marjanovic, Olivera; Nomura, Daniel K; Riley, Lee W

2015-11-01

Mycobacterium tuberculosis disrupted in a 13-gene operon (mce1) accumulates free mycolic acids (FM) in its cell wall and causes accelerated death in mice. Here, to more comprehensively analyze differences in their cell wall lipid composition, we used an untargeted metabolomics approach to compare the lipid profiles of wild-type and mce1 operon mutant strains. By liquid chromatography-mass spectrometry, we identified >400 distinct lipids significantly altered in the mce1 mutant compared to wild type. These lipids included decreased levels of saccharolipids and glycerophospholipids, and increased levels of alpha-, methoxy- and keto mycolic acids (MA), and hydroxyphthioceranic acid. The mutant showed reduced expression of mmpL8, mmpL10, stf0, pks2 and papA2 genes involved in transport and metabolism of lipids recognized to induce proinflammatory response; these lipids were found to be decreased in the mutant. In contrast, the transcripts of mmpL3, fasI, kasA, kasB, acpM and RV3451 involved in MA transport and metabolism increased; MA inhibits inflammatory response in macrophages. Since the mce1 operon is known to be regulated in intracellular M. tuberculosis, we speculate that the differences we observed in cell wall lipid metabolism and composition may affect host response to M. tuberculosis infection and determine the clinical outcome of such an infection. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Identification of ABC transporters acting in vitamin B12 metabolism in Caenorhabditis elegans.

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McDonald, Megan K; Fritz, Julie-Anne; Jia, Dongxin; Scheuchner, Deborah; Snyder, Floyd F; Stanislaus, Avalyn; Curle, Jared; Li, Liang; Stabler, Sally P; Allen, Robert H; Mains, Paul E; Gravel, Roy A

2017-12-01

Vitamin B 12 (cobalamin, Cbl) is a micronutrient essential to human health. Cbl is not utilized as is but must go through complex subcellular and metabolic processing to generate two cofactor forms: methyl-Cbl for methionine synthase, a cytosolic enzyme; and adenosyl-Cbl for methylmalonyl-CoA mutase, a mitochondrial enzyme. Some 10-12 human genes have been identified responsible for the intracellular conversion of Cbl to cofactor forms, including genes that code for ATP-binding cassette (ABC) transporters acting at the lysosomal and plasma membranes. However, the gene for mitochondrial uptake is not known. We hypothesized that ABC transporters should be candidates for other uptake and efflux functions, including mitochondrial transport, and set out to screen ABC transporter mutants for blocks in Cbl utilization using the nematode roundworm Caenorhabditis elegans. Thirty-seven mutant ABC transporters were screened for the excretion of methylmalonic acid (MMA), which should result from loss of Cbl transport into the mitochondria. One mutant, wht-6, showed elevated MMA excretion and reduced [ 14 C]-propionate incorporation, pointing to a functional block in methylmalonyl-CoA mutase. In contrast, the wht-6 mutant appeared to have a normal cytosolic pathway based on analysis of cystathionine excretion, suggesting that cytosolic methionine synthase was functioning properly. Further, the MMA excretion in wht-6 could be partially reversed by including vitamin B 12 in the assay medium. The human ortholog of wht-6 is a member of the G family of ABC transporters. We propose wht-6 as a candidate for the transport of Cbl into mitochondria and suggest that a member of the corresponding ABCG family of ABC transporters has this role in humans. Our ABC transporter screen also revealed that mrp-1 and mrp-2 mutants excreted lower MMA than wild type, suggesting they were concentrating intracellular Cbl, consistent with the cellular efflux defect proposed for the mammalian MRP1 ABC

Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

International Nuclear Information System (INIS)

Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M.; Wehnert, Manfred; Huebner, Stefan

2009-01-01

Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.

Tritium Suicide Selection Identifies Proteins Involved in the Uptake and Intracellular Transport of Sterols in Saccharomyces cerevisiae▿

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Sullivan, David P.; Georgiev, Alexander; Menon, Anant K.

2009-01-01

Sterol transport between the plasma membrane (PM) and the endoplasmic reticulum (ER) occurs by a nonvesicular mechanism that is poorly understood. To identify proteins required for this process, we isolated Saccharomyces cerevisiae mutants with defects in sterol transport. We used Upc2-1 cells that have the ability to take up sterols under aerobic conditions and exploited the observation that intracellular accumulation of exogenously supplied [3H]cholesterol in the form of [3H]cholesteryl ester requires an intact PM-ER sterol transport pathway. Upc2-1 cells were mutagenized using a transposon library, incubated with [3H]cholesterol, and subjected to tritium suicide selection to isolate mutants with a decreased ability to accumulate [3H]cholesterol. Many of the mutants had defects in the expression and trafficking of Aus1 and Pdr11, PM-localized ABC transporters that are required for sterol uptake. Through characterization of one of the mutants, a new role was uncovered for the transcription factor Mot3 in controlling expression of Aus1 and Pdr11. A number of mutants had transposon insertions in the uncharacterized Ydr051c gene, which we now refer to as DET1 (decreased ergosterol transport). These mutants expressed Aus1 and Pdr11 normally but were severely defective in the ability to accumulate exogenously supplied cholesterol. The transport of newly synthesized sterols from the ER to the PM was also defective in det1Δ cells. These data indicate that the cytoplasmic protein encoded by DET1 is involved in intracellular sterol transport. PMID:19060182

NhaA Na+/H+ antiporter mutants that hardly react to the membrane potential.

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Dudu Alkoby

Full Text Available pH and Na+ homeostasis in all cells requires Na+/H+ antiporters. The crystal structure, obtained at pH 4, of NhaA, the main antiporter of Escherichia coli, has provided general insights into an antiporter mechanism and its unique pH regulation. Here, we describe a general method to select various NhaA mutants from a library of randomly mutagenized NhaA. The selected mutants, A167P and F267C are described in detail. Both mutants are expressed in Escherichia coli EP432 cells at 70-95% of the wild type but grow on selective medium only at neutral pH, A167P on Li+ (0.1 M and F267C on Na+ (0.6 M. Surprising for an electrogenic secondary transporter, and opposed to wild type NhaA, the rates of A167P and F267C are almost indifferent to membrane potential. Detailed kinetic analysis reveals that in both mutants the rate limiting step of the cation exchange cycle is changed from an electrogenic to an electroneutral reaction.

An extra early mutant of pigeonpea

International Nuclear Information System (INIS)

Ravikesavan, R.; Kalaimagal, T.; Rathnaswamy, R.

2001-01-01

The redgram (Cajanus cajan (L.) Huth) variety 'Prabhat DT' was gamma irradiated with 100, 200, 300 and 400 Gy doses. Several mutants have been identified viz., extra early mutants, monostem mutants, obcordifoliate mutants and bi-stigmatic mutants. The extra early mutant was obtained when treated with 100 Gy dose. The mutant was selfed and forwarded from M 2 to M 4 generation. In the M 4 generation the mutant line was raised along with the parental variety. Normal cultural practices were followed and the biometrical observations were recorded. It was observed that for the characters viz., total number of branches per plant, number of pods per plants, seeds per pod, 100 seed weight and seed yield per plant there was no difference between the mutant and parent variety. Whereas, regarding the days to flowering and maturity the mutants were earlier than the parents. The observation was recorded from two hundred plants each. The mutant gives the same yield in 90 days as that of the parent variety in 107 days, which make it an economic mutant

Genetic analysis of amino acid transport in the facultatively heterotrophic cyanobacterium Synechocystis sp. Strain 6803

International Nuclear Information System (INIS)

Labarre, J.; Thuriaux, P.; Chauvat, F.

1987-01-01

The existence of active transport systems (permeases) operating on amino acids in the photoautotrophic cyanobacterium Synechocystis sp. strain 6803 was demonstrated by following the initial rates of uptake with 14 C-labeled amino acids, measuring the intracellular pools of amino acids, and isolating mutants resistant to toxic amino acids. One class of mutants (Pfa1) corresponds to a regulatory defect in the biosynthesis of the aromatic amino acids, but two other classes (Can1 and Aza1) are defective in amino acid transport. The Can1 mutants are defective in the active transport of three basic amino acids (arginine, histidine, and lysine) and in one of two transport systems operating on glutamine. The Aza1 mutants are not affected in the transport of the basic amino acids but have lost the capacity to transport all other amino acids except glutamate. The latter amino acid is probably transported by a third permease which could be identical to the Can1-independent transport operating on glutamine. Thus, genetic evidence suggests that strain 6803 has only a small number of amino acid transport systems with fairly broad specificity and that, with the exception of glutamine, each amino acid is accumulated by only one major transport system. Compared with heterotrophic bacteria such as Escherichia coli, these permeases are rather inefficient in terms of affinity (apparent K/sub m/ ranging from 6 to 60 μM) and of V/sub max/

A temperature-sensitive winter wheat chlorophyll mutant derived from space mutagenesis

International Nuclear Information System (INIS)

Zhao Hongbin; Guo Huijun; Zhao Linshu; Gu Jiayu; Zhao Shirong; Li Junhui; Liu Luxiang

2010-01-01

A temperature-sensitive winter wheat (Triticum aestivum L.) chlorophyll mutant Mt18, induced by spaceflight mutagenesis, was studied on agronomic traits, ultrastructure of chloroplast and photosynthesis characteristics. The leaf color of the mutant Mt18 showed changes from green to albino and back to green during the whole growth period. Plant height, productive tillers, spike length, grains and grain weight per plant, and 1000-grain weight of the mutant were lower than those of the wild type. The ultrastructural observation showed that no significant difference was found between the mutant and the wild type during prior albino stage, however, at the albino stage the number of granum-thylakoids and grana lamellae became fewer or completely disappeared, but the strom-thylakoid was obviously visible. After turning green,the structure of most chloroplasts recovered to normal, but number of chloroplast was still lower than that of the wild type. When exposed to photosynthetic active radiation (PAR) of 110 μmol·m -2 ·s -1 , the non-photochemical quenching (NPQ) of mutant was significantly lower than that of the wild type, and the non-regulated energy dissipation (Y NO ) was significantly higher than that of the wild type, while the change of the maximum photosystem II quantum yield (F v /F m ), potential activity of photosystem II (F v /F o ), photochemical quenching (q P ), effective quantum yield (Y PSI I) and regulated non-photochemical energy dissipation (Y NPQ ) were different at various stages. In addition, the differences of the electron transport rate (ETR), photochemical quenching (q P ), and effective quantum yield (Y PSI I) between mutant and wild type varied under different PAR conditions. It was concluded that with the change of chloroplast ultrastructure, the leaf color and photosynthesis of the wheat mutant Mt18 change correspondingly. The chloroplast ultrastructure was obviously different from that of wild type, and the photosynthetic efficiency

Mutant heterosis in rice

International Nuclear Information System (INIS)

1987-01-01

In the variety TKM6 a high yielding semidwarf mutant has been induced. This TKM6 mutant was used in test crosses with a number of other varieties and mutants to examine the extent of heterosis of dwarfs in rice and to select superior crosses. An excerpt of the published data is given. It appears from the backcross of the mutant with its original variety, that an increase in number of productive tillers occurs in the hybrid, leading to a striking grain yield increase, while the semi-dwarf culm length (the main mutant character) reverts to the normal phenotype. In the cross with IR8 on the other hand, there is only a minimal increase in tiller number but a substantial increase in TGW leading to more than 30% yield increase over the better parent

Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate.

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Sorrenson, Brie; Suetani, Rachel J; Williams, Michael J A; Bickley, Vivienne M; George, Peter M; Jones, Gregory T; McCormick, Sally P A

2013-01-01

Mutations in the ATP-binding cassette transporter A1 (ABCA1) are a major cause of decreased HDL cholesterol (HDL-C), which infers an increased risk of cardiovascular disease (CVD). Many ABCA1 mutants show impaired localization to the plasma membrane. The aim of this study was to investigate whether the chemical chaperone, sodium 4-phenylbutyrate (4-PBA) could improve cellular localization and function of ABCA1 mutants. Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Treatment restored localization to the plasma membrane and increased cholesterol efflux function for the majority of mutants. Treatment with 4-PBA also increased ABCA1 protein expression in all transfected cell lines. In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Our study is the first to investigate the effect of the chemical chaperone, 4-PBA on ABCA1 and shows that it is capable of restoring plasma membrane localization and enhancing the cholesterol efflux function of mutant ABCA1s both in vitro and ex vivo. These results suggest 4-PBA may warrant further investigation as a potential therapy for increasing cholesterol efflux and HDL-C levels.

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

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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

Mutations that alter the transport function of the LamB protein in Escherichia coli.

OpenAIRE

Wandersman, C; Schwartz, M

1982-01-01

Some Escherichia coli K-12 lamB mutants, those producing reduced amounts of LamB protein (one-tenth the wild type amount), grow normally on dextrins but transport maltose when present at a concentration of 1 microM at about one-tenth the normal rate. lamB Dex- mutants were found as derivatives of these strains. These Dex- mutants are considerably impaired in the transport of maltose at low concentrations (below 10 microM), and they have a structurally altered LamB protein which is impaired in...

Intraflagellar Transport (IFT) Role in Ciliary Assembly, Resorption and Signalling

DEFF Research Database (Denmark)

Pedersen, Lotte B; Rosenbaum, Joel L

2008-01-01

Cilia and flagella have attracted tremendous attention in recent years as research demonstrated crucial roles for these organelles in coordinating a number of physiologically and developmentally important signaling pathways, including the platelet-derived growth factor receptor (PDGFR) alpha, Sonic...... hedgehog, polycystin, and Wnt pathways. In addition, the realization that defective assembly or function of cilia can cause a plethora of diseases and developmental defects ("ciliopathies") has increased focus on the mechanisms by which these antenna-like, microtubular structures assemble. Ciliogenesis...... mechanisms and functions of IFT. In addition to a general, up-to-date description of IFT, we discuss mechanisms by which proteins are selectively targeted to the ciliary compartment, with special focus on the ciliary transition zone. Finally, we briefly review the role of IFT in cilia-mediated signaling...

Caenorhabditis elegans ABCRNAi transporters interact genetically with rde-2 and mut-7.

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Sundaram, Prema; Han, Wang; Cohen, Nancy; Echalier, Benjamin; Albin, John; Timmons, Lisa

2008-02-01

RNA interference (RNAi) mechanisms are conserved and consist of an interrelated network of activities that not only respond to exogenous dsRNA, but also perform endogenous functions required in the fine tuning of gene expression and in maintaining genome integrity. Not surprisingly, RNAi functions have widespread influences on cellular function and organismal development. Previously, we observed a reduced capacity to mount an RNAi response in nine Caenorhabditis elegans mutants that are defective in ABC transporter genes (ABC(RNAi) mutants). Here, we report an exhaustive study of mutants, collectively defective in 49 different ABC transporter genes, that allowed for the categorization of one additional transporter into the ABC(RNAi) gene class. Genetic complementation tests reveal functions for ABC(RNAi) transporters in the mut-7/rde-2 branch of the RNAi pathway. These second-site noncomplementation interactions suggest that ABC(RNAi) proteins and MUT-7/RDE-2 function together in parallel pathways and/or as multiprotein complexes. Like mut-7 and rde-2, some ABC(RNAi) mutants display transposon silencing defects. Finally, our analyses reveal a genetic interaction network of ABC(RNAi) gene function with respect to this part of the RNAi pathway. From our results, we speculate that the coordinated activities of ABC(RNAi) transporters, through their effects on endogenous RNAi-related mechanisms, ultimately affect chromosome function and integrity.

Mutant INS-gene induced diabetes of youth: proinsulin cysteine residues impose dominant-negative inhibition on wild-type proinsulin transport.

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Ming Liu

2010-10-01

Full Text Available Recently, a syndrome of Mutant INS-gene-induced Diabetes of Youth (MIDY, derived from one of 26 distinct mutations has been identified as a cause of insulin-deficient diabetes, resulting from expression of a misfolded mutant proinsulin protein in the endoplasmic reticulum (ER of insulin-producing pancreatic beta cells. Genetic deletion of one, two, or even three alleles encoding insulin in mice does not necessarily lead to diabetes. Yet MIDY patients are INS-gene heterozygotes; inheritance of even one MIDY allele, causes diabetes. Although a favored explanation for the onset of diabetes is that insurmountable ER stress and ER stress response from the mutant proinsulin causes a net loss of beta cells, in this report we present three surprising and interlinked discoveries. First, in the presence of MIDY mutants, an increased fraction of wild-type proinsulin becomes recruited into nonnative disulfide-linked protein complexes. Second, regardless of whether MIDY mutations result in the loss, or creation, of an extra unpaired cysteine within proinsulin, Cys residues in the mutant protein are nevertheless essential in causing intracellular entrapment of co-expressed wild-type proinsulin, blocking insulin production. Third, while each of the MIDY mutants induces ER stress and ER stress response; ER stress and ER stress response alone appear insufficient to account for blockade of wild-type proinsulin. While there is general agreement that ultimately, as diabetes progresses, a significant loss of beta cell mass occurs, the early events described herein precede cell death and loss of beta cell mass. We conclude that the molecular pathogenesis of MIDY is initiated by perturbation of the disulfide-coupled folding pathway of wild-type proinsulin.

Feedback inhibition of ammonium (methylammonium) ion transport in Escherichia coli by glutamine and glutamine analogs

International Nuclear Information System (INIS)

Jayakumar, A.; Hong, J.S.; Barnes, E.M. Jr.

1987-01-01

When cultured with glutamate or glutamine as the nitrogen source, Escherichia coli expresses a specific ammonium (methylammonium) transport system. Over 95% of the methylammonium transport activity in washed cells was blocked by incubation with 100 μM L-glutamine in the presence of chloramphenicol (100 μg/ml). The inhibition of transport by L-glutamine was noncompetitive with respect to the [ 14 C]methylammonium substrate. D-Glutamine had no significant effect. The glutamine analogs γ-L-glutamyl hydroxamate and γ-L-glutamyl hydrazide were also noncompetitive inhibitors of methylammonium transport, suggesting that glutamine metabolism is not required. The role of the intracellular glutamine pool in the regulation of ammonium transport was investigated by using mutants carrying defects in the operon of glnP, the gene for the glutamine transporter. The glnP mutants had normal rates of methylammonium transport but were refractory to glutamine inhibition. Glycylglycine, a noncompetitive inhibitor of methylammonium uptake in wild-type cells, was equipotent in blocking transport in glnP mutants. Although ammonium transport is also subject to repression by growth of E. coli in the presence of ammonia, this phenomenon is unrelated to glutamine inhibition

Sulfhydryl oxidation of mutants with cysteine in place of acidic residues in the lactose permease.

Science.gov (United States)

Voss, J; Sun, J; Venkatesan, P; Kaback, H R

1998-06-02

To examine further the role of charge-pair interactions in the structure and function of lactose permease, Asp237 (helix VII), Asp240 (helix VII), Glu126 (cytoplasmic loop IV/V), Glu269 (helix VIII), and Glu325 (helix X) were replaced individually with Cys in a functional mutant devoid of Cys residues. Each mutant was then oxidized with H2O2 in order to generate a sulfinic and/or sulfonic acid at these positions. Due to the isosteric relationship between aspartate and sulfinate, in particular, and the lower pKa of the sulfinic and sulfonic acid side chains, oxidized derivatives of Cys are useful probes for examining the role of carboxylates. Asp237-->Cys or Asp240-->Cys permease is inactive, as shown previously, but H2O2 oxidation restores activity to an extent similar to that observed when a negative charge is reintroduced by other means. Glu126-->Cys, Glu269-->Cys, or Glu325-->Cys permease is inactive, but oxidation does not restore active lactose transport. The data are consistent with previous observations indicating that Asp237 and Asp240 are not critical for active lactose transport, while Glu126, Glu269, and Glu325 are irreplaceable. Although Glu269-->Cys permease does not transport lactose, the oxidized mutant exhibits significant transport of beta,D-galactosylpyranosyl 1-thio-beta,D-galactopyranoside, a property observed with Glu269-->Asp permease. The observation supports the idea that an acidic residue at position 269 is important for substrate recognition. Finally, oxidized Glu325-->Cys permease catalyzes equilibrium exchange with an apparent pKa of about 6.5, more than a pH unit lower than that observed with Glu325-->Asp permease, thereby providing strong confirmatory evidence that a negative charge at position 325 determines the rate of translocation of the ternary complex between the permease, substrate, and H+.

Indy mutants: live long and prosper

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Stewart eFrankel

2012-02-01

Full Text Available Indy encodes the fly homologue of a mammalian transporter of di and tricarboxylatecomponents of the Krebs cycle. Reduced expression of fly Indy or two of the C. elegansIndy homologs leads to an increase in life span. Fly and worm tissues that play key roles inintermediary metabolism are also the places where Indy genes are expressed. One of themouse homologs of Indy (mIndy is mainly expressed in the liver. It has been hypothesizedthat decreased INDY activity creates a state similar to caloric restriction (CR. Thishypothesis is supported by the physiological similarities between Indy mutant flies on highcalorie food and control flies on CR, such as increased physical activity and decreases inweight, egg production, triglyceride levels, starvation resistance, and insulin signaling. Inaddition, Indy mutant flies undergo changes in mitochondrial biogenesis also observed inCR animals. Recent findings with mIndy knockout mice support and extend the findingsfrom flies. mIndy-/- mice display an increase in hepatic mitochondrial biogenesis, lipidoxidation and decreased hepatic lipogenesis. When mIndy-/- mice are fed high calorie foodthey are protected from adiposity and insulin resistance. These findings point to INDY as apotential drug target for the treatment of metabolic syndrome, type 2 diabetes and obesity.

DCL2- and RDR6-dependent transitive silencing of SMXL4 and SMXL5 in Arabidopsis dcl4 mutants causes defective phloem transport and carbohydrate over-accumulation.

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Wu, Yu-Yi; Hou, Bo-Han; Lee, Wen-Chi; Lu, Shin-Hua; Yang, Chen-Jui; Vaucheret, Hervé; Chen, Ho-Ming

2017-06-01

DICER-LIKE (DCL) enzymes process double-stranded RNA into small RNAs that act as regulators of gene expression. Arabidopsis DCL4 and DCL2 each allow the post-transcriptional gene silencing (PTGS) of viruses and transgenes, but primary PTGS-prone DCL4 outcompetes transitive PTGS-prone DCL2 in wild-type plants. This hierarchy likely prevents DCL2 having any detrimental effects on endogenous genes. Indeed, dcl4 mutants exhibit developmental defects and increased sensitivity to genotoxic stress. In this study, the mechanism underlying dcl4 defects was investigated using genetic, biochemical and high-throughput sequencing approaches. We show that the purple phenotype of dcl4 leaves correlates with carbohydrate over-accumulation and defective phloem transport, and depends on the activity of SUPPRESSOR OF GENE SILENCING 3, RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and DCL2. This phenotype correlates with the downregulation of two genes expressed in the apex and the vasculature, SMAX1-LIKE 4 (SMXL4) and SMXL5, and the accumulation of DCL2- and RDR6-dependent small interfering RNAs derived from these two genes. Supporting a causal effect, smxl4 smxl5 double mutants exhibit leaf pigmentation, enhanced starch accumulation and defective phloem transport, similar to dcl4 plants. Overall, this study elucidates the detrimental action of DCL2 when DCL4 is absent, and indicates that DCL4 outcompeting DCL2 in wild-type plants is crucial to prevent the degradation of endogenous transcripts by DCL2- and RDR6-dependent transitive PTGS. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport.

Science.gov (United States)

Lin, Shan-Hua; Kuo, Hui-Fen; Canivenc, Geneviève; Lin, Choun-Sea; Lepetit, Marc; Hsu, Po-Kai; Tillard, Pascal; Lin, Huey-Ling; Wang, Ya-Yun; Tsai, Chyn-Bey; Gojon, Alain; Tsay, Yi-Fang

2008-09-01

Little is known about the molecular and regulatory mechanisms of long-distance nitrate transport in higher plants. NRT1.5 is one of the 53 Arabidopsis thaliana nitrate transporter NRT1 (Peptide Transporter PTR) genes, of which two members, NRT1.1 (CHL1 for Chlorate resistant 1) and NRT1.2, have been shown to be involved in nitrate uptake. Functional analysis of cRNA-injected Xenopus laevis oocytes showed that NRT1.5 is a low-affinity, pH-dependent bidirectional nitrate transporter. Subcellular localization in plant protoplasts and in planta promoter-beta-glucuronidase analysis, as well as in situ hybridization, showed that NRT1.5 is located in the plasma membrane and is expressed in root pericycle cells close to the xylem. Knockdown or knockout mutations of NRT1.5 reduced the amount of nitrate transported from the root to the shoot, suggesting that NRT1.5 participates in root xylem loading of nitrate. However, root-to-shoot nitrate transport was not completely eliminated in the NRT1.5 knockout mutant, and reduction of NRT1.5 in the nrt1.1 background did not affect root-to-shoot nitrate transport. These data suggest that, in addition to that involving NRT1.5, another mechanism is responsible for xylem loading of nitrate. Further analyses of the nrt1.5 mutants revealed a regulatory loop between nitrate and potassium at the xylem transport step.

Gravity-dependent differentiation and root coils in Arabidopsis thaliana wild type and phospholipase-A-I knockdown mutant grown on the International Space Station.

Science.gov (United States)

Scherer, G F E; Pietrzyk, P

2014-01-01

Arabidopsis roots on 45° tilted agar in 1-g grow in wave-like figures. In addition to waves, formation of root coils is observed in several mutants compromised in gravitropism and/or auxin transport. The knockdown mutant ppla-I-1 of patatin-related phospholipase-A-I is delayed in root gravitropism and forms increased numbers of root coils. Three known factors contribute to waving: circumnutation, gravisensing and negative thigmotropism. In microgravity, deprivation of wild type (WT) and mutant roots of gravisensing and thigmotropism and circumnutation (known to slow down in microgravity, and could potentially lead to fewer waves or increased coiling in both WT and mutant). To resolve this, mutant ppla-I-1 and WT were grown in the BIOLAB facility in the International Space Station. In 1-g, roots of both types only showed waving. In the first experiment in microgravity, the mutant after 9 days formed far more coils than in 1-g but the WT also formed several coils. After 24 days in microgravity, in both types the coils were numerous with slightly more in the mutant. In the second experiment, after 9 days in microgravity only the mutant formed coils and the WT grew arcuated roots. Cell file rotation (CFR) on the mutant root surface in microgravity decreased in comparison to WT, and thus was not important for coiling. Several additional developmental responses (hypocotyl elongation, lateral root formation, cotyledon expansion) were found to be gravity-influenced. We tentatively discuss these in the context of disturbances in auxin transport, which are known to decrease through lack of gravity. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

The yeast complex I equivalent NADH dehydrogenase rescues pink1 mutants.

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Sven Vilain

2012-01-01

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

Gamma-radiation Mutagenesis in Genetically Unstable Barley Mutants. Pt. 2. Comparison of Various Mutants

International Nuclear Information System (INIS)

Balchiuniene, L.

1995-01-01

Spontaneous and gamma-induced mutability was compared in two groups of genetically unstable barley ear structure mutants - tweaky spike (tw) and branched ear (be). Instability in different loci causes different levels of spontaneous and gamma-induced mutability. A high spontaneous level of chlorophyll mutations is peculiar to be-ust mutants. It is suggested that the high level of induced chlorophyll mutations in allelic tw mutants is a result of better surviving of chlorophyll mutation carriers in the genotypical-physiological environment created by mutant tw alleles. (author). 6 refs., 2 tabs

Promising rice mutants

International Nuclear Information System (INIS)

Hakim, L.; Azam, M.A.; Miah, A.J.; Mansur, M.A.; Akanda, H.R.

1988-01-01

Two induced mutants namely, Mut NS 1 (tall) and Mut NS 5 (semi-dwarf) derived from rice variety Nizersail were evaluated for various agronomic characters at four locations in Bangladesh. Both the mutants matured about three weeks earlier and yielded significantly higher than the parent variety Nizersail. (author). 3 tabs., 9 refs

Mutational analysis of the respiratory nitrate transporter NarK2 of Mycobacterium tuberculosis.

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Michelle M Giffin

Full Text Available Mycobacterium tuberculosis induces nitrate reductase activity in response to decreasing oxygen levels. This is due to regulation of both the transcription and the activity of the nitrate transporter NarK2. A model of NarK2 structure is proposed containing 12 membrane spanning regions consistent with other members of the major facilitator superfamily. The role of the proton gradient was determined by exposing M. tuberculosis to uncouplers. Nitrite production decreased indicating that the importation of nitrate involved an H(+/nitrate symporter. The addition of nitrite before nitrate had no effect, suggesting no role for a nitrate/nitrite antiporter. In addition the NarK2 knockout mutant showed no defect in nitrite export. NarK2 is proposed to be a Type I H(+/nitrate symporter. Site directed mutagenesis was performed changing 23 amino acids of NarK2. This allowed the identification of important regions and amino acids of this transporter. Five of these mutants were inactive for nitrate transport, seven produced reduced activity and eleven mutants retained wild type activity. NarK2 is inactivated in the presence of oxygen by an unknown mechanism. However none of the mutants, including those with mutated cysteines, were altered in their response to oxygen levels. The assimilatory nitrate transporter NasA of Bacillus subtilis was expressed in the M. tuberculosis NarK2 mutant. It remained active during aerobic incubation showing that the point of oxygen control is NarK2.

Multidrug ATP-binding cassette transporters are essential for hepatic development of Plasmodium sporozoites.

Science.gov (United States)

Rijpma, Sanna R; van der Velden, Maarten; González-Pons, Maria; Annoura, Takeshi; van Schaijk, Ben C L; van Gemert, Geert-Jan; van den Heuvel, Jeroen J M W; Ramesar, Jai; Chevalley-Maurel, Severine; Ploemen, Ivo H; Khan, Shahid M; Franetich, Jean-Francois; Mazier, Dominique; de Wilt, Johannes H W; Serrano, Adelfa E; Russel, Frans G M; Janse, Chris J; Sauerwein, Robert W; Koenderink, Jan B; Franke-Fayard, Blandine M

2016-03-01

Multidrug resistance-associated proteins (MRPs) belong to the C-family of ATP-binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown. To elucidate the role of MRP throughout the parasite life cycle, Plasmodium berghei and Plasmodium falciparum mutants lacking MRP expression were generated. P. berghei mutants lacking expression of the single MRP as well as P. falciparum mutants lacking MRP1, MRP2 or both proteins have similar blood stage growth kinetics and drug-sensitivity profiles as wild type parasites. We show that MRP1-deficient parasites readily invade primary human hepatocytes and develop into mature liver stages. In contrast, both P. falciparum MRP2-deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during Plasmodium liver stage development. © 2015 John Wiley & Sons Ltd.

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

Science.gov (United States)

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.

Gamma ray induced mutants in Coleus

Energy Technology Data Exchange (ETDEWEB)

Vasudevan, K; Jos, J S [Central Tuber Crops Research Institute, Trivandrum, Kerala (India)

1988-07-01

The germplasm collection of Chinese potato (Coleus parviflorus Benth) contains almost no variation for yield contributing traits. The crop does not produce seeds. Treatment of underground tubers with 1 kR, 2 kR, 3 kR and 4 kR gamma rays resulted in 50 morphologically different mutants which are maintained as mutant clones. In the M{sub 1}V{sub 1} generation, suspected mutant sprouts, were carefully removed and grown separately. The most interesting mutant types are the following: (i) erect mutant with spoon shaped light green leaves, 30 cm long inflorescences against 20 cm in the control, cylindrical tubers measuring ca. 7.0 cm long and 3 cm girth against 4 cm and 2.5 cm in the control (ii) early mutants 1 and 2, one having less leaf serration, the other having light green small leaves and dwarf type (iii) fleshy leaf mutant, dark green, thick and smooth leaves. Control plants spread almost in 1 m{sup 2} area and bear tubers from the nodes of branches. In the early mutants tuber formation is mainly restricted to the base of the plant, which makes harvest easier. The crop usually matures within 150 - 160 days, the early mutants are ready for harvest 100 days after planting. As the mutants are less spreading, the yield could be increased by closer spacing.

Gamma ray induced mutants in Coleus

International Nuclear Information System (INIS)

Vasudevan, K.; Jos, J.S.

1988-01-01

The germplasm collection of Chinese potato (Coleus parviflorus Benth) contains almost no variation for yield contributing traits. The crop does not produce seeds. Treatment of underground tubers with 1 kR, 2 kR, 3 kR and 4 kR gamma rays resulted in 50 morphologically different mutants which are maintained as mutant clones. In the M 1 V 1 generation, suspected mutant sprouts, were carefully removed and grown separately. The most interesting mutant types are the following: (i) erect mutant with spoon shaped light green leaves, 30 cm long inflorescences against 20 cm in the control, cylindrical tubers measuring ca. 7.0 cm long and 3 cm girth against 4 cm and 2.5 cm in the control (ii) early mutants 1 and 2, one having less leaf serration, the other having light green small leaves and dwarf type (iii) fleshy leaf mutant, dark green, thick and smooth leaves. Control plants spread almost in 1 m 2 area and bear tubers from the nodes of branches. In the early mutants tuber formation is mainly restricted to the base of the plant, which makes harvest easier. The crop usually matures within 150 - 160 days, the early mutants are ready for harvest 100 days after planting. As the mutants are less spreading, the yield could be increased by closer spacing

Membrane composition and physiological activity of plastids from an oenothera plastome mutator-induced chloroplast mutant.

Science.gov (United States)

Johnson, E M; Sears, B B

1990-01-01

Plastids were isolated from a plastome mutator-induced mutant (pm7) of Oenothera hookeri and were analyzed for various physiological and biochemical attributes. No photosynthetic electron transport activity was detected in the mutant plastids. This is consistent with previous ultrastructural analysis showing the absence of thylakoid membranes in the pm7 plastids and with the observation of aberrant processing and accumulation of chloroplast proteins in the mutant. In comparison to wild type, the mutant tissue lacks chlorophyll, and has significant differences in levels of four fatty acids. The analyses did not reveal any differences in carotenoid levels nor in the synthesis of several chloroplast lipids. The consequences of the altered composition of the chloroplast membrane are discussed in terms of their relation to the aberrant protein processing of the pm7 plastids. The pigment, fatty acid, and lipid measurements were also performed on two distinct nuclear genotypes (A/A and A/C) which differ in their compatibility with the plastid genome (type I) contained in these lines. In these cases, only chlorophyll concentrations differed significantly.

Position of the third Na+ site in the aspartate transporter GltPh and the human glutamate transporter, EAAT1.

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Turgut Bastug

Full Text Available Glutamate transport via the human excitatory amino acid transporters is coupled to the co-transport of three Na(+ ions, one H(+ and the counter-transport of one K(+ ion. Transport by an archaeal homologue of the human glutamate transporters, Glt(Ph, whose three dimensional structure is known is also coupled to three Na(+ ions but only two Na(+ ion binding sites have been observed in the crystal structure of Glt(Ph. In order to fully utilize the Glt(Ph structure in functional studies of the human glutamate transporters, it is essential to understand the transport mechanism of Glt(Ph and accurately determine the number and location of Na(+ ions coupled to transport. Several sites have been proposed for the binding of a third Na(+ ion from electrostatic calculations and molecular dynamics simulations. In this study, we have performed detailed free energy simulations for Glt(Ph and reveal a new site for the third Na(+ ion involving the side chains of Threonine 92, Serine 93, Asparagine 310, Aspartate 312, and the backbone of Tyrosine 89. We have also studied the transport properties of alanine mutants of the coordinating residues Threonine 92 and Serine 93 in Glt(Ph, and the corresponding residues in a human glutamate transporter, EAAT1. The mutant transporters have reduced affinity for Na(+ compared to their wild type counterparts. These results confirm that Threonine 92 and Serine 93 are involved in the coordination of the third Na(+ ion in Glt(Ph and EAAT1.

The Swedish mutant barley collection

Energy Technology Data Exchange (ETDEWEB)

NONE

1989-07-01

Full text: The Swedish mutation research programme in barley began about 50 years ago and has mainly been carried out at Svaloev in co-operation with the institute of Genetics at the University of Lund. The collection has been produced from different Swedish high-yielding spring barley varieties, using the following mutagens: X-rays, neutrons, several organic chemical compounds such as ethyleneimine, several sulfonate derivatives and the inorganic chemical mutagen sodium azide. Nearly 10,000 barley mutants are stored in the Nordic Gene Bank and documented in databases developed by Udda Lundquist, Svaloev AB. The collection consists of the following nine categories with 94 different types of mutants: 1. Mutants with changes in the spike and spikelets; 2. Changes in culm length and culm composition; 3. Changes in growth types; 4. Physiological mutants; 5. Changes in awns; 6. Changes in seed size and shape; 7. Changes in leaf blades; 8. Changes in anthocyanin and colour; 9. Resistance to barley powdery mildew. Barley is one of the most thoroughly investigated crops in terms of induction of mutations and mutation genetics. So far, about half of the mutants stored at the Nordic Gene Bank, have been analysed genetically; They constitute, however, only a minority of the 94 different mutant types. The genetic analyses have given valuable insights into the mutation process but also into the genetic architecture of various characters. A number of mutants of two-row barley have been registered and commercially released. One of the earliest released, Mari, an early maturing, daylength neutral, straw stiff mutant, is still grown in Iceland. The Swedish mutation material has been used in Sweden, but also in other countries, such as Denmark, Germany, and USA, for various studies providing a better understanding of the barley genome. The collection will be immensely valuable for future molecular genetical analyses of clone mutant genes. (author)

The Swedish mutant barley collection

International Nuclear Information System (INIS)

1989-01-01

Full text: The Swedish mutation research programme in barley began about 50 years ago and has mainly been carried out at Svaloev in co-operation with the institute of Genetics at the University of Lund. The collection has been produced from different Swedish high-yielding spring barley varieties, using the following mutagens: X-rays, neutrons, several organic chemical compounds such as ethyleneimine, several sulfonate derivatives and the inorganic chemical mutagen sodium azide. Nearly 10,000 barley mutants are stored in the Nordic Gene Bank and documented in databases developed by Udda Lundquist, Svaloev AB. The collection consists of the following nine categories with 94 different types of mutants: 1. Mutants with changes in the spike and spikelets; 2. Changes in culm length and culm composition; 3. Changes in growth types; 4. Physiological mutants; 5. Changes in awns; 6. Changes in seed size and shape; 7. Changes in leaf blades; 8. Changes in anthocyanin and colour; 9. Resistance to barley powdery mildew. Barley is one of the most thoroughly investigated crops in terms of induction of mutations and mutation genetics. So far, about half of the mutants stored at the Nordic Gene Bank, have been analysed genetically; They constitute, however, only a minority of the 94 different mutant types. The genetic analyses have given valuable insights into the mutation process but also into the genetic architecture of various characters. A number of mutants of two-row barley have been registered and commercially released. One of the earliest released, Mari, an early maturing, daylength neutral, straw stiff mutant, is still grown in Iceland. The Swedish mutation material has been used in Sweden, but also in other countries, such as Denmark, Germany, and USA, for various studies providing a better understanding of the barley genome. The collection will be immensely valuable for future molecular genetical analyses of clone mutant genes. (author)

Mutants of alfalfa mosaic virus

International Nuclear Information System (INIS)

Roosien, J.

1983-01-01

In this thesis the isolation and characterization of a number of mutants of alfalfa mosaic virus, a plant virus with a coat protein dependent genome, is described. Thermo-sensitive (ts) mutants were selected since, at least theoretically, ts mutations can be present in all virus coded functions. It was found that a high percentage of spontaneous mutants, isolated because of their aberrant symptoms, were ts. The majority of these isolates could grow at the non-permissive temperature in the presence of a single wild type (wt) component. To increase the mutation rate virus preparations were treated with several mutagens. After nitrous acid treatment or irradiation with ultraviolet light, an increase in the level of mutations was observed. UV irradiation was preferred since it did not require large amounts of purified viral components. During the preliminary characterization of potential ts mutants the author also obtained one structural and several symptom mutants which were analysed further (chapter 7, 8 and 9). The properties of the ts mutants are described in chapter 3-7. (Auth.)

Brucella abortus ure2 region contains an acid-activated urea transporter and a nickel transport system

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García-Lobo Juan M

2010-04-01

Full Text Available Abstract Background Urease is a virulence factor that plays a role in the resistance of Brucella to low pH conditions, both in vivo and in vitro. Brucella contains two separate urease gene clusters, ure1 and ure2. Although only ure1 codes for an active urease, ure2 is also transcribed, but its contribution to Brucella biology is unknown. Results Re-examination of the ure2 locus showed that the operon includes five genes downstream of ureABCEFGDT that are orthologs to a nikKMLQO cluster encoding an ECF-type transport system for nickel. ureT and nikO mutants were constructed and analyzed for urease activity and acid resistance. A non-polar ureT mutant was unaffected in urease activity at neutral pH but showed a significantly decreased activity at acidic pH. It also showed a decreased survival rate to pH 2 at low concentration of urea when compared to the wild type. The nikO mutant had decreased urease activity and acid resistance at all urea concentrations tested, and this phenotype could be reverted by the addition of nickel to the growth medium. Conclusions Based on these results, we concluded that the operon ure2 codes for an acid-activated urea transporter and a nickel transporter necessary for the maximal activity of the urease whose structural subunits are encoded exclusively by the genes in the ure1 operon.

myo-Inositol-1-phosphate synthase is required for polar auxin transport and organ development

KAUST Repository

Chen, Hao; Xiong, Liming

2010-01-01

, cotyledon venation patterning, root growth, and root cap development. The mutant roots are also agravitropic and have reduced basipetal auxin transport. mips1 mutants have significantly reduced levels of major phosphatidylinositols and exhibit much slower

Localization of a guanylyl cyclase to chemosensory cilia requires the novel ciliary MYND domain protein DAF-25.

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Victor L Jensen

2010-11-01

Full Text Available In harsh conditions, Caenorhabditis elegans arrests development to enter a non-aging, resistant diapause state called the dauer larva. Olfactory sensation modulates the TGF-β and insulin signaling pathways to control this developmental decision. Four mutant alleles of daf-25 (abnormal DAuer Formation were isolated from screens for mutants exhibiting constitutive dauer formation and found to be defective in olfaction. The daf-25 dauer phenotype is suppressed by daf-10/IFT122 mutations (which disrupt ciliogenesis, but not by daf-6/PTCHD3 mutations (which prevent environmental exposure of sensory cilia, implying that DAF-25 functions in the cilia themselves. daf-25 encodes the C. elegans ortholog of mammalian Ankmy2, a MYND domain protein of unknown function. Disruption of DAF-25, which localizes to sensory cilia, produces no apparent cilia structure anomalies, as determined by light and electron microscopy. Hinting at its potential function, the dauer phenotype, epistatic order, and expression profile of daf-25 are similar to daf-11, which encodes a cilium-localized guanylyl cyclase. Indeed, we demonstrate that DAF-25 is required for proper DAF-11 ciliary localization. Furthermore, the functional interaction is evolutionarily conserved, as mouse Ankmy2 interacts with guanylyl cyclase GC1 from ciliary photoreceptors. The interaction may be specific because daf-25 mutants have normally-localized OSM-9/TRPV4, TAX-4/CNGA1, CHE-2/IFT80, CHE-11/IFT140, CHE-13/IFT57, BBS-8, OSM-5/IFT88, and XBX-1/D2LIC in the cilia. Intraflagellar transport (IFT (required to build cilia is not defective in daf-25 mutants, although the ciliary localization of DAF-25 itself is influenced in che-11 mutants, which are defective in retrograde IFT. In summary, we have discovered a novel ciliary protein that plays an important role in cGMP signaling by localizing a guanylyl cyclase to the sensory organelle.

Metal ion transporters and homeostasis.

OpenAIRE

Nelson, N

1999-01-01

Transition metals are essential for many metabolic processes and their homeostasis is crucial for life. Aberrations in the cellular metal ion concentrations may lead to cell death and severe diseases. Metal ion transporters play a major role in maintaining the correct concentrations of the various metal ions in the different cellular compartments. Recent studies of yeast mutants revealed key elements in metal ion homeostasis, including novel transport systems. Several of the proteins discover...

Aerobic dive limits of seals with mutant myoglobin using combined thermochemical and physiological data

DEFF Research Database (Denmark)

Dasmeh, Pouria; Davis, Randall W.; Kepp, Kasper Planeta

2013-01-01

This paper presents an integrated model of convective O2-transport, aerobic dive limits (ADL), and thermochemical data for oxygen binding to mutant myoglobin (Mb), used to quantify the impact of mutations in Mb on the dive limits of Weddell seals (Leptonychotes weddellii). We find that wild-type ...... that such conditions are mostly selected upon in seals. The model is capable of roughly quantifying the physiological impact of single-protein mutations and thus bridges an important gap between animal physiology and molecular (protein) evolution.......This paper presents an integrated model of convective O2-transport, aerobic dive limits (ADL), and thermochemical data for oxygen binding to mutant myoglobin (Mb), used to quantify the impact of mutations in Mb on the dive limits of Weddell seals (Leptonychotes weddellii). We find that wild-type Mb...... traits are only superior under specific behavioral and physiological conditions that critically prolong the ADL, action radius, and fitness of the seals. As an extreme example, the mutations in the conserved His-64 reduce ADL up to 14±2 min for routine aerobic dives, whereas many other mutations...

Genetic fingerprinting of mutant rose cultivars

Energy Technology Data Exchange (ETDEWEB)

Kumar, S; Prasad, K V; Singh, K P; Singh, A.P. [Division of Floriculture and Landscaping, Indian Agricultural Research Institute, Pusa, New Delhi (India)], E-mail: kvprasad66@gmail.com

2008-07-01

Six rose mutants evolved at the Indian Agricultural Research Institute, New Delhi from four parent cultivars were characterized based on RAPD markers. Contrary to the earlier findings our effort has conclusively proven that the RAPD markers are indeed robust tools to discern the mutants from their parents. Among 40 primers screened, 7 primers produced inconsistent banding pattern. The number of polymorphic bands varied between 4 (OPA 14) and 10 (OPA1) with an average of 6.5 bands per primer. The percentage polymorphism ranged from 62.5 (OPM 9) to 100 percent (OPA 1). Most of the primers produced monomorphic bands between parent and mutant rose cultivars. When primer OPA 2 was used a specific band of 2.5 kb was noticed in mutant cv. Pusa Urmil and cv. Pusa Abhishek but was absent in parent cv. Jantar Mantar. A polymorphic band of 750 bp was noticed in the parent Kiss of Fire and helped in differentiating the parent from its mutant when amplified with OPK 3. Primer OPS 16 produced discriminatory band of 800 bp in mutant cv. Pink Sport of Montezuma while it was absent in its parent cv. Montezuma. Another specific band of 650 bp was present in parent cv. Montezuma and absent in its mutant cv. Pink Sport of Montezuma signifying the uniqueness of the mutant. Primer OPM 5 brought out distinct polymorphism among the parent Jantar Mantar and its three mutants with absence of a specific band of 1.5 kb in the parent. The four parents and 6 mutants were divided into four distinct groups in the Dendogram constructed by UPGMA method. The most genetically similar cultivar among the 10 cultivars analyzed are Montezuma and its pink sport of Montezuma whereas Abhisarika a mutant of cv. Kiss of Fire was distinctly different and formed a separate cluster. (author)

From one body mutant to one cell mutant. A progress of radiation breeding in crops

International Nuclear Information System (INIS)

Nagatomi, Shigeki

1996-01-01

An effective method was established to obtain non-chimeral mutants with wide spectrum of flower colors, regenerated from floral organs on which mutated sectors were come out on chronic irradiated plants. By this way, six mutant varieties of flower colors have been selected from one pink flower of chrysanthemum, and cultivated for cut-flower production. By the same method, 3 mutant varieties with small and spray type flowers were selected in Eustoma. Mutant varieties such as a rust disease resistant in sugarcane, 6 dwarfs in Cytisus and pure-white mushroom in velvet shank have been selected successively for short period. (J.P.N.)

Endoplasmic Reticulum-Targeted Subunit Toxins Provide a New Approach to Rescue Misfolded Mutant Proteins and Revert Cell Models of Genetic Diseases

OpenAIRE

Adnan, Humaira; Zhang, Zhenbo; Park, Hyun-Joo; Tailor, Chetankumar; Che, Clare; Kamani, Mustafa; Spitalny, George; Binnington, Beth; Lingwood, Clifford

2016-01-01

Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER) 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD), to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant) subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subu...

Identification of a transporter Slr0982 involved in ethanol tolerance in cyanobacterium Synechocystis sp. PCC 6803

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Yanan eZhang

2015-05-01

Full Text Available Cyanobacteria have been engineered to produce ethanol through recent synthetic biology efforts. However, one major challenge to the cyanobacterial systems for high-efficiency ethanol production is their low tolerance to the ethanol toxicity. With a major goal to identify novel transporters involved in ethanol tolerance, we constructed gene knockout mutants for 58 transporter-encoding genes of Synechocystis sp. PCC 6803 and screened their tolerance change under ethanol stress. The efforts allowed discovery of a mutant of slr0982 gene encoding an ATP-binding cassette transporter which grew poorly in BG11 medium supplemented with 1.5% (v/v ethanol when compared with the wild type, and the growth loss could be recovered by complementing slr0982 in the ∆slr0982 mutant, suggesting that slr0982 is involved in ethanol tolerance in Synechocystis. To decipher the tolerance mechanism involved, a comparative metabolomic and network-based analysis of the wild type and the ethanol-sensitive ∆slr0982 mutant was performed. The analysis allowed the identification of four metabolic modules related to slr0982 deletion in the ∆slr0982 mutant, among which metabolites like sucrose and L-pyroglutamic acid which might be involved in ethanol tolerance, were found important for slr0982 deletion in the ∆slr0982 mutant. This study reports on the first transporter related to ethanol tolerance in Synechocystis, which could be a useful target for further tolerance engineering. In addition, metabolomic and network analysis provides important findings for better understanding of the tolerance mechanism to ethanol stress in Synechocystis.

Identification, cloning and characterization of sis7 and sis10 sugar-insensitive mutants of Arabidopsis

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Biddle Kelly D

2008-10-01

Full Text Available Abstract Background The levels of soluble sugars, such as glucose and sucrose, help regulate many plant metabolic, physiological and developmental processes. Genetic screens are helping identify some of the loci involved in plant sugar response and reveal extensive cross-talk between sugar and phytohormone response pathways. Results A forward genetic screen was performed to identify mutants with increased resistance to the inhibitory effects of high levels of exogenous sugars on early Arabidopsis seedling development. The positional cloning and characterization of two of these sugar insensitive (sis mutants, both of which are also involved in abscisic acid (ABA biosynthesis or response, are reported. Plants carrying mutations in SIS7/NCED3/STO1 or SIS10/ABI3 are resistant to the inhibitory effects of high levels of exogenous Glc and Suc. Quantitative RT-PCR analyses indicate transcriptional upregulation of ABA biosynthesis genes by high concentrations of Glc in wild-type germinating seeds. Gene expression profiling revealed that a significant number of genes that are expressed at lower levels in germinating sis7-1/nced3-4/sto1-4 seeds than in wild-type seeds are implicated in auxin biosynthesis or transport, suggesting cross-talk between ABA and auxin response pathways. The degree of sugar insensitivity of different sis10/abi3 mutant seedlings shows a strong positive correlation with their level of ABA insensitivity during seed germination. Conclusion Mutations in the SIS7/NCED3/STO1 gene, which is primarily required for ABA biosynthesis under drought conditions, confer a sugar-insensitive phenotype, indicating that a constitutive role in ABA biosynthesis is not necessary to confer sugar insensitivity. Findings presented here clearly demonstrate that mutations in ABI3 can confer a sugar-insensitive phenotype and help explain previous, mixed reports on this topic by showing that ABA and sugar insensitivity exhibit a strong positive correlation in

Identification of a crucial histidine involved in metal transport activity in the Arabidopsis cation/H+ exchanger CAX1.

Science.gov (United States)

Shigaki, Toshiro; Barkla, Bronwyn J; Miranda-Vergara, Maria Cristina; Zhao, Jian; Pantoja, Omar; Hirschi, Kendal D

2005-08-26

In plants, yeast, and bacteria, cation/H+ exchangers (CAXs) have been shown to translocate Ca2+ and other metal ions utilizing the H+ gradient. The best characterized of these related transporters is the plant vacuolar localized CAX1. We have used site-directed mutagenesis to assess the impact of altering the seven histidine residues to alanine within Arabidopsis CAX1. The mutants were expressed in a Saccharomyces cerevisiae strain that is sensitive to Ca2+ and other metals. By utilizing a yeast growth assay, the H338A mutant was the only mutation that appeared to alter Ca2+ transport activity. The CAX1 His338 residue is conserved among various CAX transporters and may be located within a filter for cation selection. We proceeded to mutate His338 to every other amino acid residue and utilized yeast growth assays to estimate the transport properties of the 19 CAX mutants. Expression of 16 of these His338 mutants could not rescue any of the metal sensitivities. However, expression of H338N, H338Q, and H338K allowed for some growth on media containing Ca2+. Most interestingly, H338N exhibited increased tolerance to Cd2+ and Zn2+. Endomembrane fractions from yeast cells were used to measure directly the transport of H338N. Although the H338N mutant demonstrated 25% of the wild type Ca2+/H+ transport, it showed an increase in transport for both Cd2+ and Zn2+ reflected in a decrease in the Km for these substrates. This study provides insights into the CAX cation filter and novel mechanisms by which metals may be partitioned across membranes.

Membrane Composition and Physiological Activity of Plastids from an Oenothera Plastome Mutator-Induced Chloroplast Mutant 1

Science.gov (United States)

Johnson, Ellen M.; Sears, Barbara B.

1990-01-01

Plastids were isolated from a plastome mutator-induced mutant (pm7) of Oenothera hookeri and were analyzed for various physiological and biochemical attributes. No photosynthetic electron transport activity was detected in the mutant plastids. This is consistent with previous ultrastructural analysis showing the absence of thylakoid membranes in the pm7 plastids and with the observation of aberrant processing and accumulation of chloroplast proteins in the mutant. In comparison to wild type, the mutant tissue lacks chlorophyll, and has significant differences in levels of four fatty acids. The analyses did not reveal any differences in carotenoid levels nor in the synthesis of several chloroplast lipids. The consequences of the altered composition of the chloroplast membrane are discussed in terms of their relation to the aberrant protein processing of the pm7 plastids. The pigment, fatty acid, and lipid measurements were also performed on two distinct nuclear genotypes (A/A and A/C) which differ in their compatibility with the plastid genome (type I) contained in these lines. In these cases, only chlorophyll concentrations differed significantly. PMID:16667256

Iron-hydroxamate transport in Escherichia coli K12

International Nuclear Information System (INIS)

Prody, C.A.

1984-01-01

FhuB mutants, which are deficient in ferrichrome transport, were isolated and characterized. They were found to be deficient in the utilization of all hydroxamate-type siderophores. They were, however, able to transport enterobactin. A number of analogs of hydroxamate-type siderophores were tested for biological activity in E. coli, and about half of these were active. In addition, two rhodotorulic acid analogs were able to supply iron to fhuB mutants. A search for the fhuB gene product, using one and two-dimensional polyacrylamide gels of proteins from fhuB and wild type strains proved fruitless, and it appeared that the fhuB gene product is expressed at a very low level. Therefore, the fhuB gene was subcloned from a plasmid in the Carbon bank onto plasmid vectors containing the E. coli lac UV-5 and tacI promoters as a device to amplify the fhuB gene. One of these recombinant plasmids carried an 8Kb insert which contained both the tonA and fhuB genes. This plasmid synthesized five proteins of molecular weights 78,000, 40,000, 30,000, 24,000, and 13,700 in maxicell strain CSR603. By use of deletions, the approximate order of the genes for these proteins was determined. Although 3 He-ferrichrome is transported into E. coli cells and vesicles, 3 He-ferric rhodotorulate is not, and so the mechanism of transport for these two siderophores must be different. To examine this further, mutants were obtained that could transport ferrichrome but not rhodotorulic acid. These map in the region between tonA and fhuB, and most are able to transport aerobactin, when carrying the ColV plasmid, but not schizokinen

Induction of Mutants in Durum Wheat

International Nuclear Information System (INIS)

AL-Ubaidi, M.; Ibrahim, I.; AL-Hadithi, A.

2002-01-01

This investigation presents a breeding program for induction and development of a new genotype of durum wheat, resistant to lodging with high yield, by irradiation durum wheat hybrids (F2) with gamma rays 100 Gy, during 1990-1997 cultivation seasons. This program involves: induction of variability, selection evaluation of the mutants at three locations: Twaitha (Baghdad) Latifya ( Babylon) and Swari (Kutt). All mutants showed resistance to lodging and there was a significant reduction in plant height. Mutant SIXIZ-22 surpassed other mutants and its origin in lodging resistance and plant height (83.5,82.8 and 89.4 cm) in the three locations at generation M5 and M6, respectively. Also, there were significant differences between mutant and their origin in the number of spikes/M 2 and grain yild during the two successive generation. On the other hand, mutant IZxCO-105 surpassed other mutants in the number of spikes/M 2 (231.8,242.3 and 292) and grain yield (4336,3376 and 5232 kg/ha) in all testing location, respectively . (authors) 14 refs., 4 tabs

Agp2, a Member of the Yeast Amino Acid Permease Family, Positively Regulates Polyamine Transport at the Transcriptional Level

KAUST Repository

Aouida, Mustapha

2013-06-03

Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3? nor sam3? single mutant is defective in polyamine transport, while the dur3? sam3? double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2? mutant. Studies of Agp2 localization indicate that in the double mutant dur3? sam3?, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport. © 2013 Aouida et al.

Agp2, a Member of the Yeast Amino Acid Permease Family, Positively Regulates Polyamine Transport at the Transcriptional Level

KAUST Repository

Aouida, Mustapha; Texeira, Marta Rubio; Thevelein, Johan M.; Poulin, Richard; Ramotar, Dindial

2013-01-01

Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3? nor sam3? single mutant is defective in polyamine transport, while the dur3? sam3? double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2? mutant. Studies of Agp2 localization indicate that in the double mutant dur3? sam3?, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport. © 2013 Aouida et al.

Root hair mutants of barley

International Nuclear Information System (INIS)

Engvild, K.C.; Rasmussen, K.

2005-01-01

Barley mutants without root hairs or with short or reduced root hairs were isolated among M 2 seeds of 'Lux' barley (Hordeum vulgare L.) after acidified sodium azide mutagenesis. Root hair mutants are investigated intensively in Arabidopsis where about 40 genes are known. A few root hair mutants are known in maize, rice, barley and tomato. Many plants without root hairs grow quite well with good plant nutrition, and mutants have been used for investigations of uptake of strongly bound nutrients like phosphorus, iron, zinc and silicon. Seed of 'Lux' barley (Sejet Plant Breeding, Denmark) were soaked overnight, and then treated with 1.5-millimolarsodium azide in 0.1 molar sodium phosphate buffer, pH 3, for 2.5 hours according to the IAEA Manual on Mutation Breeding (2nd Ed.). After rinsing in tap water and air-drying, the M 2 seeds were sown in the field the same day. Spikes, 4-6 per M 1 plant, were harvested. The mutation frequency was similar to that obtained with other barley cultivars from which low-phytate mutants were isolated [5]. Seeds were germinated on black filter paper in tap water for 3 or 4 days before scoring for root hair mutants

Functional analysis of ABC transporter genes from Botrytis cinerea identifies BcatrB as a transporter of eugenol

NARCIS (Netherlands)

Schoonbeek, H.; Nistelrooy, van J.G.M.; Waard, de M.A.

2003-01-01

The role of multiple ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporter genes from the plant pathogenic fungus Botrytis cinerea in protection against natural fungitoxic compounds was studied by expression analysis and phenotyping of gene-replacement mutants. The

Arg188 in rice sucrose transporter OsSUT1 is crucial for substrate transport

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Sun Ye

2012-11-01

Full Text Available Abstract Background Plant sucrose uptake transporters (SUTs are H+/sucrose symporters related to the major facilitator superfamily (MFS. SUTs are essential for plant growth but little is known about their transport mechanism. Recent work identified several conserved, charged amino acids within transmembrane spans (TMS in SUTs that are essential for transport activity. Here we further evaluated the role of one of these positions, R188 in the fourth TMS of OsSUT1, a type II SUT. Results The OsSUT1(R188K mutant, studied by expression in plants, yeast, and Xenopus oocytes, did not transport sucrose but showed a H+ leak that was blocked by sucrose. The H+ leak was also blocked by β-phenyl glucoside which is not translocated by OsSUT1. Replacing the corresponding Arg in type I and type III SUTs, AtSUC1(R163K and LjSUT4(R169K, respectively, also resulted in loss of sucrose transport activity. Fluorination at the glucosyl 3 and 4 positions of α-phenyl glucoside greatly decreased transport by wild type OsSUT1 but did not affect the ability to block H+ leak in the R188K mutant. Conclusion OsSUT1 R188 appears to be essential for sucrose translocation but not for substrate interaction that blocks H+ leak. Therefore, we propose that an additional binding site functions in the initial recognition of substrates. The corresponding Arg in type I and III SUTs are equally important. We propose that R188 interacts with glucosyl 3-OH and 4-OH during translocation.

Missense Mutations Allow a Sequence-Blind Mutant of SpoIIIE to Successfully Translocate Chromosomes during Sporulation.

Science.gov (United States)

Bose, Baundauna; Reed, Sydney E; Besprozvannaya, Marina; Burton, Briana M

2016-01-01

SpoIIIE directionally pumps DNA across membranes during Bacillus subtilis sporulation and vegetative growth. The sequence-reading domain (γ domain) is required for directional DNA transport, and its deletion severely impairs sporulation. We selected suppressors of the spoIIIEΔγ sporulation defect. Unexpectedly, many suppressors were intragenic missense mutants, and some restore sporulation to near-wild-type levels. The mutant proteins are likely not more abundant, faster at translocating DNA, or sequence-sensitive, and rescue does not involve the SpoIIIE homolog SftA. Some mutants behave differently when co-expressed with spoIIIEΔγ, consistent with the idea that some, but not all, variants may form mixed oligomers. In full-length spoIIIE, these mutations do not affect sporulation, and yet the corresponding residues are rarely found in other SpoIIIE/FtsK family members. The suppressors do not rescue chromosome translocation defects during vegetative growth, indicating that the role of the γ domain cannot be fully replaced by these mutations. We present two models consistent with our findings: that the suppressors commit to transport in one arbitrarily-determined direction or delay spore development. It is surprising that missense mutations somehow rescue loss of an entire domain with a complex function, and this raises new questions about the mechanism by which SpoIIIE pumps DNA and the roles SpoIIIE plays in vivo.

Missense Mutations Allow a Sequence-Blind Mutant of SpoIIIE to Successfully Translocate Chromosomes during Sporulation.

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Baundauna Bose

Full Text Available SpoIIIE directionally pumps DNA across membranes during Bacillus subtilis sporulation and vegetative growth. The sequence-reading domain (γ domain is required for directional DNA transport, and its deletion severely impairs sporulation. We selected suppressors of the spoIIIEΔγ sporulation defect. Unexpectedly, many suppressors were intragenic missense mutants, and some restore sporulation to near-wild-type levels. The mutant proteins are likely not more abundant, faster at translocating DNA, or sequence-sensitive, and rescue does not involve the SpoIIIE homolog SftA. Some mutants behave differently when co-expressed with spoIIIEΔγ, consistent with the idea that some, but not all, variants may form mixed oligomers. In full-length spoIIIE, these mutations do not affect sporulation, and yet the corresponding residues are rarely found in other SpoIIIE/FtsK family members. The suppressors do not rescue chromosome translocation defects during vegetative growth, indicating that the role of the γ domain cannot be fully replaced by these mutations. We present two models consistent with our findings: that the suppressors commit to transport in one arbitrarily-determined direction or delay spore development. It is surprising that missense mutations somehow rescue loss of an entire domain with a complex function, and this raises new questions about the mechanism by which SpoIIIE pumps DNA and the roles SpoIIIE plays in vivo.

The role of transition metal transporters for iron, zinc, manganese, and copper in the pathogenesis of Yersinia pestis.

Science.gov (United States)

Perry, Robert D; Bobrov, Alexander G; Fetherston, Jacqueline D

2015-06-01

Yersinia pestis, the causative agent of bubonic, septicemic and pneumonic plague, encodes a multitude of Fe transport systems. Some of these are defective due to frameshift or IS element insertions, while others are functional in vitro but have no established role in causing infections. Indeed only 3 Fe transporters (Ybt, Yfe and Feo) have been shown to be important in at least one form of plague. The yersiniabactin (Ybt) system is essential in the early dermal/lymphatic stages of bubonic plague, irrelevant in the septicemic stage, and critical in pneumonic plague. Two Mn transporters have been characterized (Yfe and MntH). These two systems play a role in bubonic plague but the double yfe mntH mutant is fully virulent in a mouse model of pneumonic plague. The same in vivo phenotype occurs with a mutant lacking two (Yfe and Feo) of four ferrous transporters. A role for the Ybt siderophore in Zn acquisition has been revealed. Ybt-dependent Zn acquisition uses a transport system completely independent of the Fe-Ybt uptake system. Together Ybt components and ZnuABC play a critical role in Zn acquisition in vivo. Single mutants in either system retain high virulence in a mouse model of septicemic plague while the double mutant is completely avirulent.

Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection

International Nuclear Information System (INIS)

Newman, A.P.; Ferro-Novick, S.

1987-01-01

We have adapted a [ 3 H]mannose suicide selection to identify mutations in additional genes which function in the early part of the yeast secretory pathway. Thus far this protocol has led to the identification of two new genes which are implicated in this process, as well as additional alleles of previously identified genes. The new mutants, bet1 and bet2, are temperature sensitive for growth and protein transport. Thin section analysis has revealed the accumulation of a network of endoplasmic reticulum (ER) at the restrictive temperature (37 0 C). Precursors of exported proteins that accumulate in the cell at 37 0 C are terminally core glycosylated. These observations suggest that the transport of precursors is blocked subsequent to translocation into the ER but before entry into the Golgi apparatus. The bet1 and bet2 mutants define two new complementation groups which have the same properties as previously identified ER-accumulating mutants. This and previous findings suggest that protein exit from the ER and entry into the Golgi apparatus is a complex process requiring at least 11 genes

Isozyme differences in barley mutants

International Nuclear Information System (INIS)

AI-Jibouri, A.A.M.; Dham, K.M.

1990-01-01

Full text: Thirty mutants (M 11 ) of barley (Hordeum vulgare L.) induced by physical and chemical mutagens were analysed for isozyme composition using polyacrylamide gel electrophoresis. Results show that these mutants were different in the isozymes leucine aminopeptidase, esterase and peroxidase. The differences included the number of forms of each enzyme, relative mobility value and their intensity on the gel. Glutamate oxaloacetate transaminase isozyme was found in six molecular forms and these forms were similar in all mutants. (author)

Ascertainment of the effect of differential growth rates of mutants on observed mutant frequencies in X-irradiated mammalian cells

International Nuclear Information System (INIS)

Knaap, A.G.A.C.; Simons, J.W.I.M.

1983-01-01

As it is not known to what extent differential growth rates of induced mutants lead to over- and under-representation of mutants in treated populations and thereby affect the determination of mutant frequencies, the mutation induction in X-irradiated L5178Y mouse lymphoma cells was determined via two methods. The first method involves the standard protocol which may suffer from the effect of differential growth rates, while the second method is based upon the fluctuation test in which the differential growth rates can be actually measured. It appeared that the standard protocol led to a mutant frequency that was similar to the mutant frequency determined in the fluctuation test. Therefore, the standard protocol appears to lead to only a minor under-estimation if any. Substantial heterogeneity in growth rates of induced mutants was observed, but the mutants with a selective advantage appear largely to compensate for the mutants that are lost because of selective disadvantage. It was calculated that the chance for isolating the same mutant twice from a treated population had been increased 2.2-fold because of the observed differential growth rates. (orig./AJ)

RAB-like 2 has an essential role in male fertility, sperm intra-flagellar transport, and tail assembly.

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Jennifer C Y Lo

Full Text Available A significant percentage of young men are infertile and, for the majority, the underlying cause remains unknown. Male infertility is, however, frequently associated with defective sperm motility, wherein the sperm tail is a modified flagella/cilia. Conversely, a greater understanding of essential mechanisms involved in tail formation may offer contraceptive opportunities, or more broadly, therapeutic strategies for global cilia defects. Here we have identified Rab-like 2 (RABL2 as an essential requirement for sperm tail assembly and function. RABL2 is a member of a poorly characterized clade of the RAS GTPase superfamily. RABL2 is highly enriched within developing male germ cells, where it localizes to the mid-piece of the sperm tail. Lesser amounts of Rabl2 mRNA were observed in other tissues containing motile cilia. Using a co-immunoprecipitation approach and RABL2 affinity columns followed by immunochemistry, we demonstrated that within developing haploid germ cells RABL2 interacts with intra-flagella transport (IFT proteins and delivers a specific set of effector (cargo proteins, including key members of the glycolytic pathway, to the sperm tail. RABL2 binding to effector proteins is regulated by GTP. Perturbed RABL2 function, as exemplified by the Mot mouse line that contains a mutation in a critical protein-protein interaction domain, results in male sterility characterized by reduced sperm output, and sperm with aberrant motility and short tails. Our data demonstrate a novel function for the RABL protein family, an essential role for RABL2 in male fertility and a previously uncharacterised mechanism for protein delivery to the flagellum.

Hydrogen is a preferred intermediate in the energy-conserving electron transport chain of Methanosarcina barkeri.

Science.gov (United States)

Kulkarni, Gargi; Kridelbaugh, Donna M; Guss, Adam M; Metcalf, William W

2009-09-15

Methanogens use an unusual energy-conserving electron transport chain that involves reduction of a limited number of electron acceptors to methane gas. Previous biochemical studies suggested that the proton-pumping F(420)H(2) dehydrogenase (Fpo) plays a crucial role in this process during growth on methanol. However, Methanosarcina barkeri Delta fpo mutants constructed in this study display no measurable phenotype on this substrate, indicating that Fpo plays a minor role, if any. In contrast, Delta frh mutants lacking the cytoplasmic F(420)-reducing hydrogenase (Frh) are severely affected in their ability to grow and make methane from methanol, and double Delta fpo/Delta frh mutants are completely unable to use this substrate. These data suggest that the preferred electron transport chain involves production of hydrogen gas in the cytoplasm, which then diffuses out of the cell, where it is reoxidized with transfer of electrons into the energy-conserving electron transport chain. This hydrogen-cycling metabolism leads directly to production of a proton motive force that can be used by the cell for ATP synthesis. Nevertheless, M. barkeri does have the flexibility to use the Fpo-dependent electron transport chain when needed, as shown by the poor growth of the Delta frh mutant. Our data suggest that the rapid enzymatic turnover of hydrogenases may allow a competitive advantage via faster growth rates in this freshwater organism. The mutant analysis also confirms the proposed role of Frh in growth on hydrogen/carbon dioxide and suggests that either Frh or Fpo is needed for aceticlastic growth of M. barkeri.

Isozyme differences in barley mutants

Energy Technology Data Exchange (ETDEWEB)

AI-Jibouri, A A.M.; Dham, K M [Department of Botany, Nuclear Research Centre, Baghdad (Iraq)

1990-01-01

Full text: Thirty mutants (M{sub 11}) of barley (Hordeum vulgare L.) induced by physical and chemical mutagens were analysed for isozyme composition using polyacrylamide gel electrophoresis. Results show that these mutants were different in the isozymes leucine aminopeptidase, esterase and peroxidase. The differences included the number of forms of each enzyme, relative mobility value and their intensity on the gel. Glutamate oxaloacetate transaminase isozyme was found in six molecular forms and these forms were similar in all mutants. (author)

Identification of a disulfide bridge important for transport function of SNAT4 neutral amino acid transporter.

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Rugmani Padmanabhan Iyer

Full Text Available SNAT4 is a member of system N/A amino acid transport family that primarily expresses in liver and muscles and mediates the transport of L-alanine. However, little is known about the structure and function of the SNAT family of transporters. In this study, we showed a dose-dependent inhibition in transporter activity of SNAT4 with the treatment of reducing agents, dithiothreitol (DTT and Tris(2-carboxyethylphosphine (TCEP, indicating the possible involvement of disulfide bridge(s. Mutation of residue Cys-232, and the two highly conserved residues Cys-249 and Cys-321, compromised the transport function of SNAT4. However, this reduction was not caused by the decrease of SNAT4 on the cell surface since the cysteine-null mutant generated by replacing all five cysteines with alanine was equally capable of being expressed on the cell surface as wild-type SNAT4. Interestingly, by retaining two cysteine residues, 249 and 321, a significant level of L-alanine uptake was restored, indicating the possible formation of disulfide bond between these two conserved residues. Biotinylation crosslinking of free thiol groups with MTSEA-biotin provided direct evidence for the existence of a disulfide bridge between Cys-249 and Cys-321. Moreover, in the presence of DTT or TCEP, transport activity of the mutant retaining Cys-249 and Cys-321 was reduced in a dose-dependent manner and this reduction is gradually recovered with increased concentration of H2O2. Disruption of the disulfide bridge also decreased the transport of L-arginine, but to a lesser degree than that of L-alanine. Together, these results suggest that cysteine residues 249 and 321 form a disulfide bridge, which plays an important role in substrate transport but has no effect on trafficking of SNAT4 to the cell surface.

Problem-Solving Test: Tryptophan Operon Mutants

Science.gov (United States)

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…

Revealing the Molecular Structure and the Transport Mechanism at the Base of Primary Cilia Using Superresolution STED Microscopy

Science.gov (United States)

Yang, Tung-Lin

The primary cilium is an organelle that serves as a signaling center of the cell and is involved in the hedgehog signaling, cAMP pathway, Wnt pathways, etc. Ciliary function relies on the transportation of molecules between the primary cilium and the cell, which is facilitated by intraflagellar transport (IFT). IFT88, one of the important IFT proteins in complex B, is known to play a role in the formation and maintenance of cilia in various types of organisms. The ciliary transition zone (TZ), which is part of the gating apparatus at the ciliary base, is home to a large number of ciliopathy molecules. Recent studies have identified important regulating elements for TZ gating in cilia. However, the architecture of the TZ region and its arrangement relative to intraflagellar transport (IFT) proteins remain largely unknown, hindering the mechanistic understanding of the regulation processes. One of the major challenges comes from the tiny volume at the ciliary base packed with numerous proteins, with the diameter of the TZ close to the diffraction limit of conventional microscopes. Using a series of stimulated emission depletion (STED) superresolution images mapped to electron microscopy images, we analyzed the structural organization of the ciliary base. Subdiffraction imaging of TZ components defines novel geometric distributions of RPGRIP1L, MKS1, CEP290, TCTN2 and TMEM67, shedding light on their roles in TZ structure, assembly, and function. We found TCTN2 at the outmost periphery of the TZ close to the ciliary membrane, with a 227+/-18 nm diameter. TMEM67 was adjacent to TCTN2, with a 205+/-20 nm diameter. RPGRIP1L was localized toward the axoneme at the same axial level as TCTN2 and TMEM67, with a 165+/-8 nm diameter. MKS1 was situated between TMEM67 and RPGRIP1L, with an 186+/-21 nm diameter. Surprisingly, CEP290 was localized at the proximal side of the TZ close to the distal end of the centrin-labeled basal body. The lateral width was unexpectedly close to

Dwarf mutant of rice variety Seratus Malam

International Nuclear Information System (INIS)

Mugiono, P. S.; Soemanggono, A.M.R.

1989-01-01

Full text: Seeds of 'Seratus Malam', a local tall upland variety with long panicles and high yield potential were irradiated with 10-50 krad gamma rays in 1983. From 50,000 M 2 plants, 130 semidwarf mutants and 1 dwarf mutant were selected. The dwarf mutant M-362 was obtained from the 10 krad treatment. The mutant shows about 50% reduction in plant height, but also in number of productive tillers. Thus the yield per plant is also significantly less. However, the mutant gene is not allelic to DGWG and therefore may be useful in cross breeding. (author)

Identification of an Efflux Transporter LmrB Regulating Stress Response and Extracellular Polysaccharide Synthesis in Streptococcus mutans

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Jia Liu

2017-06-01

Full Text Available Efflux transporters have been implicated in regulating bacterial virulence properties such as resistance to antibiotics, biofilm formation and colonization. The pathogenicity of Streptococcus mutans, the primary etiologic agent of human dental caries, relies on the bacterium’s ability to form biofilms on tooth surface. However, the studies on efflux transporters in S. mutans are scare and the function of these transporters remained to be clarified. In this study, we identified an efflux transporter (LmrB in S. mutans through cloning the lmrB gene into Escherichia coli. Introducing lmrB into E. coli conferred a multidrug-resistant phenotype and resulted in higher EtBr efflux activity which could be suppressed by efflux inhibitor. To explore whether LmrB was involved in S. mutans virulence properties regulation, we constructed the lmrB inactivation mutant and examined the phenotypes of the mutant. It was found that LmrB deficiency resulted in increased IPS storage and prolonged acid production. Enhanced biofilm formation characterized by increased extracellular polysaccharides (EPS production and elevated resistance to hydrogen peroxide and antimicrobials were also observed in lmrB mutant. To gain a better understanding of the global role of LmrB, a transcriptome analysis was performed using lmrB mutant strain. The expression of 107 genes was up- or down-regulated in the lmrB mutant compared with the wild type. Notably, expression of genes in several genomic islands was differentially modulated, such as stress-related GroELS and scnRK, sugar metabolism associated glg operons and msmREFGK transporter. The results presented here indicate that LmrB plays a vital global role in the regulation of several important virulence properties in S. mutans.

Intracellular transport and sorting of mutant human proinsulins that fail to form hexamers.

Science.gov (United States)

Quinn, D; Orci, L; Ravazzola, M; Moore, H P

1991-06-01

Human proinsulin and insulin oligomerize to form dimers and hexamers. It has been suggested that the ability of prohormones to self associate and form aggregates may be responsible for the sorting process at the trans-Golgi. To examine whether insulin oligomerization is required for proper sorting into regulated storage granules, we have constructed point mutations in human insulin B chain that have been previously shown to prevent formation of insulin hexamers (Brange, J., U. Ribel, J. F. Hansen, G. Dodson, M. T. Hansen, S. Havelund, S. G. Melberg, F. Norris, K. Norris, L. Snel, A. R. Sorensen, and H. O. Voight. 1988. Nature [Lond.]. 333:679-682). One mutant (B10His----Asp) allows formation of dimers but not hexamers and the other (B9Ser----Asp) prevents formation of both dimers and hexamers. The mutants were transfected into the mouse pituitary AtT-20 cells, and their ability to be sorted into regulated secretory granules was compared to wild-type insulin. We found that while B10His----Asp is sorted somewhat less efficiently than wild-type insulin as reported previously (Carroll, R. J., R. E. Hammer, S. J. Chan, H. H. Swift, A. H. Rubenstein, and D. F. Steiner. 1988. Proc. Natl. Acad. Sci. USA. 85:8943-8947; Gross, D. J., P. A. Halban, C. R. Kahn, G. C. Weir, and L. Villa-Kumaroff. 1989. Proc. Natl. Acad. Sci. USA. 86:4107-4111). B9Ser----Asp is targeted to granules as efficiently as wild-type insulin. These results indicate that self association of proinsulin into hexamers is not required for its targeting to the regulated secretory pathway.

Limits of transforming competence of SV40 nuclear and cytoplasmic large T mutants with altered Rb binding sequences.

Science.gov (United States)

Tedesco, D; Fischer-Fantuzzi, L; Vesco, C

1993-03-01

Multiple amino acid substitutions were introduced into the SV40 large T region that harbors the retinoblastoma protein (Rb) binding site and the nuclear transport signal, changing either one or both of these determinants. Mutant activities were examined in a set of assays allowing different levels of transforming potential to be distinguished; phenotypic changes in established and pre-crisis rat embryo fibroblasts (REFs) were detected under isogenic cell conditions, and comparisons made with other established rodent cells. The limit of the transforming ability of mutants with important substitutions in the Rb binding site fell between two transformation levels of the same established rat cells. Such cells could be induced to form dense foci but not agar colonies (their parental pre-crises REFs, as expected, were untransformed either way). Nonetheless, agar colony induction was possible in other cell lines, such as mouse NIH3T3 and (for one of the mutants) rat F2408. All these mutants efficiently immortalized pre-crisis REFs. The transforming ability of cytoplasmic mutants appeared to depend on the integrity of the Rb-binding sequence to approximately the same extent as that of the wild-type large T, although evidence of in vivo Rb-cytoplasmic large T complexes was not found. The presence or absence of small t was critical when the transforming task of mutants was near the limit of their abilities.

Lactose metabolism in Streptococcus lactis: studies with a mutant lacking glucokinase and mannose-phosphotransferase activities

International Nuclear Information System (INIS)

Thompson, J.; Chassy, B.M.; Egan, W.

1985-01-01

A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose- phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of [ 14 C]lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same. During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution 31 P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM

Lactose metabolism in Streptococcus lactis: studies with a mutant lacking glucokinase and mannose-phosphotransferase activities

Energy Technology Data Exchange (ETDEWEB)

Thompson, J.; Chassy, B.M.; Egan, W.

1985-04-01

A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose- phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of (/sup 14/C)lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same. During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution /sup 31/P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM.

Genome-wide analysis of the Pho regulon in a pstCA mutant of Citrobacter rodentium.

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Catherine Cheng

Full Text Available The phosphate-specific transport operon, pstSCAB-phoU, of Gram-negative bacteria is an essential part of the Pho regulon. Its key roles are to encode a high-affinity inorganic phosphate transport system and to prevent activation of PhoB in phosphate-rich environments. In general, mutations in pstSCAB-phoU lead to the constitutive expression of the Pho regulon. Previously, we constructed a pstCA deletion mutant of Citrobacter rodentium and found it to be attenuated for virulence in mice, its natural host. This attenuation was dependent on PhoB or PhoB-regulated gene(s because a phoB mutation restored virulence for mice to the pstCA mutant. To investigate how downstream genes may contribute to the virulence of C. rodentium, we used microarray analysis to investigate global gene expression of C. rodentium strain ICC169 and its isogenic pstCA mutant when grown in phosphate-rich medium. Overall 323 genes of the pstCA mutant were differentially expressed by at least 1.5-fold compared to the wild-type C. rodentium. Of these 145 were up-regulated and 178 were down-regulated. Differentially expressed genes included some involved in phosphate homoeostasis, cellular metabolism and protein metabolism. A large number of genes involved in stress responses and of unknown function were also differentially expressed, as were some virulence-associated genes. Up-regulated virulence-associated genes in the pstCA mutant included that for DegP, a serine protease, which appeared to be directly regulated by PhoB. Down-regulated genes included those for the production of the urease, flagella, NleG8 (a type III-secreted protein and the tad focus (which encodes type IVb pili in Yersinia enterocolitica. Infection studies using C57/BL6 mice showed that DegP and NleG8 play a role in bacterial virulence. Overall, our study provides evidence that Pho is a global regulator of gene expression in C. rodentium and indicates the presence of at least two previously unrecognized

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

Science.gov (United States)

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.

Allosteric Mutant IDH1 Inhibitors Reveal Mechanisms for IDH1 Mutant and Isoform Selectivity

Energy Technology Data Exchange (ETDEWEB)

Xie, Xiaoling; Baird, Daniel; Bowen, Kimberly; Capka, Vladimir; Chen, Jinyun; Chenail, Gregg; Cho, YoungShin; Dooley, Julia; Farsidjani, Ali; Fortin, Pascal; Kohls, Darcy; Kulathila, Raviraj; Lin, Fallon; McKay, Daniel; Rodrigues, Lindsey; Sage, David; Touré, B. Barry; van der Plas, Simon; Wright, Kirk; Xu, Ming; Yin, Hong; Levell, Julian; Pagliarini, Raymond A. (Novartis)

2017-03-01

Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 “regulatory segment,” which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

Semi-dwarf mutants for rice improvement

International Nuclear Information System (INIS)

Othman, Ramli; Osman, Mohammad; Ibrahim, Rusli

1990-01-01

Full text: MARDI and the National University of Malaysia embarked on a programme to induce resistance against blast in rice in 1978. MARDI also obtained semi dwarf mutants of cvs 'Mahsuri', 'Muda', 'Pongsu seribu' and 'Jarum Mas', which are under evaluation. The popular local rice variety 'Manik' was subjected to gamma irradiation (15-40 krad) and 101 promising semidwarf mutants have been obtained following selection in M 2 -M 6 . 29 of them show grain yields of 6.0-7.3 t/ha, compared with 5.7t for 'Manik'. Other valuable mutants were found showing long grain, less shattering, earlier maturity, and glutinous endosperm. One mutant, resistant to brown plant hopper yields 6.3t/ha. (author)

Transcriptomic profiling-based mutant screen reveals three new transcription factors mediating menadione resistance in Neurospora crassa.

Science.gov (United States)

Zhu, Jufen; Yu, Xinxu; Xie, Baogui; Gu, Xiaokui; Zhang, Zhenying; Li, Shaojie

2013-06-01

To gain insight into the regulatory mechanisms of oxidative stress responses in filamentous fungi, the genome-wide transcriptional response of Neurospora crassa to menadione was analysed by digital gene expression (DGE) profiling, which identified 779 upregulated genes and 576 downregulated genes. Knockout mutants affecting 130 highly-upregulated genes were tested for menadione sensitivity, which revealed that loss of the transcription factor siderophore regulation (SRE) (a transcriptional repressor for siderophore biosynthesis), catatase-3, cytochrome c peroxidase or superoxide dismutase 1 copper chaperone causes hypersensitivity to menadione. Deletion of sre dramatically increased transcription of the siderophore biosynthesis gene ono and the siderophore iron transporter gene sit during menadione stress, suggesting that SRE is required for repression of iron uptake under oxidative stress conditions. Contrary to its phenotype, the sre deletion mutant showed higher transcriptional levels of genes encoding reactive oxygen species (ROS) scavengers than wild type during menadione stress, which implies that the mutant suffers a higher level of oxidative stress than wild type. Uncontrolled iron uptake in the sre mutant might exacerbate cellular oxidative stress. This is the first report of a negative regulator of iron assimilation participating in the fungal oxidative stress response. In addition to SRE, eight other transcription factor genes were also menadione-responsive but their single gene knockout mutants showed wild-type menadione sensitivity. Two of them, named as mit-2 (menadione induced transcription factor-2) and mit-4 (menadione induced transcription factor-4), were selected for double mutant analysis. The double mutant was hypersensitive to menadione. Similarly, the double mutation of mit-2 and sre also had additive effects on menadione sensitivity, suggesting multiple transcription factors mediate oxidative stress resistance in an additive manner

Evaluation of tall rice mutant

International Nuclear Information System (INIS)

Hakim, L.; Azam, M.A.; Miah, A.J.; Mansur, M.A.; Akanda, H.R.

1989-01-01

One tall mutant (Mut NS1) of rice variety Nizersail was put to multilocation on-farm trial. It showed improvement over the parent in respect of by earlier maturity and higher grain yield at all locations and thus it appears as an improved mutant of Nizersail. (author). 6 refs

Amphitrite ornata dehaloperoxidase (DHP): investigations of structural factors that influence the mechanism of halophenol dehalogenation using "peroxidase-like" myoglobin mutants and "myoglobin-like" DHP mutants.

Science.gov (United States)

Du, Jing; Huang, Xiao; Sun, Shengfang; Wang, Chunxue; Lebioda, Lukasz; Dawson, John H

2011-09-27

Dehaloperoxidase (DHP), discovered in the marine terebellid polychaete Amphitrite ornata, is the first heme-containing globin with a peroxidase activity. The sequence and crystal structure of DHP argue that it evolved from an ancient O(2) transport and storage globin. Thus, DHP retains an oxygen carrier function but also has the ability to degrade halophenol toxicants in its living environment. Sperm whale myoglobin (Mb) in the ferric state has a peroxidase activity ∼10 times lower than that of DHP. The catalytic activity enhancement observed in DHP appears to have been generated mainly by subtle changes in the positions of the proximal and distal histidine residues that appeared during DHP evolution. Herein, we report investigations into the mechanism of action of DHP derived from examination of "peroxidase-like" Mb mutants and "Mb-like" DHP mutants. The dehalogenation ability of wild-type Mb is augmented in the peroxidase-like Mb mutants (F43H/H64L, G65T, and G65I Mb) but attenuated in the Mb-like T56G DHP variant. X-ray crystallographic data show that the distal His residues in G65T Mb and G65I are positioned ∼0.3 and ∼0.8 Å, respectively, farther from the heme iron compared to that in the wild-type protein. The H93K/T95H double mutant Mb with the proximal His shifted to the "DHP-like" position has an increased peroxidase activity. In addition, a better dehaloperoxidase (M86E DHP) was generated by introducing a negative charge near His89 to enhance the imidazolate character of the proximal His. Finally, only minimal differences in dehalogenation activities are seen among the exogenous ligand-free DHP, the acetate-bound DHP, and the distal site blocker L100F DHP mutant. Thus, we conclude that binding of halophenols in the internal binding site (i.e., distal cavity) is not essential for catalysis. This work provides a foundation for a new structure-function paradigm for peroxidases and for the molecular evolution of the dual-function enzyme DHP.

Amphitrite ornata Dehaloperoxidase (DHP): Investigations of Structural Factors That Influence the Mechanism of Halophenol Dehalogenation Using ;Peroxidase-like; Myoglobin Mutants and ;Myoglobin-like; DHP Mutants

Energy Technology Data Exchange (ETDEWEB)

Du, Jing; Huang, Xiao; Sun, Shengfang; Wang, Chunxue; Lebioda, Lukasz; Dawson, John H. (SC)

2012-05-14

Dehaloperoxidase (DHP), discovered in the marine terebellid polychaete Amphitrite ornata, is the first heme-containing globin with a peroxidase activity. The sequence and crystal structure of DHP argue that it evolved from an ancient O{sub 2} transport and storage globin. Thus, DHP retains an oxygen carrier function but also has the ability to degrade halophenol toxicants in its living environment. Sperm whale myoglobin (Mb) in the ferric state has a peroxidase activity {approx}10 times lower than that of DHP. The catalytic activity enhancement observed in DHP appears to have been generated mainly by subtle changes in the positions of the proximal and distal histidine residues that appeared during DHP evolution. Herein, we report investigations into the mechanism of action of DHP derived from examination of 'peroxidase-like' Mb mutants and 'Mb-like' DHP mutants. The dehalogenation ability of wild-type Mb is augmented in the peroxidase-like Mb mutants (F43H/H64L, G65T, and G65I Mb) but attenuated in the Mb-like T56G DHP variant. X-ray crystallographic data show that the distal His residues in G65T Mb and G65I are positioned {approx}0.3 and {approx}0.8 {angstrom}, respectively, farther from the heme iron compared to that in the wild-type protein. The H93K/T95H double mutant Mb with the proximal His shifted to the 'DHP-like' position has an increased peroxidase activity. In addition, a better dehaloperoxidase (M86E DHP) was generated by introducing a negative charge near His89 to enhance the imidazolate character of the proximal His. Finally, only minimal differences in dehalogenation activities are seen among the exogenous ligand-free DHP, the acetate-bound DHP, and the distal site blocker L100F DHP mutant. Thus, we conclude that binding of halophenols in the internal binding site (i.e., distal cavity) is not essential for catalysis. This work provides a foundation for a new structure-function paradigm for peroxidases and for the

Using heterologous expression systems to characterize potassium and sodium transport activities.

Science.gov (United States)

Rodríguez, Alonso; Benito, Begoña; Cagnac, Olivier

2012-01-01

The expression of plant transporters in simple well-characterized cell systems is an irreplaceable technique for gaining insights into the kinetic and energetic features of plant transporters. Among all the available expression systems, yeast cells offer the highest simplicity and have the capacity to mimic the in vivo properties of plant transporters. Here, we describe the use of yeast mutants to express K(+) and Na(+) plant transporters and discuss some experimental problems that can produce misleading results.

Homologous series of induced early mutants in indican rice. Pt.1. The production of homologous series of early mutants

International Nuclear Information System (INIS)

Chen Xiulan; Yang Hefeng; He Zhentian; Han Yuepeng; Liu Xueyu

1999-01-01

The percentage of homologous series of early mutants induced from the same Indican rice variety were almost the same (1.37%∼1.64%) in 1983∼1993, but the ones from the different eco-typical varieties were different. The early variety was 0.73%, the mid variety was 1.51%, and the late variety was 1.97%. The percentage of homologous series of early mutants from the varieties with the same pedigree and relationship were similar, but the one from the cog nation were lower than those from distant varieties. There are basic laws and characters in the homologous series of early mutants: 1. The inhibited phenotype is the basic of the homologous series of early mutants; 2. The production of the homologous series of early mutants is closely related with the growing period of the parent; 3. The parallel mutation of the stem and leaves are simultaneously happened with the variation of early or late maturing; 4. The occurrence of the homologous series of early mutants is in a state of imbalance. According to the law of parallel variability, the production of homologous series of early mutants can be predicted as long as the parents' classification of plant, pedigree and ecological type are identified. Therefore, the early breeding can be guided by the law of homologous series of early mutants

Human NKCC2 cation–Cl– co-transporter complements lack of Vhc1 transporter in yeast vacuolar membranes.

Science.gov (United States)

Petrezselyova, Silvia; Dominguez, Angel; Herynkova, Pavla; Macias, Juan F; Sychrova, Hana

2013-10-01

Cation–chloride co-transporters serve to transport Cl– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co-transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co-transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt-sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma-membrane alkali–metal cation exporters Nha1 and Ena1-5 and the vacuolar cation–chloride co-transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild-type and mutated cation–chloride co-transporters. Copyright © 2013 John Wiley & Sons, Ltd.

Studies on reduced height mutants in rice

International Nuclear Information System (INIS)

Narahari, P.; Bhagwat, S.G.

1984-01-01

Two cross-bred derivatives of the mutant TR5xTR17 and TR21 continued to show promise and were advanced to wider scale testing. TR5 was found to carry a semi-dwarfing gene different from that in IR8. New semi-dwarf mutants were screened from M 2 through M 4 from two separate radiation experiments. The gibberellin response of seedlings of mutant and tester strains was evaluated and crosses of tester stocks and mutant semi-dwarfs were made for genetic analyses. (author)

Transport activity of the sodium bicarbonate cotransporter NBCe1 is enhanced by different isoforms of carbonic anhydrase.

Directory of Open Access Journals (Sweden)

Christina Schueler

Full Text Available Transport metabolons have been discussed between carbonic anhydrase II (CAII and several membrane transporters. We have now studied different CA isoforms, expressed in Xenopus oocytes alone and together with the electrogenic sodium bicarbonate cotransporter 1 (NBCe1, to determine their catalytic activity and their ability to enhance NBCe1 transport activity. pH measurements in intact oocytes indicated similar activity of CAI, CAII and CAIII, while in vitro CAIII had no measurable activity and CAI only 30% of the activity of CAII. All three CA isoforms increased transport activity of NBCe1, as measured by the transport current and the rate of intracellular sodium rise in oocytes. Two CAII mutants, altered in their intramolecular proton pathway, CAII-H64A and CAII-Y7F, showed significant catalytic activity and also enhanced NBCe1 transport activity. The effect of CAI, CAII, and CAII mutants on NBCe1 activity could be reversed by blocking CA activity with ethoxyzolamide (EZA, 10 µM, while the effect of the less EZA-sensitive CAIII was not reversed. Our results indicate that different CA isoforms and mutants, even if they show little enzymatic activity in vitro, may display significant catalytic activity in intact cells, and that the ability of CA to enhance NBCe1 transport appears to depend primarily on its catalytic activity.

[Construction of FANCA mutant protein from Fanconi anemia patient and analysis of its function].

Science.gov (United States)

Chen, Fei; Zhang, Ke-Jian; Zuo, Xue-Lan; Zeng, Xian-Chang

2007-11-01

To study FANCA protein expression in Fanconi anemia patient's (FA) cells and explore its function. FANCA protein expression was analyzed in 3 lymphoblast cell lines derived from 3 cases of type A FA (FA-A) patients using Western blot. Nucleus and cytoplasm localization of FANCA protein was analyzed in one case of FA-A which contained a truncated FANCA (exon 5 deletion). The FANCA mutant was constructed from the same patient and its interaction with FANCG was evaluated by mammalian two-hybrid (M2H) assay. FANCA protein was not detected in the 3 FA-A patients by rabbit anti-human MoAb, but a truncated FANCA protein was detected in 1 of them by mouse anti-human MoAb. The truncated FANCA could not transport from cytoplasm into nucleus. The disease-associated FANCA mutant was defective in binding to FANCG in M2H system. FANCA proteins are defective in the 3 FA-A patients. Disfunction of disease-associated FANCA mutant proved to be the pathogenic mutations in FANCA gene. Exon 5 of FANCA gene was involved in the interaction between FANCA and FANCG.

Evc is a positive mediator of Ihh-regulated bone growth that localises at the base of chondrocyte cilia.

Science.gov (United States)

Ruiz-Perez, Victor L; Blair, Helen J; Rodriguez-Andres, M Elena; Blanco, Maria Jose; Wilson, Amy; Liu, Yu-Ning; Miles, Colin; Peters, Heiko; Goodship, Judith A

2007-08-01

EVC is a novel protein mutated in the human chondroectodermal dysplasia Ellis-van Creveld syndrome (EvC; OMIM: 225500). We have inactivated Evc in the mouse and show that Evc(-/-) mice develop an EvC-like syndrome, including short ribs, short limbs and dental abnormalities. lacZ driven by the Evc promoter revealed that Evc is expressed in the developing bones and the orofacial region. Antibodies developed against Evc locate the protein at the base of the primary cilium. The growth plate of Evc(-/-) mice shows delayed bone collar formation and advanced maturation of chondrocytes. Indian hedgehog (Ihh) is expressed normally in the growth plates of Evc(-/-) mice, but expression of the Ihh downstream genes Ptch1 and Gli1 was markedly decreased. Recent studies have shown that Smo localises to primary cilia and that Gli3 processing is defective in intraflagellar transport mutants. In vitro studies using Evc(-/-) cells demonstrate that the defect lies downstream of Smo. Chondrocyte cilia are present in Evc(-/-) mice and Gli3 processing appears normal by western blot analysis. We conclude that Evc is an intracellular component of the hedgehog signal transduction pathway that is required for normal transcriptional activation of Ihh target genes.

Mainzer-Saldino Syndrome Is a Ciliopathy Caused by IFT140 Mutations

Science.gov (United States)

Perrault, Isabelle; Saunier, Sophie; Hanein, Sylvain; Filhol, Emilie; Bizet, Albane A.; Collins, Felicity; Salih, Mustafa A.M.; Gerber, Sylvie; Delphin, Nathalie; Bigot, Karine; Orssaud, Christophe; Silva, Eduardo; Baudouin, Véronique; Oud, Machteld M.; Shannon, Nora; Le Merrer, Martine; Roche, Olivier; Pietrement, Christine; Goumid, Jamal; Baumann, Clarisse; Bole-Feysot, Christine; Nitschke, Patrick; Zahrate, Mohammed; Beales, Philip; Arts, Heleen H.; Munnich, Arnold; Kaplan, Josseline; Antignac, Corinne; Cormier-Daire, Valérie; Rozet, Jean-Michel

2012-01-01

Mainzer-Saldino syndrome (MSS) is a rare disorder characterized by phalangeal cone-shaped epiphyses, chronic renal failure, and early-onset, severe retinal dystrophy. Through a combination of ciliome resequencing and Sanger sequencing, we identified IFT140 mutations in six MSS families and in a family with the clinically overlapping Jeune syndrome. IFT140 is one of the six currently known components of the intraflagellar transport complex A (IFT-A) that regulates retrograde protein transport in ciliated cells. Ciliary abundance and localization of anterograde IFTs were altered in fibroblasts of affected individuals, a result that supports the pivotal role of IFT140 in proper development and function of ciliated cells. PMID:22503633

Osmotic water transport in aquaporins

DEFF Research Database (Denmark)

Zeuthen, Thomas; Alsterfjord, Magnus; Beitz, Eric

2013-01-01

Abstract  We test a novel, stochastic model of osmotic water transport in aquaporins. A solute molecule present at the pore mouth can either be reflected or permeate the pore. We assume that only reflected solute molecules induce osmotic transport of water through the pore, while permeating solute...... molecules give rise to no water transport. Accordingly, the rate of water transport is proportional to the reflection coefficient σ, while the solute permeability, P(S), is proportional to 1 - σ. The model was tested in aquaporins heterologously expressed in Xenopus oocytes. A variety of aquaporin channel...... sizes and geometries were obtained with the two aquaporins AQP1 and AQP9 and mutant versions of these. Osmotic water transport was generated by adding 20 mM of a range of different-sized osmolytes to the outer solution. The osmotic water permeability and the reflection coefficient were measured...

(Methyl)ammonium Transport in the Nitrogen-Fixing Bacterium Azospirillum brasilense

Science.gov (United States)

Van Dommelen, Anne; Keijers, Veerle; Vanderleyden, Jos; de Zamaroczy, Miklos

1998-01-01

An ammonium transporter of Azospirillum brasilense was characterized. In contrast to most previously reported putative prokaryotic NH4+ transporter genes, A. brasilense amtB is not part of an operon with glnB or glnZ which, in A. brasilense, encode nitrogen regulatory proteins PII and PZ, respectively. Sequence analysis predicts the presence of 12 transmembrane domains in the deduced AmtB protein and classifies AmtB as an integral membrane protein. Nitrogen regulates the transcription of the amtB gene in A. brasilense by the Ntr system. amtB is the first gene identified in A. brasilense whose expression is regulated by NtrC. The observation that ammonium uptake is still possible in mutants lacking the AmtB protein suggests the presence of a second NH4+ transport mechanism. Growth of amtB mutants at low ammonium concentrations is reduced compared to that of the wild type. This suggests that AmtB has a role in scavenging ammonium at low concentrations. PMID:9573149

Functional and immunochemical characterization of a mutant of Escherichia coli energy uncoupled for lactose transport

International Nuclear Information System (INIS)

Herzlinger, D.; Carrasco, N.; Kaback, H.R.

1985-01-01

Right-side-out cytoplasmic membrane vesicles from Escherichia coli ML 308-22, a mutant ''uncoupled'' for beta-galactoside/H + symport are specifically defective in the ability to catalyze accumulation of methyl 1-thio-beta-D-galactopyranoside (TMG) in the presence of an H + electrochemical gradient (interior negative and alkaline). Furthermore, the rate of carrier-mediated efflux under nonenergized conditions is slow and unaffected by ambient pH from pH 5.5 to 7.5, and TMG-induced H + influx is only about 15% of that observed in vesicles containing wild-type lac permease (ML 308-225). Alternatively, ML 308-22 vesicles bind p-nitrophenyl alpha-D-galactopyranoside and monoclonal antibody 4B1 to the same extent as ML 308-225 vesicles and catalyze facilitated diffusion and equilibrium exchange as well as ML 308-225 vesicles. When entrance counterflow is studied with external substrate at saturating and subsaturating concentrations, it is apparent that the mutation simulates the effects of deuterium oxide. That is, the mutation has no effect on the rate or extent of counterflow when external substrate is saturating but stimulates the efficiency of counterflow when external substrate is below the apparent K/sub m/. Moreover, although replacement of protium with deuterium stimulates counterflow in ML 308-225 vesicles when external substrate is subsaturating, the isotope has no effect on the mutant vesicles under the same conditions

Molecular analysis of waxy mutants in rice

International Nuclear Information System (INIS)

Yatou, O.; Amano, E.

1990-01-01

Full text: The 'waxy' gene is a structural gene coding a glycosyl transferase which synthesises amylose in the endosperm tissue. 'Non-waxy' rice cultivars have an active gene and their amylose content is 18-25% depending upon gene performance and modifier genes. In 'waxy' rice, no amylose is found because the enzyme is absent. In mutants induced by gamma rays, neutrons, EI or EMS, amylose content ranged from 0 to 20%, i.e. there are intermediate phenotypes as well. Some of them had the same amount of the enzyme as a 'non-waxy' cultivar, even fully 'waxy' mutants showed a certain amount of the enzyme. This suggests that in mutants there may be no structural change in the enzyme gene but the enzyme produced might be less active. By molecular analysis of the mutants' genes it was found that only two mutants induced by thermal neutrons show structural alterations, the changes in other mutants are either too small to be detected by Southern analysis or are outside the structural gene in question. (author)

X-rays sensitive mammalian cell mutant

International Nuclear Information System (INIS)

Utsumi, Hiroshi

1982-01-01

A phenomenon that in x-ray-sensitive mammalian-cell mutants, cellular death due to x-ray radiation was not increased by caffeine, but on the contrary, the dead cells were resuscitated by it was discussed. The survival rate of mutant cells increased by caffein in a low concentration. This suggested that caffeine may have induced some mechanism to produce x-ray resistant mutant cells. Postirradiation treatment with caffeine increased considerably the survival rate of the mutant cells, and this suggested the existence of latent caffeine-sensitive potentially lethal damage repair system. This system, after a few hours, is thought to be substituted by caffeine-resistant repair system which is induced by caffeine, and this may be further substituted by x-ray-resistant repair system. The repair system was also induced by adenine. (Ueda, J.)

Radiation induced mutants in cassava (Manihot esculenta Crantz)

International Nuclear Information System (INIS)

Nayar, G.G.; Rajendran, P.G.

1987-01-01

Full text: Stem cuttings and true seeds of three promising cultivars of cassava were exposed respectively to 1 to 5 kR and 10 to 50 kR acute gamma rays from a 60 Co source. Treatments of stem cuttings beyond 5 kR and seeds beyond 50 kR were lethal. One mutant each in the cultivars M4, H-165 and H-2304 was obtained from the stem irradiated populations. Another mutant was found in the seed irradiated progeny of H-2304. The mutant of M4 is characterised by light green (chlorina) leaves. The mutant of H-165 shows significantly shorter petiole (22,5 against 35.2 cm) and narrow leaf lobes, while the H-2304 mutant shows speckled leaves, branching and early flowering. The mutant found in the seed irradiated progeny of H-2304 is having yellow tuber flesh indicating the presence of carotene. The mutants may be useful in studies related to basic information as well as in practical breeding. The chlorina mutant in M4 showed slow growth and high HCN content in leaves. Late branching may be a useful trait in the traditionally non-branching clones of cassava to maintain the desirable leaf area index during high leaf fall period. Early flowering could be useful in a recombinant breeding programme. The tuber yield of the short petiole mutant in H-165 increased by 20% - 25% through closer planting. The narrow leaf lobes of this mutant permit better light penetration to lower leaves. (author)

Inactivation of the Ecs ABC transporter of Staphylococcus aureus attenuates virulence by altering composition and function of bacterial wall.

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Ing-Marie Jonsson

2010-12-01

Full Text Available Ecs is an ATP-binding cassette (ABC transporter present in aerobic and facultative anaerobic gram-positive Firmicutes. Inactivation of Bacillus subtilis Ecs causes pleiotropic changes in the bacterial phenotype including inhibition of intramembrane proteolysis. The molecule(s transported by Ecs is (are still unknown.In this study we mutated the ecsAB operon in two Staphylococcus aureus strains, Newman and LS-1. Phenotypic and functional characterization of these Ecs deficient mutants revealed a defect in growth, increased autolysis and lysostaphin sensitivity, altered composition of cell wall proteins including the precursor form of staphylokinase and an altered bacterial surface texture. DNA microarray analysis indicated that the Ecs deficiency changed expression of the virulence factor regulator protein Rot accompanied by differential expression of membrane transport proteins, particularly ABC transporters and phosphate-specific transport systems, protein A, adhesins and capsular polysaccharide biosynthesis proteins. Virulence of the ecs mutants was studied in a mouse model of hematogenous S. aureus infection. Mice inoculated with the ecs mutant strains developed markedly milder infections than those inoculated with the wild-type strains and had consequently lower mortality, less weight loss, milder arthritis and decreased persistence of staphylococci in the kidneys. The ecs mutants had higher susceptibility to ribosomal antibiotics and plant alkaloids chelerythrine and sanguinarine.Our results show that Ecs is essential for staphylococcal virulence and antimicrobial resistance probably since the transport function of Ecs is essential for the normal structure and function of the cell wall. Thus targeting Ecs may be a new approach in combating staphylococcal infection.

Inactivation of the Ecs ABC transporter of Staphylococcus aureus attenuates virulence by altering composition and function of bacterial wall.

Science.gov (United States)

Jonsson, Ing-Marie; Juuti, Jarmo T; François, Patrice; AlMajidi, Rana; Pietiäinen, Milla; Girard, Myriam; Lindholm, Catharina; Saller, Manfred J; Driessen, Arnold J M; Kuusela, Pentti; Bokarewa, Maria; Schrenzel, Jacques; Kontinen, Vesa P

2010-12-02

Ecs is an ATP-binding cassette (ABC) transporter present in aerobic and facultative anaerobic gram-positive Firmicutes. Inactivation of Bacillus subtilis Ecs causes pleiotropic changes in the bacterial phenotype including inhibition of intramembrane proteolysis. The molecule(s) transported by Ecs is (are) still unknown. In this study we mutated the ecsAB operon in two Staphylococcus aureus strains, Newman and LS-1. Phenotypic and functional characterization of these Ecs deficient mutants revealed a defect in growth, increased autolysis and lysostaphin sensitivity, altered composition of cell wall proteins including the precursor form of staphylokinase and an altered bacterial surface texture. DNA microarray analysis indicated that the Ecs deficiency changed expression of the virulence factor regulator protein Rot accompanied by differential expression of membrane transport proteins, particularly ABC transporters and phosphate-specific transport systems, protein A, adhesins and capsular polysaccharide biosynthesis proteins. Virulence of the ecs mutants was studied in a mouse model of hematogenous S. aureus infection. Mice inoculated with the ecs mutant strains developed markedly milder infections than those inoculated with the wild-type strains and had consequently lower mortality, less weight loss, milder arthritis and decreased persistence of staphylococci in the kidneys. The ecs mutants had higher susceptibility to ribosomal antibiotics and plant alkaloids chelerythrine and sanguinarine. Our results show that Ecs is essential for staphylococcal virulence and antimicrobial resistance probably since the transport function of Ecs is essential for the normal structure and function of the cell wall. Thus targeting Ecs may be a new approach in combating staphylococcal infection.

Officially released mutant varieties in China

International Nuclear Information System (INIS)

Liu, L.; Van Zanten, L.; Shu, Q.Y.; Maluszynski, M.

2004-01-01

The use of mutation techniques for crop improvement in China has a long and well-established tradition of more than 50 years. As the result of intensive research in many institutes dealing with application of nuclear technologies more than 620 cultivars of 44 crop species have been released. Numerous mutant varieties have been grown on a large scale bringing significant economic impact, sustaining crop production and greatly contributing to increase of food production also in stress prone areas of the country. However, there is still missing information not only on the number of mutant varieties released in particular crop species but also on mutagens applied, selection approaches and on the use of mutants in cross breeding. Numerous Chinese scientists collected and systematized this information. Results of their work were often published in local scientific journals in the Chinese language and as such were unavailable to breeders from other countries. Having this in mind, we requested Dr. Liu Luxiang, the Director of the Department of Plant Mutation Breeding and Genetics, Institute for Application of Atomic Energy, Chinese Academy of Agricultural Sciences in Beijing to help us in finding as much information as possible on mutant varieties officially released in China. The data has been collected in close collaboration with his colleagues from various institutions all over the country and then evaluated, edited and prepared for publication by our team responsible for the FAO/IAEA Database of Officially Released Mutant Varieties. We would like to thank all Chinese colleagues who contributed to this list of Chinese mutant varieties. We hope that this publication will stimulate plant breeders in China to collect more information on released mutant varieties and especially on the use of mutated genes in cross breeding. (author)

Kharon1 null mutants of Leishmania mexicana are avirulent in mice and exhibit a cytokinesis defect within macrophages.

Directory of Open Access Journals (Sweden)

Khoa D Tran

Full Text Available In a variety of eukaryotes, flagella play important roles both in motility and as sensory organelles that monitor the extracellular environment. In the parasitic protozoan Leishmania mexicana, one glucose transporter isoform, LmxGT1, is targeted selectively to the flagellar membrane where it appears to play a role in glucose sensing. Trafficking of LmxGT1 to the flagellar membrane is dependent upon interaction with the KHARON1 protein that is located at the base of the flagellar axoneme. Remarkably, while Δkharon1 null mutants are viable as insect stage promastigotes, they are unable to survive as amastigotes inside host macrophages. Although Δkharon1 promastigotes enter macrophages and transform into amastigotes, these intracellular parasites are unable to execute cytokinesis and form multinucleate cells before dying. Notably, extracellular axenic amastigotes of Δkharon1 mutants replicate and divide normally, indicating a defect in the mutants that is only exhibited in the intra-macrophage environment. Although the flagella of Δkharon1 amastigotes adhere to the phagolysomal membrane of host macrophages, the morphology of the mutant flagella is often distorted. Additionally, these null mutants are completely avirulent following injection into BALB/c mice, underscoring the critical role of the KHARON1 protein for viability of intracellular amastigotes and disease in the animal model of leishmaniasis.

Thylakoid redox signals are integrated into organellar-gene-expression-dependent retrograde signalling in the prors1-1 mutant

Directory of Open Access Journals (Sweden)

Luca eTadini

2012-12-01

Full Text Available Perturbations in organellar gene expression (OGE and the thylakoid redox state (TRS activate retrograde signalling pathways that adaptively modify nuclear gene expression (NGE, according to developmental and metabolic needs. The prors1-1 mutation in Arabidopsis down-regulates the expression of the nuclear gene Prolyl-tRNA Synthetase1 (PRORS1 which acts in both plastids and mitochondria, thereby impairing protein synthesis in both organelles and triggering OGE-dependent retrograde signalling. Because the mutation also affects thylakoid electron transport, TRS-dependent signals may likewise have an impact on the changes in NGE observed in this genotype. In this study, we have investigated whether signals related to TRS are actually integrated into the OGE-dependent retrograde signalling pathway. To this end, the chaos mutation (for chlorophyll a/b binding protein harvesting-organelle specific, which shows a partial loss of PSII antennae proteins and thus a reduction in PSII light absorption capability, was introduced into the prors1-1 mutant background. The resulting double mutant displayed a prors1-1-like reduction in plastid translation rate and a chaos-like decrease in PSII antenna size, whereas the hyper-reduction of the thylakoid electron transport chain, caused by the prors1-1 mutation, was alleviated, as determined by monitoring chlorophyll (Chl fluorescence and thylakoid phosphorylation. Interestingly, a substantial fraction of the nucleus-encoded photosynthesis genes down-regulated in the prors1-1 mutant are expressed at nearly wild-type rates in prors1-1 chaos leaves, and this recovery is reflected in the steady-state levels of their protein products in the chloroplast. We therefore conclude that signals related to photosynthetic electron transport and TRS, and indirectly to carbohydrate metabolism and energy balance, are indeed fed into the OGE-dependent retrograde pathway to modulate NGE and adjust the abundance of chloroplast proteins.

Temperature sensitive riboflavin mutants of Penicillium vermiculatum Dangeard

International Nuclear Information System (INIS)

Mitra, J.; Chaudhari, K.L.

1974-01-01

Two temperature sensitive UV induced riboflavin mutants rib 1 and rib 6 have been physiologically and genetically characterized. The two mutants behave differently with regard to their temperature sensitivity. The rib 1 mutant exhibits a leaky growth in minimal medium between 15 0 C and 30 0 C but grows well when the medium is supplemented with riboflavin. At 35 0 C the growth response of the mutant is at its max. and at 40 0 C and below 15 0 C it ceases to grow. The rib 6 mutant which is red brown in colour shows wild type character at temp. below 25 0 C in minimal medium but requires riboflavin at 30 0 C and above. Heterokaryotic analysis revealed the nonallelic nature of the two temperature mutants. Genetic tests of allelic relationship between riboflavin markers by crossing were also done. (author)

The γ-Aminobutyrate Permease GabP Serves as the Third Proline Transporter of Bacillus subtilis

Science.gov (United States)

Zaprasis, Adrienne; Hoffmann, Tamara; Stannek, Lorena; Gunka, Katrin; Commichau, Fabian M.

2014-01-01

PutP and OpuE serve as proline transporters when this imino acid is used by Bacillus subtilis as a nutrient or as an osmostress protectant, respectively. The simultaneous inactivation of the PutP and OpuE systems still allows the utilization of proline as a nutrient. This growth phenotype pointed to the presence of a third proline transport system in B. subtilis. We took advantage of the sensitivity of a putP opuE double mutant to the toxic proline analog 3,4-dehydro-dl-proline (DHP) to identify this additional proline uptake system. DHP-resistant mutants were selected and found to be defective in the use of proline as a nutrient. Whole-genome resequencing of one of these strains provided the lead that the inactivation of the γ-aminobutyrate (GABA) transporter GabP was responsible for these phenotypes. DNA sequencing of the gabP gene in 14 additionally analyzed DHP-resistant strains confirmed this finding. Consistently, each of the DHP-resistant mutants was defective not only in the use of proline as a nutrient but also in the use of GABA as a nitrogen source. The same phenotype resulted from the targeted deletion of the gabP gene in a putP opuE mutant strain. Hence, the GabP carrier not only serves as an uptake system for GABA but also functions as the third proline transporter of B. subtilis. Uptake studies with radiolabeled GABA and proline confirmed this conclusion and provided information on the kinetic parameters of the GabP carrier for both of these substrates. PMID:24142252

The Lack of the Essential LptC Protein in the Trans-Envelope Lipopolysaccharide Transport Machine Is Circumvented by Suppressor Mutations in LptF, an Inner Membrane Component of the Escherichia coli Transporter

KAUST Repository

Benedet, Mattia; Falchi, Federica A.; Puccio, Simone; Di Benedetto, Cristiano; Peano, Clelia; Polissi, Alessandra; Deho, Gianni

2016-01-01

The lipopolysaccharide (LPS) transport (Lpt) system is responsible for transferring LPS from the periplasmic surface of the inner membrane (IM) to the outer leaflet of the outer membrane (OM), where it plays a crucial role in OM selective permeability. In E. coli seven essential proteins are assembled in an Lpt trans-envelope complex, which is conserved in gamma-Proteobacteria. LptBFG constitute the IMABC transporter, LptDE form the OM translocon for final LPS delivery, whereas LptC, an IM-anchored protein with a periplasmic domain, interacts with the IM ABC transporter, the periplasmic protein LptA, and LPS. Although essential, LptC can tolerate several mutations and its role in LPS transport is unclear. To get insights into the functional role of LptC in the Lpt machine we searched for viable mutants lacking LptC by applying a strong double selection for lptC deletion mutants. Genome sequencing of viable Delta lptC mutants revealed single amino acid substitutions at a unique position in the predicted large periplasmic domain of the IM component LptF (LptF(SupC)). In complementation tests, lptF(SupC) mutants suppress lethality of both Delta lptC and lptC conditional expressionmutants. Our data show that mutations in a specific residue of the predicted LptF periplasmic domain can compensate the lack of the essential protein LptC, implicate such LptF domain in the formation of the periplasmic bridge between the IM and OM complexes, and suggest that LptC may have evolved to improve the performance of an ancestral six-component Lpt machine.

The Lack of the Essential LptC Protein in the Trans-Envelope Lipopolysaccharide Transport Machine Is Circumvented by Suppressor Mutations in LptF, an Inner Membrane Component of the Escherichia coli Transporter

KAUST Repository

Benedet, Mattia

2016-08-16

The lipopolysaccharide (LPS) transport (Lpt) system is responsible for transferring LPS from the periplasmic surface of the inner membrane (IM) to the outer leaflet of the outer membrane (OM), where it plays a crucial role in OM selective permeability. In E. coli seven essential proteins are assembled in an Lpt trans-envelope complex, which is conserved in gamma-Proteobacteria. LptBFG constitute the IMABC transporter, LptDE form the OM translocon for final LPS delivery, whereas LptC, an IM-anchored protein with a periplasmic domain, interacts with the IM ABC transporter, the periplasmic protein LptA, and LPS. Although essential, LptC can tolerate several mutations and its role in LPS transport is unclear. To get insights into the functional role of LptC in the Lpt machine we searched for viable mutants lacking LptC by applying a strong double selection for lptC deletion mutants. Genome sequencing of viable Delta lptC mutants revealed single amino acid substitutions at a unique position in the predicted large periplasmic domain of the IM component LptF (LptF(SupC)). In complementation tests, lptF(SupC) mutants suppress lethality of both Delta lptC and lptC conditional expressionmutants. Our data show that mutations in a specific residue of the predicted LptF periplasmic domain can compensate the lack of the essential protein LptC, implicate such LptF domain in the formation of the periplasmic bridge between the IM and OM complexes, and suggest that LptC may have evolved to improve the performance of an ancestral six-component Lpt machine.

High yielding mutants of blackgram variety 'PH-25'

International Nuclear Information System (INIS)

Misra, R.C.; Mohapatra, B.D.; Panda, B.S.

2001-01-01

Seeds of blackgram (Vigna mungo L.) variety 'PH-5' were treated with chemical mutagens ethyl methanesulfonate (EMS), nitrosoguanidine (NG), maleic hydrazide (MH) and sodium azide (NaN 3 ), each at 3 different concentrations. Thirty six mutant lines developed from mutagenic treatments along with parent varieties were tested in M 4 generation. The mutants showed wide variation in most of the traits and multivariante D 2 analysis showed genetic divergence among themselves. Twenty of the thirty mutants showed genetic divergence from parent. Ten selected high yielding mutants were tested in M 5 . Yield and other productive traits of five high yielding mutants in M 4 and M 5 are presented

The two Na+ sites in the human serotonin transporter play distinct roles in the ion coupling and electrogenicity of transport.

Science.gov (United States)

Felts, Bruce; Pramod, Akula Bala; Sandtner, Walter; Burbach, Nathan; Bulling, Simon; Sitte, Harald H; Henry, L Keith

2014-01-17

Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na(+), Cl(-), and K(+) gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na(+)-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca(2+) (but not other cations) to functionally replace Na(+) for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca(2+) and Na(+) illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca(2+) promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na(+)-binding sites.

The Two Na+ Sites in the Human Serotonin Transporter Play Distinct Roles in the Ion Coupling and Electrogenicity of Transport*

Science.gov (United States)

Felts, Bruce; Pramod, Akula Bala; Sandtner, Walter; Burbach, Nathan; Bulling, Simon; Sitte, Harald H.; Henry, L. Keith

2014-01-01

Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na+, Cl−, and K+ gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na+-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca2+ (but not other cations) to functionally replace Na+ for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca2+ and Na+ illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca2+ promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na+-binding sites. PMID:24293367

Endoplasmic Reticulum-Targeted Subunit Toxins Provide a New Approach to Rescue Misfolded Mutant Proteins and Revert Cell Models of Genetic Diseases.

Directory of Open Access Journals (Sweden)

Humaira Adnan

Full Text Available Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD, to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subunit has evolved to mimic a misfolded protein and hijack the ERAD membrane translocon (dislocon, to effect cytosolic access and cytopathology. We show such toxins compete for ERAD to rescue endogenous misfolded proteins. Cholera toxin or verotoxin (Shiga toxin containing genetically inactivated (± an N-terminal polyleucine tail A subunit can, within 2-4 hrs, temporarily increase F508delCFTR protein, the major cystic fibrosis (CF mutant (5-10x, F508delCFTR Golgi maturation (<10x, cell surface expression (20x and chloride transport (2x in F508del CFTR transfected cells and patient-derived F508delCFTR bronchiolar epithelia, without apparent cytopathology. These toxoids also increase glucocerobrosidase (GCC in N370SGCC Gaucher Disease fibroblasts (3x, another ERAD-exacerbated misfiling disease. We identify a new, potentially benign approach to the treatment of certain genetic protein misfolding diseases.

Gamma-radiation Mutagenesis in Genetically Unstable Barley Mutants. Pt. 1. Chlorophyll Mutations in Allelic tw Mutants and Their Revertants

International Nuclear Information System (INIS)

Vaitkuniene, V.

1995-01-01

Genotypical environment is an essential factor determining the mutability of mutants of the same type. Decreased chlorophyll mutant frequency was a common characteristic of all tested tw type (tw, tw 1 , tw 2 ) mutants induced in barley c. 'Auksiniai II'. The mutability of all the tested revertants was close to that of the initial c. 'Auksiniai II'. (author). 9 refs., 2 tabs

Molecular analysis of mutants of the Neurospora adenylosuccinate ...

Indian Academy of Sciences (India)

2012-08-07

Aug 7, 2012 ... and mutants induced with X-ray, UV or chemical mutagens. ... We have sequenced the ad-8 locus from 13 of these mutants and identified the molecular nature ..... mutants in yeast by selection for constitutive behavior in pig-.

Gamma-ray induced mutants in castor (Ricinus communis L.)

International Nuclear Information System (INIS)

Janila, P.; Ashok Kumar, A.; Rajashekar Reddy, N.; Hemalatha, V.

2007-01-01

We report isolation of three recessive mutants in castor using dry seed irradiation with gamma rays. The crinkled leaf mutant (crf) was identified in K-55-112 M2 family and leafy mutant (lea) in H-55-577 M2 family; both are recessive lethal and thus maintained as heterozygotes. The cri mutant has highly wrinkled leaves resembling finger millet head and failed to enter reproductive phase, consequently did not produce seeds. The number of leaf lobes is reduced in lea mutant and though it produced spikes, the male and female flowers are converted to leafy appendages. The third mutant, fused (Ius) stem identified in H-55-617 M2 family is a recessive mutant. The branches of which are fused at the base and though each branch terminates in to monoceous spike like normal plant, the spike is highly condensed. The three mutants under report are valuable genetic stocks for development of linkage maps in castor, which is at infancy. (author)

Productive mutants of niger

International Nuclear Information System (INIS)

Misra, R.C.

2001-01-01

Seeds of six niger (Guizotia abyssinica Cass.) varieties ('GA-10', 'ONS-8', 'IGP-72', 'N-71', 'NB-9' and 'UN-4') were treated with 0.5, 0.75 and 1% ethyl methanesulphonate. After four generations of selection, 29 mutant lines were developed and those were evaluated from 1990-92 during Kharif (July to October) and Rabi (December to March) seasons. Average plant characteristics and yield data of four high yielding mutants along with 'IGP-76' (National Check), GA-10 (Zonal Check) and 'Semiliguda Local' (Local Check) are presented

Isolation and characterization of MMS-sensitive mutants of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Prakash, L.; Prakash, S.

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

Uncoupling in Secondary Transport Proteins. A Mechanistic Explanation for Mutants of lac Permease with an Uncoupled Phenotype

NARCIS (Netherlands)

Lolkema, J.S.; Poolman, B.

1995-01-01

The kinetic behavior of a H+-substrate symporter has been studied in which in addition to the unloaded (E) and fully loaded states (E.S.H) of the carrier also one of the binary complexes (E.S or E.H) may reorient its binding sites. This results in two types of uncoupled mutants, the ES leak and the

Isoenzymes performance of some rice varieties and their mutants

International Nuclear Information System (INIS)

Winarno, Ermin; Suliwarno, Ambyah; Ismachin, M.

1992-01-01

Isoenzymes performance of some rice varieties and their mutants. Genetics studies on alcohol dehydrogenase, malic enzyme, peroxidase, acid phosphase, and aminopeptidase isoenzymes were carried out on several groups of rice varieties and their mutant lines. The first groups consisted of Atomita I, Pelita I/1, A227/5, Mudgo, TN-1, and IR-26. The second group was Cisadane variety and its five mutants, namely OBS 18, OBS 208, OBS 297, OBS 306, and OBS 330. The third group was mutants line 627-10-3 and its mutants, namely 1063, 1066, 1067, 1076, and 1090. Isoenzymes extracts of the rice leaves were fractionated using polyacrylamide gel disc electrophoresis. The pattern of acid phosphate isoenzyme shows the specific character of rice mutants susceptible to brown plant hopper biotype 1. The gene(s) controlling malic enzyme in Cisadane's mutants is (are) estimated more resistant toward gamma irradiation than gene(s) responsible for controlling the other enzymes. Generally, the isoenzymes zymograms show that gene(s) controlling the mutants enzyme have undergone mutation. This case is shown by the changes of Rm value, as well as the amount and intensity of mutants bands. (authors). 7 refs., 7 figs

Mutants of Escherichia coli K-12 with enhanced resistance to ionizing radiation. 4. Peculiarities of recombination in Gamsup(r) mutants

International Nuclear Information System (INIS)

Bresler, S.E.; Kalinin, V.L.; Laneeva, N.I.

1984-01-01

Radioresistant mutant Gam sup(r) 444 differs from a wild type and from Gam sup(r) 445 mutant in decreased frequency of long episome heritage ORF 1 (pur E + -tsx + -proC + -lac + ) and F 14 (ilv + -argE + ), containing hot points of RecRecF - depending recombination and in increased frequency of chromosome mobilization and integrative suppression of temperature sensitive dna A46 mutation by sexual factor F. In this respect Gam sup(r) 444 mutant resembles rec BC sbs B mutant with RecF - recombination type

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

Science.gov (United States)

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.

Characterization of a Weak Allele of Zebrafish cloche Mutant

Science.gov (United States)

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

Analysis of Yellow Striped Mutants of Zea mays Reveals Novel Loci Contributing to Iron Deficiency Chlorosis

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David Chan-Rodriguez

2018-02-01

Full Text Available The micronutrient iron (Fe is essential for photosynthesis, respiration, and many other processes, but it is only sparingly soluble in aqueous solution, making adequate acquisition by plants a serious challenge. Fe is a limiting factor for plant growth on approximately 30% of the world’s arable lands. Moreover, Fe deficiency in humans is a global health issue, affecting 1.62 billion people, or about 25% of the world’s population. It is imperative that we gain a better understanding of the mechanisms that plants use to regulate iron homeostasis, since these will be important targets for future biofortification and crop improvement strategies. Grasses and non-grasses have evolved independent mechanisms for primary iron uptake from the soil. The grasses, which include most of the world’s staple grains, have evolved a distinct ‘chelation’ mechanism to acquire iron from the soil. Strong iron chelators called phytosiderophores (PSs are synthesized by grasses and secreted into the rhizosphere where they bind and solubilize Fe(III. The Fe(III-PS complex is then taken up into root cells via transporters specific for the Fe(III-PS complex. In this study, 31 novel, uncharacterized striped maize mutants available through the Maize Genetics Cooperation Stock Center (MGCSC were analyzed to determine whether their mutant phenotypes are caused by decreased iron. Many of these proved to be either pale yellow or white striped mutants. Complementation tests were performed by crossing the MGCSC mutants to ys1 and ys3 reference mutants. This allowed assignment of 10 ys1 alleles and 4 ys3 alleles among the novel mutants. In addition, four ys∗ mutant lines were identified that are not allelic to either ys1 or ys3. Three of these were characterized as being non-allelic to each other and as having low iron in leaves. These represent new genes involved in iron acquisition by maize, and future cloning of these genes may reveal novel aspects of the grass iron

Prevention of GABA reduction during dough fermentation using a baker's yeast dal81 mutant.

Science.gov (United States)

Ando, Akira; Nakamura, Toshihide

2016-10-01

γ-Aminobutyric acid (GABA) is consumed by yeasts during fermentation. To prevent GABA reduction in bread dough, a baker's yeast mutant AY77 deficient in GABA assimilation was characterized and utilized for wheat dough fermentation. An amber mutation in the DAL81 gene, which codes for a positive regulator of multiple nitrogen degradation pathways, was found in the AY77 strain. The qPCR analyses of genes involved in nitrogen utilization showed that transcriptional levels of the UGA1 and DUR3 genes encoding GABA transaminase and urea transporter, respectively, are severely decreased in the AY77 cells. The AY77 strain cultivated by fed-batch culture using cane molasses exhibited inferior gas production during dough fermentation compared to that of wild-type strain AY13. However, when fed with molasses containing 0.5% ammonium sulfate, the mutant strain exhibited gas production comparable to that of the AY13 strain. In contrast to the AY13 strain, which completely consumed GABA in dough within 5 h, the AY77 strain consumed no GABA under either culture condition. Dough fermentation with the dal81 mutant strain should be useful for suppression of GABA reduction in breads. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Commercialization Of Orchid Mutants For Floriculture Industry

International Nuclear Information System (INIS)

Sakinah Ariffin; Zaiton Ahmad

2014-01-01

Orchids are the main contributors to cut flower industry in Malaysia with an existing good market and a huge business potential. Orchid industry has been established in Malaysia since 1960s but only started to develop and expand since 1980s. Continuous development of new orchid varieties is essential to meet customers' demands. Orchid mutagenesis research using gamma irradiation at Malaysian Nuclear Agency has successfully generated a number of new orchid varieties with commercial potentials. Therefore, Nuclear Malaysia has collaborated with an industrial partner, Hexagon Green Sdn Bhd (HGSB), to carry out commercialization research on these mutants under a Technofund project entitled 'Pre-Commercialization of Mutant Orchids for Cut Flowers Industry' from July 2011 to July 2014. Through this collaboration, Dendrobium orchid mutant plants developed by Nuclear Malaysia were transferred to HGSB's commercial orchid nursery at Bukit Changgang Agrotechnology Park, Banting, Selangor, for mass-propagation. The activities include evaluations on plant growth performance, flower quality, post harvest and market potential of these mutants. Mutants with good field performance have been identified and filed for Plant Variety Protection (PVP) with Department of Agriculture Malaysia. This paper describes outputs from this collaboration and activities undertaken in commercializing these mutants. (author)

highroad Is a Carboxypetidase Induced by Retinoids to Clear Mutant Rhodopsin-1 in Drosophila Retinitis Pigmentosa Models

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Huai-Wei Huang

2018-02-01

Full Text Available Rhodopsins require retinoid chromophores for their function. In vertebrates, retinoids also serve as signaling molecules, but whether these molecules similarly regulate gene expression in Drosophila remains unclear. Here, we report the identification of a retinoid-inducible gene in Drosophila, highroad, which is required for photoreceptors to clear folding-defective mutant Rhodopsin-1 proteins. Specifically, knockdown or genetic deletion of highroad blocks the degradation of folding-defective Rhodopsin-1 mutant, ninaEG69D. Moreover, loss of highroad accelerates the age-related retinal degeneration phenotype of ninaEG69D mutants. Elevated highroad transcript levels are detected in ninaEG69D flies, and interestingly, deprivation of retinoids in the fly diet blocks this effect. Consistently, mutations in the retinoid transporter, santa maria, impairs the induction of highroad in ninaEG69D flies. In cultured S2 cells, highroad expression is induced by retinoic acid treatment. These results indicate that cellular quality-control mechanisms against misfolded Rhodopsin-1 involve regulation of gene expression by retinoids.

Identification of key uric acid synthesis pathway in a unique mutant silkworm Bombyx mori model of Parkinson's disease.

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Hiroko Tabunoki

Full Text Available Plasma uric acid (UA levels decrease following clinical progression and stage development of Parkinson's disease (PD. However, the molecular mechanisms underlying decreases in plasma UA levels remain unclear, and the potential to apply mutagenesis to a PD model has not previously been discovered. We identified a unique mutant of the silkworm Bombyx mori (B.mori op. Initially, we investigated the causality of the phenotypic "op" by microarray analysis using our constructed KAIKO functional annotation pipeline. Consequently, we found a novel UA synthesis-modulating pathway, from DJ-1 to xanthine oxidase, and established methods for large-scale analysis of gene expression in B. mori. We found that the mRNA levels of genes in this pathway were significantly lower in B. mori op mutants, indicating that downstream events in the signal transduction cascade might be prevented. Additionally, levels of B.mori tyrosine hydroxylase (TH and DJ-1 mRNA were significantly lower in the brain of B. mori op mutants. UA content was significantly lower in the B. mori op mutant tissues and hemolymph. The possibility that the B. mori op mutant might be due to loss of DJ-1 function was supported by the observed vulnerability to oxidative stress. These results suggest that UA synthesis, transport, elimination and accumulation are decreased by environmental oxidative stress in the B. mori op mutant. In the case of B. mori op mutants, the relatively low availability of UA appears to be due both to the oxidation of DJ-1 and to its expenditure to mitigate the effects of environmental oxidative stress. Our findings are expected to provide information needed to elucidate the molecular mechanism of decreased plasma UA levels in the clinical stage progression of PD.

Arabidopsis mutants lacking asparaginases develop normally but exhibit enhanced root inhibition by exogenous asparagine.

Science.gov (United States)

Ivanov, Ana; Kameka, Alexander; Pajak, Agnieszka; Bruneau, Luanne; Beyaert, Ronald; Hernández-Sebastià, Cinta; Marsolais, Frédéric

2012-06-01

Asparaginase catalyzes the degradation of L-asparagine to L-aspartic acid and ammonia, and is implicated in the catabolism of transported asparagine in sink tissues of higher plants. The Arabidopsis genome includes two genes, ASPGA1 and ASPGB1, belonging to distinct asparaginase subfamilies. Conditions of severe nitrogen limitation resulted in a slight decrease in seed size in wild-type Arabidopsis. However, this response was not observed in a homozygous T-DNA insertion mutant where ASPG genes had been inactivated. Under nitrogen-sufficient conditions, the ASPG mutant had elevated levels of free asparagine in mature seed. This phenotype was observed exclusively under conditions of low illumination, when a low ratio of carbon to nitrogen was translocated to the seed. Mutants deficient in one or both asparaginases were more sensitive than wild-type to inhibition of primary root elongation and root hair emergence by L-asparagine as a single nitrogen source. This enhanced inhibition was associated with increased accumulation of asparagine in the root of the double aspga1-1/-b1-1 mutant. This indicates that inhibition of root growth is likely elicited by asparagine itself or an asparagine-derived metabolite, other than the products of asparaginase, aspartic acid or ammonia. During germination, a fusion between the ASPGA1 promoter and beta-glucuronidase was expressed in endosperm cells starting at the micropylar end. Expression was initially high throughout the root and hypocotyl, but became restricted to the root tip after three days, which may indicate a transition to nitrogen-heterotrophic growth.

Serrated leaf mutant in mungbean (Vigna radiata (L) Wilczek)

International Nuclear Information System (INIS)

Malik, I.A.; Ghulam, Sarwar; Yousaf, Ali; Saleem, M.

1988-01-01

Dry dormant seeds of mungbean (Vigna radiata (L) Wilczek) were treated with gamma rays (15, 30 and 60 kR). The serrated leaf mutation was noticed in M 2 of cultivar Pak 32 treated with 60 kR. Cf 14 plants, 3 showed the altered leaf structure and the others were normal. The feature of this mutant was the deep serration of leaflet margins. The mutant had large thick leaflets with prominent venation. The mutant bred true in the M 3 and successive generation. Details of the morphological characteristics of the mutant are presented. The mutant exhibited slower growth particularly during the early stages of development, flowered later and attained shorter height. There was an increase in the number of pods, in seed weight and in seed protein content, but number of seed per pod was considerably reduced. The seed coat colour showed a change from green to yellowish green. In the mutant's flowers the stamina were placed much below the stigma level and the stigma sometimes protruded the corolla. Outcrossing of 4% recorded in some of the mutant lines revealed a reduced cleistogamy. The low number of seeds per pod in the mutant could be due to reduced pollen fertility. The mutant behaved as monogenic recessive. The symbols SL/sl are proposed for this allelic pair. The mutant may have use as a green manure crop because of its large foliage and for the breeders as a genetic marker

Leukemia-Associated Mutations in Nucleophosmin Alter Recognition by CRM1: Molecular Basis of Aberrant Transport.

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Igor Arregi

Full Text Available Nucleophosmin (NPM is a nucleocytoplasmic shuttling protein, normally enriched in nucleoli, that performs several activities related to cell growth. NPM mutations are characteristic of a subtype of acute myeloid leukemia (AML, where mutant NPM seems to play an oncogenic role. AML-associated NPM mutants exhibit altered subcellular traffic, being aberrantly located in the cytoplasm of leukoblasts. Exacerbated export of AML variants of NPM is mediated by the nuclear export receptor CRM1, and due, in part, to a mutationally acquired novel nuclear export signal (NES. To gain insight on the molecular basis of NPM transport in physiological and pathological conditions, we have evaluated the export efficiency of NPM in cells, and present new data indicating that, in normal conditions, wild type NPM is weakly exported by CRM1. On the other hand, we have found that AML-associated NPM mutants efficiently form complexes with CRM1HA (a mutant CRM1 with higher affinity for NESs, and we have quantitatively analyzed CRM1HA interaction with the NES motifs of these mutants, using fluorescence anisotropy and isothermal titration calorimetry. We have observed that the affinity of CRM1HA for these NESs is similar, which may help to explain the transport properties of the mutants. We also describe NPM recognition by the import machinery. Our combined cellular and biophysical studies shed further light on the determinants of NPM traffic, and how it is dramatically altered by AML-related mutations.

Bacterio-opsin mutants of Halobacterium halobium

Science.gov (United States)

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

Drugs, ionophoric peptides, and steroids as substrates of the yeast multidrug transporter Pdr5p

NARCIS (Netherlands)

Kolaczkowski, M; vanderRest, M; CybularzKolaczkowska, A; Soumillion, JP; Konings, WN; Goffeau, A

1996-01-01

Pdr5p is the yeast Saccharomyces cerevisiae ATP-binding cassette transporter conferring resistance to several unrelated drugs. Its high overproduction in Pdr1p transcription factor mutants allows us to study the molecular mechanism of multidrug transport and substrate specificity. We have developed

Characterization of the ZAT1p zinc transporter from Arabidopsis thaliana in microbial model organisms and reconstituted proteoliposomes.

Science.gov (United States)

Bloss, Tanja; Clemens, Stephan; Nies, Dietrich H

2002-03-01

The ZAT1p zinc transporter from Arabidopsis thaliana (L.) Heynh. is a member of the cation diffusion facilitator (CDF) protein family. When heterologously expressed in Escherichia coli, ZAT1p bound zinc in a metal blot. Binding of zinc occurred mainly to the hydrophilic amino acid region from H182 to H232. A ZAT1p/ZAT1p*Delta(M1-I25) protein mixture was purified and reconstituted into proteoliposomes. Uptake of zinc into the proteoliposomes did not require a proton gradient across the liposomal membrane. ZAT1p did not transport cobalt, and transported cadmium at only 1% of the zinc transport rate. ZAT1p functioned as an uptake system for 65Zn2+ in two strains of the Gram-negative bacterium Ralstonia metallidurans, which were different in their content of zinc-efflux systems. The ZAT1 gene did not rescue increased zinc sensitivity of a Delta ZRC1single-mutant strain or of a Delta ZRC1 Delta COT1 double-mutant strain of Saccharomyces cerevisiae, but ZAT1 complemented this phenotype in a Delta SpZRC1 mutant strain of Schizosaccharomyces pombe.

Daptomycin Tolerance in the Staphylococcus aureus pitA6 Mutant Is Due to Upregulation of the dlt Operon.

Science.gov (United States)

Mechler, Lukas; Bonetti, Eve-Julie; Reichert, Sebastian; Flötenmeyer, Matthias; Schrenzel, Jacques; Bertram, Ralph; François, Patrice; Götz, Friedrich

2016-05-01

Understanding the mechanisms of how bacteria become tolerant toward antibiotics during clinical therapy is a very important object. In a previous study, we showed that increased daptomycin (DAP) tolerance of Staphylococcus aureus was due to a point mutation in pitA (inorganic phosphate transporter) that led to intracellular accumulation of both inorganic phosphate (Pi) and polyphosphate (polyP). DAP tolerance in the pitA6 mutant differs from classical resistance mechanisms since there is no increase in the MIC. In this follow-up study, we demonstrate that DAP tolerance in the pitA6 mutant is not triggered by the accumulation of polyP. Transcriptome analysis revealed that 234 genes were at least 2.0-fold differentially expressed in the mutant. Particularly, genes involved in protein biosynthesis, carbohydrate and lipid metabolism, and replication and maintenance of DNA were downregulated. However, the most important change was the upregulation of the dlt operon, which is induced by the accumulation of intracellular Pi The GraXRS system, known as an activator of the dlt operon (d-alanylation of teichoic acids) and of the mprF gene (multiple peptide resistance factor), is not involved in DAP tolerance of the pitA6 mutant. In conclusion, DAP tolerance of the pitA6 mutant is due to an upregulation of the dlt operon, triggered directly or indirectly by the accumulation of Pi. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The reaction of hydroxylamine with bacteriorhodopsin studied with mutants that have altered photocycles: selective reactivity of different photointermediates.

Science.gov (United States)

Subramaniam, S; Marti, T; Rösselet, S J; Rothschild, K J; Khorana, H G

1991-01-01

The reaction of the retinylidene Schiff base in bacteriorhodopsin (bR) to the water-soluble reagent hydroxylamine is enhanced by greater than 2 orders of magnitude under illumination. We have used this reaction as a probe for changes in Schiff base reactivity during the photocycle of wild-type bR and mutants defective in proton transport. We report here that under illumination at pH 6, the D85N mutant has a 20-fold lower rate and the D212N mutant has a greater than 4-fold higher rate for the light-dependent reaction with hydroxylamine compared with wild-type bR. In contrast, the reactivities of wild-type bR and the D96N and T46V mutants are similar. It has been previously shown that the D96N and T46V replacements have no significant effect on the kinetics of "M" formation but have dramatic effects on rate of the decay of M. We therefore conclude that the hydroxylamine reaction occurs before formation of the M intermediate. Most likely it occurs at the "L" stage of the cycle and reflects increased water accessibility to the Schiff base due to a light-driven change in protein conformation. PMID:2006195

vph6 mutants of Saccharomyces cerevisiae require calcineurin for growth and are defective in vacuolar H(+)-ATPase assembly.

Science.gov (United States)

Hemenway, C S; Dolinski, K; Cardenas, M E; Hiller, M A; Jones, E W; Heitman, J

1995-11-01

We have characterized a Saccharomyces cerevisiae mutant strain that is hypersensitive to cyclosporin A (CsA) and FK506, immunosuppressants that inhibit calcineurin, a serine-threonine-specific phosphatase (PP2B). A single nuclear mutation, designated cev1 for calcineurin essential for viability, is responsible for the CsA-FK506-sensitive phenotype. The peptidyl-prolyl cis-trans isomerases cyclophilin A and FKBP12, respectively, mediate CsA and FK506 toxicity in the cev1 mutant strain. We demonstrate that cev1 is an allele of the VPH6 gene and that vph6 mutant strains fail to assemble the vacuolar H(+)-ATPase (V-ATPase). The VPH6 gene was mapped on chromosome VIII and is predicted to encode a 181-amino acid (21 kD) protein with no identity to other known proteins. We find that calcineurin is essential for viability in many mutant strains with defects in V-ATPase function or vacuolar acidification. In addition, we find that calcineurin modulates extracellular acidification in response to glucose, which we propose occurs via calcineurin regulation of the plasma membrane H(+)-ATPase PMA1. Taken together, our findings suggest calcineurin plays a general role in the regulation of cation transport and homeostasis.

The role of cysteine residues in the sulphate transporter, SHST1: construction of a functional cysteine-less transporter.

Science.gov (United States)

Howitt, Susan M

2005-05-20

We investigated the role of cysteine residues in the sulphate transporter, SHST1, with the aim of generating a functional cysteine-less variant. SHST1 contains five cysteine residues and none was essential for function. However, replacement of C421 resulted in a reduction in transport activity. Sulphate transport by C205 mutants was dependent on the size of the residue at this position. Alanine at position 205 resulted in a complete loss of function whereas leucine resulted in a 3-fold increase in sulphate transport relative to wild type SHST1. C205 is located in a putative intracellular loop and our results suggest that this loop may be important for sulphate transport. By replacing C205 with leucine and the other four cysteine residues with alanine, we constructed a cysteine-less variant of SHST1 that has transport characteristics indistinguishable from wild type. This construct will be useful for further structure and function studies of SHST1.

The ferrous iron transporter FtrABCD is required for the virulence of Brucella abortus 2308 in mice.

Science.gov (United States)

Elhassanny, Ahmed E M; Anderson, Eric S; Menscher, Evan A; Roop, R Martin

2013-06-01

Iron transport has been linked to the virulence of Brucella strains in both natural and experimental hosts. The genes designated BAB2_0837-0840 in the Brucella abortus 2308 genome sequence are predicted to encode a CupII-type ferrous iron transporter homologous to the FtrABCD transporter recently described in Bordetella. To study the role of the Brucella FtrABCD in iron transport, an isogenic ftrA mutant was constructed from B. abortus 2308. Compared with the parental strain, the B. abortus ftrA mutant displays a decreased capacity to use non-haem iron sources in vitro, a growth defect in a low iron medium that is enhanced at pH 6, and studies employing radiolabelled FeCl3 confirmed that FtrABCD transports ferrous iron. Transcription of the ftrA gene is induced in B. abortus 2308 in response to iron deprivation and exposure to acid pH, and similar to other Brucella iron acquisition genes that have been examined the iron-responsiveness of ftrA is dependent upon the iron response regulator Irr. The B. abortus ftrA mutant exhibits significant attenuation in both cultured murine macrophages and experimentally infected mice, supporting the proposition that ferrous iron is a critical iron source for these bacteria in the mammalian host. © 2013 John Wiley & Sons Ltd.

The application of shortened upper leaf mutant in barley breeding

International Nuclear Information System (INIS)

Jin Hua

2004-01-01

The shortened upper leaf mutant was induced from Fuji Nigo by γ-ray irradiation. Fuji Nigo, the mutant, cross-cut F 1 , F 2 and back-cross F 1 , F 2 were used to analyze mutant heredity by comparative study. The yield, chlorophyll content, light intensity, dry matter of mutant were investigated. The results showed that (1) the mutant character was controlled by a couple of nuclear genes which were partial dominance; (2) the transmittance of the mutant colony was better than that of Fuji Nigo and bottom dry matter was much more than that of Fuji Nigo; (3) under the condition of high fertilizer and high plant population , the yield of mutant was higher than that of Fuji Nigo; (4) the content of chlorophyll a in the mutant was higher than that in Fuji Nigo

Genetical, cytological and physiological studies on the induced mutants with special regard to effective methods for obtaining useful mutants in perennial woody plants

International Nuclear Information System (INIS)

Kukimura, H.; Ikeda, F.; Fujita, H.; Maeta, T.; Nakajima, K.; Katagiri, K.; Nakahira, K.; Somegou, M.

1975-01-01

The study was aimed at elucidating the biological aspects of artificially induced mutations in perennial tree crops and at promoting the utilization of such mutations in a practical breeding programme. A number of mutants obtained particularly in Cryptomeria and mulberry (Morus spp.) by means of gamma radiation were examined for their practical usefulness. Doses from 7.5 to 15.0 kR were used. In mulbery, some mutant strains showed increased shoot growth, and one mutant strain showed a remarkable increase also in rooting ability. Entire leaf mutants were investigated for their breeding behaviour. None of the mutant strains showed acquired disease resistance. Changes in the number of isozyme bands and different staining intensity was observed in all the mutant strains compared to the original strains

IFT20 modulates ciliary PDGFRα signaling by regulating the stability of Cbl E3 ubiquitin ligases

DEFF Research Database (Denmark)

Schmid, Fabian Marc; Schou, Kenneth Bødtker; Vilhelm, Martin Juel

2018-01-01

ciliogenesis, and ciliary localization of the receptor is required for its appropriate ligand-mediated activation by PDGF-AA. However, the mechanisms regulating sorting of PDGFRα and feedback inhibition of PDGFRα signaling at the cilium are unknown. Here, we provide evidence that intraflagellar transport...... protein 20 (IFT20) interacts with E3 ubiquitin ligases c-Cbl and Cbl-b and is required for Cbl-mediated ubiquitination and internalization of PDGFRα for feedback inhibition of receptor signaling. In wild-type cells treated with PDGF-AA, c-Cbl becomes enriched in the cilium, and the receptor...

Semi-dwarf mutants in triticale and wheat breeding

International Nuclear Information System (INIS)

Driscoll, C.J.

1984-01-01

The triticale lines Beagle and DR-IRA have been subjected to ionizing irradiation and chemical mutagenesis in order to produce semi-dwarf mutants. Beagle is 100 cm tall and DR-IRA 80 cm under average field conditions. A bulk then pedigree method is currently represented by 158 single plots of M 6 (or in some cases M 7 ) mutants that are from 5 to 35 cm shorter than the control variety. The shortest mutants are 65 cm in height. Forty of these mutants are also earlier flowering than the control varieties. Replicated yield testing will be conducted on confirmed mutants in 1983. Response to gibberellic acid of these mutants will also be determined. The Cornerstone male-sterility mutant (ms1c) on chromosome arm 4Aα has been combined with the GA-insensitive/reduced height gene Gai/Rht1 which is also on chromosome arm 4Aα. The ms1c mutant has also been combined with Gai/Rht2 on chromosome 4D and with both Gai/Rht1 and Gai/Rht2. The combination ms1c and Gai/Rht1 has been chosen as the basis of a composite cross. Thirteen varieties were tested with GA 3 and seven (Warigal, Aroona, Oxley, Banks, Avocet, Matipo and Toquifen) which contain Gai/Rht1 were crossed with ms1c Gai/Rht1 and entered into an interpollinating F 2 . The entire composite is homozygous for this semi-dwarf allele and selection will be practiced for increased height on a GA-insensitive background. (author)

Functional characterization of dopamine transporter in vivo using Drosophila melanogaster behavioral analysis.

Directory of Open Access Journals (Sweden)

Taro eUeno

2014-09-01

Full Text Available Dopamine mediates diverse functions such as motivation, reward, attention, learning/memory and sleep/arousal. Recent studies using model organisms including the fruit fly, have elucidated various physiological functions of dopamine, and identified specific neural circuits for these functions. Flies with mutations in the Drosophila dopamine transporter (dDAT gene show enhanced dopamine signaling, and short sleep and memory impairment phenotypes. However, understanding the mechanism by which dopamine signaling causes these phenotypes requires an understanding of the dynamics of dopamine release. Here we report the effects of dDAT expression on behavioral traits. We show that dDAT expression in a subset of dopaminergic neurons is sufficient for normal sleep. dDAT expression in other cell types such as Kenyon cells and glial cells can also rescue the short sleep phenotype of dDAT mutants. dDAT mutants also show a down-regulation of the D1-like dopamine receptor dDA1, and this phenotype is rescued when dDAT is expressed in the same cell types in which it rescues sleep. On the other hand, dDAT overexpression in mushroom bodies, which are the target of memory forming dopamine neurons, abolishes olfactory aversive memory. Our data demonstrate that expression of extrasynaptic dopamine transporters can rescue some aspects of dopamine signaling in dopamine transporter mutants. These results provide novel insights into regulatory systems that modulate dopamine signaling.

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

Science.gov (United States)

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.

Choline transporter mutations in severe congenital myasthenic syndrome disrupt transporter localization.

Science.gov (United States)

Wang, Haicui; Salter, Claire G; Refai, Osama; Hardy, Holly; Barwick, Katy E S; Akpulat, Ugur; Kvarnung, Malin; Chioza, Barry A; Harlalka, Gaurav; Taylan, Fulya; Sejersen, Thomas; Wright, Jane; Zimmerman, Holly H; Karakaya, Mert; Stüve, Burkhardt; Weis, Joachim; Schara, Ulrike; Russell, Mark A; Abdul-Rahman, Omar A; Chilton, John; Blakely, Randy D; Baple, Emma L; Cirak, Sebahattin; Crosby, Andrew H

2017-11-01

The presynaptic, high-affinity choline transporter is a critical determinant of signalling by the neurotransmitter acetylcholine at both central and peripheral cholinergic synapses, including the neuromuscular junction. Here we describe an autosomal recessive presynaptic congenital myasthenic syndrome presenting with a broad clinical phenotype due to homozygous choline transporter missense mutations. The clinical phenotype ranges from the classical presentation of a congenital myasthenic syndrome in one patient (p.Pro210Leu), to severe neurodevelopmental delay with brain atrophy (p.Ser94Arg) and extend the clinical outcomes to a more severe spectrum with infantile lethality (p.Val112Glu). Cells transfected with mutant transporter construct revealed a virtually complete loss of transport activity that was paralleled by a reduction in transporter cell surface expression. Consistent with these findings, studies to determine the impact of gene mutations on the trafficking of the Caenorhabditis elegans choline transporter orthologue revealed deficits in transporter export to axons and nerve terminals. These findings contrast with our previous findings in autosomal dominant distal hereditary motor neuropathy of a dominant-negative frameshift mutation at the C-terminus of choline transporter that was associated with significantly reduced, but not completely abrogated choline transporter function. Together our findings define divergent neuropathological outcomes arising from different classes of choline transporter mutation with distinct disease processes and modes of inheritance. These findings underscore the essential role played by the choline transporter in sustaining acetylcholine neurotransmission at both central and neuromuscular synapses, with important implications for treatment and drug selection. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Cytogenetic characteristics of soft wheat mutants under x-irradiation

International Nuclear Information System (INIS)

Shakaryan, Zh.O.; Avakyan, V.A.; Amirbekyan, V.A.

1981-01-01

Radiosensitivity of induced mutants of soft wheat is studied by criteria of frequency and character of changes in 1 and 2 divisions of meiosis. Two constant induced mutant forms of soft wheat were investigated. Mutant lines of squareheads with red ear (re) and erectoids 37/1 were obtained by X-ray irradiating hydride seeds F 1 of hybride combination of Alty-Agach Awnless 1. Seeds of mutants and initial kinds were exposed to X-rays at a dose of 10 kR. A conclusion may be drawn on the basis of studying the meiosis process in mutants and initial kinds of soft wheat on X-ray radiation that the mutants are more radiosensitive. This testifies to that that the induced mutants of soft wheat represent new genotypes in comparison with the initial kinds and differ from the latter not only in morphological characters but in the reaction norm with respect to external medium factors, i.e. the limit of possible changeability of the genotype has been extended [ru

Genetics of Ustilago violacea. I. Carotenoid mutants and carotenogenesis

International Nuclear Information System (INIS)

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

1975-01-01

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, γ-carotene; and one yellow mutant, β-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 white 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

Characterization of placental cholesterol transport

DEFF Research Database (Denmark)

Lindegaard, Marie L; Wassif, Christopher A; Vaisman, Boris

2008-01-01

Patients with Smith-Lemli-Opitz syndrome (SLOS) are born with multiple congenital abnormalities. Postnatal cholesterol supplementation is provided; however, it cannot correct developmental malformations due to in utero cholesterol deficit. Increased transport of cholesterol from maternal to fetal...... circulation might attenuate congenital malformations. The cholesterol transporters Abca1, Abcg1, and Sr-b1 are present in placenta; however, their potential role in placental transport remains undetermined. In mice, expression analyses showed that Abca1 and Abcg1 transcripts increased 2-3-fold between...... embryonic days 13.5 and 18.5 in placental tissue; whereas, Sr-b1 expression decreased. To examine the functional role of Abca1, Abcg1 and Sr-b1 we measured the maternal-fetal transfer of (14)C-cholesterol in corresponding mutant embryos. Disruption of either Abca1 or Sr-b1 decreased cholesterol transfer...

Growth of desferrioxamine-deficient Streptomyces mutants through xenosiderophore piracy of airborne fungal contaminations.

Science.gov (United States)

Arias, Anthony Argüelles; Lambert, Stéphany; Martinet, Loïc; Adam, Delphine; Tenconi, Elodie; Hayette, Marie-Pierre; Ongena, Marc; Rigali, Sébastien

2015-07-01

Due to the necessity of iron for housekeeping functions, nutrition, morphogenesis and secondary metabolite production, siderophore piracy could be a key strategy in soil and substrate colonization by microorganisms. Here we report that mutants of bacterium Streptomyces coelicolor unable to produce desferrioxamine siderophores could recover growth when the plates were contaminated by indoor air spores of a Penicillium species and Engyodontium album. UPLC-ESI-MS analysis revealed that the HPLC fractions with the extracellular 'resuscitation' factors of the Penicillium isolate were only those that contained siderophores, i.e. Fe-dimerum acid, ferrichrome, fusarinine C and coprogen. The restored growth of the Streptomyces mutants devoid of desferrioxamine is most likely mediated through xenosiderophore uptake as the cultivability depends on the gene encoding the ABC-transporter-associated DesE siderophore-binding protein. That a filamentous fungus allows the growth of desferrioxamine non-producing Streptomyces in cocultures confirms that xenosiderophore piracy plays a vital role in nutritional interactions between these taxonomically unrelated filamentous microorganisms. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A Nostoc punctiforme sugar transporter necessary to establish a Cyanobacterium-plant symbiosis.

Science.gov (United States)

Ekman, Martin; Picossi, Silvia; Campbell, Elsie L; Meeks, John C; Flores, Enrique

2013-04-01

In cyanobacteria-plant symbioses, the symbiotic nitrogen-fixing cyanobacterium has low photosynthetic activity and is supplemented by sugars provided by the plant partner. Which sugars and cyanobacterial sugar uptake mechanism(s) are involved in the symbiosis, however, is unknown. Mutants of the symbiotically competent, facultatively heterotrophic cyanobacterium Nostoc punctiforme were constructed bearing a neomycin resistance gene cassette replacing genes in a putative sugar transport gene cluster. Results of transport activity assays using (14)C-labeled fructose and glucose and tests of heterotrophic growth with these sugars enabled the identification of an ATP-binding cassette-type transporter for fructose (Frt), a major facilitator permease for glucose (GlcP), and a porin needed for the optimal uptake of both fructose and glucose. Analysis of green fluorescent protein fluorescence in strains of N. punctiforme bearing frt::gfp fusions showed high expression in vegetative cells and akinetes, variable expression in hormogonia, and no expression in heterocysts. The symbiotic efficiency of N. punctiforme sugar transport mutants was investigated by testing their ability to infect a nonvascular plant partner, the hornwort Anthoceros punctatus. Strains that were specifically unable to transport glucose did not infect the plant. These results imply a role for GlcP in establishing symbiosis under the conditions used in this work.

Spectrum of induced floral mutants in Petunia

International Nuclear Information System (INIS)

Padmaja, V.; Sudhakar, P.

1987-01-01

A total of six floral mutants of garden Petunia isolated from the populations raised from the seed treatment with γ-rays, 2, 4-D and sodium azide are described. Five of the mutants viz. stellata, Campyloflora, Rubriflora mixed, Grandiflora and Albiflora mixed originated as segregants in M 2 generation while the chimeral floral phenotype was expressed in M 1 generation itself. Breeding behaviour of these horticulturally interesting altered floral phenotypes were studied in subsequent generations and appropriate conclusions were drawn regarding mode of inheritance of the mutant traits. 15 refs., 4 figures, 1 table. (author)

Chemotaxis-defective mutants of the nematode Caenorhabditis elegans.

Science.gov (United States)

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)

Science.gov (United States)

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

Promising semi-dwarf mutant in wheat variety K68

Energy Technology Data Exchange (ETDEWEB)

Kumar, D [Banaras Hindu Univ. (India). Dept. of Genetics and Plant Breeding

1977-04-01

A semi-dwarf mutant (HUW-SDf 1) was induced from common wheat Var. K68 through the exposure of /sup 60/Co ..gamma..-rays at 15 kR. This mutant along with other induced mutants and control was assessed for yield components, yield and grain quality (M/sub 4/ generation); internode length reduction pattern and the yielding ability at three levels of nitrogen (M/sub 5/ generation). The mutant was significantly shorter in height and almost equal in tillers per plant and grains per spike to K68. However, it showed marked reduction in spike length and spikelets per spike. On the other hand, it possessed significantly higher (50.04 g) 1000-grain weight against control (41.15 g). The mutant gave 56.0% higher yield than the control. Grain quality studies indicated that the mutant possessed significantly higher (14.15%) total protein than K68. It was equally as good as K68 in lysine content. Pelshenke value (62.5 min) of the mutant indicated medium hard nature of gluten as compared to hard nature (198.0) of the control. The mutant showed 24.0% reduction in total culm length compared to K68. Reduction occurred due to maximum and almost equal reduction in 5th and 4th internodes (ca 34.0%) followed by 3rd, 2nd and 1st. The mutant showed similar yield and yield response to increasing nitrogen levels (80 to 160 kg per ha.) as for current commercial semi-dwarf varieties.

Spectrum of mutant characters utilized in developing improved cultivars

International Nuclear Information System (INIS)

Donini, B.; Kawai, T.; Micke, A.

1984-01-01

Although about 500 cultivars are known to have been developed by using induced mutations, the range of mutant traits seems to be rather narrow. Mutant traits have mostly been used that can be detected visually on an individual plant basis. However, in the background of such mutants other valuable mutations have been found in later generations. In cross-breeding with mutants valuable characteristics occurred, which could not be predicted from the phenotypes of the parents. It is concluded that improved attributes in the released mutant varieties do not comprise the entire genetic variation that could derive from mutagenesis. Current selection techniques are inadequate to exploit the full potential of mutagenesis for plant breeding. (author)

Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors

Science.gov (United States)

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.

Circulation of Pneumocystis dihydropteroate synthase mutants in France.

Science.gov (United States)

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 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.

Mutant number distribution in an exponentially growing population

Science.gov (United States)

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.

Characteristics of mutant lines of sweet potato flour

International Nuclear Information System (INIS)

Aryanti

2012-01-01

Research on mutation induction of sweet potato Sari variety has been conducted. Flour mutant lines were obtained from selection of M1V5 tubers irradiated by gamma rays at the dose of 10 Gy. Flour was made by peeling of tubers, then dried, blended and sieved. The quality test of flour have been done by measuring degree of whiteness, proximate, amylose contents, water content, soluble water, swelling power, and flour characteristics. The result of this work showed that flour of C6.26.13 mutant line had higher protein content than the parent plant with concentration of 3.62 % and its amylose content was also higher than the other mutant lines. The soluble water value of mutant lines were significant different compared to the parent plant from 1.82 to 2.25 % and swelling power from 4.28 to 5.55 %. The flour granule of the mutant line was different compared to the parent plant. (author)

Snapin-regulated late endosomal transport is critical for efficient autophagy-lysosomal function in neurons.

Science.gov (United States)

Cai, Qian; Lu, Li; Tian, Jin-Hua; Zhu, Yi-Bing; Qiao, Haifa; Sheng, Zu-Hang

2010-10-06

Neuron maintenance and survival require late endocytic transport from distal processes to the soma where lysosomes are predominantly localized. Here, we report a role for Snapin in attaching dynein to late endosomes through its intermediate chain (DIC). snapin(-/-) neurons exhibit aberrant accumulation of immature lysosomes, clustering and impaired retrograde transport of late endosomes along processes, reduced lysosomal proteolysis due to impaired delivery of internalized proteins and hydrolase precursors from late endosomes to lysosomes, and impaired clearance of autolysosomes, combined with reduced neuron viability and neurodegeneration. The phenotypes are rescued by expressing the snapin transgene, but not the DIC-binding-defective Snapin-L99K mutant. Snapin overexpression in wild-type neurons enhances late endocytic transport and lysosomal function, whereas expressing the mutant defective in Snapin-DIC coupling shows a dominant-negative effect. Altogether, our study highlights new mechanistic insights into how Snapin-DIC coordinates retrograde transport and late endosomal-lysosomal trafficking critical for autophagy-lysosomal function, and thus neuronal homeostasis. Copyright © 2010 Elsevier Inc. All rights reserved.

Functional characterization of the Bradyrhizobium japonicum modA and modB genes involved in molybdenum transport.

Science.gov (United States)

Delgado, María J; Tresierra-Ayala, Alvaro; Talbi, Chouhra; Bedmar, Eulogio J

2006-01-01

A modABC gene cluster that encodes an ABC-type, high-affinity molybdate transporter from Bradyrhizobium japonicum has been isolated and characterized. B. japonicum modA and modB mutant strains were unable to grow aerobically or anaerobically with nitrate as nitrogen source or as respiratory substrate, respectively, and lacked nitrate reductase activity. The nitrogen-fixing ability of the mod mutants in symbiotic association with soybean plants grown in a Mo-deficient mineral solution was severely impaired. Addition of molybdate to the bacterial growth medium or to the plant mineral solution fully restored the wild-type phenotype. Because the amount of molybdate required for suppression of the mutant phenotype either under free-living or under symbiotic conditions was dependent on sulphate concentration, it is likely that a sulphate transporter is also involved in Mo uptake in B. japonicum. The promoter region of the modABC genes has been characterized by primer extension. Reverse transcription and expression of a transcriptional fusion, P(modA)-lacZ, was detected only in a B. japonicum modA mutant grown in a medium without molybdate supplementation. These findings indicate that transcription of the B. japonicum modABC genes is repressed by molybdate.

Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

OpenAIRE

Hirata, Hiromi; Carta, Eloisa; Yamanaka, Iori; Harvey, Robert J.; Kuwada, John Y.

2010-01-01

Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Recently, in vivo analysis of glycinergic synaptic transmission has been pursued in zebrafish using molecular genetics. An ENU mutagenesis screen identified two behavioral mutants that are defective in glycinergic synaptic transmission. Zebrafish bandoneon (beo) mutants have a defect in glrbb, one of the duplicated glycine receptor (GlyR) β subunit genes. These mutants exhibit a loss of glycinergic synaptic ...

X-ray-induced mutants resistant to 8-azaguanine

International Nuclear Information System (INIS)

Carver, J.H.; Dewey, W.C.; Hopwood, L.E.

1976-01-01

Asynchronous Chinese hamster ovary cells were irradiated and colony survival in Alpha MEM medium with dialyzed serum was determined with or without 15 μg/ml 8-Azaguanine (AG). Data indicated that a reproducible assay for the system was dependent upon controlling cell density at least two days prior to induction as well as throughout the expression period. Generally, spontaneous and radiation-induced mutant frequencies decreased when cell densities exceeded a critical density of 3-6 x 10 4 cells/cm 2 . Infrequently, the critical density was exceeded by a factor of two with no observed decrease, possibly correlated with a longer cell doubling time. Drug depletion artifacts can occur because of drug degradation, or because wild-type cells utilize the drug or produce conditions which reduce uptake of the drug. Thus, as the effective drug concentration is lowered, the observed mutant frequency increases because a spectrum of mutants resistant to only low concentrations can now survive. In fact, refeeding with AG at intervals during the incubation period lowered spontaneous and radiation-induced frequencies approx. 5-fold. Therefore, to standardize conditions, cells were trypsinized at the end of the expression time and replated at a constant cell number for mutant selection by AG. Over two generations of growth during the expression period were required for optimal manifestation of induced mutants, and when densities were kept below 4 x 10 4 cells/cm 2 at all times, observed mutant frequencies did not change significantly over a period between 80 and 140 h post-induction (over 4 generations for irradiated cells and over 6 generations for controls). Previous reports of observed mutant frequencies decreasing beyond three generations may be due to cell interaction prior to mutant selection

Dynamic microtubule organization and mitochondrial transport are regulated by distinct Kinesin-1 pathways

Directory of Open Access Journals (Sweden)

Anna Melkov

2015-12-01

Full Text Available The microtubule (MT plus-end motor kinesin heavy chain (Khc is well known for its role in long distance cargo transport. Recent evidence showed that Khc is also required for the organization of the cellular MT network by mediating MT sliding. We found that mutations in Khc and the gene of its adaptor protein, kinesin light chain (Klc resulted in identical bristle morphology defects, with the upper part of the bristle being thinner and flatter than normal and failing to taper towards the bristle tip. We demonstrate that bristle mitochondria transport requires Khc but not Klc as a competing force to dynein heavy chain (Dhc. Surprisingly, we demonstrate for the first time that Dhc is the primary motor for both anterograde and retrograde fast mitochondria transport. We found that the upper part of Khc and Klc mutant bristles lacked stable MTs. When following dynamic MT polymerization via the use of GFP-tagged end-binding protein 1 (EB1, it was noted that at Khc and Klc mutant bristle tips, dynamic MTs significantly deviated from the bristle parallel growth axis, relative to wild-type bristles. We also observed that GFP-EB1 failed to concentrate as a focus at the tip of Khc and Klc mutant bristles. We propose that the failure of bristle tapering is due to defects in directing dynamic MTs at the growing tip. Thus, we reveal a new function for Khc and Klc in directing dynamic MTs during polarized cell growth. Moreover, we also demonstrate a novel mode of coordination in mitochondrial transport between Khc and Dhc.

The research progress on plant mutant germplasm resources in China

International Nuclear Information System (INIS)

He Cexi; Ji Linzhen; Zhao Shirong

1991-07-01

Mutants induced by nuclear radiation or other mutagens are new artificial germplasm resources. Some mutants have been applied in plant breeding and great achievements have been reached. The status and progress on the collection, identification and utilization of mutants in China are introduced. A proposal for developing mutant germplasm resources with good agronomic characters is suggested

Radiation studies in Cajanus cajan: meiotic behaviour in some M/sub 2/ mutants

Energy Technology Data Exchange (ETDEWEB)

Sinha, S.S.N.; Akhaury, S.B. (Ranchi Univ. (India). Dept. of Botany)

1982-01-01

A qualitative study of the mutants produced in M/sub 2/ generation has been made. The mutants were classified as: (1) chlorophyll mutant, (2) morphological mutant, (3) pollen mutant, (4) semi-sterile and (5) sterile mutant. Cytological investigations of pollen mutants, sterile and semi-sterile mutants have revealed that these mutants generally arise at higher dose levels (20 Kr and 25 Kr).

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

Directory of Open Access Journals (Sweden)

Edson Jiovany Ramírez-Nava

2017-10-01

Full Text Available 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.

Phanerochaete mutants with enhanced ligninolytic activity

International Nuclear Information System (INIS)

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

1994-01-01

In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organo pollutants in soils and aqueous media. Most of the organic compounds are degraded under ligninolytic conditions with the involvement of the extracellular enzymes, lignin peroxidases, and manganese-dependent peroxidases, which are produced as secondary metabolites triggered by conditions of nutrient starvation (e.g., nitrogen limitation). The fungus and its enzymes can thus provide alternative technologies for bioremediation, bio pulping, bio bleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated, or are hyper producers or super secretors of key enzymes under enriched conditions. Through UV-light and γ-ray mutagenesis, we have isolated a variety of mutants, some of which produce key enzymes of the ligninolytic system under high-nitrogen growth conditions. One of the mutants, 76UV, produced 272 U of lignin peroxidases enzyme activity/L after 9 d under high nitrogen (although the parent strain does not produce this enzyme under these conditions). The mutant and the parent strains produced up to 54 and 62 U/L, respectively, of the enzyme activity under low nitrogen growth conditions during this period. In some experiments, the mutant showed 281 U/L of enzyme activity under high nitrogen after 17 d

Isolation of New Gravitropic Mutants under Hypergravity Conditions.

Science.gov (United States)

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 .

Inactivation of carbenicillin by some radioresistant mutant strains

International Nuclear Information System (INIS)

Zahiera, T.S.; Mahmoud, M.I.; Bashandy, A.A.

1990-01-01

Sensitivity test of five bacterial species to carbenicillin was performed microbiologically. The bacterial species were previously isolated from high level radiation environment. All the studied species could either highly decrease the antibiotic activity or even inactivate it completely. Detailed study of the inactivation of carbenicillin by the radioresistant mutant strains B. Laterosporus, B. firmus and M. roseus was performed, in the present study. Using high performace liquid chromatography technique. The gram-positive m. roseus mutant strain seemed to be the most active mutant in degrading the antibiotic. The left over of the antibiotic attained a value of 9% of the original amount after 14 day incubation of the antibiotic with this mutant strain, while the value of the left over reached 36% and 32% after the same period of incubation with the mutants B. laterosporus and B. firmus respectively. In the case of bacillus species, the degradation of the antibiotic started at the same moment when it was added to the bacterial cultures. This fact may indicate that the inactivation of the studied antibiotic by these bacillus species was due to extracellular enzymes extracted rapidly in the surrounding medium. In the case of M. roseus the inactivation process started later. after the addition of the antibiotic to the mutant culture

Agronomic performance of old soybean variety 'Altona' derived mutants

International Nuclear Information System (INIS)

Hodosne, K.G.; Heszky, L.E.

2001-01-01

An induced mutation program has been initiated at the Department of Genetics and Plant Breeding to develop early maturing cultivars with good yielding capacity. Some new mutants have been produced by irradiation of variety Altona with 60 Co gamma rays. Ten years of breeding resulted in two new mutant varieties named 'Noventa' and 'Gate 511'. The present study deals with agronomic performance of these mutants. Registered soybean varieties Altona and 'McCall' as well as Altona derived mutants (Gate 511 and Noventa) have been compared

Analysis of AtCry1 and Mutants

Science.gov (United States)

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.

IFT46 plays an essential role in cilia development

Science.gov (United States)

Lee, Mi-Sun; Hwang, Kyu-Seok; Oh, Hyun-Woo; Ji-Ae, Kim; Kim, Hyun-Taek; Cho, Hyun-Soo; Lee, Jeong-Ju; Ko, Je Yeong; Choi, Jung-Hwa; Jeong, Yun-Mi; You, Kwan-Hee; Kim, Joon; Park, Doo-Sang; Nam, Ki-Hoan; Aizawa, Shinichi; Kiyonari, Hiroshi; Shioi, Go; Park, Jong-Hoon; Zhou, Weibin; Kim, Nam-Soon; Kim, Cheol-Hee

2015-01-01

Cilia are microtubule-based structures that project into the extracellular space. Ciliary defects are associated with several human diseases, including polycystic kidney disease, primary ciliary dyskinesia, left-right axis patterning, hydrocephalus and retinal degeneration. However, the genetic and cellular biological control of ciliogenesis remains poorly understood. The IFT46 is one of the highly conserved intraflagellar transport complex B proteins. In zebrafish, ift46 is expressed in various ciliated tissues such as Kupffer’s vesicle, pronephric ducts, ears and spinal cord. We show that ift46 is localized to the basal body. Knockdown of ift46 gene results in multiple phenotypes associated with various ciliopathies including kidney cysts, pericardial edema and ventral axis curvature. In ift46 morphants, cilia in kidney and spinal canal are shortened and abnormal. Similar ciliary defects are observed in otic vesicles, lateral line hair cells, olfactory pits, but not in Kupffer’s vesicle. To explore the functions of Ift46 during mouse development, we have generated Ift46 knock-out mice. The Ift46 mutants have developmental defects in brain, neural tube and heart. In particular Ift46(−/−) homozygotes displays randomization of the embryo heart looping, which is a hallmark of defective left-right (L/R) axis patterning. Taken together, our results demonstrated that IFT46 has an essential role in vertebrate ciliary development. PMID:25722189

Biochemical characteristics of mutant lines of currant tomato

International Nuclear Information System (INIS)

Gorbatenko, I.Yu.; Khrustaleva, V.V.; Shcherbakov, V.K.

1988-01-01

The currant tomato is used in breeding for fruit quality. It contains up to 50 mg% ascorbic acid, a large quantity of sugar and 8-10% of dry matter. The weight of the fruit, however, does not exceed 1.2-1.5 g. The plants have long, spreading and very branchy stems. Gamma ray induced mutants of currant tomato were used, as initial material in breeding for fruit quality in varieties suitable for mechanized harvesting. The research was carried out mainly at the Department of Vegetable Growing Ukrainian Scientific Research Institute of Irrigation Farming. The regional variety Lebyazhinskij (suitable for mechanized harvesting) was adopted as the standard. Its fruits contain: 5.6% dry matter, 2.7% sugars, 0.543% titrated acidity, 26.6 mg/100 g ascorbic acid, 0.425 mg% carotene and 0.35% cellulose. The biochemical characteristics of the tomato mutants are shown. In terms of fruit dry matter, all mutants surpassed the standard. The acidity and the ascorbic acid content varied considerably. Most noteworthy in terms of carotene were the lines GP-5, GP-9 and GP-12. An important factor in the production of tomato paste is the fruit cellulose content. The lowest cellulose content is found in mutant GP-3. As shown, all of the mutants were early ripening. The mutants surpassed the standard in simultaneous fruit ripening. Mutant lines GP-3, GP-6, GP-9 and GP-12 will be used in the breeding programme for improving fruit quality of varieties suitable for mechanized harvesting

Genetic analyses of nonfluorescent root mutants induced by mutagenesis in soybean

International Nuclear Information System (INIS)

Sawada, S.; Palmer, R.G.

1987-01-01

Nonfluorescent root mutants in soybean [Glycine max (L.) Merr.] are useful as markers in genetic studies and in tissue culture research. Our objective was to obtain mutagen-induced nonfluorescent root mutants and to conduct genetic studies with them. Thirteen nonfluorescent mutants were detected among 154016 seedlings derived from soybean lines treated with six mutagens. One of these mutants, derived from Williams treated with 20 kR gamma rays, did not correspond to any of the known (standard) nonfluorescent spontaneous mutants. This is the first mutagen-induced nonfluorescent root mutant in soybean. It was assigned Genetic Type Collection no. T285 and the gene symbol fr5 fr5. The fr5 allele was not located on trisomics A, B, or C and was not linked to five chlorophyll-deficient mutants (y9, y11, y12, y13, and y20-k2) or flower color mutant w1. The remaining nonfluorescent root mutants were at the same loci as known spontaneous mutants; i.e., four had the fr1 allele, five had the fr2 allele, and three had the fr4 allele

Genetical, cytological and physiological studies on the induced mutants with special regard to effective methods for obtaining useful mutants in perennial woody plant

International Nuclear Information System (INIS)

Kukimura, H.; Ikeda, F.; Fujita, H.; Maeta, T.; Nakajima, K.; Katagiri, K.; Nakahira, K.; Somegou, M.

1976-01-01

The plants studied included apple trees, cryptomeria (japanese cedar) and mulberry. In apple, dwarf and compact types of mutants from cv. Fuji were found to be graft incompatible on Maruba-kaido(Malus prunifolia) rootstock. In Sunki mandarin(Citrus sunki), the number of nucellar embryo per seed was affected by gamma-irradiation, and morphological mutants from nucellar seedlings were obtained at high rate by irradiation at floral bud stage with 2kR exposure. In Cryptomeria, re-irradiated waxless mutants by gamma-rays showed very high rate of somatic mutation when compared to other morphological mutants. Pollen sterility and pollen shaped PMC were found in the most of gamma-induced-mutants. Mutants forming pollen shaped PMC had a genetical tendency of continuous male flower bud formation for a longer term. With mulberry, time of sprouting of induced mutants differed from the originals. Ability of root initiation of semi-softwood cuttings in morphological mutants were tested. Cytochimera induction were found at considerably high rate when actively growing diploid plants were irradiated by gamma-rays. Eight kinds of cytochimeras were induced. Frequency of 2-4-4 was extremely high(approx. 50%), then 4-2-2 and 2-4-2 chimeras followed. Seven kinds were induced by semi-acute irradiation(200R/h), while 4 kinds by acute irradiation(5kR/h). By breeding test it was cleared that the elongate and entire leaf was sexually transmissible, whereas the 'dwarf' was not obvious and the 'marginally curledleaf' was not transmissible. Pyronin-methylgreen staining method proved to be useful in some morphological mutants to distinguish the histo-genetical differences which exist in the shoot apex.

Development of high yielding mutants in lentil

International Nuclear Information System (INIS)

Rajput, M.A.; Sarwar, G.; Siddiqui, K.A.

2001-01-01

Full text: Lentil (Lens culinaris Medik.) locally known as Masoor, is the second most important rabi pulse crop, after chickpea, in Pakistan. It is cultivated on an area of over 63,400 ha, which constitutes about 4.83% of the total area under pulses. The annual production of the crop is 28,200 tones with an average yield of 445 kg/ha. Yield at the national level is very low, about one-half of the world's yield, which is mainly due to non-availability of high yield potential genotypes. Keeping in view the importance of mutants in developing a large number of new varieties, an induced mutations programme was initiated at AEARC, Tandojam during 1987-88, to develop high yielding varieties in lentil. For this, seeds of two lentil varieties, 'Masoor-85' and 'ICARDA-8' had been irradiated with gamma-rays ranging from 100-600 Gy in NIAB, Faisalabad during 1990. Selections were made in M2 on the basis of earliness, plant height, branches/plant and 100 grain weight. After confirming these mutants in M3 they were promoted in station yield trials and studied continuously for three consecutive years (1993- 1995). Overall results revealed that these mutants have consistent improvement of earliness in flowering and maturity. Plant height also increased in all mutant lines except AEL 23/40/91 where reduction in this attribute was observed as compared to parent variety. Mutant lines AEL 49/20/91 and AEL 13/30/91 showed improvement in 100 grain weight. The improvement of some agronomic characters enhanced the yield of mutant lines in comparison to parent varieties (Masoor-85 and ICARDA-8). The diversity in yield over the respective parents was computed from 6.94 to 60.12%. From these encouraging results it is hoped that mutant lines like AEL 12/30/91 and AEL 49/20/91 may serve as potential lentil genotypes in future. (author)

Enterobactin-mediated iron transport in Pseudomonas aeruginosa.

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Poole, K; Young, L; Neshat, S

1990-01-01

A pyoverdine-deficient strain of Pseudomonas aeruginosa was unable to grow in an iron-deficient minimal medium in the presence of the nonmetabolizable iron chelator ethylene diamine-di(omega-hydroxyphenol acetic acid) (EDDHA), although addition of enterobactin to EDDHA-containing minimal media did restore growth of the pyoverdine-deficient P. aeruginosa. Consistent with the apparent ability of enterobactin to provide iron to P. aeruginosa, enterobactin-dependent 55Fe3+ uptake was observed in cells of P. aeruginosa previously grown in an iron-deficient medium containing enterobactin (or enterobactin-containing Escherichia coli culture supernatant). This uptake was energy dependent, was observable at low concentrations (60 nM) of FeCl3, and was absent in cells cultured without enterobactin. A novel protein with a molecular weight of approximately 80,000 was identified in the outer membranes of cells grown in iron-deficient minimal medium containing enterobactin, concomitant with the induction of enterobactin-dependent iron uptake. A Tn501 insertion mutant lacking this protein was isolated and shown to be deficient in enterobactin-mediated iron transport at 60 nM FeCl3, although it still exhibited enterobactin-dependent growth in iron-deficient medium containing EDDHA. It was subsequently observed that the mutant was, however, capable of enterobactin-mediated iron transport at much higher concentrations (600 nM) of FeCl3. Indeed, enterobactin-dependent iron uptake at this concentration of iron was observed in both the mutant and parent strains irrespective of whether they had been cultured in the presence of enterobactin.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2174865

Intracellular distribution of a speech/language disorder associated FOXP2 mutant

International Nuclear Information System (INIS)

Mizutani, Akifumi; Matsuzaki, Ayumi; Momoi, Mariko Y.; Fujita, Eriko; Tanabe, Yuko; Momoi, Takashi

2007-01-01

Although a mutation (R553H) in the forkhead box (FOX)P2 gene is associated with speech/language disorder, little is known about the function of FOXP2 or its relevance to this disorder. In the present study, we identify the forkhead nuclear localization domains that contribute to the cellular distribution of FOXP2. Nuclear localization of FOXP2 depended on two distally separated nuclear localization signals in the forkhead domain. A truncated version of FOXP2 lacking the leu-zip, Zn 2+ finger, and forkhead domains that was observed in another patient with speech abnormalities demonstrated an aggregated cytoplasmic localization. Furthermore, FOXP2 (R553H) mainly exhibited a cytoplasmic localization despite retaining interactions with nuclear transport proteins (importin α and β). Interestingly, wild type FOXP2 promoted the transport of FOXP2 (R553H) into the nucleus. Mutant and wild type FOXP2 heterodimers in the nucleus or FOXP2 R553H in the cytoplasm may underlie the pathogenesis of the autosomal dominant speech/language disorder

An allosteric binding site at the human serotonin transporter mediates the inhibition of escitalopram by R-citalopram: kinetic binding studies with the ALI/VFL-SI/TT mutant.

Science.gov (United States)

Zhong, Huailing; Hansen, Kasper B; Boyle, Noel J; Han, Kiho; Muske, Galina; Huang, Xinyan; Egebjerg, Jan; Sánchez, Connie

2009-10-25

The human serotonin transporter (hSERT) has primary and allosteric binding sites for escitalopram and R-citalopram. Previous studies have established that the interaction of these two compounds at a low affinity allosteric binding site of hSERT can affect the dissociation of [(3)H]escitalopram from hSERT. The allosteric binding site involves a series of residues in the 10th, 11th, and 12th trans-membrane domains of hSERT. The low affinity allosteric activities of escitalopram and R-citalopram are essentially eliminated in a mutant hSERT with changes in some of these residues, namely A505V, L506F, I507L, S574T, I575T, as measured in dissociation binding studies. We confirm that in association binding experiments, R-citalopram at clinically relevant concentrations reduces the association rate of [(3)H]escitalopram as a ligand to wild type hSERT. We demonstrate that the ability of R-citalopram to reduce the association rate of escitalopram is also abolished in the mutant hSERT (A505V, L506F, I507L, S574T, I575T), along with the expected disruption the low affinity allosteric function on dissociation binding. This suggests that the allosteric binding site mediates both the low affinity and higher affinity interactions between R-citalopram, escitalopram, and hSERT. Our data add an additional structural basis for the different efficacies of escitalopram compared to racemic citalopram reported in animal studies and clinical trials, and substantiate the hypothesis that hSERT has complex allosteric mechanisms underlying the unexplained in vivo activities of its inhibitors.

Methods of producing protoporphyrin IX and bacterial mutants therefor

Science.gov (United States)

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.

Genetic studies with morphological mutants of Aspergillus niger

International Nuclear Information System (INIS)

Roy, Ponty; Das, Arati

1979-01-01

Three classes of coloured mutations, viz., fawn, yellow and green, occurred recurrently among the population following UV- and γ-radiation from Co 60 of a wild Aspergillus niger strain 350. Ten mutants were picked up and complementation tests were performed by growing them in pairwise combinations. In two cases, allelic mutants of the same colour were observed. All these mutants were again grown in pairwise crosses with a brown A. niger mutant of different lineage. A poor heterokaryotic growth was, however, observed in one combination which later produced a diploid heterozygous nucleus. It segregated spontaneously to develop a large variety of colonies ranging from haploidy to diploidy including aneuploids. These have been analysed genetically and the possible explanations have been given. (auth.)

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

Science.gov (United States)

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.

Pollen irradiation method to obtain mutants in cucumber

International Nuclear Information System (INIS)

Iida, S.; Amano, E.

1988-01-01

Seed irradiation for mutation induction in dioecious crops like cucumber is not very useful because chimerism of the mutated tissues makes the segregation of mutants in the M 2 generation nearly impossible. This problem does not exist with pollen irradiation. Cucumber (Cucumis sativus L. var. Nishikisuyo) was used for a model experiment. The petals of male and female flowers were closed by pinching with binding wire before flowering to prevent pollination by insects. On the flowering day, the male flowers were collected and irradiated with 1kR to 10 kR of acute gamma rays (137-Cs), then used to pollinate the female flowers. The M 1 seeds thus obtained are not chimeric but heterozygous for induced mutations. When planted, no mutant phenotype appeared. Selfing within a plant lead to segregation of mutants in the M 2 generation. Seedling examination revealed eight mutants. One mutant line, in which the shape of leaves changed from pentagonal to round heart shape, was found under field conditions. The optimal dose for pollen irradiation seems to be between 2 kR and 4kR

Mutant number distribution in an exponentially growing population

International Nuclear Information System (INIS)

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. (paper)

A wheat cold resistance mutant derived from space mutagenesis

International Nuclear Information System (INIS)

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

2012-01-01

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

A wheat cold resistance mutant derived from space mutagenesis

International Nuclear Information System (INIS)

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

2011-01-01

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

Arabidopsis thickvein mutation affects vein thickness and organ vascularization, and resides in a provascular cell-specific spermine synthase involved in vein definition and in polar auxin transport.

Science.gov (United States)

Clay, Nicole K; Nelson, Timothy

2005-06-01

Polar auxin transport has been implicated in the induction of vascular tissue and in the definition of vein positions. Leaves treated with chemical inhibitors of polar auxin transport exhibited vascular phenotypes that include increased vein thickness and vascularization. We describe a recessive mutant, thickvein (tkv), which develops thicker veins in leaves and in inflorescence stems. The increased vein thickness is attributable to an increased number of vascular cells. Mutant plants have smaller leaves and shorter inflorescence stems, and this reduction in organ size and height is accompanied by an increase in organ vascularization, which appears to be attributable to an increase in the recruitment of cells into veins. Furthermore, although floral development is normal, auxin transport in the inflorescence stem is significantly reduced in the mutant, suggesting that the defect in auxin transport is responsible for the vascular phenotypes. In the primary root, the veins appear morphologically normal, but root growth in the tkv mutant is hypersensitive to exogenous cytokinin. The tkv mutation was found to reside in the ACL5 gene, which encodes a spermine synthase and whose expression is specific to provascular cells. We propose that ACL5/TKV is involved in vein definition (defining the boundaries between veins and nonvein regions) and in polar auxin transport, and that polyamines are involved in this process.

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH.

Science.gov (United States)

Yun, Jihye; Mullarky, Edouard; Lu, Changyuan; Bosch, Kaitlyn N; Kavalier, Adam; Rivera, Keith; Roper, Jatin; Chio, Iok In Christine; Giannopoulou, Eugenia G; Rago, Carlo; Muley, Ashlesha; Asara, John M; Paik, Jihye; Elemento, Olivier; Chen, Zhengming; Pappin, Darryl J; Dow, Lukas E; Papadopoulos, Nickolas; Gross, Steven S; Cantley, Lewis C

2015-12-11

More than half of human colorectal cancers (CRCs) carry either KRAS or BRAF mutations and are often refractory to approved targeted therapies. We found that cultured human CRC cells harboring KRAS or BRAF mutations are selectively killed when exposed to high levels of vitamin C. This effect is due to increased uptake of the oxidized form of vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased DHA uptake causes oxidative stress as intracellular DHA is reduced to vitamin C, depleting glutathione. Thus, reactive oxygen species accumulate and inactivate glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Inhibition of GAPDH in highly glycolytic KRAS or BRAF mutant cells leads to an energetic crisis and cell death not seen in KRAS and BRAF wild-type cells. High-dose vitamin C impairs tumor growth in Apc/Kras(G12D) mutant mice. These results provide a mechanistic rationale for exploring the therapeutic use of vitamin C for CRCs with KRAS or BRAF mutations. Copyright © 2015, American Association for the Advancement of Science.

Transcriptome-based analysis on carbon metabolism of Haematococcus pluvialis mutant under 15% CO2.

Science.gov (United States)

Li, Ke; Cheng, Jun; Lu, Hongxiang; Yang, Weijuan; Zhou, Junhu; Cen, Kefa

2017-06-01

To elucidate the mechanism underlying the enhanced growth rate in the Haematococcus pluvialis mutated with 60 Co-γ rays and domesticated with 15% CO 2 , transcriptome sequencing was conducted to clarify the carbon metabolic pathways of mutant cells. The CO 2 fixation rate of mutant cells increased to 2.57gL -1 d -1 under 15% CO 2 due to the enhanced photosynthesis, carbon fixation, glycolysis pathways. The upregulation of PetH, ATPF0A and PetJ related to photosynthetic electron transport, ATP synthase and NADPH generation promoted the photosynthesis. The upregulation of genes related to Calvin cycle and ppdK promoted carbon fixation in both C3 and C4 photosynthetic pathways. The reallocation of carbon was also enhanced under 15% CO 2 . The 19-, 14- and 3.5-fold upregulation of FBA, TPI and PK genes, respectively, remarkably promoted the glycolysis pathways. This accelerated the conversion of photosynthetic carbon to pyruvate, which was an essential precursor for astaxanthin and lipids biosynthesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolation of new gravitropic mutants under hypergravity conditions

Directory of Open Access Journals (Sweden)

Akiko Mori

2016-09-01

Full Text Available 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 upwards. 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.

Structural determinants of NH3 and NH4+ transport by mouse Rhbg, a renal Rh glycoprotein.

Science.gov (United States)

Abdulnour-Nakhoul, Solange; Le, Trang; Rabon, Edd; Hamm, L Lee; Nakhoul, Nazih L

2016-12-01

Renal Rhbg is localized to the basolateral membrane of intercalated cells and is involved in NH 3 /NH 4 + transport. The structure of Rhbg is not yet resolved; however, a high-resolution crystal structure of AmtB, a bacterial homolog of Rh, has been determined. We aligned the sequence of Rhbg to that of AmtB and identified important sites of Rhbg that may affect transport. Our analysis positioned three conserved amino acids, histidine 183 (H183), histidine 342 (H342), and tryptophan 230 (W230), within the hydrophobic pore where they presumably serve to control NH 3 transport. A fourth residue, phenylalanine 128 (F128) was positioned at the upper vestibule, presumably contributing to recruitment of NH 4 + We generated three mutations each of H183, H342, W230, and F128 and expressed them in frog oocytes. Immunolabeling showed that W230 and F128 mutants were localized to the cell membrane, whereas H183 and H342 staining was diffuse and mostly intracellular. To determine function, we compared measurements of NH 3 /NH 4 + and methyl amine (MA)/methyl ammonium (MA + )-induced currents, intracellular pH, and surface pH (pHs) among oocytes expressing the mutants, Rhbg, or injected with H 2 O. In H183 and W230 mutants, NH 4 + -induced current and intracellular acidification were inhibited compared with that of Rhbg, and MA-induced intracellular alkalinization was completely absent. Expression of H183A or W230A mutants inhibited NH 3 /NH 4 + - and MA/MA + -induced decrease in pHs to the level observed in H 2 O-injected oocytes. Mutations of F128 did not significantly affect transport of NH 3 or NH 4 + These data demonstrated that mutating H183 or W230 caused loss of function but not F128. H183 and H342 may affect membrane expression of the transporter.

X-ray-sensitive mutants of Chinese hamster ovary cell line

International Nuclear Information System (INIS)

Jeggo, P.A.; Kemp, L.M.

1983-01-01

A standard technique of microbial genetics, which involves the transfer of cells from single colonies by means of sterile toothpicks, has been adapted to somatic cell genetics. Its use has been demonstrated in the isolation of X-ray-sensitive mutants of CHO cells. 9000 colonies have been tested and 6 appreciably X-ray-sensitive mutants were isolated. (D 10 values 5-10-fold of wild-type D 10 value.) A further 6 mutants were obtained which showed a slight level of sensitivity (D 10 values less than 2-fold of wild-type D 10 value). The 6 more sensitive mutants were also sensitive to bleomycin, a chemotherapeutic agent inducing X-ray-like damage. Cross-sensitivity to UV-irradiation and treatment with the alkylating agents, MMS, EMS and MNNG, was investigated for these mutants. Some sensitivity to these other agents was observed, but in all cases it was less severe than the level of sensitivity to X-irradiation. Each mutant showed a different overall response to the spectrum of agents examined and these appear to represent new mutant phenotypes derived from cultured mammalian cell lines. One mutant strain, xrs-7, was cross-sensitive to all the DNA-damaging agents, but was proficient in the repair of single-strand breaks. (Auth.)

Histological and Molecular Characterization of Grape Early Ripening Bud Mutant

Directory of Open Access Journals (Sweden)

Da-Long Guo

2016-01-01

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

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

Science.gov (United States)

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.

Chinese hamster ovary cell mutants defective in heparan sulfate biosynthesis

International Nuclear Information System (INIS)

Bame, K.J.; Kiser, C.S.; Esko, J.D.

1987-01-01

The authors have isolated Chinese hamster ovary cell mutants defective in proteoglycan synthesis by radiographic screening for cells unable to incorporate 35 SO 4 into acid-precipitable material. Some mutants did not incorporate 35 SO 4 into acid-precipitable material, whereas others incorporated about 3-fold less radioactivity. HPLC anion exchange chromatographic analysis of radiolabelled glycosaminoglycans isolated from these mutants revealed many are defective in heparan sulfate biosynthesis. Mutants 803 and 677 do not synthesize heparan sulfate, although they produce chondroitin sulfate: strain 803 makes chondroitin sulfate normally, whereas 677 overaccumulates chondroitin sulfate by a factor of three. These mutants fall into the same complementation group, suggesting that the mutations are allelic. A second group of heparan sulfate biosynthetic mutants, consisting of cell lines 625, 668 and 679, produce undersulfated heparan sulfate and normal chondroitin sulfate. Treatment of the chains with nitrous acid should determine the position of the sulfate groups along the chain. These mutants may define a complementation group that is defective in the enzymes which modify the heparan sulfate chain. To increase the authors repertoire of heparan sulfate mutants, they are presently developing an in situ enzyme assay to screen colonies replica plated on filter discs for sulfotransferase defects

Primary study on lesion mimic mutants of rice (oryza sativa L.)

International Nuclear Information System (INIS)

Hao Zhongna; Zhang Hongzhi; Tao Rongixang

2007-01-01

Nineteen lesion mimic mutants (xsl1-19) of japonica rice Xiushui11 were obtained by γ-rays irradiation treatment. All mutants belonged to whole life lesion mimic. Lesion mimic of mutants didn't largen after tillering stage, leaves didn't wither, and no effect on the plants exsert spikes and seed. When the highest temperature in day exceeded 32 degree C in seedling stage, lesion mimic of all mutant expect xsl19 disappeared. Under 32 degree C, lesion mimic would appear gradually, and symptoms weren't inhibited by high temperature after 5 leaf stage. The plant heights of all lesion mimic mutants were 47.56-63.54 cm in the tillering stage, and that of CK was 83.75 cm; but the dwarf phenomenon of mutants only appeared before tillering stage, and didn't affect plant heights finally; the heading dates of mutants were the same to the CK, the ear length of all mutants were 9.43-15.19 cm, and that of CK was 16.41 cm; the total grain quantity per spike of all mutants were 88.17-165.33, and those of xsl19 and CK were 49.50 and 76.17. The results showed all lesion mimic mutants except xsl19 had short spikes and total grain quantity per spike increasing. All lesion mimic mutants were susceptible to Magnaporthe grisea, and they had no relationship with resistance. (authors)

Grain product of 34 soya mutant lines;Rendimiento de grano de 34 lineas mutantes de soya

Energy Technology Data Exchange (ETDEWEB)

Salmeron E, J.; Mastache L, A. A.; Valencia E, F.; Diaz V, G. E. [Colegio Superior Agropecuario del Estado de Guerrero, Vicente Guerrero No. 81, Col. Centro, 40000 Iguala, Guerrero (Mexico); Cervantes S, T. [Instituto de Recursos Geneticos y Productividad, Colegio de Posgraduados, Carretera Mexico-Texcoco Km. 36.5, Montecillo, 56230 Texcoco, Estado de Mexico (Mexico); De la Cruz T, E.; Garcia A, J. M.; Falcon B, T.; Gatica T, M. A. [ININ, Departamento de Biologia, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)

2009-07-01

This work was development with the objective of obtaining information of the agronomic behavior of 34 soya mutant lines (R{sub 4}M{sub 18}) for human consumption and this way to select the 2 better lines. The genetic materials were obtained starting from the variety ISAAEG-B M2 by means of the application of recurrent radiation with Co{sup 60} gammas, to a dose of 350 Gray for the first two generations and both later to 200 Gray and selection during 17 cycles, being obtained the 34 better lines mutants with agronomic characteristic wanted and good flavor. The obtained results were that the mutant lines L{sub 25} and L{sub 32} produced the major quantity in branches/plant number with 7.5 and 7.25, pods/plant number with 171.25 and 167, grains/plant number with 350.89 and 333.07 and grain product (ton/ha) to 15% of humidity 5.15 and 4.68 ton/ha, respectively. (Author)

Molecular and biochemical analysis of the plastidic ADP-glucose transporter (ZmBT1) from Zea mays.

NARCIS (Netherlands)

Kirchberger, S.; Leroch, M.; Huynen, M.A.; Wahl, M.; Neuhaus, H.E.; Tjaden, J.

2007-01-01

Physiological studies on the Brittle1 maize mutant have provided circumstantial evidence that ZmBT1 (Zea mays Brittle1 protein) is involved in the ADP-Glc transport into maize endosperm plastids, but up to now, no direct ADP-Glc transport mediated by ZmBT1 has ever been shown. The heterologous

Molecular and biochemical analysis of the plastidic ADP-glucose transporter (ZmBT1) from Zea mays

NARCIS (Netherlands)

Kirchberger, S.; Leroch, M.; Huynen, M.A.; Wahl, M.; Neuhaus, H.E.; Tjaden, J.

2007-01-01

Physiological studies on the Brittle1 maize mutant have provided circumstantial evidence that ZmBT1 (Zea mays Brittle1 protein) is involved in the ADP-Glc transport into maize endosperm plastids, but up to now, no direct ADP-Glc transport mediated by ZmBT1 has ever been shown. The heterologous

Arabidopsis thickvein Mutation Affects Vein Thickness and Organ Vascularization, and Resides in a Provascular Cell-Specific Spermine Synthase Involved in Vein Definition and in Polar Auxin Transport1

Science.gov (United States)

Clay, Nicole K.; Nelson, Timothy

2005-01-01

Polar auxin transport has been implicated in the induction of vascular tissue and in the definition of vein positions. Leaves treated with chemical inhibitors of polar auxin transport exhibited vascular phenotypes that include increased vein thickness and vascularization. We describe a recessive mutant, thickvein (tkv), which develops thicker veins in leaves and in inflorescence stems. The increased vein thickness is attributable to an increased number of vascular cells. Mutant plants have smaller leaves and shorter inflorescence stems, and this reduction in organ size and height is accompanied by an increase in organ vascularization, which appears to be attributable to an increase in the recruitment of cells into veins. Furthermore, although floral development is normal, auxin transport in the inflorescence stem is significantly reduced in the mutant, suggesting that the defect in auxin transport is responsible for the vascular phenotypes. In the primary root, the veins appear morphologically normal, but root growth in the tkv mutant is hypersensitive to exogenous cytokinin. The tkv mutation was found to reside in the ACL5 gene, which encodes a spermine synthase and whose expression is specific to provascular cells. We propose that ACL5/TKV is involved in vein definition (defining the boundaries between veins and nonvein regions) and in polar auxin transport, and that polyamines are involved in this process. PMID:15894745

Cytoembryologic study of gamma-ray induced sterile Pisum sativum L. mutants

International Nuclear Information System (INIS)

Molkhova, E.; Vasileva, M.

1977-01-01

Three new pea mutant forms are described - 1878, Crampled petal Waxless type, and Lathyrus type - which were induced by different gamma-ray ( 60 Co) doses and rates. The flowers of the 1878 and Crampled petal Waxless type mutants were very much deformed, while those of the Lathyrus type had smaller flowers with normal morphology. The three mutant forms were entirely sterile and were propagated by segregation in the progeny of heterozygous sister plants. PMC meiosis and the development of the male gametophyte of the Lathyrus type mutant had a normal course, while in the mutant forms Crampled petal Waxless type and 1878 slight disturbances were observed, but the pollen of all three mutants was not functional. The development of the female gametophyte of the three mutants stops at an early phase and only in the Lathyrus type mutant in single cases embryosacks were formed with differentiated sex apparatus and early stages of embryo and endosperm development were scored, but they also soon degenerate. It is pointed out that sterility of the three pea mutant forms studied depends on factors, which stop at different stages the normal development of the generative organs, of the female gametophyte and of embryogenesis. (author)

Collection of rice mutants and application studies of their agronomic characters

International Nuclear Information System (INIS)

Sun Shuxiang; Jin Wei; Luo Qian; Sheng Ping; Huang Rongmin

1993-01-01

More than 1600 accessions of rice mutant germplasm have been collected since 1980, and 1142 accessions of mutants have been identified according to their agronomy and pattern characters. A part of mutants were compared with their original cultivars in eight main agronomic characters. The results showed that the agronomic characters of mutants induced by ionizing radiations changed to both positive and negative directions compared with their original cultivars. Only 6.3% mutants varied in single agronomic character, and 91.1% mutants varied in two to six agronomic characters. Tenetic analysis and Cellular observations were carried out for two kinds of early mutants. It showed that early mutants 'Yuan Feng Zao' are controlled by two independent and incomplete dominant genes. For the dwarf, the reduction of the number of longitudinal cell layers causes the stem shorter and the increase of the number of horizontal cell layers causes the stem wall thicker. More than 100 preserved accessions of mutants were supplied to breeding units as parents or for genetic studies. Sixteen cultivars (lines) were bred from the parents which played an important role in raising the output of rice production

SERCA mutant E309Q binds two Ca ions but adopts a catalytically incompetent conformation

DEFF Research Database (Denmark)

Clausen, Johannes D.; Bublitz, Maike; Arnou, Bertrand

2013-01-01

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) couples ATP hydrolysis to transport of Ca2+. This directed energy transfer requires cross-talk between the two Ca2+ sites and the phosphorylation site over 50 Å distance. We have addressed the mechano-structural basis for this intramolecular...... a shift of transmembrane segment M1 into an ‘up and kinked position’. This transition is impaired in the E309Q mutant, most likely due to a lack of charge neutralization and altered hydrogen binding capacities at Ca2+ site II....

Strigolactone Inhibition of Branching Independent of Polar Auxin Transport1[OPEN

Science.gov (United States)

Mason, Michael G.; Beveridge, Christine A.

2015-01-01

The outgrowth of axillary buds into branches is regulated systemically via plant hormones and the demand of growing shoot tips for sugars. The plant hormone auxin is thought to act via two mechanisms. One mechanism involves auxin regulation of systemic signals, cytokinins and strigolactones, which can move into axillary buds. The other involves suppression of auxin transport/canalization from axillary buds into the main stem and is enhanced by a low sink for auxin in the stem. In this theory, the relative ability of the buds and stem to transport auxin controls bud outgrowth. Here, we evaluate whether auxin transport is required or regulated during bud outgrowth in pea (Pisum sativum). The profound, systemic, and long-term effects of the auxin transport inhibitor N-1-naphthylphthalamic acid had very little inhibitory effect on bud outgrowth in strigolactone-deficient mutants. Strigolactones can also inhibit bud outgrowth in N-1-naphthylphthalamic acid-treated shoots that have greatly diminished auxin transport. Moreover, strigolactones can inhibit bud outgrowth despite a much diminished auxin supply in in vitro or decapitated plants. These findings demonstrate that auxin sink strength in the stem is not important for bud outgrowth in pea. Consistent with alternative mechanisms of auxin regulation of systemic signals, enhanced auxin biosynthesis in Arabidopsis (Arabidopsis thaliana) can suppress branching in yucca1D plants compared with wild-type plants, but has no effect on bud outgrowth in a strigolactone-deficient mutant background. PMID:26111543

The agronomic characters of a high protein rice mutant

International Nuclear Information System (INIS)

Harn, C.; Won, J.L.; Choi, K.T.

1975-01-01

Mutant lines (M 5 -M 9 ) of macro-phenotypic traits from several varieties were screened for the protein content. Mutant 398 (M 9 ) is one of the high protein mutants selected from Hokwang. Three years' tests revealed that it has a high protein line under any condition of cultivation. Except for early maturity and short culmness, other agronomic and yield characters were similar to the original variety. There was no difference between the mutant 398 and its mother variety in grain shape and weight, and also the size and protein content of the embryo. The high protein content of the mutant is attributable to the increase of protein in the endosperm. About 150 normal-looking or a few days-earlier-maturing selections were made from Jinheung variety in the M 3 and screened for protein. Promising lines in terms of the plant type, yield and protein were obtained. (author)

Induction and characterization of Arabidopsis mutants by Ion beam

International Nuclear Information System (INIS)

Yoon, Y. H.; Choi, J. D.; Park, J. Y.; Lee, J. R.; Sohn, H. S.

2008-03-01

This study was conducted to search the proper conditions and times for irradiating proton beam to seeds generally used for induction of mutant. Arabidopsis as model plants has good characters that is a short generation time, producing a lot of seeds, sequenced genome, developed maker. This points were the best materials for plant breeding for this study. The data of inducing mutants of Arabidopsis is used to be applicate to crops have more longer generation that is the final goals of this study. The goals of this project were to inducing and characterizing arabidopsis mutants by the proton ion beam and γ-ray. As well as, the purpose of this study was securing more than 10 lines of arabidopsis mutants in this project and also to know the changed DNA structure of the mutants using the basic data for applying to the more study

Induction and characterization of Arabidopsis mutants by Ion beam

Energy Technology Data Exchange (ETDEWEB)

Yoon, Y. H.; Choi, J. D.; Park, J. Y.; Lee, J. R.; Sohn, H. S. [Gyeongbuk Institute for Bio Industry, Andong (Korea, Republic of)

2008-03-15

This study was conducted to search the proper conditions and times for irradiating proton beam to seeds generally used for induction of mutant. Arabidopsis as model plants has good characters that is a short generation time, producing a lot of seeds, sequenced genome, developed maker. This points were the best materials for plant breeding for this study. The data of inducing mutants of Arabidopsis is used to be applicate to crops have more longer generation that is the final goals of this study. The goals of this project were to inducing and characterizing arabidopsis mutants by the proton ion beam and {gamma}-ray. As well as, the purpose of this study was securing more than 10 lines of arabidopsis mutants in this project and also to know the changed DNA structure of the mutants using the basic data for applying to the more study

Multivariate analysis for selecting apple mutants

International Nuclear Information System (INIS)

Faedi, W.; Bagnara, G.L.; Rosati, P.; Cecchini, M.

1992-01-01

The mutlivariate analysis of four year records on several vegetative and productive traits of twenty-one apple mutants (3 of 'Jonathan', 3 of 'Ozark Gold', 14 of 'Mollie's Delicious', 1 of 'Neipling's Early Stayman)' induced by gamma radiations showed that observation of some traits of one-year-old shoots is the most efficient way to reveal compact growing apple mutants. In particular, basal cross-section area, total length and leaf area resulted the most appropriate parameters, while internode length together with conopy height and width are less appropriate. The most interesting mutants we found are: one of 'Mollie's Delicious for the best balance among tree and fruit traits and for high skin color; one of 'Neipling's Early Stayman' with an earlier and more extensively red colored apple than the original clone. (author)

Biocontrol potential of salinity tolerant mutants of Trichoderma harzianum against Fusarium oxysporum Potencial de biocontrole de mutantes sal-tolerantes de Trichoderma harzianum contra Fusarium oxysporum

Directory of Open Access Journals (Sweden)

Hassan Abdel-Latif A. Mohamed

2006-06-01

Full Text Available Exposing a wild-type culture of Trichoderma harzianum to gamma irradiation induced two stable salt-tolerant mutants (Th50M6 and Th50M11. Under saline conditions, both mutants greatly surpassed their wild type strain in growth rate, sporulation and biological proficiency against Fusarium oxysporum, the causal agent of tomato wilt disease. Tolerant T. harzianum mutants detained a capability to grow and convinced sporulation in growth media containing up to 69 mM NaCl. In comparison with their parent strain, characterization of both mutants confirmed that they have reinforced contents of proline and hydroxyproline, relatively higher sodium content compared to potassium, calcium or magnesium contents, higher level of total phenols. Electrophoretic analysis of total soluble proteins in the salt tolerance mutant Th50M6 showed different bands accumulated in response to 69 mM NaCl. Data also showed that mutants produce certain active metabolites, such as chitinases, cellulases, beta-galactosidases, as well as, some antibiotics i.e., trichodermin, gliotoxin and gliovirin. Trichoderma mutants significantly reduced wilt disease incidence and improved yield and mineral contents of tomato plants under both saline and non-saline soil conditions, as well as, under infested and natural conditions. T. harzianum mutants were also more efficient in dropping the F. oxysporum growth in rhizosphere compared to the wild type strain. Population density of both mutants in rhizosphere far exceeded that of T. harzianum wild type strain.A exposição de uma cepa selvagem de Trichoderma harzianum à irradiação gama induziu dois mutantes tolerantes a sal (Th50M6 e Th50M11. Em condições salinas, os dois mutantes foram muito superiores à cepa selvagem em relação à velocidade de multiplicação, esporulação e eficiência contra Fusarium oxysporum, o agente causador da doença wilt do tomate. Os mutantes tolerantes foram capazes de multiplicação e esporulação em

Characterization of MMS-sensitive mutants of Neurospora crassa

Energy Technology Data Exchange (ETDEWEB)

DeLange, A.M.; Mishra, N.C.

1982-01-01

Several MMS-sensitive mutants of Neurospora crassa were compared with the wild-type strain for their relative sensitivities to UV, X-ray, and histidine. They were also compared for the frequency of spontaneous mutation at the loci which confer resistance to p-fluorophenylalanine. The mutants were also examined for possible defects in meiotic behavior in homozygous crosses and for any change in the inducible DNA salvage pathways. On the basis of these characterizations, the present MMS-sensitive mutants of Neurospora can be placed into three groups. On the basis of data presented, the MMS sensitivity of the first group mutants cannot be ascertained to arise from a defect in the DNA repair pathways; instead, it may stem from altered cell permeability or other pleotropic effects of the mus mutations. However, it can be suggested that the second and third group of mus mutants may indeed result from a defect in the DNA repair pathways controlled by the mus genes; this conclusion is based on their cross-sensitivity to a number of DNA-damaging agents such as MMS, UV and/or X-rays, high frequencies of spontaneous mutation and defects in meiotic behavior.

Productive mutants in lemongrass induced by gamma rays

International Nuclear Information System (INIS)

Gopinathan Nair, V.

1980-01-01

Seeds of the lemongrass variety O.D. 19 were irradiated with gamma rays at a dose range of 5 to 30 krad. M 1 plants with one or a few tillers differing from the standard plants of O.D. 19 were selected, split into single slips and planted as clonal progenies. Mutants were isolated in M 1 V 1 and carried forward. Forty two M 1 V 2 mutant clones differing from O.D. 19 in morphological characters such as vigour, plant height, growth habit, pigmentation and number of tillers have been established. These were evaluated for tiller number, grass yield and oil content. Six clones gave higher grass yield, the highest being 556 gm per plant per cutting as against 360 gm in the standard. Five clones gave higher oil yield, the highest being 0.42% as against 0.23% in the standard. Isolation of viable mutants with high grass yield and essential oil content indicate the scope for evolving productive mutant varieties in this perennial aromatic grass. The eleven M 1 V 2 mutant clones are being critically evaluated by estimating oil yield per hectare per year. (author)

Generation and characterization of pigment mutants of ...

African Journals Online (AJOL)

Compared to the wild CC-124, these mutants are characterized by a decrease in chlorophyll a & b content and an increase in carotenoids. The lowest decrease in chlorophyll a was 3 to 4 folds, while the highest increase in carotenoids was 2 to 4 folds. The result of bio-test, using the resulting pigment mutant of C. reinhardtii ...

Study on ionizing radiosensitivity of respiratory deficiency yeast mutants

International Nuclear Information System (INIS)

Mao Shuhong; Chinese Academy of Sciences, Beijing; Jin Genming; Wei Zengquan; Xie Hongmei

2006-01-01

The radiosensitivity of respiratory deficiency yeast mutants has been studied in this work. The mutants which were screened from the yeasts after ionizing irradiation were irradiated with 12 C 6+ at different doses. Because of the great change in its mitochondria and mitochondrial DNA, the respiratory deficiency yeast mutants show radio-sensitivity at dose less than 1 Gy and radioresistance at doses higher than 1 Gy. (authors)

Expression of SPEF2 During Mouse Spermatogenesis and Identification of IFT20 as an Interacting Protein

DEFF Research Database (Denmark)

Sironen, Anu; Hansen, Jeanette; Thomsen, Bo

2010-01-01

SPEF2 is expressed in all ciliated cells and is essential for correct sperm tail development and male fertility. We have previously identified a mutation within the SPEF2 gene as the cause for infertility due to immotile and malformed sperm tails in pigs. This mutation in pigs alters the testis...... in the distal part of the sperm tail midpiece. Using yeast two hybrid assay and co-immunoprecipitation experiments we identified an interaction between SPEF2 and the intra-flagellar transport protein IFT20 in the testis. Furthermore, these two proteins co-localize in differentiating male germ cells...

Identification and functional analysis of two Golgi-localized UDP-galactofuranose transporters with overlapping functions in Aspergillus niger.

Science.gov (United States)

Park, Joohae; Tefsen, Boris; Heemskerk, Marc J; Lagendijk, Ellen L; van den Hondel, Cees A M J J; van Die, Irma; Ram, Arthur F J

2015-11-02

Galactofuranose (Galf)-containing glycoconjugates are present in numerous microbes, including filamentous fungi where they are important for morphology, virulence and maintaining cell wall integrity. The incorporation of Galf-residues into galactomannan, galactomannoproteins and glycolipids is carried out by Golgi-localized Galf transferases. The nucleotide sugar donor used by these transferases (UDP-Galf) is produced in the cytoplasm and has to be transported to the lumen of the Golgi by a dedicated nucleotide sugar transporter. Based on homology with recently identified UDP-Galf-transporters in A. fumigatus and A. nidulans, two putative UDP-Galf-transporters in A. niger were found. Their function and localization was determined by gene deletions and GFP-tagging studies, respectively. The two putative UDP-Galf-transporters in A. niger are homologous to each other and are predicted to contain eleven transmembrane domains (UgtA) or ten transmembrane domains (UgtB) due to a reduced length of the C-terminal part of the UgtB protein. The presence of two putative UDP-Galf-transporters in the genome was not unique for A. niger. From the twenty Aspergillus species analysed, nine species contained two additional putative UDP-Galf-transporters. Three of the nine species were outside the Aspergillus section nigri, indication an early duplication of UDP-Galf-transporters and subsequent loss of the UgtB copy in several aspergilli. Deletion analysis of the single and double mutants in A. niger indicated that the two putative UDP-Galf-transporters (named UgtA and UgtB) have a redundant function in UDP-Galf-transport as only the double mutant displayed a Galf-negative phenotype. The Galf-negative phenotype of the double mutant could be complemented by expressing either CFP-UgtA or CFP-UgtB fusion proteins from their endogenous promoters, indicating that both CFP-tagged proteins are functional. Both Ugt proteins co-localize with each other as well as with the GDP

Development of Database Software with Plant Mutant Resources

International Nuclear Information System (INIS)

Namgoong, Won; Lee, M. J.; Kim, J. D.; Ma, N. K.

2007-03-01

In this research, mutants induced by nuclear radiation are developed information computerised system. The status and progress on the collection, identification and utilization of mutants in Korea are introduced. And it was produced home page, manual, test record, construction of system

Construindo Marcas Mutantes

Directory of Open Access Journals (Sweden)

Elizete De Azevedo Kreutz

2012-09-01

Full Text Available O presente artigo é o resultado de estudos realizados desde 2000 e busca instrumentalizar os proñssionals para a construção de Marcas Mutantes, que é   uma tendência contemporânea nas estratégias comunicacionais e de branding. Embora esta estratégia ainda não esteja consolidada, observamos que a mesma tem obtido um crescimento constante e tem sido adotadas pelas mais diferentes categorias de marcas e não apenas por aquelas direcionadas aos jovens, ao esporte, ao entretenimento, como era no principia. Com base na Hermenêutica de Profundidade de Thompson (1995, alicerçada nas pesquisas bibliográficas, de intemet, entrevistas e análise semiótica, desenhamos um método de construção de Marcas Mutantes dividido em sete fases. Como resultado, esperamos que este estudo possa auxiliar na compreensão dos processos envolvidos, ao mesmo tempo que provoque a discussão sobreo mesmo e, por consequência, o seu aprimoramento.

An auxin transport independent pathway is involved in phosphate stress-induced root architectural alterations in Arabidopsis. Identification of BIG as a mediator of auxin in pericycle cell activation.

Science.gov (United States)

López-Bucio, José; Hernández-Abreu, Esmeralda; Sánchez-Calderón, Lenin; Pérez-Torres, Anahí; Rampey, Rebekah A; Bartel, Bonnie; Herrera-Estrella, Luis

2005-02-01

Arabidopsis (Arabidopsis thaliana) plants display a number of root developmental responses to low phosphate availability, including primary root growth inhibition, greater formation of lateral roots, and increased root hair elongation. To gain insight into the regulatory mechanisms by which phosphorus (P) availability alters postembryonic root development, we performed a mutant screen to identify genetic determinants involved in the response to P deprivation. Three low phosphate-resistant root lines (lpr1-1 to lpr1-3) were isolated because of their reduced lateral root formation in low P conditions. Genetic and molecular analyses revealed that all lpr1 mutants were allelic to BIG, which is required for normal auxin transport in Arabidopsis. Detailed characterization of lateral root primordia (LRP) development in wild-type and lpr1 mutants revealed that BIG is required for pericycle cell activation to form LRP in both high (1 mm) and low (1 microm) P conditions, but not for the low P-induced alterations in primary root growth, lateral root emergence, and root hair elongation. Exogenously supplied auxin restored normal lateral root formation in lpr1 mutants in the two P treatments. Treatment of wild-type Arabidopsis seedlings with brefeldin A, a fungal metabolite that blocks auxin transport, phenocopies the root developmental alterations observed in lpr1 mutants in both high and low P conditions, suggesting that BIG participates in vesicular targeting of auxin transporters. Taken together, our results show that auxin transport and BIG function have fundamental roles in pericycle cell activation to form LRP and promote root hair elongation. The mechanism that activates root system architectural alterations in response to P deprivation, however, seems to be independent of auxin transport and BIG.

Missense mutations in the WD40 domain of AHI1 cause non-syndromic retinitis pigmentosa.

Science.gov (United States)

Nguyen, Thanh-Minh T; Hull, Sarah; Roepman, Ronald; van den Born, L Ingeborgh; Oud, Machteld M; de Vrieze, Erik; Hetterschijt, Lisette; Letteboer, Stef J F; van Beersum, Sylvia E C; Blokland, Ellen A; Yntema, Helger G; Cremers, Frans P M; van der Zwaag, Paul A; Arno, Gavin; van Wijk, Erwin; Webster, Andrew R; Haer-Wigman, Lonneke

2017-09-01

Recent findings suggesting that Abelson helper integration site 1 ( AHI1 ) is involved in non-syndromic retinal disease have been debated, as the functional significance of identified missense variants was uncertain. We assessed whether AHI1 variants cause non-syndromic retinitis pigmentosa (RP). Exome sequencing was performed in three probands with RP. The effects of the identified missense variants in AHI1 were predicted by three-dimensional structure homology modelling. Ciliary parameters were evaluated in patient's fibroblasts, and recombinant mutant proteins were expressed in ciliated retinal pigmented epithelium cells. In the three patients with RP, three sets of compound heterozygous variants were detected in AHI1 (c.2174G>A; p.Trp725* and c.2258A>T; p.Asp753Val, c.660delC; p.Ser221Glnfs*10 and c.2090C>T; p.Pro697Leu, c.2087A>G; p.His696Arg and c.2429C>T; p.Pro810Leu). All four missense variants were present in the conserved WD40 domain of Jouberin, the ciliary protein encoded by AHI1 , with variable predicted implications for the domain structure. No significant changes in the percentage of ciliated cells, nor in cilium length or intraflagellar transport were detected. However, expression of mutant recombinant Jouberin in ciliated cells showed a significantly decreased enrichment at the ciliary base. This report confirms that mutations in AHI1 can underlie autosomal recessive RP. Moreover, it structurally and functionally validates the effect of the RP-associated AHI1 variants on protein function, thus proposing a new genotype-phenotype correlation for AHI1 mutation associated retinal ciliopathies. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Mutants of Cercospora kikuchii altered in cercosporin synthesis and pathogenicity

International Nuclear Information System (INIS)

Upchurch, R.G.; Walker, D.C.; Rollins, J.A.; Ehrenshaft, M.; Daub, M.E.

1991-01-01

The authors have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus

Connexin mutants and cataracts

Directory of Open Access Journals (Sweden)

Eric C Beyer

2013-04-01

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

Brucella abortusΔcydCΔcydD and ΔcydCΔpurD double-mutants are highly attenuated and confer long-term protective immunity against virulent Brucella abortus.

Science.gov (United States)

Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Kim, Kiju; Hahn, Tae-Wook

2016-01-04

We constructed double deletion (ΔcydCΔcydD and ΔcydCΔpurD) mutants from virulent Brucella abortus biovar 1 field isolate (BA15) by deleting the genes encoding an ATP-binding cassette-type transporter (cydC and cydD genes) and a phosphoribosylamine-glycine ligase (purD). Both BA15ΔcydCΔcydD and BA15ΔcydCΔpurD double-mutants exhibited significant attenuation of virulence when assayed in murine macrophages or in BALB/c mice. Both double-mutants were readily cleared from spleens by 4 weeks post-inoculation even when inoculated at the dose of 10(8) CFU per mouse. Moreover, the inoculated mice showed no splenomegaly, which indicates that the mutants are highly attenuated. Importantly, the attenuation of in vitro and in vivo growth did not impair the ability of these mutants to confer long-term protective immunity in mice against challenge with B. abortus strain 2308. Vaccination of mice with either mutant induced humoral and cell-mediated immune responses, and provided significantly better protection than commercial B. abortus strain RB51 vaccine. These results suggest that highly attenuated BA15ΔcydCΔcydD and BA15ΔcydCΔpurD mutants can be used effectively as potential live vaccine candidates against bovine brucellosis. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Hyperpolarized 13C MR imaging detects no lactate production in mutant IDH1 gliomas: Implications for diagnosis and response monitoring

Directory of Open Access Journals (Sweden)

Myriam M. Chaumeil

2016-01-01

Full Text Available Metabolic imaging of brain tumors using 13C Magnetic Resonance Spectroscopy (MRS of hyperpolarized [1-13C] pyruvate is a promising neuroimaging strategy which, after a decade of preclinical success in glioblastoma (GBM models, is now entering clinical trials in multiple centers. Typically, the presence of GBM has been associated with elevated hyperpolarized [1-13C] lactate produced from [1-13C] pyruvate, and response to therapy has been associated with a drop in hyperpolarized [1-13C] lactate. However, to date, lower grade gliomas had not been investigated using this approach. The most prevalent mutation in lower grade gliomas is the isocitrate dehydrogenase 1 (IDH1 mutation, which, in addition to initiating tumor development, also induces metabolic reprogramming. In particular, mutant IDH1 gliomas are associated with low levels of lactate dehydrogenase A (LDHA and monocarboxylate transporters 1 and 4 (MCT1, MCT4, three proteins involved in pyruvate metabolism to lactate. We therefore investigated the potential of 13C MRS of hyperpolarized [1-13C] pyruvate for detection of mutant IDH1 gliomas and for monitoring of their therapeutic response. We studied patient-derived mutant IDH1 glioma cells that underexpress LDHA, MCT1 and MCT4, and wild-type IDH1 GBM cells that express high levels of these proteins. Mutant IDH1 cells and tumors produced significantly less hyperpolarized [1-13C] lactate compared to GBM, consistent with their metabolic reprogramming. Furthermore, hyperpolarized [1-13C] lactate production was not affected by chemotherapeutic treatment with temozolomide (TMZ in mutant IDH1 tumors, in contrast to previous reports in GBM. Our results demonstrate the unusual metabolic imaging profile of mutant IDH1 gliomas, which, when combined with other clinically available imaging methods, could be used to detect the presence of the IDH1 mutation in vivo.

High-Protein Soybean Mutants by Using Irradiation Technique

International Nuclear Information System (INIS)

Yathaputanon, C.; Kumsueb, B.; Srisombun, S.

2009-07-01

Full text: Soybean variety improvement for high seed protein using induced mutation was initiated. Approximately 5,000 seeds of soybean variety Chiang Mai 60 were irradiated with gamma rays at the dose of 200 Grays at Kasetsart University. High-protein seed mutants in M2 to M4 generations were selected at Nakhon Ratchasima Field Crops Research Center during 2004-2008. The Pedigree method of selection was used. Kjeldahl method was used to analyze seed protein percentages. The M2 seeds protein content of the M2 generation was 45.2% while that of the original parent was 43.0%. M3s were seeded plant to row. In each row, the best four plants were selected for protein analysis. The average protein content of selected mutant lines was 3.9% while the check variety had average protein content of 42.4%. In the M4 generation, the result showed that the average protein contents of the selected mutant lines and the check variety were 42.8% and 42.0%, respectively. In the 2007-2008 trials, four promising mutants had and average protein content of 428%, while the check variety had and average protein content of 41.1%. The four mutants produced the mean grain yield of 2.20-2.42 t/Ha, which was 10.21% higher than that of Chiang Mai 60. The mutant lines produced both a high grain protein content and a high grain yield. They will be further tested their adaptability in the research centers and farmer fields

Gamma ray induced male sterility mutant in lentil

International Nuclear Information System (INIS)

Srivastava, A.; Yadav, A.K.

2001-01-01

Full text: Male sterility refers to the failure of pollen grains to bring about effective fertilization, either due to structural default or physiological disfunctioning and has special significance in hybridization programmes. Male steriles have been produced in a number of crop plants like red gram, pigeon pea, mung bean, khesari and lentil. A completely male sterile mutant was isolated in Lens culinaris Medik, after seed treatment with 100 Gy dose of gamma rays. The male sterile mutant showed 100% pollen sterility but was morphologically more vigorous than the parent plants. It showed more branches and its leaves were bigger, more oblong and dark green. The number of flowers borne by the mutant was significantly higher than any other plant of the treatment. The size of the flowers was also increased but the anthers were smaller in size. Pollen grains were few in number, round in shape but empty and did not take up any stain, indicating that normal microsporogenesis had not taken place. This male sterile mutant was used as the female parent and pollinated with pollen of a parent. Four pods with one seed in each were formed indicating that the mutant was female fertile. The seeds were smaller than those of the parent variety and also dark coloured. The mutant showed increased vigour and flower number as compared to parental plants. Lentil is an important pulse crop and induction of variability in its germplasm is necessary for its improvement. Male steriles can be used conveniently in lentil hybridization programmes. (author)

Phosphorus-31 nuclear magnetic resonance studies of wild-type and glycolytic pathway mutants of Saccharomyces cerevisiae.

Science.gov (United States)

Navon, G; Shulman, R G; Yamane, T; Eccleshall, T R; Lam, K B; Baronofsky, J J; Marmur, J

1979-10-16

High-resolution phosphorus-31 nuclear magnetic resonance (31P NMR) spectra of wild-type and mutant strains of Saccharomyces cerevisiae were observed at a frequency of 145.7 MHz. Levels of various phosphorus metabolites were investigated upon addition of glucose under both aerobic and anaerobic conditions. Three mutant strains were isolated and their biochemical defects characterized: pfk lacked phosphofructokinase activity; pgi lacked phosphoglucose isomerase activity; and cif had no glucose catabolite repression of the fructose bisphosphatase activity. Each mutant strain was found to accumulate characteristic sugar phosphates when glucose was added to the cell suspension. In the case of the phosphofructokinase deficient mutant, the appearance of a pentose shunt metabolite was observed. 31P NMR peak assignments were made by a pH titration of the acid extract of the cells. Separate signals for terminal, penultimate, and central phosphorus atoms in intracellular polyphosphates allowed the estimation of their average molecular weight. Signals for glycero(3)phosphochline, glycero(3)phosphoserine, and glycero(3) phosphoethanolamine as well as three types of nucleotide diphosphate sugars could be observed. The intracellular pH in resting and anaerobic cells was in the range 6.5--6.8 and the level of adenosine 5'-triphosphate (ATP) low. Upon introduction of oxygen, the ATP level increased considerably and the intracellular pH reached a value of pH 7.2--7.3, irrespective of the external medium pH, indicating active proton transport in these cells. A new peak representing the inorganic phosphate of one of the cellular organelles, whose pH differed from the cytoplasmic pH, could be detected under appropriate conditions.

Temperature-sensitive host range mutants of herpes simplex virus type 2

International Nuclear Information System (INIS)

Koment, R.W.; Rapp, F.

1975-01-01

Herpesviruses are capable of several types of infection of a host cell. To investigate the early events which ultimately determine the nature of the virus-host cell interaction, a system was established utilizing temperature-sensitive mutants of herpes simplex virus type 2. Four mutants have been isolated which fail to induce cytopathic effects and do not replicate at 39 C in hamster embryo fibroblast cells. At least one mutant is virus DNA negative. Since intracellular complementation is detectable between pairs of mutants, a virus function is known to be temperature sensitive. However, all four mutants induce cytopathic effects and replicate to parental virus levels in rabbit kidney cells at 39 C. This suggests that a host cell function, lacking or nonfunctional in HEF cells but present in rabbit kidney cells at 39 C, is required for the replication of these mutants in hamster embryo fibroblast cells at 39 C. Therefore, we conclude that these mutants are both temperature sensitive and exhibit host range properties

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

Science.gov (United States)

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

2017-11-15

common features of Parkinson's disease (PD), causing significant disability. Mutations in LRRK2 represent the most common known genetic cause of PD. We found that PD-linked LRRK2 mutations increased dendritic and mitochondrial calcium uptake in cortical neurons and familial PD patient fibroblasts, accompanied by increased expression of the mitochondrial calcium transporter MCU. Blocking the ERK1/2-dependent upregulation of MCU conferred protection against mutant LRRK2-elicited dendrite shortening, as did inhibiting MCU-mediated calcium import. Conversely, stimulating the export of calcium from mitochondria was also neuroprotective. These results implicate increased susceptibility to mitochondrial calcium overload in LRRK2-driven neurodegeneration, and suggest possible interventions that may slow the progression of cognitive dysfunction in PD. Copyright © 2017 the authors 0270-6474/17/3711152-15$15.00/0.

Induced mutants for the improvement of sesame and hybrid seed production

International Nuclear Information System (INIS)

Murty, G.S.S.

2001-01-01

With an overall objective to develop hybrids in sesame, induced mutants were used in cross breeding and five initial yield trials were conducted. For obtaining the mutant hybrids, recessive morphological mutants were used as female, and check varieties as male parents. In each trial, seed yields of mutant hybrids were compared with: i) the original parent in which the mutants were induced, ii) best check variety and iii) best cultivar hybrid. Among 138 mutant hybrids evaluated between 1994 and 1997, 18 showed superiority. In the development of hybrids, it is also desirable to have male sterile lines. By irradiating seeds with 400 Gy gamma rays, four genetic male sterile mutants were isolated. One of them, TMST-11 appears to be promising for breeding programme showing 100% male sterility and characterised by dark green foliage. To study the percent outcrossing, a monogenic chlorina mutant which can be identified from the seedling stage, was used in experiments conducted for two years. Among open pollinated plants, 92-98% plants were found outcrossed. Based on plant to row progenies, percent outcrossing ranged between 0.0 to 13.8%. (author)

Molecular Genetic Identification Of Some Flax Mutants

International Nuclear Information System (INIS)

AMER, I.M.; MOUSTAFA, H.A.M.

2009-01-01

Five flax genotypes (Linum usitatissimum L.) i.e., commercial cultivar Sakha 2, the mother variety Giza 4 and three mutant types induced by gamma rays, were screened for their salinity tolerance in field experiments (salinity concentration was 8600 and 8300 ppm for soil and irrigation water, respectively). Mutation 6 was the most salt tolerant as compared to the other four genotypes.RAPD technique was used to detect some molecular markers associated with salt tolerance in flax (Mut 6), RAPD-PCR results using 12 random primers exhibited 149 amplified fragments; 91.9% of them were polymorphic and twelve molecular markers (8.1%) for salt tolerant (mutant 6) were identified with molecular size ranged from 191 to 4159 bp and only eight primers successes to amplify these specific markers. Concerning the other mutants, Mut 15 and Mut 25 exhibited 4.3% and 16.2% specific markers, respectively. The induced mutants exhibited genetic similarity to the parent variety were about 51%, 58.3% and 61.1% for Mut 25, Mut 6 and Mut 15, respectively. These specific markers (SM) are used for identification of the induced mutations and it is important for new variety registration.

Semi-dwarf mutant lines of hexaploid triticale

International Nuclear Information System (INIS)

Pidra, M.

1989-01-01

A spring form of hexaploid secondary triticale ADD 143/71, bred by MOGILEVA at the Plant Breeding Station at Uhretice was used for the mutagen treatment. The mutation experiment started in 1979. Seeds were treated with a 0.8 mM water solution of N-methyl-N-nitrosourea (MNH) (CETL and RELICHOVA, unpublished). From 180 M 1 plants, one spike was harvested per plant. A random sample of these seeds was sown as M 2 in 1980 and several plants with shorter main culm were selected. Selfed progenies of eight mutant plants designated ADD 143-m1, ADD 143-m2, ADD 143-m3 etc. were further tested in M 3 and M 4 . There were significant differences in culm length and in some other characters between the original line and the mutant lines. Especially the line m8 looks like a promising source of semi-dwarfness for breeding programmes of hexaploid triticale. During 1985-1987 genetic analysis was performed on the ADD 143/71 and the mutant lines m2, m6, m7 and m8, which suggest that their mutant genes are allelic and recessive

Subunit-specific phenotypes of Salmonella typhimurium HU mutants.

OpenAIRE

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

1990-01-01

Salmonella hupA and hupB mutants were studied to determine the reasons for the high degree of conservation in HU structure in bacteria. We found one HU-1-specific effect; the F'128 plasmid was 25-fold less stable in hupB compared with hupA or wild-type cells. F' plasmids were 120-fold more unstable in hupA hupB double mutants compared with wild-type cells, and the double mutant also had a significant alteration in plasmid DNA structure. pBR322 DNA isolated from hupA hupB strains was deficient...

Mutation induction and evaluation of high yield rice mutants

International Nuclear Information System (INIS)

Abdul Rahim Harun; Sobri Husein; Rusli Ibrahim

2006-01-01

The successful use of plant breeding for improving crops requires the existence of genetic variation of useful traits. Unfortunately, the desired variation is often lacking. However, radiation has been used to induce mutations and thereby generate genetic variation from which desired mutants may be selected. Mutation induction has become a proven way of creating variation within a crop variety. It offers the possibility of inducing desired attributes that either cannot be expressed in nature or have been lost during evolution. Rice is security food crop in Malaysia. Efforts were undertaken to enhance rice yield from 4.0 tones per hectare in 1995 to 5.5 tones per hectare in 2010. Proper management and good varieties are two factors that require for enhancing yield of rice. In this research, purified seeds of MR211 and MR219 were gamma irradiated at 100 to 400 Gray and sown for planting as M1 generation at MARDI experimental plot. The M2 population was sown in bulk with population size around 15,000 to 20,000 plants. Individual plant selection was carried out at maturity and each selected plant became a mutant line of M3 generation. Agronomic trial of M3 mutants lines were conducted in Mardi, Tanjung Karang, Selangor. About 115 of selected mutant lines were evaluated. Each row of those mutant lines were planted in two rows at planting distance of 25cm within and between rows. These mutant lines were visually observed and data were recorded in each of every mutant line. (Author)

Endoplasmic Reticulum-Targeted Subunit Toxins Provide a New Approach to Rescue Misfolded Mutant Proteins and Revert Cell Models of Genetic Diseases.

Science.gov (United States)

Adnan, Humaira; Zhang, Zhenbo; Park, Hyun-Joo; Tailor, Chetankumar; Che, Clare; Kamani, Mustafa; Spitalny, George; Binnington, Beth; Lingwood, Clifford

2016-01-01

Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER) 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD), to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant) subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subunit has evolved to mimic a misfolded protein and hijack the ERAD membrane translocon (dislocon), to effect cytosolic access and cytopathology. We show such toxins compete for ERAD to rescue endogenous misfolded proteins. Cholera toxin or verotoxin (Shiga toxin) containing genetically inactivated (± an N-terminal polyleucine tail) A subunit can, within 2-4 hrs, temporarily increase F508delCFTR protein, the major cystic fibrosis (CF) mutant (5-10x), F508delCFTR Golgi maturation (glucocerobrosidase (GCC) in N370SGCC Gaucher Disease fibroblasts (3x), another ERAD-exacerbated misfiling disease. We identify a new, potentially benign approach to the treatment of certain genetic protein misfolding diseases.

Hypoxic resistance of KRAS mutant tumor cells to 3-Bromopyruvate is counteracted by Prima-1 and reversed by N-acetylcysteine.

Science.gov (United States)

Orue, Andrea; Chavez, Valery; Strasberg-Rieber, Mary; Rieber, Manuel

2016-11-18

The metabolic inhibitor 3-bromopyruvate (3-BrPA) is a promising anti-cancer alkylating agent, shown to inhibit growth of some colorectal carcinoma with KRAS mutation. Recently, we demonstrated increased resistance to 3-BrPA in wt p53 tumor cells compared to those with p53 silencing or mutation. Since hypoxic microenvironments select for tumor cells with diminished therapeutic response, we investigated whether hypoxia unequally increases resistance to 3-BrPA in wt p53 MelJuso melanoma harbouring (Q61L)-mutant NRAS and wt BRAF, C8161 melanoma with (G12D)-mutant KRAS (G464E)-mutant BRAF, and A549 lung carcinoma with a KRAS (G12S)-mutation. Since hypoxia increases the toxicity of the p53 activator, Prima-1 against breast cancer cells irrespective of their p53 status, we also investigated whether Prima-1 reversed hypoxic resistance to 3-BrPA. In contrast to the high susceptibility of hypoxic mutant NRAS MelJuso cells to 3-BrPA or Prima-1, KRAS mutant C8161 and A549 cells revealed hypoxic resistance to 3-BrPA counteracted by Prima-1. In A549 cells, Prima-1 increased p21CDKN1mRNA, and reciprocally inhibited mRNA expression of the SLC2A1-GLUT1 glucose transporter-1 and ALDH1A1, gene linked to detoxification and stem cell properties. 3-BrPA lowered CAIX and VEGF mRNA expression. Death from joint Prima-1 and 3-BrPA treatment in KRAS mutant A549 and C8161 cells seemed mediated by potentiating oxidative stress, since it was antagonized by the anti-oxidant and glutathione precursor N-acetylcysteine. This report is the first to show that Prima-1 kills hypoxic wt p53 KRAS-mutant cells resistant to 3-BrPA, partly by decreasing GLUT-1 expression and exacerbating pro-oxidant stress.

Biological changes in Barley mutants resistant to powdery mildew disease

International Nuclear Information System (INIS)

Amer, I. M.; Fahim, M. M.; Moustafa, N. A.

2012-12-01

physiological studies showed that all kinds of chlorophyll (a), (b) and (a + b) content in infected plant were decreased while, the carotenes pigment were increased. Infection generally reduced total sugars content of all resistant mutants. Infected resistant mutant showed more phenols content and peroxidase, polyphenoloxidase activities than healthy ones of the mutants. (Author)

CorA, the magnesium/nickel/cobalt transporter, affects virulence and extracellular enzyme production in the soft rot pathogen Pectobacterium carotovorum.

Science.gov (United States)

Kersey, Caleb M; Agyemang, Paul A; Dumenyo, C Korsi

2012-01-01

Pectobacterium carotovorum (formerly Erwinia carotovora ssp. carotovora) is a phytopathogenic bacterium that causes soft rot disease, characterized by water-soaked soft decay, resulting from the action of cell wall-degrading exoenzymes secreted by the pathogen. Virulence in soft rot bacteria is regulated by environmental factors, host and bacterial chemical signals, and a network of global and gene-specific bacterial regulators. We isolated a mini-Tn5 mutant of P. carotovorum that is reduced in the production of extracellular pectate lyase, protease, polygalacturonase and cellulase. The mutant is also decreased in virulence as it macerates less host tissues than its parent and is severely impaired in multiplication in planta. The inactivated gene responsible for the reduced virulent phenotype was identified as corA. CorA, a magnesium/nickel/cobalt membrane transporter, is the primary magnesium transporter for many bacteria. Compared with the parent, the CorA(-) mutant is cobalt resistant. The mutant phenotype was confirmed in parental strain P. carotovorum by marker exchange inactivation of corA. A functional corA(+) DNA from P. carotovorum restored exoenzyme production and pathogenicity to the mutants. The P. carotovorum corA(+) clone also restored motility and cobalt sensitivity to a CorA(-) mutant of Salmonella enterica. These data indicate that CorA is required for exoenzyme production and virulence in P. carotovorum. © 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.

The PaAlr1 magnesium transporter is required for ascospore development in Podospora anserina.

Science.gov (United States)

Grognet, Pierre; Lalucque, Hervé; Silar, Philippe

2012-10-01

The PaAlr1 gene encoding a putative plasma membrane magnesium (Mg) transporter in Podospora anserina was inactivated. The PaAlr1(Δ) mutants showed sensitivity to deprivation and excess Mg(2+) and Ca(2+). They also exhibited an autonomous ascospore maturation defect. Mutant ascospores were arrested at an early stage when they contained two nuclei. These data emphasize the role of Mg ions during sexual development in a filamentous fungus. Copyright © 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

A Nostoc punctiforme Sugar Transporter Necessary to Establish a Cyanobacterium-Plant Symbiosis1[C][W

Science.gov (United States)

Ekman, Martin; Picossi, Silvia; Campbell, Elsie L.; Meeks, John C.; Flores, Enrique

2013-01-01

In cyanobacteria-plant symbioses, the symbiotic nitrogen-fixing cyanobacterium has low photosynthetic activity and is supplemented by sugars provided by the plant partner. Which sugars and cyanobacterial sugar uptake mechanism(s) are involved in the symbiosis, however, is unknown. Mutants of the symbiotically competent, facultatively heterotrophic cyanobacterium Nostoc punctiforme were constructed bearing a neomycin resistance gene cassette replacing genes in a putative sugar transport gene cluster. Results of transport activity assays using 14C-labeled fructose and glucose and tests of heterotrophic growth with these sugars enabled the identification of an ATP-binding cassette-type transporter for fructose (Frt), a major facilitator permease for glucose (GlcP), and a porin needed for the optimal uptake of both fructose and glucose. Analysis of green fluorescent protein fluorescence in strains of N. punctiforme bearing frt::gfp fusions showed high expression in vegetative cells and akinetes, variable expression in hormogonia, and no expression in heterocysts. The symbiotic efficiency of N. punctiforme sugar transport mutants was investigated by testing their ability to infect a nonvascular plant partner, the hornwort Anthoceros punctatus. Strains that were specifically unable to transport glucose did not infect the plant. These results imply a role for GlcP in establishing symbiosis under the conditions used in this work. PMID:23463784

Phage Pl mutants with altered transducing abilities for Escherichia coli

International Nuclear Information System (INIS)

Wall, J.D.; Harriman, P.D.

1974-01-01

A search was made for mutants of the coliphage P1 with altered transducing frequencies. A method was developed for the rapid assay of transducing frequencies in single plaques using prophage lambda as the transduced bacterial marker. This procedure selects for mutants altered in their ability to package host DNA. Mutants with 5 to 10 times higher or 10 to 20 times lower frequencies than those of wild-type P1 were found. Not only are the markers used for the detection of the mutants affected, but all other markers are similarly affected (not always to the same extent). One of the high transducing frequency mutants is a suppressible amber, indicating that loss of a function increases P1's ability to package host DNA preferentially. (U.S.)

Photosynthetic and nitrogen fixation capability in several soybean mutant lines

International Nuclear Information System (INIS)

Gandanegara, S.; Hendratno, K.

1987-01-01

Photosynthetic and nitrogen fixation capability in several soybean mutant lines. A greenhouse experiment has been carried out to study photosynthetic and nitrogen fixation capability of five mutant lines and two soybean varieties. An amount of 330 uCi of 14 CO 2 was fed to the plants including of the non-fixing reference crop (Chippewa non-nodulating isoline). Nitrogen fixation measurements was carried out using 15 N isotope dilution technique according to A-value concept. Results showed that beside variety/mutant lines, plant growth also has important role in photosynthetic and N fixing capability. Better growth and a higher photosynthetic capability in Orba, mutant lines nos. 63 and 65 resulted in a greater amount of N 2 fixed (mg N/plant) than other mutant lines. (author). 12 refs.; 5 figs

Isolation and characterization of stable mutants of Streptomyces

Indian Academy of Sciences (India)

Daunorubicin and its derivative doxorubicin are antitumour anthracycline antibiotics produced by Streptomyces peucetius. In this study we report isolation of stable mutants of S. peucetius blocked in different steps of the daunorubicin biosynthesis pathway. Mutants were screened on the basis of colony colour since producer ...

Identification of Potential Calorie Restriction-Mimicking Yeast Mutants with Increased Mitochondrial Respiratory Chain and Nitric Oxide Levels

Directory of Open Access Journals (Sweden)

Bin Li

2011-01-01

Full Text Available Calorie restriction (CR induces a metabolic shift towards mitochondrial respiration; however, molecular mechanisms underlying CR remain unclear. Recent studies suggest that CR-induced mitochondrial activity is associated with nitric oxide (NO production. To understand the role of mitochondria in CR, we identify and study Saccharomyces cerevisiae mutants with increased NO levels as potential CR mimics. Analysis of the top 17 mutants demonstrates a correlation between increased NO, mitochondrial respiration, and longevity. Interestingly, treating yeast with NO donors such as GSNO (S-nitrosoglutathione is sufficient to partially mimic CR to extend lifespan. CR-increased NO is largely dependent on mitochondrial electron transport and cytochrome c oxidase (COX. Although COX normally produces NO under hypoxic conditions, CR-treated yeast cells are able to produce NO under normoxic conditions. Our results suggest that CR may derepress some hypoxic genes for mitochondrial proteins that function to promote the production of NO and the extension of lifespan.

Compact type mutants in apple and sour cherries

International Nuclear Information System (INIS)

Zagaja, S.W.; Przybyla, A.

1976-01-01

Induction of mutations in deciduous fruits is considered complementary to the conventional breeding methods. Several promissing mutants, particularly in apples, were described and some of them were introduced to commercial orchards. Studies described herein are aimed at developing compact type mutants in apple cultivars, apple rootstocks and in sour cherry cultivars. Data obtained so far confirm the results of the other authors, who developed compact type mutants in apples and sweet cherries. Physiological studies have shown that the leaves of spontaneous apple mutants of compact type are more efficient in photosynthesis than the leaves of respective standards. In spite of this, using branch ringing techniques, it was found that the leaves of compacts and those of standards do not differ in their productivity. There seem to be several advantages in employing tissue culture technique in mutation breeding. That is why a project was started to work out a method of growing apple shoots from adventitious buds developed on sections of roots. (author)

early maturing mutants in Indica rice and their traits

International Nuclear Information System (INIS)

Chen Xiulan; He Zhentian; Han Yuepeng; Liu Xueyu; Yang Hefeng; Xu Chenwu; Gu Shiliang

1998-01-01

The correlation and genetic parameters of eleven agronomic characters of 50 early mature lines induced from late mature cultivar, IR 1529-68-3-2 were studied by morphological classification and correlation and regression analysis. The results showed that: 1. The early mutants could be divided into two ecotype: early mature type and medium mature type of mid-maturity rice. 2. The 1000-grain weight of early mutants negatively correlated with the length of growing period. 3. According to direct path coefficients, the relation with heading period of early mutants was in order of 1000-grain-weight>plant height>seed sterility. 4.The higher heritability in broad sense were found in plant height, 1000 grain weight and heading period of the early mutants

Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo.

Directory of Open Access Journals (Sweden)

Suneeta Chimalapati

Full Text Available Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt, deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection.

Neurofilament subunit (NFL) head domain phosphorylation regulates axonal transport of neurofilaments.

LENUS (Irish Health Repository)

Yates, Darran M

2009-04-01

Neurofilaments are the intermediate filaments of neurons and are synthesised in neuronal cell bodies and then transported through axons. Neurofilament light chain (NFL) is a principal component of neurofilaments, and phosphorylation of NFL head domain is believed to regulate the assembly of neurofilaments. However, the role that NFL phosphorylation has on transport of neurofilaments is poorly understood. To address this issue, we monitored axonal transport of phosphorylation mutants of NFL. We mutated four known phosphorylation sites in NFL head domain to either preclude phosphorylation, or mimic permanent phosphorylation. Mutation to preclude phosphorylation had no effect on transport but mutation of three sites to mimic permanent phosphorylation inhibited transport. Mutation of all four sites together to mimic permanent phosphorylation proved especially potent at inhibiting transport and also disrupted neurofilament assembly. Our results suggest that NFL head domain phosphorylation is a regulator of neurofilament axonal transport.

Identification of residues in ABCG2 affecting protein trafficking and drug transport, using co-evolutionary analysis of ABCG sequences.

Science.gov (United States)

Haider, Ameena J; Cox, Megan H; Jones, Natalie; Goode, Alice J; Bridge, Katherine S; Wong, Kelvin; Briggs, Deborah; Kerr, Ian D

2015-07-17

ABCG2 is an ABC (ATP-binding cassette) transporter with a physiological role in urate transport in the kidney and is also implicated in multi-drug efflux from a number of organs in the body. The trafficking of the protein and the mechanism by which it recognizes and transports diverse drugs are important areas of research. In the current study, we have made a series of single amino acid mutations in ABCG2 on the basis of sequence analysis. Mutant isoforms were characterized for cell surface expression and function. One mutant (I573A) showed disrupted glycosylation and reduced trafficking kinetics. In contrast with many ABC transporter folding mutations which appear to be 'rescued' by chemical chaperones or low temperature incubation, the I573A mutation was not enriched at the cell surface by either treatment, with the majority of the protein being retained in the endoplasmic reticulum (ER). Two other mutations (P485A and M549A) showed distinct effects on transport of ABCG2 substrates reinforcing the role of TM helix 3 in drug recognition and transport and indicating the presence of intracellular coupling regions in ABCG2. © 2015 Authors.

Promising mutant variety of rice evolved through gamma irradiation

International Nuclear Information System (INIS)

Prasad, S.C.; Sinha, S.K.

1980-01-01

Rice occupies a major share in crop production in the Chotanagpur plateau of Bihar State. Uplands are roughly 40% in area where traditional low yielding rice, known as ''gora'' is cultivated as directly sown crop. Despite introduction of high yielding rice varieties, gora group of rices continue to prevail. It is therefore desired to increase the productivity level of the gora rice by mutation breeding. One such mutant known as ''gora mutant'' was obtained through gamma irradiation (10 kR) of variety Brown gora. The maturity of both parent and mutant remaining constant (ie. 100 days), there is some improvement in other characteristics like plant height, tillering capacity and kernel character. The parent being tall, shy in tillering and red bold kernel, the mutant has dwarfish characteristics, profuse tillering habit and white kernel with fine grains. The yielding capacity of mutant derivative is 30-40% higher than the parent Brown gora. This variety is in pre-release stage, and the farmers have taken great liking for it. (author)

A preliminary yield trial of some soybean mutant lines

International Nuclear Information System (INIS)

Ratma, Rivaie

1985-01-01

A preliminary yield trial of some soybean mutant lines, derived from irradiated Orba variety with dose of 0.40 kGy, were carried out during the wet and dry season in 1979-1982 in Muara and Citayam, Bogor. The result obtained showed that yield potential of mutant lines no. M6/40/10 was higher than that of the control in dry season in 1979 as well as in the wet season of 1979/80 in Muara. Whereas, the yield potential of the mutant lines no. M6/40/8 and no. M6/40/14 were higher than that of the control only in the wet season. The yield potential of semi dwarf mutant lines no. M6/40/68 was highly significant compared to that of the control in dry season in Muara and the wet season in 1981/82 in Citayam. Whereas, the yield potential of the mutant lines no. M6/40/69 was higher yield compared to that of the control in dry season in 1981 in Muara. (author). 10 refs

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

Science.gov (United States)

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.

Evaluation of some mutant lines of rice induced by gamma radiation treatment 1. mean performance of rice mutants in M4 generation

International Nuclear Information System (INIS)

El-Banna, M.N.; El-Wakil, H.M.F.; Ebaid, R.A.; Sallam, R.A.

2009-01-01

Grains of eight rice mutants; SC 1, SC 6, RTY 1, RTY 3, HY 14, HYI 17, EH 4 and HYPI 22 were secured from Botany Department Faculty of Agriculture Cairo university. The procedures and the methodology for induction these mutants as well as the original mean performance of such mutants are presented else where; Sabbour, (1989) and Sabbour etal. (2002). Grains were sown (M4 generation) at the experimental farm in Itai EI-Baroud Agricultural Research Station Behaira Governorate Agricultural Research Center (ARC) in the summer season (2007). The mean performance of such mutants was studied during M4 generation. The most exciting results were as follows: the selected line SC 1 showed in M4 generation superior agronomic and yield traits. Sc 1 mutant line is not bred truly and it need more generations to reach stability. SC 6 in M4 generation showed considerable number of individuals scored low mean values toward the negative direction and lowering the overall trait mean performance. The rice lines RTY 1 and RTY 3 proved that, the average number of fertile tillers per plant of the selected lines maintained previously recorded mean values of M3 generation in M4. The traits showed significant differences among their progeny that recorded high CV% values as compared with those showed no significant differences. The rice lines HY 14 and HYI 17 showed a true breeding signs and no more breeding generations are required. Rice lines EH 4, showed a considerable reduction in number of days elapsed from date of cultivation till harvest. As, this mutant maintained 86.58 days till heading. Rice mutant line HYPI 22 did not bred truly for the original selected traits (high yield and high protein content) and it still need more generations of selection to reach considerable stability

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

Science.gov (United States)

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

Identification of a novel ga-related bush mutant in pumpkin (cucurbita moschata duchesne)

International Nuclear Information System (INIS)

Wu, T.; Cao, J.

2015-01-01

Pumpkin (Cucurbita moschata Duchesne) bush mutant plants were characterized by short stems. The sensitivity of pumpkin bush mutant plants to exogenous hormones was identified in this study. Results revealed that internode elongation of bush mutant plants could respond to gibberellins (GA4+7 and GA3), but not to indole acetic acid (IAA) and brassinosteroids (BR); by contrast, the mutant phenotype of bush mutant plants could not be fully rescued by GA4+7 and GA3. The internode of bush mutant plants yielded a lower KS expression level than that of vine plants. Therefore, pumpkin bush mutant plants were designated as GA-related mutant plants eliciting a partial response to GAs; the action of IAA and BR might not be involved in the internode growth of pumpkin bush mutant plants, specifically Cucurbita moschata Duch. (author)

Mutant lines of currant tomato, valuable germplasm with multiple disease resistance

International Nuclear Information System (INIS)

Govorova, G.F.; Khrustaleva, V.V.; Shcherbakov, V.K.

1987-01-01

Studies were carried out for two years on eight mutant lines of currant tomato at the Krymsk Experimental Breeding Station of the N.I. Vavilov All-Union Scientific Research Institute of Plant-Growing (VIR). The station is situated in an area of commercial field tomato growing (Krasnodar region). The mutant lines of currant tomato (VIR specimen No. k-4053) were obtained through chronic gamma-irradiation. A disease resistance evaluation of the mutants was carried out for Verticillium wilt (Verticillium albo-atrum Rein. and Berth.), for black bacterial spotting (Xanthomonas vesicatoria Dows.), for tobacco mosaic virus Nicotiana 1 Smith), for streak virus (Nicotiana 1), for the combination TMV with X and Y potato viruses, for cucumber virus (Cucumis 1), and also for top rot. Fifty plants of each mutant line were evaluated and checks were made three times in each season. A comparison of the currant tomato mutants with the standard tomato varieties demonstrates the better resistance shown by the mutant germplasm to the main pathogens. The degree to which some currant tomato mutants were affected by Verticillium was lower than that of the most VerticiIlium-resistant samples of tomato evaluated between 1975 and 1981. The mutants of currant tomato should therefore be of interest as germplasm in breeding tomatoes for improved multiple disease resistance

A chilling sensitive mutant of Arabidopsis with altered steryl-ester metabolism

International Nuclear Information System (INIS)

Hugly, S.; McCourt, P.; Somerville, C.; Browse, J.; Patterson, G.W.

1990-01-01

A chilling-sensitive mutant of Arabidopsis thaliana was isolated and subjected to genetic, physiological, and biochemical analysis. The chilling-sensitive nature of the mutant line is due to a single recessive nuclear mutation at a locus designated chs1. In contrast to wild-type plants, which are not adversely affected by low temperatures, the chs1 mutant is killed by several days of exposure to temperatures below 18 degree C. Following exposure to chilling temperatures, the mutant displays two common symptoms of chilling injury - leaf chlorosis and electrolyte leakage. In these respects, the physiological response of the mutant to low temperatures mimics the response observed in some naturally occurring chilling sensitive species. The biochemical basis of chilling sensitivity was explored by examining the pattern of incorporation of 14 CO 2 into soluble metabolites and lipids in wild-type and mutant plants. The only difference observed between the mutant and wild type was that following low temperature treatment, the mutant accumulated 10-fold more radioactivity in a specific class of neutral lipids which were identified by a variety of criteria to be steryl-esters. The accumulation of radioactivity in the steryl-ester fraction occurs 24 hours before there is any visible evidence of chilling injury

Sodium azide mutagenesis in wheat: Mutants with golden glumes

International Nuclear Information System (INIS)

Siddiqui, K.A.; Jafri, K.A.; Arain, M.A.

1989-01-01

In bread wheat, Triticum aestivum L. (2n=6x=42, AABBDD), detection of induced mutations is hampered by the presence of duplicate and triplicate genes. Induced changes in spike characteristics are known, but mutants with changed glume colour do not seem to have been reported. Physical mutagens such as gamma rays, thermal neutrons and fast neutrons, and chemical mutagens like EMS, El, dES and NEH have been extensively used for induction of mutations in bread wheat but it seems as if these mutagens did not induce mutants with changed glume colour. We used sodium azide for inducing mutations in the widely adapted cultivar 'Sonalika', which is characterized by brown glume colour. Presoaked seeds were treated with 0.2M sodium azide for 3 hours. Three spikes were harvested from each M 1 plant. M 2 generation was space-planted as spike progeny. We were successful in identifying 3 mutants with golden glumes. The mutants resemble 'Sonalika' in other spike characteristics. The mutants glume colour was confirmed in M 3 . The mutants were also evaluated for agronomically important characteristics. Some characters were significantly different from the parent. Glume colours may be useful as genetic markers since such characters are less influenced by the environment. Our investigation confirms that also agronomically useful genetic variation may be readily induced in bread wheat through sodium azide

Production and characterization of radiation-sensitive meiotic mutants of Coprinus cinereus

International Nuclear Information System (INIS)

Zolan, M.E.; Tremel, C.J.; Pukkila, P.J.

1988-01-01

We have isolated four gamma-sensitive mutants of the basidiomycete Coprinus cinereus. When homozygous, two of these (rad 3-1 and rad 9-1) produce fruiting bodies with very few viable basidiospores, the products of meiosis in this organism. A less radiation-sensitive allele of RAD 3, rad 3-2, causes no apparent meiotic defect in homozygous strains. Quantitative measurements of oidial survival of rad 3-1;rad 9-1 double mutants compared to the single mutants indicated that rad 3-1 and rad 9-1 mutants are defective in the same DNA repair pathway. In the pew viable basidiospores that are produced by these two strains, essentially normal levels of meiotic recombination can be detected. None of the mutants exhibits increased sensitivity to UV radiation. Cytological examination of meiotic chromosomes from mutant and wild-type fruiting bodies showed that rad 3-1 homozygous strains fail to condense and pair homologous chromosomes during prophase I. Although rad 9-1 strains are successful at chromosome pairing, meiosis is usually not completed in these mutants

Seed protein and nitrogen fixation in chickpea mutant variety Hyprosola

Energy Technology Data Exchange (ETDEWEB)

Schroeder, H E; Gibson, A H; Oram, R N [CSIRO, Division of Plant Industry, Canberra ACT (Australia); Shaikh, M A.Q. [Bangladesh Institute of Nuclear Agriculture, Mymensingh (Bangladesh)

1989-01-01

Full text: 'Hyprosola' is a high yielding, high protein mutant cultivar obtained after gamma irradiation from the variety 'Faridpur-1'. The mutant yields 45 % more protein per unit area. The essential amino acid index is unchanged. It is likely that the high nutritional value in 'Hyprosola' seed protein arises from an increase in the albumin:globulin ratio. Nitrogen fixation rates of the mutant during the first 7 weeks of growth were found to be similar to 'Faridpur-1'. Under field conditions, the mutant may be able to nodulate more rapidly and more extensively than the parent variety. (author)

Morphological and physiological investigations on mutants of Fusarium monoliforme IM

International Nuclear Information System (INIS)

Gancheva, V.

1996-01-01

High-producing mutants of Fusarium moniliforme IM are obtained as a result of gamma irradiation. The cultural characteristics of mutant strains 3284, 3211 and 76 following incubation of the producers for 14 days on potato-glucose agar are described. The colour of the aerial and substrate mycelium and the ability of the mutant strains to form conidiae and pigments are discussed in detail. The differences in the ability of mutants to assimilate different carbon and nitrogen sources are of specific importance for modelling nutrient media for submerged cultivation of F. moniliforme. 2 tabs., 2 figs. 7 refs

Brain-derived neurotrophic factor (BDNF) enhances GABA transport by modulating the trafficking of GABA transporter-1 (GAT-1) from the plasma membrane of rat cortical astrocytes

DEFF Research Database (Denmark)

Vaz, Sandra H; Jørgensen, Trine Nygaard; Cristóvão-Ferreira, Sofia

2011-01-01

/MAPK pathway and requires active adenosine A(2A) receptors. Transport through GAT-3 is not affected by BDNF. To elucidate if BDNF affects trafficking of GAT-1 in astrocytes, we generated and infected astrocytes with a functional mutant of the rat GAT-1 (rGAT-1) in which the hemagglutinin (HA) epitope...

CHX14 is a plasma membrane K-efflux transporter that regulates K(+) redistribution in Arabidopsis thaliana.

Science.gov (United States)

Zhao, Jian; Li, Penghui; Motes, Christy M; Park, Sunghun; Hirschi, Kendal D

2015-11-01

Potassium (K(+) ) is essential for plant growth and development, yet the molecular identity of many K(+) transporters remains elusive. Here we characterized cation/H(+) exchanger (CHX) 14 as a plasma membrane K(+) transporter. CHX14 expression was induced by elevated K(+) and histochemical analysis of CHX14 promoter::GUS transgenic plants indicated that CHX14 was expressed in xylem parenchyma of root and shoot vascular tissues of seedlings. CHX14 knockout (chx14) and CHX14 overexpression seedlings displayed different growth phenotypes during K(+) stress as compared with wild-type seedlings. Roots of mutant seedlings displayed higher K(+) uptake rates than wild-type roots. CHX14 expression in yeast cells deficient in K(+) uptake renders the mutant cells more sensitive to deficiencies of K(+) in the medium. CHX14 mediates K(+) efflux in yeast cells loaded with high K(+) . Uptake experiments using (86) Rb(+) as a tracer for K(+) with both yeast and plant mutants demonstrated that CHX14 expression in yeast and in planta mediated low-affinity K(+) efflux. Functional green fluorescent protein (GFP)-tagged versions of CHX14 were localized to both the yeast and plant plasma membranes. Taken together, we suggest that CHX14 is a plasma membrane K(+) efflux transporter involved in K(+) homeostasis and K(+) recirculation. © 2015 John Wiley & Sons Ltd.

Mildew resistant and less lodging wheat mutants induced in Iran

International Nuclear Information System (INIS)

Naghedi-Ahmadi, I.

1989-01-01

''Tabassi'' is a lodging and mildew susceptible cultivar. To induce mutations, seeds were gamma irradiated (50 to 150 Gy) in 1982 and selection for lodging resistance was carried out in M 2 . During field experiments with the mutant lines in 1985/86 there has been a heavy mildew epidemic under which mutant 63-5-I (derived from 50 Gy treatment) exhibited considerable resistance and as a consequence, higher yield. The control was 100% infected, the mutant only 40%. The mutant yielded 31% more grain, 7.5% less straw and 4.5% more protein than the control. Lodging of 63-5-I was only 60% in an experiment under rainfed conditions in the same season, resulting in a relative yield increase of about 11%. In 1986/87 there was no mildew epidemic and the mutant yielded the same as ''Tabassi''

TAF-4 is required for the life extension of isp-1, clk-1 and tpk-1 Mit mutants.

Science.gov (United States)

Khan, Maruf H; Ligon, Melissa; Hussey, Lauren R; Hufnal, Bryce; Farber, Robert; Munkácsy, Erin; Rodriguez, Amanda; Dillow, Andy; Kahlig, Erynn; Rea, Shane L

2013-10-01

While numerous life-extending manipulations have been discovered in the nematode Caenorhabditis elegans, one that remains most enigmatic is disruption of oxidative phosphorylation. In order to unravel how such an ostensibly deleterious manipulation can extend lifespan, we sought to identify the ensemble of nuclear transcription factors that are activated in response to defective mitochondrial electron transport chain (ETC) function. Using a feeding RNAi approach, we targeted over 400 transcription factors and identified 15 that, when reduced in function, reproducibly and differentially altered the development, stress response, and/or fecundity of isp-1(qm150) Mit mutants relative to wild-type animals. Seven of these transcription factors--AHA-1, CEH-18, HIF-1, JUN-1, NHR-27, NHR-49 and the CREB homolog-1 (CRH-1)-interacting protein TAF-4--were also essential for isp-1 life extension. When we tested the involvement of these seven transcription factors in the life extension of two other Mit mutants, namely clk-1(qm30) and tpk-1(qm162), TAF-4 and HIF-1 were consistently required. Our findings suggest that the Mit phenotype is under the control of multiple transcriptional responses, and that TAF-4 and HIF-1 may be part of a general signaling axis that specifies Mit mutant life extension.

Mutants of GABA transaminase (POP2 suppress the severe phenotype of succinic semialdehyde dehydrogenase (ssadh mutants in Arabidopsis.

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Frank Ludewig

Full Text Available BACKGROUND: The gamma-aminubutyrate (GABA shunt bypasses two steps of the tricarboxylic acid cycle, and is present in both prokaryotes and eukaryotes. In plants, the pathway is composed of the calcium/calmodulin-regulated cytosolic enzyme glutamate decarboxylase (GAD, the mitochondrial enzymes GABA transaminase (GABA-T; POP2 and succinic semialdehyde dehydrogenase (SSADH. We have previously shown that compromising the function of the GABA-shunt, by disrupting the SSADH gene of Arabidopsis, causes enhanced accumulation of reactive oxygen intermediates (ROIs and cell death in response to light and heat stress. However, to date, genetic investigations of the relationships between enzymes of the GABA shunt have not been reported. PRINCIPAL FINDINGS: To elucidate the role of succinic semialdehyde (SSA, gamma-hydroxybutyrate (GHB and GABA in the accumulation of ROIs, we combined two genetic approaches to suppress the severe phenotype of ssadh mutants. Analysis of double pop2 ssadh mutants revealed that pop2 is epistatic to ssadh. Moreover, we isolated EMS-generated mutants suppressing the phenotype of ssadh revealing two new pop2 alleles. By measuring thermoluminescence at high temperature, the peroxide contents of ssadh and pop2 mutants were evaluated, showing that only ssadh plants accumulate peroxides. In addition, pop2 ssadh seedlings are more sensitive to exogenous SSA or GHB relative to wild type, because GHB and/or SSA accumulate in these plants. SIGNIFICANCE: We conclude that the lack of supply of succinate and NADH to the TCA cycle is not responsible for the oxidative stress and growth retardations of ssadh mutants. Rather, we suggest that the accumulation of SSA, GHB, or both, produced downstream of the GABA-T transamination step, is toxic to the plants, resulting in high ROI levels and impaired development.

A new human NHERF1 mutation decreases renal phosphate transporter NPT2a expression by a PTH-independent mechanism.

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Marie Courbebaisse

Full Text Available BACKGROUND: The sodium-hydrogen exchanger regulatory factor 1 (NHERF1 binds to the main renal phosphate transporter NPT2a and to the parathyroid hormone (PTH receptor. We have recently identified mutations in NHERF1 that decrease renal phosphate reabsorption by increasing PTH-induced cAMP production in the renal proximal tubule. METHODS: We compared relevant parameters of phosphate homeostasis in a patient with a previously undescribed mutation in NHERF1 and in control subjects. We expressed the mutant NHERF1 protein in Xenopus Oocytes and in cultured cells to study its effects on phosphate transport and PTH-induced cAMP production. RESULTS: We identified in a patient with inappropriate renal phosphate reabsorption a previously unidentified mutation (E68A located in the PDZ1 domain of NHERF1.We report the consequences of this mutation on NHERF1 function. E68A mutation did not modify cAMP production in the patient. PTH-induced cAMP synthesis and PKC activity were not altered by E68A mutation in renal cells in culture. In contrast to wild-type NHERF1, expression of the E68A mutant in Xenopus oocytes and in human cells failed to increase phosphate transport. Pull down experiments showed that E68A mutant did not interact with NPT2a, which robustly interacted with wild type NHERF1 and previously identified mutants. Biotinylation studies revealed that E68A mutant was unable to increase cell surface expression of NPT2a. CONCLUSIONS: Our results indicate that the PDZ1 domain is critical for NHERF1-NPT2a interaction in humans and for the control of NPT2a expression at the plasma membrane. Thus we have identified a new mechanism of renal phosphate loss and shown that different mutations in NHERF1 can alter renal phosphate reabsorption via distinct mechanisms.

Point mutations in a nucleoside transporter gene from Leishmania donovani confer drug resistance and alter substrate selectivity

OpenAIRE

Vasudevan, Gayatri; Ullman, Buddy; Landfear, Scott M.

2001-01-01

Leishmania parasites lack a purine biosynthetic pathway and depend on surface nucleoside and nucleobase transporters to provide them with host purines. Leishmania donovani possess two closely related genes that encode high affinity adenosine-pyrimidine nucleoside transporters LdNT1.1 and LdNT1.2 and that transport the toxic adenosine analog tubercidin in addition to the natural substrates. In this study, we have characterized a drug-resistant clonal mutant of L. do...

Characterization of Lactococcus lactis mutants with improved performance at high temperatures and potential dairy applications

DEFF Research Database (Denmark)

Chen, Jun

microarray analysis revealed apparent differences in the transcriptional response to heat between the mutant and parent. It was found that SNPs preceding gene groESL and ribU resulted in over-expression of chaperone proteins GroES-GroEL, and membrane associated riboflavin transporter protein RibU in TM29......, respectively. Moreover, a large deletion in TM29 caused the inactivation of 10 genes (llmg_1349-llmg_1358). Through allelic replacement and gene knockout followed by fitness assessment, four main positive mutations were eventually discovered. The SNP preceding groESL and deletion of llmg_1349-llmg_1358...

Yeast carboxypeptidase Y requires glycosylation for efficient intracellular transport, but not for vacuolar sorting, in vivo stability, or activity

DEFF Research Database (Denmark)

Winther, Jakob R.; Stevens, T H; Kielland-Brandt, Morten

1991-01-01

the formation of asparagine-linked glycosylation, had a similar effect on the transport of CPY at 23 degrees C. However, the absence of N-linked carbohydrate in general had the more dramatic result of blocking the transport of CPY altogether at an increased temperature (37 degrees C). The unglycosylated mutant...

Isozyme patterns of powdery mildew resistant wheat mutants

International Nuclear Information System (INIS)

Xia Wengau; Li Zhengkui; Wang Kefeng

1989-01-01

Full Text: Wheat mutants induced by gamma irradiation and showing improved resistance to powdery mildew were analysed for isozymes. The peroxidase band 3A could be related to the disease reaction. The band 3A is absent in resistant mutants, the higher the activity of band 3A the greater the susceptibility. (author)

Characterization of membrane protein trafficking and cellular signaling at the primary cilium

DEFF Research Database (Denmark)

Mogensen, Johanne Bay

Primary cilia are microtubule-based, non-motile, sensory organelles emerging in a single copy from the surface of most quiescent cells in vertebrates. They emanate from the centrosomal mother centriole and are assembled and maintained by a bidirectional transport process termed intraflagellar...... transport. Specific receptors, ion channels and downstream signaling components are localized along the cilium-centrosome axis, enabling the cilium to function as a hot spot for the balanced coordination of multiple signaling pathways to control cell cycle entry, differentiation and migration during...... differentiation of mouse stem cells into cardiomyocytes. These results support the conclusion that Tab2 functions at the primary cilium to coordinate specified signaling events, which when defective may lead to congenital heart disease Collectively, the results presented in this PhD thesis provide new insights...

Nature of mutants induced by ionizing radiation in cultured hamster cells. II. Antigenic response and reverse mutation of HPRT-deficient mutants induced by. gamma. -rays or ethyl methanesulphonate

Energy Technology Data Exchange (ETDEWEB)

Brown, R; Stretch, A; Thacker, J

1986-04-01

A large series of independent mutants deficient in HPRT enzyme activity, isolated from V79-4 hamster cells, were assessed for properties which reflect the nature of the genetic changes induced. A total of 88 mutants were screened, 43 isolated from ..gamma..-ray-treated cultures and 45 induced by ethyl methanesulphonate (EMS). Firstly, each mutant was assayed for the presence of protein with the antigenic response of HPRT. In a competitive inhibition assay, 31% of EMS-induced mutants were CRM-positive compared to 7% of the ..gamma..-ray series. Secondly, each mutant was tested for ability to revert to HPRT proficiency. All except 2 of the EMS-induced mutants reverted with ethyl nitrosourea ENU, and many reverted spontaneously, under the given conditions. However reversion was not detected in about 80% of ..gamma..-ray-induced mutants, suggesting that the types of forward mutation caused by ionizing radiation differ qualitatively from those caused by EMS. (Auth.). 30 refs.; 6 figs.; 2 tabs.

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

Science.gov (United States)

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 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.

Purine Restriction Induces Pronounced Translational Upregulation of the NT1 Adenosine/Pyrimidine Nucleoside Transporter in Leishmania major

OpenAIRE

Ortiz, Diana; Valdés, Raquel; Sanchez, Marco A.; Hayenga, Johanna; Elya, Carolyn; Detke, Siegfried; Landfear, Scott M.

2010-01-01

Leishmania and other parasitic protozoa are unable to synthesize purines de novo and are reliant upon purine nucleoside and nucleobase transporters to import preformed purines from their hosts. To study the roles of the four purine permeases NT1-NT4 in Leishmania major, null mutants in each transporter gene were prepared and the effect of each gene deletion on purine uptake was monitored. Deletion of the NT3 purine nucleobase transporter gene or both NT3 and the NT2 nucleoside transporter gen...

Nitrate levels and stages of growth in hypernodulating mutants of Lupinus albus. II. Enzymatic activity and transport of N in the xylem sap Diferentes níveis de nitrato e estágio de crescimento em mutantes hipernodulantes de Lupinus albus II. Atividade enzimática e transporte de N na seiva do xilema

Directory of Open Access Journals (Sweden)

Hélio Almeida Burity

1999-04-01

Full Text Available The enzymatic study and transport of N in the xylem sap was carried out with a view to observing the influence of different nitrate levels and growth stages of the plant in chemically treated mutants of Lupinus albus. Several stresses induce a reduction in plant growth, resulting in the accumulation of free amino acids, amides or ureides, not only in the shoot, but also in the roots and nodules. Although enzyme activity is decisive in avoiding products that inhibit nitrogenase by ammonium, little is known about the mechanism by which the xylem carries these products. However, this process may be the key to the function of avoiding the accumulation of amino acids in the cells of infected nodules. The behaviour of the enzymes nitrate reductase (NR, phosphoenolpyruvate carboxylase (PEPC, glutamine synthetase (GS and nitrogen compounds derived from fixation, such as N-a-amino, N-ureides and N-amide in mutant genotypes were observed. The NR enzyme activity was highly influenced by the application of nitrate showing much higher values than those in the non-application of nitrate, independently of genotype, being that the NR, the best evaluation period was in the tenth week. The L-62 genotype characterized with nitrate- resistance, clearly showed that the enzyme PEPC is inhibited by presence of nitrate. The L-135 genotype (nod- fix- showed GS activity extremely low, thus demonstrating that GS is an enzyme highly correlated with fixation. With regard to the best growth stage for GS, Lupinus albus should be evaluated in the seventh week.O estudo enzimático e o transporte de N na seiva do xilema foi realizado visando observar a influência de diferentes níveis de nitrato e estágios de crescimento da planta em mutantes tratadas quimicamente. Vários estresses induzem a redução no crescimento da planta da qual resulta na acumulação de aminoácidos livres, amidas ou ureídos, tanto na parte aérea como nas raízes e nódulos. A atividade de enzimas �

Establishment of HeLa cell mutants deficient in sphingolipid-related genes using TALENs.

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Toshiyuki Yamaji

Full Text Available Sphingolipids are essential components in eukaryotes and have various cellular functions. Recent developments in genome-editing technologies have facilitated gene disruption in various organisms and cell lines. We here show the disruption of various sphingolipid metabolic genes in human cervical carcinoma HeLa cells by using transcription activator-like effector nucleases (TALENs. A TALEN pair targeting the human CERT gene (alternative name COL4A3BP encoding a ceramide transport protein induced a loss-of-function phenotype in more than 60% of HeLa cells even though the cell line has a pseudo-triploid karyotype. We have isolated several loss-of-function mutant clones for CERT, UGCG (encoding glucosylceramide synthase, and B4GalT5 (encoding the major lactosylceramide synthase, and also a CERT/UGCG double-deficient clone. Characterization of these clones supported previous proposals that CERT primarily contributes to the synthesis of SM but not GlcCer, and that B4GalT5 is the major LacCer synthase. These newly established sphingolipid-deficient HeLa cell mutants together with our previously established stable transfectants provide a 'sphingolipid-modified HeLa cell panel,' which will be useful to elucidate the functions of various sphingolipid species against essentially the same genomic background.

Enhancement of yellow pigment production by intraspecific protoplast fusion of Monascus spp. yellow mutant (ade(-)) and white mutant (prototroph).

Science.gov (United States)

Klinsupa, Worawan; Phansiri, Salak; Thongpradis, Panida; Yongsmith, Busaba; Pothiratana, Chetsada

2016-01-10

To breed industrially useful strains of a slow-growing, yellow pigment producing strain of Monascus sp., protoplasts of Monascus purpureus yellow mutant (ade(-)) and rapid-growing M. purpureus white mutant (prototroph) were fused and fusants were selected on minimal medium (MM). Preliminary conventional protoplast fusion of the two strains was performed and the result showed that only white colonies were detected on MM. It was not able to differentiate the fusants from the white parental prototroph. To solve this problem, the white parental prototroph was thus pretreated with 20mM iodoacetamide (IOA) for cytoplasm inactivation and subsequently taken into protoplast fusion with slow-growing Monascus yellow mutant. Under this development technique, only the fusants, with viable cytoplasm from Monascus yellow mutant (ade(-)), could thus grow on MM, whereas neither IOA pretreated white parental prototroph nor yellow auxotroph (ade(-)) could survive. Fifty-three fusants isolated from yellow colonies obtained through this developed technique were subsequently inoculated on complete medium (MY agar). Fifteen distinguished yellow colonies from their parental yellow mutant were then selected for biochemical, morphological and fermentative properties in cassava starch and soybean flour (SS) broth. Finally, three most stable fusants (F7, F10 and F43) were then selected and compared in rice solid culture. Enhancement of yellow pigment production over the parental yellow auxotroph was found in F7 and F10, while enhanced glucoamylase activity was found in F43. The formation of fusants was further confirmed by monacolin K content, which was intermediate between the two parents (monacolin K-producing yellow auxotroph and non-monacolin K producing white prototroph). Copyright © 2015 Elsevier B.V. All rights reserved.

Repair-defective mutants of Alteromonas espejiana, the host for bacteriophage PM2

International Nuclear Information System (INIS)

Zerler, B.R.; Wallace, S.S.

1984-01-01

The in vivo repair processes of Alteromonas espejiana, the host for bacteriophage PM2, were characterized, and UV- and methyl methanesulfonate (MMS)-sensitive mutants were isolated. Wild-type A. espejiana cells were capable of photoreactivation, excision, recombination, and inducible repair. There was no detecttable pyrimidine dimer-DNA N-glycosylase activity, and pyrimidine dimer removal appeared to occur by a pathway analogous to the Escherichia coli Uvr pathway. The UV- and MMS-sensitive mutants of A. espejiana included three groups, each containing at least one mutation involved with excision, recombination, or inducible repair. One group that was UV sensitive but not sensitive to MMS or X rays showed a decreased ability to excise pyrimidine dimers. Mutants in this group were also sensitive to psoralen plus near-UV light and were phenotypically analogous to the E. coli uvr mutants. A second group was UV and MMS sensitive but not sensitive to X rays and appeared to contain mutations in a gene(s) involved in recombination repair. These recombination-deficient mutants differed from the E. coli rec mutants, which are MMS and X-ray sensitive. The third group of A. espejiana mutants was sensitive to UV, MMS, and X rays. These mutants were recombination deficient, lacked inducible repair, and were phenotypically similar to E. coli recA mutants

Survival and mutant production induced by mutagenic agents in Metarhizium anisopliae Sobrevivência e obtenção de mutantes induzidos por agentes mutagênicos em Metarhizium anisopliae

Directory of Open Access Journals (Sweden)

V. Kava - Cordeiro

1995-12-01

Full Text Available A wild strain of Metarhizium anisopliae, an entomopathogenic fungus, was submitted to three mutagenic agents: gamma radiation, ultraviolet light and nitrous acid. Survival curves were obtained and mutants were selected using different mutagenic doses which gave 1 to 5% survival. Morphological and auxotrophic mutants were isolated. Morphological mutants were grouped in a class with yellow conidia and other with pale vinaceous conidia as opposed to the green wild type conidia. Auxotrophic mutants had requirements for vitamin and aminoacid biosynthesis. More than 58% of the total auxotrophk mutants required proline/aipnine. Gamma radiation showed to be the most efficient mutagenic agent giving 0.2% of auxotrophk mutants followed by ultraviolet light (0.12% and nitrous acid (0.06%.The conidial colour and auxotrophk mutants isolated until now from M. anisopliae were reviewed.Uma linhagem selvagem do fungo entomopatogênico Metarhizium anisopliae foi submetida à ação de três agentes mutagênicos: radiação gama, luz ultravioleta e ácido nitroso. Curvas de sobrevivência foram obtidas para cada mutagênicos utilizado e mutantes foram selecionados a partir de doses dos mutagênicos que proporcionassem de 1 a 5% de sobrevivência. Mutantes morfológicos para a coloração de conídios e mutantes auxotróficos foram isolados. Mutantes para coloração de conidios foram agrupados em duas classes, uma com conídios amarelos e outra com conídios vinho pálido. Os mutantes auxotróficos obtidos foram deficientes para aminoácidos e vitaminas e mais de 58% deles eram auxotróficos para prolina/argmina. Radiação gama foi o mutagênico mais eficiente com uma porcentagem de obtenção de mulantes auxotróficos de aproximadamente 0,2%, seguido pela luz ultravioleta (0.12% e pelo ácido nitroso (0.06%.Os mulantes morfológicos e auxotróficos obtidos até o momento em Metarhizium anisopliae foram revistos.

Relationship between conformational changes in the dopamine transporter and cocaine-like subjective effects of uptake inhibitors

DEFF Research Database (Denmark)

Løland, Claus Juul; Desai, Rajeev I; Zou, Mu-Fa

2007-01-01

Cocaine exerts its stimulatory effect by inhibiting the dopamine transporter (DAT). However, novel benztropine- and rimcazole-based inhibitors show reduced stimulant effects compared with cocaine, despite higher affinity and selectivity for DAT. To investigate possible mechanisms, we compared...... the extracellular transporter gate is open but inaccessible when it is closed. The data indicated that cocaine analogs bind an open conformation, whereas benztropine and rimcazole analogs bind a closed conformation. Next, we investigated the changes in inhibition potency of [(3)H]dopamine uptake of the compounds...... at a mutant DAT (Y335A) characterized by a global change in the conformational equilibrium. We observed a close relationship between the decrease in potencies of inhibitors at this mutant and cocaine-like responding in rats trained to discriminate cocaine from saline injections. Our data suggest...

Study on the early and late mutants of radiation induced rice

International Nuclear Information System (INIS)

Yang Hefeng; Chen Xiulan; He Zhentian; Gu Shiliang; Xu Chenwu

1990-12-01

After three years of consecutive experiments for the irradiated M 2 generations of 53 different varieties of rice, the following results have been obtained: (1) The average of early mutant plant rate is 1.4%. The rate in the early-maturing varieties is lower than that in the late-maturing varieties. It is in proportion to the length of growing period of these varieties tested. The shortened days of growing period of early mutants are 3 to 32 days (the average was 9.5 days), and it is increasing as the growing period increases. (2) In the irradiated M 2 generation of same variety, the early mutants and late mutants could be simultaneously happened, but the rate of the late mutants is 2.67%, which is higher than the rate of early mutants (1.39%). The shortened and prolonged days of growing period are 11.5 and 10.5 days respectively. These early and late mutants have some changes, both good and bad, in agronomical traits such as plant height, weight per kilo-grains and grains number per tassel. In some extent these changes are significant

Development Of New Chrysanthemum Mutants For Malaysian Floriculture Industry

International Nuclear Information System (INIS)

Zaiton Ahmad; Affrida Abu Hassan; Shakinah Salleh; Nurul Hidayah Mahmud; Shuhaimi Shamsudin; Mohamed Najli Mohamed Yasin

2014-01-01

This five-year project was in collaboration with Japan Atomic Energy Agency (JAEA) under the Bilateral Cooperative Research Program and was partly funded by Ministry of Agriculture and Agro-Based Industry (MOA) under Agriculture R&D Fund. The main objective was to produce new chrysanthemum varieties with good horticultural traits especially for cut flower production. In this project, tissue culture samples of chrysanthemum (red and pink varieties) were sent to JAEA for ion beam irradiations. Plant regeneration and multiplication were carried out at Nuclear Malaysia whilst field screenings for morphological characteristics were done at MARDI Cameron Highlands. Through this project, a number of stable chrysanthemum mutants with various new features have been generated and of these, 8 mutants were selected based on their uniqueness and/or suitability for cut flower production. In preparation for future commercialization process, five of these mutants have been filed for plant variety protection with Department of Agriculture Malaysia and a similar process in Japan is also under consideration. In addition, molecular marker work to fingerprint these mutants has also been initiated and future research may also include development of markers for selected horticultural traits and isolation of unique mutant genes. (author)

Molybdate transporter ModABC is important for Pseudomonas aeruginosa chronic lung infection.

Science.gov (United States)

Périnet, Simone; Jeukens, Julie; Kukavica-Ibrulj, Irena; Ouellet, Myriam M; Charette, Steve J; Levesque, Roger C

2016-01-12

Mechanisms underlying the success of Pseudomonas aeruginosa in chronic lung infection among cystic fibrosis (CF) patients are poorly defined. The modA gene was previously linked to in vivo competitiveness of P. aeruginosa by a genetic screening in the rat lung. This gene encodes a subunit of transporter ModABC, which is responsible for extracellular uptake of molybdate. This compound is essential for molybdoenzymes, including nitrate reductases. Since anaerobic growth conditions are known to occur during CF chronic lung infection, inactivation of a molybdate transporter could inhibit proliferation through the inactivation of denitrification enzymes. Hence, we performed phenotypic characterization of a modA mutant strain obtained by signature-tagged mutagenesis (STM_modA) and assessed its virulence in vivo with two host models. The STM_modA mutant was in fact defective for anaerobic growth and unable to use nitrates in the growth medium for anaerobic respiration. Bacterial growth and nitrate usage were restored when the medium was supplemented with molybdate. Most significantly, the mutant strain showed reduced virulence compared to wild-type strain PAO1 according to a competitive index in the rat model of chronic lung infection and a predation assay with Dictyostelium discoideum amoebae. As the latter took place in aerobic conditions, the in vivo impact of the mutation in modA appears to extend beyond its effect on anaerobic growth. These results support the modABC-encoded transporter as important for the pathogenesis of P. aeruginosa, and suggest that enzymatic machinery implicated in anaerobic growth during chronic lung infection in CF merits further investigation as a potential target for therapeutic intervention.

Gamma ray induced mutants in Colocasia with improved storability

International Nuclear Information System (INIS)

Vasudevan, K.; Jos, J.S.; Padmaja, G.

1989-01-01

Our mutation induction experiments with Colocasia esculenta (taro) were described before. Poor storability of tubers and acridity of tuber flesh in tubers are problems in taro. While screening for induced mutants, variability in shelf-life of tubers was observed. Tubers of the mutant CM 17 did neither spoil nor lose their viability even after storing for 180 days. Yield and results of quality analyses are presented in the Table in comparison with the control variety C 9 (locally known as ''Thamarakkannan''), the check variety Rasmi (well accepted in Kerala) and another mutant CM 1. Besides high yield and long storability, the mutant CM 17 shows a reduction in phenol and sugar, but an increase in dry matter and starch content which were found to be excellent characteristics for making taro chips as the usual browning phenomenon did not occur

Unravelling molecular responses to moderate dehydration in harvested fruit of sweet orange (Citrus sinensis L. Osbeck) using a fruit-specific ABA-deficient mutant.

Science.gov (United States)

Romero, Paco; Rodrigo, María J; Alférez, Fernando; Ballester, Ana-Rosa; González-Candelas, Luis; Zacarías, Lorenzo; Lafuente, María T

2012-04-01

Water stress affects many agronomic traits that may be regulated by the phytohormone abscisic acid (ABA). Within these traits, loss of fruit quality becomes important in many citrus cultivars that develop peel damage in response to dehydration. To study peel dehydration transcriptional responsiveness in harvested citrus fruit and the putative role of ABA in this process, this study performed a comparative large-scale transcriptional analysis of water-stressed fruits of the wild-type Navelate orange (Citrus sinesis L. Osbeck) and its spontaneous ABA-deficient mutant Pinalate, which is more prone to dehydration and to developing peel damage. Major changes in gene expression occurring in the wild-type line were impaired in the mutant fruit. Gene ontology analysis revealed the ability of Navelate fruits to induce the response to water deprivation and di-, tri-valent inorganic cation transport biological processes, as well as repression of the carbohydrate biosynthesis process in the mutant. Exogenous ABA triggered relevant transcriptional changes and repressed the protein ubiquitination process, although it could not fully rescue the physiological behaviour of the mutant. Overall, the results indicated that dehydration responsiveness requires ABA-dependent and -independent signals, and highlight that the ability of citrus fruits to trigger molecular responses against dehydration is an important factor in reducing their susceptibility to developing peel damage.

Genetic analysis of plant height in induced mutants of aromatic rice

International Nuclear Information System (INIS)

Kole, P.C.

2005-01-01

Inheritance of plant height in five gamma-ray induced mutants of aromatic rice cultivar Gobindabhog was studied through 6 x 6 diallel cross and segregation analyses. Diallel analysis revealed presence of additive and non-additive gene action with the preponderance of the latter. Proportion of dominant and recessive alleles was distributed unequally among the parents. The direction of dominance was towards tallness. The number of groups of genes was found to be three. The segregation analysis indicated the role of a single major recessive gene for height reduction in three mutants and, in another mutant, a single major recessive gene with negative modifiers. The other semi-dwarf mutant had two major recessive genes with almost equal effect in height reduction. The mutant allele(s) of the latter two mutants were non-allelic to sd sub(1) gene, which could be used as an alternative source of Dee Gee Woo Gen to widen the genetic diversity in semi-dwarfism [it

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

Science.gov (United States)

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.

Fusion genetic analysis of jasmonate-signalling mutants in Arabidopsis

DEFF Research Database (Denmark)

Jensen, Anders Bøgh; Raventos, D.; Mundy, John Williams

2002-01-01

as two recessive mutants, designated joe1 and 2, that overexpress the reporter. Genetic analysis indicated that reporter overexpression in the joe mutants requires COI. joe1 responded to MeJA with increased anthocyanin accumulation, while joe2 responded with decreased root growth inhibition. In addition...... activity was also induced by the protein kinase inhibitor staurosporine and antagonized by the protein phosphatase inhibitor okadaic acid. FLUC bio-imaging, RNA gel-blot analysis and progeny analyses identified three recessive mutants that underexpress the FLUC reporter, designated jue1, 2 and 3, as well...

Radiation induced mutants in cape-gooseberry (Physalis peruviana L.)

International Nuclear Information System (INIS)

Gupta, S.K.; Roy, S.K.

1986-01-01

Dry seeds of Physalis peruviana (n=24) were irradiated with different doses of gamma-rays. The M 1 plants were grown to maturity and their seeds collected and sown separately for M 2 generation. Mutants were isolated from M 2 seedlings and plants. Mutant characters obtained were virido-albino chlorophyllous, high yielding, small leaf and fruit, semi-sterile and curly leaf type etc. The high yielding and small leaf and fruit mutants bred true in M 3 and M 4 generation reproducing the characters of the M 2 generation. (author)

An inorganic carbon transport system responsible for acclimation specific to air levels of CO2 in Chlamydomonas reinhardtii.

Science.gov (United States)

Wang, Yingjun; Spalding, Martin H

2006-06-27

Many photosynthetic microorganisms acclimate to CO(2) limited environments by induction and operation of CO(2)-concentrating mechanisms (CCMs). Despite their central role in CCM function, inorganic carbon (Ci) transport systems never have been identified in eukaryotic photosynthetic organisms. In the green alga Chlamydomonas reinhardtii, a mutant, pmp1, was described in 1983 with deficiencies in Ci transport, and a Pmp1 protein-associated Ci uptake system has been proposed to be responsible for Ci uptake in low CO(2) (air level)-acclimated cells. However, even though pmp1 represents the only clear genetic link to Ci transport in microalgae and is one of only a very few mutants directly affecting the CCM itself, the identity of Pmp1 has remained unknown. Physiological analyses indicate that C. reinhardtii possesses multiple Ci transport systems responsible for acclimation to different levels of limiting CO(2) and that the Pmp1-associated transport system is required specifically for low (air level) CO(2) acclimation. In the current study, we identified and characterized a pmp1 allelic mutant, air dier 1 (ad1) that, like pmp1, cannot grow in low CO(2) (350 ppm) but can grow either in high CO(2) (5% CO(2)) or in very low CO(2) (<200 ppm). Molecular analyses revealed that the Ad1/Pmp1 protein is encoded by LciB, a gene previously identified as a CO(2)-responsive gene. LciB and three related genes in C. reinhardtii compose a unique gene family that encode four closely related, apparently soluble plastid proteins with no clearly identifiable conserved motifs.

ESKIMO1 disruption in Arabidopsis alters vascular tissue and impairs water transport.

Directory of Open Access Journals (Sweden)

Valérie Lefebvre

Full Text Available Water economy in agricultural practices is an issue that is being addressed through studies aimed at understanding both plant water-use efficiency (WUE, i.e. biomass produced per water consumed, and responses to water shortage. In the model species Arabidopsis thaliana, the ESKIMO1 (ESK1 gene has been described as involved in freezing, cold and salt tolerance as well as in water economy: esk1 mutants have very low evapo-transpiration rates and high water-use efficiency. In order to establish ESK1 function, detailed characterization of esk1 mutants has been carried out. The stress hormone ABA (abscisic acid was present at high levels in esk1 compared to wild type, nevertheless, the weak water loss of esk1 was independent of stomata closure through ABA biosynthesis, as combining mutant in this pathway with esk1 led to additive phenotypes. Measurement of root hydraulic conductivity suggests that the esk1 vegetative apparatus suffers water deficit due to a defect in water transport. ESK1 promoter-driven reporter gene expression was observed in xylem and fibers, the vascular tissue responsible for the transport of water and mineral nutrients from the soil to the shoots, via the roots. Moreover, in cross sections of hypocotyls, roots and stems, esk1 xylem vessels were collapsed. Finally, using Fourier-Transform Infrared (FTIR spectroscopy, severe chemical modifications of xylem cell wall composition were highlighted in the esk1 mutants. Taken together our findings show that ESK1 is necessary for the production of functional xylem vessels, through its implication in the laying down of secondary cell wall components.

Strain improvement in dye decolourising mutants of Mucor mucedo ...

African Journals Online (AJOL)

STORAGESEVER

2009-12-15

Dec 15, 2009 ... M. mucedo {MMM1-U.V. irradiated mutant and MMM2-EMS (ethyl methyl sulfonate) treated ... tions were induced and two positive mutants (MMM1, .... yeast biofilter for the treatment of a Nigerian fertilizer plant effluent. World J.

Molecular analysis of mutant and wild type alcohol dehydrogenase alleles from Drosophila

International Nuclear Information System (INIS)

Batzer, M.A.

1988-01-01

Wild type alcohol dehydrogenase polypeptides (ADH) from Drosophila melanogaster transformants were examined using western blots and polyclonal antiserum specific for Drosophila melanogaster ADH. Mutants induced in Drosophila spermatozoa at the alcohol dehydrogenase (Adh) locus using X-rays, 1-ethyl-1-nitrosourea (ENU) or ethyl methanesulfonate (EMS) were characterized using genetic complementation tests, western blots, Southern blots, northern blots and enzymatic amplification of the Adh locus. Genetic complementation tests showed that 22/30 X-ray-induced mutants, and 3/13 ENU and EMS induced mutants were multi-locus deficiencies. Western blot analysis of the intragenic mutations showed that 4/7 X-ray-induced mutants produced detectable polypeptides, one of which was normal in molecular weight and charge. In contrast 8/10 intragenic ENU and EMS induced mutants produced normal polypeptides. Southern blot analysis showed that 5/7 intragenic X-ray induced mutants and all 10 of the intragenic ENU and EMS induced mutants were normal with respect to the alleles they were derived from

Normal aging modulates the neurotoxicity of mutant huntingtin.

Directory of Open Access Journals (Sweden)

Elsa Diguet

Full Text Available Aging likely plays a role in neurodegenerative disorders. In Huntington's disease (HD, a disorder caused by an abnormal expansion of a polyglutamine tract in the protein huntingtin (Htt, the role of aging is unclear. For a given tract length, the probability of disease onset increases with age. There are mainly two hypotheses that could explain adult onset in HD: Either mutant Htt progressively produces cumulative defects over time or "normal" aging renders neurons more vulnerable to mutant Htt toxicity. In the present study, we directly explored whether aging affected the toxicity of mutant Htt in vivo. We studied the impact of aging on the effects produced by overexpression of an N-terminal fragment of mutant Htt, of wild-type Htt or of a beta-Galactosidase (beta-Gal reporter gene in the rat striatum. Stereotaxic injections of lentiviral vectors were performed simultaneously in young (3 week and old (15 month rats. Histological evaluation at different time points after infection demonstrated that the expression of mutant Htt led to pathological changes that were more severe in old rats, including an increase in the number of small Htt-containing aggregates in the neuropil, a greater loss of DARPP-32 immunoreactivity and striatal neurons as assessed by unbiased stereological counts.The present results support the hypothesis that "normal" aging is involved in HD pathogenesis, and suggest that age-related cellular defects might constitute potential therapeutic targets for HD.

Mutant genes in pea breeding

International Nuclear Information System (INIS)

Swiecicki, W.K.

1990-01-01

Full text: Mutations of genes Dpo (dehiscing pods) and A (anthocyanin synthesis) played a role in pea domestication. A number of other genes were important in cultivar development for 3 types of usage (dry seeds, green vegetable types, fodder), e.g. fn, fna, le, p, v, fas and af. New genes (induced and spontaneous), are important for present ideotypes and are registered by the Pisum Genetics Association (PGA). Comparison of a pea variety ideotype with the variation available in gene banks shows that breeders need 'new' features. In mutation induction experiments, genotype, mutagen and method of treatment (e.g. combined or fractionated doses) are varied for broadening the mutation spectrum and selecting more genes of agronomic value. New genes are genetically analysed. In Poland, some mutant varieties with the gene afila were registered, controlling lodging by a shorter stem and a higher number of internodes. Really non-lodging pea varieties could strongly increase seed yield. But the probability of detecting a major gene for lodging resistance is low. Therefore, mutant genes with smaller influence on plant architecture are sought, to combine their effect by crossing. Promising seem to be the genes rogue, reductus and arthritic as well as a number of mutant genes not yet genetically identified. The gene det for terminal inflorescence - similarly to Vicia faba - changes plant development. Utilisation of assimilates and ripening should be better. Improvement of harvest index should give higher seed yield. A number of genes controlling disease resistance are well known (eg. Fw, Fnw, En, mo and sbm). Important in mass screening of resistance are closely linked gene markers. Pea gene banks collect respective lines, but mutants induced in highly productive cultivars would be better. Inducing gene markers sometimes seems to be easier than transfer by crossing. Mutation induction in pea breeding is probably more important because a high number of monogenic features are

Study on homologous series of induced early mutants in Indica rice Ⅱ. the relationship between the homologous series of early mutants induced and the ecotype in Indica rice

International Nuclear Information System (INIS)

Chen Xiulan; Yang Hefeng; He Zhentian; Han Yuepeng; Liu Xueyu

2001-01-01

The induced mutation in light sensitivity of the Indica rice leads to induction of the homologous series of early mutants along with the variation of ecological character and the ecoclimate. The induction of mutants was closely related to the ecotype of Indica rice, the homologous series of early mutants in different level were derived from the different ecotype of the Indica rice, otherwise, the similar homologous series of early mutants were derived from the same ecotypic variety. The induction of the early ecotypic variety derived from the homologous series of early mutants provides the basis and possibility for accelerating the development of the new cultivars. (authors)

Leaf and canopy photosynthesis of a chlorophyll deficient soybean mutant.

Science.gov (United States)

Sakowska, Karolina; Alberti, Giorgio; Genesio, Lorenzo; Peressotti, Alessandro; Delle Vedove, Gemini; Gianelle, Damiano; Colombo, Roberto; Rodeghiero, Mirco; Panigada, Cinzia; Juszczak, Radosław; Celesti, Marco; Rossini, Micol; Haworth, Matthew; Campbell, Benjamin W; Mevy, Jean-Philippe; Vescovo, Loris; Cendrero-Mateo, M Pilar; Rascher, Uwe; Miglietta, Franco

2018-03-02

The photosynthetic, optical, and morphological characteristics of a chlorophyll-deficient (Chl-deficient) "yellow" soybean mutant (MinnGold) were examined in comparison with 2 green varieties (MN0095 and Eiko). Despite the large difference in Chl content, similar leaf photosynthesis rates were maintained in the Chl-deficient mutant by offsetting the reduced absorption of red photons by a small increase in photochemical efficiency and lower non-photochemical quenching. When grown in the field, at full canopy cover, the mutants reflected a significantly larger proportion of incoming shortwave radiation, but the total canopy light absorption was only slightly reduced, most likely due to a deeper penetration of light into the canopy space. As a consequence, canopy-scale gross primary production and ecosystem respiration were comparable between the Chl-deficient mutant and the green variety. However, total biomass production was lower in the mutant, which indicates that processes other than steady state photosynthesis caused a reduction in biomass accumulation over time. Analysis of non-photochemical quenching relaxation and gas exchange in Chl-deficient and green leaves after transitions from high to low light conditions suggested that dynamic photosynthesis might be responsible for the reduced biomass production in the Chl-deficient mutant under field conditions. © 2018 John Wiley & Sons Ltd.

High lysine and high yielding mutants in wheat (Triticum aestivum) L

International Nuclear Information System (INIS)

Mohammad, T.; Mahmood, F.; Ahmad, A.; Sattar, A.; Khan, I.

1988-01-01

The dry seeds of the variety Lu-26 were irradiated with 20 krad of gamma rays. In M 2 about 300 mutant plants were selected for short stature, rust resistance and other desirable traits. As a result of further selection, in M 6 , eight superior lines were finally identified. The agronomic characteristics of these mutants, the parent variety (Lu-26) and a standard check variety (Pak-81) are shown. The selected mutants and commercially grown cultivars (Lu-26 and Pak-81) were studied for total protein content and amino acid pattern. The mutants WM-89-1, WM-6-17 and WM-81-2 showing high yield also contained comparatively high amounts of methionine and lysine. The lysine contents were 565, 410, and 370 mg/100g in the mutants WM-89-1, WM-6-17 and WM-81-2, respectively against a range value of 210-370 mg/100g in other mutants and 250-320 in the commercial cultivars. The mutant WM-81-2 was comparable to WM-56-1-5 in lysine content. The results of these experiments show a possibility of developing varieties having high yield and high amounts of essential amino acids such as lysine and methionine

Development of compact mutants in apple and sour cherry

International Nuclear Information System (INIS)

Zagaja, S.W.; Przybyla, A.; Machnik, B.

1982-01-01

During the period 1973 - 79 studies were conducted with the aim of developing compact mutants in apple and cherry cultivars and in apple vegetative rootstocks. During the investigations the effect of the dose of gamma rays on frequency of the mutants was studied. Attempts were also made to evolve a micropropagation technique adapted to propagate P 2 and P 22 apple rootstocks, as an aid in mutation breeding. Several mutants were produced in all the material studied, but none of them have yet reached a sufficient developmental stage to enable their complete assessment. On the basis of the results obtained so far the following conclusions can be drawn: higher doses of irradiation resulted in higher frequency of mutants in most apple cultivars and apple rootstocks; in sour cherries the effect of dose depended on the cultivars. Among V 1 shoots developed from sleeping buds on irradiated scion wood, compact mutants were found; their frequency, however, was about 60% lower than among V 1 shoots developed directly from irradiated dormant buds. In apple rootstocks A 2 and M 26 several dwarfed mutants were found; some of these produced thorny plants and some had lower rooting ability; both these characteristics are inferior from the practical point of view. Multiplication and rooting media for in vitro propagation of apple rootstocks, worked out for M 26, were found unsuitable for the rootstocks P 2 and P 22; modifications made in the growth substance composition of the above media enabled satisfactory propagation to be obtained. (author)

High protein mutants of winter fodder barley induced by radiation and chemical mutagens

Energy Technology Data Exchange (ETDEWEB)

Yankulov, M.; Genchev, K.; Nikolov, Kh.

1982-01-01

Several induced mutants of winter fodder barley with higher rpotein content are described. These mutants were produced by treating seeds of cvs. Vogelsaenger Gold, Ager and 468 with gamma-rays, sodium azide and ethyl methanesulfonate (alone and in combinations) and with ethylene and formamide. The gamma-ray induced mutants of winter fodder barley have 1-4% higher protein content. The mutant line 109 has, besides high protein content (17,37%), 5.96 lysine per 100 g protein, but its endosperm is wrinkeled. Mutants produced by chemical mutagens have 6-7% higher protein content than the initial cultivars. All induced mutants have 85-95 cm high stems, i.e. they are by 10-20 cm shorter than the initial cultivars. Some of these mutants are now resistant to the diseases Helminthosporium gramineum and Ustilago nuda. The recommended mutants could be successfully used in breeding programs for producing of higher protein content and quality in winter fodder barley.

High protein mutants of winter fodder barley induced by radiation and chemical mutagens

International Nuclear Information System (INIS)

Yankulov, M.; Genchev, K.; Nikolov, Kh.

1982-01-01

Several induced mutants of winter fodder barley with higher rpotein content are described. These mutants were produced by treating seeds of cvs. Vogelsaenger Gold, Ager and 468 with gamma-rays, sodium azide and ethyl methanesulfonate (alone and in combinations) and with ethylene and formamide. The gamma-ray induced mutants of winter fodder barley have 1-4% higher protein content. The mutant line 109 has, besides high protein content (17,37%), 5.96 lysine per 100 g protein, but its endosperm is wrinkeled. Mutants produced by chemical mutagens have 6-7% higher protein content than the initial cultivars. All induced mutants have 85-95 cm high stems, i.e. they are by 10-20 cm shorter than the initial cultivars. Some of these mutants are now resistant to the diseases Helminthosporium gramineum and Ustilago nuda. The recommended mutants could be successfully used in breeding programs for producing of higher protein content and quality in winter fodder barley

The pht4;1-3 mutant line contains a loss of function allele in the Fatty Acid Desaturase 7 gene caused by a remnant inactivated selection marker-a cautionary tale.

Science.gov (United States)

Nilsson, Anders K; Andersson, Mats X

2017-01-01

A striking and unexpected biochemical phenotype was found in an insertion mutant line in the model plant Arabidopsis thaliana . One of two investigated insertion mutant lines in the gene encoding the phosphate transporter PHT4;1 demonstrated a prominent loss of trienoic fatty acids, whereas the other insertion line was indistinguishable from wild type in this aspect. We demonstrate that the loss of trienoic fatty acids was due to a remnant inactive negative selection marker gene in this particular transposon tagged line, pht4;1-3 . This constitutes a cautionary tale that warns of the importance to confirm the loss of this type of selection markers and the importance of verifying the relationship between a phenotype and genotype by more than one independent mutant line or alternatively genetic complementation.

The absence of chlorophyll b affects lateral mobility of photosynthetic complexes and lipids in grana membranes of Arabidopsis and barley chlorina mutants.

Science.gov (United States)

Tyutereva, Elena V; Evkaikina, Anastasiia I; Ivanova, Alexandra N; Voitsekhovskaja, Olga V

2017-09-01

The lateral mobility of integral components of thylakoid membranes, such as plastoquinone, xanthophylls, and pigment-protein complexes, is critical for the maintenance of efficient light harvesting, high rates of linear electron transport, and successful repair of damaged photosystem II (PSII). The packaging of the photosynthetic pigment-protein complexes in the membrane depends on their size and stereometric parameters which in turn depend on the composition of the complexes. Chlorophyll b (Chlb) is an important regulator of antenna size and composition. In this study, the lateral mobility (the mobile fraction size) of pigment-protein complexes and lipids in grana membranes was analyzed in chlorina mutants of Arabidopsis and barley lacking Chlb. In the Arabidopsis ch1-3 mutant, diffusion of membrane lipids decreased as compared to wild-type plants, but the diffusion of photosynthetic complexes was not affected. In the barley chlorina f2 3613 mutant, the diffusion of pigment-protein complexes significantly decreased, while the diffusion of lipids increased, as compared to wild-type plants. We propose that the size of the mobile fractions of pigment-protein complexes in grana membranes in vivo is higher than reported previously. The data are discussed in the context of the protein composition of antennae, characteristics of the plastoquinone pool, and production of reactive oxygen species in leaves of chlorina mutants.

Plasma membrane lipid–protein interactions affect signaling processes in sterol-biosynthesis mutants in Arabidopsis thaliana

Science.gov (United States)

Zauber, Henrik; Burgos, Asdrubal; Garapati, Prashanth; Schulze, Waltraud X.

2014-01-01

The plasma membrane is an important organelle providing structure, signaling and transport as major biological functions. Being composed of lipids and proteins with different physicochemical properties, the biological functions of membranes depend on specific protein–protein and protein–lipid interactions. Interactions of proteins with their specific sterol and lipid environment were shown to be important factors for protein recruitment into sub-compartmental structures of the plasma membrane. System-wide implications of altered endogenous sterol levels for membrane functions in living cells were not studied in higher plant cells. In particular, little is known how alterations in membrane sterol composition affect protein and lipid organization and interaction within membranes. Here, we conducted a comparative analysis of the plasma membrane protein and lipid composition in Arabidopsis sterol-biosynthesis mutants smt1 and ugt80A2;B1. smt1 shows general alterations in sterol composition while ugt80A2;B1 is significantly impaired in sterol glycosylation. By systematically analyzing different cellular fractions and combining proteomic with lipidomic data we were able to reveal contrasting alterations in lipid–protein interactions in both mutants, with resulting differential changes in plasma membrane signaling status. PMID:24672530

Gamma rays induced bold seeded high yielding mutant in chickpea

International Nuclear Information System (INIS)

Wani, A.A.; Anis, M.

2001-01-01

In pulses especially in chickpea (Cicer arietinum L.), genetic variability has been exhausted due to natural selection and hence conventional breeding methods are not very fruitful. Mutation techniques are the best methods to enlarge the genetically conditioned variability of a species within a short time and have played a significant role in the development of many crop varieties. Investigations on the effects of ionizing radiations and chemical mutagens in induction of macro-mutations have received much attention owing to their utmost importance in plant breeding. The present study reports a bold seeded mutant in chickpea, the most dominating pulse crop on the Indian subcontinent. Fresh seeds of chickpea variety 'Pusa-212' were procured from IARI, New Delhi and treated with different doses/concentrations of gamma rays ( 60 Co source at NBRI, Lucknow) and ethyl methanesulphonate (EMS), individually as well as in combination, to raise the M1 generation. Seeds of M 1 plants were sown to raise M2 plant progenies. A bold seeded mutant was isolated from 400 Gy gamma ray treatments. The mutant was confirmed as true bred, all the mutant seeds gave rise to morphologically similar plants in M 3 , which were quite distinct from the control. The bold seeded mutant showed 'gigas' characteristics and vigorous growth. The plant remained initially straight but later on attained a trailing habit due to heavy secondary branching. The leaves, petioles, flowers, pods and seeds were almost double that of the parent variety, in size. The flowering occurred 10 days later than the parent and maturity was also delayed accordingly. Observations were recorded on various quantitative traits. Plant height and number of primary branches showed a significant improvement over the parent. It is interesting to note that the number of pods and number of seeds per pod significantly decreased. However, the hundred seed weight (31.73±0.59g) in the mutant plants was more than double in the parent

Radiation-induced mutagenicity in repair deficient Chinese hamster ovary (CHO) mutants

International Nuclear Information System (INIS)

Tesmer, J.G.; Saunders, E.H.; Chen, D.J.

1987-01-01

To determine if there is a relationship between DNA double-strand break repair and mutagenicity the authors utilized two x-ray sensitive mutants of Chinese hamster ovary cells along with the parental line K1. The two mutant lines xrs-5 and xrs-6, which have different DSB repair capabilities, were used to determine cell killing and 6-thioguanine resistance (6TG/sup r/) mutation frequencies induced by either x-rays of α-particles, x-ray survival data indicated the two mutant lines have similar sensitivity and are 5-7 fold more sensitive than the parental line K1. The mutant lines are also sensitive to α-particles but to a lesser extent. The authors' 6TG mutation data indicated that the two mutant lines are hypermutable. When mutation frequencies were plotted against the log of survival, mutation frequency at a given survival level was greater in mutant cell population than in parental K1 cells. Their results support the notion that repair of DSB play an important role in the expression of radiation-induced cell killing and mutagenicity

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

Science.gov (United States)

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.

Grain product of 34 soya mutant lines

International Nuclear Information System (INIS)

Salmeron E, J.; Mastache L, A. A.; Valencia E, F.; Diaz V, G. E.; Cervantes S, T.; De la Cruz T, E.; Garcia A, J. M.; Falcon B, T.; Gatica T, M. A.

2009-01-01

This work was development with the objective of obtaining information of the agronomic behavior of 34 soya mutant lines (R 4 M 18 ) for human consumption and this way to select the 2 better lines. The genetic materials were obtained starting from the variety ISAAEG-B M2 by means of the application of recurrent radiation with Co 60 gammas, to a dose of 350 Gray for the first two generations and both later to 200 Gray and selection during 17 cycles, being obtained the 34 better lines mutants with agronomic characteristic wanted and good flavor. The obtained results were that the mutant lines L 25 and L 32 produced the major quantity in branches/plant number with 7.5 and 7.25, pods/plant number with 171.25 and 167, grains/plant number with 350.89 and 333.07 and grain product (ton/ha) to 15% of humidity 5.15 and 4.68 ton/ha, respectively. (Author)

Officially released mutant varieties - the FAO/IAEA Database

International Nuclear Information System (INIS)

Maluszynski, M.; Nichterlein, K.; Zanten, L. van; Ahloowalia, B.S.

2000-01-01

In the approximately 70 year-old history of induced mutations, there are many examples on the development of new and valuable alteration in plant characters significantly contributing to increased yield potential of specific crops. However, knowledge on the success of induced mutations in crop improvement among geneticists and breeders is usually limited to species of their interest. The present paper contains a comprehensive list of officially released mutant varieties, based on information from plant breeders. The number of mutant varieties officially released and recorded in the FAO/IAEA Mutant Varieties Database before the end of 2000 is 2,252. Almost half of these varieties have been released during the last 15 years. Considering a significant delay in the dissemination of information on newly released varieties and difficulties in the collection of such data, there has been a renaissance in the use of mutation techniques in crop improvement. At the demand of geneticists, plant breeders, and more recently molecular geneticists, for information on released mutant varieties of specific crops, the MVD was transferred to the web site of the FAO/IAEA Joint Division. The MVD will be available on our web pages early in 2001. (author)

Selection of mutants of capsicum annuum induced by gamma ray

Energy Technology Data Exchange (ETDEWEB)

Lee, Y. I.; Lee, Y. B. [Korea Atomic Energy Research Institute, Taejeon (Korea, Republic of); Lee, E. K. [Chungnam National Univ., Taejeon (Korea, Republic of)

1998-06-01

For induction and selection of mutations of Capsicum annuum L., dry seeds of pure lines No.1 and No.2 were irradiated with gamma ray of 150Gy, 200Gy and 250Gy. Various mutants were selected such as showing early maturity, short plant height, long fruit and chlorophyll mutations. Mutation frequency of No.1 line was 3.4% in the dose of 150Gy, while the frequency of No.2 line was 2.7% in the dose of 250Gy. For selection of resistant mutant to amino acid analog, the optimum concentration of 5-methyltryptophan (5-MT) and S-(2-aminoethyl)-L-cysteine were 25 ppm and 30 ppm, respectively. Four resistant mutant lines to 5-MT were selected among 400 mutant lines.

Andrographolide induces degradation of mutant p53 via activation of Hsp70.

Science.gov (United States)

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.

Isolation of glutathione-deficient mutants of the yeast Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Kistler, M.; Eckardt, F.; Summer, K.-H.

1986-01-01

Glutathione-deficient (gsh - ) mutants of the yeast Saccharomyces cerevisiae were isolated after UV treatment using MNNG as selective agent. For genetic and biochemical characterization 5 mutant strains were chosen which exhibited considerably decreased residual GSH contents varying from 2 to 6% of the wild-type levels. All 5 isolates showed a 2:2 segregation of the gsh - :GSH + phenotypes alluding to a monogenic recessive mutation. Complementation analysis indicates that all gsh - mutants belong to one complementation group. (Auth.)

Is auxin involved in the induction of genetic instability in barley homeotic double mutants?

Science.gov (United States)

Šiukšta, Raimondas; Vaitkūnienė, Virginija; Rančelis, Vytautas

2018-02-01

The triggers of genetic instability in barley homeotic double mutants are tweaky spike -type mutations associated with an auxin imbalance in separate spike phytomeres. Barley homeotic tweaky spike;Hooded (tw;Hd) double mutants are characterized by an inherited instability of spike and flower development, which is absent in the single parental constituents. The aim of the present study was to show that the trigger of genetic instability in the double mutants is the tw mutations, which are associated with an auxin imbalance in the developing spikes. Their pleiotropic effects on genes related to spike/flower development may cause the genetic instability of double mutants. The study of four double-mutant groups composed of different mutant alleles showed that the instability arose only if the mutant allele tw was a constituent of the double mutants. Application of auxin inhibitors and 2,4-dichlorophenoxyacetic acid (2,4-D) demonstrated the relationship of the instability of the double mutants and the phenotype of the tw mutants to auxin imbalance. 2,4-D induced phenocopies of the tw mutation in wild-type plants and rescued the phenotypes of three allelic tw mutants. The differential display (dd-PCR) method allowed the identification of several putative candidate genes in tw that may be responsible for the initiation of instability in the double mutants by pleiotropic variations of their expression in the tw mutant associated with auxin imbalance in the developing spikes. The results of the present study linked the genetic instability of homeotic double mutants with an auxin imbalance caused by one of the constituents (tw). The genetic instability of the double mutants in relation to auxin imbalance was studied for the first time. A matrocliny on instability expression was also observed.

Sequential super-stereotypy of an instinctive fixed action pattern in hyper-dopaminergic mutant mice: a model of obsessive compulsive disorder and Tourette's

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Houchard Kimberly R

2005-02-01

Full Text Available Abstract Background Excessive sequential stereotypy of behavioral patterns (sequential super-stereotypy in Tourette's syndrome and obsessive compulsive disorder (OCD is thought to involve dysfunction in nigrostriatal dopamine systems. In sequential super-stereotypy, patients become trapped in overly rigid sequential patterns of action, language, or thought. Some instinctive behavioral patterns of animals, such as the syntactic grooming chain pattern of rodents, have sufficiently complex and stereotyped serial structure to detect potential production of overly-rigid sequential patterns. A syntactic grooming chain is a fixed action pattern that serially links up to 25 grooming movements into 4 predictable phases that follow 1 syntactic rule. New mutant mouse models allow gene-based manipulation of brain function relevant to sequential patterns, but no current animal model of spontaneous OCD-like behaviors has so far been reported to exhibit sequential super-stereotypy in the sense of a whole complex serial pattern that becomes stronger and excessively rigid. Here we used a hyper-dopaminergic mutant mouse to examine whether an OCD-like behavioral sequence in animals shows sequential super-stereotypy. Knockdown mutation of the dopamine transporter gene (DAT causes extracellular dopamine levels in the neostriatum of these adult mutant mice to rise to 170% of wild-type control levels. Results We found that the serial pattern of this instinctive behavioral sequence becomes strengthened as an entire entity in hyper-dopaminergic mutants, and more resistant to interruption. Hyper-dopaminergic mutant mice have stronger and more rigid syntactic grooming chain patterns than wild-type control mice. Mutants showed sequential super-stereotypy in the sense of having more stereotyped and predictable syntactic grooming sequences, and were also more likely to resist disruption of the pattern en route, by returning after a disruption to complete the pattern from the

Isolation and functional analysis of Thmfs1, the first major facilitator superfamily transporter from the biocontrol fungus Trichoderma harzianum.

Science.gov (United States)

Liu, Mu; Liu, Jun; Wang, Wei Min

2012-10-01

A novel major facilitator superfamily (MFS) transporter gene, Thmfs1, was isolated from Trichoderma harzianum (T. harzianum). A Thmfs1 over-expressing mutant displayed enhanced antifungal activity and fungicide tolerance, while the Thmfs1 disruption mutant showed the opposite trend. Trichodermin production in Thmfs1 disruption group (185 mg l(-1)) was decreased by less than 17 % compared to the parental strain, suggesting that Thmfs1 is not mainly responsible for trichodermin secretion. Real-time PCR showed that Thmfs1 transcript level could be induced by a certain range of trichodermin concentrations, while expression of Tri5, encoding a trichodiene synthase, was strongly inhibited under these conditions. To our knowledge, Thmfs1 is the first MFS transporter gene identified in T. harzianum.

Proteomic analysis of the flooding tolerance mechanism in mutant soybean.

Science.gov (United States)

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.

The AbcA Transporter of Staphylococcus aureus Affects Cell Autolysis

Science.gov (United States)

Schrader-Fischer, Gesine; Berger-Bächi, Brigitte

2001-01-01

Increased production of penicillin-binding protein PBP 4 is known to increase peptidoglycan cross-linking and contributes to methicillin resistance in Staphylococcus aureus. The pbp4 gene shares a 400-nucleotide intercistronic region with the divergently transcribed abcA gene, encoding an ATP-binding cassette transporter of unknown function. Our study revealed that methicillin stimulated abcA transcription but had no effects on pbp4 transcription. Analysis of abcA expression in mutants defective for global regulators showed that abcA is under the control of agr. Insertional inactivation of abcA by an erythromycin resistance determinant did not influence pbp4 transcription, nor did it alter resistance to methicillin and other cell wall-directed antibiotics. However, abcA mutants showed spontaneous partial lysis on plates containing subinhibitory concentrations of methicillin due to increased spontaneous autolysis. Since the autolytic zymograms of cell extracts were identical in mutants and parental strains, we postulate an indirect role of AbcA in control of autolytic activities and in protection of the cells against methicillin. PMID:11158733

Genetic and agronomic evaluation of induced semi-dwarf mutants of rice

International Nuclear Information System (INIS)

Rutger, J.N.

1984-01-01

Induced semi-dwarf mutants have played an important role in California's rapid shift from nearly all tall rice varieties in 1978 to nearly all semi-dwarf varieties at present. In 1981 over half of the California rice area was planted with semi-dwarf varieties carrying the induced mutant semi-dwarfing gene sd 1 , while much of the other half was planted to a variety deriving its semi-dwarfism from IR8. The sd 1 mutant is allelic to the major semi-dwarfing gene in DGWG and IR8. Current objectives are to determine the inheritance of new semi-dwarf mutants, including allelism tests with sd 1 , and to evaluate the agronomic potential of nonallelic sources and of double-dwarfs. To date semi-dwarf mutants from 10 varieties have been partially or completely evaluated. At least three nonallelic semi-dwarfing genes, sd 1 , sd 2 , and sd 4 , have been described. Rather than attempt to determine all possible allelic relationships of new mutants, crosses are being made only to the reference sd 1 source, since sd 1 , still seems to be the most productive semi-dwarfing gene source. However, nonallelic semi-dwarf mutants in the varieties M5 and Labelle may be useful if genetic vulnerability from widespread usage of the sd 1 source becomes a problem. (author)

Induction of mutants in Durum Wheat by hybridization and irradiation techniques

International Nuclear Information System (INIS)

Al Ubaidi, M.O.; Ibrahim, I.F.

2001-01-01

This investigation presents a breeding program for induction and development a new genotypes of durum wheat, resistant to lodging with high yield, by irradiated seeds (F2) of durum wheat hybrid's (Sin Al-jemal X Izraa, Sin Al-Jemal X Cocorat and Izraa X Cocorat) with gamma rays 100 Gy dose. This program involves: Induction of variability, selection, evaluation of the best mutants at three different locations, Twaitha(Baghdad), Latifya (Babylon) and Swari (Kutt), for the period 1990-1999. Results revealed that the mutants ( Si X Iz-7, Si X Iz-22, Si X Co-43, Si X Co-48, Si X Co-50, Si X Co- 87, Iz X Co-95 and Iz X Co-105) showed resistance to lodging with a significant reduction in plant heigth, but mutant Si X Iz-22 surpassed the other mutants and it is origin in lodging resistance and reduction in plant heigth (84.8, 81.9 and 86.3 cm) at Twaitha, Latifya and Babylon respectively in M7 and M8 generations. Also there were a significant differences between the mutants and their origin in yield and yield components during the two successive generations, on the other hand mutant Iz X Co-105 surpassed the other mutants in spikes/m2 ( 278.8, 263.3 and 289) and grain yield (4950, 4820 and 5320 kg/ha) in the testing locations respectively

Structure and expression of cytochrome f in an Oenothera plastome mutant.

Science.gov (United States)

Johnson, E M; Sears, B B

1990-06-01

The chloroplast mutant pm7 is one of a number of mutants derived from the plastome mutator (pm) line of Oenothera hookeri, strain Johansen. Immunoblotting showed that this mutant accumulates a protein that is cross-antigenic with cytochrome f, but five kilodaltons larger than the mature wild-type protein. Since cytochrome f is known to be translated on plastid ribosomes as a precursor with an amino-terminal extension, it is proposed that the unprocessed cytochrome f precursor accumulates in pm7. In addition to this precursor-sized cytochrome f protein, some mature-sized cytochrome f was also found in the mutant plastids. The pm7 mutation is inherited in a non-Mendelian fashion; but no alterations in chloroplast DNA restriction patterns, or differences in DNA sequence in the region encoding cytochrome f, were found in a comparison of the wild-type and pm7 chloroplast DNAs. Although the mutant was capable of synthesizing heme, no covalently-bound heme, normally found associated with mature, functional, cytochrome f was detected in the mutant at sizes expected for the presumed precursor, or for mature cytochrome f. These results indicate that the aberrant accumulation of a precursor-sized cytochrome f in pm7 is not due to a lesion directly in the plastid gene encoding cytochrome f, petA, or to a deficiency in the ability of the mutant plastids to synthesize or accumulate heme.

Splicing factor SR34b mutation reduces cadmium tolerance in Arabidopsis by regulating iron-regulated transporter 1 gene

International Nuclear Information System (INIS)

Zhang, Wentao; Du, Bojing; Liu, Di; Qi, Xiaoting

2014-01-01

Highlights: • Arabidopsis splicing factor SR34b gene is cadmium-inducible. • SR34b T-DNA insertion mutant is sensitive to cadmium due to high cadmium uptake. • SR34b is a regulator of cadmium transporter IRT1 at the posttranscription level. • These results highlight the roles of splicing factors in cadmium tolerance of plant. - Abstract: Serine/arginine-rich (SR) proteins are important splicing factors. However, the biological functions of plant SR proteins remain unclear especially in abiotic stresses. Cadmium (Cd) is a non-essential element that negatively affects plant growth and development. In this study, we provided clear evidence for SR gene involved in Cd tolerance in planta. Systemic expression analysis of 17 Arabidopsis SR genes revealed that SR34b is the only SR gene upregulated by Cd, suggesting its potential roles in Arabidopsis Cd tolerance. Consistent with this, a SR34b T-DNA insertion mutant (sr34b) was moderately sensitive to Cd, which had higher Cd 2+ uptake rate and accumulated Cd in greater amounts than wild-type. This was due to the altered expression of iron-regulated transporter 1 (IRT1) gene in sr34b mutant. Under normal growth conditions, IRT1 mRNAs highly accumulated in sr34b mutant, which was a result of increased stability of IRT1 mRNA. Under Cd stress, however, sr34b mutant plants had a splicing defect in IRT1 gene, thus reducing the IRT1 mRNA accumulation. Despite of this, sr34b mutant plants still constitutively expressed IRT1 proteins under Cd stress, thereby resulting in Cd stress-sensitive phenotype. We therefore propose the essential roles of SR34b in posttranscriptional regulation of IRT1 expression and identify it as a regulator of Arabidopsis Cd tolerance

Splicing factor SR34b mutation reduces cadmium tolerance in Arabidopsis by regulating iron-regulated transporter 1 gene

Energy Technology Data Exchange (ETDEWEB)

Zhang, Wentao; Du, Bojing; Liu, Di; Qi, Xiaoting, E-mail: qixiaoting@cnu.edu.cn

2014-12-12

Highlights: • Arabidopsis splicing factor SR34b gene is cadmium-inducible. • SR34b T-DNA insertion mutant is sensitive to cadmium due to high cadmium uptake. • SR34b is a regulator of cadmium transporter IRT1 at the posttranscription level. • These results highlight the roles of splicing factors in cadmium tolerance of plant. - Abstract: Serine/arginine-rich (SR) proteins are important splicing factors. However, the biological functions of plant SR proteins remain unclear especially in abiotic stresses. Cadmium (Cd) is a non-essential element that negatively affects plant growth and development. In this study, we provided clear evidence for SR gene involved in Cd tolerance in planta. Systemic expression analysis of 17 Arabidopsis SR genes revealed that SR34b is the only SR gene upregulated by Cd, suggesting its potential roles in Arabidopsis Cd tolerance. Consistent with this, a SR34b T-DNA insertion mutant (sr34b) was moderately sensitive to Cd, which had higher Cd{sup 2+} uptake rate and accumulated Cd in greater amounts than wild-type. This was due to the altered expression of iron-regulated transporter 1 (IRT1) gene in sr34b mutant. Under normal growth conditions, IRT1 mRNAs highly accumulated in sr34b mutant, which was a result of increased stability of IRT1 mRNA. Under Cd stress, however, sr34b mutant plants had a splicing defect in IRT1 gene, thus reducing the IRT1 mRNA accumulation. Despite of this, sr34b mutant plants still constitutively expressed IRT1 proteins under Cd stress, thereby resulting in Cd stress-sensitive phenotype. We therefore propose the essential roles of SR34b in posttranscriptional regulation of IRT1 expression and identify it as a regulator of Arabidopsis Cd tolerance.

Chemical Genomic Screening of a Saccharomyces cerevisiae Genomewide Mutant Collection Reveals Genes Required for Defense against Four Antimicrobial Peptides Derived from Proteins Found in Human Saliva

Science.gov (United States)

Bhatt, Sanjay; Schoenly, Nathan E.; Lee, Anna Y.; Nislow, Corey; Bobek, Libuse A.

2013-01-01

To compare the effects of four antimicrobial peptides (MUC7 12-mer, histatin 12-mer, cathelicidin KR20, and a peptide containing lactoferricin amino acids 1 to 11) on the yeast Saccharomyces cerevisiae, we employed a genomewide fitness screen of combined collections of mutants with homozygous deletions of nonessential genes and heterozygous deletions of essential genes. When an arbitrary fitness score cutoffs of 1 (indicating a fitness defect, or hypersensitivity) and −1 (indicating a fitness gain, or resistance) was used, 425 of the 5,902 mutants tested exhibited altered fitness when treated with at least one peptide. Functional analysis of the 425 strains revealed enrichment among the identified deletions in gene groups associated with the Gene Ontology (GO) terms “ribosomal subunit,” “ribosome biogenesis,” “protein glycosylation,” “vacuolar transport,” “Golgi vesicle transport,” “negative regulation of transcription,” and others. Fitness profiles of all four tested peptides were highly similar, particularly among mutant strains exhibiting the greatest fitness defects. The latter group included deletions in several genes involved in induction of the RIM101 signaling pathway, including several components of the ESCRT sorting machinery. The RIM101 signaling regulates response of yeasts to alkaline and neutral pH and high salts, and our data indicate that this pathway also plays a prominent role in regulating protective measures against all four tested peptides. In summary, the results of the chemical genomic screens of S. cerevisiae mutant collection suggest that the four antimicrobial peptides, despite their differences in structure and physical properties, share many interactions with S. cerevisiae cells and consequently a high degree of similarity between their modes of action. PMID:23208710

EMS mutant spectra generated by multi-parameter flow cytometry

Energy Technology Data Exchange (ETDEWEB)

Keysar, Stephen B. [Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO (United States); Fox, Michael H., E-mail: michael.fox@colostate.edu [Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO (United States); Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO (United States)

2009-12-01

The CHO A{sub L} cell line contains a single copy of human chromosome 11 that encodes several cell surface proteins including glycosyl phosphatidylinositol (GPI) linked CD59 and CD90, as well as CD98, CD44 and CD151 which are not GPI-linked. The flow cytometry mutation assay (FCMA) measures mutations of the CD59 gene by the absence of fluorescence when stained with antibodies against the CD59 cell surface protein. We have measured simultaneous mutations in CD59, CD44, CD90, CD98 and CD151 to generate a mutant spectrum for ionizing radiation. After treatment with ethyl methanesulfonate (EMS) many cells have an intermediate level of CD59 staining. Single cells were sorted from CD59{sup -} regions with varying levels of fluorescence and the resulting clonal populations had a stable phenotype for CD59 expression. Mutant spectra were generated by flow cytometry using the isolated clones and nearly all clones were mutated in CD59 only. Interestingly, about 60% of the CD59 negative clones were actually GPI mutants determined by staining with the GPI specific fluorescently labeled bacterial toxin aerolysin (FLAER). The GPI negative cells are most likely caused by mutations in the X-linked pigA gene important in GPI biosynthesis. Small mutations of pigA and CD59 were expected for the alkylating agent EMS and the resulting spectra are significantly different than the large deletions found when analyzing radiation mutants. After analyzing the CD59{sup -} clonal populations we have adjusted the FCMA mutant regions from 1% to 10% of the mean of the CD59 positive peak to include the majority of CD59 mutants.

Nanoformulated cell-penetrating survivin mutant and its dual actions

Directory of Open Access Journals (Sweden)

Sriramoju B

2014-07-01

Full Text Available Bhasker Sriramoju, Rupinder K Kanwar, Jagat R Kanwar Nanomedicine Laboratory of Immunology and Molecular Biomedical Research (NLIMBR, School of Medicine, Faculty of Health, Deakin University, Geelong, Australia Abstract: In this study, we investigated the differential actions of a dominant-negative survivin mutant (SurR9-C84A against cancerous SK-N-SH neuroblastoma cell lines and differentiated SK-N-SH neurons. In both the cases, the mutant protein displayed dual actions, where its effects were cytotoxic toward cancerous cells and proliferative toward the differentiated neurons. This can be explained by the fact that tumorous (undifferentiated SK-N-SH cells have a high endogenous survivin pool and upon treatment with mutant SuR9-C84A causes forceful survivin expression. These events significantly lowered the microtubule dynamics and stability, eventually leading to apoptosis. In the case of differentiated SK-N-SH neurons that express negligible levels of wild-type survivin, the mutant indistinguishably behaved in a wild-type fashion. It also favored cell-cycle progression, forming the chromosome-passenger complex, and stabilized the microtubule-organizing center. Therefore, mutant SurR9-C84A represents a novel therapeutic with its dual actions (cytotoxic toward tumor cells and protective and proliferative toward neuronal cells, and hence finds potential applications against a variety of neurological disorders. In this study, we also developed a novel poly(lactic-co-glycolic acid nanoparticulate formulation to surmount the hurdles associated with the delivery of SurR9-C84A, thus enhancing its effective therapeutic outcome. Keywords: survivin mutant, neurological disorders, protein therapeutics, inhibitor of apoptosis protein family, poly(lactic-co-glycolic acid

Some mutants in maize obtained by irradiation with thermal neutrons

International Nuclear Information System (INIS)

Diaconu, P.

1993-01-01

Irradiation was carried out at the Bucharest Institute of Atomic Physics and the National Laboratory Brookhaven, USA. A description is given of 22 genic mutants affecting leaf color, plant size, and branching capacity. Characteristics related to pollen fertility and the vegetative period were affected in all the mutants. Improvement of pollen fertility was attempted over four generations without success. The maize mutants obtained by irradiation may be considered as being without practical significance. (author). 7 figs., 1 tab. 11 ref

Heterozygous inactivation of tsc2 enhances tumorigenesis in p53 mutant zebrafish

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Seok-Hyung Kim

2013-07-01

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.

Variation of several agronomic and biochemical traits in γ-ray induced mutant soybeans

International Nuclear Information System (INIS)

Shim, Kyo Moon; Kim, Sun Hyung; Kim, Nam Soo; Son, Hi Sup; Rhee, Hae Ik

1996-01-01

Two soybean cultivars(Paldalkong and Bangsakong) were irradiated with gamma-ray to reduce seed size, which is an important trait for soybean sprout and the derived mutant soybeans were analyzed in several agronomic and biochemical traits. There were high levels of variations in quantitative traits among the mutants. Several mutant lines showed higher yield and smaller seed than their parents. Qualitative traits such as seed coat color or pubescent color were also changed in a few lines. Biochemical variations were also observed among the mutants. In seed storage protein analysis, many mutant lines showed reduced intensities in β-subunits in 7S globulin than their parents and an additional band in the acidic subunit at 31KDa. Two mutant lines derived from Paldalkong showed an additional band and a shifted band of protease inhibitor by electrophoretic analysis. Variations in isozymes and RAPD were also observed among the mutants. Six isozymes(Adh, Est, Gdh, Idh, Mdh and Pgm) among eleven isozymes showed some variations and six out of ten primers showed polymorphic amplified DNA fragments among the mutants. (author)

Behavioral and other phenotypes in a cytoplasmic Dynein light intermediate chain 1 mutant mouse.

Science.gov (United States)

Banks, Gareth T; Haas, Matilda A; Line, Samantha; Shepherd, Hazel L; Alqatari, Mona; Stewart, Sammy; Rishal, Ida; Philpott, Amelia; Kalmar, Bernadett; Kuta, Anna; Groves, Michael; Parkinson, Nicholas; Acevedo-Arozena, Abraham; Brandner, Sebastian; Bannerman, David; Greensmith, Linda; Hafezparast, Majid; Koltzenburg, Martin; Deacon, Robert; Fainzilber, Mike; Fisher, Elizabeth M C

2011-04-06

The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system.

Application Of Database Program in selecting Sorghum (Sorghum bicolor L) Mutant Lines

International Nuclear Information System (INIS)

H, Soeranto

2000-01-01

Computer database software namely MSTAT and paradox have been exercised in the field of mutation breeding especially in the process of selecting plant mutant lines of sorghum. In MSTAT, selecting mutant lines can be done by activating the SELECTION function and then followed by entering mathematical formulas for the selection criterion. Another alternative is by defining the desired selection intensity to the analysis results of subprogram SORT. Including the selected plant mutant lines in BRSERIES program, it will make their progenies be easier to be traced in subsequent generations. In paradox, an application program for selecting mutant lines can be made by combining facilities of Table, form and report. Selecting mutant lines with defined selection criterion can easily be done through filtering data. As a relation database, paradox ensures that the application program for selecting mutant lines and progeny trachings, can be made easier, efficient and interactive

A Simple and Rapid Gene Disruption Strategy in Mycobacterium abscessus: On the Design and Application of Glycopeptidolipid Mutants.

Science.gov (United States)

Viljoen, Albertus; Gutiérrez, Ana Victoria; Dupont, Christian; Ghigo, Eric; Kremer, Laurent

2018-01-01

Little is known about the disease-causing genetic determinants that are used by Mycobacterium abscessus , increasingly acknowledged as an important emerging pathogen, notably in cystic fibrosis. The presence or absence of surface exposed glycopeptidolipids (GPL) conditions the smooth (S) or rough (R) M. abscessus subsp. abscessus ( M. abscessus ) variants, respectively, which are characterized by distinct infective programs. However, only a handful of successful gene knock-out and conditional mutants have been reported in M. abscessus , testifying that genetic manipulation of this mycobacterium is difficult. To facilitate gene disruption and generation of conditional mutants in M. abscessus , we have designed a one-step single cross-over system that allows the rapid and simple generation of such mutants. Cloning of as small as 300 bp of the target gene allows for efficient homologous recombination to occur without additional exogenous recombination-promoting factors. The presence of tdTomato on the plasmids allows easily sifting out the large background of mutants spontaneously resistant to antibiotics. Using this strategy in the S genetic background and the target gene mmpL4a , necessary for GPL synthesis and transport, nearly 100% of red fluorescent clones exhibited a rough morphotype and lost GPL on the surface, suggesting that most red fluorescent colonies obtained after transformation incorporated the plasmid through homologous recombination into the chromosome. This system was further exploited to generate another strain with reduced GPL levels to explore how the presence of these cell wall-associated glycolipids influences M. abscessus hydrophobicity as well as virulence in the zebrafish model of infection. This mutant exhibited a more pronounced killing phenotype in zebrafish embryos compared to its S progenitor and this effect correlated with the production of abscesses in the central nervous system. Overall, these results suggest that the near

Characterization of mutants of yeast sensitive to x rays

International Nuclear Information System (INIS)

Strike, T.L.

1978-01-01

This study deals with the characterization of mutants at the rad50 to rad57 loci selected on the basis of their sensitivity to x rays. They were also examined for sensitivity to uv and mms and for characteristics of mutation induction, heteroallelic reversion (gene conversion), liquid holding recovery from x rays, and sporulation. All the mutants were slightly to moderately sensitive to uv though they did not show the extreme sensitivity of the rad1 to rad22 mutations, and all demonstrated cross sensitivity to both x rays and MMS. If a mutant was very sensitive to x-rays, it was usually very sensitive to MMS also

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

Science.gov (United States)

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.

Clear Plaque Mutants of Lactococcal Phage TP901-1

DEFF Research Database (Denmark)

Kot, Witold; Kilstrup, Mogens; Vogensen, Finn K.

2016-01-01

We report a method for obtaining turbid plaques of the lactococcal bacteriophage TP901-1 and its derivative TP901-BC1034. We have further used the method to isolate clear plaque mutants of this phage. Analysis of 8 such mutants that were unable to lysogenize the host included whole genome...

myo-Inositol-1-phosphate synthase is required for polar auxin transport and organ development

KAUST Repository

Chen, Hao

2010-06-01

myo-Inositol-1-phosphate synthase is a conserved enzyme that catalyzes the first committed and rate-limiting step in inositol biosynthesis. Despite its wide occurrence in all eukaryotes, the role of myo-inositol-1-phosphate synthase and de novo inositol biosynthesis in cell signaling and organism development has been unclear. In this study, we isolated loss-of-function mutants in the Arabidopsis MIPS1 gene from different ecotypes. It was found that all mips1 mutants are defective in embryogenesis, cotyledon venation patterning, root growth, and root cap development. The mutant roots are also agravitropic and have reduced basipetal auxin transport. mips1 mutants have significantly reduced levels of major phosphatidylinositols and exhibit much slower rates of endocytosis. Treatment with brefeldin A induces slower PIN2 protein aggregation in mips1, indicating altered PIN2 trafficking. Our results demonstrate that MIPS1 is critical for maintaining phosphatidylinositol levels and affects pattern formation in plants likely through regulation of auxin distribution. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Chlorophyll mutants in Phaseolus vulgaris (L.) Savi

International Nuclear Information System (INIS)

Svetleva, D.; Petkova, S.

1991-01-01

Three-year investigations were conducted on chlorophyll mutants of three type: viridissima, claroviridis, flavoviridis, viridocostata and xanthomarginata produced post gamma irradiation ( 60 Co, 8 krad, 280 rad/min). Cell division rate in spectrum and in quantity of induced aberrations was found to have no significant differences with the control. Chlorophyll mutations compared to the control are less developed and their productive characters are less manifested. Cell division rate and the quantity of induced aberrations have no relation to the elements of productivity in the mutants investigated. 3 tabs., 12 refs

Characterization of mitomycin-C-sensitive mouse lymphoma L5178Y cell mutants

International Nuclear Information System (INIS)

Inaba, Hiroko; Shiomi, Naoko; Shiomi, Tadahiro; Sato, Koki; Yoshida, Michihiro.

1985-01-01

Twenty-six mutants showing high sensitivity to mytomicin-C (MMC) were isolated from mouse lymphoma L5178Y cells by a replica-plating technique. Twenty-five of the mutants were 5 - 10 times more sensitive to MMC than were parental cells, and showed normal sensitivity to U.V. light and x-rays. From a complementation analysis, 5 mutants (MC s ) isolated from independently mutagenized cell populations were classified into two groups. These mutants possessed recessive character for MMC-sensitivity and there were at least two genes involved in the MMC-sensitivity. As for DNA-damaging factors, such as photoadducts of 8-methoxypsoralen (8-MOP) and 3-carbethoxysoralen (3-CPs), MC s mutants showed higher sensitivity to photoadducts of 8-MOP than to (3-CPs). MC s mutants were also highly sensitive to a DNA cross-linking agent, cisplatin. Characterization of the sensitivity of mouse MC s mutants was analogous to that of Fanconi's anemia (FA)-derived cells. Low concentrations (10 ng/ml) of MMC induced chromosome aberration in a high incidence in mouse MC s cells, as well as in FA cells. The frequency of MMC-induced chromosome aberrations was normal in hybrid cells between normal human diploid somatic cells and mouse mutants and between FA cells and mouse wild cells, and hereditary deficiency became normal by hybrization. (Namekawa, K.)

Agronomic performance of rape seed (brassica napus L.) mutant lines under drought conditions

International Nuclear Information System (INIS)

Shah, S.A.; Ali, I.; Shah, S.J.A.; Rehman, K.; Rashid, A.

1995-01-01

Oil seed forms of Brassica napus are not well adapted to drought and the warner environments of Pakistan. Induced mutations were, therefore, utilized for improving drought tolerance efficiency of two napus cultivars. Induction of genetic variability, selection of desirable mutants and stabilization of mutants in acceptable agronomic background were carried out during 1988-1991. Fourteen promising mutants each of cv. Pak-cheen and Tower were evaluated for different agronomic characters in separate yield trials, under extremely drought conditions. The results demonstrated that yield potential of some mutants was very high and 9 mutants of cv. Pak-cheen and 8 mutants of cv. Tower significantly (P<0.05) out yield the local commercial cultivar. Eleven mutants in both the trials matured significantly earlier than the check. Nevertheless, more extensive testing of the drought tolerant lines under diversified environs of the country will help confirm these findings. (author)

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

Science.gov (United States)

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.

Functional analysis RaZIP1 transporter of the ZIP family from the ectomycorrhizal Zn-accumulating Russula atropurpurea.

Science.gov (United States)

Leonhardt, Tereza; Sácký, Jan; Kotrba, Pavel

2018-04-01

A search of R. atropurpurea transcriptome for sequences encoding the transporters of the Zrt-, Irt-like Protein (ZIP) family, which are in eukaryotes integral to Zn supply into cytoplasm, allowed the identification of RaZIP1 cDNA with a predicted product belonging to ZIP I subfamily; it was subjected to functional studies in mutant Saccharomyces cerevisiae strains. The expression of RaZIP1, but not RaZIP1 H208A or RaZIP1 H232A mutants lacking conserved-among-ZIPs transmembrane histidyls, complemented Zn uptake deficiency in zrt1Δzrt2Δ yeasts. RaZIP1 substantially increased cellular Zn uptake in this strain and added to Zn sensitivity in zrc1Δcot1Δ mutant. The Fe uptake deficiency in ftr1Δ strain was not rescued and Mn uptake was insufficient for toxicity in Mn-sensitive pmr1Δ yeasts. By contrast, RaZIP1 increased Cd sensitivity in yap1Δ strain and conferred Cd transport activity in yeasts, albeit with substantially lower efficiency compared to Zn transport. In metal uptake assays, the accumulation of Zn in zrt1Δzrt2Δ strain remained unaffected by Cd, Fe, and Mn present in 20-fold molar excess over Zn. Immunofluorescence microscopy detected functional hemagglutinin-tagged HA::RaZIP1 on the yeast cell protoplast periphery. Altogether, these data indicate that RaZIP1 is a high-affinity plasma membrane transporter specialized in Zn uptake, and improve the understanding of the cellular and molecular biology of Zn in R. atropurpurea that is known for its ability to accumulate remarkably high concentrations of Zn.

Evaluation of short stature mutants of Basmati-370 for yield and grain quality characteristics

International Nuclear Information System (INIS)

Awan, M.A.; Ahmad, M.; Cheema, A.A.

1982-01-01

Three short stature mutants were induced in an indica rice cultivar by gamma irradiation. The mutants were assessed for their yielding ability and grain quality characteristics. All the mutants out yielded the parent variety, Basmati-370. The increase in yield of the mutants ranged from 19.37% to 29.66%. DM-2 gave the highest yield (3587.96 kg/ha) among the mutants. As regards physical, cooking and eating quality characteristics, there was no significant difference in water absorption, volume expansion ratios and stickiness among the mutants and Basmati-370. However, Basmati-370 was scored best for flavour as this variety had strong aroma as compared to its mutants which were scored for moderately strong aroma. (authors)

Characterization of a bacteriophage T4 mutant lacking DNA-dependent ATPase

International Nuclear Information System (INIS)

Behme, M.T.; Ebisuzaki, K.

1975-01-01

A DNA-dependent ATPase has previously been purified from bacteriophage T4-infected Escherichia coli. A mutant phage strain lacking this enzyme has been isolated and characterized. Although the mutant strain produced no detectable DNA-dependent ATPase, growth properties were not affected. Burst sizes were similar for the mutant phage and T4D in polAl, recB, recC, uvrA, uvrB, uvrC, and various DNA-negative E. coli. UV sensitivity and genetic recombination were normal in a variety of E. coli hosts. Mapping data indicate that the genetic locus controlling the mutant occurs near gene 56. The nonessential nature of this gene is discussed

Radiation-sensitive mutant of hypertoxinogenic strain 569B of Vibro cholerae

International Nuclear Information System (INIS)

Das, G.; Das, J.

1983-01-01

A radiation-sensitive mutant of the hypertoxinogenic strain 569B of Vibrio cholerae was isolated and characterized. The mutant, designated V. cholerae 569Bsub(s), lacks both excision- and medium-dependent dark-repair mechanisms of UV-induced DNA damage while retaining the wild-type photoreactivating capability. Analysis of the UV-irradiated cell DNA by velocity sedimentation in alkaline sucrose gradient suggests that UV-induced pyrimidine dimers may not be incised in these cells. In contrast to the wild-type cells, the mutant cell DNA was degraded after treatment with nalidixic acid. The mutant cells failed to produce any detectable amount of cholera toxin as measured by ileal-loop assay. (orig.)

Global alteration of the drug-binding pocket of human P-glycoprotein (ABCB1) by substitution of fifteen conserved residues reveals a negative correlation between substrate size and transport efficiency.

Science.gov (United States)

Vahedi, Shahrooz; Chufan, Eduardo E; Ambudkar, Suresh V

2017-11-01

P-glycoprotein (P-gp), an ATP-dependent efflux pump, is linked to the development of multidrug resistance in cancer cells. However, the drug-binding sites and translocation pathways of this transporter are not yet well-characterized. We recently demonstrated the important role of tyrosine residues in regulating P-gp ATP hydrolysis via hydrogen bond formations with high affinity modulators. Since tyrosine is both a hydrogen bond donor and acceptor, and non-covalent interactions are key in drug transport, in this study we investigated the global effect of enrichment of tyrosine residues in the drug-binding pocket on the drug binding and transport function of P-gp. By employing computational analysis, 15 conserved residues in the drug-binding pocket of human P-gp that interact with substrates were identified and then substituted with tyrosine, including 11 phenylalanine (F72, F303, F314, F336, F732, F759, F770, F938, F942, F983, F994), two leucine (L339, L975), one isoleucine (I306), and one methionine (M949). Characterization of the tyrosine-rich P-gp mutant in HeLa cells demonstrated that this major alteration in the drug-binding pocket by introducing fifteen additional tyrosine residues is well tolerated and has no measurable effect on total or cell surface expression of this mutant. Although the tyrosine-enriched mutant P-gp could transport small to moderate size (transport large (>1000 Daltons) substrates such as NBD-cyclosporine A, Bodipy-paclitaxel and Bodipy-vinblastine was significantly decreased. This was further supported by the physico-chemical characterization of seventeen tested substrates, which revealed a negative correlation between drug transport and molecular size for the tyrosine-enriched P-gp mutant. Published by Elsevier Inc.

Therapeutic Targeting of Spliceosomal-Mutant Acquired Bone Marrow Failure Disorders

Science.gov (United States)

2017-05-01

spliceosomal mutant cells . This effort has also highlighted a requirement for innate immune signaling in SF3B1-mutant MDS and has implicated a few specific...relative to single-mutant cells (Figure 5A). As innate immune signaling has been implicated in MDS pathogenesis (Basiorka et al., 2016; Fang et al...Sato et al., 2005; Tang et al., 2008; Vink et al., 2013; Xin et al., 2017). Here, we observed that SF3B1K700E/+ human lymphoid leukemia cells (NALM-6

Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus

International Nuclear Information System (INIS)

Mesa, Rosana; Magadan, Javier; Barbieri, Alejandro; Lopez, Cecilia; Stahl, Philip D.; Mayorga, Luis S.

2005-01-01

The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN)

Structure-Function Correlation Analysis of Connexin50 Missense Mutations Causing Congenital Cataract: Electrostatic Potential Alteration Could Determine Intracellular Trafficking Fate of Mutants

Directory of Open Access Journals (Sweden)

Devroop Sarkar

2014-01-01

Full Text Available Connexin50 (Cx50 mutations are reported to cause congenital cataract probably through the disruption of intercellular transport in the lens. Cx50 mutants that undergo mistrafficking have generally been associated with failure to form functional gap junction channels; however, sometimes even properly trafficked mutants were found to undergo similar consequences. We hereby wanted to elucidate any structural bases of the varied functional consequences of Cx50 missense mutations through in silico approach. Computational studies have been done based on a Cx50 homology model to assess conservation, solvent accessibility, and 3-dimensional localization of mutated residues as well as mutation-induced changes in surface electrostatic potential, H-bonding, and steric clash. This was supplemented with meta-analysis of published literature on the functional properties of connexin missense mutations. Analyses revealed that the mutation-induced critical alterations of surface electrostatic potential in Cx50 mutants could determine their fate in intracellular trafficking. A similar pattern was observed in case of mutations involving corresponding conserved residues in other connexins also. Based on these results the trafficking fates of 10 uncharacterized Cx50 mutations have been predicted. Further experimental analyses are needed to validate the observed correlation.

Tyrosine hydroxylase immunoreactivity and [3H]WIN 35,428 binding to the dopamine transporter in a hamster model of idiopathic paroxysmal dystonia

International Nuclear Information System (INIS)

Nobrega, J.N.; Gernert, M.; Loescher, W.; Raymond, R.; Belej, T.; Richter, A.

1999-01-01

Recent pharmacological studies and receptor analyses have suggested that dopamine neurotransmission is enhanced in mutant dystonic hamsters (dt sz ), a model of idiopathic paroxysmal dystonia which displays attacks of generalized dystonia in response to mild stress. In order to further characterize the nature of dopamine alterations, the present study investigated possible changes in the number of dopaminergic neurons, as defined by tyrosine hydroxylase immunohistochemistry, as well as binding to the dopamine transporter labelled with [ 3 H]WIN 35,428 in dystonic hamsters. No differences in the number of tyrosine hydroxylase-immunoreactive neurons were found within the substantia nigra and ventral tegmental area of mutant hamsters compared to non-dystonic control hamsters. Similarly, under basal conditions, i.e. in the absence of a dystonic episode, no significant changes in [ 3 H]WIN 35,428 binding were detected in dystonic brains. However, in animals killed during the expression of severe dystonia, significant decreases in dopamine transporter binding became evident in the nucleus accumbens and ventral tegmental area in comparison to controls exposed to the same external stimulation. Since stimulation tended to increase [ 3 H]WIN 35,428 binding in control brains, the observed decrease in the ventral tegmental area appeared to be due primarily to the fact that binding was increased less in dystonic brains than in similarly stimulated control animals.This finding could reflect a diminished ability of the dopamine transporter to undergo adaptive changes in response to external stressful stimulation in mutant hamsters. The selective dopamine uptake inhibitor GBR 12909 (20 mg/kg) aggravated dystonia in mutant hamsters, further suggesting that acute alterations in dopamine transporter function during stimulation may be an important component of dystonia in this model. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

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

International Nuclear Information System (INIS)

Chandra, P. Manish; Brannigan, James A.; Prabhune, Asmita; Pundle, Archana; Turkenburg, Johan P.; Dodson, G. Guy; Suresh, C. G.

2004-01-01

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 will provide three-dimensional structures of precursor PVA forms, plus open a route to the study of enzyme–substrate complexes for this industrially important enzyme

PedonnanceofE3rly MatUring MutantS Derived from ''SuPa'~ Rice ...

African Journals Online (AJOL)

Vienna, Austria in 1994. The dry seeds were in-adiated with gamma rays using three doses (170, 210. --iifid 24OC;Y).frOm C.obalt 60 (lCO) in order shorten the plant height and maturity period. From the resulting mutant. PoPulations ortgindtiriifroni modified single seed descent method, five Jery early maturing lines plus the ...

Molecular determinants of transport stimulation of EAAT2 are located at interface between the trimerization and substrate transport domains.

Science.gov (United States)

Mortensen, Ole V; Liberato, José L; Coutinho-Netto, Joaquim; Dos Santos, Wagner F; Fontana, Andréia C K

2015-04-01

Excitatory amino acid transporters (EAATs) regulate glutamatergic signal transmission by clearing extracellular glutamate. Dysfunction of these transporters has been implicated in the pathogenesis of various neurological disorders. Previous studies have shown that venom from the spider Parawixia bistriata and a purified compound (Parawixin1) stimulate EAAT2 activity and protect retinal tissue from ischemic damage. In the present study, the EAAT2 subtype specificity of this compound was explored, employing chimeric proteins between EAAT2 and EAAT3 transporter subtypes and mutants to characterize the structural region targeted by the compound. This identified a critical residue (Histidine-71 in EAAT2 and Serine-45 in EAAT3) in transmembrane domain 2 (TM2) to be important for the selectivity between EAAT2 and EAAT3 and for the activity of the venom. Using the identified residue in TM2 as a structural anchor, several neighboring amino acids within TM5 and TM8 were identified to also be important for the activity of the venom. This structural domain of the transporter lies at the interface of the rigid trimerization domain and the central substrate-binding transport domain. Our studies suggest that the mechanism of glutamate transport enhancement involves an interaction with the transporter that facilitates the movement of the transport domain. We identified a domain (purple star) in the glutamate transporter EAAT2 that is important for transport stimulation through a spider venom, and suggest a mechanism for enhanced transporter function through facilitated substrate translocation (arrow). Because the dysfunction of glutamate transporters is implicated in the pathogenesis of neurological disorders, understanding the mechanisms of enhanced transport could have therapeutic implications. © 2015 International Society for Neurochemistry.

Studies on induced partially resistant mutants of barley against powdery mildew

International Nuclear Information System (INIS)

Roebbelen, G.; Abdel-Hafez, A.G.; Reinhold, M.; Kwon, H.J.; Neuhaus-Steinmetz, J.P.; Heun, M.

1983-01-01

After mutagenic seed treatment of three partially resistant cultivars of spring barley with EMS and NaN 3 , 45 mutants in a first and 16 in a second experiment were selected in the M 2 -M 4 generations. The screening was done alternatively under natural infection in the field or controlled infection with a single pathotype in the greenhouse. These mutants exhibited a higher resistance and a higher susceptibility, respectively, than the initial cultivars Asse, Bomi and Vada. Some mutants expressed their altered resistance behaviour particularly during certain stages of development. High-level resistance was conditioned by mutation in the ml-o locus in three cases. For several Bomi mutants pathotype specificity with and without reversed ranking was proven as well as pathotype non-specificity in comparison with the reaction of the original cultivar. In 14 cases studied the inheritance of the involved mutants was monogenic recessive. The laevigatum locus responsible for the intermediate mildew resistance of Bomi was not affected by the mutations. Detection of groups of allelic mutants showed that there are at least two regions in the barley genome in which mutations for mildew resistance can occur rather frequently. In total, the past ten years of this mutation research have given convincing evidence that the strategies of mutant screening applied have yielded promising new material both for breeding and for progress in basic understanding of host-pathogen interactions. (author)

Characteristics and use of wheat mutants tolerant or resistant to Septoria nodorum Berk

International Nuclear Information System (INIS)

Fossati, A.; Kleijer, G.; Fried, P.M.

1983-01-01

Mutation induction was used to obtain mutants tolerant or resistant to Septoria nodorum. This technique is valuable but many genotypes had to be treated because mutants could not be selected from all the genotypes. Short tolerant mutants could be obtained from 3 of the 15 treated tall tolerant lines. Induction of tolerance in susceptible lines of good agronomic value succeeded for 2 of 5 treated varieties. All these mutants showed a reduction in yield potential. One mutant showed partial resistance to S. nodorum. The disease development on the leaves and the spikes of this mutant was much slower than on the original variety. The characteristics of this mutant are discussed in detail. The genetics of tolerance proved to be polygenic and additive, which has consequences on the breeding method. A good way of obtaining a stable system would be the combination of high tolerance and partial resistance in the same cultivar. (author)

The ABC transporter Tba of Amycolatopsis balhimycina is required for efficient export of the glycopeptide antibiotic balhimycin.

Science.gov (United States)

Menges, R; Muth, G; Wohlleben, W; Stegmann, E

2007-11-01

All known gene clusters for glycopeptide antibiotic biosynthesis contain a conserved gene supposed to encode an ABC-transporter. In the balhimycin-producer Amycolatopsis balhimycina this gene (tba) is localised between the prephenate dehydrogenase gene pdh and the peptide synthetase gene bpsA. Inactivation of tba in A. balhimycina by gene replacement did not interfere with growth and did not affect balhimycin resistance. However, in the supernatant of the tba mutant RM43 less balhimycin was accumulated compared to the wild type; and the intra-cellular balhimycin concentration was ten times higher in the tba mutant RM43 than in the wild type. These data suggest that the ABC transporter encoded in the balhimycin biosynthesis gene cluster is not involved in resistance but is required for the efficient export of the antibiotic. To elucidate the activity of Tba it was heterologously expressed in Escherichia coli with an N-terminal His-tag and purified by nickel chromatography. A photometric assay revealed that His(6)-Tba solubilised in dodecylmaltoside possesses ATPase activity, characteristic for ABC-transporters.

Transmembrane transporter expression regulated by the glucosylceramide pathway in Cryptococcus neoformans.

Science.gov (United States)

Singh, Arpita; Rella, Antonella; Schwacke, John; Vacchi-Suzzi, Caterina; Luberto, Chiara; Del Poeta, Maurizio

2015-11-16

The sphingolipid glucosylceramide (GlcCer) and factors involved in the fungal GlcCer pathways were shown earlier to be an integral part of fungal virulence, especially in fungal replication at 37 °C, in neutral/alkaline pH and 5 % CO2 environments (e.g. alveolar spaces). Two mutants, ∆gcs 1 lacking glucosylceramide synthase 1 gene (GCS1) which catalyzes the formation of sphingolipid GlcCer from the C9-methyl ceramide and ∆smt1 lacking sphingolipid C9 methyltransferase gene (SMT1), which adds a methyl group to position nine of the sphingosine backbone of ceramide, of this pathway were attenuated in virulence and have a growth defect at the above-mentioned conditions. These mutants with either no or structurally modified GlcCer located on the cell-membrane have reduced membrane rigidity, which may have altered not only the physical location of membrane proteins but also their expression, as the pathogen's mode of adaptation to changing need. Importantly, pathogens are known to adapt themselves to the changing host environments by altering their patterns of gene expression. By transcriptional analysis of gene expression, we identified six genes whose expression was changed from their wild-type counterpart grown in the same conditions, i.e. they became either down regulated or up regulated in these two mutants. The microarray data was validated by real-time PCR, which confirmed their fold change in gene expression. All the six genes we identified, viz siderochrome-iron transporter (CNAG_02083), monosaccharide transporter (CNAG_05340), glucose transporter (CNAG_03772), membrane protein (CNAG_03912), membrane transport protein (CNAG_00539), and sugar transporter (CNAG_06963), are membrane-localized and have significantly altered gene expression levels. Therefore, we hypothesize that these genes function either independently or in tandem with a structurally modified cell wall/plasma membrane resulting from the modifications of the GlcCer pathway and thus possibly

An early maturing rice mutant released as a variety

International Nuclear Information System (INIS)

Azam, M.A.; Imtiaz Uddin, Md.

2001-01-01

In the content of food grain production deficiency (about 1.0-1.5 million tons of rice per year according to the Bangladesh Bureau of Statistics, 1998) an induced mutation programme was undertaken in 1985. One moderate early maturing and high yielding rice mutant line (BINA6-84-4-115) has been developed by irradiating F 2 seeds of the cross 'BR4' x 'Iratom 38'. Three treatments viz., 250, 300 and 350 Gy were given to the F 2 seeds. Finally, this line was selected in M 6 generation for advanced yield trial. The line was evaluated in comparative trials with another mutant line BINA6-84-4-163. These two mutant lines had been selected earlier from 300 Gy originated lines. The two check varieties, 'BR 11' and 'BR 22' were also included in the trial, which was conducted in two consecutive T. aman seasons (July to December) during 1994 and 1995 at five locations in Bangladesh. From the results, it was evident that the mutant BINA6-84-4-115 did not differ much with the other mutant lines or check varieties in respect to plant height, number of effective tillers and panicle length but it was 10-18 days earlier than the other 3 entries. It produced a similar yield as the check BR 11 in 1994 and a higher yield than the check BR 11 and BR 22 in 1995. This mutant line gave the highest yield per day among all the entries. In addition to this, the grains are long, fine and possess a high L/B ratio, which are of high commercial value. This line has been released by the National Seed Board of Bangladesh in 1998 as a commercial variety under the name 'BINADHAN-4' for cultivation throughout Bangladesh

Isolation of new gravitropic mutants under hypergravity conditions

OpenAIRE

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

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 upwards. 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 ...

A higher yielding mutant of black gram with improved nodule formation

International Nuclear Information System (INIS)

Singh, R.K.; Raghuvanshi, S.S.

1987-01-01

Dry seeds of black gram (Vigna mungo (L) Hopper) var. T 9 with 12.2% moisture content were irradiated at 10, 20 and 30 krad of gamma rays. This was followed by combined treatment of one set in each dose with freshly prepared 0. 25% EMS in phosphate buffer at 7.0 pH at 30± deg. C for 6 hours. In M 2 population of 20 krad two mutants with pentafoliate instead of trifoliate leaves were found. This character was true breeding in M 3 M 6 generation. Crosses revealed monogenic recessive inheritance of this character. The proposed gene symbol is p5. This mutant has normal maturity period and the plant height is the same as T 9 (ca. 50 cm). Preliminary yield trials indicate superiority of the mutant line over control. The mutant line also shows a significant improvement in number and weight of root nodules, potentially improving green manuring value. Improvement of root nodulation in mungbean mutants was reported before by others

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

Science.gov (United States)

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.

Sensorimotor learning in Dab1(scm) (scrambler) mutant mice.

Science.gov (United States)

Lalonde, R; Strazielle, C

2011-04-15

Homozygous Dab1(scm) mouse mutants with cell ectopias in cerebellar cortex and neocortex were compared with non-ataxic controls on two tests of motor coordination: rotorod and grid climbing. Even at the minimal speed of 4 rpm and unlike controls, none of the Dab1(scm) mutants reached criterion on the constant speed rotorod. In contrast, Dab1(scm) mutants improved their performances on the vertical grid over the course of the same number of trials. Thus, despite massive cerebellar degeneration, sensorimotor learning for equilibrium is still possible, indicating the potential usefulness of the grid-climbing test in determining residual functions in mice with massive cerebellar damage. Copyright © 2010. Published by Elsevier B.V.

Molecular analyses of in vivo hprt mutant T cells from atomic bomb survivors

International Nuclear Information System (INIS)

Hakoda, M.; Hirai, Y.; Kyoizumi, S.; Akiyama, M.

1989-01-01

In vivo-derived hprt-deficient mutant T cells isolated from three nonirradiated controls and two atomic bomb survivors were studied by Southern blot analysis to investigate the molecular spectra of the mutations. Mutant frequencies for the three controls were 1.8, 2.3, and 7.3 x 10(-6), and those for the two survivors (who had received radiation doses of 2.46 and 2.15 Gy, based upon the revised atomic bomb shielded kerma estimates) were 9.3 and 14.4 x 10(-6), respectively. Fourteen (13%) of 105 mutant T-cell colonies from the controls showed various structural changes in the hprt gene. The frequency of mutants with hprt gene structural changes in one atomic bomb survivor, who exhibited a mutant frequency of 9.3 x 10(-6), was 26% (16/61), which was significantly higher than that of the controls. However, the frequency of structural changes in the other survivor (14%, 8/59) was not higher than that of the controls. Two sets of mutants (in total, eight mutants) from the survivor, who showed a significantly higher frequency of mutants with hprt gross alterations than did the controls, had the same hprt changes and the same rearrangements of T-cell receptor (TcR) beta- and gamma-chain genes, indicating a clonal expansion from one progenitor mutant. This phenomenon may reflect an in vivo recovery process of T cells in the periphery after exposure to atomic bomb radiation. However, when comparing the frequency of mutations, these two sets of mutants should be reduced. After reducing the total number of mutants from the number of gross hprt changes, the frequency was not significantly higher than that of the controls

Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

DEFF Research Database (Denmark)

Hove-Jensen, Bjarne; Nygaard, Per

1982-01-01

, stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib...

Increased spontaneous mitotic segregation in MMS-sensitive mutants of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Prakash, S.; Prakash, L.

1977-01-01

Methyl methanesulfonate (MMS)-sensitive mutants of Saccharomyces cerevisiae belonging to four different complementation groups, when homozygous, increase the rate of spontaneous mitotic segregation to canavanine resistance from heterozygous sensitive (can/sup r//+) diploids by 13- to 170-fold. The mms8-1 mutant is MMS and x-ray sensitive and increases the rate of spontaneous mitotic segregation 170-fold. The mms9-1 and mms13-1 mutants are sensitive to x rays and uv, respectively, in addition to MMS, and increase the rate of spontaneous mitotic segregation by 13-fold and 85-fold, respectively. The mutant mms21-1 is sensitive to MMS, x rays and uv and increases the rate of spontaneous mitotic segregation 23-fold

Identification of dominant male sterile mutants in rice (Oryza sativa L.)

International Nuclear Information System (INIS)

Zhu Xudong; Rutger, J.N.

2000-01-01

Genetic male sterile mutants 1783 and 1789 were selected from US variety Orion and Kaybonnet seeds treated by gamma irradiation. Investigation of fertility characterization of pollen and spikelets of these mutants were made by progeny tests in 1783 M 7 and 1789 M 6 generations. The results showed that genetic male sterile mutants 1783 and 1789 with the fertility segregating of 1 sterile: 1 fertile were controlled by a single dominant gene. The pollen staining of mutants characterized partial sterility. Open-pollinated seed set was about 30% and bagged seed set was only 0.3%-3.5%. It is concluded that dominant genetic male sterile is a useful tool in improvement of population for rice breeding

Root and Nodulation Phenotypes of the Ethylene-Insensitive Sickle Mutant of Medicago truncatula

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JOKO PRAYITNO

2010-09-01

Full Text Available The sickle (skl mutant of the model legume Medicago truncatula is an ethylene-sensitive mutant that have a ten-fold increase in nodule numbers. The nodulation and root phenotypes of the skl mutant were investigated and further characterised. The skl mutant had longer roots than the wild type, but when inoculated with Sinorhizobium, its root length was reduced to the level of wild type. Furthermore, lateral root numbers in uninoculated skl were similar to those in uninoculated wild type. However, when the root tips were decapitated, fewer lateral roots formed in skl than in wild type. Nodule numbers of the skl mutant were significantly reduced by low nitrate concentration (2.5 mM. These results suggest that skl mutant has alterations in both root and nodule development.

Disruption of the rice nitrate transporter OsNPF2.2 hinders root-to-shoot nitrate transport and vascular development

Science.gov (United States)

Li, Yuge; Ouyang, Jie; Wang, Ya-Yun; Hu, Rui; Xia, Kuaifei; Duan, Jun; Wang, Yaqin; Tsay, Yi-Fang; Zhang, Mingyong

2015-01-01

Plants have evolved to express some members of the nitrate transporter 1/peptide transporter family (NPF) to uptake and transport nitrate. However, little is known of the physiological and functional roles of this family in rice (Oryza sativa L.). Here, we characterized the vascular specific transporter OsNPF2.2. Functional analysis using cDNA-injected Xenopus laevis oocytes revealed that OsNPF2.2 is a low-affinity, pH-dependent nitrate transporter. Use of a green fluorescent protein tagged OsNPF2.2 showed that the transporter is located in the plasma membrane in the rice protoplast. Expression analysis showed that OsNPF2.2 is nitrate inducible and is mainly expressed in parenchyma cells around the xylem. Disruption of OsNPF2.2 increased nitrate concentration in the shoot xylem exudate when nitrate was supplied after a deprivation period; this result suggests that OsNPF2.2 may participate in unloading nitrate from the xylem. Under steady-state nitrate supply, the osnpf2.2 mutants maintained high levels of nitrate in the roots and low shoot:root nitrate ratios; this observation suggests that OsNPF2.2 is involved in root-to-shoot nitrate transport. Mutation of OsNPF2.2 also caused abnormal vasculature and retarded plant growth and development. Our findings demonstrate that OsNPF2.2 can unload nitrate from the xylem to affect the root-to-shoot nitrate transport and plant development. PMID:25923512

Mutant of Japanese pear resistant to Black Spot Disease

International Nuclear Information System (INIS)

Sanada, T.; Nishida, T.; Ikeda, F.

1987-01-01

Full text: Nijisseike is one of the leading cultivars of Japanese pear (Pyrus serotinea Rehd.), but susceptible to black spot disease. Farmers try to prevent this disease by wrapping the fruit with a paper bag and by repeated spraying of fungicides. The disease is caused by a Japanese pear pathotype of Alternaria alternata (Fr.) Keissler. Susceptibility is controlled by a single dominant gene. In 1962, grafted trees of this cultivar were planted at a distance between 53 and 93 m from the 60 Co source in the gamma-field (daily dose 15-4 rad). One branch on a tree planted at 53 m was detected as resistant in 1981. Under field conditions, black spots were observed on many fruits and leaves of the original trees by natural infection in early July, however, they were not observed on the mutant. To examine the resistance of the mutant, artificial inoculations were made using spores of the pathogen and the host specific toxin produced by germinating spores. When some drops of the spore suspension are placed on leaves, the formation of black spots depends upon the leaf age. In a resistant cv. as Chojuro, black spot symptoms are formed only when inoculated on young leaves. An intermediate reaction was observed in the mutant, whereas the original Nijisseiki showed severe symptoms. When inoculation was made on matured fruit skins, no black spot was formed on the mutant just like on the resistant cv. Chojuro, while many small black spots were formed and grew into large spots overlapping each other on the susceptible cv. Nijisseiki. In case of the crude toxin inoculation (4-0.04 ppm) of cv. Nijisseiki black spots were formed on the surface of the susceptible fruit skin, and necrotic lesions at the cut end of detached small pieces of leaves, although reaction on fruit skins was weaker compared with inoculation by spores. However, no symptoms were observed from the toxin application on the mutant and the resistant cv. Chojuro. That the resistance of the mutant is classified as

UV and gamma-ray sensitivity of meiosis-deficient mutants in Podospora anserina

International Nuclear Information System (INIS)

Simonet, J.M.

1976-01-01

Two mutants, mei1 and mei2, were isolated by screening for deficiencies occurring in the meiotic process. The sensitivity of mei1 and mei2 mutant strains to UV irradiation showed a significant increase as compared with that of the wild-type stock, hwhereas the sensitivity to γ-rays remained unchanged. The double-mutant strains were no more sensitive than each single mutant. The data indicate that both mei1 and mei2 loci are probably involved in the same pathway of excision-repair of UV-induced lesions

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

Science.gov (United States)

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.

Secretion and activation of the Serratia marcescens hemolysin by structurally defined ShlB mutants.

Science.gov (United States)

Pramanik, Avijit; Könninger, Ulrich; Selvam, Arun; Braun, Volkmar

2014-05-01

The ShlA hemolysin of Serratia marcescens is secreted across the outer membrane by the ShlB protein; ShlB belongs to the two-partner secretion system (type Vb), a subfamily of the Omp85 outer membrane protein assembly and secretion superfamily. During secretion, ShlA is converted from an inactive non-hemolytic form into an active hemolytic form. The structure of ShlB is predicted to consist of the N-terminal α-helix H1, followed by the two polypeptide-transport-associated domains POTRA P1 and P2, and the β-barrel of 16 β-strands. H1 is inserted into the pore of the β-barrel in the outer membrane; P1 and P2 are located in the periplasm. To obtain insights into the secretion and activation of ShlA by ShlB, we isolated ShlB mutants impaired in secretion and/or activation. The triple H1 P1 P2 mutant did not secrete ShlA. The P1 and P2 deletion derivatives secreted reduced amounts of ShlA, of which P1 showed some hemolysis, whereas P2 was inactive. Deletion of loop 6 (L6), which is conserved among exporters of the Omp85 family, compromised activation but retained low secretion. Secretion-negative mutants generated by random mutagenesis were located in loop 6. The inactive secreted ShlA derivatives were complemented in vitro to active ShlA by an N-terminal ShlA fragment (ShlA242) secreted by ShlB. Deletion of H1 did not impair secretion of hemolytic ShlA. The study defines domains of ShlB which are important for ShlA secretion and activation. Copyright © 2013 Elsevier GmbH. All rights reserved.

Preliminary Study of the Characteristics of Several Glossy Cabbage (Brassica oleracea var. capitata L. Mutants

Directory of Open Access Journals (Sweden)

Tang Jun

2015-09-01

Full Text Available To determine the characteristics and potential practical applications of glossy cabbage (Brassica oleracea var. capitata L. mutants, five different glossy mutants were studied. The amount of epicuticular wax covering the mutant leaves was only approximately 30% that of the wild-type (WT leaves. The wax crystals of WT plants were columnar and linear, while they were granular and rod-shaped in the mutants. Additionally, in WT cabbage, the primary wax components were alkanes, alcohols, fatty acids, ketones, and aldehydes. There was a significant decrease in the abundance of alkanes and ketones in the wax of the mutants. The glossy-green trait of the mutants may be the result of an inhibited alkane-forming pathway. Higher rates of chlorophyll leaching and water loss demonstrate that the mutant leaves were more permeable and sensitive to drought stress than the WT leaves. Growth curve results indicated that the growth rate of mutant-1 and mutant-3 was slower than that of the corresponding WT cabbage, resulting in shorter plants. However, the growth rate of mutant-2 was not influenced by the lack of coating wax. An investigation of the agronomic traits and heterosis of the glossy cabbage mutants indicated that all five mutants had glossy-green leaves, which was a favorable characteristic. The F1 plants derived from crosses involving mutant-2 exhibited obvious heterosis, suggesting the observed glossy-green trait is controlled by a dominant gene. Therefore, mutant-2 may be useful as a source of genetic material for future cabbage breeding experiments.

Behaviour of UV-sensitive mutants of Proteus mirabilis to repair incision breaks

International Nuclear Information System (INIS)

Stoerl, K.; Mund, C.

1977-01-01

In U.V.-sensitive mutants of P. mirabilis with the phenotype HCR, REC and EXR single-strand breaks appeared immediately after UV-irradiation. The behaviour of REC- and EXR-mutants was similar to the wildtype. The number of incision breaks observed by sedimentation analysis in these strains was very low. They could be joined during the excision repair process. From the ability of REC- and EXR-strains to rejoin most of the induced single-strand breaks it can be concluded that these strains have approximately the same capacity for excision repair as the wildtype. HCR-mutants of P. mirabilis produced single-strand breaks after UV-irradiation in contrast to HCR-mutants of E. coli. Therefore we suggest that HCR-mutants of P. mirabilis are not completely inhibited in the incision step. The single-strand breaks introduced in the DNA at the beginning of the repair process were not rejoined during further incubation. Experiments with toluenized cells led to the same results. The newly synthesized daughter DNA-strands of UV-irradiated HCR-mutants were of low molecular weight in comparison with those from unirradiated control cells during the repair period. This result is in agreement with the incapability of HCR-mutants to remove the pyrimidine dimers from the parental template strand. (author)

Development mutants of anabaena doliolum defective in repair of UV-damage

International Nuclear Information System (INIS)

Tiwari, D.N.; Singh, C.B.

1980-01-01

Nitrosoguanidine induced 'blue' pigment mutants of the blue-green alga anabaena doliolum were isolated. The blue-mutants on further characterization were grouped into three developmental phenotypes - (i) those forming doli-form blue-spores of heterogenous size i.e., Ad 011, (ii) those forming spheroidal cells in the stationary phase, some of which behave like spores on transfer to fresh medium i.e., Ad 012, and (iii) those showing no sporulation and conditionally producing abnormal cells in the presence of combined nitrogen only i.e., Ad 007. The former two classes of mutants showed the formation of abnormal cells irrespective of the presence or absence of combined nitrogen sources in the medium. The formation of abnormal cells in the filaments of the above mutants were distinguished by their larger size and irregular mode of division leading to true-branch formation. The comparative characterization of these mutant strains with the parental one showed sluggish growth, increased UV-sensitivity, almost unchanged photorepair capacity, a marked change in the pigment composition and relative resistance to nitrosoguanidine. Irregular cell division in both space and time in the mutant strains and their increased sensitivity to ultraviolet irradiation indicate the possible involvement of dark repair system in maintaining the precision of cell cylce in this alga. (orig.) 891 AJ/orig. 892 HIS

Selection of high hectolitre weight mutants of winter wheat

International Nuclear Information System (INIS)

Crowley, C.; Jones, P.

1989-01-01

Grain quality in wheat includes hectolitre weight (HLW) besides protein content and thousand-grain weight (TGW). The British winter wheat variety ''Guardian'' has a very high yield potential. Although the long grain of ''Guardian'' results in a desirable high TGW the HLW is too low. To select mutants exhibiting increased HLW the character was first analyzed to identify traits that could more easily be screened for using M 2 seeds. In comparison of 6 wheat cultivars, correlation analyses with HLW resulted in coefficients of -0.86 (grain length, L:P 2 seeds for shorter, less prolate grains. Mutagenesis was carried out using EMS sulphonate (1.8 or 3.6%), sodium azide (2 or 20 mM) or X-rays (7.5 or 20 kR). 69 M 2 grains with altered shape were selected. Examination of the M 3 progeny confirmed 6 grain-shape mutants, most of them resulting from EMS treatment (Table). Two of the mutants showed TGW values significantly below the parental variety, but three mutants exhibited HLW and TGW values significantly greater than those of the parental variety. Microplot yield trails on selected M 3 lines are in progress. The influence of physical grain characteristics on HLW offers prospects for mechanical fractionation of large M 2 populations. The application of gravity separators (fractionation on the basis of grain density) and sieves (fractionation on the basis of grain length) in screening mutants possessing improved grain quality is being investigated