Science.gov

Sample records for response mutants defective

  1. Defective responsiveness of adenylate cyclase to forskolin in the Drosophila memory mutant rutabaga.

    PubMed

    Dudai, Y; Sher, B; Segal, D; Yovell, Y

    1985-12-01

    The Drosophila memory mutant rutabaga (rut) has been previously shown to have a defective subpopulation (or functional state) of the enzyme adenylate cyclase. We report here that the reduced adenylate cyclase activity is also associated with a defective responsiveness of the enzyme to forskolin. Forskolin activation isotherms of the enzyme in normal membranes reveal low- and high-affinity forskolin-interacting components; the residual enzyme in the mutant shows a smaller proportion of the high-affinity response. In addition, in mutant membrane preparations, forskolin fails to shift the Km of the enzyme for free Mg2+ and for MgATP, in contrast to the situation in the normal tissue. The defect in the responsiveness to forskolin in rut is even more pronounced in a Lubrol-solubilized enzyme preparation, and is due to intrinsic properties of the cyclase system rather than to the absence (or presence) of a soluble, or detergent solubilized, factor in rut. The reduced forskolin responsiveness maps to the X chromosomal segment 12F5-6 to 13A1-5, within the region previously reported to span the locus that controls both the abortive memory and the lack of Ca2+-stimulation of adenylate cyclase in rut17. The possible relevance of the findings to postulated molecular mechanisms of short-term memory formation is discussed. PMID:3935769

  2. A Medicago truncatula mutant hyper-responsive to mycorrhiza and defective for nodulation.

    PubMed

    Morandi, Dominique; le Signor, Christine; Gianinazzi-Pearson, Vivienne; Duc, Gérard

    2009-08-01

    One key strategy for the identification of plant genes required for mycorrhizal development is the use of plant mutants affected in mycorrhizal colonisation. In this paper, we report a new Medicago truncatula mutant defective for nodulation but hypermycorrhizal for symbiosis development and response. This mutant, called B9, presents a poor shoot and, especially, root development with short laterals. Inoculation with Glomus intraradices results in significantly higher root colonisation of the mutant than the wild-type genotype A17 (+20% for total root length, +16% for arbuscule frequency in the colonised part of the root, +39% for arbuscule frequency in the total root system). Mycorrhizal effects on shoot and root biomass of B9 plants are about twofold greater than in the wild-type genotype. The B9 mutant of M. truncatula is characterised by considerably higher root concentrations of the phytoestrogen coumestrol and by the novel synthesis of the coumestrol conjugate malonyl glycoside, absent from roots of wild-type plants. In conclusion, this is the first time that a hypermycorrhizal plant mutant affected negatively for nodulation (Myc(++), Nod (-/+) phenotype) is reported. This mutant represents a new tool for the study of plant genes differentially regulating mycorrhiza and nodulation symbioses, in particular, those related to autoregulation mechanisms.

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

    PubMed Central

    2013-01-01

    Background Brucella abortus is an intracellular zoonotic pathogen which causes undulant fever, endocarditis, arthritis and osteomyelitis in human and abortion and infertility in cattle. This bacterium is able to invade and replicate in host macrophage instead of getting removed by this defense mechanism. Therefore, understanding the interaction between virulence of the bacteria and the host cell is important to control brucellosis. Previously, we generated internalization defective mutants and analyzed the envelope proteins. The present study was undertaken to evaluate the changes in early transcriptional responses between wild type and internalization defective mutants infected mouse macrophage, RAW 264.7. Results Both of the wild type and mutant infected macrophages showed increased expression levels in proinflammatory cytokines, chemokines, apoptosis and G-protein coupled receptors (Gpr84, Gpr109a and Adora2b) while the genes related with small GTPase which mediate intracellular trafficking was decreased. Moreover, cytohesin 1 interacting protein (Cytip) and genes related to ubiquitination (Arrdc3 and Fbxo21) were down-regulated, suggesting the survival strategy of this bacterium. However, we could not detect any significant changes in the mutant infected groups compared to the wild type infected group. Conclusions In summary, it was very difficult to clarify the alterations in host cellular transcription in response to infection with internalization defective mutants. However, we found several novel gene changes related to the GPCR system, ubiquitin-proteosome system, and growth arrest and DNA damages in response to B. abortus infection. These findings may contribute to a better understanding of the molecular mechanisms underlying host-pathogen interactions and need to be studied further. PMID:23802650

  4. Epididymis Response Partly Compensates for Spermatozoa Oxidative Defects in snGPx4 and GPx5 Double Mutant Mice

    PubMed Central

    Noblanc, Anaïs; Peltier, Manon; Damon-Soubeyrand, Christelle; Kerchkove, Nicolas; Chabory, Eléonore; Vernet, Patrick; Saez, Fabrice; Cadet, Rémi; Janny, Laurent; Pons-Rejraji, Hanae; Conrad, Marcus; Drevet, Joël R.; Kocer, Ayhan

    2012-01-01

    We report here that spermatozoa of mice lacking both the sperm nucleaus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H2O2-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice. PMID:22719900

  5. Egg-Laying Defective Mutants of the Nematode CAENORHABDITIS ELEGANS

    PubMed Central

    Trent, Carol; Tsung, Nancy; Horvitz, H. Robert

    1983-01-01

    We have isolated 145 fertile mutants of C. elegans that are defective in egg laying and have characterized 59 of them genetically, behaviorally and pharmacologically. These 59 mutants define 40 new genes called egl, for egg-laying abnormal. Most of the other mutants are defective in previously identified genes. The egl mutants differ with respect to the severity of their egg-laying defects and the presence of behavioral or morphological pleiotropies. We have defined four distinct categories of mutants based on their responses to the pharmacological agents serotonin and imipramine, which stimulate egg laying by wild-type hermaphrodites. These drugs test the functioning of the vulva, the vulval and uterine muscles and the hermaphrodite-specific neurons (HSNs), which innervate the vulval muscles. Mutants representing 14 egl genes fail to respond to serotonin and to imipramine and are likely to be defective in the functioning of the vulva or the vulval and uterine muscles. Four mutants (representing four different genes) lay eggs in response to serotonin but not to imipramine and appear to be egg-laying defective because of defects in the HSNs; three of these four were selected specifically for these drug responses. Mutants representing seven egl genes lay eggs in response to serotonin and to imipramine. One egl mutant responds to imipramine but not to serotonin. The remaining egl mutants show variable or intermediate responses to the drugs. Two of the HSN-defective mutants, egl-1 and her-1(n695), lack HSN cell bodies and are likely to be expressing the normally male-specific program of HSN cell death. Whereas egl-1 animals appear to be defective specifically in HSN development, her-1(n695) animals exhibit multiple morphological pleiotropies, displaying partial transformation of the sexual phenotype of many cells and tissues. At least two of the egl mutants appear to be defective in the processing of environmental signals that modulate egg laying and may define new

  6. Biofilm formation-defective mutants in Pseudomonas putida.

    PubMed

    López-Sánchez, Aroa; Leal-Morales, Antonio; Jiménez-Díaz, Lorena; Platero, Ana I; Bardallo-Pérez, Juan; Díaz-Romero, Alberto; Acemel, Rafael D; Illán, Juan M; Jiménez-López, Julia; Govantes, Fernando

    2016-07-01

    Out of 8000 candidates from a genetic screening for Pseudomonas putida KT2442 mutants showing defects in biofilm formation, 40 independent mutants with diminished levels of biofilm were analyzed. Most of these mutants carried insertions in genes of the lap cluster, whose products are responsible for synthesis, export and degradation of the adhesin LapA. All mutants in this class were strongly defective in biofilm formation. Mutants in the flagellar regulatory genes fleQ and flhF showed similar defects to that of the lap mutants. On the contrary, transposon insertions in the flagellar structural genes fliP and flgG, that also impair flagellar motility, had a modest defect in biofilm formation. A mutation in gacS, encoding the sensor element of the GacS/GacA two-component system, also had a moderate effect on biofilm formation. Additional insertions targeted genes involved in cell envelope function: PP3222, encoding the permease element of an ABC-type transporter and tolB, encoding the periplasmic component of the Tol-OprL system required for outer membrane stability. Our results underscore the central role of LapA, suggest cross-regulation between motility and adhesion functions and provide insights on the role of cell envelope trafficking and maintenance for biofilm development in P. putida.

  7. Activation of the Rcs Signal Transduction System Is Responsible for the Thermosensitive Growth Defect of an Escherichia coli Mutant Lacking Phosphatidylglycerol and Cardiolipin

    PubMed Central

    Shiba, Yasuhiro; Yokoyama, Yasuko; Aono, Yoshiko; Kiuchi, Takashi; Kusaka, Jin; Matsumoto, Kouji; Hara, Hiroshi

    2004-01-01

    The lethal effect of an Escherichia coli pgsA null mutation, which causes a complete lack of the major acidic phospholipids, phosphatidylglycerol and cardiolipin, is alleviated by a lack of the major outer membrane lipoprotein encoded by the lpp gene, but an lpp pgsA strain shows a thermosensitive growth defect. Using transposon mutagenesis, we found that this thermosensitivity was suppressed by disruption of the rcsC, rcsF, and yojN genes, which code for a sensor kinase, accessory positive factor, and phosphotransmitter, respectively, of the Rcs phosphorelay signal transduction system initially identified as regulating the capsular polysaccharide synthesis (cps) genes. Disruption of the rcsB gene coding for the response regulator of the system also suppressed the thermosensitivity, whereas disruption of cpsE did not. By monitoring the expression of a cpsB′-lac fusion, we showed that the Rcs system is activated in the pgsA mutant and is reverted to a wild-type level by the rcs mutations. These results indicate that envelope stress due to an acidic phospholipid deficiency activates the Rcs phosphorelay system and thereby causes the thermosensitive growth defect independent of the activation of capsule synthesis. PMID:15375134

  8. Chinese hamster ovary cell mutants defective in heparan sulfate biosynthesis

    SciTech Connect

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

    1987-05-01

    The authors have isolated Chinese hamster ovary cell mutants defective in proteoglycan synthesis by radiographic screening for cells unable to incorporate TVSO4 into acid-precipitable material. Some mutants did not incorporate TVSO4 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.

  9. 2-O-methylation of fucosyl residues of a rhizobial lipopolysaccharide is increased in response to host exudate and is eliminated in a symbiotically defective mutant.

    PubMed

    Noel, K Dale; Box, Jodie M; Bonne, Valerie J

    2004-03-01

    When Rhizobium etli CE3 was grown in the presence of Phaseolus vulgaris seed extracts containing anthocyanins, its lipopolysaccharide (LPS) sugar composition was changed in two ways: greatly decreased content of what is normally the terminal residue of the LPS, di-O-methylfucose, and a doubling of the 2-O-methylation of other fucose residues in the LPS O antigen. R. etli strain CE395 was isolated after Tn5 mutagenesis of strain CE3 by screening for mutant colonies that did not change antigenically in the presence of seed extract. The LPS of this strain completely lacked 2-O-methylfucose, regardless of whether anthocyanins were present during growth. The mutant gave only pseudonodules in association with P. vulgaris. Interpretation of this phenotype was complicated by a second LPS defect exhibited by the mutant: its LPS population had only about 50% of the normal amount of O-antigen-containing LPS (LPS I). The latter defect could be suppressed genetically such that the resulting strain (CE395 alpha 395) synthesized the normal amount of an LPS I that still lacked 2-O-methylfucose residues. Strain CE395 alpha 395 did not elicit pseudonodules but resulted in significantly slower nodule development, fewer nodules, and less nitrogenase activity than lps(+) strains. The relative symbiotic deficiency was more severe when seeds were planted and inoculated with bacteria before they germinated. These results support previous conclusions that the relative amount of LPS I on the bacterial surface is crucial in symbiosis, but LPS structural features, such as 2-O-methylation of fucose, also may facilitate symbiotic interactions. PMID:15006776

  10. 2-O-Methylation of Fucosyl Residues of a Rhizobial Lipopolysaccharide Is Increased in Response to Host Exudate and Is Eliminated in a Symbiotically Defective Mutant

    PubMed Central

    Noel, K. Dale; Box, Jodie M.; Bonne, Valerie J.

    2004-01-01

    When Rhizobium etli CE3 was grown in the presence of Phaseolus vulgaris seed extracts containing anthocyanins, its lipopolysaccharide (LPS) sugar composition was changed in two ways: greatly decreased content of what is normally the terminal residue of the LPS, di-O-methylfucose, and a doubling of the 2-O-methylation of other fucose residues in the LPS O antigen. R. etli strain CE395 was isolated after Tn5 mutagenesis of strain CE3 by screening for mutant colonies that did not change antigenically in the presence of seed extract. The LPS of this strain completely lacked 2-O-methylfucose, regardless of whether anthocyanins were present during growth. The mutant gave only pseudonodules in association with P. vulgaris. Interpretation of this phenotype was complicated by a second LPS defect exhibited by the mutant: its LPS population had only about 50% of the normal amount of O-antigen-containing LPS (LPS I). The latter defect could be suppressed genetically such that the resulting strain (CE395α395) synthesized the normal amount of an LPS I that still lacked 2-O-methylfucose residues. Strain CE395α395 did not elicit pseudonodules but resulted in significantly slower nodule development, fewer nodules, and less nitrogenase activity than lps+ strains. The relative symbiotic deficiency was more severe when seeds were planted and inoculated with bacteria before they germinated. These results support previous conclusions that the relative amount of LPS I on the bacterial surface is crucial in symbiosis, but LPS structural features, such as 2-O-methylation of fucose, also may facilitate symbiotic interactions. PMID:15006776

  11. Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

    PubMed Central

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

    2009-01-01

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

  12. Mutant strains of Tetrahymena thermophila defective in thymidine kinase activity: Biochemical and genetic characterization

    SciTech Connect

    Cornish, K.V.; Pearlman, R.E.

    1982-08-01

    Three mutant strains, one conditional, of Tetrahymena thermophila were defective in thymidine phosphorylating activity in vivo and in thymidine kinase activity in vitro. Nucleoside phosphotransferase activity in mutant cell extracts approached wild-type levels, suggesting that thymidine kinase is responsible for most, if not all, thymidine phosphorylation in vivo. Thymidine kinase activity in extracts of the conditional mutant strain was deficient when the cells were grown or assayed or both at the permissive temperature, implying a structural enzyme defect. Analysis of the reaction products from in vitro assays with partially purified enzymes showed that phosphorylation by thymidine kinase and nucleoside phosphotransferase occurred at the 5' position. Genetic analyses showed that the mutant phenotype was recessive and that mutations in each of the three mutant strains did not complement, suggesting allelism.

  13. Starvation Induced Cell Death in Autophagy-Defective Yeast Mutants Is Caused by Mitochondria Dysfunction

    PubMed Central

    Suzuki, Sho W.; Onodera, Jun; Ohsumi, Yoshinori

    2011-01-01

    Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg) mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT) cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species) scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA). We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants. PMID:21364763

  14. Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction.

    PubMed

    Suzuki, Sho W; Onodera, Jun; Ohsumi, Yoshinori

    2011-02-25

    Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg) mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT) cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species) scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA). We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants.

  15. Mutants of Saccharomyces cerevisiae with defective vacuolar function

    SciTech Connect

    Kitamoto, K.; Yoshizawa, K.; Ohsumi, Y.; Anraku, Y.

    1988-06-01

    Mutants of the yeast Saccharomyces cerevisiae that have a small vacuolar lysine pool were isolated and characterized. Mutant KL97 (lys1 slp1-1) and strain KL197-1A (slp1-1), a prototrophic derivative of KL97, did not grow well in synthetic medium supplemented with 10 mM lysine. Genetic studies indicated that the slp1-1mutation (for small lysine pool) is recessive and is due to a single chromosomal mutation. Mutant KL97 shows the following pleiotropic defects in vacuolar functions. (i) It has small vacuolar pools for lysine, arginine, and histidine. (ii) Its growth is sensitive to lysine, histidine, Ca/sup 2 +/, heavy metal ions, and antibiotics. (iii) It has many small vesicles but no large central vacuole. (iv) It has a normal amount of the vacuolar membrane marker ..cap alpha..-mannosidase but shows reduced activities of the vacuole sap markers proteinase A, proteinase B, and carboxypeptidase Y.

  16. TBP mutants defective in activated transcription in vivo.

    PubMed Central

    Arndt, K M; Ricupero-Hovasse, S; Winston, F

    1995-01-01

    The TATA box binding protein (TBP) plays a central and essential role in transcription initiation. At TATA box-containing genes transcribed by RNA polymerase II, TBP binds to the promoter and initiates the assembly of a multiprotein preinitiation complex. Several studies have suggested that binding of TBP to the TATA box is an important regulatory step in transcription initiation in vitro. To determine whether TBP is a target of regulatory factors in vivo, we performed a genetic screen in yeast for TBP mutants defective in activated transcription. One class of TBP mutants identified in this screen comprises inositol auxotrophs that are also defective in using galactose as a carbon source. These phenotypes are due to promoter-specific defects in transcription initiation that are governed by the upstream activating sequence (UAS) and apparently not by the sequence of the TATA element. The finding that these TBP mutants are severely impaired in DNA binding in vitro suggests that transcription initiation at certain genes is regulated at the level of TATA box binding by TBP in vivo. Images PMID:7729424

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

    PubMed Central

    McCammon, M. T.

    1996-01-01

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

  18. Mutants of Saccharomyces cerevisiae with defects in acetate metabolism: isolation and characterization of Acn- mutants.

    PubMed

    McCammon, M T

    1996-09-01

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

  19. Temperature Sensitivity of Neural Tube Defects in Zoep Mutants.

    PubMed

    Ma, Phyo; Swartz, Morgan R; Kindt, Lexy M; Kangas, Ashley M; Liang, Jennifer Ostrom

    2015-12-01

    Neural tube defects (NTD) occur when the flat neural plate epithelium fails to fold into the neural tube, the precursor to the brain and spinal cord. Squint (Sqt/Ndr1), a Nodal ligand, and One-eyed pinhead (Oep), a component of the Nodal receptor, are required for anterior neural tube closure in zebrafish. The NTD in sqt and Zoep mutants are incompletely penetrant. The penetrance of several defects in sqt mutants increases upon heat or cold shock. In this project, undergraduate students tested whether temperature influences the Zoep open neural tube phenotype. Single pairs of adults were spawned at 28.5°C, the normal temperature for zebrafish, and one half of the resulting embryos were moved to 34°C at different developmental time points. Analysis of variance indicated temperature and clutch/genetic background significantly contributed to the penetrance of the open neural tube phenotype. Heat shock affected the embryos only at or before the midblastula stage. Many factors, including temperature changes in the mother, nutrition, and genetic background, contribute to NTD in humans. Thus, sqt and Zoep mutants may serve as valuable models for studying the interactions between genetics and the environment during neurulation.

  20. Isolation and phenotypic characterization of Lotus japonicus mutants specifically defective in arbuscular mycorrhizal formation.

    PubMed

    Kojima, Tomoko; Saito, Katsuharu; Oba, Hirosuke; Yoshida, Yuma; Terasawa, Junya; Umehara, Yosuke; Suganuma, Norio; Kawaguchi, Masayoshi; Ohtomo, Ryo

    2014-05-01

    Several symbiotic mutants of legume plants defective in nodulation have also been shown to be mutants related to arbuscular mycorrhizal (AM) symbiosis. The origin of the AM symbiosis can be traced back to the early land plants. It has therefore been postulated that the older system of AM symbiosis was partially incorporated into the newer system of legume-rhizobium symbiosis. To unravel the genetic basis of the establishment of AM symbiosis, we screened about 34,000 plants derived from ethyl methanesulfonate (EMS)-mutagenized Lotus japonicus seeds by microscopic observation. As a result, three lines (ME778, ME966 and ME2329) were isolated as AM-specific mutants that exhibit clear AM-defective phenotypes but form normal effective root nodules with rhizobial infection. In the ME2329 mutant, AM fungi spread their hyphae into the intercellular space of the cortex and formed trunk hyphae in the cortical cells, but the development of fine branches in the arbuscules was arrested. The ME2329 mutant carried a nonsense mutation in the STR-homolog gene, implying that the line may be an str mutant in L. japonicus. On the ME778 and ME966 mutant roots, the entry of AM fungal hyphae was blocked between two adjacent epidermal cells. Occasionally, hyphal colonization accompanied by arbuscules was observed in the two mutants. The genes responsible for the ME778 and ME966 mutants were independently located on chromosome 2. These results suggest that the ME778 and ME966 lines are symbiotic mutants involved in the early stage of AM formation in L. japonicus.

  1. Isolation and characterization of Pichia heedii mutants defective in xylose uptake

    SciTech Connect

    Does, A.L.; Bisson, L.F. )

    1990-11-01

    To investigate the role of xylose uptake in xylose metabolism in yeasts, we isolated a series of mutated strains of the yeast Pichia heedii which are defective in xylose utilization. Four of these demonstrated defects in xylose uptake. Overlaps between the functional or regulatory mechanisms for glucose and xylose uptake may exist in this yeast since some of the mutants defective in xylose uptake were also defective in glucose transport. None of the mutants were defective in xylose reductase or xylitol dehydrogenase activities.

  2. Auditory and vestibular defects in the circling (ci2) rat mutant.

    PubMed

    Kaiser, A; Fedrowitz, M; Ebert, U; Zimmermann, E; Hedrich, H J; Wedekind, D; Löscher, W

    2001-10-01

    The circling rat is an autosomal recessive mutant (homozygous ci2/ci2) that displays lateralized circling behaviour, locomotor hyperactivity, ataxia and stereotypic head-movement. These abnormal behaviours occur in phases or bursts either spontaneously or in response to stress. Heterozygous (ci2/+) littermates display normal spontaneous behaviours. We have previously found that ci2/ci2 rats of both genders have a lower tissue content of dopamine in the striatum ipsilateral to the preferred direction of rotation, indicating that the rats turn away from the brain hemisphere with higher striatal dopaminergic activity. In view of the similarities of the motor syndrome of the ci2/ci2 mutant rat to that of mouse deafness mutants, the present study evaluated the hearing ability of the circling rat mutant by recording brainstem auditory-evoked potentials. To test for vestibular dysfunction, a swimming test was conducted. Histological methods were used to examine the cochlear and vestibular parts of the inner ear and the cochlear and vestibular brainstem nuclei for defects. The absence of auditory-evoked potentials demonstrated a complete hearing loss in the adult ci2/ci2 mutant rat, whereas heterozygous littermates exhibited auditory-evoked potentials with thresholds resembling those of other laboratory strains. Furthermore, the mutant rats were unable to swim. Histological analysis of the inner ear of adult mutants revealed virtually complete loss of the cochlear neuroepithelium, while no such hair cell degeneration was seen in the vestibular parts of the inner ear. However, part of the vestibular hair cells showed protrusions into the endolymphatic space, suggesting alterations in the cytoskeletal architecture. The histological findings in mutant circling rats strongly indicate that the hearing loss of the mutants is of the sensory neural type, the most prevalent type of hearing loss. In the cochlear nuclei of the brain stem of mutant rats, neurons exhibited an abnormal

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

    PubMed Central

    Duchen, Michael R.

    2016-01-01

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

  4. Cuticular Defects in Oryza sativa ATP-binding Cassette Transporter G31 Mutant Plants Cause Dwarfism, Elevated Defense Responses and Pathogen Resistance.

    PubMed

    Garroum, Imène; Bidzinski, Przemyslaw; Daraspe, Jean; Mucciolo, Antonio; Humbel, Bruno M; Morel, Jean-Benoit; Nawrath, Christiane

    2016-06-01

    The cuticle covers the surface of the polysaccharide cell wall of leaf epidermal cells and forms an essential diffusion barrier between plant and environment. Homologs of the ATP-binding cassette (ABC) transporter AtABCG32/HvABCG31 clade are necessary for the formation of a functional cuticle in both monocots and dicots. Here we characterize the osabcg31 knockout mutant and hairpin RNA interference (RNAi)-down-regulated OsABCG31 plant lines having reduced plant growth and a permeable cuticle. The reduced content of cutin in leaves and structural alterations in the cuticle and at the cuticle-cell wall interface in plants compromised in OsABCG31 expression explain the cuticle permeability. Effects of modifications of the cuticle on plant-microbe interactions were evaluated. The cuticular alterations in OsABCG31-compromised plants did not cause deficiencies in germination of the spores or the formation of appressoria of Magnaporthe oryzae on the leaf surface, but a strong reduction of infection structures inside the plant. Genes involved in pathogen resistance were constitutively up-regulated in OsABCG31-compromised plants, thus being a possible cause of the resistance to M. oryzae and the dwarf growth phenotype. The findings show that in rice an abnormal cuticle formation may affect the signaling of plant growth and defense. PMID:27121976

  5. A rhizobium leguminosarum mutant defective in symbiotic iron acquisition

    SciTech Connect

    Nadler, K.D.; Chen, Jing-Wen; John, T.R. ); Johnston, A.W.B. )

    1990-02-01

    Iron acquisition by symbiotic Rhizobium spp. is essential for nitrogen fixation in the legume root nodule symbiosis. Rhizobium leguminosarum 116, an ineffective mutant strain with a defect in iron acquisition, was isolated after nitrosoguanidine mutagenesis of the effective strain 1062. The pop-1 mutation in strain 116 imparted to it a complex phenotype, characteristic of iron deficiency. Several iron(III)-solubilizing agents, such as citrate, hydroxyquinoline, and dihydroxybenzoate, stimulated growth of 116 on low-iron solid medium; anthranilic acid, the R. leguminosarum siderophore, inhibited low-iron growth of 116. The initial rate of {sup 55}Fe uptake by suspensions of iron-starved 116 cells was 10-fold less than that of iron-starved wild-type cells. Electron microscopic observations revealed no morphological abnormalities in the small, white nodules induced by 116. Nodule cortical cells were filled with vesicles containing apparently normal bacteroids. No premature degeneration of bacteroids or of plant cell organelles was evident. The authors mapped pop-1 by R plasmid-mediated conjugation and recombination to the ade-27-rib-2 region of the R. leguminosarum chromosome. No segregation of pop-1 and the symbiotic defect was observed among the recombinants from these crosses. Cosmid pKN1, a pLAFR1 derivative containing a 24-kilobase-pair fragment of R. leguminosarum DNA, conferred on 116 the ability to grow on dipyridyl medium and to fix nitrogen symbiotically.

  6. Co-occurence of filamentation defects and impaired biofilms in Candida albicans protein kinase mutants.

    PubMed

    Konstantinidou, Nina; Morrissey, John Patrick

    2015-12-01

    Pathogenicity of Candida albicans is linked with its developmental stages, notably the capacity switch from yeast-like to hyphal growth, and to form biofilms on surfaces. To better understand the cellular processes involved in C. albicans development, a collection of 63 C. albicans protein kinase mutants was screened for biofilm formation in a microtitre plate assay. Thirty-eight mutants displayed some degree of biofilm impairment, with 20 categorised as poor biofilm formers. All the poor biofilm formers were also defective in the switch from yeast to hyphae, establishing it as a primary defect. Five genes, VPS15, IME2, PKH3, PGA43 and CEX1, encode proteins not previously reported to influence hyphal development or biofilm formation. Network analysis established that individual components of some processes, most interestingly MAP kinase pathways, are not required for biofilm formation, most likely indicating functional redundancy. Mutants were also screened for their response to bacterial supernatants and it was found that Pseudomonas aeruginosa supernatants inhibited biofilm formation in all mutants, regardless of the presence of homoserine lactones (HSLs). In contrast, Candida morphology was only affected by supernatant containing HSLs. This confirms the distinct HSL-dependent inhibition of filamentation and the HSL-independent impairment of biofilm development by P. aeruginosa.

  7. Germination-defective mutant of Neurospora crassa that responds to siderophores

    NASA Technical Reports Server (NTRS)

    Charlang, G.; Williams, N. P.

    1977-01-01

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

  8. The SIR1 gene of Saccharomyces cerevisiae and its role as an extragenic suppressor of several mating-defective mutants.

    PubMed Central

    Stone, E M; Swanson, M J; Romeo, A M; Hicks, J B; Sternglanz, R

    1991-01-01

    The SIR1 gene product of Saccharomyces cerevisiae is one of several proteins involved in repressing transcription of the silent mating-type genes. Strains with mutations in the genes coding for these proteins are defective in mating due to derepression of the silent loci. We have found that overexpression of the SIR1 gene suppresses the mating defects of several of these mutants, including nat1 and ard1 mutants (the products of these two genes are responsible for N-terminal acetylation of a subset of yeast proteins), certain sir3 mutants, and a histone H4 mutant. The SIR1 gene has been sequenced and found to contain an open reading frame coding for a 678-amino-acid protein. Images PMID:2005909

  9. Genetic and biochemical evidence that gastrulation defects in Pofut2 mutants result from defects in ADAMTS9 secretion.

    PubMed

    Benz, Brian A; Nandadasa, Sumeda; Takeuchi, Megumi; Grady, Richard C; Takeuchi, Hideyuki; LoPilato, Rachel K; Kakuda, Shinako; Somerville, Robert P T; Apte, Suneel S; Haltiwanger, Robert S; Holdener, Bernadette C

    2016-08-01

    Protein O-fucosyltransferase 2 (POFUT2) adds O-linked fucose to Thrombospondin Type 1 Repeats (TSR) in 49 potential target proteins. Nearly half the POFUT2 targets belong to the A Disintegrin and Metalloprotease with ThromboSpondin type-1 motifs (ADAMTS) or ADAMTS-like family of proteins. Both the mouse Pofut2 RST434 gene trap allele and the Adamts9 knockout were reported to result in early embryonic lethality, suggesting that defects in Pofut2 mutant embryos could result from loss of O-fucosylation on ADAMTS9. To address this question, we compared the Pofut2 and Adamts9 knockout phenotypes and used Cre-mediated deletion of Pofut2 and Adamts9 to dissect the tissue-specific role of O-fucosylated ADAMTS9 during gastrulation. Disruption of Pofut2 using the knockout (LoxP) or gene trap (RST434) allele, as well as deletion of Adamts9, resulted in disorganized epithelia (epiblast, extraembryonic ectoderm, and visceral endoderm) and blocked mesoderm formation during gastrulation. The similarity between Pofut2 and Adamts9 mutants suggested that disruption of ADAMTS9 function could be responsible for the gastrulation defects observed in Pofut2 mutants. Consistent with this prediction, CRISPR/Cas9 knockout of POFUT2 in HEK293T cells blocked secretion of ADAMTS9. We determined that Adamts9 was dynamically expressed during mouse gastrulation by trophoblast giant cells, parietal endoderm, the most proximal visceral endoderm adjacent to the ectoplacental cone, extraembryonic mesoderm, and anterior primitive streak. Conditional deletion of either Pofut2 or Adamts9 in the epiblast rescues the gastrulation defects, and identified a new role for O-fucosylated ADAMTS9 during morphogenesis of the amnion and axial mesendoderm. Combined, these results suggested that loss of ADAMTS9 function in the extra embryonic tissue is responsible for gastrulation defects in the Pofut2 knockout. We hypothesize that loss of ADAMTS9 function in the most proximal visceral endoderm leads to slippage of

  10. Proton suicide: general method for direct selection of sugar transport- and fermentation-defective mutants

    SciTech Connect

    Winkelman, J.W.; Clark, D.P.

    1984-11-01

    A positive selection procedure was devised for bacterial mutants incapable of producing acid from sugars by fermentation. The method relied on the production of elemental bromine from a mixture of bromide and bromate under acidic conditions. When wild-type Escherichia coli cells were plated on media containing a fermentable sugar and an equimolar mixture of bromide and bromate, most of the cells were killed but a variety of mutants unable to produce acid from the sugar survived. Among these mutants were those defective in (i) sugar uptake, (ii) the glycolytic pathway, and (iii) the excretion. There were also novel mutants with some presumed regulatory defects affecting fermentation.

  11. Insights into prevention of human neural tube defects by folic acid arising from consideration of mouse mutants.

    PubMed

    Harris, Muriel J

    2009-04-01

    Almost 30 years after the initial study by Richard W. Smithells and coworkers, it is still unknown how maternal periconceptional folic acid supplementation prevents human neural tube defects (NTDs). In this article, questions about human NTD prevention are considered in relation to three groups of mouse models: NTD mutants that respond to folate, NTD mutants and strains that do not respond to folate, and mutants involving folate-pathway genes. Of the 200 mouse NTD mutants, only a few have been tested with folate; half respond and half do not. Among responsive mutants, folic acid supplementation reduces exencephaly and/or spina bifida aperta frequency in the Sp(2H), Sp, Cd, Cited2, Cart1, and Gcn5 mutants. Prevention ranges from 35 to 85%. The responsive Sp(2H) (Pax3) mutant has abnormal folate metabolism, but the responsive Cited2 mutant does not. Neither folic nor folinic acid reduces NTD frequency in Axd, Grhl3, Fkbp8, Map3k4, or Nog mutants or in the curly tail or SELH/Bc strains. Spina bifida frequency is reduced in Axd by methionine and in curly tail by inositol. Exencephaly frequency is reduced in SELH/Bc by an alternative commercial ration. Mutations in folate-pathway genes do not cause NTDs, except for 30% exencephaly in folate-treated Folr1. Among folate-pathway mutants, neural tube closure is normal in Cbs, Folr2, Mthfd1, Mthfd2, Mthfr, and Shmt1 mutants. Embryos die by midgestation in Folr1, Mtr, Mtrr, and RFC1 mutants. The mouse models point to genetic heterogeneity in the ability to respond to folic acid and also to heterogeneity in genetic cause of NTDs that can be prevented by folic acid.

  12. Profiling of the toxicity mechanisms of coated and uncoated silver nanoparticles to yeast Saccharomyces cerevisiae BY4741 using a set of its 9 single-gene deletion mutants defective in oxidative stress response, cell wall or membrane integrity and endocytosis.

    PubMed

    Käosaar, Sandra; Kahru, Anne; Mantecca, Paride; Kasemets, Kaja

    2016-09-01

    The widespread use of nanosilver in various antibacterial, antifungal, and antiviral products warrants the studies of the toxicity pathways of nanosilver-enabled materials toward microbes and viruses. We profiled the toxicity mechanisms of uncoated, casein-coated, and polyvinylpyrrolidone-coated silver nanoparticles (AgNPs) using Saccharomyces cerevisiae wild-type (wt) and its 9 single-gene deletion mutants defective in oxidative stress (OS) defense, cell wall/membrane integrity, and endocytosis. The 48-h growth inhibition assay in organic-rich growth medium and 24-h cell viability assay in deionized (DI) water were applied whereas AgNO3, H2O2, and SDS served as positive controls. Both coated AgNPs (primary size 8-12nm) were significantly more toxic than the uncoated (~85nm) AgNPs. All studied AgNPs were ~30 times more toxic if exposed to yeast cells in DI water than in the rich growth medium: the IC50 based on nominal concentration of AgNPs in the growth inhibition test ranged from 77 to 576mg Ag/L and in the cell viability test from 2.7 to 18.7mg Ag/L, respectively. Confocal microscopy showed that wt but not endocytosis mutant (end3Δ) internalized AgNPs. Comparison of toxicity patterns of wt and mutant strains defective in OS defense and membrane integrity revealed that the toxicity of the studied AgNPs to S. cerevisiae was not caused by the OS or cell wall/membrane permeabilization. PMID:27260961

  13. Detecting Functional Groups of Arabidopsis Mutants by Metabolic Profiling and Evaluation of Pleiotropic Responses

    PubMed Central

    Hofmann, Jörg; Börnke, Frederik; Schmiedl, Alfred; Kleine, Tatjana; Sonnewald, Uwe

    2011-01-01

    Metabolic profiles and fingerprints of Arabidopsis thaliana plants with various defects in plastidic sugar metabolism or photosynthesis were analyzed to elucidate if the genetic mutations can be traced by comparing their metabolic status. Using a platform of chromatographic and spectrometric tools data from untargeted full MS scans as well as from selected metabolites including major carbohydrates, phosphorylated intermediates, carboxylates, free amino acids, major antioxidants, and plastidic pigments were evaluated. Our key observations are that by multivariate statistical analysis each mutant can be separated by a unique metabolic signature. Closely related mutants come close. Thus metabolic profiles of sugar mutants are different but more similar than those of photosynthesis mutants. All mutants show pleiotropic responses mirrored in their metabolic status. These pleiotropic responses are typical and can be used for separating and grouping of the mutants. Our findings show that metabolite fingerprints can be taken to classify mutants and hence may be used to sort genes into functional groups. PMID:22639613

  14. Cancer predisposition in mutant mice defective in multiple genetic pathways: uncovering important genetic interactions.

    PubMed

    Meira, L B; Reis, A M; Cheo, D L; Nahari, D; Burns, D K; Friedberg, E C

    2001-06-01

    Mouse models that mimic the human skin cancer-prone disease xeroderma pigmentosum (XP) provide an useful experimental system with which to study the relationship between the DNA repair process of nucleotide excision repair (NER) and ultraviolet- (UV) induced skin carcinogenesis. We have generated Xpc mutant mice and documented their deficiency in the process of NER of UV-induced DNA damage. Xpc mutant mice are highly predisposed to UV-B radiation-induced skin cancer, both in the homozygous and the heterozygous state. The combination of Xpc and Trp53 mutations enhances this predisposition and alters the tumor spectrum observed in single mutant mice. These results suggest a synergism between NER and the function of Trp53 in suppression of cancer. We have examined the mutational spectrum in the Trp53 gene from skin cancers in Trp53+/+ and Trp53+/- mice of all three Xpc genotypes and have found evidence for signature mutations associated with defective NER. In addition, we have demonstrated that Xpc mutant mice are highly predisposed to the induction of lung and liver cancers by treatment with 2-acetylaminofluorene (2-AAF) and N-OH-2-AAF. By combining the Xpc mutation with other mutations in genes involved in repair of DNA damage we have identified additional genetic interactions important in carcinogenesis. The mouse Apex gene is a critical component of the base excision repair (BER) pathway as well as the redox regulation of transcription factors important in growth control and the cellular response to DNA damage. By combining mutations in Xpc, Trp53 and Apex we have obtained genetic evidence for a functional interaction between Apex and Trp53 which probably involves the activation of the Trp53 protein by Apex. Mutations in the mismatch repair (MMR) gene Msh2 also influence the carcinogenesis observed in Xpc Trp53 mutant mice. Our results demonstrate that multiple repair pathways operate in prevention of tumor formation. PMID:11376686

  15. Identification and Characterization of Aspergillus Nidulans Mutants Defective in Cytokinesis

    PubMed Central

    Harris, S. D.; Morrell, J. L.; Hamer, J. E.

    1994-01-01

    Filamentous fungi undergo cytokinesis by forming crosswalls termed septa. Here, we describe the genetic and physiological controls governing septation in Aspergillus nidulans. Germinating conidia do not form septa until the completion of their third nuclear division. The first septum is invariantly positioned at the basal end of the germ tube. Block-and-release experiments of nuclear division with benomyl or hydroxyurea, and analysis of various nuclear division mutants demonstrated that septum formation is dependent upon the third mitotic division. Block-and-release experiments with cytochalasin A and the localization of actin in germlings by indirect immunofluorescence showed that actin participated in septum formation. In addition to being concentrated at the growing hyphal tips, a band of actin was also apparent at the site of septum formation. Previous genetic analysis in A. nidulans identified four genes involved in septation (sepA-D). We have screened a new collection of temperature sensitive (ts) mutants of A. nidulans for strains that failed to form septa at the restrictive temperature but were able to complete early nuclear divisions. We identified five new genes designated sepE, G, H, I and J, along with one additional allele of a previously identified septation gene. On the basis of temperature shift experiments, nuclear counts and cell morphology, we sorted these cytokinesis mutants into three phenotypic classes. Interestingly, one class of mutants fails to form septa and fails to progress past the third nuclear division. This class of mutants suggests the existence of a regulatory mechanism in A. nidulans that ensures the continuation of nuclear division following the initiation of cytokinesis. PMID:8150280

  16. Lipopolysaccharide mutants of Rhizobium meliloti are not defective in symbiosis

    SciTech Connect

    Clover, R.H.; Kieber, J.; Signer, E.R. )

    1989-07-01

    Mutants of Rhizobium meliloti selected primarily for bacteriophage resistance fall into 13 groups. Mutants in the four best-characterized groups (class A, lpsB, lpsC, and class D), which map to the rhizobial chromosome, appear to affect lipopolysaccharide (LPS) as judged by the reactivity with monoclonal antibodies and behavior on sodium dodecyl sulfate-polyacrylamide gels of extracted LPS. Mutations in all 13 groups, in an otherwise wild-type genetic background, are Fix{sup +} on alfalfa. This suggests that LPS does not play a major role in symbiosis. Mutations in lpsB, however, are Fix{sup {minus}} in one particular genetic background, evidently because of the cumulative effect of several independent background mutations. In addition, an auxotrophic mutation evidently equivalent to Escherichia coli carAB is Fix{sup {minus}} on alfalfa.

  17. Molecular and biochemical characterization of xrs mutants defective in Ku80.

    PubMed Central

    Singleton, B K; Priestley, A; Steingrimsdottir, H; Gell, D; Blunt, T; Jackson, S P; Lehmann, A R; Jeggo, P A

    1997-01-01

    The gene product defective in radiosensitive CHO mutants belonging to ionizing radiation complementation group 5, which includes the extensively studied xrs mutants, has recently been identified as Ku80, a subunit of the Ku protein and a component of DNA-dependent protein kinase (DNA-PK). Several group 5 mutants, including xrs-5 and -6, lack double-stranded DNA end-binding and DNA-PK activities. In this study, we examined additional xrs mutants at the molecular and biochemical levels. All mutants examined have low or undetectable levels of Ku70 and Ku80 protein, end-binding, and DNA-PK activities. Only one mutant, xrs-6, has Ku80 transcript levels detectable by Northern hybridization, but Ku80 mRNA was detectable by reverse transcription-PCR in most other mutants. Two mutants, xrs-4 and -6, have altered Ku80 transcripts resulting from mutational changes in the genomic Ku80 sequence affecting RNA splicing, indicating that the defects in these mutants lie in the Ku80 gene rather than a gene controlling its expression. Neither of these two mutants has detectable wild-type Ku80 transcript. Since the mutation in both xrs-4 and xrs-6 cells results in severely truncated Ku80 protein, both are likely candidates to be null mutants. Azacytidine-induced revertants of xrs-4 and -6 carried both wild-type and mutant transcripts. The results with these revertants strongly support our model proposed earlier, that CHO-K1 cells carry a copy of the Ku80 gene (XRCC5) silenced by hypermethylation. Site-directed mutagenesis studies indicate that previously proposed ATP-binding and phosphorylation sites are not required for Ku80 activity, whereas N-terminal deletions of more than the first seven amino acids result in severe loss of activities. PMID:9032253

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

    PubMed

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

    2016-01-01

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

  19. Craniofacial skeletal defects of adult zebrafish glypican 4 (knypek) mutants

    PubMed Central

    LeClair, Elizabeth E.; Mui, Stephanie R.; Huang, Angela; Topczewska, Jolanta M.; Topczewski, Jacek

    2010-01-01

    The heparan sulfate proteoglycan Glypican 4 (Gpc4) is part of the Wnt/planar cell polarity pathway, which is required for convergence and extension during zebrafish gastrulation. To observe Glypican 4-deficient phenotypes at later stages, we rescued gpc4−/− (knypek) homozygotes and raised them for more than one year. Adult mutants showed diverse cranial malformations of both dermal and endochondral bones, ranging from shortening of the rostral-most skull to loss of the symplectic. Additionally, the adult palatoquadrate cartilage was disorganized, with abnormal chondrocyte orientation. To understand how the palatoquadrate cartilage normally develops, we examined a juvenile series of wild type and mutant specimens. This identified two novel domains of elongated chondrocytes in the larval palatoquadrate, which normally form prior to endochondral ossification. In contrast, gpc4−/− larvae never form these domains, suggesting a failure of chondrocyte orientation, though not differentiation. Our findings implicate Gpc4 in the regulation of zebrafish cartilage and bone morphogenesis. PMID:19777561

  20. Podospora mutant defective in glucose-dependent growth control.

    PubMed Central

    Durrens, P

    1983-01-01

    A mutation (modE), previously described as a membrane mutation, results in several modifications of the female developmental cycle: a high density of protoperithecia, the unscheduled development of protoperithecia into sterile perithecia on the homokaryons of each mating type, and the independence of ascospore outgrowth from the substances normally required for germination. Cultured in liquid medium, the modE strain showed two additional specific features: a higher growth yield than that of wild-type cultures (plus 10% of dry weight) and an extreme reduction of cell life span. Both mutant traits were specific to glucose limitation. Despite the large difference existing in the sensitivity of cells to glucose starvation, the glycogen and trehalose reserves of mutant and wild-type cells were nearly identical. Considered together, these results suggest that the primary effect of the mutation lies in the disruption of a glucose-dependent regulation controlling the transition of the metabolic pattern of cells from growth to quiescence. PMID:6841314

  1. A novel histone H4 mutant defective in nuclear division and mitotic chromosome transmission.

    PubMed Central

    Smith, M M; Yang, P; Santisteban, M S; Boone, P W; Goldstein, A T; Megee, P C

    1996-01-01

    The histone proteins are essential for the assembly and function of th e eukaryotic chromosome. Here we report the first isolation of a temperature-sensitive lethal histone H4 mutant defective in mitotic chromosome transmission Saccharomyces cerevisiae. The mutant requires two amino acid substitutions in histone H4: a lethal Thr-to-Ile change at position 82, which lies within one of the DNA-binding surfaces of the protein, and a substitution of Ala to Val at position 89 that is an intragenic suppressor. Genetic and biochemical evidence shows that the mutant histone H4 is temperature sensitive for function but not for synthesis, deposition, or stability. The chromatin structure of 2 micrometer circle minichromosomes is temperature sensitive in vivo, consistent with a defect in H4-DNA interactions. The mutant also has defects in transcription, displaying weak Spt- phenotypes. At the restrictive temperature, mutant cells arrest in the cell cycle at nuclear division, with a large bud, a single nucleus with 2C DNA content, and a short bipolar spindle. At semipermissive temperatures, the frequency of chromosome loss is elevated 60-fold in the mutant while DNA recombination frequencies are unaffected. High-copy CSE4, encoding an H3 variant related to the mammalian CENP-A kinetochore antigen, was found to suppress the temperature sensitivity of the mutant without suppressing the Spt- transcription defect. These genetic, biochemical, and phenotypic results indicate that this novel histone H4 mutant defines one or more chromatin-dependent steps in chromosome segregation. PMID:8622646

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

    PubMed Central

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

    2005-01-01

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

  3. Lanthionine ketimine ethyl ester partially rescues neurodevelopmental defects in unc-33 (DPYSL2/CRMP2) mutants.

    PubMed

    Hubbard, Caleb; Benda, Erica; Hardin, Tyler; Baxter, Taylor; St John, Elizabeth; O'Brien, Sean; Hensley, Kenneth; Holgado, Andrea M

    2013-09-01

    Lanthionine ketimine (LK) is a natural sulfur amino acid metabolite with potent neurotrophic activity. Proteomics indicate that LK interacts with collapsin response mediator protein-2 (CRMP2/DPYSL2/UNC-33), a brain-enriched protein that was shown to regulate cytoskeletal remodeling, neuronal morphology, and synaptic function. To elucidate further the molecular interplay and biological action of LK and UNC-33, we began examining the nervous system of Caenorhabditis elegans nematodes in which both LK concentrations and UNC-33 protein were manipulated. To this end, a cell-permeable LK-ester (LKE) was administered to developing C. elegans engineered to express yellow fluorescent protein (YFP) in cholinergic neurons (strain RM3128) or green fluorescent protein (GFP) in GABAergic neurons (strain CZ1200), and neural morphology was assessed. Fluorescent imaging analyses show that LKE exposure to wild-type animals induced neural commissure outgrowth, crossing over, and bundling in both neurites from GABAergic and cholinergic motor neurons. Additionally, when unc-33(e204) hypomorph mutant nematodes (D389N substitution mutants) were exposed to LKE, both the neuroanatomical defects of incomplete dorsoventral neural commissures and the ventral nerve cord gaps were partially rescued. In contrast, LKE did not rescue ventral nerve cord gaps found in unc-33(mn407) null mutant. Together these data suggest possible functions for LK as a regulator of neuritic elongation, corroborate roles for UNC-33/CRMP2 in the mechanism of LKE activity, and suggest the potential of LKE as a therapeutic molecule for neurological diseases involving CRMP2 dysfunction. PMID:23825043

  4. Drosophila calmodulin mutants with specific defects in the musculature or in the nervous system.

    PubMed Central

    Wang, Bo; Sullivan, Kathleen M C; Beckingham, Kathy

    2003-01-01

    We have studied lethal mutations in the single calmodulin gene (Cam) of Drosophila to gain insight into the in vivo functions of this important calcium sensor. As a result of maternal calmodulin (CaM) in the mature egg, lethality is delayed until the postembryonic stages. Prior to death in the first larval instar, Cam nulls show a striking behavioral abnormality (spontaneous backward movement) whereas a mutation, Cam7, that results in a single amino acid change (V91G) produces a very different phenotype: short indented pupal cases and pupal death with head eversion defects. We show here that the null behavioral phenotype originates in the nervous system and involves a CaM function that requires calcium binding to all four sites of the protein. Further, backward movement can be induced in hypomorphic mutants by exposure to high light levels. In contrast, the V91G mutation specifically affects the musculature and causes abnormal calcium release in response to depolarization of the muscles. Genetic interaction studies suggest that failed regulation of the muscle calcium release channel, the ryanodine receptor, is the major defect underlying the Cam7 phenotype. PMID:14668380

  5. Twin-arginine translocation system (tat) mutants of Salmonella are attenuated due to envelope defects, not respiratory defects.

    PubMed

    Craig, Maureen; Sadik, Adam Y; Golubeva, Yekaterina A; Tidhar, Avital; Slauch, James M

    2013-09-01

    The twin-arginine translocation system (Tat) transports folded proteins across the cytoplasmic membrane and is critical to virulence in Salmonella and other pathogens. Experimental and bioinformatic data indicate that 30 proteins are exported via Tat in Salmonella Typhimurium. However, there are no data linking specific Tat substrates with virulence. We inactivated every Tat-exported protein and determined the virulence phenotype of mutant strains. Although a tat mutant is highly attenuated, no single Tat-exported substrate accounts for this virulence phenotype. Rather, the attenuation is due primarily to envelope defects caused by failure to translocate three Tat substrates, the N-acetylmuramoyl-l-alanine amidases, AmiA and AmiC, and the cell division protein, SufI. Strikingly, neither the amiA amiC nor the sufI mutations alone conferred any virulence defect. Although AmiC and SufI have previously been localized to the divisome, the synthetic phenotypes observed are the first to suggest functional overlap. Many Tat substrates are involved in anaerobic respiration, but we show that a mutant completely deficient in anaerobic respiration retains full virulence in both the oral and systemic phases of infection. Similarly, an obligately aerobic mutant is fully virulent. These results suggest that in the classic mouse model of infection, S. Typhimurium is replicating only in aerobic environments.

  6. Twin-arginine Translocation System (tat) Mutants of Salmonella are Attenuated Due to Envelope Defects, not Respiratory Defects

    PubMed Central

    Craig, Maureen; Sadik, Adam Y.; Golubeva, Yekaterina A.; Tidhar, Avital; Slauch, James M.

    2013-01-01

    Summary The twin-arginine translocation system (Tat) transports folded proteins across the cytoplasmic membrane and is critical to virulence in Salmonella and other pathogens. Experimental and bioinformatic data indicate that 30 proteins are exported via Tat in Salmonella Typhimurium. However, there are no data linking specific Tat substrates with virulence. We inactivated every Tat-exported protein and determined the virulence phenotype of mutant strains. Though a tat mutant is highly attenuated, no single Tat-exported substrate accounts for this virulence phenotype. Rather, the attenuation is due primarily to envelope defects caused by failure to translocate three Tat substrates, the N-acetylmuramoyl-L-alanine amidases, AmiA and AmiC, and the cell division protein, SufI. Strikingly, neither the amiA amiC nor the sufI mutations alone conferred any virulence defect. Although AmiC and SufI have previously been localized to the divisome, the synthetic phenotypes observed are the first to suggest functional overlap. Many Tat substrates are involved in anaerobic respiration, but we show that a mutant completely deficient in anaerobic respiration retains full virulence in both the oral and systemic phases of infection. Similarly, an obligately aerobic mutant is fully virulent. These results suggest that in the classic mouse model of infection, S. Typhimurium is replicating only in aerobic environments. PMID:23822642

  7. Molecular basis for the spontaneous generation of colonization-defective mutants of Streptococcus mutans.

    PubMed

    Ueda, S; Kuramitsu, H K

    1988-01-01

    Spontaneous mutants of Streptococcus mutans GS-5 defective in sucrose-dependent colonization of smooth surfaces are generated at frequencies above the spontaneous mutation rate. Southern blot analysis of such mutants suggested rearrangement of the genes coding for glucosyltransferase (GTF) activity. Two strain GS-5 homologous tandem genes, gtfB and gtfC, coding for GTF-I and GTF-S activities respectively, were demonstrated to undergo recombination when introduced into recombination-proficient Escherichia coli transformants. However, the two genes were quite stable when transformed on a single DNA fragment into a recA mutant of E. coli. The DNA fragment coding for GTF activity from one S. mutans colonization-defective mutant, SP2, was isolated and shown also to have undergone recombination between the gtfB and gtfC genes, resulting in reduced GTF activity. These results are discussed relative to the in vivo generation of colonization-defective mutants in cultures of S. mutans.

  8. Mutant Chinese hamster ovary cells pleiotropically defective in receptor-mediated endocytosis

    PubMed Central

    1983-01-01

    Populations of Chinese hamster ovary cells selected for resistance to diphtheria toxin were found to be highly enriched for mutants deficient in the uptake of lysosomal hydrolases via the mannose 6-phosphate receptor. One doubly defective mutant, DTF 1-5-1, exhibited increased resistance to Sindbis virus, although it was able to bind and internalize virus normally. Normal production of virus was obtained when, subsequent to virus binding, the mutant was exposed for 2 min to acidic pH. Similarly, a shift to acidic pH increased the sensitivity of DTF 1-5-1 to diphtheria toxin 12-fold. Decreased uptake of lysosomal hydrolases by the mutant correlated with decreased mannose 6-phosphate receptor activity at the cell surface; results of lactoperoxidase- catalyzed iodination indicated that the surface-associated receptor was present but inactive on DTF 1-5-1. Total mannose 6-phosphate receptor activity was also decreased in the mutant and this decrease was reflected by increased secretion of lysosomal hydrolases. The phenotype of DTF 1-5-1 resembles in many ways that of cells treated with ammonia. We suggest that the defect in DTF 1-5-1 stems from an inability to deliver virus, diphtheria toxin, and lysosomal hydrolases to an acidic compartment. Other ligands may be endocytosed through a different pathway since the defect of DTF 1-5-1 did not decrease the endocytosis of ricin, modeccin, or Pseudomonas toxin and had minimal effects on uptake and degradation of low density lipoprotein. PMID:6300143

  9. Identification and characterization of biofilm formation-defective mutants of Xanthomonas citri subsp. citri.

    PubMed

    Malamud, Florencia; Homem, Rafael Augusto; Conforte, Valeria Paola; Yaryura, Pablo Marcelo; Castagnaro, Atilio Pedro; Marano, María Rosa; do Amaral, Alexandre Morais; Vojnov, Adrián Alberto

    2013-09-01

    Xanthomonas citri subsp. citri (Xcc) develops a biofilm structure both in vitro and in vivo. Despite all the progress achieved by studies regarding biofilm formation, many of its mechanisms remain poorly understood. This work focuses on the identification of new genes involved in biofilm formation and how they are related to motility, virulence and chemotaxis in Xcc. A Tn5 library of approximately 6000 Xcc (strain 306) mutants was generated and screened to search for biofilm formation defective strains. We identified 23 genes not previously associated with biofilm formation. The analysis of the 23 mutants not only revealed the involvement of new genes in biofilm formation, but also reinforced the importance of exopolysaccharide production, motility and cell surface structures in this process. This collection of biofilm-defective mutants underscores the multifactorial genetic programme underlying the establishment of biofilm in Xcc. PMID:23813675

  10. Identification and characterization of biofilm formation-defective mutants of Xanthomonas citri subsp. citri.

    PubMed

    Malamud, Florencia; Homem, Rafael Augusto; Conforte, Valeria Paola; Yaryura, Pablo Marcelo; Castagnaro, Atilio Pedro; Marano, María Rosa; do Amaral, Alexandre Morais; Vojnov, Adrián Alberto

    2013-09-01

    Xanthomonas citri subsp. citri (Xcc) develops a biofilm structure both in vitro and in vivo. Despite all the progress achieved by studies regarding biofilm formation, many of its mechanisms remain poorly understood. This work focuses on the identification of new genes involved in biofilm formation and how they are related to motility, virulence and chemotaxis in Xcc. A Tn5 library of approximately 6000 Xcc (strain 306) mutants was generated and screened to search for biofilm formation defective strains. We identified 23 genes not previously associated with biofilm formation. The analysis of the 23 mutants not only revealed the involvement of new genes in biofilm formation, but also reinforced the importance of exopolysaccharide production, motility and cell surface structures in this process. This collection of biofilm-defective mutants underscores the multifactorial genetic programme underlying the establishment of biofilm in Xcc.

  11. Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas

    PubMed Central

    Tam, Lai-Wa; Dentler, William L.; Lefebvre, Paul A.

    2003-01-01

    Four long-flagella (LF) genes are important for flagellar length control in Chlamydomonas reinhardtii. Here, we characterize two new null lf3 mutants whose phenotypes are different from previously identified lf3 mutants. These null mutants have unequal-length flagella that assemble more slowly than wild-type flagella, though their flagella can also reach abnormally long lengths. Prominent bulges are found at the distal ends of short, long, and regenerating flagella of these mutants. Analysis of the flagella by electron and immunofluorescence microscopy and by Western blots revealed that the bulges contain intraflagellar transport complexes, a defect reported previously (for review see Cole, D.G., 2003. Traffic. 4:435–442) in a subset of mutants defective in intraflagellar transport. We have cloned the wild-type LF3 gene and characterized a hypomorphic mutant allele of LF3. LF3p is a novel protein located predominantly in the cell body. It cosediments with the product of the LF1 gene in sucrose density gradients, indicating that these proteins may form a functional complex to regulate flagellar length and assembly. PMID:14610061

  12. Exogenous Boron supplementation partially rescues fertilization defect of osbor4 mutant.

    PubMed

    Tanaka, Nobuhiro; Uraguchi, Shimpei; Fujiwara, Toru

    2014-01-01

    Arabidopsis thaliana BOR1 is the first boron (B) transporter identified in the living systems. In the rice genome, there are four AtBOR1-like genes, OsBOR1, 2, 3 and 4. We have previously demonstrated that OsBOR4 is a B efflux transporter gene specifically expressed in rice pollen. OsBOR4 heterozygous lines showed abnormal segregation ratio, suggesting the significance of OsBOR4 in rice pollen tube germination/elongation process. To obtain further insights into the mechanisms underlying fertilization defects by osbor4 mutations, we examined if the mutant pollen exhibits morphological changes. The cross section of the pollen of the mutant was similar to those of the wild type. We also determined B concentrations in brown rice of three osbor4 mutants and found that B levels were comparable. These results suggest that osbor4 mutation does not affect B transport to pollen and seeds. We then examined if exogenous B supplementation can rescue segregation defect of osbor4. As reported previously, a OsBOR4 heterozygous lines showed abnormal segregation rate under the normal growth condition in this present study, too. Importantly, this abnormality in segregation was partially rescued by application of six-times higher B concentration to roots, providing further evidence that the fertilization defect of osbor4 is due to the defect in B transport process. Taken together we propose that osbor4 causes defect in B transport process during pollen germination to fertilization.

  13. Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20.

    PubMed Central

    Priestley, A; Beamish, H J; Gell, D; Amatucci, A G; Muhlmann-Diaz, M C; Singleton, B K; Smith, G C; Blunt, T; Schalkwyk, L C; Bedford, J S; Jackson, S P; Jeggo, P A; Taccioli, G E

    1998-01-01

    The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is a member of a sub-family of phosphatidylinositol (PI) 3-kinases termed PIK-related kinases. A distinguishing feature of this sub-family is the presence of a conserved C-terminal region downstream of a PI 3-kinase domain. Mutants defective in DNA-PKcs are sensitive to ionising radiation and are unable to carry out V(D)J recombination. Irs-20 is a DNA-PKcs-defective cell line with milder gamma-ray sensitivity than two previously characterised mutants, V-3 and mouse scid cells. Here we show that the DNA-PKcs protein from irs-20 cells can bind to DNA but is unable to function as a protein kinase. To verify the defect in irs-20 cells and provide insight into the function and expression of DNA-PKcs in double-strand break repair and V(D)J recombination we introduced YACs encoding human and mouse DNA-PKcs into defective mutants and achieved complementation of the defective phenotypes. Furthermore, in irs-20 we identified a mutation in DNA-PKcs that causes substitution of a lysine for a glutamic acid in the fourth residue from the C-terminus. This represents a strong candidate for the inactivating mutation and provides supportive evidence that the extreme C-terminal motif is important for protein kinase activity. PMID:9518490

  14. How deeply does your mutant sleep? Probing arousal to better understand sleep defects in Drosophila

    PubMed Central

    Faville, R.; Kottler, B.; Goodhill, G. J.; Shaw, P. J.; van Swinderen, B.

    2015-01-01

    The fruitfly, Drosophila melanogaster, has become a critical model system for investigating sleep functions. Most studies use duration of inactivity to measure sleep. However, a defining criterion for sleep is decreased behavioral responsiveness to stimuli. Here we introduce the Drosophila ARousal Tracking system (DART), an integrated platform for efficiently tracking and probing arousal levels in animals. This video-based platform delivers positional and locomotion data, behavioral responsiveness to stimuli, sleep intensity measures, and homeostatic regulation effects – all in one combined system. We show how insight into dynamically changing arousal thresholds is crucial for any sleep study in flies. We first find that arousal probing uncovers different sleep intensity profiles among related genetic background strains previously assumed to have equivalent sleep patterns. We then show how sleep duration and sleep intensity can be uncoupled, with distinct manipulations of dopamine function producing opposite effects on sleep duration but similar sleep intensity defects. We conclude by providing a multi-dimensional assessment of combined arousal and locomotion metrics in the mutant and background strains. Our approach opens the door for deeper insights into mechanisms of sleep regulation and provides a new method for investigating the role of different genetic manipulations in controlling sleep and arousal. PMID:25677943

  15. Genetic analysis of endocytosis in Caenorhabditis elegans: coelomocyte uptake defective mutants.

    PubMed Central

    Fares, H; Greenwald, I

    2001-01-01

    The coelomocytes of Caenorhabditis elegans are scavenger cells that continuously and nonspecifically endocytose fluid from the pseudocoelom (body cavity). Green fluorescent protein (GFP) secreted into the pseudocoelom from body wall muscle cells is endocytosed and degraded by coelomocytes. We show that toxin-mediated ablation of coelomocytes results in viable animals that fail to endocytose pseudocoelomic GFP, indicating that endocytosis by coelomocytes is not essential for growth or survival of C. elegans under normal laboratory conditions. We examined known viable endocytosis mutants, and performed RNAi for other known endocytosis genes, for coelomocyte uptake defective (Cup) phenotypes. We also screened for new genes involved in endocytosis by isolating viable mutants with Cup defects; this screen identified 14 different genes, many with multiple alleles. A variety of Cup terminal phenotypes were observed, consistent with defects at various steps in the endocytic pathway. Available molecular information indicates that the Cup mutant screen has identified novel components of the endocytosis machinery that are conserved in mammals but not in Saccharomyces cerevisiae, the only other organism for which large-scale genetic screens for endocytosis mutants have been performed. PMID:11560892

  16. Characterization of zebrafish mutants with defects in bone calcification during development.

    PubMed

    Xi, Yang; Chen, Dongyan; Sun, Lei; Li, Yuhao; Li, Lei

    2013-10-11

    Using the fluorescent dyes calcein and alcian blue, we stained the F3 generation of chemically (ENU) mutagenized zebrafish embryos and larvae, and screened for mutants with defects in bone development. We identified a mutant line, bone calcification slow (bcs), which showed delayed axial vertebra calcification during development. Before 4-5 days post-fertilization (dpf), the bcs embryos did not display obvious abnormalities in bone development (i.e., normal number, size and shape of cartilage and vertebrae). At 5-6 dpf, when vertebrae calcification starts, bcs embryos began to show defects. At 7 dpf, for example, in most of the bcs embryos examined, calcein staining revealed no signals of vertebrae mineralization, whereas during the same developmental stages, 2-14 mineralized vertebrae were observed in wild-type animals. Decreases in the number of calcified vertebrae were also observed in bcs mutants when examined at 9 and 11 dpf, respectively. Interestingly, by 13 dpf the defects in bcs mutants were no longer evident. There were no significant differences in the number of calcified vertebrae between wild-type and mutant animals. We examined the expression of bone development marker genes (e.g., Sox9b, Bmp2b, and Cyp26b1, which play important roles in bone formation and calcification). In mutant fish, we observed slight increases in Sox9b expression, no alterations in Bmp2b expression, but significant increases in Cyp26b1 expression. Together, the data suggest that bcs delays axial skeletal calcification, but does not affect bone formation and maturation.

  17. Aberrant Fat Metabolism in Caenorhabditis elegans Mutants with Defects in the Defecation Motor Program

    PubMed Central

    Sheng, Ming; Gaur, Rahul; Selstam, Eva; Tuck, Simon

    2015-01-01

    The molecular mechanisms by which dietary fatty acids are absorbed by the intestine, and the way in which the process is regulated are poorly understood. In a genetic screen for mutations affecting fat accumulation in the intestine of Caenorhabditis elegans, nematode worms, we have isolated mutations in the aex-5 gene, which encodes a Kex2/subtilisin-family, Ca2+-sensitive proprotein convertase known to be required for maturation of certain neuropeptides, and for a discrete step in an ultradian rhythmic phenomenon called the defecation motor program. We demonstrate that aex-5 mutants have markedly lower steady-state levels of fat in the intestine, and that this defect is associated with a significant reduction in the rate at which labeled fatty acid derivatives are taken up from the intestinal lumen. Other mutations affecting the defecation motor program also affect steady-state levels of triglycerides, suggesting that the program is required per se for the proper accumulation of neutral lipids. Our results suggest that an important function of the defecation motor program in C. elegans is to promote the uptake of an important class of dietary nutrients. They also imply that modulation of the program might be one way in which worms adjust nutrient uptake in response to altered metabolic status. PMID:25849533

  18. Isolation and characterization of Escherichia coli mutants defective for phenylpropionate degradation.

    PubMed Central

    Burlingame, R P; Wyman, L; Chapman, P J

    1986-01-01

    Mutants of Escherichia coli defective in catabolism of 3-phenylpropionate, 3-(3-hydroxyphenyl)propionate, or both were isolated after mutagenesis with ethylmethane sulfonate. Nine phenotypically distinct classes of mutants were identified, including strains lacking each of the first five enzyme activities for the degradation of these compounds and mutants pleiotropically negative for some of these activities. Characterization of these mutants was greatly facilitated by the use of indicator media in which accumulation of 3-(2,3-dihydroxyphenyl)propionate or 2-hydroxy-6-ketononadienedioic acid led to the formation of dark red or bright yellow colors, respectively, in the medium. Assays with wild-type and mutant strains indicated that 3-phenylpropionate (or its dihydrodiol), but none of the hydroxylated derivatives tested, induced the synthesis of enzymes for its conversion to 3-(2,3-dihydroxyphenyl)propionate. The remaining enzymes were induced by the 2- or 3-hydroxy or 2,3-dihydroxy derivatives of 3-phenylpropionate, with the 2-hydroxy compound acting as an apparent gratuitous inducer. Metabolism to nonaromatic intermediates appeared to be unnecessary for full induction of any pathway enzyme. One unusual class of mutants, in which 2-keto-4-pentenoate hydratase appeared to be uninducible, indicated a level of control not previously shown in meta-fission catabolic pathways. PMID:3531186

  19. Isolation and characterization of Arabidopsis mutants defective in the induction of ethylene biosynthesis by cytokinin

    NASA Technical Reports Server (NTRS)

    Vogel, J. P.; Schuerman, P.; Woeste, K.; Brandstatter, I.; Kieber, J. J.; Evans, M. L. (Principal Investigator)

    1998-01-01

    Cytokinins elevate ethylene biosynthesis in etiolated Arabidopsis seedlings via a post-transcriptional modification of one isoform of the key biosynthetic enzyme ACC synthase. In order to begin to dissect the signaling events leading from cytokinin perception to this modification, we have isolated a series of mutants that lack the ethylene-mediated triple response in the presence of cytokinin due to their failure to increase ethylene biosynthesis. Analysis of genetic complementation and mapping revealed that these Cin mutants (cytokinin-insensitive) represent four distinct complementation groups, one of which, cin4, is allelic to the constitutive photomorphogenic mutant fus9/cop10. The Cin mutants have subtle effects on the morphology of adult plants. We further characterized the Cin mutants by analyzing ethylene biosynthesis in response to various other inducers and in adult tissues, as well as by assaying additional cytokinin responses. The cin3 mutant did not disrupt ethylene biosynthesis under any other conditions, nor did it disrupt any other cytokinin responses. Only cin2 disrupted ethylene biosynthesis in multiple circumstances. cin1 and cin2 made less anthocyanin in response to cytokinin. cin1 also displayed reduced shoot initiation in tissue culture in response to cytokinin, suggesting that it affects a cytokinin signaling element.

  20. Cartilage Abnormalities Associated with Defects of Chondrocytic Primary Cilia in Bardet-Biedl Syndrome Mutant Mice

    PubMed Central

    Kaushik, Anjan P.; Martin, James A.; Zhang, Qihong; Sheffield, Val C.; Morcuende, Jose A.

    2013-01-01

    SUMMARY Primary cilia are found on nearly every mammalian cell, including osteocytes, fibroblasts, and chondrocytes. However, the functions of primary cilia have not been extensively studied in these cells, particularly chondrocytes. Interestingly, defects in the primary cilium result in skeletal defects such as polydactyly in Bardet-Biedl Syndrome (BBS), a ciliary disorder that also results in obesity, retinopathy, and cognitive impairments (1–4). Wild-type mice and mutant mice of the ciliary proteins Bbs1, Bbs2, and Bbs6 were evaluated with respect to histological and biochemical differences in chondrocytes from articular cartilage and xiphoid processes. Using immunofluorescence microscopy, chondrocytic cilia were visualized from the load-bearing joints and non-load-bearing xiphoid processes. Significant differences in ciliary morphology were not identified between mutant and wild-type mice. However, after expanding chondrocytes in cell culture and implanting them in solid agarose matrix, it was seen that the fraction of ciliated cells in cultures from mutant mice was significantly lower than in the wild-type cultures (p<.05). In addition, in Safranin-O-stained whole joint sections, Bbs mutant mice had significantly lower articular joint thickness (p<.05) and lower proteoglycan content saturation (p<.05) than wild-type mice. Moreover, there were statistically significant differences of cell distribution between Bbs mutant and wild-type mice (p<.05), indicating that mutant articular cartilage had changes consistent with early signs of osteoarthritis. These data indicate that Bbs genes and their functions in the chondrocytic primary cilium are important for normal articular cartilage maintenance. PMID:19195025

  1. Expression of chicken vinculin complements the adhesion-defective phenotype of a mutant mouse F9 embryonal carcinoma cell

    PubMed Central

    1993-01-01

    A mutant cell line, derived from the mouse embryonal carcinoma cell line F9, is defective in cell-cell adhesion (compaction) and in cell- substrate adhesion. We have previously shown that neither uvomorulin (E- cadherin) nor integrins are responsible for the mutant phenotype (Calogero, A., M. Samuels, T. Darland, S. A. Edwards, R. Kemler, and E. D. Adamson. 1991. Dev. Biol. 146:499-508). Several cytoskeleton proteins were assayed and only vinculin was found to be absent in mutant (5.51) cells. A chicken vinculin expression vector was transfected into the 5.51 cells together with a neomycin-resistance vector. Clones that were adherent to the substrate were selected in medium containing G418. Two clones, 5.51Vin3 and Vin4, were analyzed by Nomarski differential interference contrast and laser confocal microscopy as well as by biochemical and molecular biological techniques. Both clones adhered well to substrates and both exhibited F- actin stress fibers with vinculin localized at stress fiber tips in focal contacts. This was in marked contrast to 5.51 parental cells, which had no stress fibers and no vinculin. The mutant and complemented F9 cell lines will be useful models for examining the complex interactions between cytoskeletal and cell adhesion proteins. PMID:8491782

  2. Incomplete Splicing, Cell Division Defects and Hematopoietic Blockage in dhx8 Mutant Zebrafish

    PubMed Central

    English, Milton A.; Lei, Lin; Blake, Trevor; Wincovitch, Stephen M.; Sood, Raman; Azuma, Mizuki; Hickstein, Dennis; Liu, P. Paul

    2012-01-01

    Vertebrate hematopoiesis is a complex developmental process that is controlled by genes in diverse pathways. To identify novel genes involved in early hematopoiesis, we conducted an ENU (N-ethyl-N-nitrosourea) mutagenesis screen in zebrafish. The mummy (mmy) line was investigated because of its multiple hematopoietic defects. Homozygous mmy embryos lacked circulating blood cells types and were dead by 30 hours post-fertilization (hpf). The mmy mutants did not express myeloid markers and had significantly decreased expression of progenitor and erythroid markers in primitive hematopoiesis. Through positional cloning, we identified a truncation mutation in dhx8 in the mmy fish. dhx8 is the zebrafish ortholog of the yeast splicing factor prp22, which is a DEAH-box RNA helicase. Mmy mutants had splicing defects in many genes, including several hematopoietic genes. Mmy embryos also showed cell division defects as characterized by disorganized mitotic spindles and formation of multiple spindle poles in mitotic cells. These cell division defects were confirmed by DHX8 knockdown in HeLa cells. Together, our results confirm that dhx8 is involved in mRNA splicing and suggest that it is also important for cell division during mitosis. This is the first vertebrate model for dhx8, whose function is essential for primitive hematopoiesis in developing embryos. PMID:22411201

  3. Temperature-sensitive mutants of fowl plague virus defective in the intracellular transport of the hemagglutinin.

    PubMed

    Naruse, H; Scholtissek, C; Klenk, H D

    1986-08-01

    Nine mutants of fowl plague virus with temperature-sensitive defects in the biosynthesis of the hemagglutinin have been characterized by analyzing the processing and the intracellular location of this glycoprotein in MDCK and chick embryo cells. It was found that with all of these mutants the transport of the hemagglutinin to the cell surface was impeded at the non-permissive temperature. There were differences, however, in the site of the block. With mutants tsl, ts227, ts478 and ts658 the precursor HA was not cleaved and the oligosaccharide side chains remained sensitive to endoglucosaminidase H. When the hemagglutinin was analyzed in permeabilized cells by immunofluorescence, usually only cytoplasmic labeling was seen. Immunofluorescence of non-permeabilized cells and hemadsorption revealed that the hemagglutinin did not reach the cell surface. In contrast, the hemagglutinin of mutants ts79, ts482, ts532, ts546 and ts651 was cleaved and oligosaccharides were processed to the endoglucosaminidase H-resistant form at non-permissive temperature. In permeabilized cells, the cytoplasm and juxtanuclear regions typical for the Golgi apparatus were labeled by immunofluorescence. Except for ts482, ts532 and ts546 which were leaky, hemagglutinin could not be detected at the cell surface. These observations indicate that, with the first group of mutants, hemagglutinin transport is usually arrested already in the rough endoplasmic reticulum, whereas with the second group it is inhibited at a late stage between the Golgi apparatus and the plasma membrane. PMID:3765827

  4. Yeast mutants of glucose metabolism with defects in the coordinate regulation of carbon assimilation.

    PubMed

    Dennis, R A; Rhodey, M; McCammon, M T

    1999-05-15

    The enzymes of the glyoxylate cycle and gluconeogenesis are tightly regulated by transcriptional, posttranscriptional, and posttranslational mechanisms in Saccharomyces cerevisiae. We have previously identified four genes, ACN8, ACN9, ACN17, and ACN18, whose mutant phenotype includes two- to fourfold elevated levels of enzymes of the glyoxylate cycle, gluconeogenesis, and acetyl-CoA metabolism. The affected enzymes are elevated on nonfermentable carbon sources but are still fully repressed by glucose. Catabolite inactivation of the cytosolic malate dehydrogenase is not affected in the mutants. Instead, the phenotype appeared to be manifested primarily at the level of transcription. The ACN8, ACN17, and ACN18 genes were isolated by functional complementation of the respective mutant's inability to utilize acetate as a carbon and energy source, and these genes were shown to encode subunits of metabolic enzymes. ACN8 was identical to FBP1, which encodes the gluconeogenic enzyme, fructose 1,6-bisphosphatase, while ACN17 and ACN18 were identical to the SDH2 and SDH4 genes, respectively, that encode subunits of the respiratory chain and tricarboxylic acid cycle enzyme, succinate dehydrogenase. Mutants defective in other glyoxylate cycle and gluconeogenic enzymes also display the elevated enzyme phenotype, indicating that the enzyme superinduction is a general property of gluconeogenic dysfunction. Glucose 6-phosphate levels were diminished in the mutants, suggesting that endogenous glucose synthesis can regulate the expression of gluconeogenic enzymes.

  5. Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa.

    PubMed Central

    Sung, C H; Schneider, B G; Agarwal, N; Papermaster, D S; Nathans, J

    1991-01-01

    Thirteen mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa (ADRP) have been produced by transfection of cloned cDNA into tissue culture cells. Three mutants [class I: Phe-45----Leu, Gln-344----termination (deletion of C-terminal positions 344-348), and Pro-347----Leu] resemble wild-type rhodopsin in yield, regenerability with 11-cis-retinal, and plasma membrane localization. Ten mutants [class II: Thr-17----Met, Pro-23----His, Thr-58----Arg, Val-87----Asp, Gly-89----Asp, Gly-106----Trp, Arg-135----Leu, Arg-135----Trp, Tyr-178----Cys, and Asp-190----Gly] accumulate to significantly lower levels, regenerate with 11-cis-retinal variably or not at all, and are transported inefficiently to the plasma membrane, remaining primarily in the endoplasmic reticulum. These data suggest that there are at least two distinct biochemical defects associated with different rhodopsin mutants in ADRP. Images PMID:1924344

  6. Tissue-specific Expression of Dominant Negative Mutant Drosophila HSC70 Causes Developmental Defects and Lethality

    PubMed Central

    Elefant, Felice; Palter, Karen B.

    1999-01-01

    The Drosophila melanogaster HSC3 and HSC4 genes encode Hsc70 proteins homologous to the mammalian endoplasmic reticulum (ER) protein BiP and the cytoplasmic clathrin uncoating ATPase, respectively. These proteins possess ATP binding/hydrolysis activities that mediate their ability to aid in protein folding by coordinating the sequential binding and release of misfolded proteins. To investigate the roles of HSC3 (Hsc3p) and HSC4 (Hsc4p) proteins during development, GAL4-targeted gene expression was used to analyze the effects of producing dominant negatively acting Hsc3p (D231S, K97S) and Hsc4p (D206S, K71S) proteins, containing single amino acid substitutions in their ATP-binding domains, in specific tissues of Drosophila throughout development. We show that the production of each mutant protein results in lethality over a range of developmental stages, depending on the levels of protein produced and which tissues are targeted. We demonstrate that the functions of both Hsc3p and Hsc4p are required for proper tissue establishment and maintenance. Production of mutant Hsc4p, but not Hsc3p, results in induction of the stress-inducible Hsp70 at normal temperatures. Evidence is presented that lethality is caused by tissue-specific defects that result from a global accumulation of misfolded protein caused by lack of functional Hsc70. We show that both mutant Hsc3ps are defective in ATP-induced substrate release, although Hsc3p(D231S) does undergo an ATP-induced conformational change. We believe that the amino acid substitutions in Hsc3p interfere with the structural coupling of ATP binding to substrate release, and this defect is the basis for the mutant proteins’ dominant negative effects in vivo. PMID:10397752

  7. Defective calmodulin-dependent rapid apical endocytosis in zebrafish sensory hair cell mutants.

    PubMed

    Seiler, C; Nicolson, T

    1999-11-15

    Vertebrate mechanosensory hair cells contain a narrow "pericuticular" zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1-43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function. PMID:10526320

  8. Modulation of lipid metabolic defects rescues cleft palate in Tgfbr2 mutant mice.

    PubMed

    Iwata, Junichi; Suzuki, Akiko; Pelikan, Richard C; Ho, Thach-Vu; Sanchez-Lara, Pedro A; Chai, Yang

    2014-01-01

    Mutations in transforming growth factor beta (TGFβ) receptor type II (TGFBR2) cause Loeys-Dietz syndrome, characterized by craniofacial and cardiovascular abnormalities. Mice with a deletion of Tgfbr2 in cranial neural crest cells (Tgfbr2(fl/fl);Wnt1-Cre mice) develop cleft palate as the result of abnormal TGFβ signaling activation. However, little is known about metabolic processes downstream of TGFβ signaling during palatogenesis. Here, we show that Tgfbr2 mutant palatal mesenchymal cells spontaneously accumulate lipid droplets, resulting from reduced lipolysis activity. Tgfbr2 mutant palatal mesenchymal cells failed to respond to the cell proliferation stimulator sonic hedgehog, derived from the palatal epithelium. Treatment with p38 mitogen-activated protein kinase (MAPK) inhibitor or telmisartan, a modulator of p38 MAPK activation and lipid metabolism, blocked abnormal TGFβ-mediated p38 MAPK activation, restoring lipid metabolism and cell proliferation activity both in vitro and in vivo. Our results highlight the influence of alternative TGFβ signaling on lipid metabolic activities, as well as how lipid metabolic defects can affect cell proliferation and adversely impact palatogenesis. This discovery has broader implications for the understanding of metabolic defects and potential prevention of congenital birth defects. PMID:23975680

  9. Ocular Coloboma and Dorsoventral Neuroretinal Patterning Defects in Lrp6 Mutant Eyes

    PubMed Central

    Zhou, Cheng-Ji; Molotkov, Andrei; Song, Lanying; Li, Yunhong; Pleasure, David E.; Pleasure, Samuel J.; Wang, Ya-Zhou

    2009-01-01

    Coloboma, an ocular birth defect seen in humans and other species, is caused by incomplete closure of the optic fissure. Here, we demonstrate that genetic deletion of Lrp6, a bottleneck coreceptor in the canonical Wnt signaling pathway, results in ocular coloboma and neuroretinal patterning defects in mice. The expression of ventral neuroretinal patterning gene Vax2 was conserved but with dorsally shifted expression domains; however, the dorsal neuroretinal patterning gene Tbx5 was lost in the Lrp6-mutant eyes at embryonic day 10.5. Both Bmp4 and phosphorylated Smad 1/5/8 were also significantly attenuated in the dorsal neuroretina. In addition, the retinoic acid synthesizing enzymes Raldh1 and Raldh3 were significantly changed in the mutant eyes. Our findings suggest that defective retinal patterning causes coloboma in the Lrp6-deficient mice, and that canonical Wnt signaling plays a primary role in dorsal neuroretinal patterning and related morphogenetic movements by regulation of both Bmp and retinoic acid signaling pathways. PMID:18985738

  10. Isolation and characterization of a mutant defective in triacylglycerol accumulation in nitrogen-starved Chlamydomonas reinhardtii.

    PubMed

    Hung, Chun-Hsien; Kanehara, Kazue; Nakamura, Yuki

    2016-09-01

    Triacylglycerol (TAG), a major source of biodiesel production, accumulates in nitrogen-starved Chlamydomonas reinhardtii. However, the metabolic pathway of starch-to-TAG conversion remains elusive because an enzyme that affects the starch degradation is unknown. Here, we isolated a new class of mutant bgal1, which expressed an overaccumulation of starch granules and defective photosynthetic growth. The bgal1 was a null mutant of a previously uncharacterized β-galactosidase-like gene (Cre02.g119700), which decreased total β-galactosidase activity 40% of the wild type. Upon nitrogen starvation, the bgal1 mutant showed decreased TAG accumulation mainly due to the reduced flux of de novo TAG biosynthesis evidenced by increased unsaturation of fatty acid composition in TAG and reduced TAG accumulation by additional supplementation of acetate to the culture media. Metabolomic analysis of the bgal1 mutant showed significantly reduced levels of metabolites following the hydrolysis of starch and substrates for TAG accumulation, whereas metabolites in TCA cycle were unaffected. Upon nitrogen starvation, while levels of glucose 6-phosphate, fructose 6-phosphate and acetyl-CoA remained lower, most of the other metabolites in glycolysis were increased but those in the TCA cycle were decreased, supporting TAG accumulation. We suggest that BGAL1 may be involved in the degradation of starch, which affects TAG accumulation in nitrogen-starved C. reinhardtii. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. PMID:27060488

  11. Isolation and characterization of a mutant defective in triacylglycerol accumulation in nitrogen-starved Chlamydomonas reinhardtii.

    PubMed

    Hung, Chun-Hsien; Kanehara, Kazue; Nakamura, Yuki

    2016-09-01

    Triacylglycerol (TAG), a major source of biodiesel production, accumulates in nitrogen-starved Chlamydomonas reinhardtii. However, the metabolic pathway of starch-to-TAG conversion remains elusive because an enzyme that affects the starch degradation is unknown. Here, we isolated a new class of mutant bgal1, which expressed an overaccumulation of starch granules and defective photosynthetic growth. The bgal1 was a null mutant of a previously uncharacterized β-galactosidase-like gene (Cre02.g119700), which decreased total β-galactosidase activity 40% of the wild type. Upon nitrogen starvation, the bgal1 mutant showed decreased TAG accumulation mainly due to the reduced flux of de novo TAG biosynthesis evidenced by increased unsaturation of fatty acid composition in TAG and reduced TAG accumulation by additional supplementation of acetate to the culture media. Metabolomic analysis of the bgal1 mutant showed significantly reduced levels of metabolites following the hydrolysis of starch and substrates for TAG accumulation, whereas metabolites in TCA cycle were unaffected. Upon nitrogen starvation, while levels of glucose 6-phosphate, fructose 6-phosphate and acetyl-CoA remained lower, most of the other metabolites in glycolysis were increased but those in the TCA cycle were decreased, supporting TAG accumulation. We suggest that BGAL1 may be involved in the degradation of starch, which affects TAG accumulation in nitrogen-starved C. reinhardtii. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

  12. Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators.

    PubMed

    Loo, T W; Clarke, D M

    1997-01-10

    There is growing evidence that abnormal protein folding or trafficking (protein kinesis) leads to diseases. We have used P-glycoprotein as a model protein to develop strategies to overcome defects in protein kinesis. Misprocessed mutants of the human P-glycoprotein are retained in the endoplasmic reticulum as core-glycosylated biosynthetic intermediates and rapidly degraded. Synthesis of the mutant proteins in the presence of drug substrates or modulators such as capsaicin, cyclosporin, vinblastine, or verapamil, however, resulted in the appearance of a fully glycosylated and functional protein at the cell surface. These effects were dose-dependent and occurred within a few hours after the addition of substrate. The ability to facilitate processing of the misfolded mutants appeared to be independent of the cell lines used and location of the mutation. P-glycoproteins with mutations in transmembrane segments, extracellular or cytoplasmic loops, the nucleotide-binding domains, or the linker region were processed to the fully mature form in the presence of these substrates. These drug substrates or modulators acted as specific chemical chaperones for P-glycoprotein because they were ineffective on the deltaF508 mutant of cystic fibrosis transmembrane conductance regulator. Therefore, one possible strategy to prevent protein misfolding is to carry out synthesis in the presence of specific substrates or modulators of the protein.

  13. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases.

    PubMed Central

    Robinson, J S; Klionsky, D J; Banta, L M; Emr, S D

    1988-01-01

    Using a selection for spontaneous mutants that mislocalize a vacuolar carboxypeptidase Y (CPY)-invertase fusion protein to the cell surface, we identified vacuolar protein targeting (vpt) mutants in 25 new vpt complementation groups. Additional alleles in each of the eight previously identified vpt complementation groups (vpt1 through vpt8) were also obtained. Representative alleles from each of the 33 vpt complementation groups (vpt1 through vpt33) were shown to exhibit defects in the sorting and processing of several native vacuolar proteins, including the soluble hydrolases CPY, proteinase A, and proteinase B. Of the 33 complementation groups, 19 were found to contain mutant alleles that led to extreme defects. In these mutants, CPY accumulated in its Golgi complex-modified precursor form which was secreted by the mutant cells. Normal protein secretion appeared to be unaffected in the vpt mutants. The lack of significant leakage of cytosolic markers from the vpt mutant cells indicated that the vacuolar protein-sorting defects associated with these mutants do not result from cell lysis. In addition, the observation that the precursor rather than the mature forms of CPY, proteinase A, proteinase B were secreted from the vpt mutants was consistent with the fact that mislocalization occurred at a stage after Golgi complex-specific modification, but before final vacuolar sorting of these enzymes. Vacuolar membrane protein sorting appeared to be unaffected in the majority of the vpt mutants. However, a subset of the vpt mutants (vpt11, vpt16, vpt18, and vpt33) was found to exhibit defects in the sorting of a vacuolar membrane marker enzyme, alpha-mannosidase. Up to 50% of the alpha-mannosidase enzyme activity was found to be mislocalized to the cell surface in these vpt mutants. Seven of the vpt complementation groups (vpt3, vpt11, vpt15, vpt16, vpt18, vpt29, and vpt33) contained alleles that led to a conditional lethal phenotype; the mutants were temperature

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

    PubMed Central

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

    1987-01-01

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

  15. Analysis of mutants of Salmonella typhimurium defective in the synthesis of the nucleotide loop of cobalamin.

    PubMed Central

    O'Toole, G A; Rondon, M R; Escalante-Semerena, J C

    1993-01-01

    The CobIII region of the cobalamin (CBL) biosynthetic (cob) operon of Salmonella typhimurium encodes functions necessary for the synthesis of the nucleotide loop of CBL and comprises three genes, designated cobU, cobS, and cobT (26). Complementation studies identified two classes of CobIII mutants: (i) 34 mutants were complemented by a plasmid carrying the cobU+ gene, and (ii) 27 mutants were complemented by a plasmid carrying the cobS+ gene; none of the mutants tested was complemented by the cobT+ clone, a result suggesting that no cobT mutations were isolated. These data were consistent with those of complementation studies done with F' cobUST plasmids, which also suggested that the CobIII region comprises two complementation groups. A plasmid carrying cobUS+ was sufficient to complement a deletion of the entire CobIII region, a result suggesting that CobT was not required for CBL biosynthesis. Nutritional studies done with synthetic putative intermediates of the CobIII pathway were performed to further classify cobIII mutants. A subset of cobU mutants were found to be responsive to exogenous dicyano-cobinamide-GDP, while cobS mutants were found to be responsive only to CBL. These results are consistent with the adenosyl-cobinamide kinase-GTP:adenosyl-cobinamide-phosphate guanylyltransferase and CBL synthase activities proposed for CobU and CobS, respectively. The cobIII genes under the control of the T7 promoter were overexpressed, and the resulting polypeptides were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three polypeptides with apparent molecular masses of 22, 26 and 39 kDa, consistent with the predicted masses for CobU, CobS, and CobT, respectively, were detected. Images PMID:8501035

  16. Abelson murine leukemia virus transformation-defective mutants with impaired P120-associated protein kinase activity.

    PubMed Central

    Reynolds, F H; Van de Ven, W J; Stephenson, J R

    1980-01-01

    Several transformation-defective (td) mutants of Abelson murine leukemia virus (AbLV) are described. Cells nonproductively infected with such mutants exhibited a high degree of growth contact inhibition, failed to form colonies in soft agar, lacked rescuable transforming virus, and were as susceptible as uninfected control cells to transformation by wild-type (wt) AbLV pseudotype virus. In addition, each of several td AbLV nonproductively infected cell clones analyzed was found to be nontumorigenic in vivo. Biochemical analysis of td mutant AbLV-infected clones revealed levels of expression of the major AbLV translational product, P120, and a highly related 80,000-Mr AbLV-encoded protein, P80, at concentrations analogous to those in wt AbLV-transformed cells. Although the AbLV-specific 120,000-Mr polyproteins expressed in td mutant AbLV-infected clones were indistinguishable from those in wt AbLV-transformed lines with respect to molecular weight and [35S]methionine tryptic peptide composition, they each differed from wt AbLV P120 in their patterns of post-translational phosphorylation. A previously described AbLV-associated protein kinase activity is shown to recognize as substrate a major tyrosine-specific acceptor site(s) contained within a single well-resolved tryptic peptide common to both AbLV P120 and P80. In vitro [gamma-32P]ATP-mediated labeling of this phosphorylation site was reduced to below detectable levels in td mutant nonproductively infected cell clones. These findings establish that the AbLV-encoded polyprotein P120 and its associated protein kinase activity are involved in AbLV tumorigenesis. Images PMID:6253663

  17. Cortico-striatal synaptic defects and OCD-like behaviors in SAPAP3 mutant mice

    PubMed Central

    Welch, Jeffrey M.; Lu, Jing; Rodriguiz, Ramona M.; Trotta, Nicholas C.; Peca, Joao; Ding, Jin-Dong; Feliciano, Catia; Chen, Meng; Adams, J. Paige; Luo, Jianhong; Dudek, Serena M.; Weinberg, Richard J.; Calakos, Nicole; Wetsel, William C.; Feng, Guoping

    2008-01-01

    Obsessive-compulsive disorder (OCD) is an anxiety-spectrum disorder characterized by persistent intrusive thoughts (obsessions) and repetitive actions (compulsions). Dysfunction of cortico-striato-thalamo-cortical circuitry is implicated in OCD, though the underlying pathogenic mechanisms are unknown. SAP90/PSD95-associated protein 3 (SAPAP3) is a postsynaptic scaffolding protein at excitatory synapses that is highly expressed in the striatum. Here we show that mice with genetic deletion of SAPAP3 exhibit increased anxiety and compulsive grooming behavior leading to facial hair loss and skin lesions; both behaviors are alleviated by a selective serotonin reuptake inhibitor. Electrophysiological, structural, and biochemical studies of SAPAP3 mutant mice reveal defects in cortico-striatal synapses. Furthermore, lentiviral-mediated selective expression of SAPAP3 in the striatum rescues the synaptic and behavioral defects of SAPAP3 mutant mice. These findings demonstrate a critical role for SAPAP3 at cortico-striatal synapses and emphasize the importance of cortico-striatal circuitry in OCD-like behaviors. PMID:17713528

  18. Brucella abortus Cyclic β-1,2-Glucan Mutants Have Reduced Virulence in Mice and Are Defective in Intracellular Replication in HeLa Cells

    PubMed Central

    Briones, Gabriel; Iñón de Iannino, Nora; Roset, Mara; Vigliocco, Ana; Paulo, Patricia Silva; Ugalde, Rodolfo A.

    2001-01-01

    Null cyclic β-1,2-glucan synthetase mutants (cgs mutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic β-1,2-glucan plays an important role in the residual virulence of the attenuated B. abortus S19 strain. Although the cgs mutant was cleared from the spleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected with B. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by the cgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response. PMID:11401996

  19. Impaired embryonic motility in dusp27 mutants reveals a developmental defect in myofibril structure

    PubMed Central

    Fero, Kandice; Bergeron, Sadie A.; Horstick, Eric J.; Codore, Hiba; Li, Grace H.; Ono, Fumihito; Dowling, James J.; Burgess, Harold A.

    2014-01-01

    ABSTRACT An essential step in muscle fiber maturation is the assembly of highly ordered myofibrils that are required for contraction. Much remains unknown about the molecular mechanisms governing the formation of the contractile apparatus. We identified an early embryonic motility mutant in zebrafish caused by integration of a transgene into the pseudophosphatase dual specificity phosphatase 27 (dusp27) gene. dusp27 mutants exhibit near complete paralysis at embryonic and larval stages, producing extremely low levels of spontaneous coiling movements and a greatly diminished touch response. Loss of dusp27 does not prevent somitogenesis but results in severe disorganization of the contractile apparatus in muscle fibers. Sarcomeric structures in mutants are almost entirely absent and only rare triads are observed. These findings are the first to implicate a functional role of dusp27 as a gene required for myofiber maturation and provide an animal model for analyzing the mechanisms governing myofibril assembly. PMID:24203884

  20. Ceramides And Stress Signalling Intersect With Autophagic Defects In Neurodegenerative Drosophila blue cheese (bchs) Mutants

    PubMed Central

    Hebbar, Sarita; Sahoo, Ishtapran; Matysik, Artur; Argudo Garcia, Irene; Osborne, Kathleen Amy; Papan, Cyrus; Torta, Federico; Narayanaswamy, Pradeep; Fun, Xiu Hui; Wenk, Markus R; Shevchenko, Andrej; Schwudke, Dominik; Kraut, Rachel

    2015-01-01

    Sphingolipid metabolites are involved in the regulation of autophagy, a degradative recycling process that is required to prevent neuronal degeneration. Drosophila blue cheese mutants neurodegenerate due to perturbations in autophagic flux, and consequent accumulation of ubiquitinated aggregates. Here, we demonstrate that blue cheese mutant brains exhibit an elevation in total ceramide levels; surprisingly, however, degeneration is ameliorated when the pool of available ceramides is further increased, and exacerbated when ceramide levels are decreased by altering sphingolipid catabolism or blocking de novo synthesis. Exogenous ceramide is seen to accumulate in autophagosomes, which are fewer in number and show less efficient clearance in blue cheese mutant neurons. Sphingolipid metabolism is also shifted away from salvage toward de novo pathways, while pro-growth Akt and MAP pathways are down-regulated, and ER stress is increased. All these defects are reversed under genetic rescue conditions that increase ceramide generation from salvage pathways. This constellation of effects suggests a possible mechanism whereby the observed deficit in a potentially ceramide-releasing autophagic pathway impedes survival signaling and exacerbates neuronal death. PMID:26639035

  1. RNA polymerase II mutants defective in transcription of a subset of genes.

    PubMed Central

    Scafe, C; Nonet, M; Young, R A

    1990-01-01

    Saccharomyces cerevisiae RNA polymerase II conditional mutants that selectively disrupt the synthesis of specific mRNAs were isolated. At the permissive temperature, several of the mutants were inositol auxotrophs as a result of inadequate induction of INO1 transcription. The transcriptional defects exhibited by one of these Ino- mutants (rpb2-2) were further investigated. The induction of GAL10 and HIS4 transcription in rpb2-2 strains was similar to that of wild-type strains, in contrast to the lack of induction of INO1 transcription. When shifted to the nonpermissive temperature, cells containing rpb2-2 continued to accumulate some mRNAs but not others. Together, these results indicate that transcription of specific genes can be disrupted by RNA polymerase II mutations. The rpb2-2 allele alters an amino acid residue that occurs in a highly conserved segment of the RPB2 protein and that is shared by homologous subunits in other species. Images PMID:2406558

  2. Yeast lsm pro-apoptotic mutants show defects in S-phase entry and progression.

    PubMed

    Palermo, Vanessa; Cundari, Enrico; Mangiapelo, Eleonora; Falcone, Claudio; Mazzoni, Cristina

    2010-10-01

    Expression of the histone genes is tightly coupled to rates of DNA synthesis in yeast and histone mRNAs are modulated both transcriptionally and post-transcriptionally. Trf4 and Trf5, poly(A) polymerases, that mediates polyadenylation and consequent degradation) and Rrp6, an exosome component, play a role in the regulation of histone mRNA levels. In this paper we show that in the mRNA degradation mutant Kllsm4Δ1, histone mRNAs are induced early in the S-phase and maintained at high level all along the entire cell cycle due to a delay in the exit from S-phase and/or entry into M-phase. The overexpression of the HIR1 gene (Histone transcriptional repressor), previously isolated as a multicopy suppressor of the apoptotic phenotypes observed in Kllsm4Δ1, can also restore the normal cycling of histone genes expression. We also found that low doses of hydroxyurea neutralize the onset of the apoptotic phenotypes in Kllsm4Δ1, as well in another mRNA decapping mutants (lsm1) and, in addition, increase the chronological lifespan in both strains suggesting that an entry delay into the S phase can recover some cellular defects in decapping mutants.

  3. Survival Defects of Cryptococcus neoformans Mutants Exposed to Human Cerebrospinal Fluid Result in Attenuated Virulence in an Experimental Model of Meningitis▿ †

    PubMed Central

    Lee, Anthony; Toffaletti, Dena L.; Tenor, Jennifer; Soderblom, Erik J.; Thompson, J. Will; Moseley, M. Arthur; Price, Michael; Perfect, John R.

    2010-01-01

    Cryptococcus neoformans is a fungal pathogen that encounters various microenvironments during growth in the mammalian host, including intracellular vacuoles, blood, and cerebrospinal fluid (CSF). Because the CSF is isolated by the blood-brain barrier, we hypothesize that CSF presents unique stresses that C. neoformans must overcome to establish an infection. We assayed 1,201 mutants for survival defects in growth media, saline, and human CSF. We assessed CSF-specific mutants for (i) mutant survival in both human bronchoalveolar lavage (BAL) fluid and fetal bovine serum (FBS), (ii) survival in macrophages, and (iii) virulence using both Caenorhabditis elegans and rabbit models of cryptococcosis. Thirteen mutants exhibited significant survival defects unique to CSF. The mutations of three of these mutants were recreated in the clinical serotype A strain H99: deletions of the genes for a cation ATPase transporter (ena1Δ), a putative NEDD8 ubiquitin-like protein (rub1Δ), and a phosphatidylinositol 4-kinase (pik1Δ). Mutant survival rates in yeast media, saline, and BAL fluid were similar to those of the wild type; however, survival in FBS was reduced but not to the levels in CSF. These mutant strains also exhibited decreased intracellular survival in macrophages, various degrees of virulence in nematodes, and severe attenuation of survival in a rabbit meningitis model. We analyzed the CSF by mass spectrometry for candidate compounds responsible for the survival defect. Our findings indicate that the genes required for C. neoformans survival in CSF ex vivo are necessary for survival and infection in this unique host environment. PMID:20696827

  4. Hyperproduction of β-Glucanase Exg1 Promotes the Bioconversion of Mogrosides in Saccharomyces cerevisiae Mutants Defective in Mannoprotein Deposition.

    PubMed

    Wang, Reuben; Lin, Pei-Yin; Huang, Shyue-Tsong; Chiu, Chun-Hui; Lu, Ting-Jang; Lo, Yi-Chen

    2015-12-01

    Bacteria and fungi can secrete extracellular enzymes to convert macromolecules into smaller units. Hyperproduction of extracellular enzymes is often associated with alterations in cell wall structure in fungi. Recently, we identified that Saccharomyces cerevisiae kre6Δ mutants can efficiently convert mogroside V into mogroside III E, which has antidiabetic properties. However, the underlying efficient bioconversion mechanism is unclear. In the present study, the mogroside (MG) bioconversion properties of several cell wall structure defective mutants were analyzed. We also compared the cell walls of these mutants by transmission electron microscopy, a zymolyase sensitivity test, and a mannoprotein release assay. We found zymolyase-sensitive mutants (including kre1Δ, las21Δ, gas1Δ, and kre6Δ), with defects in mannoprotein deposition, exhibit efficient MG conversion and excessive leakage of Exg1; such defects were not observed in wild-type cells, or mutants with abnormal levels of glucans in the cell wall. Thus, yeast mutants defective in mannoprotein deposition may be employed to convert glycosylated bioactive compounds.

  5. Enhanced Biofilm Formation by Escherichia coli LPS Mutants Defective in Hep Biosynthesis

    PubMed Central

    Nakao, Ryoma; Ramstedt, Madeleine; Wai, Sun Nyunt; Uhlin, Bernt Eric

    2012-01-01

    Lipopolysaccharide (LPS) is the major component of the surface of Gram-negative bacteria and its polysaccharide portion is situated at the outermost region. We investigated the relationship between the polysaccharide portion of LPS and biofilm formation using a series of Escherichia coli mutants defective in genes earlier shown to affect the LPS sugar compositions. Biofilm formation by a deep rough LPS mutant, the hldE strain, was strongly enhanced in comparison with the parental strain and other LPS mutants. The hldE strain also showed a phenotype of increased auto-aggregation and stronger cell surface hydrophobicity compared to the wild-type. Similar results were obtained with another deep rough LPS mutant, the waaC strain whose LPS showed same molecular mass as that of the hldE strain. Confocal laser scanning microscopy (CLSM) analysis and biofilm formation assay using DNase I revealed that biofilm formation by the hldE strain was dependent on extracellular DNA. Furthermore, a loss of flagella and an increase in amount of outer membrane vesicles in case of the hldE strain were also observed by transmission electron microscopy and atomic force microscopy, respectively. In addition, we demonstrated that a mutation in the hldE locus, which alters the LPS structure, caused changes in both expression and properties of several surface bacterial factors involved in biofilm formation and virulence. We suggest that the implication of these results should be considered in the context of biofilm formation on abiotic surfaces, which is frequently associated with nosocominal infections such as the catheter-associated infections. PMID:23284671

  6. Enhanced biofilm formation by Escherichia coli LPS mutants defective in Hep biosynthesis.

    PubMed

    Nakao, Ryoma; Ramstedt, Madeleine; Wai, Sun Nyunt; Uhlin, Bernt Eric

    2012-01-01

    Lipopolysaccharide (LPS) is the major component of the surface of Gram-negative bacteria and its polysaccharide portion is situated at the outermost region. We investigated the relationship between the polysaccharide portion of LPS and biofilm formation using a series of Escherichia coli mutants defective in genes earlier shown to affect the LPS sugar compositions. Biofilm formation by a deep rough LPS mutant, the hldE strain, was strongly enhanced in comparison with the parental strain and other LPS mutants. The hldE strain also showed a phenotype of increased auto-aggregation and stronger cell surface hydrophobicity compared to the wild-type. Similar results were obtained with another deep rough LPS mutant, the waaC strain whose LPS showed same molecular mass as that of the hldE strain. Confocal laser scanning microscopy (CLSM) analysis and biofilm formation assay using DNase I revealed that biofilm formation by the hldE strain was dependent on extracellular DNA. Furthermore, a loss of flagella and an increase in amount of outer membrane vesicles in case of the hldE strain were also observed by transmission electron microscopy and atomic force microscopy, respectively. In addition, we demonstrated that a mutation in the hldE locus, which alters the LPS structure, caused changes in both expression and properties of several surface bacterial factors involved in biofilm formation and virulence. We suggest that the implication of these results should be considered in the context of biofilm formation on abiotic surfaces, which is frequently associated with nosocominal infections such as the catheter-associated infections.

  7. A defective mutant of Salmonella enterica Serovar Gallinarum in cobalamin biosynthesis is avirulent in chickens

    PubMed Central

    de Paiva, Jacqueline Boldrin; Penha Filho, Rafael Antonio Casarin; Arguello, Yuli Melisa Sierra; Berchieri Junior, Ângelo; Lemos, Manuel Victor Franco; Barrow, Paul A.

    2009-01-01

    Salmonella enterica serovar Gallinarum (SG) is a fowl typhoid agent in chickens and is a severe disease with worldwide economic impact as its mortality may reach up to 80%. It is one of a small group of serovars that typically produces typhoid-like infections in a narrow range of host species and which therefore represents a good model for human typhoid. The survival mechanisms are not considered to be virulent mechanisms but are essential for the life of the bacterium. Mutants of Salmonella Gallinarum containing defective genes, related to cobalamin biosynthesis and which Salmonella spp. has to be produced to survive when it is in an anaerobic environment, were produced in this study. Salmonella Gallinarum is an intracellular parasite. Therefore, this study could provide information about whether vitamin B12 biosynthesis might be essential to its survival in the host. The results showed that the singular deletion in cbiA or cobS genes did not interfere in the life of Salmonella Gallinarum in the host, perhaps because single deletion is not enough to impede vitamin B12 biosynthesis. It was noticed that diluted SG mutants with single deletion produced higher mortality than the wild strain of SG. When double mutation was carried out, the Salmonella Gallinarum mutant was unable to provoke mortality in susceptible chickens. This work showed that B12 biosynthesis is a very important step in the metabolism of Salmonella Gallinarum during the infection of the chickens. Further research on bacterium physiology should be carried out to elucidate the events described in this research and to assess the mutant as a vaccine strain. PMID:24031393

  8. Striking Complexity of Lipopolysaccharide Defects in a Collection of Sinorhizobium meliloti Mutants

    PubMed Central

    Campbell, Gordon R. O.; Sharypova, Larissa A.; Scheidle, Heiko; Jones, Kathryn M.; Niehaus, Karsten; Becker, Anke; Walker, Graham C.

    2003-01-01

    Although the role that lipopolysaccharide (LPS) plays in the symbiosis between Sinorhizobium meliloti and alfalfa has been studied for over a decade, its function in this process remains controversial and poorly understood. This is largely due to a lack of mutants affected by its synthesis. In one of the definitive studies concerning this issue, Clover et al. (R. H. Clover, J. Kieber, and E. R. Signer, J. Bacteriol. 171:3961-3967, 1989) identified a series of mutants with putative LPS defects, judged them to be symbiotically proficient on Medicago sativa, and concluded that LPS might not have a symbiotic function in S. meliloti. The mutations in these strains were never characterized at the molecular level nor was the LPS from most of them analyzed. We have transduced these mutations from the Rm2011 background from which they were originally isolated into the sequenced strain Rm1021 and have characterized the resulting strains in greater detail. We found the LPS from these mutants to display a striking complexity of phenotypes on polyacrylamide electrophoresis gels, including additional rough LPS bands and alterations in the molecular weight distribution of the smooth LPS. We found that some of the mutants contain insertions in genes that are predicted to be involved in the synthesis of carbohydrate components of LPS, including ddhB, lpsB, lpsC, and lpsE. The majority, however, code for proteins predicted to be involved in a wide variety of functions not previously recognized to play a role in LPS synthesis, including a possible transcription elongation factor (GreA), a possible queuine synthesis protein, and a possible chemotaxis protein. Furthermore, using more extensive assays, we have found that most of these strains have symbiotic deficiencies. These results support more recent findings that alterations in LPS structure can affect the ability of S. meliloti to form an effective symbiosis. PMID:12813079

  9. Generation of a homozygous fertilization-defective gcs1 mutant by heat-inducible removal of a rescue gene.

    PubMed

    Nagahara, Shiori; Takeuchi, Hidenori; Higashiyama, Tetsuya

    2015-03-01

    Key message: New gametic homozygous mutants. In angiosperms, a haploid male gamete (sperm cell) fuses with a haploid female gamete (egg cell) during fertilization to form a zygote carrying paternally and maternally derived chromosomes. Several fertilization-defective mutants in Arabidopsis thaliana, including a generative cell-specific 1 (gcs1)/hapless 2 mutant, the sperm cells of which are unable to fuse with female gametes, can only be maintained as heterozygous lines due to the infertile male or female gametes. Here, we report successful generation of a gcs1 homozygous mutant by heat-inducible removal of the GCS1 transgene. Using the gcs1 homozygous mutant as male, the defect in gamete fusion was observed with great frequency; in our direct observation by semi-in vivo fertilization assay using ovules, 100 % of discharged sperm cells in culture failed to show gamete fusion. More than 70 % of ovules in the pistil received a second pollen tube as attempted fertilization recovery. Moreover, gcs1 mutant sperm cells could fertilize female gametes at a low frequency in the pistil. This strategy to generate homozygous fertilization-defective mutants will facilitate novel approaches in plant reproduction research.

  10. Most mutant OccR proteins that are defective in positive control hold operator DNA in a locked high-angle bend.

    PubMed

    Tsai, Ching-Sung; Chen, Chia-Sui; Winans, Stephen C

    2011-10-01

    OccR is a LysR-type transcriptional regulator of Agrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid. Positive control of the occ genes occurs in response to octopine, a nutrient released from crown gall tumors. OccR also functions as an autorepressor in the presence or absence of octopine. OccR binds to a site between occQ and occR in the presence or absence of octopine, although octopine triggers a conformational change that shortens the DNA footprint and relaxes a DNA bend. In order to determine the roles of this conformational change in transcriptional activation, we isolated 11 OccR mutants that were defective in activation of the occQ promoter but were still capable of autorepression. The mutations in these mutants spanned most of the length of the protein. Two additional positive-control mutants were isolated using site-directed mutagenesis. Twelve mutant proteins displayed a high-angle DNA bend in the presence or absence of octopine. One mutant, the L26A mutant, showed ligand-responsive DNA binding similar to that of wild-type OccR and therefore must be impaired in a subsequent step in activation. PMID:21804007

  11. Defective copper transport in the copt5 mutant affects cadmium tolerance.

    PubMed

    Carrió-Seguí, Angela; Garcia-Molina, Antoni; Sanz, Amparo; Peñarrubia, Lola

    2015-03-01

    Cadmium toxicity interferes with essential metal homeostasis, which is a problem for both plant nutrition and the consumption of healthy food by humans. Copper uptake is performed by the members of the Arabidopsis high affinity copper transporter (COPT) family. One of the members, COPT5, is involved in copper recycling from the vacuole toward the cytosolic compartment. We show herein that copt5 mutants are more sensitive to cadmium stress than wild-type plants, as indicated by reduced growth. Exacerbated cadmium toxicity in copt5 mutants is due specifically to altered copper traffic through the COPT5 transporter. Three different processes which have been shown to affect cadmium tolerance are altered in copt5 mutants. First, ethylene biosynthesis diminishes under copper deficiency and, in the presence of cadmium, ethylene production diminishes further. Copper deficiency responses are also attenuated under cadmium treatment. Remarkably, while copt5 roots present higher oxidative stress toxicity symptoms than controls, aerial copt5 parts display lower oxidative stress, as seen by reduced cadmium delivery to shoots. Taken together, these results demonstrate that copper transport plays a key role in cadmium resistance, and suggest that oxidative stress triggers an NADPH oxidase-mediated signaling pathway, which contributes to cadmium translocation and basal plant resistance. The slightly lower cadmium levels that reach aerial parts in the copt5 mutants, irrespective of the copper content in the media, suggest a new biotechnological approach to minimize toxic cadmium entry into food chains.

  12. Defects in the N-linked oligosaccharide biosynthetic pathway in a Trypanosoma brucei glycosylation mutant.

    PubMed

    Acosta-Serrano, Alvaro; O'Rear, Jessica; Quellhorst, George; Lee, Soo Hee; Hwa, Kuo-Yuan; Krag, Sharon S; Englund, Paul T

    2004-04-01

    Concanavalin A (ConA) kills the procyclic (insect) form of Trypanosoma brucei by binding to its major surface glycoprotein, procyclin. We previously isolated a mutant cell line, ConA 1-1, that is less agglutinated and more resistant to ConA killing than are wild-type (WT) cells. Subsequently we found that the ConA resistance phenotype in this mutant is due to the fact that the procyclin either has no N-glycan or has an N-glycan with an altered structure. Here we demonstrate that the alteration in procyclin N-glycosylation correlates with two defects in the N-linked oligosaccharide biosynthetic pathway. First, ConA 1-1 has a defect in activity of polyprenol reductase, an enzyme involved in synthesis of dolichol. Metabolic incorporation of [3H]mevalonate showed that ConA 1-1 synthesizes equal amounts of dolichol and polyprenol, whereas WT cells make predominantly dolichol. Second, we found that ConA 1-1 synthesizes and accumulates an oligosaccharide lipid (OSL) precursor that is smaller in size than that from WT cells. The glycan of OSL in WT cells is apparently Man9GlcNAc2, whereas that from ConA 1-1 is Man7GlcNAc2. The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man4GlcNAc2 modified with a terminal N-acetyllactosamine group, which is poorly recognized by ConA.

  13. Isolation and Characterization of Mutants Defective in Seed Coat Mucilage Secretory Cell Development in Arabidopsis1

    PubMed Central

    Western, Tamara L.; Burn, Joanne; Tan, Wei Ling; Skinner, Debra J.; Martin-McCaffrey, Luke; Moffatt, Barbara A.; Haughn, George W.

    2001-01-01

    In Arabidopsis, fertilization induces the epidermal cells of the outer ovule integument to differentiate into a specialized seed coat cell type producing extracellular pectinaceous mucilage and a volcano-shaped secondary cell wall. Differentiation involves a regulated series of cytological events including growth, cytoplasmic rearrangement, mucilage synthesis, and secondary cell wall production. We have tested the potential of Arabidopsis seed coat epidermal cells as a model system for the genetic analysis of these processes. A screen for mutants defective in seed mucilage identified five novel genes (MUCILAGE-MODIFIED [MUM]1–5). The seed coat development of these mutants, and that of three previously identified ones (TRANSPARENT TESTA GLABRA1, GLABRA2, and APETALA2) were characterized. Our results show that the genes identified define several events in seed coat differentiation. Although APETALA2 is needed for differentiation of both outer layers of the seed coat, TRANSPARENT TESTA GLABRA1, GLABRA2, and MUM4 are required for complete mucilage synthesis and cytoplasmic rearrangement. MUM3 and MUM5 may be involved in the regulation of mucilage composition, whereas MUM1 and MUM2 appear to play novel roles in post-synthesis cell wall modifications necessary for mucilage extrusion. PMID:11706181

  14. A receptor-like kinase mutant with absent endodermal diffusion barrier displays selective nutrient homeostasis defects

    PubMed Central

    Pfister, Alexandre; Barberon, Marie; Alassimone, Julien; Kalmbach, Lothar; Lee, Yuree; Vermeer, Joop EM; Yamazaki, Misako; Li, Guowei; Maurel, Christophe; Takano, Junpei; Kamiya, Takehiro; Salt, David E; Roppolo, Daniele; Geldner, Niko

    2014-01-01

    The endodermis represents the main barrier to extracellular diffusion in plant roots, and it is central to current models of plant nutrient uptake. Despite this, little is known about the genes setting up this endodermal barrier. In this study, we report the identification and characterization of a strong barrier mutant, schengen3 (sgn3). We observe a surprising ability of the mutant to maintain nutrient homeostasis, but demonstrate a major defect in maintaining sufficient levels of the macronutrient potassium. We show that SGN3/GASSHO1 is a receptor-like kinase that is necessary for localizing CASPARIAN STRIP DOMAIN PROTEINS (CASPs)—major players of endodermal differentiation—into an uninterrupted, ring-like domain. SGN3 appears to localize into a broader band, embedding growing CASP microdomains. The discovery of SGN3 strongly advances our ability to interrogate mechanisms of plant nutrient homeostasis and provides a novel actor for localized microdomain formation at the endodermal plasma membrane. DOI: http://dx.doi.org/10.7554/eLife.03115.001 PMID:25233277

  15. A cytoplasmically inherited barley mutant is defective in photosystem I assembly due to a temperature-sensitive defect in ycf3 splicing.

    PubMed

    Landau, Alejandra Mabel; Lokstein, Heiko; Scheller, Henrik Vibe; Lainez, Verónica; Maldonado, Sara; Prina, Alberto Raúl

    2009-12-01

    A cytoplasmically inherited chlorophyll-deficient mutant of barley (Hordeum vulgare) termed cytoplasmic line 3 (CL3), displaying a viridis (homogeneously light-green colored) phenotype, has been previously shown to be affected by elevated temperatures. In this article, biochemical, biophysical, and molecular approaches were used to study the CL3 mutant under different temperature and light conditions. The results lead to the conclusion that an impaired assembly of photosystem I (PSI) under higher temperatures and certain light conditions is the primary cause of the CL3 phenotype. Compromised splicing of ycf3 transcripts, particularly at elevated temperature, resulting from a mutation in a noncoding region (intron 1) in the mutant ycf3 gene results in a defective synthesis of Ycf3, which is a chaperone involved in PSI assembly. The defective PSI assembly causes severe photoinhibition and degradation of PSII.

  16. Agenesis of the Corpus Callosum Due to Defective Glial Wedge Formation in Lhx2 Mutant Mice.

    PubMed

    Chinn, Gregory A; Hirokawa, Karla E; Chuang, Tony M; Urbina, Cecilia; Patel, Fenil; Fong, Jeanette; Funatsu, Nobuo; Monuki, Edwin S

    2015-09-01

    Establishment of the corpus callosum involves coordination between callosal projection neurons and multiple midline structures, including the glial wedge (GW) rostrally and hippocampal commissure caudally. GW defects have been associated with agenesis of the corpus callosum (ACC). Here we show that conditional Lhx2 inactivation in cortical radial glia using Emx1-Cre or Nestin-Cre drivers results in ACC. The ACC phenotype was characterized by aberrant ventrally projecting callosal axons rather than Probst bundles, and was 100% penetrant on 2 different mouse strain backgrounds. Lhx2 inactivation in postmitotic cortical neurons using Nex-Cre mice did not result in ACC, suggesting that the mutant phenotype was not autonomous to the callosal projection neurons. Instead, ACC was associated with an absent hippocampal commissure and a markedly reduced to absent GW. Expression studies demonstrated strong Lhx2 expression in the normal GW and in its radial glial progenitors, with absence of Lhx2 resulting in normal Emx1 and Sox2 expression, but premature exit from the cell cycle based on EdU-Ki67 double labeling. These studies define essential roles for Lhx2 in GW, hippocampal commissure, and corpus callosum formation, and suggest that defects in radial GW progenitors can give rise to ACC.

  17. Mutant Huntingtin and Elusive Defects in Oxidative Metabolism and Mitochondrial Calcium Handling.

    PubMed

    Brustovetsky, Nickolay

    2016-07-01

    Elongation of a polyglutamine (polyQ) stretch in huntingtin protein (Htt) is linked to Huntington's disease (HD) pathogenesis. The mutation in Htt correlates with neuronal dysfunction in the striatum and cerebral cortex and eventually leads to neuronal cell death. The exact mechanisms of the injurious effect of mutant Htt (mHtt) on neurons are not completely understood but might include aberrant gene transcription, defective autophagy, abnormal mitochondrial biogenesis, anomalous mitochondrial dynamics, and trafficking. In addition, deficiency in oxidative metabolism and defects in mitochondrial Ca(2+) handling are considered essential contributing factors to neuronal dysfunction in HD and, consequently, in HD pathogenesis. Since the discovery of the mutation in Htt, the questions whether mHtt affects oxidative metabolism and mitochondrial Ca(2+) handling and, if it does, what mechanisms could be involved were in focus of numerous investigations. However, despite significant research efforts, the detrimental effect of mHtt and the mechanisms by which mHtt might impair oxidative metabolism and mitochondrial Ca(2+) handling remain elusive. In this paper, I will briefly review studies aimed at clarifying the consequences of mHtt interaction with mitochondria and discuss experimental results supporting or arguing against the mHtt effects on oxidative metabolism and mitochondrial Ca(2+) handling.

  18. Positive selection of mutants defective in transcriptional repression of riboflavin synthesis by iron in the flavinogenic yeast Pichia guilliermondii.

    PubMed

    Boretsky, Yuriy R; Kapustyak, Kostyantyn Y; Fayura, Lyubov R; Stasyk, Oleh V; Stenchuk, Mykola M; Bobak, Yaroslav P; Drobot, Lyudmyla B; Sibirny, Andriy A

    2005-06-01

    It is known for many years that iron represses synthesis of riboflavin (RF) and most of RF-synthesizing enzymes in several yeast species, known as flavinogenic yeasts. However, the mechanism of such repression is not known. We have found that iron represses transcription of RIB1 and RIB7 genes coding for the first and the last enzymes of RF biosynthesis in the model flavinogenic organism Pichia guilliermondii. To decipher molecular mechanisms of iron-dependent repression, isolation and study of the regulatory mutants defective in corresponding regulation is desirable. However, no suitable methods for isolation of such mutants were previously available. We have produced a single-point transition mutation in the RIB1 gene. The corresponding rib1-86 mutant exhibits leaky phenotype and is unable to grow in iron-sufficient minimal medium without exogenous RF. However, it can grow in minimal iron-deficient medium without RF, or in iron-sufficient medium upon introduction of the previously-isolated regulatory mutation rib81, which leads to increase in RF production. Using the rib1-86 mutant as parental strain, a collection of mutants able to grow in iron-sufficient medium without exogenous RF has been isolated. The mutants appeared to be defective in regulation of RF biosynthesis and iron homeostasis and were divided into six new complementation groups. Study of one corresponding mutant, red6, showed derepression of RIB1 mRNA synthesis in iron-sufficient medium. PMID:15925311

  19. Mus308 Mutants of Drosophila Exhibit Hypersensitivity to DNA Cross-Linking Agents and Are Defective in a Deoxyribonuclease

    PubMed Central

    Boyd, J. B.; Sakaguchi, K.; Harris, P. V.

    1990-01-01

    Mutagen-sensitive strains that identify 16 different Drosophila genes have been screened for alterations in DNA metabolic enzymes. A characteristic defect in an acid-active deoxyribonuclease was observed in strains carrying the six available mutant alleles of the mus308 gene. Since that enzyme is detected at normal levels in a mutant strain that is deficient in the previously identified enzymes DNase 1 and DNase 2, it represents a new Drosophila nuclease that is designated Nuclease 3. The mus308 mutants were originally distinguished from all other mutagen-sensitive mutants of Drosophila because they exhibit hypersensitivity to the DNA cross-linking agent nitrogen mustard without expressing a concurrent sensitivity to the monofunctional agent methyl methanesulfonate. Further observations of hypersensitivity to the mutagens trimethylpsoralen, diepoxybutane and cis-platinum now establish a more general sensitivity of these mutants to agents capable of generating DNA cross-links. In spite of the hypersensitivity of the mus308 mutants to DNA cross-linking agents, the initial incision step of DNA cross-link repair is normal in mus308 cells as assayed by the alkaline elution procedure. The Drosophila mus308 mutants show promise of providing a useful model for analogous defects in other organisms including man. PMID:2397884

  20. The heterozygous abp1/ABP1 insertional mutant has defects in functions requiring polar auxin transport and in regulation of early auxin-regulated genes.

    PubMed

    Effendi, Yunus; Rietz, Steffen; Fischer, Urs; Scherer, Günther F E

    2011-01-01

    AUXIN-BINDING PROTEIN 1 (ABP1) is not easily accessible for molecular studies because the homozygous T-DNA insertion mutant is embryo-lethal. We found that the heterozygous abp1/ABP1 insertion mutant has defects in auxin physiology-related responses: higher root slanting angles, longer hypocotyls, agravitropic roots and hypocotyls, aphototropic hypocotyls, and decreased apical dominance. Heterozygous plants flowered earlier than wild-type plants under short-day conditions. The length of the main root, the lateral root density and the hypocotyl length were little altered in the mutant in response to auxin. Compared to wild-type plants, transcription of early auxin-regulated genes (IAA2, IAA11, IAA13, IAA14, IAA19, IAA20, SAUR9, SAUR15, SAUR23, GH3.5 and ABP1) was less strongly up-regulated in the mutant by 0.1, 1 and 10 μm IAA. Surprisingly, ABP1 was itself an early auxin-up-regulated gene. IAA uptake into the mutant seedlings during auxin treatments was indistinguishable from wild-type. Basipetal auxin transport in young roots was slower in the mutant, indicating a PIN2/EIR1 defect, while acropetal transport was indistinguishable from wild-type. In the eir1 background, three of the early auxin-regulated genes tested (IAA2, IAA13 and ABP1) were more strongly induced by 1 μm IAA in comparison to wild-type, but eight of them were less up-regulated in comparison to wild-type. Similar but not identical disturbances in regulation of early auxin-regulated genes indicate tight functional linkage of ABP1 and auxin transport regulation. We hypothesize that ABP1 is involved in the regulation of polar auxin transport, and thus affects local auxin concentration and early auxin gene regulation. In turn, ABP1 itself is under the transcriptional control of auxin.

  1. 'Green revolution' genes encode mutant gibberellin response modulators.

    PubMed

    Peng, J; Richards, D E; Hartley, N M; Murphy, G P; Devos, K M; Flintham, J E; Beales, J; Fish, L J; Worland, A J; Pelica, F; Sudhakar, D; Christou, P; Snape, J W; Gale, M D; Harberd, N P

    1999-07-15

    World wheat grain yields increased substantially in the 1960s and 1970s because farmers rapidly adopted the new varieties and cultivation methods of the so-called 'green revolution'. The new varieties are shorter, increase grain yield at the expense of straw biomass, and are more resistant to damage by wind and rain. These wheats are short because they respond abnormally to the plant growth hormone gibberellin. This reduced response to gibberellin is conferred by mutant dwarfing alleles at one of two Reduced height-1 (Rht-B1 and Rht-D1) loci. Here we show that Rht-B1/Rht-D1 and maize dwarf-8 (d8) are orthologues of the Arabidopsis Gibberellin Insensitive (GAI) gene. These genes encode proteins that resemble nuclear transcription factors and contain an SH2-like domain, indicating that phosphotyrosine may participate in gibberellin signalling. Six different orthologous dwarfing mutant alleles encode proteins that are altered in a conserved amino-terminal gibberellin signalling domain. Transgenic rice plants containing a mutant GAI allele give reduced responses to gibberellin and are dwarfed, indicating that mutant GAI orthologues could be used to increase yield in a wide range of crop species.

  2. pistillata-5, an Arabidopsis B class mutant with strong defects in petal but not in stamen development.

    PubMed

    Yang, Yingzhen; Xiang, Hongjun; Jack, Thomas

    2003-01-01

    The Arabidopsis floral organ identity genes APETALA3 (AP3) and PISTILLATA (PI) encode related DNA-binding proteins of the MADS family. Considerable evidence supports the hypothesis that a heterodimer of AP3 and PI is an essential component of B class activity. All ap3 and pi alleles characterized to date exhibit equivalent phenotypic defects in both whorls 2 and 3. In strong ap3 and pi mutants, petals and stamens are missing and sepals and carpels develop in their place. Weak ap3 and pi mutants exhibit partial conversions of petals to sepals and stamens to carpels. In this report, we describe the isolation and characterization of pi-5, an unusual B class mutant that exhibits defects in whorl 2 where sepals develop in place of petals, but third whorl stamens are most often normal. pi-5 flowers resemble those from 35S::SEP3 antisense plants. pi-5 contains missense mutation in the K domain (PIE125K). PIE125K exhibits defects in heterodimerization with its partner protein AP3. Via a reverse yeast two-hybrid screen, AP3K139E was isolated as a compensatory mutant of PIE125K. The compensatory interaction between PIE125K and AP3K139E is observed both in yeast two-hybrid assays and in planta. On its own, AP3K139E exhibits defects in specifying both petal and stamen identity. In addition, PIE125K is defective in interaction with SEPALLATA proteins in both two- and three-hybrid assays suggesting that PIE125K is defective in forming higher order complexes of MADS proteins. The decreased concentration of PI/AP3/SEP complexes offers an explanation for the petal defects observed in both pi-5 and 35S::SEP3 antisense plants. PMID:12943551

  3. Cancer-associated mutants of RNA helicase DDX3X are defective in RNA-stimulated ATP hydrolysis

    PubMed Central

    Partridge, Janet F.; Enemark, Eric J.

    2015-01-01

    The DEAD-box RNA helicase DDX3X is frequently mutated in pediatric medulloblastoma. We dissect how these mutants affect DDX3X function with structural, biochemical, and genetic experiments. We identify an N-terminal extension (“ATP-binding loop”, ABL) that is critical for the stimulation of ATP hydrolysis by RNA. We present crystal structures that suggest the ABL interacts dynamically with ATP and confirm the interaction occurs in solution by NMR chemical shift perturbation (CSP) and isothermal calorimetry (ITC). DEAD-box helicases require interaction between two conserved RecA-like helicase domains, D1 and D2 for function. We use NMR CSP to show that DDX3X interacts specifically with double-stranded RNA (dsRNA) through its D1 domain, with contact mediated by residues G302 and G325. Mutants of these residues, G302V and G325E, are associated with pediatric medulloblastoma. These mutants are defective in RNA-stimulated ATP hydrolysis. We show that DDX3X complements the growth defect in a ded1 temperature-sensitive strain of S. pombe, but the cancer-associated mutants G302V and G325E do not complement and exhibit protein expression defects. Taken together, our results suggest that impaired translation of important mRNA targets by mutant DDX3X represents a key step in the development of medulloblastoma. PMID:25724843

  4. Temperature-Sensitive Mutants of a Chinese Hamster Cell Line. I. Selection of Clones with Defective Macromolecular Biosynthesis

    PubMed Central

    Roufa, Donald J.; Reed, Susan J.

    1975-01-01

    Temperature-sensitive clones have been selected from a mutagenized culture of Chinese hamster lung cells by a procedure involving bromodeoxy-uridine (BrdU) incorporation and irradiation with black light. The selection procedure used in these studies was adapted from methods developed by others to yield mutants that cease DNA replication within a short time after they are transferred to nonpermissive temperature. After mutagenesis with ethyl methanosulfonate ten clones survived the selection procedure. Three of the clones (mutants) were temperature-sensitive as measured by growth properties. Two mutants ceased DNA synthesis within six hours of being shifted to 39° and the third mutant continued to synthesize DNA at nonpermissive temperature at a reduced rate for at least 24 hours. Thus, all three mutants survived the selection procedure for understandable reasons, since each was unable to incorporate sufficient BrdU at 39° to lethally protosensitize its DNA during the standard exposure period. The two mutants that cease DNA synthesis at high temperature (clones 115–47 and 115–53) also stop incorporating radioactive amino acids and uridine within six hours at 39°. Their complex phenotype, i.e. defective DNA, RNA and protein biosynthesis, is reversible. When these mutants were returned to 33° after 8 hours at 39°, both resumed DNA synthesis immediately (< 1 hour). Reversal of defective DNA synthesis in both mutants was sensitive to drugs that inhibit protein biosynthesis specifically. Those same drugs, as well as toxic amino acids analogs, also effected a striking mutant phenocopy in wild-type cells. The phenocopy produced by amino acid analogs that are incorporated into mammalian proteins suggested that one or more proteins must be synthesized continuously to support mammalian cells engaged in programmed DNA replication. PMID:1232024

  5. Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects

    NASA Technical Reports Server (NTRS)

    Ruegger, M.; Dewey, E.; Hobbie, L.; Brown, D.; Bernasconi, P.; Turner, J.; Muday, G.; Estelle, M.

    1997-01-01

    Polar auxin transport plays a key role in the regulation of plant growth and development. To identify genes involved in this process, we have developed a genetic procedure to screen for mutants of Arabidopsis that are altered in their response to auxin transport inhibitors. We recovered a total of 16 independent mutants that defined seven genes, called TRANSPORT INHIBITOR RESPONSE (TIR) genes. Recessive mutations in one of these genes, TIR3, result in altered responses to transport inhibitors, a reduction in polar auxin transport, and a variety of morphological defects that can be ascribed to changes in indole-3-acetic acid distribution. Most dramatically, tir3 seedlings are strongly deficient in lateral root production, a process that is known to depend on polar auxin transport from the shoot into the root. In addition, tir3 plants display a reduction in apical dominance as well as decreased elongation of siliques, pedicels, roots, and the inflorescence. Biochemical studies indicate that tir3 plants have a reduced number of N-1-naphthylphthalamic (NPA) binding sites, suggesting that the TIR3 gene is required for expression, localization, or stabilization of the NPA binding protein (NBP). Alternatively, the TIR3 gene may encode the NBP. Because the tir3 mutants have a substantial defect in NPA binding, their phenotype provides genetic evidence for a role for the NBP in plant growth and development.

  6. Photocurrent response from photonic crystal defect modes.

    PubMed

    Schartner, Stephan; Nobile, Michele; Schrenk, Werner; Andrews, Aaron M; Klang, Pavel; Strasser, Gottfried

    2008-03-31

    The authors use a quantum well intersubband photodetector fabricated into a two dimensional photonic crystal to investigate the optical defect modes of a single missing hole defect. The modes appear as a local enhancement in spectral photocurrent due to an increased in-coupling of surface incident light when a defect mode is present. The frequencies of these localized modes are tracked as they are varied by the defect geometry and compared to simulations. PMID:18542578

  7. Reversion of a transcriptionally defective MHC class II-negative human B-cell mutant.

    PubMed Central

    Ombra, M N; Perfetto, C; Autiero, M; Anzisi, A M; Pasquinelli, R; Maffei, A; Del Pozzo, G; Guardiola, J

    1993-01-01

    RJ2.2.5, a mutant derived from the human B-lymphoma cell, Raji, is unable to express the MHC class II genes because of a recessive transcriptional defect attributed to the lack of an activator function. We report the isolation of a RJ2.2.5 revertant, namely AR, in which the expression of the mRNAs encoded by these genes is restored. Comparison of the binding of nuclear extracts or of partially purified nuclear preparations from the wild-type, the mutant and the revertant cells to a conserved MHC class II promoter element, the X-box, showed no alteration in the mobility of the complexes thus formed. However, in extracts from RJ2.2.5, and other MHC class II negative cell lines, such as HeLa, the amount of complex observed was significantly higher than in wild-type Raji cells. Furthermore, the binding activity exhibited by the AR revertant was lower than that of the RJ2.2.5 and higher than that of Raji. The use of specific monoclonal antibodies indicated that in all cases c-Jun and c-Fos or antigenically related proteins were required for binding. An inverse correlation between the level of DNA-protein complex formed and the level of MHC class II gene mRNA expressed in the three cell lines was apparent, suggesting that overexpression of a DNA binding factor forming complexes with class II promoter elements may cause repression of MHC class II transcription. A model which reconciles the previously ascertained recessivity of the phenotype of the mutation carried by RJ2.2.5 with the findings reported here is discussed. Images PMID:8441650

  8. Analysis of the DNA replication competence of the xrs-5 mutant cells defective in Ku86.

    PubMed

    Matheos, Diamanto; Novac, Olivia; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2003-01-01

    The radiosensitive mutant xrs-5, a derivative of the Chinese hamster ovary (CHO) K1 cell line, is defective in DNA double-strand break repair and V(D)J recombination. The defective phenotypes of xrs-5 cells are complemented by the 86 kDa subunit of Ku antigen. OBA is a protein, previously purified from HeLa cells, that binds in a sequence-specific manner to mammalian origins of DNA replication. The DNA-binding subunit of OBA has been identified as Ku86. We tested the xrs-5 cell line for its ability to replicate a mammalian origin-containing plasmid, p186, in vivo and in vitro. In vivo, the p186 episomal DNA replication in transfected xrs-5 cells was reduced by 45% when compared with the CHO K1 cells transfected with p186. In vitro, although total and cytoplasmic cell extracts from xrs-5 cells replicated the p186 with the same efficiency as the parental CHO K1 cell extracts, xrs-5 nuclear extracts did not possess any detectable replication activity. Addition of affinity-purified OBA/Ku restored replication in the xrs-5 nuclear extract reaction. Western blot analyses showed that the levels of other replication proteins (Orc2, PCNA, DNA polymerase epsilon and delta, Primase and Topoisomerase IIalpha) were comparable in both the xrs-5 mutant and CHO K1 wild-type cell lines. In addition, the in vivo association of Ku with the DHFR origin-containing sequence (oribeta) was examined in both the CHO K1 and xrs-5 cell lines by a chromatin immunoprecipitation (ChIP) assay. Anti-Ku antibodies did not immunoprecipitate a detectable amount of Ku from the xrs-5 cells in the origin-containing sequence, in contrast to the CHO K1 cells, wherein Ku was found to be associated with the oribeta origin. The data implicate Ku antigen in in vivo and in vitro DNA replication and suggest the existence of another protein with Ku-like functions in the xrs-5 cells.

  9. Molecular basis for the transformation defects in mutants of Haemophilus influenzae.

    PubMed

    Notani, N K; Setlow, J K; Joshi, V R; Allison, D P

    1972-06-01

    To determine the molecular basis of transformation defects in Haemophilus influenzae, the fate of genetically marked, (32)P-labeled, heavy deoxyribonucleic acid (DNA) was examined in three mutant strains (rec(1) (-), rec(2) (-), and KB6) and in wild type having (3)H-labeled DNA and a second genetic marker. Transforming cells upon lysis with digitonin followed by low-speed centrifugation are separable into the supernatant fraction, containing mainly the unintegrated donor DNA, and the pellet, containing most of the resident DNA along with integrated donor DNA. Electron micrographs of digitonin-treated cells also indicate that the resident DNA is trapped inside a cellular structure but that cytoplasmic elements such as ribosomes are extensively released. DNA synthesis in digitonin-treated cells is immediately blocked, as is any further integration of donor DNA into the resident genome. Isopycnic and sedimentation analysis of supernatant fluids and pellets revealed that in strains rec(2) (-) and KB6 there is little or no association between donor and resident DNA, and thus there is negligible transfer of donor DNA genetic information. In these strains, the donor DNA is not broken into pieces of lower molecular weight as it is in strain rec(1) (-) and in the wild type, both of which show association between donor and recipient DNA. In strain rec(1) (-), although some donor DNA atoms become covalently linked to resident DNA, the incorporated material does not have the donor DNA transforming activity. PMID:4537421

  10. A defective signal peptide in the maize high-lysine mutant floury 2.

    PubMed Central

    Coleman, C E; Lopes, M A; Gillikin, J W; Boston, R S; Larkins, B A

    1995-01-01

    The maize floury 2 (fl2) mutation enhances the lysine content of the grain, but the soft texture of the endosperm makes it unsuitable for commercial production. The mutant phenotype is linked with the appearance of a 24-kDa alpha-zein protein and increased synthesis of binding protein, both of which are associated with irregularly shaped protein bodies. We have cloned the gene encoding the 24-kDa protein and show that it is expressed as a 22-kDa alpha-zein with an uncleaved signal peptide. Comparison of the deduced N-terminal amino acid sequence of the 24-kDa alpha-zein protein with other alpha-zeins revealed an alanine to valine substitution at the C-terminal position of the signal peptide, a histidine insertion within the seventh alpha-helical repeat, and an alanine to threonine substitution with the same alpha-helical repeat of the protein. Structural defects associated with this alpha-zein explain many of the phenotypic effects of the fl2 mutation. Images Fig. 1 Fig. 2 Fig. 5 PMID:7624327

  11. Disrupting ER-associated protein degradation suppresses the abscission defect of a weak hae hsl2 mutant in Arabidopsis

    PubMed Central

    Baer, John; Taylor, Isaiah; Walker, John C.

    2016-01-01

    In Arabidopsis thaliana, the process of abscission, or the shedding of unwanted organs, is mediated by two genes, HAESA (HAE) and HAESA-LIKE 2 (HSL2), encoding receptor-like protein kinases (RLKs). The double loss-of-function mutant hae-3 hsl2-3 is completely deficient in floral abscission, but, interestingly, the hae-3 hsl2-9 mutant displays a less severe defect. This mutant was chosen for an ethyl methanesulfonate (EMS) screen to isolate enhancer and suppressor mutants, and two such suppressors are the focus of this study. Pooled DNA from the F2 generation of a parental backcross was analyzed by genome sequencing to reveal candidate genes, two of which complement the suppressor phenotype. These genes, EMS-MUTAGENIZED BRI1 SUPPRESSOR 3 (EBS3) and EBS4, both encode mannosyltransferases involved in endoplasmic reticulum (ER)-associated degradation (ERAD) of proteins. Further analysis of these suppressor lines revealed that suppressor mutations are acting solely on the partially functional hsl2-9 mutant receptor to modify the abscission phenotype. Expressing a hsl2-9–yellow fluorescent protein (YFP) transgene in ebs3 mutants yields a higher fluorescent signal than in EBS3/ebs3, suggesting that these mutants restore abscission by disrupting ERAD to allow accumulation of the hsl2-9 receptor, which probably escapes degradation to be trafficked to the plasma membrane to regain signaling. PMID:27566817

  12. Purification of a. beta. -amylase that accumulates in Arabidopsis thaliana mutants defective in starch metabolism. [Arabidopsis thaliana

    SciTech Connect

    Monroe, J.D.; Preiss, J. )

    1990-11-01

    Amylase activity is elevated 5- to 10-fold in leaves of several different Arabidopsis thaliana mutants defective in starch metabolism when they are grown under a 12-hour photoperiod. Activity is also increased when plants are grown under higher light intensity. It was previously determined that the elevated activity was an extrachloroplastic {beta}-(exo)amylase. Due to the location of this enzyme outside the chloroplast, its function is not known. The enzyme was purified to homogeneity from leaves of both a starchless mutant deficient in plastid phosphoglucomutase and from the wild type using polyethylene glycol fractionation and cyclohexaamylose affinity chromatography. The molecular mass of the {beta}-amylase from both sources was 55,000 daltons as determined by denaturing gel electrophoresis. Gel filtration studies indicated that the enzyme was a monomer. The specific activities of the purified protein from mutant and wild-type sources, their substrate specificities, and K{sub m} for amylopectin were identical. Based on these results it was concluded that the mutant contained an increased level of {beta}-amylase protein. Enzyme neutralization studies using a polyclonal antiserum raised to purified {beta}-amylase showed that in each of two starchless mutants, one starch deficient mutant and one starch overproducing mutant, the elevated amylase activity was due to elevated {beta}-amylase protein.

  13. 49 CFR 573.5 - Defect and noncompliance responsibility.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 7 2011-10-01 2011-10-01 false Defect and noncompliance responsibility. 573.5 Section 573.5 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY... RESPONSIBILITY AND REPORTS § 573.5 Defect and noncompliance responsibility. (a) Each manufacturer of a...

  14. 49 CFR 573.5 - Defect and noncompliance responsibility.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 7 2010-10-01 2010-10-01 false Defect and noncompliance responsibility. 573.5 Section 573.5 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY... RESPONSIBILITY AND REPORTS § 573.5 Defect and noncompliance responsibility. (a) Each manufacturer of a...

  15. A mutant of Escherichia coli defective in penicillin-binding protein 5 and lacking D-alanine carboxypeptidase IA.

    PubMed Central

    Nishimura, Y; Suzuki, H; Hirota, Y; Park, J T

    1980-01-01

    A mutant of Escherichia coli defective in penicillin-binding protein 5 activity was isolated. The mutation (pfv) was shown to be located at 14.0 min on the E. coli chromosome map. Loss of penicillin-binding protein 5 in the pfv mutant was associated with the loss of D-alanine carboxypeptidase IA activity and increased sensitivity to beta-lactam antibiotics. We conclude that penicillin-binding protein 5 catalyzes the major D-alanine carboxypeptidase IA activity and that the enzyme activity, in vivo, protects E. coli cells from killing by low inhibitory concentrations of beta-lactam antibiotics. PMID:6995448

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

    PubMed Central

    Wright, David J.; Tate, Christopher G.

    2015-01-01

    The tetracycline antiporter TetA(B) is a member of the Major Facilitator Superfamily which confers tetracycline resistance to cells by coupling the efflux of tetracycline to the influx of protons down their chemical potential gradient. Although it is a medically important transporter, its structure has yet to be determined. One possibility for why this has proven difficult is that the transporter may be conformationally heterogeneous in the purified state. To overcome this, we developed two strategies to rapidly identify TetA(B) mutants that were transport-defective and that could still bind tetracycline. Up to 9 amino acid residues could be deleted from the loop between transmembrane α-helices 6 and 7 with only a slight decrease in affinity of tetracycline binding as measured by isothermal titration calorimetry, although the mutant was transport-defective. Scanning mutagenesis where all the residues between 2 and 389 were mutated to either valine, alanine or glycine (VAG scan) identified 15 mutants that were significantly impaired in tetracycline transport. Of these mutants, 12 showed no evidence of tetracycline binding by isothermal titration calorimetry performed on the purified transporters. In contrast, the mutants G44V and G346V bound tetracycline 4–5 fold more weakly than TetA(B), with Kds of 28 μM and 36 μM, respectively, whereas the mutant R70G bound tetracycline 3-fold more strongly (Kd 2.1 μM). Systematic mutagenesis is thus an effective strategy for isolating transporter mutants that may be conformationally constrained and which represent attractive targets for crystallisation and structure determination. PMID:26143388

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

    PubMed

    Wright, David J; Tate, Christopher G

    2015-10-01

    The tetracycline antiporter TetA(B) is a member of the Major Facilitator Superfamily which confers tetracycline resistance to cells by coupling the efflux of tetracycline to the influx of protons down their chemical potential gradient. Although it is a medically important transporter, its structure has yet to be determined. One possibility for why this has proven difficult is that the transporter may be conformationally heterogeneous in the purified state. To overcome this, we developed two strategies to rapidly identify TetA(B) mutants that were transport-defective and that could still bind tetracycline. Up to 9 amino acid residues could be deleted from the loop between transmembrane α-helices 6 and 7 with only a slight decrease in affinity of tetracycline binding as measured by isothermal titration calorimetry, although the mutant was transport-defective. Scanning mutagenesis where all the residues between 2 and 389 were mutated to either valine, alanine or glycine (VAG scan) identified 15 mutants that were significantly impaired in tetracycline transport. Of these mutants, 12 showed no evidence of tetracycline binding by isothermal titration calorimetry performed on the purified transporters. In contrast, the mutants G44V and G346V bound tetracycline 4-5 fold more weakly than TetA(B), with Kds of 28 μM and 36 μM, respectively, whereas the mutant R70G bound tetracycline 3-fold more strongly (Kd 2.1 μM). Systematic mutagenesis is thus an effective strategy for isolating transporter mutants that may be conformationally constrained and which represent attractive targets for crystallisation and structure determination. PMID:26143388

  18. Bone defects and future regenerative nanomedicine approach using stem cells in the mutant Tabby mouse model.

    PubMed

    Noordijk, Mélanie; Davideau, Jean-Luc; Eap, Sandy; Huck, Olivier; Fioretti, Florence; Stoltz, Jean-François; Bacon, William; Benkirane-Jessel, Nadia; Clauss, François

    2015-01-01

    X-linked Hypohidrotic Ectodermal Dysplasia (XLHED) is associated to a large spectrum of ectodermal and extra-ectodermal symptoms, especially craniofacial bone morphological, structural and metabolic anomalies. This skeletal phenotype described in affected patients and in the Ta mutant mouse model leads to craniofacial dysmorphies, endosseous implants and jaw bone grafts complications. Bone tissue bioengineering based on the use of PCL synthetic nanofibrous membrane and BMP nanoreservoirs appears as an original and promising approach to prevent such complications in the context of dysfunctional bone. Use of osteoblasts or stem cells seeded biomembranes appears as another strategy developed on the Tabby (Ta) model of XLHED. The Ta mouse experimental model is used to study the jaw bone response during the post-operative period after bone lesion and placement of synthetic PCL membrane functionalized with nanoreservoirs embedding different BMPs dimers or seeded with living cells. PMID:25538062

  19. Medicago truncatula Mtha1-2 mutants loose metabolic responses to mycorrhizal colonization.

    PubMed

    Hubberten, Hans-Michael; Sieh, Daniela; Zöller, Daniela; Hoefgen, Rainer; Krajinski, Franziska

    2015-01-01

    Bidirectional nutrient transfer is one of the key features of the arbuscular mycorrhizal symbiosis. Recently we were able to identify a Medicago truncatula mutant (mtha1-2) that is defective in the uptake of phosphate from the periarbuscular space due to a lack of the energy providing proton gradient provided by the symbiosis specific proton ATPase MtHA1 In order to further characterize the impact of fungal colonization on the plant metabolic status, without the beneficial aspect of improved mineral nutrition, we performed leaf ion analyses in mutant and wildtype plants with and without fungal colonization. Although frequency of fungal colonization was unaltered, the mutant did not show a positive growth response to mycorrhizal colonization. This indicates that nutrient transfer into the plant cell fails in the truncated arbuscules due to lacking expression of a functional MtHA1 protein. The leaves of wildtype plants showed clear metabolic responses to root mycorrhizal colonization, whereas no changes of leaf metabolite levels of mycorrhizal mtha1-2 plants were detected, even though they were colonized. These results show that MtHa1 is indispensable for a functional mycorrhizal symbiosis and, moreover, suggest that fungal root colonization per se does not depend on nutrient transfer to the plant host. PMID:25751449

  20. Diversification of Photoperiodic Response Patterns in a Collection of Early-Flowering Mutants of Arabidopsis1[W

    PubMed Central

    Pouteau, Sylvie; Carré, Isabelle; Gaudin, Valérie; Ferret, Valérie; Lefebvre, Delphine; Wilson, Melanie

    2008-01-01

    Many plant species exhibit seasonal variation of flowering time in response to daylength. Arabidopsis (Arabidopsis thaliana) flowers earlier under long days (LDs) than under short days (SDs). This quantitative response to photoperiod is characterized by two parameters, the critical photoperiod (Pc), below which there is a delay in flowering, and the ceiling photoperiod (Pce), below which there is no further delay. Thus Pc and Pce define the thresholds beyond which maximum LD and SD responses are observed, respectively. We studied the quantitative response to photoperiod in 49 mutants selected for early flowering in SDs. Nine of these mutants exhibited normal Pce and Pc, showing that their precocious phenotype was not linked to abnormal measurement of daylength. However, we observed broad diversification in the patterns of quantitative responses in the other mutants. To identify factors involved in abnormal measurement of daylength, we analyzed the association of these various patterns with morphogenetic and rhythmic defects. A high proportion of mutants with altered Pce exhibited abnormal hypocotyl elongation in the dark and altered circadian periods of leaf movements. This suggested that the circadian clock and negative regulators of photomorphogenesis may contribute to the specification of SD responses. In contrast, altered Pc correlated with abnormal hypocotyl elongation in the light and reduced photosynthetic light-input requirements for bolting. This indicated that LD responses may be specified by positive elements of light signal transduction pathways and by regulators of resource allocation. Furthermore, the frequency of circadian defects in mutants with normal photoperiodic responses suggested that the circadian clock may regulate the number of leaves independently of its effect on daylength perception. PMID:18799658

  1. High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis.

    PubMed

    Sridharan, Vinod; Heimiller, Joseph; Robida, Mark D; Singh, Ravinder

    2016-01-01

    The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during spermatogenesis. The heph2 mutation in this gene results in a specific defect in spermatogenesis, causing aberrant spermatid individualization and male sterility. However, the array of molecular defects in the mutant remains uncharacterized. Using an unbiased high throughput sequencing approach, we have identified transcripts that are misregulated in this mutant. Aberrant transcripts show altered expression levels, exon skipping, and alternative 5' ends. We independently verified these findings by reverse-transcription and polymerase chain reaction (RT-PCR) analysis. Our analysis shows misregulation of transcripts that have been connected to spermatogenesis, including components of the actomyosin cytoskeletal apparatus. We show, for example, that the Myosin light chain 1 (Mlc1) transcript is aberrantly spliced. Furthermore, bioinformatics analysis reveals that Mlc1 contains a high affinity binding site(s) for dmPTB and that the site is conserved in many Drosophila species. We discuss that Mlc1 and other components of the actomyosin cytoskeletal apparatus offer important molecular links between the loss of dmPTB function and the observed developmental defect in spermatogenesis. This study provides the first comprehensive list of genes misregulated in vivo in the heph2 mutant in Drosophila and offers insight into the role of dmPTB during spermatogenesis.

  2. High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis

    PubMed Central

    Sridharan, Vinod; Heimiller, Joseph; Robida, Mark D.; Singh, Ravinder

    2016-01-01

    The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during spermatogenesis. The heph2 mutation in this gene results in a specific defect in spermatogenesis, causing aberrant spermatid individualization and male sterility. However, the array of molecular defects in the mutant remains uncharacterized. Using an unbiased high throughput sequencing approach, we have identified transcripts that are misregulated in this mutant. Aberrant transcripts show altered expression levels, exon skipping, and alternative 5’ ends. We independently verified these findings by reverse-transcription and polymerase chain reaction (RT-PCR) analysis. Our analysis shows misregulation of transcripts that have been connected to spermatogenesis, including components of the actomyosin cytoskeletal apparatus. We show, for example, that the Myosin light chain 1 (Mlc1) transcript is aberrantly spliced. Furthermore, bioinformatics analysis reveals that Mlc1 contains a high affinity binding site(s) for dmPTB and that the site is conserved in many Drosophila species. We discuss that Mlc1 and other components of the actomyosin cytoskeletal apparatus offer important molecular links between the loss of dmPTB function and the observed developmental defect in spermatogenesis. This study provides the first comprehensive list of genes misregulated in vivo in the heph2 mutant in Drosophila and offers insight into the role of dmPTB during spermatogenesis. PMID:26942929

  3. Arabidopsis RAB geranylgeranyl transferase beta-subunit mutant is constitutively photomorphogenic, and has shoot growth and gravitropic defects.

    PubMed

    Hála, Michal; Soukupová, Hana; Synek, Lukás; Zárský, Viktor

    2010-05-01

    RAB GTPases are important directional regulators of intracellular vesicle transport. Membrane localization of RAB GTPases is mediated by C-terminal double geranylgeranylation. This post-translational modification is catalyzed by the alpha-beta-heterodimer catalytic core of RAB geranylgeranyl transferase (RAB-GGT), which cooperates with the RAB escort protein (REP) that presents a nascent RAB. Here, we show that RAB-geranylgeranylation activity is significantly reduced in two homozygous mutants of the major Arabidopsis beta-subunit of RAB-GGT (AtRGTB1), resulting in unprenylated RAB GTPases accumulation in the cytoplasm. Both endocytosis and exocytosis are downregulated in rgtb1 homozygotes defective in shoot growth and morphogenesis. Root gravitropism is normal in rgtb1 roots, but is significantly compromised in shoots. Mutants are defective in etiolation and show constitutive photomorphogenic phenotypes that cannot be rescued by brassinosteroid treatment, similarly to the det3 mutant that is also defective in the secretory pathway. Transcriptomic analysis revealed an upregulation of specific RAB GTPases in etiolated wild-type plants. Taken together, these data suggest that the downregulation of the secretory pathway is interpreted as a photomorphogenic signal in Arabidopsis.

  4. Brucellosis Vaccines: Assessment of Brucella melitensis Lipopolysaccharide Rough Mutants Defective in Core and O-Polysaccharide Synthesis and Export

    PubMed Central

    González, David; Grilló, María-Jesús; De Miguel, María-Jesús; Ali, Tara; Arce-Gorvel, Vilma; Delrue, Rose-May; Conde-Álvarez, Raquel; Muñoz, Pilar; López-Goñi, Ignacio; Iriarte, Maite; Marín, Clara-M.; Weintraub, Andrej; Widmalm, Göran; Zygmunt, Michel; Letesson, Jean-Jacques; Gorvel, Jean-Pierre; Blasco, José-María; Moriyón, Ignacio

    2008-01-01

    Background The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. Methodology/Principal Findings To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. Conclusions/Significance The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are

  5. Suppression of Proliferative Defects Associated with Processing-defective Lamin A Mutants by hTERT or Inactivation of p53

    PubMed Central

    Kudlow, Brian A.; Stanfel, Monique N.; Burtner, Christopher R.; Johnston, Elijah D.

    2008-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare, debilitating disease with early mortality and rapid onset of aging-associated pathologies. It is linked to mutations in LMNA, which encodes A-type nuclear lamins. The most frequent HGPS-associated LMNA mutation results in a protein, termed progerin, with an internal 50 amino acid deletion and, unlike normal A-type lamins, stable farnesylation. The cellular consequences of progerin expression underlying the HGPS phenotype remain poorly understood. Here, we stably expressed lamin A mutants, including progerin, in otherwise identical primary human fibroblasts to compare the effects of different mutants on nuclear morphology and cell proliferation. We find that expression of progerin leads to inhibition of proliferation in a high percentage of cells and slightly premature senescence in the population. Expression of a stably farnesylated mutant of lamin A phenocopied the immediate proliferative defects but did not result in premature senescence. Either p53 inhibition or, more surprisingly, expression of the catalytic subunit of telomerase (hTERT) suppressed the early proliferative defects associated with progerin expression. These findings lead us to propose that progerin may interfere with telomere structure or metabolism in a manner suppressible by increased telomerase levels and possibly link mechanisms leading to progeroid phenotypes to those of cell immortalization. PMID:18843043

  6. Mutants of PC12 cells with altered cyclic AMP responses

    SciTech Connect

    Block, T.; Kon, C.; Breckenridge, B.M.

    1984-10-01

    PCl2 cells, derived from a rat pheochromocytoma, were mutagenized and selected in media containing agents known to elevate intracellular concentrations of cyclic AMP (cAMP). More than 40 clones were isolated by selection with cholera toxin or 2-chloroadenosine or both. The variants that were deficient in accumulating cAMP were obtained by using a protocol in which 1 ..mu..m 8-bromo-cAMP was included in addition to the agonist. Certain of these variants were partially characterized with respect to the site of altered cAMP metabolism. The profiles of adenylate cyclase activity responsiveness of certain variants to guanosine-5'-(BETA,..gamma..-imido) triphosphate and to forskolin resembled those of UNC and cyc phenotypes of S49 lymphoma cells, which are functionally deficient in the GTP-sensitive coupling protein, N/sub s/. Other variants were characterized by increased cyclic nucleotide phosphodiesterase activity at low substrate concentration. Diverse morphological traits were observed among the variants, but it was not possible to assign them to a particular cAMP phenotype. Two revertants of a PCl2 mutant were isolated and observed to have regained a cellular cAMP response to 2-chloroadenosine and to forskolin. It is hoped that these PCl2 mutants will have utility for defining cAMP-mediated functions, including any links to the action of nerve growth factor, in cells derived from the neural crest.

  7. Mirror movement-like defects in startle behavior of zebrafish dcc mutants are caused by aberrant midline guidance of identified descending hindbrain neurons.

    PubMed

    Jain, Roshan A; Bell, Hannah; Lim, Amy; Chien, Chi-Bin; Granato, Michael

    2014-02-19

    Mirror movements are involuntary movements on one side of the body that occur simultaneously with intentional movements on the contralateral side. Humans with heterozygous mutations in the axon guidance receptor DCC display such mirror movements, where unilateral stimulation results in inappropriate bilateral motor output. Currently, it is unclear whether mirror movements are caused by incomplete midline crossing and reduced commissural connectivity of DCC-dependent descending pathways or by aberrant ectopic ipsilateral axonal projections of normally commissural neurons. Here, we show that in response to unilateral tactile stimuli, zebrafish dcc mutant larvae perform involuntary turns on the inappropriate body side. We show that these mirror movement-like deficits are associated with axonal guidance defects of two identified groups of commissural reticulospinal hindbrain neurons. Moreover, we demonstrate that in dcc mutants, axons of these identified neurons frequently fail to cross the midline and instead project ipsilaterally. Whereas laser ablation of these neurons in wild-type animals does not affect turning movements, their ablation in dcc mutants restores turning movements. Thus, our results demonstrate that in dcc mutants, turns on the inappropriate side of the body are caused by aberrant ipsilateral axonal projections, and suggest that aberrant ipsilateral connectivity of a very small number of descending axons is sufficient to induce incorrect movement patterns.

  8. A Carotenoid-Deficient Mutant in Pantoea sp. YR343, a Bacteria Isolated from the Rhizosphere of Populus deltoides, Is Defective in Root Colonization.

    PubMed

    Bible, Amber N; Fletcher, Sarah J; Pelletier, Dale A; Schadt, Christopher W; Jawdy, Sara S; Weston, David J; Engle, Nancy L; Tschaplinski, Timothy; Masyuko, Rachel; Polisetti, Sneha; Bohn, Paul W; Coutinho, Teresa A; Doktycz, Mitchel J; Morrell-Falvey, Jennifer L

    2016-01-01

    The complex interactions between plants and their microbiome can have a profound effect on the health and productivity of the plant host. A better understanding of the microbial mechanisms that promote plant health and stress tolerance will enable strategies for improving the productivity of economically important plants. Pantoea sp. YR343 is a motile, rod-shaped bacterium isolated from the roots of Populus deltoides that possesses the ability to solubilize phosphate and produce the phytohormone indole-3-acetic acid (IAA). Pantoea sp. YR343 readily colonizes plant roots and does not appear to be pathogenic when applied to the leaves or roots of selected plant hosts. To better understand the molecular mechanisms involved in plant association and rhizosphere survival by Pantoea sp. YR343, we constructed a mutant in which the crtB gene encoding phytoene synthase was deleted. Phytoene synthase is responsible for converting geranylgeranyl pyrophosphate to phytoene, an important precursor to the production of carotenoids. As predicted, the ΔcrtB mutant is defective in carotenoid production, and shows increased sensitivity to oxidative stress. Moreover, we find that the ΔcrtB mutant is impaired in biofilm formation and production of IAA. Finally we demonstrate that the ΔcrtB mutant shows reduced colonization of plant roots. Taken together, these data suggest that carotenoids are important for plant association and/or rhizosphere survival in Pantoea sp. YR343. PMID:27148182

  9. A Carotenoid-Deficient Mutant in Pantoea sp. YR343, a Bacteria Isolated from the Rhizosphere of Populus deltoides, Is Defective in Root Colonization

    PubMed Central

    Bible, Amber N.; Fletcher, Sarah J.; Pelletier, Dale A.; Schadt, Christopher W.; Jawdy, Sara S.; Weston, David J.; Engle, Nancy L.; Tschaplinski, Timothy; Masyuko, Rachel; Polisetti, Sneha; Bohn, Paul W.; Coutinho, Teresa A.; Doktycz, Mitchel J.; Morrell-Falvey, Jennifer L.

    2016-01-01

    The complex interactions between plants and their microbiome can have a profound effect on the health and productivity of the plant host. A better understanding of the microbial mechanisms that promote plant health and stress tolerance will enable strategies for improving the productivity of economically important plants. Pantoea sp. YR343 is a motile, rod-shaped bacterium isolated from the roots of Populus deltoides that possesses the ability to solubilize phosphate and produce the phytohormone indole-3-acetic acid (IAA). Pantoea sp. YR343 readily colonizes plant roots and does not appear to be pathogenic when applied to the leaves or roots of selected plant hosts. To better understand the molecular mechanisms involved in plant association and rhizosphere survival by Pantoea sp. YR343, we constructed a mutant in which the crtB gene encoding phytoene synthase was deleted. Phytoene synthase is responsible for converting geranylgeranyl pyrophosphate to phytoene, an important precursor to the production of carotenoids. As predicted, the ΔcrtB mutant is defective in carotenoid production, and shows increased sensitivity to oxidative stress. Moreover, we find that the ΔcrtB mutant is impaired in biofilm formation and production of IAA. Finally we demonstrate that the ΔcrtB mutant shows reduced colonization of plant roots. Taken together, these data suggest that carotenoids are important for plant association and/or rhizosphere survival in Pantoea sp. YR343. PMID:27148182

  10. luxS mutants of Serratia defective in autoinducer-2-dependent 'quorum sensing' show strain-dependent impacts on virulence and production of carbapenem and prodigiosin.

    PubMed

    Coulthurst, Sarah J; Kurz, C Léopold; Salmond, George P C

    2004-06-01

    The enzyme LuxS is responsible for the production of autoinducer-2 (AI-2), a molecule that has been implicated in quorum sensing in many bacterial species. This study investigated whether there is a luxS-dependent signalling system in the Gram-negative bacteria Serratia spp. Serratia marcescens is a broad-host-range pathogen and an important cause of nosocomial infections. Production of AI-2 activity was detected in S. marcescens ATCC 274 and Serratia ATCC 39006 and their luxS genes were sequenced. luxS mutants were constructed in these strains and were analysed to determine which phenotypes are regulated by luxS and therefore, potentially, by AI-2. The phenotypes of the luxS mutants included decreased carbapenem antibiotic production in Serratia ATCC 39006 and decreased prodigiosin and secreted haemolysin production in S. marcescens ATCC 274. The luxS mutant of S. marcescens ATCC 274 was also found to exhibit modestly reduced virulence in a Caenorhabditis elegans model. Finally, it was shown that the culture supernatant of a wild-type strain contains a signal, presumably AI-2, capable of complementing the prodigiosin defect of the luxS mutant of another strain, even when substantially diluted. It is concluded that luxS modulates virulence and antibiotic production in Serratia, in a strain-dependent manner, and that, for at least one phenotype, this regulation is via extracellular signalling.

  11. A Carotenoid-Deficient Mutant in Pantoea sp. YR343, a Bacteria Isolated from the Rhizosphere of Populus deltoides, Is Defective in Root Colonization

    DOE PAGES

    Bible, Amber; Fletcher, Sarah J; Pelletier, Dale A; Schadt, Christopher Warren; Jawdy, Sara; Weston, David; Engle, Nancy L.; Tschaplinski, Timothy J.; Masyuko, Rachel; Polisetti, Sneha; et al

    2016-04-18

    The complex interactions between plants and their microbiome can have a profound effect on the health and productivity of the plant host. A better understanding of the microbial mechanisms that promote plant health and stress tolerance will enable strategies for improving the productivity of economically-important plants. Pantoea sp. YR343 is a motile, rod-shaped bacterium isolated from the roots of Populus deltoides that possesses the ability to solubilize phosphate and produce the phytohormone indole-3-acetic acid. Pantoea sp. YR343 readily colonizes plant roots and does not appear to be pathogenic when applied to the leaves or roots of selected plant hosts. Tomore » better understand the molecular mechanisms involved in plant association and rhizosphere survival by Pantoea sp. YR343, we constructed a mutant in which the crtB gene encoding phytoene synthase was deleted. Phytoene synthase is responsible for converting geranylgeranyl pyrophosphate to phytoene, an important precursor to the production of carotenoids. As predicted, the ΔcrtB mutant is defective in carotenoid production, and shows increased sensitivity to oxidative stress. Moreover, we find that the ΔcrtB mutant is impaired in biofilm formation and production of indole-3-acetic acid. Finally we demonstrate that the ΔcrtB mutant shows reduced colonization of plant roots. Taken together, these data suggest that carotenoids are important for plant association and/or rhizosphere survival in Pantoea sp. YR343.« less

  12. SPN1, a conserved gene identified by suppression of a postrecruitment-defective yeast TATA-binding protein mutant.

    PubMed Central

    Fischbeck, Julie A; Kraemer, Susan M; Stargell, Laurie A

    2002-01-01

    Little is known about TATA-binding protein (TBP) functions after recruitment to the TATA element, although several TBP mutants display postrecruitment defects. Here we describe a genetic screen for suppressors of a postrecruitment-defective TBP allele. Suppression was achieved by a single point mutation in a previously uncharacterized Saccharomyces cerevisiae gene, SPN1 (suppresses postrecruitment functions gene number 1). SPN1 is an essential yeast gene that is highly conserved throughout evolution. The suppressing mutation in SPN1 substitutes an asparagine for an invariant lysine at position 192 (spn1(K192N)). The spn1(K192N) strain is able to suppress additional alleles of TBP that possess postrecruitment defects, but not a TBP allele that is postrecruitment competent. In addition, Spn1p does not stably associate with TFIID in vivo. Cells containing the spn1(K192N) allele exhibit a temperature-sensitive phenotype and some defects in activated transcription, whereas constitutive transcription appears relatively robust in the mutant background. Consistent with an important role in postrecruitment functions, transcription from the CYC1 promoter, which has been shown to be regulated by postrecruitment mechanisms, is enhanced in spn1(K192N) cells. Moreover, we find that SPN1 is a member of the SPT gene family, further supporting a functional requirement for the SPN1 gene product in transcriptional processes. PMID:12524336

  13. The mur4 mutant of arabidopsis is partially defective in the de novo synthesis of uridine diphospho L-arabinose

    SciTech Connect

    Burget, E.G.; Reiter, W.D.

    1999-10-01

    To obtain information on the synthesis and function of arabinosylated glycans, the mur4 mutant of arabidopsis was characterized. This mutation leads to a 50% reduction in the monosaccharide L-arabinose in most organs and affects arabinose-containing pectic cell wall polysaccharides and arabinogalactan proteins. Feeding L-arabinose to mur4 plants restores the cell wall composition to wild-type levels, suggesting a partial defect in the de novo synthesis of UDP-L-arabinose, the activated sugar used by arabinosyltransferases. The defect was traced to the conversion of UDP-D-xylose to UDP-L-arabinose in the microsome fraction of leaf material, indicating that mur4 plants are defective in a membrane-bound UDP-D-xylose 4-epimerase.

  14. Yoghurt fermented by Lactobacillus delbrueckii subsp. bulgaricus H+ -ATPase-defective mutants exhibits enhanced viability of Bifidobacterium breve during storage.

    PubMed

    Ongol, Martin Patrick; Sawatari, Yuki; Ebina, Yoshiko; Sone, Teruo; Tanaka, Michiko; Tomita, Fusao; Yokota, Atsushi; Asano, Kozo

    2007-05-30

    Persistent acid production by Lactobacillus delbrueckii subsp. bulgaricus during refrigerated storage is a major cause of reduced viability of probiotic strains such as Bifidobacterium breve in yoghurt. It was established that H+ -ATPase-defective mutants of lactic acid bacteria have reduced growth and metabolism in low pH environments. Therefore, the aim of this study was to evaluate inhibition of post-acidification and maintenance of B. breve viability in yoghurt fermented by L. delbrueckii subsp. bulgaricus mutants with reduced membrane-bound H+ -ATPase activity during refrigerated storage. Spontaneous neomycin mutants of L. delbrueckii subsp. bulgaricus that had a significantly (P < or = 0.05) reduced H+ -ATPase activity were successfully isolated. Yoghurt fermented using L. delbrueckii subsp. bulgaricus SBT0164 No. 55-1 (mutant) starter culture had markedly reduced post-acidification and maintained viability (> or = 10(8) CFU/ml) of both Bifidobacteruim breve JCM 1192(T) and Bifidobacteruim breve JCM 7017 during storage at 10 degrees C for 21 days. These results clearly showed that yoghurt fermented by mutants of L. delbrueckii subsp. bulgaricus with reduced membrane-bound H+ -ATPase activity has reduced post-acidification that prolongs viability of B. breve in yoghurt during refrigerated storage.

  15. Kharon1 Null Mutants of Leishmania mexicana Are Avirulent in Mice and Exhibit a Cytokinesis Defect within Macrophages

    PubMed Central

    Sanchez, Marco A.; Valli, Jessica; Gluenz, Eva; Landfear, Scott M.

    2015-01-01

    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. PMID:26266938

  16. [Catabolyte repression of Escherichia coli K12 mutants with defects in different systems of glucose transport].

    PubMed

    Gershanovich, V N; Iurovitskaia, N V; Komissarova, L V; Bol'shakova, T N; Erlagaeva, R S

    1976-01-01

    The phenomenon of glucose catabolite repression was studied in E. coli mutants inable to transport this carbohydrate. The pts 1, H mutant P34 was much less sensitive to the repressive effect of glucose on beta-galactosidase synthesis than the parent type. The 1103 mutant devoid of enzyme 1 of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) behaves in the same way as P34 mutant after addition of glucose to casamino acid mineral medium. However, in minimal medium with succinate as the sole source of carbon, cells of the 1103 mutant show enhanced sensibility to transient glucose repression. The effect of hypersensibility disappears when the lac I mutation leading to constitutive the beta-galactosidase synthesis is introduced in 1103 mutant. It is shown that the enhanced sensibility of beta-galactosidase synthesis to glucose transient repression in 1103 mutant is an effect of the aburpt decrease in its growth rate in the presence of succinate and most probably this decrease leads to "inducer exclusion" of the lac operon. It is also shown that if one introduces the P34 mutation in strain JD3 devoid of one of the enzymes II for glucose (and due to this resistant to glucose catabolite respression) then the level of resistance in double mutant does not increase in spite of considerable supression of 14C glucose accumulation. In connection with this the role is discussed of separate components of the E. coli K 12 glucose transport system in realization of the phenomenon of catabolite repression. PMID:785237

  17. A Laboratory Exercise for Isolation and Characterizing Microbial Mutants with Metabolic Defects.

    ERIC Educational Resources Information Center

    Doe, Frank J.; Leslie, John F.

    1993-01-01

    Describes science experiments for undergraduate biology instruction on the concepts of mutation and characterization of the resulting mutant strains. The filamentous fungi "Fusarium moniliforme" is used to illustrate the induction of mutants (mutagenesis), identification of the mutated gene, construction of a biochemical pathway, and genetic…

  18. Neurophysiological defects in temperature-sensitive paralytic mutants of Drosophila melanogaster.

    PubMed Central

    Siddiqi, O; Benzer, S

    1976-01-01

    A new temperature-sensitive paralytic mutant of Drosophila, comatose, is compared behaviorally and physiologically with the previously known types, para and shi. All three have different properties with respect to kinetics of paralysis at high temperature and recovery from paralysis; com is hypersensitive to paralysis by cooling. Neurophysiological experimeents indicate different mechanisms for paralysis in each of the mutants. PMID:184469

  19. Two distinct genetic loci regulating class II gene expression are defective in human mutant and patient cell lines.

    PubMed Central

    Yang, Z; Accolla, R S; Pious, D; Zegers, B J; Strominger, J L

    1988-01-01

    Heterokaryons were prepared and analyzed shortly after cell fusion using two mutant class-II-negative human B cell lines (RJ 2.2.5 and 6.1.6) and a cell line (TF) from a patient with a class-II-negative Bare Lymphocyte Syndrome. The resulting transient heterokaryons were analyzed by using an anti-HLA-DR monoclonal antibody to assess the cell surface expression of HLA-DR (the major subtype of class II antigens) by immunofluorescence microscopy and by using uniformly 32P-labeled SP6 RNA probes in Northern blots and RNase protection assays to assess mRNA synthesis. We find that class II gene expression in a B cell line from a Bare Lymphocyte Syndrome patient (TF) is rescued by a B cell line which expresses class II antigens indicating that this disease, at least in part, is caused by a defect(s) in a genetic locus encoding a factor(s) necessary for class II gene expression. Secondly, reciprocal genetic complementation was demonstrated in the heterokaryons 6.1.6 x RJ 2.2.5 and TF x RJ 2.2.5 (but not in TF x 6.1.6) by detection of cell surface DR by immunofluorescence microscopy and by a novel class II mRNA typing technique which allows characterization of distinct class II alleles. Thus, the two mutants generated in vitro have defects at two different genetic loci encoding specific regulatory factors necessary for human class II gene expression. One of these mutant cell lines, but not the other, complements the defect in the patient cell line, TF. Images PMID:2458252

  20. A poliovirus 2A(pro) mutant unable to cleave 3CD shows inefficient viral protein synthesis and transactivation defects.

    PubMed Central

    Ventoso, I; Carrasco, L

    1995-01-01

    Four poliovirus mutants with modifications of tyrosine 88 in 2A(pro) were generated and introduced into the cloned poliovirus genome. Mutants Y88P and Y88L were nonviable, mutant Y88F showed a wild-type (WT) phenotype, and mutant Y88S showed a delayed cytopathic effect and formed small plaques in HeLa cells. Growth of Y88S in HeLa cells was restricted, giving rise to about 20% of the PFU production of the WT poliovirus. The 2A (Y88S) mutant synthesized significantly lower levels of viral proteins in HeLa cells than did the WT poliovirus, while the kinetics of p220 cleavage were identical for both viruses. Strikingly, the 2A (Y88S) mutant was unable to cleave 3CD, as shown by analysis of poliovirus proteins labeled with [35S]methionine or immunoblotted with a specific anti-3C serum. The ability of the Y88S mutant to form infectious virus and cleave 3CD can be complemented by the WT poliovirus. Synthesis of viral RNA was diminished in the Y88S mutant but less than the inhibition of translation of viral RNA. Experiments in which guanidine was used to inhibit poliovirus RNA synthesis suggest that the primary defect of the Y88S mutant virus is at the level of poliovirus RNA translation, while viral genome replication is much less affected. Transfection of HeLa cells infected with the WT poliovirus with a luciferase mRNA containing the poliovirus 5' untranslated sequence gives rise to a severalfold increase in luciferase activity. This enhanced translation of leader-luc mRNA was not observed when the transfected cells were infected with the 2A (Y88S) mutant. Moreover, cotransfection with mRNA encoding WT poliovirus 2A(pro) enhanced translation of leader-luc mRNA. This enhancement was much lower upon transfection with mRNA encoding 2A(Y88S), 2A(Y88L), or 2A(Y88P). These findings support the view that 2A(pro) itself, rather than the 3C' and/or 3D' products, is necessary for efficient translation of poliovirus RNA in HeLa cells. PMID:7666528

  1. Alginic acid synthesis in Pseudomonas aeruginosa mutants defective in carbohydrate metabolism.

    PubMed Central

    Banerjee, P C; Vanags, R I; Chakrabarty, A M; Maitra, P K

    1983-01-01

    Mutant cells of mucoid Pseudomonas aeruginosa isolated from cystic fibrosis patients were examined for their ability to synthesize alginic acid in resting cell suspensions. Unlike the wild-type strain which synthesizes alginic acid from glycerol, fructose, mannitol, glucose, gluconate, glutamate, or succinate, mutants lacking specific enzymes of carbohydrate metabolism are uniquely impaired. A phosphoglucose isomerase mutant did not synthesize the polysaccharide from mannitol, nor did a glucose 6-phosphate dehydrogenase mutant synthesize the polysaccharide from mannitol or glucose. Mutants lacking the Entner-Doudoroff pathway dehydrase or aldolase failed to produce alginate from mannitol, glucose, or gluconate, as a 3-phosphoglycerate kinase or glyceraldehyde 3-phosphate dehydrogenase mutant failed to produce from glutamate or succinate. These results demonstrate the primary role of the Entner-Doudoroff pathway enzymes in the synthesis of alginate from glucose, mannitol, or gluconate and the role of glyceraldehyde 3-phosphate dehydrogenase reaction for the synthesis from gluconeogenic precursors such as glutamate. The virtual absence of any activity of phosphomannose isomerase in cell extracts of several independent mucoid bacteria and the impairment of alginate synthesis from mannitol in mutants lacking phosphoglucose isomerase or glucose 6-phosphate dehydrogenase rule out free mannose 6-phosphate as an intermediate in alginate biosynthesis. PMID:6408061

  2. pH-Responsive Pharmacological Chaperones for Rescuing Mutant Glycosidases.

    PubMed

    Mena-Barragán, Teresa; Narita, Aya; Matias, Dino; Tiscornia, Gustavo; Nanba, Eiji; Ohno, Kousaku; Suzuki, Yoshiyuki; Higaki, Katsumi; Garcia Fernández, José Manuel; Ortiz Mellet, Carmen

    2015-09-28

    A general approach is reported for the design of small-molecule competitive inhibitors of lysosomal glycosidases programmed to 1) promote correct folding of mutant enzymes at the endoplasmic reticulum, 2) facilitate trafficking, and 3) undergo dissociation and self-inactivation at the lysosome. The strategy is based on the incorporation of an orthoester segment into iminosugar conjugates to switch the nature of the aglycone moiety from hydrophobic to hydrophilic in the pH 7 to pH 5 window, which has a dramatic effect on the enzyme binding affinity. As a proof of concept, new highly pH-responsive glycomimetics targeting human glucocerebrosidase or α-galactosidase with strong potential as pharmacological chaperones for Gaucher or Fabry disease, respectively, were developed. PMID:26386364

  3. Defective initiation in an Escherichia coli dnaA(Cs,Sx) mutant.

    PubMed

    Boye, E; Blinkova, A; Walker, J R

    2001-01-01

    Mutations in the Escherichia coli gene for initiation of DNA replication, dnaA, which suppress the polymerization defect and growth inhibition caused by temperature-sensitive (Ts) mutations in the polymerization gene, dnaX, are called Cs,Sx. We show here that these mutations, on their own, also cause defects in initiation, including inhibition of initiation at both low (20 degrees C) and high (44 degrees C) temperatures and asynchronous initiation at both the permissive (34 degrees C) and suppression (39 degrees C) temperatures. These findings suggests a relationship between partially defective initiation and suppression of the polymerization defect, both of which occur at 39 degrees C.

  4. Arabidopsis DNA polymerase lambda mutant is mildly sensitive to DNA double strand breaks but defective in integration of a transgene

    PubMed Central

    Furukawa, Tomoyuki; Angelis, Karel J.; Britt, Anne B.

    2015-01-01

    The DNA double-strand break (DSB) is a critical type of damage, and can be induced by both endogenous sources (e.g., errors of oxidative metabolism, transposable elements, programmed meiotic breaks, or perturbation of the DNA replication fork) and exogenous sources (e.g., ionizing radiation or radiomimetic chemicals). Although higher plants, like mammals, are thought to preferentially repair DSBs via nonhomologous end joining (NHEJ), much remains unclear about plant DSB repair pathways. Our reverse genetic approach suggests that DNA polymerase λ is involved in DSB repair in Arabidopsis. The Arabidopsis T-DNA insertion mutant (atpolλ-1) displayed sensitivity to both gamma-irradiation and treatment with radiomimetic reagents, but not to other DNA damaging treatments. The atpolλ-1 mutant showed a moderate sensitivity to DSBs, while Arabidopsis Ku70 and DNA ligase 4 mutants (atku70-3 and atlig4-2), both of which play critical roles in NHEJ, exhibited a hypersensitivity to these treatments. The atpolλ-1/atlig4-2 double mutant exhibited a higher sensitivity to DSBs than each single mutant, but the atku70/atpolλ-1 showed similar sensitivity to the atku70-3 mutant. We showed that transcription of the DNA ligase 1, DNA ligase 6, and Wee1 genes was quickly induced by BLM in several NHEJ deficient mutants in contrast to wild-type. Finally, the T-DNA transformation efficiency dropped in NHEJ deficient mutants and the lowest transformation efficiency was scored in the atpolλ-1/atlig4-2 double mutant. These results imply that AtPolλ is involved in both DSB repair and DNA damage response pathway. PMID:26074930

  5. Process of Cellular Division in Escherichia coli: Physiological Study on Thermosensitive Mutants Defective in Cell Division

    PubMed Central

    Ricard, Matthieu; Hirota, Yukinori

    1973-01-01

    Thermosensitive fts mutants of Escherichia coli belonging to seven previously identified genetic classes (ftsA to ftsG) were studied from a physiological standpoint. These mutants immediately stopped dividing and formed multinucleated filaments when the temperature was shifted to 41 C. Macromolecular syntheses (deoxyribonucleic acid), ribonucleic acid, cell mass, and murein) continued exponentially for at least 40 to 120 min. The number of surviving bacteria remained constant during the time of incubation, and this number began to decrease exponentially, as the rate of cell mass increase leveled off from the initial rate. The recovery of cell division at 30 C in these filamentous cells was studied after 60 min of incubation at 41 C. The existence of three types of mutants was shown. The ftsA and ftsE mutants resumed cell division without new protein synthesis; ftsD mutants resumed cell division only if new protein synthesis occured, while ftsB, C, F and G mutants did not resume cell division at all. No alteration in the cell envelope was detected by the method used here, although the ftsA, B, D, F and G mutations, in contrast with ftsC and E, caused an increased resistance to penicillin G. It was also shown that the recA mutation did not suppress the effect of the fts mutations and that none of the lysogenic fts mutants induced prophage multiplication while forming filaments. The effects of osmotic pressure and salts which rescue the mutant phenotype is described. PMID:4583216

  6. Characterization and virulence properties of Erwinia chrysanthemi lipopolysaccharide-defective, phi EC2-resistant mutants.

    PubMed

    Schoonejans, E; Expert, D; Toussaint, A

    1987-09-01

    Outer membrane alterations were characterized in spontaneous mutants of the Erwinia chrysanthemi 3937jRH, which were selected for resistance to bacteriophage phi EC2. All but one of the mutants analyzed were affected in their lipopolysaccharide (LPS) structure, lacking the entire heterogeneous region of apparent high molecular weight present in the wild-type E. chrysanthemi LPS. At least two 3937jRH mutants, one selected as phi EC2 resistant (RH6065) and the other previously selected (D. Expert and A. Toussaint, J. Bacteriol. 163:221-227, 1985) as bacteriocin resistant (R1456), were cross-resistant to bacteriophage Mu and had rough LPSs with an altered core structure. Two phi EC2r mutants (RH6053 and RH6065) were most severely affected in their outer membrane integrity and also lost their virulence on saintpaulia plants, although they still possessed normal extracellular levels of pectinolytic and cellulolytic activities. The two Mur mutants RH6065 and R1456 were also able to induce systemic resistance in the challenged plant. All the other phi EC2r mutants retained the virulence of 393jRH. PMID:3624200

  7. Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation.

    PubMed Central

    Manos, M M; Gluzman, Y

    1984-01-01

    The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen. Images PMID:6330530

  8. A Porphyromonas gingivalis Mutant Defective in a Putative Glycosyltransferase Exhibits Defective Biosynthesis of the Polysaccharide Portions of Lipopolysaccharide, Decreased Gingipain Activities, Strong Autoaggregation, and Increased Biofilm Formation▿ †

    PubMed Central

    Yamaguchi, Mikiyo; Sato, Keiko; Yukitake, Hideharu; Noiri, Yuichiro; Ebisu, Shigeyuki; Nakayama, Koji

    2010-01-01

    The Gram-negative anaerobic bacterium Porphyromonas gingivalis is a major pathogen in periodontal disease, one of the biofilm-caused infectious diseases. The bacterium possesses potential virulence factors, including fimbriae, proteinases, hemagglutinin, lipopolysaccharide (LPS), and outer membrane vesicles, and some of these factors are associated with biofilm formation; however, the precise mechanism of biofilm formation is still unknown. Colonial pigmentation of the bacterium on blood agar plates is related to its virulence. In this study, we isolated a nonpigmented mutant that had an insertion mutation within the new gene PGN_1251 (gtfB) by screening a transposon insertion library. The gene shares homology with genes encoding glycosyltransferase 1 of several bacteria. The gtfB mutant was defective in biosynthesis of both LPSs containing O side chain polysaccharide (O-LPS) and anionic polysaccharide (A-LPS). The defect in the gene resulted in a complete loss of surface-associated gingipain proteinases, strong autoaggregation, and a marked increase in biofilm formation, suggesting that polysaccharide portions of LPSs influence attachment of gingipain proteinases to the cell surface, autoaggregation, and biofilm formation of P. gingivalis. PMID:20624909

  9. A Glycosylation Mutant of Trypanosoma brucei Links Social Motility Defects In Vitro to Impaired Colonization of Tsetse Flies In Vivo.

    PubMed

    Imhof, Simon; Vu, Xuan Lan; Bütikofer, Peter; Roditi, Isabel

    2015-06-01

    Transmission of African trypanosomes by tsetse flies requires that the parasites migrate out of the midgut lumen and colonize the ectoperitrophic space. Early procyclic culture forms correspond to trypanosomes in the lumen; on agarose plates they exhibit social motility, migrating en masse as radial projections from an inoculation site. We show that an Rft1(-/-) mutant needs to reach a greater threshold number before migration begins, and that it forms fewer projections than its wild-type parent. The mutant is also up to 4 times less efficient at establishing midgut infections. Ectopic expression of Rft1 rescues social motility defects and restores the ability to colonize the fly. These results are consistent with social motility reflecting movement to the ectoperitrophic space, implicate N-glycans in the signaling cascades for migration in vivo and in vitro, and provide the first evidence that parasite-parasite interactions determine the success of transmission by the insect host. PMID:25862152

  10. A Toxoplasma MORN1 Null Mutant Undergoes Repeated Divisions but Is Defective in Basal Assembly, Apicoplast Division and Cytokinesis

    PubMed Central

    Lorestani, Alexander; Sheiner, Lilach; Yang, Kevin; Robertson, Seth D.; Sahoo, Nivedita; Brooks, Carrie F.; Ferguson, David J. P.; Striepen, Boris; Gubbels, Marc-Jan

    2010-01-01

    The membrane occupation and recognition nexus protein 1 (MORN1) is highly conserved among apicomplexan parasites and is associated with several structures that have a role in cell division. Here we dissected the role of MORN1 using the relatively simple budding process of Toxoplasma gondii as a model. Ablation of MORN1 in a conditional null mutant resulted in pronounced defects suggesting a central role for MORN1 in apicoplast segregation and in daughter cell budding. Lack of MORN1 resulted in double-headed parasites. These Janus-headed parasites form two complete apical complexes but fail to assemble a basal complex. Moreover, these parasites were capable of undergoing several more budding rounds resulting in the formation of up to 16-headed parasites conjoined at the basal end. Despite this segregation defect, the mother's cytoskeleton was completely disassembled in every budding round. Overall this argues that successful completion of the budding is not required for cell cycle progression. None of the known basal complex components, including a set of recently identified inner membrane complex (IMC) proteins, localized correctly in these multi-headed parasites. These data suggest that MORN1 is essential for assembly of the basal complex, and that lack of the basal complex abolishes the contractile capacity assigned to the basal complex late in daughter formation. Consistent with this hypothesis we observe that MORN1 mutants fail to efficiently constrict and divide the apicoplast. We used the null background provided by the mutant to dissect the function of subdomains of the MORN1 protein. This demonstrated that deletion of a single MORN domain already prevented the function of MORN1 whereas a critical role for the short linker between MORN domains 6 and 7 was identified. In conclusion, MORN1 is required for basal complex assembly and loss of MORN1 results in defects in apicoplast division and daughter segregation. PMID:20808817

  11. Escherichia coli mutants defective in membrane phospholipid synthesis: binding and metabolism of 1-oleoylglycerol 3-phosphate by a plsB deep rough mutant.

    PubMed Central

    McIntyre, T M; Bell, R M

    1978-01-01

    Mutants of Escherichia coli containing a defective sn-glycerol 3-phosphate acyltransferase are conditionally defective in the synthesis of acylglycerol phosphate (acylglycerol-P). Incubation of a deep rough derivative of one of these plsB strains with 1-[3H]oleoylglycerol-32P resulted in the binding of up to 70 nmol of oleoylglycerol-P per 100 nmol of cellular phospholipid. The binding was dependent on time, oleoylglycerol-P concentration, and the quantity of cells employed. The rate and extent of oleoylglycerol-P binding was affected by the deep rough mutation. The altered phospholipid composition due to oleoylglycerol-P binding was without consequence on cell growth and viability, but caused the appearance of intracellular multilamellar structures. Use of the double-labeled oleoylglycerol P demonstrated that the entire molecule was bound to the cell. Intact [3H]-oleoylglycerol-32P was converted to phosphatidylethanolamine and phosphotidyl-glycerol at a rate about 40% of that of de novo phospholipid synthesis. These data demonstrate the transmembrane movement of oleoylglycerol-P to the inner surface of the cytoplasmic membrane and suggest that it may become possible to supplement plsB strains of E. coli with acylglycerol-P's. Images PMID:353031

  12. FGT-1-mediated glucose uptake is defective in insulin/IGF-like signaling mutants in Caenorhabditis elegans.

    PubMed

    Kitaoka, Shun; Morielli, Anthony D; Zhao, Feng-Qi

    2016-06-01

    Insulin signaling plays a central role in the regulation of facilitative glucose transporters (GLUTs) in humans. To establish Caenorhabditis elegans (C. elegans) as a model to study the mechanism underlying insulin regulation of GLUT, we identified that FGT-1 is most likely the only functional GLUT homolog in C. elegans and is ubiquitously expressed. The FGT-1-mediated glucose uptake was almost completely defective in insulin/IGF-like signaling (IIS) mutants daf-2 and age-1, and this defect mainly resulted from the down-regulated FGT-1 protein expression. However, glycosylation may also be involved because OGA-1, an O-GlcNAcase, was essential for the function of FGT-1. Thus, our study showed that C. elegans can be a new powerful model system to study insulin regulation of GLUT. PMID:27419060

  13. Mouse Slc9a8 Mutants Exhibit Retinal Defects Due to Retinal Pigmented Epithelium Dysfunction

    PubMed Central

    Jadeja, Shalini; Barnard, Alun R.; McKie, Lisa; Cross, Sally H.; White, Jacqueline K.; Robertson, Morag; Budd, Peter S.; MacLaren, Robert E.; Jackson, Ian J.

    2015-01-01

    Purpose. As part of a large scale systematic screen to determine the effects of gene knockout mutations in mice, a retinal phenotype was found in mice lacking the Slc9a8 gene, encoding the sodium/hydrogen ion exchange protein NHE8. We aimed to characterize the mutant phenotype and the role of sodium/hydrogen ion exchange in retinal function. Methods. Detailed histology characterized the pathological consequences of Slc9a8 mutation, and retinal function was assessed by electroretinography (ERG). A conditional allele was used to identify the cells in which NHE8 function is critical for retinal function, and mutant cells analyzed for the effect of the mutation on endosomes. Results. Histology of mutant retinas reveals a separation of photoreceptors from the RPE and infiltration by macrophages. There is a small reduction in photoreceptor length and a mislocalization of visual pigments. The ERG testing reveals a deficit in rod and cone pathway function. The RPE shows abnormal morphology, and mutation of Slc9a8 in only RPE cells recapitulates the mutant phenotype. The NHE8 protein localizes to endosomes, and mutant cells have much smaller recycling endosomes. Conclusions. The NHE8 protein is required in the RPE to maintain correct regulation of endosomal volume and/or pH which is essential for the cellular integrity and subsequent function of RPE. PMID:25736793

  14. [Salt Stress Response in Arabidopsis thaliana Plants with Defective Jasmonate Signaling].

    PubMed

    Yastreb, T O; Kolupayev, Yu E; Shvidenko, A A; Lugovaya, A A; Dmitriev, A P

    2015-01-01

    The effects of exogenous jasmonic acid (JA) on antioxidant enzymes in four-week-old leaves of wild-type Arabidopsis thaliana L. (Columbia-0) and jin1 (jasmonate insensitive 1) mutant plants with defective jasmonate signaling were investigated under normal conditions and under salt stress (200 mM NaCl, 24 h). The wild-type plants responded to JA by an increase in the activities of Cu/Zn superoxide dismutase, catalase, and guaiacol peroxidase, while there was no change in the case of the mutant plants. In response to the salt stress of both the wild-type and mutant genotypes, the activities of superoxide dismutase, catalase, and guaiacol peroxidase were unchanged, decreased, and increased, respectively. The JA-treated wild type plants showed the highest activity of all three enzymes as compared with the mutant plants. Salinity caused a decrease in chlorophyll content in the wild-type and jin 1 plants. Preliminary JA treatment of the Col-0 plants resulted in a normal content of photosynthetic pigments after the salt stress, while the positive JA effect was insignificant in the jin 1 mutants. It was concluded that the MYC2/JIN 1 protein is involved in the JA signal transduction and plant adaptation to salt stress.

  15. Defective Dissociation of a “Slow” RecA Mutant Protein Imparts an Escherichia coli Growth Defect*

    PubMed Central

    Cox, Julia M.; Li, Hao; Wood, Elizabeth A.; Chitteni-Pattu, Sindhu; Inman, Ross B.; Cox, Michael M.

    2008-01-01

    The RecA and some related proteins possess a simple motif, called (KR)X(KR), that (in RecA) consists of two lysine residues at positions 248 and 250 at the subunit-subunit interface. This study and previous work implicate this RecA motif in the following: (a) catalyzing ATP hydrolysis in trans,(b) coordinating the ATP hydrolytic cycles of adjacent subunits, (c) governing the rate of ATP hydrolysis, and (d) coupling the ATP hydrolysis to work (in this case DNA strand exchange). The conservative K250R mutation leaves RecA nucleoprotein filament formation largely intact. However, ATP hydrolysis is slowed to less than 15% of the wild-type rate. DNA strand exchange is also slowed commensurate with the rate of ATP hydrolysis. The results reinforce the idea of a tight coupling between ATP hydrolysis and DNA strand exchange. When a plasmid-borne RecA K250R protein is expressed in a cell otherwise lacking RecA protein, the growth of the cells is severely curtailed. The slow growth defect is alleviated in cells lacking RecFOR function, suggesting that the defect reflects loading of RecA at stalled replication forks. Suppressors occur as recA gene alterations, and their properties indicate that limited dissociation by RecA K250R confers the slow growth phenotype. Overall, the results suggest that recombinational DNA repair is a common occurrence in cells. RecA protein plays a sufficiently intimate role in the bacterial cell cycle that its properties can limit the growth rate of a bacterial culture. PMID:18603529

  16. Surveillance of Different Recombination Defects in Mouse Spermatocytes Yields Distinct Responses despite Elimination at an Identical Developmental Stage

    PubMed Central

    Barchi, Marco; Mahadevaiah, Shantha; Di Giacomo, Monica; Baudat, Frédéric; de Rooij, Dirk G.; Burgoyne, Paul S.; Jasin, Maria; Keeney, Scott

    2005-01-01

    Fundamentally different recombination defects cause apoptosis of mouse spermatocytes at the same stage in development, stage IV of the seminiferous epithelium cycle, equivalent to mid-pachynema in normal males. To understand the cellular response(s) that triggers apoptosis, we examined markers of spermatocyte development in mice with different recombination defects. In Spo11−/− mutants, which lack the double-strand breaks (DSBs) that initiate recombination, spermatocytes express markers of early to mid-pachynema, forming chromatin domains that contain sex body-associated proteins but that rarely encompass the sex chromosomes. Dmc1−/− spermatocytes, impaired in DSB repair, appear to arrest at or about late zygonema. Epistasis analysis reveals that this earlier arrest is a response to unrepaired DSBs, and cytological analysis implicates the BRCT-containing checkpoint protein TOPBP1. Atm−/− spermatocytes show similarities to Dmc1−/− spermatocytes, suggesting that ATM promotes meiotic DSB repair. Msh5−/− mutants display a set of characteristics distinct from these other mutants. Thus, despite equivalent stages of spermatocyte elimination, different recombination-defective mutants manifest distinct responses, providing insight into surveillance mechanisms in male meiosis. PMID:16055729

  17. Maize reas1 Mutant Stimulates Ribosome Use Efficiency and Triggers Distinct Transcriptional and Translational Responses1[OPEN

    PubMed Central

    Qi, Weiwei; Zhu, Jie; Wu, Qiao; Wang, Qun; Li, Xia; Yao, Dongsheng; Jin, Ying; Wang, Gang; Wang, Guifeng

    2016-01-01

    Ribosome biogenesis is a fundamental cellular process in all cells. Impaired ribosome biogenesis causes developmental defects; however, its molecular and cellular bases are not fully understood. We cloned a gene responsible for a maize (Zea mays) small seed mutant, dek* (for defective kernel), and found that it encodes Ribosome export associated1 (ZmReas1). Reas1 is an AAA-ATPase that controls 60S ribosome export from the nucleus to the cytoplasm after ribosome maturation. dek* is a weak mutant allele with decreased Reas1 function. In dek* cells, mature 60S ribosome subunits are reduced in the nucleus and cytoplasm, but the proportion of actively translating polyribosomes in cytosol is significantly increased. Reduced phosphorylation of eukaryotic initiation factor 2α and the increased elongation factor 1α level indicate an enhancement of general translational efficiency in dek* cells. The mutation also triggers dramatic changes in differentially transcribed genes and differentially translated RNAs. Discrepancy was observed between differentially transcribed genes and differentially translated RNAs, indicating distinct cellular responses at transcription and translation levels to the stress of defective ribosome processing. DNA replication and nucleosome assembly-related gene expression are selectively suppressed at the translational level, resulting in inhibited cell growth and proliferation in dek* cells. This study provides insight into cellular responses due to impaired ribosome biogenesis. PMID:26645456

  18. Uncovering DELLA-Independent Gibberellin Responses by Characterizing New Tomato procera Mutants

    PubMed Central

    Livne, Sivan; Lor, Vai S.; Nir, Ido; Eliaz, Natanella; Aharoni, Asaph; Olszewski, Neil E.; Eshed, Yuval; Weiss, David

    2015-01-01

    Gibberellin (GA) regulates plant development primarily by triggering the degradation/deactivation of the DELLA proteins. However, it remains unclear whether all GA responses are regulated by DELLAs. Tomato (Solanum lycopersicum) has a single DELLA gene named PROCERA (PRO), and its recessive pro allele exhibits constitutive GA activity but retains responsiveness to external GA. In the loss-of-function mutant proΔGRAS, all examined GA developmental responses were considerably enhanced relative to pro and a defect in seed desiccation tolerance was uncovered. As pro, but not proΔGRAS, elongation was promoted by GA treatment, pro may retain residual DELLA activity. In agreement with homeostatic feedback regulation of the GA biosynthetic pathway, we found that GA20oxidase1 expression was suppressed in proΔGRAS and was not affected by exogenous GA3. In contrast, expression of GA2oxidase4 was not affected by the elevated GA signaling in proΔGRAS but was strongly induced by exogenous GA3. Since a similar response was found in Arabidopsis thaliana plants with impaired activity of all five DELLA genes, we suggest that homeostatic GA responses are regulated by both DELLA-dependent and -independent pathways. Transcriptome analysis of GA-treated proΔGRAS leaves suggests that 5% of all GA-regulated genes in tomato are DELLA independent. PMID:26036254

  19. The cellular and molecular etiology of the craniofacial defects in the avian ciliopathic mutant talpid2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    talpid2 is an avian autosomal recessive mutant with a myriad of congenital malformations, including polydactyly and facial clefting. Although phenotypically similar to talpid3, talpid2 has a distinct facial phenotype and an unknown cellular, molecular and genetic basis. We set out to determine the e...

  20. Autonomous and Non-autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC-Derived Cardiomyocytes.

    PubMed

    Josowitz, Rebecca; Mulero-Navarro, Sonia; Rodriguez, Nelson A; Falce, Christine; Cohen, Ninette; Ullian, Erik M; Weiss, Lauren A; Rauen, Katherine A; Sobie, Eric A; Gelb, Bruce D

    2016-09-13

    Germline mutations in BRAF cause cardio-facio-cutaneous syndrome (CFCS), whereby 40% of patients develop hypertrophic cardiomyopathy (HCM). As the role of the RAS/MAPK pathway in HCM pathogenesis is unclear, we generated a human induced pluripotent stem cell (hiPSC) model for CFCS from three patients with activating BRAF mutations. By cell sorting for SIRPα and CD90, we generated a method to examine hiPSC-derived cell type-specific phenotypes and cellular interactions underpinning HCM. BRAF-mutant SIRPα(+)/CD90(-) cardiomyocytes displayed cellular hypertrophy, pro-hypertrophic gene expression, and intrinsic calcium-handling defects. BRAF-mutant SIRPα(-)/CD90(+) cells, which were fibroblast-like, exhibited a pro-fibrotic phenotype and partially modulated cardiomyocyte hypertrophy through transforming growth factor β (TGFβ) paracrine signaling. Inhibition of TGFβ or RAS/MAPK signaling rescued the hypertrophic phenotype. Thus, cell autonomous and non-autonomous defects underlie HCM due to BRAF mutations. TGFβ inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies. PMID:27569062

  1. Genetic Interaction Landscape Reveals Critical Requirements for Schizosaccharomyces pombe Brc1 in DNA Damage Response Mutants.

    PubMed

    Sánchez, Arancha; Roguev, Assen; Krogan, Nevan J; Russell, Paul

    2015-05-01

    Brc1, which was first identified as a high-copy, allele-specific suppressor of a mutation impairing the Smc5-Smc6 holocomplex in Schizosaccharomyces pombe, protects genome integrity during normal DNA replication and when cells are exposed to toxic compounds that stall or collapse replication forks. The C-terminal tandem BRCT (BRCA1 C-terminus) domain of fission yeast Brc1 docks with phosphorylated histone H2A (γH2A)-marked chromatin formed by ATR/Rad3 checkpoint kinase at arrested and damaged replication forks; however, how Brc1 functions in relation to other genome protection modules remains unclear. Here, an epistatic mini-array profile reveals critical requirements for Brc1 in mutants that are defective in multiple DNA damage response pathways, including checkpoint signaling by Rad3-Rad26/ATR-ATRIP kinase, DNA repair by Smc5-Smc6 holocomplex, replication fork stabilization by Mrc1/claspin and Swi1-Swi3/Timeless-Tipin, and control of ubiquitin-regulated proteolysis by the COP9 signalosome (CSN). Exogenous genotoxins enhance these negative genetic interactions. Rad52 and RPA foci are increased in CSN-defective cells, and loss of γH2A increases genotoxin sensitivity, indicating a critical role for the γH2A-Brc1 module in stabilizing replication forks in CSN-defective cells. A negative genetic interaction with the Nse6 subunit of Smc5-Smc6 holocomplex indicates that the DNA repair functions of Brc1 and Smc5-Smc6 holocomplex are at least partially independent. Rtt107, the Brc1 homolog in Saccharomyces cerevisiae, has a very different pattern of genetic interactions, indicating evolutionary divergence of functions and DNA damage responses.

  2. Isolation and characterization of Saccharomyces cerevisiae mutants defective in chromosome transmission in an undergraduate genetics research course.

    PubMed

    Sleister, Heidi Major

    2007-10-01

    An upper-level genetics research course was developed to expose undergraduates to investigative science. Students are immersed in a research project with the ultimate goal of identifying proteins important for chromosome transmission in mitosis. After mutagenizing yeast Saccharomyces cerevisiae cells, students implement a genetic screen that allows for visual detection of mutants with an increased loss of an ADE2-marked yeast artificial chromosome (YAC). Students then genetically characterize the mutants and begin efforts to identify the defective genes in these mutants. While engaged in this research project, students practice a variety of technical skills in both classical and molecular genetics. Furthermore, students learn to collaborate and gain experience in sharing scientific findings with others in the form of written papers, poster presentations, and oral presentations. Previous students indicated that, relative to a traditional laboratory course, this research course improved their understanding of scientific concepts and technical skills and helped them make connections between concepts. Moreover, this course allowed students to experience scientific inquiry and was influential for students as they considered future endeavors.

  3. Deletion of a Cys-His motif from the Alpharetrovirus nucleocapsid domain reveals late domain mutant-like budding defects

    SciTech Connect

    Lee, Eun-Gyung; Linial, Maxine L. . E-mail: mlinial@fhcrc.org

    2006-03-30

    The Rous sarcoma virus (RSV) Gag polyprotein is the only protein required for virus assembly and release. We previously found that deletion of either one of the two Cys-His (CH) motifs in the RSV nucleocapsid (NC) protein did not abrogate Gag-Gag interactions, RNA binding, or packaging but greatly reduced virus production (E-G. Lee, A. Alidina et al., J. Virol. 77: 2010-2020, 2003). In this report, we have further investigated the effects of mutations in the CH motifs on virus assembly and release. Precise deletion of either CH motif, without affecting surrounding basic residues, reduced virus production by approximately 10-fold, similar to levels seen for late (L) domain mutants. Strikingly, transmission electron microscopy revealed that virions of both {delta}CH1 and {delta}CH2 mutants were assembled normally at the plasma membrane but were arrested in budding. Virus particles remained tethered to the membrane or to each other, reminiscent of L domain mutants, although the release defect appears to be independent of the L domain functions. Therefore, two CH motifs are likely to be required for budding independent of a requirement for either Gag-Gag interactions or RNA packaging.

  4. Tagging ribosomal protein S7 allows rapid identification of mutants defective in assembly and function of 30 S subunits.

    PubMed

    Fredrick, K; Dunny, G M; Noller, H F

    2000-05-01

    Ribosomal protein S7 nucleates folding of the 16 S rRNA 3' major domain, which ultimately forms the head of the 30 S ribosomal subunit. Recent crystal structures indicate that S7 lies on the interface side of the 30 S subunit, near the tRNA binding sites of the ribosome. To map the functional surface of S7, we have tagged the protein with a Protein Kinase A recognition site and engineered alanine substitutions that target each exposed, conserved residue. We have also deleted conserved features of S7, using its structure to guide our design. By radiolabeling the tag sequence using Protein Kinase A, we are able to track the partitioning of each mutant protein into 30 S, 70 S, and polyribosome fractions in vivo. Overexpression of S7 confers a growth defect, and we observe a striking correlation between this phenotype and proficiency in 30 S subunit assembly among our collection of mutants. We find that the side chain of K35 is required for efficient assembly of S7 into 30 S subunits in vivo, whereas those of at least 17 other conserved exposed residues are not required. In addition, an S7 derivative lacking the N-terminal 17 residues causes ribosomes to accumulate on mRNA to abnormally high levels, indicating that our approach can yield interesting mutant ribosomes.

  5. The Cell Wall Arabinose-Deficient Arabidopsis thaliana Mutant murus5 Encodes a Defective Allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2.

    PubMed

    Dugard, Christopher K; Mertz, Rachel A; Rayon, Catherine; Mercadante, Davide; Hart, Christopher; Benatti, Matheus R; Olek, Anna T; SanMiguel, Phillip J; Cooper, Bruce R; Reiter, Wolf-Dieter; McCann, Maureen C; Carpita, Nicholas C

    2016-07-01

    Traditional marker-based mapping and next-generation sequencing was used to determine that the Arabidopsis (Arabidopsis thaliana) low cell wall arabinose mutant murus5 (mur5) encodes a defective allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2 (RGP2). Marker analysis of 13 F2 confirmed mutant progeny from a recombinant mapping population gave a rough map position on the upper arm of chromosome 5, and deep sequencing of DNA from these 13 lines gave five candidate genes with G→A (C→T) transitions predicted to result in amino acid changes. Of these five, only insertional mutant alleles of RGP2, a gene that encodes a UDP-arabinose mutase that interconverts UDP-arabinopyranose and UDP-arabinofuranose, exhibited the low cell wall arabinose phenotype. The identities of mur5 and two SALK insertional alleles were confirmed by allelism tests and overexpression of wild-type RGP2 complementary DNA placed under the control of the 35S promoter in the three alleles. The mur5 mutation results in the conversion of cysteine-257 to tyrosine-257 within a conserved hydrophobic cluster predicted to be distal to the active site and essential for protein stability and possible heterodimerization with other isoforms of RGP. PMID:27217494

  6. The Cell Wall Arabinose-Deficient Arabidopsis thaliana Mutant murus5 Encodes a Defective Allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2.

    PubMed

    Dugard, Christopher K; Mertz, Rachel A; Rayon, Catherine; Mercadante, Davide; Hart, Christopher; Benatti, Matheus R; Olek, Anna T; SanMiguel, Phillip J; Cooper, Bruce R; Reiter, Wolf-Dieter; McCann, Maureen C; Carpita, Nicholas C

    2016-07-01

    Traditional marker-based mapping and next-generation sequencing was used to determine that the Arabidopsis (Arabidopsis thaliana) low cell wall arabinose mutant murus5 (mur5) encodes a defective allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2 (RGP2). Marker analysis of 13 F2 confirmed mutant progeny from a recombinant mapping population gave a rough map position on the upper arm of chromosome 5, and deep sequencing of DNA from these 13 lines gave five candidate genes with G→A (C→T) transitions predicted to result in amino acid changes. Of these five, only insertional mutant alleles of RGP2, a gene that encodes a UDP-arabinose mutase that interconverts UDP-arabinopyranose and UDP-arabinofuranose, exhibited the low cell wall arabinose phenotype. The identities of mur5 and two SALK insertional alleles were confirmed by allelism tests and overexpression of wild-type RGP2 complementary DNA placed under the control of the 35S promoter in the three alleles. The mur5 mutation results in the conversion of cysteine-257 to tyrosine-257 within a conserved hydrophobic cluster predicted to be distal to the active site and essential for protein stability and possible heterodimerization with other isoforms of RGP.

  7. Reducing ppGpp level rescues an extreme growth defect caused by mutant EF-Tu.

    PubMed

    Bergman, Jessica M; Hammarlöf, Disa L; Hughes, Diarmaid

    2014-01-01

    Transcription and translation of mRNA's are coordinated processes in bacteria. We have previously shown that a mutant form of EF-Tu (Gln125Arg) in Salmonella Typhimurium with a reduced affinity for aa-tRNA, causes ribosome pausing, resulting in an increased rate of RNase E-mediated mRNA cleavage, causing extremely slow growth, even on rich medium. The slow growth phenotype is reversed by mutations that reduce RNase E activity. Here we asked whether the slow growth phenotype could be reversed by overexpression of a wild-type gene. We identified spoT (encoding ppGpp synthetase/hydrolase) as a gene that partially reversed the slow growth rate when overexpressed. We found that the slow-growing mutant had an abnormally high basal level of ppGpp that was reduced when spoT was overexpressed. Inactivating relA (encoding the ribosome-associated ppGpp synthetase) also reduced ppGpp levels and significantly increased growth rate. Because RelA responds specifically to deacylated tRNA in the ribosomal A-site this suggested that the tuf mutant had an increased level of deacylated tRNA relative to the wild-type. To test this hypothesis we measured the relative acylation levels of 4 families of tRNAs and found that proline isoacceptors were acylated at a lower level in the mutant strain relative to the wild-type. In addition, the level of the proS tRNA synthetase mRNA was significantly lower in the mutant strain. We suggest that an increased level of deacylated tRNA in the mutant strain stimulates RelA-mediated ppGpp production, causing changes in transcription pattern that are inappropriate for rich media conditions, and contributing to slow growth rate. Reducing ppGpp levels, by altering the activity of either SpoT or RelA, removes one cause of the slow growth and reveals the interconnectedness of intracellular regulatory mechanisms.

  8. Organogenic responses in tissue culture of srd mutants of Arabidopsis thaliana.

    PubMed

    Ozawa, S; Yasutani, I; Fukuda, H; Komamine, A; Sugiyama, M

    1998-01-01

    In Arabidopsis thaliana, shoot redifferentiation and root redifferentiation can be induced at high frequency from hypocotyl and root explants by a two-step culture method. Tissues are precultured on callus-inducing medium and then transferred onto shoot-inducing medium for shoot redifferentiation or onto root-inducing medium for root redifferentiation. In an attempt to dissect these organogenic processes genetically, we characterized the responses in tissue culture of srd1, srd2 and srd3 mutants that were originally isolated as temperature-sensitive strains with defects in shoot redifferentiation (Yasutani, I., Ozawa, S., Nishida, T., Sugiyama, M. and Komamine, A. (1994) Plant Physiol. 105, 815-822). These mutants exhibited temperature sensitivity at different steps of organogenesis, which allowed the identification of three states associated with organogenic competence: IC (incompetent); CR (competent with respect to root redifferentiation); and CSR (competent with respect to shoot and root redifferentiation). Hypocotyl explants were shown to be in the IC state at the initiation of culture and to enter the CSR state, via the CR state, during preculture on callus-inducing medium, whereas root explants seemed to be in the CR state at the initiation of culture. The transition from IC to CR and that from CR to CSR appeared to require the functions of SRD2 and SRD3, respectively. It appears that explants in the CSR state redifferentiate shoots with the aid of the products of SRD1 and SRD2 when transplanted onto shoot-inducing medium. Histological examination of the srd mutants revealed that the function of SRD2 is required not only for organogenesis but also for the reinitiation of cell proliferation in hypocotyl explants during culture on callus-inducing medium. Linkage analysis using RFLP markers indicated that SRD1, SRD2, and SRD3 are located at the lower region, the central region, and the upper region of chromosome 1, respectively.

  9. Characterization of a Salmonella typhimurium hisU mutant defective in tRNA precursor processing.

    PubMed Central

    Bossi, L; Ciampi, M S; Cortese, R

    1978-01-01

    The DA11 mutant of Salmonella typhimurium, originally isolated as derepressed for the histidine operon, carries a temperature-dependent alteration in a nucleolytic enzyme specifically involved in the maturation of tRNA. As a consequence of this alteration, no detectable synthesis of any mature tRNA species occurs in DA11 upon shift at 43 degrees C, whereas many tRNA precursors, whose sizes range between 80 and 750 nucleotides, do accumulate. Kinetic studies on the synthesis and processing of these maturation intermediates show that these molecules represent different stages in the maturation pathway, most of them being the products of previous nucleolytic events. These RNA molecules are in vivo substrates of methylation and thiolation enzymes and can be cleaved in vitro to 4S RNA by wild-type but not by DA11 cell-free extract. Evidence is presented that DA11 is very probably a ribonuclease P mutant. Images PMID:350829

  10. Negative feedback-defective PRPS1 mutants drive thiopurine resistance in relapsed childhood ALL

    PubMed Central

    Li, Benshang; Li, Hui; Bai, Yun; Kirschner-Schwabe, Renate; Yang, Jun J; Chen, Yao; Lu, Gang; Tzoneva, Gannie; Ma, Xiaotu; Wu, Tongmin; Li, Wenjing; Lu, Haisong; Ding, Lixia; Liang, Huanhuan; Huang, Xiaohang; Yang, Minjun; Jin, Lei; Kang, Hui; Chen, Shuting; Du, Alicia; Shen, Shuhong; Ding, Jianping; Chen, Hongzhuan; Chen, Jing; von Stackelberg, Arend; Gu, Longjun; Zhang, Jinghui; Ferrando, Adolfo; Tang, Jingyan; Wang, Shengyue; Zhou, Bin-Bing S.

    2015-01-01

    Relapse is the leading cause of mortality in children with acute lymphoblastic leukemia (ALL). Among chemotherapeutics, thiopurines are key drugs in the backbone of ALL combination therapy. Using whole-exome sequencing, we identified relapse-specific mutations in phosphoribosyl pyrophosphate synthetase 1 (PRPS1), a rate-limiting purine biosynthesis enzyme, in 24/358 (6.7%) relapse B-ALL cases. All individuals who harbored PRPS1 mutations relapsed early on-treatment, and mutated ALL clones expanded exponentially prior to clinical relapse. Our functional analyses of PRPS1 mutants uncovered a new chemotherapy resistance mechanism involving reduced feedback inhibition of de novo purine biosynthesis and competitive inhibition of thiopurine activation. Notably, the de novo purine synthesis inhibitor lometrexol can effectively abrogate PRPS1 mutant-driven drug resistance. Overall these results highlight the importance of constitutive activation of de novo purine pathway in thiopurine resistance, and offer therapeutic strategies for the treatment of relapsed and resistant ALL. PMID:25962120

  11. Identification and molecular characterization of a novel Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis.

    PubMed

    Grovenstein, Phillip B; Wilson, Darryel A; Lennox, Cameron G; Smith, Katherine P; Contractor, Alisha A; Mincey, Jonathan L; Lankford, Kathryn D; Smith, Jacqueline M; Haye, Tashana C; Mitra, Mautusi

    2013-01-01

    The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms and are synthesized via a common branched tetrapyrrole biosynthetic pathway. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg (2+) into protoporphyrin IX (PPIX, proto) to form magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. MgChel is a multimeric enzyme that consists of three subunits designated CHLD, CHLI and CHLH. Plants have two isozymes of CHLI (CHLI1 and CHLI2) which are 70%-81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. We have isolated a non-photosynthetic light sensitive mutant 5A7 by random DNA insertional mutagenesis that is devoid of any detectable Chl. PCR based analyses show that 5A7 is missing the CHLI1 gene and at least eight additional functionally uncharacterized genes. 5A7 has an intact CHLI2 gene. Complementation with a functional copy of the CHLI1 gene restored Chl biosynthesis, photo-autotrophic growth and light tolerance in 5A7. We have identified the first chli1 (chli1-1) mutant of Chlamydomonas reinhardtii and in green algae. Our results show that in the wild type Chlamydomonas CHLI2 protein amount is lower than that of CHLI1 and the chli1-1 mutant has a drastic reduction in CHLI2 protein levels although it possesses the CHLI2 gene. Our chli1-1 mutant opens up new avenues to explore the functional roles of CHLI1 and CHLI2 in Chl biosynthesis in Chlamydomonas, which has never been studied before.

  12. Identification and molecular characterization of a novel Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis

    PubMed Central

    Mitra, Mautusi

    2013-01-01

    The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms and are synthesized via a common branched tetrapyrrole biosynthetic pathway. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg 2+ into protoporphyrin IX (PPIX, proto) to form magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. MgChel is a multimeric enzyme that consists of three subunits designated CHLD, CHLI and CHLH. Plants have two isozymes of CHLI (CHLI1 and CHLI2) which are 70%-81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. We have isolated a non-photosynthetic light sensitive mutant 5A7 by random DNA insertional mutagenesis that is devoid of any detectable Chl. PCR based analyses show that 5A7 is missing the CHLI1 gene and at least eight additional functionally uncharacterized genes. 5A7 has an intact CHLI2 gene. Complementation with a functional copy of the CHLI1 gene restored Chl biosynthesis, photo-autotrophic growth and light tolerance in 5A7. We have identified the first chli1 (chli1-1) mutant of Chlamydomonas reinhardtii and in green algae. Our results show that in the wild type Chlamydomonas CHLI2 protein amount is lower than that of CHLI1 and the chli1-1 mutant has a drastic reduction in CHLI2 protein levels although it possesses the CHLI2 gene. Our chli1-1 mutant opens up new avenues to explore the functional roles of CHLI1 and CHLI2 in Chl biosynthesis in Chlamydomonas, which has never been studied before. PMID:24555064

  13. Using mycorrhiza-defective mutant genotypes of non-legume plant species to study the formation and functioning of arbuscular mycorrhiza: a review.

    PubMed

    Watts-Williams, Stephanie J; Cavagnaro, Timothy R

    2015-11-01

    A significant challenge facing the study of arbuscular mycorrhiza is the establishment of suitable non-mycorrhizal treatments that can be compared with mycorrhizal treatments. A number of options are available, including soil disinfection or sterilisation, comparison of constitutively mycorrhizal and non-mycorrhizal plant species, comparison of plants grown in soils with different inoculum potential and the comparison of mycorrhiza-defective mutant genotypes with their mycorrhizal wild-type progenitors. Each option has its inherent advantages and limitations. Here, the potential to use mycorrhiza-defective mutant and wild-type genotype plant pairs as tools to study the functioning of mycorrhiza is reviewed. The emphasis of this review is placed on non-legume plant species, as mycorrhiza-defective plant genotypes in legumes have recently been extensively reviewed. It is concluded that non-legume mycorrhiza-defective mutant and wild-type pairs are useful tools in the study of mycorrhiza. However, the mutant genotypes should be well characterised and, ideally, meet a number of key criteria. The generation of more mycorrhiza-defective mutant genotypes in agronomically important plant species would be of benefit, as would be more research using these genotype pairs, especially under field conditions.

  14. Using mycorrhiza-defective mutant genotypes of non-legume plant species to study the formation and functioning of arbuscular mycorrhiza: a review.

    PubMed

    Watts-Williams, Stephanie J; Cavagnaro, Timothy R

    2015-11-01

    A significant challenge facing the study of arbuscular mycorrhiza is the establishment of suitable non-mycorrhizal treatments that can be compared with mycorrhizal treatments. A number of options are available, including soil disinfection or sterilisation, comparison of constitutively mycorrhizal and non-mycorrhizal plant species, comparison of plants grown in soils with different inoculum potential and the comparison of mycorrhiza-defective mutant genotypes with their mycorrhizal wild-type progenitors. Each option has its inherent advantages and limitations. Here, the potential to use mycorrhiza-defective mutant and wild-type genotype plant pairs as tools to study the functioning of mycorrhiza is reviewed. The emphasis of this review is placed on non-legume plant species, as mycorrhiza-defective plant genotypes in legumes have recently been extensively reviewed. It is concluded that non-legume mycorrhiza-defective mutant and wild-type pairs are useful tools in the study of mycorrhiza. However, the mutant genotypes should be well characterised and, ideally, meet a number of key criteria. The generation of more mycorrhiza-defective mutant genotypes in agronomically important plant species would be of benefit, as would be more research using these genotype pairs, especially under field conditions. PMID:25862569

  15. Correlation of Recombinant Integrase Activity and Functional Preintegration Complex Formation during Acute Infection by Replication-Defective Integrase Mutant Human Immunodeficiency Virus

    PubMed Central

    Li, Xiang; Koh, Yasuhiro

    2012-01-01

    Previous studies characterized two types of replication-defective human immunodeficiency virus type 1 (HIV-1) integrase mutants: class I, which are specifically blocked at the integration step, and class II, which harbor additional virion production and/or reverse transcription defects. Class I mutant enzymes supported little if any metal ion-dependent 3′-processing and DNA strand transfer activities in vitro, whereas class II enzymes displayed partial or full catalytic function in studies with simplified assay designs, suggesting that defective interaction(s) with heterologous integrase binding proteins might underlie the class II mutant viral phenotype. To address this hypothesis, class I and II mutant enzymes were interrogated under expanded sets of in vitro conditions. The majority failed to catalyze the concerted integration of two viral DNA ends into target DNA, highlighting defective integrase function as the root cause of most class II in addition to all class I mutant virus infection defects. One mutant protein, K264E, in contrast, could support the wild-type level of concerted integration activity. After accounting for its inherent reverse transcription defect, HIV-1K264E moreover formed preintegration complexes that supported the efficient integration of endogenous viral DNA in vitro and normal levels and sequences of 2-long terminal repeat-containing circle junctions during acute infection. K264E integrase furthermore efficiently interacted in vitro with two heterologous binding partners, LEDGF/p75 and reverse transcriptase. Our results underscore the physiological relevance of concerted integration assays for tests of integrase mutant function and suggest that the K264E mutation disrupts an interaction with an intranuclear integrase binding partner that is important for HIV-1 integration. PMID:22278243

  16. Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis

    SciTech Connect

    Kishino, Hideyuki; Eguchi, Hiroki; Takagi, Keiko; Horiuchi, Hiroyuki; Fukuda, Ryouichi; Ohta, Akinori

    2014-03-07

    Highlights: • Dioctanoyl-PC (diC8PC) supported growth of a yeast mutant defective in PC synthesis. • diC8PC was converted to PC species containing longer acyl residues in the mutant. • Both acyl residues of diC8PC were replaced by longer fatty acids in vitro. • This system will contribute to the elucidation of the acyl chain remodeling of PC. - Abstract: A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipid methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of {sup 13}C-labeled diC8PC ((methyl-{sup 13}C){sub 3}-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-{sup 13}C){sub 3}-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast.

  17. Isolation of Aneuploid-Generating Mutants of ASPERGILLUS NIDULANS, One of Which Is Defective in Interphase of the Cell Cycle

    PubMed Central

    Upshall, A.; Mortimore, I. D.

    1984-01-01

    A method is described for isolating mutants potentially defective in loci involved in mitotic chromosome segregation. Conditional lethal, heat-sensitive (42°) mutants were assayed at a subrestrictive temperature of 37° for an inflated production of colonies displaying phenotypes and behavior patterns of whole chromosome aneuploids. Of 14 mutants, three showed specificity for one disomic phenotype, whereas 11 generated colonies mosaic for different aneuploid phenotypes. This latter group is designated hfa ( high frequency of aneuploid). For ten of the 11 mutants temperature sensitivity and aneuploid production cosegregated, indicating a single mutation in each. These mutations were recessive and nonallelic. Analysis was concentrated on the hfaB3 mutation which is mapped to chromosome VI tightly linked to the methB and tsB loci. The disruptive influence of hfaB3 on mitosis at 37° was shown by (1) ploidy and whole chromosome-type segregation of markers in the breakdown sectors of phenotypically aneuploid colonies obtained from multiply marked homozygous hfaB3 disploids; (2) a high frequency of haploid and nondisjunctional diploid segregants among spontaneous yellow-spored parasexual recombinants taken from green-spored homozygous hfaB3 diploids. The mutation had no effect on meiotic chromosome segregation at 37°. The single interphase nucleus in germlings at 42°, coupled with changes in the mitotic index in temperature exchange experiments, showed hfaB3 to arrest the cell cycle in interphase at restrictive temperature. A conclusion drawn is that the hfaB gene product is required both for entry into mitosis and for normal chromosome segregation in dividing nuclei. PMID:6479583

  18. Defects in tRNA Modification Associated with Neurological and Developmental Dysfunctions in Caenorhabditis elegans Elongator Mutants

    PubMed Central

    Chen, Changchun; Tuck, Simon; Byström, Anders S.

    2009-01-01

    Elongator is a six subunit protein complex, conserved from yeast to humans. Mutations in the human Elongator homologue, hELP1, are associated with the neurological disease familial dysautonomia. However, how Elongator functions in metazoans, and how the human mutations affect neural functions is incompletely understood. Here we show that in Caenorhabditis elegans, ELPC-1 and ELPC-3, components of the Elongator complex, are required for the formation of the 5-carbamoylmethyl and 5-methylcarboxymethyl side chains of wobble uridines in tRNA. The lack of these modifications leads to defects in translation in C. elegans. ELPC-1::GFP and ELPC-3::GFP reporters are strongly expressed in a subset of chemosensory neurons required for salt chemotaxis learning. elpc-1 or elpc-3 gene inactivation causes a defect in this process, associated with a posttranscriptional reduction of neuropeptide and a decreased accumulation of acetylcholine in the synaptic cleft. elpc-1 and elpc-3 mutations are synthetic lethal together with those in tuc-1, which is required for thiolation of tRNAs having the 5′methylcarboxymethyl side chain. elpc-1; tuc-1 and elpc-3; tuc-1 double mutants display developmental defects. Our results suggest that, by its effect on tRNA modification, Elongator promotes both neural function and development. PMID:19593383

  19. The human CSB (ERCC6) gene corrects the transcription-coupled repair defect in the CHO cell mutant UV61.

    PubMed

    Orren, D K; Dianov, G L; Bohr, V A

    1996-09-01

    The human CSB gene, mutated in Cockayne's syndrome group B (partially defective in both repair and transcription) was previously cloned by virtue of its ability to correct the moderate UV sensitivity of the CHO mutant UV61. To determine whether the defect in UV61 is the hamster equivalent of Cockayne's syndrome, the RNA polymerase II transcription and DNA repair characteristics of a repair-proficient CHO cell line (AA8), UV61 and a CSB transfectant of UV61 were compared. In each cell line, formation and removal of UV-induced cyclobutane pyrimidine dimers (CPDs) were measured in the individual strands of the actively transcribed DHFR gene and in a transcriptionally inactive region downstream of DHFR. AA8 cells efficiently remove CPDs from the transcribed strand, but not from either the non-transcribed strand or the inactive region. There was no detectable repair of CPDs in any region of the genome in UV61. Transfection of the human CSB gene into UV61 restores the normal repair pattern (CPD removal in only the transcribed strand), demonstrating that the DNA repair defect in UV61 is homologous to that in Cockayne's syndrome (complementation group B) cells. However, we observe no significant deficiency in RNA polymerase II-mediated transcription in UV61, suggesting that the CSB protein has independent roles in DNA repair and RNA transcription pathways. PMID:8811084

  20. Strong morphological defects in conditional Arabidopsis abp1 knock-down mutants generated in absence of functional ABP1 protein

    PubMed Central

    Perrot-Rechenmann, Catherine; Friml, Jiří

    2016-01-01

    The Auxin Binding Protein 1 (ABP1) is one of the most studied proteins in plants. Since decades ago, it has been the prime receptor candidate for the plant hormone auxin with a plethora of described functions in auxin signaling and development. The developmental importance of ABP1 has recently been questioned by identification of Arabidopsis thaliana abp1 knock-out alleles that show no obvious phenotypes under normal growth conditions. In this study, we examined the contradiction between the normal growth and development of the abp1 knock-outs and the strong morphological defects observed in three different ethanol-inducible abp1 knock-down mutants ( abp1-AS, SS12K, SS12S). By analyzing segregating populations of abp1 knock-out vs. abp1 knock-down crosses we show that the strong morphological defects that were believed to be the result of conditional down-regulation of ABP1 can be reproduced also in the absence of the functional ABP1 protein. This data suggests that the phenotypes in  abp1 knock-down lines are due to the off-target effects and asks for further reflections on the biological function of ABP1 or alternative explanations for the missing phenotypic defects in the abp1 loss-of-function alleles. PMID:26925228

  1. Unidirectional startle responses and disrupted left-right coordination of motor behaviors in robo3 mutant zebrafish

    PubMed Central

    Burgess, Harold A.; Johnson, Stephen L.; Granato, Michael

    2009-01-01

    The Roundabout (Robo) family of receptors and their Slit ligands play well-established roles in axonal guidance, including in humans where horizontal gaze palsy with progressive scoliosis (HGPPS) is caused by mutations in the robo3 gene. While significant progress has been made towards understanding the mechanism by which Robo receptors establish commissural projections in the central nervous system, less is known about how these projections contribute to neural circuits mediating behavior. Here we report cloning of the zebrafish behavioral mutant twitch twice and show that twitch twice encodes robo3. We demonstrate that in mutant hindbrains the axons of an identified pair of neurons, the Mauthner cells, fail to cross the midline. The Mauthner neurons are essential for the startle response, and in twitch twice/robo3 mutants misguidance of the Mauthner axons results in a unidirectional startle response. Moreover, we show that twitch twice mutants exhibit normal visual acuity but display defects in horizontal eye movements, suggesting a specific and critical role for twitch twice/robo3 in sensory guided behavior. PMID:19496826

  2. A proteome analysis of the response of a Pseudomonas aeruginosa oxyR mutant to iron limitation.

    PubMed

    Vinckx, Tiffany; Wei, Qing; Matthijs, Sandra; Noben, Jean-Paul; Daniels, Ruth; Cornelis, Pierre

    2011-06-01

    In Pseudomonas aeruginosa the response to oxidative stress is orchestrated by the LysR regulator OxyR by activation of the transcription of two catalase genes (katA and katB), of the alkyl-hydroxyperoxidases ahpCF and ahpB. Next to the expected high sensitivity to oxidative stress generated by reactive oxygen species (ROS: H(2)O(2), O(2)(-)), the oxyR mutant shows a defective growth under conditions of iron limitation (Vinckx et al. 2008). Although production and uptake of the siderophore pyoverdine is not affected by the absence of oxyR, the mutant is unable to satisfy its need for iron when grown under iron limiting conditions. In order to get a better insight into the effects caused by iron limitation on the physiological response of the oxyR mutant we decided to compare the proteomes of the wild type and the mutant grown in the iron-poor casamino acids medium (CAA), in CAA plus H(2)O(2), and in CAA plus the strong iron chelator ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid) (EDDHA). Especially in the presence of hydrogen peroxide the oxyR cells increase the production of stress proteins (Dps and IbpA). The superoxide dismutase SodM is produced in higher amounts in the oxyR mutant grown in CAA plus H(2)O(2). The PchB protein, a isochorismate-pyruvate lyase involved in the siderophore pyochelin biosynthesis is not detectable in the extracts from the oxyR mutant grown in the presence of hydrogen peroxide. When cells were grown in the presence of EDDHA, we observed a reduction of the ferric uptake regulator (Fur), and an increase in the two subunits of the succinyl-CoA synthetase and the fumarase FumC1. PMID:21207115

  3. Demonstration of two distinct cytopathic effects with syncytium formation-defective human immunodeficiency virus type 1 mutants.

    PubMed

    Dedera, D; Ratner, L

    1991-11-01

    The mechanism of human immunodeficiency virus type 1 (HIV-1) cytopathicity is poorly understood and might involve formation of multinucleated giant cells (syncytia), single-cell lysis, or both. In order to determine the contributions of the fusion domain to syncytium formation, single-cell lysis, and viral infectivity and to clarify the molecular details of these events, insertion mutations were made in the portion of env encoding this sequence in the functional HIV-1 proviral clone HXB2. Viruses produced from these mutant clones were found to have a partial (F3) or complete (F6) loss of syncytium-forming ability in acutely infected CEM, Sup T1, and MT4 T-cell lines. During the early stage of acute infection by F6 virus, there was a loss of the syncytial cytopathic effect, which resulted in increased cell viability, and a 1.9- to 2.6-fold increase in virus yield in the cell lines tested. In the late stage of acute infection, the single-cell cytopathic effect of F6 virus was similar to that of the parental HXB2 virus. The F3 and F6 viruses were also found to have a 1.7- to 43-fold reduction in infectivity compared with the HXB2 virus. The mutant F3 and F6 and parental HXB2 envelope proteins were expressed in vaccinia virus, and the mutant envelope proteins were observed to be defective in their ability to form syncytia. BSC-40 cells infected with vaccinia virus recombinants revealed no differences in kinetics of cleavage, cell surface expression, or CD4 binding capacity of the mutant and parental envelope proteins. These results demonstrate that a loss of syncytium formation results in an attenuation of infectivity and a loss of the syncytial cytopathic effect without a loss of single-cell lysis. These mutants may reflect in tissue culture the changes observed in the HIV isolates in vivo during disease progression, which exhibit marked differences in syncytium production.

  4. Carbon allocation and element composition in four Chlamydomonas mutants defective in genes related to the CO2 concentrating mechanism.

    PubMed

    Memmola, Francesco; Mukherjee, Bratati; Moroney, James V; Giordano, Mario

    2014-09-01

    Four mutants of Chlamydomonas reinhardtii with defects in different components of the CO2 concentrating mechanism (CCM) or in Rubisco activase were grown autotrophically at high pCO2 and then transferred to low pCO2, in order to study the role of different components of the CCM on carbon allocation and elemental composition. To study carbon allocation, we measured the relative size of the main organic pools by Fourier Transform Infrared spectroscopy. Total reflection X-ray fluorescence was used to analyze the elemental composition of algal cells. Our data show that although the organic pools increased their size at high CO2 in all strains, their stoichiometry was highly homeostatic, i.e., the ratios between carbohydrates and proteins, lipid and proteins, and carbohydrates and lipids, did not change significantly. The only exception was the wild-type 137c, in which proteins decreased relative to carbohydrates and lipids, when the cells were transferred to low CO2. It is noticeable that the two wild types used in this study responded differently to the transition from high to low CO2. Malfunctions of the CCM influenced the concentration of several elements, somewhat altering cell elemental stoichiometry: especially the C/P and N/P ratios changed appreciably in almost all strains as a function of the growth CO2 concentration, except in 137c and the Rubisco activase mutant rca1. In strain cia3, defective in the lumenal carbonic anhydrase (CA), the cell quotas of P, S, Ca, Mn, Fe, and Zn were about 5-fold higher at low CO2 than at high CO2. A Principle Components Analysis showed that, mostly because of its elemental composition, cia3 behaved in a substantially different way from all other strains, at low CO2. The lumenal CA thus plays a crucial role, not only for the correct functioning of the CCM, but also for element utilization. Not surprisingly, growth at high CO2 attenuated differences among strains.

  5. Development and analysis of a transformation-defective mutant of Harvey murine sarcoma tk virus and its gene product.

    PubMed Central

    Weeks, M O; Hager, G L; Lowe, R; Scolnick, E M

    1985-01-01

    The Harvey murine sarcoma virus has been cloned and induces focus formation on NIH 3T3 cells. Recombinants of this virus have been constructed which include the thymidine kinase gene of herpes simplex virus type 1 in a downstream linkage with the p21 ras gene of Harvey murine sarcoma virus. Harvey murine sarcoma tk virus rescued from cells transfected with this construct is both thymidine kinase positive and focus inducing in in vitro transmission studies. The hypoxanthine-aminopterin-thymidine selectability of the thymidine kinase gene carried by this virus has been exploited to develop three mutants defective in the p21 ras sequence. All three are focus negative and thymidine kinase positive when transmitted to suitable cells. Of these, only one encodes a p22 that is immunologically related to p21. This mutant has been used to explore the relationship between the known characteristics of p21 and cellular transformation. Data presented herein indicate that the p21 of Harvey murine sarcoma virus consists of at least two domains, one which specifies the guanine nucleotide-binding activity of p21 and the other which is involved in p21-membrane association in transformed cells. Images PMID:2985821

  6. Development and analysis of a transformation-defective mutant of Harvey murine sarcoma tk virus and its gene product.

    PubMed

    Weeks, M O; Hager, G L; Lowe, R; Scolnick, E M

    1985-05-01

    The Harvey murine sarcoma virus has been cloned and induces focus formation on NIH 3T3 cells. Recombinants of this virus have been constructed which include the thymidine kinase gene of herpes simplex virus type 1 in a downstream linkage with the p21 ras gene of Harvey murine sarcoma virus. Harvey murine sarcoma tk virus rescued from cells transfected with this construct is both thymidine kinase positive and focus inducing in in vitro transmission studies. The hypoxanthine-aminopterin-thymidine selectability of the thymidine kinase gene carried by this virus has been exploited to develop three mutants defective in the p21 ras sequence. All three are focus negative and thymidine kinase positive when transmitted to suitable cells. Of these, only one encodes a p22 that is immunologically related to p21. This mutant has been used to explore the relationship between the known characteristics of p21 and cellular transformation. Data presented herein indicate that the p21 of Harvey murine sarcoma virus consists of at least two domains, one which specifies the guanine nucleotide-binding activity of p21 and the other which is involved in p21-membrane association in transformed cells.

  7. Molecular characterization of V59E NIS, a Na+/I- symporter mutant that causes congenital I- transport defect.

    PubMed

    Reed-Tsur, Mia D; De la Vieja, Antonio; Ginter, Christopher S; Carrasco, Nancy

    2008-06-01

    I(-) is actively transported into thyrocytes via the Na+/I(-) symporter (NIS), a key glycoprotein located on the basolateral plasma membrane. The cDNA encoding rat NIS was identified in our laboratory, where an extensive structure/function characterization of NIS is being conducted. Several NIS mutants have been identified as causes of congenital I(-) transport defect (ITD), including V59E NIS. ITD is characterized by low thyroid I(-) uptake, low saliva/plasma I(-) ratio, hypothyroidism, and goiter and may cause mental retardation if untreated. Studies of other ITD-causing NIS mutants have revealed valuable information regarding NIS structure/function. V59E NIS was reported to exhibit as much as 30% of the activity of wild-type NIS. However, this observation was at variance with the patients' phenotype of total lack of activity. We have thoroughly characterized V59E NIS and studied several amino acid substitutions at position 59. We demonstrated that, in contrast to the previous report, V59E NIS is inactive, although it is properly targeted to the plasma membrane. Glu and all other charged amino acids or Pro at position 59 also yielded nonfunctional NIS proteins. However, I(-) uptake was rescued to different degrees by the other substitutions. Although the Km values for Na+ and I(-) were not altered in these active mutants, we found that the structural requirement for NIS function at position 59 is a neutral, helix-promoting amino acid. This result suggests that the region that contains V59 may be involved in intramembrane helix-helix interactions during the transport cycle without being in direct contact with the substrates. PMID:18339708

  8. groE mutants of Escherichia coli are defective in umuDC-dependent UV mutagenesis

    SciTech Connect

    Donnelly, C.E.; Walker, G.C. )

    1989-11-01

    Overexpression of the SOS-inducible umuDC operon of Escherichia coli results in the inability of these cells to grow at 30 degrees C. Mutations in several heat shock genes suppress this cold sensitivity. Suppression of umuD+C+-dependent cold sensitivity appears to occur by two different mechanisms. We show that mutations in lon and dnaK heat shock genes suppress cold sensitivity in a lexA-dependent manner. In contrast, mutations in groES, groEL, and rpoH heat shock genes suppress cold sensitivity regardless of the transcriptional regulation of the umuDC genes. We have also found that mutations in groES and groEL genes are defective in umuDC-dependent UV mutagenesis. This defect can be suppressed by increased expression of the umuDC operon. The mechanism by which groE mutations affect umuDC gene product function may be related to the stability of the UmuC protein, since the half-life of this protein is shortened because of mutations at the groE locus.

  9. Characterization of the Two Maize Embryo-Lethal Defective Kernel Mutants Rgh*-1210 and Fl*-1253b: Effects on Embryo and Gametophyte Development

    PubMed Central

    Clark, J. K.; Sheridan, W. F.

    1988-01-01

    We have examined the effects on embryonic and gametophytic development of two nonallelic defective-kernel mutants of maize. Earlier studies indicated that both mutants are abnormal in embryonic morphogenesis as well as in the formation of their endosperm. Mutant rgh*-1210 embryos depart from the normal embryogenic pathway at the proembryo and transition stage, by developing meristematic lobes and losing bilateral symmetry. They continue growth as irregular cell masses that enlarge and become necrotic. Somatic embryos arising in rgh*-1210 callus cultures display the rgh*-1210 mutant phenotype. Mutant fl*-1253B embryos are variably blocked from the coleoptilar stage through stage 2. Following formation of the shoot apex in the mutant embryos the leaf primordia and tissues surrounding the embryonic axis continue growth and cell division, while the scutellum ceases development and becomes hypertrophied. Mutant fl*-1253B embryos are unable to germinate, either in mutant kernels or as immature embryos in culture, and the mutant scutellar tissue does not produce regenerable callus. Expression of the fl*-1253B locus during male gametophytic development is revealed by a marked reduction in pollen transmission as a result of mutant expression during the interval between meiosis and the initiation of pollen tube growth. In both mutants, there is considerable proliferation of the aleurone cells of the endosperm. Mutant expression of rgh*-1210 in the female gametophyte is revealed by the abnormal antipodal cells of the embryo sac. These results show that these two gene loci play unique and crucial roles in normal morphogenesis of the embryo. In addition, it is evident that both mutants are pleiotropic in affecting the development of the endosperm and gametophyte as well as the embryo. These pleiotropisms suggest some commonality in the gene regulation of development in these three tissues. PMID:17246478

  10. Delayed Induction of Human NTE (PNPLA6) Rescues Neurodegeneration and Mobility Defects of Drosophila swiss cheese (sws) Mutants

    PubMed Central

    Sujkowski, Alyson; Rainier, Shirley; Fink, John K.; Wessells, Robert J.

    2015-01-01

    Human PNPLA6 gene encodes Neuropathy Target Esterase protein (NTE). PNPLA6 gene mutations cause hereditary spastic paraplegia (SPG39 HSP), Gordon-Holmes syndrome, Boucher-Neuhäuser syndromes, Laurence-Moon syndrome, and Oliver-McFarlane syndrome. Mutations in the Drosophila NTE homolog swiss cheese (sws) cause early-onset, progressive behavioral defects and neurodegeneration characterized by vacuole formation. We investigated sws5 flies and show for the first time that this allele causes progressive vacuolar formation in the brain and progressive deterioration of negative geotaxis speed and endurance. We demonstrate that inducible, neuron-specific expression of full-length human wildtype NTE reduces vacuole formation and substantially rescues mobility. Indeed, neuron-specific expression of wildtype human NTE is capable of rescuing mobility defects after 10 days of adult life at 29°C, when significant degeneration has already occurred, and significantly extends longevity of mutants at 25°C. These results raise the exciting possibility that late induction of NTE function may reduce or ameliorate neurodegeneration in humans even after symptoms begin. In addition, these results highlight the utility of negative geotaxis endurance as a new assay for longitudinal tracking of degenerative phenotypes in Drosophila. PMID:26671664

  11. Haemophilus ducreyi Lipooligosaccharide Mutant Defective in Expression of β-1,4-Glucosyltransferase Is Virulent in Humans

    PubMed Central

    Young, Royden S.; Filiatrault, Melanie J.; Fortney, Kate R.; Hood, Antoinette F.; Katz, Barry P.; Munson, Robert S.; Campagnari, Anthony A.; Spinola, Stanley M.

    2001-01-01

    The lipooligosaccharide (LOS) of Haemophilus ducreyi contains a major glycoform that is immunochemically identical to paragloboside, a glycosphingolipid precursor of major human blood group antigens. We recently identified the gene responsible for the glucosyltransferase activity and constructed an isogenic mutant (35000glu-) deficient in this activity. 35000glu- makes an LOS that consists only of the heptose trisaccharide core and 2-keto-deoxyoctulosonic acid (KDO). For this study, the mutant was reconstructed in the 35000HP (human passaged [HP]) background. Five human subjects were inoculated with 35000HP and 35000HPglu- in a dose-response trial. The pustule formation rates were 40% (95% confidence interval [CI], 13.7 to 72.6%) at 10 sites for 35000HP and 46.7% (95% CI, 24.8 to 69.9%) at 15 sites for 35000HPglu-. The histopathology and recovery rates of H. ducreyi from surface cultures and biopsies obtained from mutant and parent sites were similar. These results indicate that the expression of glycoforms with sugar moieties extending beyond the heptose trisaccharide core is not required for pustule formation by H. ducreyi in humans. PMID:11349097

  12. Fatty acid elongation in yeast--biochemical characteristics of the enzyme system and isolation of elongation-defective mutants.

    PubMed

    Dittrich, F; Zajonc, D; Hühne, K; Hoja, U; Ekici, A; Greiner, E; Klein, H; Hofmann, J; Bessoule, J J; Sperling, P; Schweizer, E

    1998-03-15

    Elongation of long-chain fatty acids was investigated in yeast mutants lacking endogenous de novo fatty acid synthesis. In this background, in vitro fatty acid elongation was dependent strictly on the substrates malonyl-CoA, NADPH and a medium-chain or long-chain acyl-CoA primer of 10 or more carbon atoms. Maximal activity was observed with primers containing 12-14 carbon atoms, while shorter-chain-length acyl-CoA were almost (octanoyl-CoA) or completely (hexanoyl-CoA, acetyl-CoA) inactive. In particular, acetyl-CoA was inactive as a primer and as extender unit. The Michaelis constants for octanoyl-CoA (0.33 mM), decanoyl-CoA (0.83 mM) lauroyl-CoA (0.05 mM), myristoyl-CoA (0.4 mM) and palmitoyl-CoA (0.13 mM) were determined and were comparable for fatty acid synthesis and elongation. In contrast, the affinity of malonyl-CoA was 17-fold lower for elongation (Km = 0.13 mM) than for the fatty acid synthase (FAS) system. With increasing chain length of the primer (> or = 12:0), fatty acid elongation becomes increasingly sensitive to substrate inhibition. Due to the activation of endogenous fatty acids, ATP exhibits a stimulatory effect at suboptimal but not at saturating substrate concentrations. In the yeast cell homogenate, the specific activity of fatty acid elongation is about 10-20-fold lower than that of de novo fatty acid synthesis. The same elongation activity is observed in respiratory competent and in mitochondrially defective cells. The products of in vitro fatty acid elongation are fatty acids of 15-17 or 22-26 carbon atoms, depending on whether tridecanoyl-CoA or stearoyl-CoA is used as a primer. In vitro, the elongation products are converted in part, by alpha-oxidation, to their odd-chain-length lower homologues or are hydrolyzed to fatty acids. In contrast, no odd-chain-length elongation products or very-long-chain fatty acids (VLCFA) shorter than 26:0 are observed in vivo. Hence, VLCFA synthesis exhibits a higher processivity in vivo than in the cell

  13. Defects of mutant DNMT1 are linked to a spectrum of neurological disorders.

    PubMed

    Baets, Jonathan; Duan, Xiaohui; Wu, Yanhong; Smith, Gordon; Seeley, William W; Mademan, Inès; McGrath, Nicole M; Beadell, Noah C; Khoury, Julie; Botuyan, Maria-Victoria; Mer, Georges; Worrell, Gregory A; Hojo, Kaori; DeLeon, Jessica; Laura, Matilde; Liu, Yo-Tsen; Senderek, Jan; Weis, Joachim; Van den Bergh, Peter; Merrill, Shana L; Reilly, Mary M; Houlden, Henry; Grossman, Murray; Scherer, Steven S; De Jonghe, Peter; Dyck, Peter J; Klein, Christopher J

    2015-04-01

    We report a broader than previously appreciated clinical spectrum for hereditary sensory and autonomic neuropathy type 1E (HSAN1E) and a potential pathogenic mechanism for DNA methyltransferase (DNMT1) mutations. The clinical presentations and genetic characteristics of nine newly identified HSAN1E kinships (45 affected subjects) were investigated. Five novel mutations of DNMT1 were discovered; p.C353F, p.T481P, p.P491L, p.Y524D and p.I531N, all within the target-sequence domain, and two mutations (p.T481P, p.P491L) arising de novo. Recently, HSAN1E has been suggested as an allelic disorder of autosomal dominant cerebellar ataxia, deafness and narcolepsy. Our results indicate that all the mutations causal for HSAN1E are located in the middle part or N-terminus end of the TS domain, whereas all the mutations causal for autosomal dominant cerebellar ataxia, deafness and narcolepsy are located in the C-terminus end of the TS domain. The impact of the seven causal mutations in this cohort was studied by cellular localization experiments. The binding efficiency of the mutant DNMT proteins at the replication foci and heterochromatin were evaluated. Phenotypic characterizations included electromyography, brain magnetic resonance and nuclear imaging, electroencephalography, sural nerve biopsies, sleep evaluation and neuropsychometric testing. The average survival of HSAN1E was 53.6 years. [standard deviation = 7.7, range 43-75 years], and mean onset age was 37.7 years. (standard deviation = 8.6, range 18-51 years). Expanded phenotypes include myoclonic seizures, auditory or visual hallucinations, and renal failure. Hypersomnia, rapid eye movement sleep disorder and/or narcolepsy were identified in 11 subjects. Global brain atrophy was found in 12 of 14 who had brain MRI. EEGs showed low frequency (delta waves) frontal-predominant abnormality in five of six patients. Marked variability in cognitive deficits was observed, but the majority of patients (89%) developed

  14. Defects of mutant DNMT1 are linked to a spectrum of neurological disorders

    PubMed Central

    Baets, Jonathan; Duan, Xiaohui; Wu, Yanhong; Smith, Gordon; Seeley, William W.; Mademan, Inès; McGrath, Nicole M.; Beadell, Noah C.; Khoury, Julie; Botuyan, Maria-Victoria; Mer, Georges; Worrell, Gregory A.; Hojo, Kaori; DeLeon, Jessica; Laura, Matilde; Liu, Yo-Tsen; Senderek, Jan; Weis, Joachim; Van den Bergh, Peter; Merrill, Shana L.; Reilly, Mary M.; Houlden, Henry; Grossman, Murray; Scherer, Steven S.; De Jonghe, Peter; Dyck, Peter J.

    2015-01-01

    We report a broader than previously appreciated clinical spectrum for hereditary sensory and autonomic neuropathy type 1E (HSAN1E) and a potential pathogenic mechanism for DNA methyltransferase (DNMT1) mutations. The clinical presentations and genetic characteristics of nine newly identified HSAN1E kinships (45 affected subjects) were investigated. Five novel mutations of DNMT1 were discovered; p.C353F, p.T481P, p.P491L, p.Y524D and p.I531N, all within the target-sequence domain, and two mutations (p.T481P, p.P491L) arising de novo. Recently, HSAN1E has been suggested as an allelic disorder of autosomal dominant cerebellar ataxia, deafness and narcolepsy. Our results indicate that all the mutations causal for HSAN1E are located in the middle part or N-terminus end of the TS domain, whereas all the mutations causal for autosomal dominant cerebellar ataxia, deafness and narcolepsy are located in the C-terminus end of the TS domain. The impact of the seven causal mutations in this cohort was studied by cellular localization experiments. The binding efficiency of the mutant DNMT proteins at the replication foci and heterochromatin were evaluated. Phenotypic characterizations included electromyography, brain magnetic resonance and nuclear imaging, electroencephalography, sural nerve biopsies, sleep evaluation and neuropsychometric testing. The average survival of HSAN1E was 53.6 years. [standard deviation = 7.7, range 43–75 years], and mean onset age was 37.7 years. (standard deviation = 8.6, range 18–51 years). Expanded phenotypes include myoclonic seizures, auditory or visual hallucinations, and renal failure. Hypersomnia, rapid eye movement sleep disorder and/or narcolepsy were identified in 11 subjects. Global brain atrophy was found in 12 of 14 who had brain MRI. EEGs showed low frequency (delta waves) frontal-predominant abnormality in five of six patients. Marked variability in cognitive deficits was observed, but the majority of patients (89%) developed

  15. Defects of mutant DNMT1 are linked to a spectrum of neurological disorders

    PubMed Central

    Baets, Jonathan; Duan, Xiaohui; Wu, Yanhong; Smith, Gordon; Seeley, William W.; Mademan, Inès; McGrath, Nicole M.; Beadell, Noah C.; Khoury, Julie; Botuyan, Maria-Victoria; Mer, Georges; Worrell, Gregory A.; Hojo, Kaori; DeLeon, Jessica; Laura, Matilde; Liu, Yo-Tsen; Senderek, Jan; Weis, Joachim; Van den Bergh, Peter; Merrill, Shana L.; Reilly, Mary M.; Houlden, Henry; Grossman, Murray; Scherer, Steven S.; De Jonghe, Peter; Dyck, Peter J.

    2015-01-01

    We report a broader than previously appreciated clinical spectrum for hereditary sensory and autonomic neuropathy type 1E (HSAN1E) and a potential pathogenic mechanism for DNA methyltransferase (DNMT1) mutations. The clinical presentations and genetic characteristics of nine newly identified HSAN1E kinships (45 affected subjects) were investigated. Five novel mutations of DNMT1 were discovered; p.C353F, p.T481P, p.P491L, p.Y524D and p.I531N, all within the target-sequence domain, and two mutations (p.T481P, p.P491L) arising de novo. Recently, HSAN1E has been suggested as an allelic disorder of autosomal dominant cerebellar ataxia, deafness and narcolepsy. Our results indicate that all the mutations causal for HSAN1E are located in the middle part or N-terminus end of the TS domain, whereas all the mutations causal for autosomal dominant cerebellar ataxia, deafness and narcolepsy are located in the C-terminus end of the TS domain. The impact of the seven causal mutations in this cohort was studied by cellular localization experiments. The binding efficiency of the mutant DNMT proteins at the replication foci and heterochromatin were evaluated. Phenotypic characterizations included electromyography, brain magnetic resonance and nuclear imaging, electroencephalography, sural nerve biopsies, sleep evaluation and neuropsychometric testing. The average survival of HSAN1E was 53.6 years. [standard deviation = 7.7, range 43–75 years], and mean onset age was 37.7 years. (standard deviation = 8.6, range 18–51 years). Expanded phenotypes include myoclonic seizures, auditory or visual hallucinations, and renal failure. Hypersomnia, rapid eye movement sleep disorder and/or narcolepsy were identified in 11 subjects. Global brain atrophy was found in 12 of 14 who had brain MRI. EEGs showed low frequency (delta waves) frontal-predominant abnormality in five of six patients. Marked variability in cognitive deficits was observed, but the majority of patients (89%) developed

  16. A mutation affecting carbon catabolite repression suppresses growth defects in pyruvate carboxylase mutants from Saccharomyces cerevisiae.

    PubMed

    Blázquez, M A; Gamo, F J; Gancedo, C

    1995-12-18

    Yeasts with disruptions in the genes PYC1 and PYC2 encoding the isoenzymes of pyruvate carboxylase cannot grow in a glucose-ammonium medium (Stucka et al. (1991) Mol. Gen. Genet. 229, 307-315). We have isolated a dominant mutation, BPC1-1, that allows growth in this medium of yeasts with interrupted PYC1 and PYC2 genes. The BPC1-1 mutation abolishes catabolite repression of a series of genes and allows expression of the enzymes of the glyoxylate cycle during growth in glucose. A functional glyoxylate cycle is necessary for suppression as a disruption of gene ICL1 encoding isocitrate lyase abolished the phenotypic effect of BPC1-1 on growth in glucose-ammonium. Concurrent expression from constitutive promoters of genes ICL1 and MLS1 (encoding malate synthase) also suppressed the growth phenotype of pyc1 pyc2 mutants. The mutation BPC1-1 is either allelic or closely linked to the mutation DGT1-1.

  17. A West Nile virus NS4B-P38G mutant strain induces cell intrinsic innate cytokine responses in human monocytic and macrophage cells

    PubMed Central

    Xie, Guorui; Luo, Huanle; Tian, Bing; Mann, Brian; Bao, Xiaoyong; McBride, Jere; Tesh, Robert; Barrett, Alan D; Wang, Tian

    2015-01-01

    Previous studies have shown that an attenuated West Nile virus (WNV) nonstructural (NS) 4B-P38G mutant induces stronger innate and adaptive immune responses than wild-type WNV in mice, which has important applications to vaccine development. To investigate the mechanism of immunogenicity, we characterized WNV NS4B-P38G mutant infection in two human cell lines--THP-1 cells and THP-1 macrophages. Although the NS4B-P38G mutant produced more viral RNA than the parental WNV NY99 in both cell types, there was no detectable infectious virus in the supernatant of either cell type. Nonetheless, the attenuated mutant boosted higher innate cytokine responses than virulent parental WNV NY99 in these cells. The NS4B-P38G mutant infection of THP-1 cells led to more diverse and robust innate cytokine responses than that seen in THP-1 macrophages, which were mediated by toll-like receptor (TLR)7 and retinoic acid-inducible gene 1(RIG-I) signaling pathways. Overall, these results suggest that a defective viral life cycle during NS4B-P38G mutant infection in human monocytic and macrophage cells leads to more potent cell intrinsic innate cytokine responses. PMID:25562791

  18. Genetically engineered SCN5A mutant pig hearts exhibit conduction defects and arrhythmias.

    PubMed

    Park, David S; Cerrone, Marina; Morley, Gregory; Vasquez, Carolina; Fowler, Steven; Liu, Nian; Bernstein, Scott A; Liu, Fang-Yu; Zhang, Jie; Rogers, Christopher S; Priori, Silvia G; Chinitz, Larry A; Fishman, Glenn I

    2015-01-01

    SCN5A encodes the α subunit of the major cardiac sodium channel Na(V)1.5. Mutations in SCN5A are associated with conduction disease and ventricular fibrillation (VF); however, the mechanisms that link loss of sodium channel function to arrhythmic instability remain unresolved. Here, we generated a large-animal model of a human cardiac sodium channelopathy in pigs, which have cardiac structure and function similar to humans, to better define the arrhythmic substrate. We introduced a nonsense mutation originally identified in a child with Brugada syndrome into the orthologous position (E558X) in the pig SCN5A gene. SCN5A(E558X/+) pigs exhibited conduction abnormalities in the absence of cardiac structural defects. Sudden cardiac death was not observed in young pigs; however, Langendorff-perfused SCN5A(E558X/+) hearts had an increased propensity for pacing-induced or spontaneous VF initiated by short-coupled ventricular premature beats. Optical mapping during VF showed that activity often began as an organized focal source or broad wavefront on the right ventricular (RV) free wall. Together, the results from this study demonstrate that the SCN5A(E558X/+) pig model accurately phenocopies many aspects of human cardiac sodium channelopathy, including conduction slowing and increased susceptibility to ventricular arrhythmias.

  19. Draft Genome Sequences for Clostridium thermocellum Wild-Type Strain YS and Derived Cellulose Adhesion-Defective Mutant Strain AD2

    SciTech Connect

    Brown, Steven D; Lamed, Raphael; Morag, Ely; Borovok, Ilya; Shoham, Yuval; Klingeman, Dawn Marie; Johnson, Courtney M; Yang, Zamin; Land, Miriam L; Utturkar, Sagar M; Keller, Martin; Bayer, Edward A

    2012-01-01

    Clostridium thermocellum wild-type strain YS is an anaerobic, thermophilic, cellulolytic bacterium capable of directly converting cellulosic substrates into ethanol. Strain YS and a derived cellulose adhesion-defective mutant strain AD2 played pivotal roles in describing the original cellulosome concept. We present their draft genome sequences.

  20. An ALS-linked mutant SOD1 produces a locomotor defect associated with aggregation and synaptic dysfunction when expressed in neurons of Caenorhabditis elegans.

    PubMed

    Wang, Jiou; Farr, George W; Hall, David H; Li, Fei; Furtak, Krystyna; Dreier, Lars; Horwich, Arthur L

    2009-01-01

    The nature of toxic effects exerted on neurons by misfolded proteins, occurring in a number of neurodegenerative diseases, is poorly understood. One approach to this problem is to measure effects when such proteins are expressed in heterologous neurons. We report on effects of an ALS-associated, misfolding-prone mutant human SOD1, G85R, when expressed in the neurons of Caenorhabditis elegans. Stable mutant transgenic animals, but not wild-type human SOD1 transgenics, exhibited a strong locomotor defect associated with the presence, specifically in mutant animals, of both soluble oligomers and insoluble aggregates of G85R protein. A whole-genome RNAi screen identified chaperones and other components whose deficiency increased aggregation and further diminished locomotion. The nature of the locomotor defect was investigated. Mutant animals were resistant to paralysis by the cholinesterase inhibitor aldicarb, while exhibiting normal sensitivity to the cholinergic agonist levamisole and normal muscle morphology. When fluorescently labeled presynaptic components were examined in the dorsal nerve cord, decreased numbers of puncta corresponding to neuromuscular junctions were observed in mutant animals and brightness was also diminished. At the EM level, mutant animals exhibited a reduced number of synaptic vesicles. Neurotoxicity in this system thus appears to be mediated by misfolded SOD1 and is exerted on synaptic vesicle biogenesis and/or trafficking. PMID:19165329

  1. Intra-host competition between nef-defective escape mutants and wild-type human immunodeficiency virus type 1.

    PubMed Central

    Altes, H K; Jansen, V A

    2000-01-01

    Various forms of nef genes with deletions at conserved positions along the sequence have been reported to persist in human immunodeficiency virus type 1 infected patients. We investigate the forces maintaining such variants in the proviral population. The main selection pressures are preservation of function and host immune response. The crippled Nef protein might have fewer epitopes, and as such be less visible to the specific immune response, but it will lose some function. Does a trade-off between avoidance of the immune response and loss of function explain the dynamics of the crippled virus found in the patients? To answer this question, we formulated a deterministic model of the virus-host interactions. We found that when the crippled protein presents few epitopes and suffers little loss of function, the two viral types can coexist. Otherwise, the wild-type comes to prevail. The mutant form might initially dominate, but as the selective pressure by the CD84+ T cells decreases over the course of infection, the advantage for the crippled form of losing epitopes disappears. Hence, we go from a situation of coexistence of wild-type and mutant, to a situation of only full-length nef. The results are discussed in the context of the suggested use of live attenuated vaccines having deletions in nef. PMID:10687825

  2. The thick aleurone1 mutant defines a negative regulation of maize aleurone cell fate that functions downstream of defective kernel1.

    PubMed

    Yi, Gibum; Lauter, Adrienne M; Scott, M Paul; Becraft, Philip W

    2011-08-01

    The maize (Zea mays) aleurone layer occupies the single outermost layer of the endosperm. The defective kernel1 (dek1) gene is a central regulator required for aleurone cell fate specification. dek1 mutants have pleiotropic phenotypes including lack of aleurone cells, aborted embryos, carotenoid deficiency, and a soft, floury endosperm deficient in zeins. Here we describe the thick aleurone1 (thk1) mutant that defines a novel negative function in the regulation of aleurone differentiation. Mutants possess multiple layers of aleurone cells as well as aborted embryos. Clonal sectors of thk1 mutant tissue in otherwise normal endosperm showed localized expression of the phenotype with sharp boundaries, indicating a localized cellular function for the gene. Sectors in leaves showed expanded epidermal cell morphology but the mutant epidermis generally remained in a single cell layer. Double mutant analysis indicated that the thk1 mutant is epistatic to dek1 for several aspects of the pleiotropic dek1 phenotype. dek1 mutant endosperm that was mosaic for thk1 mutant sectors showed localized patches of multilayered aleurone. Localized sectors were surrounded by halos of carotenoid pigments and double mutant kernels had restored zein profiles. In sum, loss of thk1 function restored the ability of dek1 mutant endosperm to accumulate carotenoids and zeins and to differentiate aleurone. Therefore the thk1 mutation defines a negative regulator that functions downstream of dek1 in the signaling system that controls aleurone specification and other aspects of endosperm development. The thk1 mutation was found to be caused by a deletion of approximately 2 megabases.

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

    PubMed Central

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

    2014-01-01

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

  4. Characterization of papillomavirus E1 helicase mutants defective for interaction with the SUMO-conjugating enzyme Ubc9

    SciTech Connect

    Fradet-Turcotte, Amelie; Brault, Karine; Titolo, Steve; Howley, Peter M.; Archambault, Jacques

    2009-12-20

    The E1 helicase from BPV and HPV16 interacts with Ubc9 to facilitate viral genome replication. We report that HPV11 E1 also interacts with Ubc9 in vitro and in the yeast two-hybrid system. Residues in E1 involved in oligomerization (353-435) were sufficient for binding to Ubc9 in vitro, but the origin-binding and ATPase domains were additionally required in yeast. Nuclear accumulation of BPV E1 was shown previously to depend on its interaction with Ubc9 and sumoylation on lysine 514. In contrast, HPV11 and HPV16 E1 mutants defective for Ubc9 binding remained nuclear even when the SUMO pathway was inhibited. Furthermore, we found that K514 in BPV E1 and the analogous K559 in HPV11 E1 are not essential for nuclear accumulation of E1. These results suggest that the interaction of E1 with Ubc9 is not essential for its nuclear accumulation but, rather, depends on its oligomerization and binding to DNA and ATP.

  5. High-Throughput Cloning of Temperature-Sensitive Caenorhabditis elegans Mutants with Adult Syncytial Germline Membrane Architecture Defects

    PubMed Central

    Lowry, Josh; Yochem, John; Chuang, Chien-Hui; Sugioka, Kenji; Connolly, Amy A.; Bowerman, Bruce

    2015-01-01

    The adult Caenorhabditis elegans hermaphrodite gonad consists of two mirror-symmetric U-shaped arms, with germline nuclei located peripherally in the distal regions of each arm. The nuclei are housed within membrane cubicles that are open to the center, forming a syncytium with a shared cytoplasmic core called the rachis. As the distal germline nuclei progress through meiotic prophase, they move proximally and eventually cellularize as their compartments grow in size. The development and maintenance of this complex and dynamic germline membrane architecture are relatively unexplored, and we have used a forward genetic screen to identify 20 temperature-sensitive mutations in 19 essential genes that cause defects in the germline membrane architecture. Using a combined genome-wide SNP mapping and whole genome sequencing strategy, we have identified the causal mutations in 10 of these mutants. Four of the genes we have identified are conserved, with orthologs known to be involved in membrane biology, and are required for proper development or maintenance of the adult germline membrane architecture. This work provides a starting point for further investigation of the mechanisms that control the dynamics of syncytial membrane architecture during adult oogenesis. PMID:26311651

  6. Herpes simplex virus mutants defective in the virion-associated shutoff of host polypeptide synthesis and exhibiting abnormal synthesis of alpha (immediate early) viral polypeptides.

    PubMed

    Read, G S; Frenkel, N

    1983-05-01

    Six mutants isolated from herpes simplex virus type 1 were judged to be defective with respect to the virion-associated function acting to rapidly shut off host polypeptide synthesis in herpes simplex virus-infected cells. The mutants were capable of proper entry into the cells, but, unlike the parent wild-type virus, they failed to shut off host polypeptide syntehsis in the presence of actinomycin D. They were consequently designated as virion-associated host shutoff (vhs) mutants. In the presence of actinomycin D, three of the mutants, vhs1, -2, and -3, failed to shut off the host at both 34 and 39 degrees C, whereas vhs4, -5, and -6 exhibited a temperature-dependent vhs phenotype. Since the mutants were capable of growth at 34 degrees C, it appeared that the vhs function was not essential for virus replication in cultured cells. Temperature-shift experiments performed with the vhs4 mutant showed that an active vhs function was required throughout the shutoff process and that, once established, the translational shutoff could not be reversed. In the absence of actinomycin D, the mutants induced a generalized, secondary shutoff of host translation, which required the synthesis of beta (early) or gamma (late) viral polypeptide(s). The vhs mutants appeared to be defective also with respect to post-transcriptional shutoff of alpha (immediate early) viral gene expression, since (i) cells infected with mutant viruses overproduced alpha viral polypeptides, (ii) there was an increased functional stability of alpha mRNA in the vhs1 mutant virus-infected cells, and (iii) superinfection of vhs1-infected cells with wild-type virus, in the presence of actinomycin D, resulted in a more pronounced shutoff of alpha polypeptide synthesis from preformed alpha mRNA than equivalent superinfection with vhs1 virus. The data suggest that the synthesis of alpha polypeptides in wild-type virus infections is subject to a negative post-transcriptional control involving viral gene product

  7. Mutants of Arabidopsis thaliana with altered responses to auxins and gravity.

    PubMed

    Maher, E P; Martindale, S J

    1980-12-01

    Auxin-resistant mutants of Arabidopsis have been induced and isolated by screening for survivors on a medium containing the herbicide 2,4-D. Thirty independently arisen mutants have been isolated in this way and one of them, P 83, has been investigated in detail. When wild type and P 83 are compared in concentration/response curves, where the response is the inhibition of root growth, the ED50 values of the auxins, 2,4-D and IAA, are 14-fold higher for the mutant. The mutant also responds differently to gravity: its roots do not show positive geotropism, but tend to grow with a clockwise curvature on agar surfaces. The seedling roots of the mutant also grow more rapidly than those of the wild type in the absence of 2,4-D, following faster germination. The F1 between P 83 and wild type is similar to the latter, but has a slightly increased resistance to 2,4-D. Results obtained from the F2, F3 and backcross generations suggest monofactorial inheritance. Most of the other 29 mutants have the P 83 phenotype, but at least five are different. Four have lower levels of resistance to 2,4-D and P 83, and their roots appear to respond normally to gravity. One mutant has an abnormal georesponse and a much higher level of resistance to 2,4-D than P 83.

  8. Diseases Associated with Defective Responses to DNA Damage

    PubMed Central

    O’Driscoll, Mark

    2012-01-01

    Within the last decade, multiple novel congenital human disorders have been described with genetic defects in known and/or novel components of several well-known DNA repair and damage response pathways. Examples include disorders of impaired nucleotide excision repair, DNA double-strand and single-strand break repair, as well as compromised DNA damage-induced signal transduction including phosphorylation and ubiquitination. These conditions further reinforce the importance of multiple genome stability pathways for health and development in humans. Furthermore, these conditions inform our knowledge of the biology of the mechanics of genome stability and in some cases provide potential routes to help exploit these pathways therapeutically. Here, I will review a selection of these exciting findings from the perspective of the disorders themselves, describing how they were identified, how genotype informs phenotype, and how these defects contribute to our growing understanding of genome stability pathways. PMID:23209155

  9. Identification of six loci in which mutations partially restore peroxisome biogenesis and/or alleviate the metabolic defect of pex2 mutants in podospora.

    PubMed Central

    Ruprich-Robert, Gwenaël; Berteaux-Lecellier, Véronique; Zickler, Denise; Panvier-Adoutte, Arlette; Picard, Marguerite

    2002-01-01

    Peroxins (PEX) are proteins required for peroxisome biogenesis. Mutations in PEX genes cause lethal diseases in humans, metabolic defects in yeasts, and developmental disfunctions in plants and filamentous fungi. Here we describe the first large-scale screening for suppressors of a pex mutation. In Podospora anserina, pex2 mutants exhibit a metabolic defect [inability to grow on medium containing oleic acid (OA medium) as sole carbon source] and a developmental defect (inability to differentiate asci in homozygous crosses). Sixty-three mutations able to restore growth of pex2 mutants on OA medium have been analyzed. They fall in six loci (suo1 to suo6) and act as dominant, allele-nonspecific suppressors. Most suo mutations have pleiotropic effects in a pex2(+) background: formation of unripe ascospores (all loci except suo5 and suo6), impaired growth on OA medium (all loci except suo4 and suo6), or sexual defects (suo4). Using immunofluorescence and GFP staining, we show that peroxisome biogenesis is partially restored along with a low level of ascus differentiation in pex2 mutant strains carrying either the suo5 or the suo6 mutations. The data are discussed with respect to beta-oxidation of fatty acids, peroxisome biogenesis, and cell differentiation. PMID:12136013

  10. Nse1-dependent recruitment of Smc5/6 to lesion-containing loci contributes to the repair defects of mutant complexes

    PubMed Central

    Tapia-Alveal, Claudia; O'Connell, Matthew J.

    2011-01-01

    Of the three structural maintenance of chromosomes (SMC) complexes, Smc5/6 remains the most poorly understood. Genetic studies have shown that Smc5/6 mutants are defective in homologous recombination (HR), and consistent with this, Smc5/6 is enriched at lesions. However, Smc5/6 is essential for viability, but HR is not, and the terminal phenotype of null Smc5/6 mutants is mitotic failure. Here we analyze the function of Nse1, which contains a variant RING domain that is characteristic of ubiquitin ligases. Whereas deletion of this domain causes DNA damage sensitivity and mitotic failure, serine mutations in conserved cysteines do not. However, these mutations suppress the DNA damage sensitivity of Smc5/6 hypomorphs but not that of HR mutants and remarkably decrease the recruitment of Smc5/6 to loci containing lesions marked for HR-mediated repair. Analysis of DNA repair pathways in suppressed double mutants suggests that lesions are channeled into recombination-dependent and error-free postreplication repair. Thus the HR defect in Smc5/6 mutants appears to be due to the presence of dysfunctional complexes at lesions rather than to reflect an absolute requirement for Smc5/6 to complete HR. PMID:21976700

  11. Genetic and biochemical interactions involving tricarboxylic acid cycle (TCA) function using a collection of mutants defective in all TCA cycle genes.

    PubMed

    Przybyla-Zawislak, B; Gadde, D M; Ducharme, K; McCammon, M T

    1999-05-01

    The eight enzymes of the tricarboxylic acid (TCA) cycle are encoded by at least 15 different nuclear genes in Saccharomyces cerevisiae. We have constructed a set of yeast strains defective in these genes as part of a comprehensive analysis of the interactions among the TCA cycle proteins. The 15 major TCA cycle genes can be sorted into five phenotypic categories on the basis of their growth on nonfermentable carbon sources. We have previously reported a novel phenotype associated with mutants defective in the IDH2 gene encoding the Idh2p subunit of the NAD+-dependent isocitrate dehydrogenase (NAD-IDH). Null and nonsense idh2 mutants grow poorly on glycerol, but growth can be enhanced by extragenic mutations, termed glycerol suppressors, in the CIT1 gene encoding the TCA cycle citrate synthase and in other genes of oxidative metabolism. The TCA cycle mutant collection was utilized to search for other genes that can suppress idh2 mutants and to identify TCA cycle genes that display a similar suppressible growth phenotype on glycerol. Mutations in 7 TCA cycle genes were capable of functioning as suppressors for growth of idh2 mutants on glycerol. The only other TCA cycle gene to display the glycerol-suppressor-accumulation phenotype was IDH1, which encodes the companion Idh1p subunit of NAD-IDH. These results provide genetic evidence that NAD-IDH plays a unique role in TCA cycle function.

  12. Ambroxol-induced rescue of defective glucocerebrosidase is associated with increased LIMP-2 and saposin C levels in GBA1 mutant Parkinson's disease cells.

    PubMed

    Ambrosi, Giulia; Ghezzi, Cristina; Zangaglia, Roberta; Levandis, Giovanna; Pacchetti, Claudio; Blandini, Fabio

    2015-10-01

    Heterozygous mutations in GBA1 gene, encoding for lysosomal enzyme glucocerebrosidase (GCase), are a major risk factor for sporadic Parkinson's disease (PD). Defective GCase has been reported in fibroblasts of GBA1-mutant PD patients and pharmacological chaperone ambroxol has been shown to correct such defect. To further explore this issue, we investigated GCase and elements supporting GCase function and trafficking in fibroblasts from sporadic PD patients--with or without heterozygous GBA1 mutations--and healthy subjects, in basal conditions and following in vitro exposure to ambroxol. We assessed protein levels of GCase, lysosomal integral membrane protein-2 (LIMP-2), which mediates GCase trafficking to lysosomes, GCase endogenous activator saposin (Sap) C and parkin, which is involved in degradation of defective GCase. We also measured activities of GCase and cathepsin D, which cleaves Sap C from precursor prosaposin. GCase activity was reduced in fibroblasts from GBA1-mutant patients and ambroxol corrected this defect. Ambroxol increased cathepsin D activity, GCase and Sap C protein levels in all groups, while LIMP-2 levels were increased only in GBA1-mutant PD fibroblasts. Parkin levels were slightly increased only in the PD group without GBA1 mutations and were not significantly modified by ambroxol. Our study confirms that GCase activity is deficient in fibroblasts of GBA1-mutant PD patients and that ambroxol corrects this defect. The drug increased Sap C and LIMP-2 protein levels, without interfering with parkin. These results confirm that chemical chaperone ambroxol modulates lysosomal markers, further highlighting targets that may be exploited for innovative PD therapeutic strategies.

  13. Ambroxol-induced rescue of defective glucocerebrosidase is associated with increased LIMP-2 and saposin C levels in GBA1 mutant Parkinson's disease cells.

    PubMed

    Ambrosi, Giulia; Ghezzi, Cristina; Zangaglia, Roberta; Levandis, Giovanna; Pacchetti, Claudio; Blandini, Fabio

    2015-10-01

    Heterozygous mutations in GBA1 gene, encoding for lysosomal enzyme glucocerebrosidase (GCase), are a major risk factor for sporadic Parkinson's disease (PD). Defective GCase has been reported in fibroblasts of GBA1-mutant PD patients and pharmacological chaperone ambroxol has been shown to correct such defect. To further explore this issue, we investigated GCase and elements supporting GCase function and trafficking in fibroblasts from sporadic PD patients--with or without heterozygous GBA1 mutations--and healthy subjects, in basal conditions and following in vitro exposure to ambroxol. We assessed protein levels of GCase, lysosomal integral membrane protein-2 (LIMP-2), which mediates GCase trafficking to lysosomes, GCase endogenous activator saposin (Sap) C and parkin, which is involved in degradation of defective GCase. We also measured activities of GCase and cathepsin D, which cleaves Sap C from precursor prosaposin. GCase activity was reduced in fibroblasts from GBA1-mutant patients and ambroxol corrected this defect. Ambroxol increased cathepsin D activity, GCase and Sap C protein levels in all groups, while LIMP-2 levels were increased only in GBA1-mutant PD fibroblasts. Parkin levels were slightly increased only in the PD group without GBA1 mutations and were not significantly modified by ambroxol. Our study confirms that GCase activity is deficient in fibroblasts of GBA1-mutant PD patients and that ambroxol corrects this defect. The drug increased Sap C and LIMP-2 protein levels, without interfering with parkin. These results confirm that chemical chaperone ambroxol modulates lysosomal markers, further highlighting targets that may be exploited for innovative PD therapeutic strategies. PMID:26094596

  14. Flocculation-Related Gene Identification by Whole-Genome Sequencing of Thauera aminoaromatica MZ1T Floc-Defective Mutants

    PubMed Central

    Allen, M. S.

    2015-01-01

    Thauera aminoaromatica MZ1T, a floc-forming bacterium isolated from an industrial activated-sludge wastewater treatment plant, overproduces exopolysaccharide (EPS), leading to viscous bulking. This phenomenon results in poor sludge settling and dewatering during the clarification process. To identify genes responsible for bacterial flocculation, a whole-genome phenotypic-sequencing technique was applied. Genomic DNA of MZ1T flocculation-deficient mutants was subjected to massively parallel sequencing. The resultant high-quality reads were assembled and compared to the reference genome of the wild type (WT). We identified nine nonsynonymous mutations and one nonsense mutation putatively involved in EPS biosynthesis. Complementation of the nonsense mutation located in an EPS deacetylase gene restored the flocculating phenotype. The Fourier transform infrared (FTIR) spectra of EPS isolated from the wild type showed a reduced C=O peak of the N-acetyl group at 1,665 cm−1 compared to the spectra of MZ1T floc-deficient mutant EPS, suggesting that the WT EPS was partially deacetylated. Gene expression analysis also demonstrated that the putative deacetylase gene transcript increased before flocculation occurred. These data suggest that targeting deacetylation processes via direct chemical modification of EPS or enzyme inhibition may prove useful in combating viscous bulking in this and related bacteria. PMID:26712552

  15. Structural Studies of Lipopolysaccharide-defective Mutants from Brucella melitensis Identify a Core Oligosaccharide Critical in Virulence*

    PubMed Central

    Fontana, Carolina; Conde-Álvarez, Raquel; Ståhle, Jonas; Holst, Otto; Iriarte, Maite; Zhao, Yun; Arce-Gorvel, Vilma; Hanniffy, Seán; Gorvel, Jean-Pierre; Moriyón, Ignacio; Widmalm, Göran

    2016-01-01

    The structures of the lipooligosaccharides from Brucella melitensis mutants affected in the WbkD and ManBcore proteins have been fully characterized using NMR spectroscopy. The results revealed that disruption of wbkD gives rise to a rough lipopolysaccharide (R-LPS) with a complete core structure (β-d-Glcp-(1→4)-α-Kdop-(2→4)[β-d-GlcpN-(1→6)-β-d-GlcpN-(1→4)[β-d-GlcpN-(1→6)]-β-d-GlcpN-(1→3)-α-d-Manp-(1→5)]-α-Kdop-(2→6)-β-d-GlcpN3N4P-(1→6)-α-d-GlcpN3N1P), in addition to components lacking one of the terminal β-d-GlcpN and/or the β-d-Glcp residues (48 and 17%, respectively). These structures were identical to those of the R-LPS from B. melitensis EP, a strain simultaneously expressing both smooth and R-LPS, also studied herein. In contrast, disruption of manBcore gives rise to a deep-rough pentasaccharide core (β-d-Glcp-(1→4)-α-Kdop-(2→4)-α-Kdop-(2→6)-β-d-GlcpN3N4P-(1→6)-α-d-GlcpN3N1P) as the major component (63%), as well as a minor tetrasaccharide component lacking the terminal β-d-Glcp residue (37%). These results are in agreement with the predicted functions of the WbkD (glycosyltransferase involved in the biosynthesis of the O-antigen) and ManBcore proteins (phosphomannomutase involved in the biosynthesis of a mannosyl precursor needed for the biosynthesis of the core and O-antigen). We also report that deletion of B. melitensis wadC removes the core oligosaccharide branch not linked to the O-antigen causing an increase in overall negative charge of the remaining LPS inner section. This is in agreement with the mannosyltransferase role predicted for WadC and the lack of GlcpN residues in the defective core oligosaccharide. Despite carrying the O-antigen essential in B. melitensis virulence, the core deficiency in the wadC mutant structure resulted in a more efficient detection by innate immunity and attenuation, proving the role of the β-d-GlcpN-(1→6)-β-d-GlcpN-(1→4)[β-d-GlcpN-(1→6)]-β-d-GlcpN-(1→3)-

  16. Severity of infantile nystagmus syndrome-like ocular motor phenotype is linked to the extent of the underlying optic nerve projection defect in zebrafish belladonna mutant.

    PubMed

    Huber-Reggi, Sabina P; Chen, Chien-Cheng; Grimm, Lea; Straumann, Dominik; Neuhauss, Stephan C F; Huang, Melody Ying-Yu

    2012-12-12

    Infantile nystagmus syndrome (INS), formerly known as congenital nystagmus, is an ocular motor disorder in humans characterized by spontaneous eye oscillations (SOs) and, in several cases, reversed optokinetic response (OKR). Its etiology and pathomechanism is largely unknown, but misrouting of the optic nerve has been observed in some patients. Likewise, optic nerve misrouting, a reversed OKR and SOs with INS-like waveforms are observed in zebrafish belladonna (bel) mutants. We aimed to investigate whether and how misrouting of the optic nerve correlates with the ocular motor behaviors in bel larvae. OKR and SOs were quantified and subsequently the optic nerve fibers were stained with fluorescent lipophilic dyes. Eye velocity during OKR was reduced in larvae with few misprojecting optic nerve fibers and reversed in larvae with a substantial fraction of misprojecting fibers. All larvae with reversed OKR also displayed SOs. A stronger reversed OKR correlated with more frequent SOs. Since we did not find a correlation between additional retinal defects and ocular motor behavior, we suggest that axon misrouting is in fact origin of INS in the zebrafish animal model. Depending on the ratio between misprojecting ipsilateral and correctly projecting contralateral fibers, the negative feedback loop normally regulating OKR can turn into a positive loop, resulting in an increase in retinal slip. Our data not only give new insights into the etiology of INS but may also be of interest for studies on how the brain deals with and adapts to conflicting inputs. PMID:23238723

  17. An efficient deletion mutant packaging system for defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal physiology

    SciTech Connect

    Geller, A.I.; Keyomarsi, K.; Bryan, J.; Pardee, A.B. )

    1990-11-01

    The authors have previously described a defective herpes simplex virus (HSV-1) vector system that permits that introduction of virtually any gene into nonmitotic cells. pHSVlac, the prototype vector, stably expresses Escherichia coli {beta}-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain. HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors. A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; ts mutants revert to wild type. In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors. They now report an efficient packaging system for HSV-1 VECTORS USING A DELETION MUTANT, d30EBA, as helper virus; virus is grown on the complementing cell line M64A. pHSVlac virus prepared using the deletion mutant packaging system stably expresses {beta}-galactosidase in cultured rat sympathetic neurons and glia. Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system.

  18. Biosynthesis of membrane-derived oligosaccharides: characterization of mdoB mutants defective in phosphoglycerol transferase I activity.

    PubMed Central

    Jackson, B J; Bohin, J P; Kennedy, E P

    1984-01-01

    Phosphoglycerol transferase I, an enzyme of the inner, cytoplasmic membrane of Escherichia coli, catalyzes the in vitro transfer of phosphoglycerol residues from phosphatidylglycerol to membrane-derived oligosaccharides or to the model substrate arbutin (p-hydroxyphenyl-beta-D-glucoside). The products are a phosphoglycerol diester derivative of membrane-derived oligosaccharides or arbutin, respectively, and sn-1,2-diglyceride (B. J. Jackson and E. P. Kennedy, J. Biol. Chem. 258:2394-2398, 1983). Because this enzyme has its active site on the outer aspect of the inner membrane, it also catalyzes the transfer of phosphoglycerol residues to arbutin added to the medium (J.-P. Bohin and E. P. Kennedy, J. Biol. Chem. 259:8388-8393, 1984). When strains bearing the dgk mutation, which are defective in the enzyme diglyceride kinase, are grown in medium containing arbutin, they accumulate large amounts of sn-1,2-diglyceride, a product of the phosphoglycerol transferase I reaction. Growth is inhibited under these conditions. A further mutation in such a dgk strain, leading to the loss of phosphoglycerol transferase I activity, should result in the phenotype of arbutin resistance. We have exploited this fact to obtain strains with such mutations, designated mdoB, that map near min 99. Such mutants lack detectable phosphoglycerol transferase I activity, cannot transfer phosphoglycerol residues to arbutin in vivo, and synthesize membrane-derived oligosaccharides devoid of phosphoglycerol residues. These findings offer strong genetic support for the function of phosphoglycerol transferase I in membrane-derived oligosaccharide biosynthesis. PMID:6094515

  19. Improved plasma membrane expression of the trafficking defective P344R mutant of muscle, skeletal, receptor tyrosine kinase (MuSK) causing congenital myasthenic syndrome.

    PubMed

    Milhem, Reham M; Al-Gazali, Lihadh; Ali, Bassam R

    2015-03-01

    Muscle, skeletal, receptor tyrosine kinase (MuSK) is a key organizer at the postsynaptic membrane and critical for proper development and maintenance of the neuromuscular junction. Mutations in MUSK result in congenital myasthenic syndrome (CMS). We hypothesized that the CMS-causing missense mutation (P344R), found within the cysteine-rich domain of the protein, will affect its conformational tertiary structure. Consequently, the protein will misfold, get retained in the endoplasmic reticulum (ER) and lose its biological function through degradation by the highly conserved ER associated degradation (ERAD) machinery. We report that P344R-MuSK mutant is trafficking-deficient when expressed at 37°C in HeLa, COS-7 and HEK293 cell lines. It colocalized with the ER marker calnexin in contrast to wild-type MuSK which localized to the plasma membrane. The N-glycosylation status of P344R-MuSK is that of an immature and not properly post-translationally modified protein. Inhibition of protein synthesis showed that the P344R mutant's half-life is shorter than wild-type MuSK protein. Proteasomal inhibition resulted in the stabilization of the mutant protein. The mutant protein is highly ubiquitinated compared to wild-type confirming targeting for proteasomal degradation. The mutant showed around 50% of its in vivo autophosphorylation activity. P344R-MuSK mutant's trafficking defect is correctable by culturing the expressing cells at 27°C. Moreover, chemical compounds namely 2.5% glycerol, 1% dimethyl sulfoxide, 10 μM thapsigargin and 1 μM curcumin improved the maturation and exit of the mutant protein from the ER. These findings open perspectives for potential therapeutic intervention for patients with CMS harboring the P344R-MuSK mutation.

  20. Construction and Characterization of Haemophilus ducreyi Lipooligosaccharide (LOS) Mutants Defective in Expression of Heptosyltransferase III and β1,4-Glucosyltransferase: Identification of LOS Glycoforms Containing Lactosamine Repeats

    PubMed Central

    Filiatrault, Melanie J.; Gibson, Bradford W.; Schilling, Birgit; Sun, Shuhua; Munson, Robert S.; Campagnari, Anthony A.

    2000-01-01

    To begin to understand the role of the lipooligosaccharide (LOS) molecule in chancroid infections, we constructed mutants defective in expression of glycosyltransferase genes. Pyocin lysis and immunoscreening was used to identify a LOS mutant of Haemophilus ducreyi 35000. This mutant, HD35000R, produced a LOS molecule that lacked the monoclonal antibody 3F11 epitope and migrated with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Structural studies indicated that the principal LOS glycoform contains lipid A, Kdo, and two of the three core heptose residues. HD35000R was transformed with a plasmid library of H. ducreyi 35000 DNA, and a clone producing the wild-type LOS was identified. Sequence analysis of the plasmid insert revealed one open reading frame (ORF) that encodes a protein with homology to the WaaQ (heptosyltransferase III) of Escherichia coli. A second ORF had homology to the LgtF (glucosyltransferase) of Neisseria meningitidis. Individual isogenic mutants lacking expression of the putative H. ducreyi heptosyltransferase III, the putative glucosyltransferase, and both glycosyltransferases were constructed and characterized. Each mutant was complemented with the representative wild-type genes in trans to restore expression of parental LOS and confirm the function of each enzyme. Matrix-assisted laser desorption ionization mass spectrometry and SDS-PAGE analysis identified several unique LOS glycoforms containing di-, tri-, and poly-N-acetyllactosamine repeats added to the terminal region of the main LOS branch synthesized by the heptosyltransferase III mutant. These novel H. ducreyi mutants provide important tools for studying the regulation of LOS assembly and biosynthesis. PMID:10816485

  1. Dynamics of SOS-Response in UVR-Mutants of Escherichia Coli Cells under Ultraviolet Irradiation

    NASA Astrophysics Data System (ADS)

    Tuchina, M. A.; Parkhomenko, A. Yu.; Belov, O. V.; Bugai, A. N.

    2010-01-01

    A mathematical model of the genetic regulatory system of the SOS-response induced by ultraviolet radiation in excision repair-deficient mutants of E. coli bacterial cells is developed. On the basis of the model, the dynamics of the SOS system regulatory proteins is analyzed. The influence of excision repair on the induction of the key gene products during the SOS-response is studied.

  2. [Mutants of bacterium Azospirillum brasilense Sp245 with Omegon insertion in mmsB or fabG genes of lipid metabolism are defective in motility and flagellation].

    PubMed

    Kovtunov, E A; Shelud'ko, A V; Chernyshova, M P; Petrova, L P; Katsy, E I

    2013-11-01

    Bacteria Azospirillum brasilense have mixed flagellation: in addition to the polar flagellum, numerous lateral flagella are formed in their cells on medium with increased density. Flagella determine the active swimming and swarming capacities of azospirilla. Using A. brasilense Sp245 as an example, we showed that the Omegon-Km artificial transposon insertion into the chromosomal gene for 3-hydroxyisobutyrate dehydrogenase (mmsB) was concurrent with the appearance of significant defects in the formation of polar flagella and with the paralysis of lateral flagella. The Sp245 mutant with the Omegon insertion into the plasmid AZOBR_p1-borne gene for 3-oxoacyl-[acyl-carrier protein]-reductase (fabG) showed the complete loss of flagella and the swarming capacity, as well as significant defects in polar flagellar assembly (though some cells are still motile in liquid medium). The viability of the A. brasilense Sp245 mutants with the Omegon insertion into the mmsB or fabG gene was not reduced. No considerable differences in the fatty acid composition of whole cell lipid extracts were found for the A. brasilense Sp245 strain and its mmsB and fabG mutants.

  3. Defects in a New Class of Sulfate/Anion Transporter Link Sulfur Acclimation Responses to Intracellular Glutathione Levels and Cell Cycle Control1[W][OPEN

    PubMed Central

    Fang, Su-Chiung; Chung, Chin-Lin; Chen, Chun-Han; Lopez-Paz, Cristina; Umen, James G.

    2014-01-01

    We previously identified a mutation, suppressor of mating type locus3 15-1 (smt15-1), that partially suppresses the cell cycle defects caused by loss of the retinoblastoma tumor suppressor-related protein encoded by the MAT3 gene in Chlamydomonas reinhardtii. smt15-1 single mutants were also found to have a cell cycle defect leading to a small-cell phenotype. SMT15 belongs to a previously uncharacterized subfamily of putative membrane-localized sulfate/anion transporters that contain a sulfate transporter domain and are found in a widely distributed subset of eukaryotes and bacteria. Although we observed that smt15-1 has a defect in acclimation to sulfur-limited growth conditions, sulfur acclimation (sac) mutants, which are more severely defective for acclimation to sulfur limitation, do not have cell cycle defects and cannot suppress mat3. Moreover, we found that smt15-1, but not sac mutants, overaccumulates glutathione. In wild-type cells, glutathione fluctuated during the cell cycle, with highest levels in mid G1 phase and lower levels during S and M phases, while in smt15-1, glutathione levels remained elevated during S and M. In addition to increased total glutathione levels, smt15-1 cells had an increased reduced-to-oxidized glutathione redox ratio throughout the cell cycle. These data suggest a role for SMT15 in maintaining glutathione homeostasis that impacts the cell cycle and sulfur acclimation responses. PMID:25361960

  4. A conditionally lethal mutant of Salmonella Typhimurium induces a protective response in mice.

    PubMed

    Hidalgo, Alejandro A; Villagra, Nicolás A; Jerez, Sebastián A; Fuentes, Juan A; Mora, Guido C

    2016-02-01

    Here we present the design of a conditionally lethal mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium) which growth depends on tetracycline (Tet). Four mutants of S. Typhimurium, with Tet-conditional growth, were created by inserting the tetRA cassette. Three of the mutants presented a conditional-lethal phenotype in vitro. One mutant in the yabB gene remained conditional inside cells and did not persisted after 24 h in cell cultures. The capacity of S. Typhimurium yabB::tetRA to invade deep organs was investigated in intraperitoneally (IP) infected mice fed with or without chlortetracycline (CTet), a Tet analog with lower antibiotic activity. The yabB::tetRA mutant was undetectable in liver or spleen of animals under normal diet, while in mice under diet including CTet, yabB::tetRA invaded at a level comparable to the WT in mice under normal diet. Moreover, yabB::tetRA produced a strong humoral-immunoresponse after one IP immunization with 10(6) bacteria, measured as serum reactivity against S. Typhimurium whole cell extract. By contrast, oral immunization with 10(6) bacteria was weaker and variable on inducing antibodies. Consistently, IP infected mice were fully protected in a challenge with 10(4) oral S. Typhimurium, while protection was partial in orally immunized mice. Our data indicate that S. Typhimurium yabB::tetRA is a conditionally attenuated strain capable of inducing a protective response in mice in non-permissive conditions.

  5. Thiamine-auxotrophic mutants of Pseudomonas fluorescens CHA0 are defective in cell-cell signaling and biocontrol factor expression.

    PubMed

    Dubuis, Christophe; Rolli, Joëlle; Lutz, Matthias; Défago, Geneviève; Haas, Dieter

    2006-04-01

    In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 x 10(-8) M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens.

  6. Failure to Target RANKL Signaling Through p38-MAPK Results in Defective Osteoclastogenesis in the Microphthalmia Cloudy-Eyed Mutant.

    PubMed

    Carey, Heather A; Bronisz, Agnieszka; Cabrera, Jennifer; Hildreth, Blake E; Cuitiño, Maria; Fu, Qi; Ahmad, Asrar; Toribio, Ramiro E; Ostrowski, Michael C; Sharma, Sudarshana M

    2016-03-01

    The Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper family factor that is essential for terminal osteoclast differentiation. Previous work demonstrates that phosphorylation of MITF by p38 MAPK downstream of Receptor Activator of NFkB Ligand (RANKL) signaling is necessary for MITF activation in osteoclasts. The spontaneous Mitf cloudy eyed (ce) allele results in production of a truncated MITF protein that lacks the leucine zipper and C-terminal end. Here we show that the Mitf(ce) allele leads to a dense bone phenotype in neonatal mice due to defective osteoclast differentiation. In response to RANKL stimulation, in vitro osteoclast differentiation was impaired in myeloid precursors derived from neonatal or adult Mitf(ce/ce) mice. The loss of the leucine zipper domain in Mitf(ce/ce) mice does not interfere with the recruitment of MITF/PU.1 complexes to target promoters. Further, we have mapped the p38 MAPK docking site within the region deleted in Mitf(ce). This interaction is necessary for the phosphorylation of MITF by p38 MAPK. Site-directed mutations in the docking site interfered with the interaction between MITF and its co-factors FUS and BRG1. MITF-ce fails to recruit FUS and BRG1 to target genes, resulting in decreased expression of target genes and impaired osteoclast function. These results highlight the crucial role of signaling dependent MITF/p38 MAPK interactions in osteoclast differentiation.

  7. Failure to Target RANKL Signaling Through p38-MAPK Results in Defective Osteoclastogenesis in the Microphthalmia Cloudy-Eyed Mutant.

    PubMed

    Carey, Heather A; Bronisz, Agnieszka; Cabrera, Jennifer; Hildreth, Blake E; Cuitiño, Maria; Fu, Qi; Ahmad, Asrar; Toribio, Ramiro E; Ostrowski, Michael C; Sharma, Sudarshana M

    2016-03-01

    The Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper family factor that is essential for terminal osteoclast differentiation. Previous work demonstrates that phosphorylation of MITF by p38 MAPK downstream of Receptor Activator of NFkB Ligand (RANKL) signaling is necessary for MITF activation in osteoclasts. The spontaneous Mitf cloudy eyed (ce) allele results in production of a truncated MITF protein that lacks the leucine zipper and C-terminal end. Here we show that the Mitf(ce) allele leads to a dense bone phenotype in neonatal mice due to defective osteoclast differentiation. In response to RANKL stimulation, in vitro osteoclast differentiation was impaired in myeloid precursors derived from neonatal or adult Mitf(ce/ce) mice. The loss of the leucine zipper domain in Mitf(ce/ce) mice does not interfere with the recruitment of MITF/PU.1 complexes to target promoters. Further, we have mapped the p38 MAPK docking site within the region deleted in Mitf(ce). This interaction is necessary for the phosphorylation of MITF by p38 MAPK. Site-directed mutations in the docking site interfered with the interaction between MITF and its co-factors FUS and BRG1. MITF-ce fails to recruit FUS and BRG1 to target genes, resulting in decreased expression of target genes and impaired osteoclast function. These results highlight the crucial role of signaling dependent MITF/p38 MAPK interactions in osteoclast differentiation. PMID:26218069

  8. Splicing defective mutants of the COXI gene of yeast mitochondrial DNA: initial definition of the maturase domain of the group II intron aI2.

    PubMed Central

    Moran, J V; Mecklenburg, K L; Sass, P; Belcher, S M; Mahnke, D; Lewin, A; Perlman, P

    1994-01-01

    Six mutations blocking the function of a seven intron form of the mitochondrial gene encoding subunit I of cytochrome c oxidase (COXI) and mapping upstream of exon 3 were isolated and characterized. A cis-dominant mutant of the group IIA intron 1 defines a helical portion of the C1 substructure of domain 1 as essential for splicing. A trans-recessive mutant confirms that the intron 1 reading frame encodes a maturase function. A cis-dominant mutant in exon 2 was found to have no effect on the splicing of intron 1 or 2. A trans-recessive mutant, located in the group IIA intron 2, demonstrates for the first time that intron 2 encodes a maturase. A genetic dissection of the five missense mutations present in the intron 2 reading frame of that strain demonstrates that the maturase defect results from one or both of the missense mutations in a newly-recognized conserved sequence called domain X. Images PMID:8029012

  9. Functional Genomics Screening Utilizing Mutant Mouse Embryonic Stem Cells Identifies Novel Radiation-Response Genes

    PubMed Central

    Loesch, Kimberly; Galaviz, Stacy; Hamoui, Zaher; Clanton, Ryan; Akabani, Gamal; Deveau, Michael; DeJesus, Michael; Ioerger, Thomas; Sacchettini, James C.; Wallis, Deeann

    2015-01-01

    Elucidating the genetic determinants of radiation response is crucial to optimizing and individualizing radiotherapy for cancer patients. In order to identify genes that are involved in enhanced sensitivity or resistance to radiation, a library of stable mutant murine embryonic stem cells (ESCs), each with a defined mutation, was screened for cell viability and gene expression in response to radiation exposure. We focused on a cancer-relevant subset of over 500 mutant ESC lines. We identified 13 genes; 7 genes that have been previously implicated in radiation response and 6 other genes that have never been implicated in radiation response. After screening, proteomic analysis showed enrichment for genes involved in cellular component disassembly (e.g. Dstn and Pex14) and regulation of growth (e.g. Adnp2, Epc1, and Ing4). Overall, the best targets with the highest potential for sensitizing cancer cells to radiation were Dstn and Map2k6, and the best targets for enhancing resistance to radiation were Iqgap and Vcan. Hence, we provide compelling evidence that screening mutant ESCs is a powerful approach to identify genes that alter radiation response. Ultimately, this knowledge can be used to define genetic variants or therapeutic targets that will enhance clinical therapy. PMID:25853515

  10. The Arabidopsis her1 mutant implicates GABA in E-2-hexenal responsiveness.

    PubMed

    Mirabella, Rossana; Rauwerda, Han; Struys, Eduard A; Jakobs, Cornelis; Triantaphylidès, Christian; Haring, Michel A; Schuurink, Robert C

    2008-01-01

    When wounded or attacked by herbivores or pathogens, plants produce a blend of six-carbon alcohols, aldehydes and esters, known as C6-volatiles. Undamaged plants, when exposed to C6-volatiles, respond by inducing defense-related genes and secondary metabolites, suggesting that C6-volatiles can act as signaling molecules regulating plant defense responses. However, to date, the molecular mechanisms by which plants perceive and respond to these volatiles are unknown. To elucidate such mechanisms, we decided to isolate Arabidopsis thaliana mutants in which responses to C6-volatiles were altered. We observed that treatment of Arabidopsis seedlings with the C6-volatile E-2-hexenal inhibits root elongation. Among C6-volatiles this response is specific to E-2-hexenal, and is not dependent on ethylene, jasmonic and salicylic acid. Using this bioassay, we isolated 18 E-2-hexenal-response (her) mutants that showed sustained root growth after E-2-hexenal treatment. Here, we focused on the molecular characterization of one of these mutants, her1. Microarray and map-based cloning revealed that her1 encodes a gamma-amino butyric acid transaminase (GABA-TP), an enzyme that degrades GABA. As a consequence of the mutation, her1 plants accumulate high GABA levels in all their organs. Based on the observation that E-2-hexenal treatment induces GABA accumulation, and that high GABA levels confer resistance to E-2-hexenal, we propose a role for GABA in mediating E-2-hexenal responses.

  11. The Arabidopsis mutant eer2 has enhanced ethylene responses in the light.

    PubMed

    De Paepe, Annelies; De Grauwe, Liesbeth; Bertrand, Sophie; Smalle, Jan; Van der Straeten, Dominique

    2005-09-01

    By screening for ethylene response mutants in Arabidopsis, a novel mutant, eer2, was isolated which displays enhanced ethylene responses. On a low nutrient medium (LNM) light-grown eer2 seedlings showed a significant hypocotyl elongation in response to low levels of 1-amino-cyclopropane-1-carboxylate (ACC), the precursor of ethylene, compared with the wild type, indicating that eer2 is hypersensitive to ethylene. Treatment with 1-MCP (1-methylcyclopropene), a competitive inhibitor of ethylene signalling, suppressed this hypersensitive response, demonstrating that it is a bona fide ethylene effect. By contrast, roots of eer2 were less sensitive than the wild type to low concentrations of ACC. The ethylene levels in eer2 did not differ from the wild type, indicating that ethylene overproduction is not the primary cause of the eer2 phenotype. In addition to its enhanced ethylene response of hypocotyls, eer2 is also affected in the pattern of senescence and its phenotype depends on the nutritional status of the growth medium. Furthermore, linkage analysis of eer2 suggests that this mutant defines a new locus in ethylene signalling.

  12. Regulation of the abscisic acid-responsive gene rab28 in maize viviparous mutants.

    PubMed

    Pla, M; Gómez, J; Goday, A; Pagès, M

    1991-12-01

    We have isolated a new maize gene, rab28, that responds to abscisic acid (ABA) treatment. This gene has been characterized by determining the sequence of the cDNA and corresponding genomic copy, and by mapping the start site of its transcript. The rab 28 gene encodes a protein of predicted molecular weight 27713 Da which shows strong homology with the Lea D-34 protein identified in cotton. The proximal promoter region contains the conserved ABA-response element, CACGTGG, reported in other plant genes to be responsible for ABA induction. rab 28 mRNA has been identified as ABA-inducible in embryos and young leaves. It is also induced by water-stress in leaves of wild-type plants. Regulation of the rab 28 gene was studied in maize viviparous mutants. The results obtained with the ABA-insensitive vp1 mutant show that rab 28 transcripts do not accumulate to a significant level during embryogenesis. Surprisingly, induction of rab 28 mRNA can be achieved in young embryos by exogenous ABA treatment. Moreover, water-stressed or ABA-treated seedlings of vp1 contain significant levels of rab 28 mRNA which is not detectable in well-watered seedlings. Regulation of the rab 28 gene in excised young embryos of ABA-deficient vp2 mutants, in which influences of the maternal environment are absent, closely resembles that found in non-mutant excised young embryos. The significance of these results is discussed.

  13. Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm.

    PubMed

    Song, Yuyu; Nagy, Maria; Ni, Weiming; Tyagi, Navneet K; Fenton, Wayne A; López-Giráldez, Francesc; Overton, John D; Horwich, Arthur L; Brady, Scott T

    2013-04-01

    Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin. Here, we observe that several different oligomeric states of a fusion protein, comprising ALS-associated human G85R SOD1 joined with yellow fluorescent protein (G85R SOD1YFP), which produces ALS in transgenic mice, inhibited anterograde transport when added to squid axoplasm. Inhibition was blocked both by an apoptosis signal-regulating kinase 1 (ASK1; MAPKKK) inhibitor and by a p38 inhibitor, indicating the transport defect is mediated through the MAPK cascade. In further incubations, we observed that addition of the mammalian molecular chaperone Hsc70, abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice, exerted partial correction of the transport defect, associated with diminished phosphorylation of p38. Most striking, the addition of the molecular chaperone Hsp110, in a concentration substoichiometric to the mutant SOD1 protein, completely rescued both the transport defect and the phosphorylation of p38. Hsp110 has been demonstrated to act as a nucleotide exchange factor for Hsc70 and, more recently, to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein, abrogating toxicity.

  14. Isolation and characterization of type III group B streptococcal mutants defective in biosynthesis of the type-specific antigen.

    PubMed Central

    Yeung, M K; Mattingly, S J

    1983-01-01

    Four classes of mutants of type III group B streptococcus were isolated by serial subculture of the wild-type strain in the presence of type III-specific rabbit antiserum. Class I mutants no longer synthesized sialic acid but still elaborated the core antigen. Class II mutants maintained the ability to synthesize sialic acid but could not attach it to the core antigen. Class III mutants did not produce the core antigen but still synthesized intracellular sialic acid. Class IV mutants synthesized the complete antigen; however, only approximately 4% of the antigen synthesized was found associated with the cell wall peptidoglycan (in the wild-type strain greater than 85% of the antigen synthesized is covalently attached to the cell wall peptidoglycan), whereas greater than 90% of the antigen was secreted into the growth medium. Production of other components (CAMP factor, group B antigen, beta-hemolysin, neuraminidase) by these mutants appeared similar to those of the wild-type strain. Mouse lethality studies of these strains indicated that all four classes have greater than 3 log10-higher 50% lethal dose values than that of the wild-type strain. To understand the basis for this variation, the invasive ability of the wild-type strain and the sialic acid-deficient mutant strain M-10 (class I) was examined. Mice received 10(5) CFU of each organism; they were then sacrificed at various times postinoculation, and viable group B streptococci from different organs were enumerated. Mice were able to clear M-10 more efficiently, with greater than 80% of M-10 cells being phagocytized by macrophages within 1 h, whereas the wild-type strain was able to evade phagocytic killing and disseminate to other tissues. These data, therefore, strongly indicate that the sialic acid moiety greatly enhances the virulence of the type III antigen. In addition, the level of cell-associated type-specific antigen appears to contribute significantly to the pathogenicity of the organism. PMID

  15. Class II-antigen-negative patient and mutant B-cell lines represent at least three, and probably four, distinct genetic defects defined by complementation analysis.

    PubMed Central

    Bénichou, B; Strominger, J L

    1991-01-01

    Expression of class II major histocompatibility complex antigens in defective B-lymphoblastoid cell lines from patients with class II antigen deficiency and from in vitro mutants generated with the same phenotype was studied. By heterogenetic fusion experiments, at least three, and probably four, complementation groups were defined. Furthermore, clone 13 (a DR-, DP-, but DQ+ cell line) appeared to belong to the RJ2.2.5 complementation group, for which all other members are DR-, DP-, and also DQ-. Thus, it is hypothesized that the cell lines of this group lack the activity of a gene that can differentially regulate the DR/DP and the DQ promoters. Images PMID:1852002

  16. Streptococcus salivarius mutants defective in mannose phosphotransferase systems show reduced sensitivity to mutacins I-T9 and R-3B.

    PubMed

    Nicolas, Guillaume G; Frenette, Michel; Lavoie, Marc C

    2010-08-01

    Twenty-four mutacin-producing Streptococcus mutans strains were screened for their propensity to produce class II one-peptide bacteriocin using a deferred antagonism assay. Streptococcus salivarius and 3 mutants defective in their mannose phosphotransferase systems (mannose-PTS) were used as sensitive strains to identify which mannose-PTS could act as the docking site for class II one-peptide bacteriocin activity. We observed that only 2 strains of S. mutans, T9 and 3B, potentially produce class II one-peptide bacteriocin, namely mutacins I-T9 and R-3B, but with no preference for any mannose-PTS complex as a target. PMID:20725132

  17. Use of Arabidopsis thaliana defense-related mutants to dissect the plant response to pathogens.

    PubMed Central

    Ausubel, F M; Katagiri, F; Mindrinos, M; Glazebrook, J

    1995-01-01

    The plant defense response to microbial pathogens had been studied primarily by using biochemical and physiological techniques. Recently, several laboratories have developed a variety of pathosystems utilizing Arabidopsis thaliana as a model host so that genetic analysis could also be used to study plant defense responses. Utilizing a pathosystem that involves the infection of Arabidopsis with pathogenic pseudomonads, we have cloned the Arabidopsis disease-resistance gene RPS2, which corresponds to the avirulence gene avrRpt2 in a gene-for-gene relationship. RPS2 encodes a 105-kDa protein containing a leucine zipper, a nucleotide binding site, and 14 imperfect leucine-rich repeats. The RPS2 protein is remarkably similar to the product of the tobacco N gene, which confers resistance to tobacco mosaic virus. We have also isolated a series of Arabidopsis mutants that synthesize decreased levels of an Arabidopsis phytoalexin called camalexin. Analysis of these mutants indicated that camalexin does not play a significant role in limiting growth of avirulent Pseudomonas syringae strains during the hypersensitive defense response but that it may play a role in limiting the growth of virulent strains. More generally, we have shown that we can utilize Arabidopsis to systematically dissect the defense response by isolation and characterization of appropriate defense-related mutants. Images Fig. 1 Fig. 3 PMID:7753782

  18. Host range and cell cycle activation properties of polyomavirus large T-antigen mutants defective in pRB binding

    SciTech Connect

    Freund, R.; Bauer, P.H.; Benjamin, T.L.; Crissman, H.A.; Bradbury, E.M. |

    1994-11-01

    The authors have examined the growth properties of polyomavirus large T-antigen mutants that ar unable to bind pRB, the product of the retinoblastoma tumor suppressor gene. These mutants grow poorly on primary mouse cells yet grow well on NIH 3T3 and other established mouse cell lines. Preinfection of primary baby mouse kidney (BMK) epithelial cells with wild-type simian virus 40 renders these cells permissive to growth of pRB-binding polyomavirus mutants. Conversely, NIH 3T3 cells transfected by and expressing wild-type human pRB become nonpermissive. Primary fibroblasts for mouse embryos that carry a homozygous knockout of the RB gene are permissive, while those from normal littermates are nonpermissive. The host range of polyomavirus pRB-binding mutants is thus determined by expression or lack of expression of functional pRB by the host. These results demonstrate the importance of pRB binding by large T antigen for productive viral infection in primary cells. Failure of pRB-binding mutants to grow well in BMK cells correlates with their failure to induce progression from G{sub 0} or G{sub 1} through the S phase of the cell cycle. Time course studies show delayed synthesis and lower levels of accumulation of large T antigen, viral DNA, and VP1 in mutant compared with wild-type virus-infected BMK cells. These results support a model in which productive infection by polyomavirus in normal mouse cells is tightly coupled to the induction and progression of the cell cycle. 48 refs., 6 figs., 5 tabs.

  19. A phenotype survey of 36 mutant mouse strains with gene-targeted defects in glycosyltransferases or glycan-binding proteins

    PubMed Central

    Orr, Sally L; Le, Dzung; Long, Jeffrey M; Sobieszczuk, Peter; Ma, Bo; Tian, Hua; Fang, Xiaoqun; Paulson, James C; Marth, Jamey D; Varki, Nissi

    2013-01-01

    The consortium for functional glycomics (CFG) was a large research initiative providing networking and resources for investigators studying the role of glycans and glycan-binding proteins in health and disease. Starting in 2001, six scientific cores were established to generate data, materials and new technologies. By the end of funding in 2011, the mouse phenotype core (MPC) submitted data to a website from the phenotype screen of 36 mutant mouse strains deficient in a gene for either a glycan-binding protein (GBP) or glycosyltransferase (GT). Each mutant strain was allotted three months for analysis and screened by standard phenotype assays used in the fields of immunology, histology, hematology, coagulation, serum chemistry, metabolism and behavior. Twenty of the deficient mouse strains had been studied in other laboratories, and additional tests were performed on these strains to confirm previous observations and discover new data. The CFG constructed 16 new homozygous mutant mouse strains and completed the initial phenotype screen of the majority of these new mutant strains. In total, >300 phenotype changes were observed, but considering the over 100 assays performed on each strain, most of the phenotypes were unchanged. Phenotype differences include abnormal testis morphology in GlcNAcT9- and Siglec-H-deficient mice and lethality in Pomgnt1-deficient mice. The numerous altered phenotypes discovered, along with the consideration of the significant findings of normality, will provide a platform for future characterization to understand the important roles of glycans and GBPs in the mechanisms of health and disease. PMID:23118208

  20. A Drosophila behavioral mutant, down and out (dao), is defective in an essential regulator of Erg potassium channels

    PubMed Central

    Fergestad, Tim; Sale, Harinath; Bostwick, Bret; Schaffer, Ashleigh; Ho, Lingling; Robertson, Gail A.; Ganetzky, Barry

    2010-01-01

    To signal properly, excitable cells must establish and maintain the correct balance of various types of ion channels that increase or decrease membrane excitability. The mechanisms by which this balance is regulated remain largely unknown. Here, we describe a regulatory mechanism uncovered by a Drosophila behavioral mutant, down and out (dao). At elevated temperatures, dao loss-of-function mutants exhibit seizures associated with spontaneous bursts of neural activity. This phenotype closely resembles that of seizure mutations, which impair activity of ether-a-go-go-related gene (Erg)-type potassium channels. Conversely, neural over-expression of wild-type Dao confers dominant temperature-sensitive paralysis with kinetics reminiscent of paralytic sodium-channel mutants. The over-expression phenotype of dao is suppressed in a seizure mutant background, suggesting that Dao acts by an effect on Erg channels. In support of this hypothesis, functional expression of Erg channels in a heterologous system is dependent on the presence of Dao. These results indicate that Dao has an important role in establishing the proper level of neuronal membrane excitability by regulating functional expression of Erg channels. PMID:20212103

  1. A Drosophila behavioral mutant, down and out (dao), is defective in an essential regulator of Erg potassium channels.

    PubMed

    Fergestad, Tim; Sale, Harinath; Bostwick, Bret; Schaffer, Ashleigh; Ho, Lingling; Robertson, Gail A; Ganetzky, Barry

    2010-03-23

    To signal properly, excitable cells must establish and maintain the correct balance of various types of ion channels that increase or decrease membrane excitability. The mechanisms by which this balance is regulated remain largely unknown. Here, we describe a regulatory mechanism uncovered by a Drosophila behavioral mutant, down and out (dao). At elevated temperatures, dao loss-of-function mutants exhibit seizures associated with spontaneous bursts of neural activity. This phenotype closely resembles that of seizure mutations, which impair activity of ether-a-go-go-related gene (Erg)-type potassium channels. Conversely, neural over-expression of wild-type Dao confers dominant temperature-sensitive paralysis with kinetics reminiscent of paralytic sodium-channel mutants. The over-expression phenotype of dao is suppressed in a seizure mutant background, suggesting that Dao acts by an effect on Erg channels. In support of this hypothesis, functional expression of Erg channels in a heterologous system is dependent on the presence of Dao. These results indicate that Dao has an important role in establishing the proper level of neuronal membrane excitability by regulating functional expression of Erg channels. PMID:20212103

  2. 49 CFR 210.7 - Responsibility for noise defective railroad equipment.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Responsibility for noise defective railroad...) FEDERAL RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION RAILROAD NOISE EMISSION COMPLIANCE REGULATIONS General Provisions § 210.7 Responsibility for noise defective railroad equipment. Any...

  3. The Arabidopsis dwf7/ste1 mutant is defective in the delta7 sterol C-5 desaturation step leading to brassinosteroid biosynthesis.

    PubMed Central

    Choe, S; Noguchi, T; Fujioka, S; Takatsuto, S; Tissier, C P; Gregory, B D; Ross, A S; Tanaka, A; Yoshida, S; Tax, F E; Feldmann, K A

    1999-01-01

    Lesions in brassinosteroid (BR) biosynthetic genes result in characteristic dwarf phenotypes in plants. Understanding the regulation of BR biosynthesis demands continued isolation and characterization of mutants corresponding to the genes involved in BR biosynthesis. Here, we present analysis of a novel BR biosynthetic locus, dwarf7 (dwf7). Feeding studies with BR biosynthetic intermediates and analysis of endogenous levels of BR and sterol biosynthetic intermediates indicate that the defective step in dwf7-1 resides before the production of 24-methylenecholesterol in the sterol biosynthetic pathway. Furthermore, results from feeding studies with 13C-labeled mevalonic acid and compactin show that the defective step is specifically the Delta7 sterol C-5 desaturation, suggesting that dwf7 is an allele of the previously cloned STEROL1 (STE1) gene. Sequencing of the STE1 locus in two dwf7 mutants revealed premature stop codons in the first (dwf7-2) and the third (dwf7-1) exons. Thus, the reduction of BRs in dwf7 is due to a shortage of substrate sterols and is the direct cause of the dwarf phenotype in dwf7. PMID:9927639

  4. Production of host shutoff-defective mutants of herpes simplex virus type 1 by inactivation of the UL13 gene.

    PubMed

    Overton, H; McMillan, D; Hope, L; Wong-Kai-In, P

    1994-07-01

    Two mutants of HSV-1(SC16) carrying disrupted UL13 genes have been generated independently by recombination of wild-type genomic DNA with a plasmid-cloned copy of the UL13 gene containing multiple stop codons. The two mutants were shown to be deficient in UL13 gene expression by Western blotting of infected cells. A revertant virus, in which UL13 expression was restored to a near-normal level, was generated by recombination of one of the UL13-negative mutants with a plasmid carrying the wild-type UL13 gene. The replication of the two UL13-negative viruses in cell culture was somewhat reduced compared to their wild-type parent, and the viruses were unable to produce shutoff of host protein synthesis. The replication of the revertant virus was intermediate between that of the UL13-negative and wild-type viruses, as was its ability to produce host shutoff. Cells infected with the UL13-negative mutants were shown to contain much lower levels than normal of the UL41 gene product, which is known to be required for virion host shutoff. However, there was no significant difference between levels of the UL41 gene product in wild-type and mutant virions. The UL13-negative viruses exhibited different patterns of protein phosphorylation from wild-type virus when infected cells were metabolically labeled with [32P]-orthophosphate and when lysates of infected cells and of virions were subjected to in vitro phosphorylation. However, the UL41 gene product could still be phosphorylated in lysates of UL13-negative virions. We conclude that the UL13 gene is necessary to produce the virion host shutoff effect, but it seems unlikely that the role of UL13 is simply to activate the UL41 gene product by phosphorylation.

  5. Trafficking defect of mutant kidney anion exchanger 1 (kAE1) proteins associated with distal renal tubular acidosis and Southeast Asian ovalocytosis.

    PubMed

    Sawasdee, Nunghathai; Udomchaiprasertkul, Wandee; Noisakran, Sansanee; Rungroj, Nanyawan; Akkarapatumwong, Varaporn; Yenchitsomanus, Pa-thai

    2006-11-24

    Compound heterozygous anion exchanger 1 (AE1) SAO/G701D mutations result in distal renal tubular acidosis with Southeast Asian ovalocytosis. Interaction, trafficking and localization of wild-type and mutant (SAO and G701D) kAE1 proteins fused with hemagglutinin, six-histidine, Myc, or green fluorescence protein (GFP) were examined in human embryonic kidney (HEK) 293 cells. When individually expressed, wild-type kAE1 was localized at cell surface while mutant kAE1 SAO and G701D were intracellularly retained. When co-expressed, wild-type kAE1 could form heterodimer with kAE1 SAO or kAE1 G701D and could rescue mutant kAE1 proteins to express on the cell surface. Co-expression of kAE1 SAO and kAE1 G701D also resulted in heterodimer formation but intracellular retention without cell surface expression, suggesting their trafficking defect and failure to rescue each other to the plasma membrane, most likely the molecular mechanism of the disease in the compound heterozygous condition. PMID:17027918

  6. Vaccinia virus temperature-sensitive mutants in the A28 gene produce non-infectious virions that bind to cells but are defective in entry

    SciTech Connect

    Turner, Peter C. Dilling, Bradley P.; Prins, Cindy; Cresawn, Steven G.; Moyer, Richard W.; Condit, Richard C.

    2007-09-15

    The vaccinia virus temperature-sensitive mutations Cts6 and Cts9 were mapped by marker rescue and DNA sequencing to the A28 gene. Cts6 and Cts9 contain an identical 2-bp deletion truncating the A28 protein and removing the fourth conserved cysteine near the C-terminus. Cts9 mutant virions produced at 40 deg. C were non-infectious and unable to cause cytopathic effect. However, the mutant A28 protein localized to purified mature virions (MV) at 31 deg. C and 40 deg. C. MV of Cts9 produced at 40 deg. C bound to cells but did not enter cells. Low pH treatment of Cts9-infected cells at 18 h p.i. failed to produce fusion from within at 40 deg. C, but gave fusion at 31 deg. C. Adsorption of Cts9 mutant virions to cells followed by low pH treatment showed a defect in fusion from without. The Cts9 phenotype suggests that the A28 protein is involved in both virus entry and cell-cell fusion, and supports the linkage between the two processes.

  7. The Cell Wall Arabinose-Deficient Arabidopsis thaliana Mutant murus5 Encodes a Defective Allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE21[OPEN

    PubMed Central

    Dugard, Christopher K.; Olek, Anna T.; Cooper, Bruce R.

    2016-01-01

    Traditional marker-based mapping and next-generation sequencing was used to determine that the Arabidopsis (Arabidopsis thaliana) low cell wall arabinose mutant murus5 (mur5) encodes a defective allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2 (RGP2). Marker analysis of 13 F2 confirmed mutant progeny from a recombinant mapping population gave a rough map position on the upper arm of chromosome 5, and deep sequencing of DNA from these 13 lines gave five candidate genes with G→A (C→T) transitions predicted to result in amino acid changes. Of these five, only insertional mutant alleles of RGP2, a gene that encodes a UDP-arabinose mutase that interconverts UDP-arabinopyranose and UDP-arabinofuranose, exhibited the low cell wall arabinose phenotype. The identities of mur5 and two SALK insertional alleles were confirmed by allelism tests and overexpression of wild-type RGP2 complementary DNA placed under the control of the 35S promoter in the three alleles. The mur5 mutation results in the conversion of cysteine-257 to tyrosine-257 within a conserved hydrophobic cluster predicted to be distal to the active site and essential for protein stability and possible heterodimerization with other isoforms of RGP. PMID:27217494

  8. Protein glycosylation defects in the Saccharomyces cerevisiae mnn7 mutant class. Support for the stop signal proposed for regulation of outer chain elongation.

    PubMed

    Ballou, L; Alvarado, E; Tsai, P K; Dell, A; Ballou, C E

    1989-07-15

    Total cell mannoprotein was isolated from Saccharomyces cerevisiae X2180 mutants that have defects in elongation of the outer chain attached to the N-linked core oligosaccharides (mnn7, mnn8, mnn9, and mnn10) (Ballou, L., Cohen, R. E., and Ballou, C. E. (1980) J. Biol. Chem. 255, 5986-5991). Comparison of the oligosaccharides released by endoglucosaminidase H digestion confirmed that the mnn9 mutation eliminates all but two mannoses of the outer chain, whereas the mnn8 and mnn10 strains produce outer chains of variable but similar lengths. The isolate designated mnn7 was found to be allelic with mnn8. Haploid mutants of the type mnn8 mnn9 or mnn9 mnn10 had the mnn9 phenotype, which established that the mnn9 defect is dominant and presumably acts at a processing step prior to the steps affected by mnn8 and mnn10. Analysis of the mnn1 mnn2 mnn10 oligosaccharides revealed that the heterogeneous outer chain contained 6-16 alpha 1----6-linked mannose units and each was terminated by a single alpha 1----2-linked mannose unit, whereas the core lacked one such unit that was present in the mnn9 oligosaccharide. The results are consistent with and support the hypothesis (Gopal, P. K., and Ballou, C. E. (1988) Proc. Natl. Acad. Sci. U.S.A. 84, 8824-8828) that addition of such a side-chain mannose unit is associated with termination of outer chain elongation in these mutants and may serve as a stop signal that regulates outer chain synthesis in the parent wild-type strain. PMID:2663843

  9. Large-scale investigation of oxygen response mutants in Saccharomyces cerevisiae.

    PubMed

    Samanfar, Bahram; Omidi, Katayoun; Hooshyar, Mohsen; Laliberte, Ben; Alamgir, Md; Seal, Andrew J; Ahmed-Muhsin, Eman; Viteri, Duber Frey; Said, Kamaleldin; Chalabian, Firoozeh; Golshani, Ardeshir; Wainer, Gabriel; Burnside, Daniel; Shostak, Kristina; Bugno, Magdalena; Willmore, William G; Smith, Myron L; Golshani, Ashkan

    2013-06-01

    A genome-wide screen of a yeast non-essential gene-deletion library was used to identify sick phenotypes due to oxygen deprivation. The screen provided a manageable list of 384 potentially novel as well as known oxygen responding (anoxia-survival) genes. The gene-deletion mutants were further assayed for sensitivity to ferrozine and cobalt to obtain a subset of 34 oxygen-responsive candidate genes including the known hypoxic gene activator, MGA2. With each mutant in this subset a plasmid based β-galactosidase assay was performed using the anoxic-inducible promoter from OLE1 gene, and 17 gene deletions were identified that inhibit induction under anaerobic conditions. Genetic interaction analysis for one of these mutants, the RNase-encoding POP2 gene, revealed synthetic sick interactions with a number of genes involved in oxygen sensing and response. Knockdown experiments for CNOT8, human homolog of POP2, reduced cell survival under low oxygen condition suggesting a similar function in human cells.

  10. Copy-up mutants of the plasmid RK2 replication initiation protein are defective in coupling RK2 replication origins.

    PubMed Central

    Blasina, A; Kittell, B L; Toukdarian, A E; Helinski, D R

    1996-01-01

    The broad host range plasmid RK2 replicates and regulates its copy number in a wide range of Gram-negative bacteria. The plasmid-encoded trans-acting replication protein TrfA and the origin of replication oriV are sufficient for controlled replication of the plasmid in all Gram-negative bacteria tested. The TrfA protein binds specifically to direct repeat sequences (iterons) at the origin of replication. A replication control model, designated handcuffing or coupling, has been proposed whereby the formation of coupled TrfA-oriV complexes between plasmid molecules results in hindrance of origin activity and, consequently, a shut-down of plasmid replication under conditions of higher than normal copy number. Therefore, according to this model, the coupling activity of an initiation protein is essential for copy number control and a copy-up initiation protein mutant should have reduced ability to form coupled complexes. To test this model for plasmid RK2, two previously characterized copy-up TrfA mutations, trfA-254D and trfA-267L, were combined and the resulting copy-up double mutant TFrfA protein TrfA-254D/267L was characterized. Despite initiating runaway (uncontrolled) replication in vivo, the copy-up double-mutant TrfA protein exhibited replication kinetics similar to the wild-type protein in vitro. Purified TrfA-254D, TrfA-267L, and TrfA-254D/267L proteins were then examined for binding to the iterons and for coupling activity using an in vitro ligase-catalyzed multimerization assay. It was found that both single and double TrfA mutant proteins exhibited substantially reduced (single mutants) or barely detectable (double mutant) levels of coupling activity while not being diminished in their capacity to bind to the origin of replication. These observations provide direct evidence in support of the coupling model of replication control. Images Fig. 1 Fig. 2 Fig. 4 PMID:8622975

  11. The Maize High-Lysine Mutant opaque7 Is Defective in an Acyl-CoA Synthetase-Like Protein

    PubMed Central

    Miclaus, Mihai; Wu, Yongrui; Xu, Jian-Hong; Dooner, Hugo K.; Messing, Joachim

    2011-01-01

    Maize (Zea mays) has a large class of seed mutants with opaque or nonvitreous endosperms that could improve the nutritional quality of our food supply. The phenotype of some of them appears to be linked to the improper formation of protein bodies (PBs) where zein storage proteins are deposited. Although a number of genes affecting endosperm vitreousness have been isolated, it has been difficult to clone opaque7 (o7), mainly because of its low penetrance in many genetic backgrounds. The o7-reference (o7-ref) mutant arose spontaneously in a W22 inbred, but is poorly expressed in other lines. We report here the isolation of o7 with a combination of map-based cloning and transposon tagging. We first identified an o7 candidate gene by map-based cloning. The putative o7-ref allele has a 12-bp in-frame deletion of codons 350–353 in a 528-codon-long acyl-CoA synthetase-like gene (ACS). We then confirmed this candidate gene by generating another mutant allele from a transposon-tagging experiment using the Activator/Dissociation (Ac/Ds) system in a W22 background. The second allele, isolated from ∼1 million gametes, presented a 2-kb Ds insertion that resembles the single Ds component of double-Ds, McClintock’s original Dissociation element, at codon 496 of the ACS gene. PBs exhibited striking membrane invaginations in the o7-ref allele and a severe number reduction in the Ds-insertion mutant, respectively. We propose a model in which the ACS enzyme plays a key role in membrane biogenesis, by taking part in protein acylation, and that altered PBs render the seed nonvitreous. PMID:21926304

  12. A Mycobacterium smegmatis mutant with a defective inositol monophosphate phosphatase gene homolog has altered cell envelope permeability.

    PubMed Central

    Parish, T; Liu, J; Nikaido, H; Stoker, N G

    1997-01-01

    A bacteriophage infection mutant (strain LIMP7) of Mycobacterium smegmatis was isolated following transposon mutagenesis. The mutant showed an unusual phenotype, in that all phages tested produced larger plaques on this strain compared to the parent strain. Other phenotypic characteristics of the mutant were slower growth, increased clumping in liquid culture, increased resistance to chloramphenicol and erythromycin, and increased sensitivity to isoniazid and several beta-lactam antibiotics. Permeability studies showed decreases in the accumulation of lipophilic molecules (norfloxacin and chenodeoxycholate) and a small increase with hydrophilic molecules (cephaloridine); taken together, these characteristics indicate an altered cell envelope. The DNA adjacent to the transposon in LIMP7 was cloned and was shown to be highly similar to genes encoding bacterial and mammalian inositol monophosphate phosphatases. Inositol is important in mycobacteria as a component of the major thiol mycothiol and also in the cell wall, with phosphatidylinositol anchoring lipoarabinomannan (LAM) in the cell envelope. In LIMP7, levels of phosphatidylinositol dimannoside, the precursor of LAM, were less than half of those in the wild-type strain, confirming that the mutation had affected the synthesis of inositol-containing molecules. The impA gene is located within the histidine biosynthesis operon in both M. smegmatis and Mycobacterium tuberculosis, lying between the hisA and hisF genes. PMID:9401044

  13. Neural crest defects in ephrin-B2 mutant mice are non-autonomous and originate from defects in the vasculature.

    PubMed

    Lewis, Ace E; Hwa, Jennifer; Wang, Rong; Soriano, Philippe; Bush, Jeffrey O

    2015-10-15

    Ephrin-B2, a member of the Eph/ephrin family of cell signaling molecules, has been implicated in the guidance of cranial and trunk neural crest cells (NCC) and development of the branchial arches(BA), but detailed examination in mice has been hindered by embryonic lethality of Efnb2 null loss of function due to a requirement in angiogenic remodeling. To elucidate the developmental roles for Efnb2, we generated a conditional rescue knock-in allele that allows rescue of ephrin-B2 specifically in the vascular endothelium (VE), but is otherwise ephrin-B2 deficient. Restoration of ephrin-B2 expression specifically to the VE completely circumvents angiogenic phenotypes, indicating that the requirement of ephrin-B2 in angiogenesis is limited to the VE. Surprisingly, we find that expression of ephrin-B2 specifically in the VE is also sufficient for normal NCC migration and that conversely, embryos in which ephrin-B2 is absent specifically from the VE exhibit NCC migration and survival defects. Disruption of vascular development independent of loss of ephrin-B2 function also leads to defects in NCC and BA development. Together, these data indicate that direct ephrin-B2 signaling to NCCs is not required for NCC guidance, which instead depends on proper organization of the embryonic vasculature.

  14. Neural crest defects in ephrin-B2 mutant mice are non-autonomous and originate from defects in the vasculature

    PubMed Central

    Lewis, Ace E.; Hwa, Jennifer; Wang, Rong; Soriano, Philippe; Bush, Jeffrey O.

    2015-01-01

    Ephrin-B2, a member of the Eph/ephrin family of cell signaling molecules, has been implicated in the guidance of cranial and trunk neural crest cells (NCC) and development of the branchial arches(BA), but detailed examination in mice has been hindered by embryonic lethality of Efnb2 null loss of function due to a requirement in angiogenic remodeling. To elucidate the developmental roles for Efnb2, we generated a conditional rescue knock-in allele that allows rescue of ephrin-B2 specifically in the vascular endothelium (VE), but is otherwise ephrin-B2 deficient. Restoration of ephrin-B2 expression specifically to the VE completely circumvents angiogenic phenotypes, indicating that the requirement of ephrin-B2 in angiogenesis is limited to the VE. Surprisingly, we find that expression of ephrin-B2 specifically in the VE is also sufficient for normal NCC migration and that conversely, embryos in which ephrin-B2 is absent specifically from the VE exhibit NCC migration and survival defects. Disruption of vascular development independent of loss of ephrin-B2 function also leads to defects in NCC and BA development. Together, these data indicate that direct ephrin-B2 signaling to NCCs is not required for NCC guidance, which instead depends on proper organization of the embryonic vasculature. PMID:26385750

  15. The Walker A motif mutation recA4159 abolishes the SOS response and recombination in a recA730 mutant of Escherichia coli.

    PubMed

    Šimatović, Ana; Mitrikeski, Petar T; Vlašić, Ignacija; Sopta, Mary; Brčić-Kostić, Krunoslav

    2016-01-01

    In bacteria, the RecA protein forms recombinogenic filaments required for the SOS response and DNA recombination. In order to form a recombinogenic filament, wild type RecA needs to bind ATP and to interact with mediator proteins. The RecA730 protein is a mutant version of RecA with superior catalytic abilities, allowing filament formation without the help of mediator proteins. The mechanism of RecA730 filament formation is not well understood, and the question remains as to whether the RecA730 protein requires ATP binding in order to become competent for filament formation. We examined two mutants, recA730,4159 (presumed to be defective for ATP binding) and recA730,2201 (defective for ATP hydrolysis), and show that they have different properties with respect to SOS induction, conjugational recombination and double-strand break repair. We show that ATP binding is essential for all RecA730 functions, while ATP hydrolysis is required only for double-strand break repair. Our results emphasize the similarity of the SOS response and conjugational recombination, neither of which requires ATP hydrolysis by RecA730.

  16. Defective response inhibition and collicular noradrenaline enrichment in mice with duplicated retinotopic map in the superior colliculus.

    PubMed

    Mathis, Chantal; Savier, Elise; Bott, Jean-Bastien; Clesse, Daniel; Bevins, Nicholas; Sage-Ciocca, Dominique; Geiger, Karin; Gillet, Anaïs; Laux-Biehlmann, Alexis; Goumon, Yannick; Lacaud, Adrien; Lelièvre, Vincent; Kelche, Christian; Cassel, Jean-Christophe; Pfrieger, Frank W; Reber, Michael

    2015-01-01

    The superior colliculus is a hub for multisensory integration necessary for visuo-spatial orientation, control of gaze movements and attention. The multiple functions of the superior colliculus have prompted hypotheses about its involvement in neuropsychiatric conditions, but to date, this topic has not been addressed experimentally. We describe experiments on genetically modified mice, the Isl2-EphA3 knock-in line, that show a well-characterized duplication of the retino-collicular and cortico-collicular axonal projections leading to hyperstimulation of the superior colliculus. To explore the functional impact of collicular hyperstimulation, we compared the performance of homozygous knock-in, heterozygous knock-in and wild-type mice in several behavioral tasks requiring collicular activity. The light/dark box test and Go/No-Go conditioning task revealed that homozygous mutant mice exhibit defective response inhibition, a form of impulsivity. This defect was specific to attention as other tests showed no differences in visually driven behavior, motivation, visuo-spatial learning and sensorimotor abilities among the different groups of mice. Monoamine quantification and gene expression profiling demonstrated a specific enrichment of noradrenaline only in the superficial layers of the superior colliculus of Isl2-EphA3 knock-in mice, where the retinotopy is duplicated, whereas transcript levels of receptors, transporters and metabolic enzymes of the monoaminergic pathway were not affected. We demonstrate that the defect in response inhibition is a consequence of noradrenaline imbalance in the superficial layers of the superior colliculus caused by retinotopic map duplication. Our results suggest that structural abnormalities in the superior colliculus can cause defective response inhibition, a key feature of attention-deficit disorders.

  17. Defective response inhibition and collicular noradrenaline enrichment in mice with duplicated retinotopic map in the superior colliculus.

    PubMed

    Mathis, Chantal; Savier, Elise; Bott, Jean-Bastien; Clesse, Daniel; Bevins, Nicholas; Sage-Ciocca, Dominique; Geiger, Karin; Gillet, Anaïs; Laux-Biehlmann, Alexis; Goumon, Yannick; Lacaud, Adrien; Lelièvre, Vincent; Kelche, Christian; Cassel, Jean-Christophe; Pfrieger, Frank W; Reber, Michael

    2015-01-01

    The superior colliculus is a hub for multisensory integration necessary for visuo-spatial orientation, control of gaze movements and attention. The multiple functions of the superior colliculus have prompted hypotheses about its involvement in neuropsychiatric conditions, but to date, this topic has not been addressed experimentally. We describe experiments on genetically modified mice, the Isl2-EphA3 knock-in line, that show a well-characterized duplication of the retino-collicular and cortico-collicular axonal projections leading to hyperstimulation of the superior colliculus. To explore the functional impact of collicular hyperstimulation, we compared the performance of homozygous knock-in, heterozygous knock-in and wild-type mice in several behavioral tasks requiring collicular activity. The light/dark box test and Go/No-Go conditioning task revealed that homozygous mutant mice exhibit defective response inhibition, a form of impulsivity. This defect was specific to attention as other tests showed no differences in visually driven behavior, motivation, visuo-spatial learning and sensorimotor abilities among the different groups of mice. Monoamine quantification and gene expression profiling demonstrated a specific enrichment of noradrenaline only in the superficial layers of the superior colliculus of Isl2-EphA3 knock-in mice, where the retinotopy is duplicated, whereas transcript levels of receptors, transporters and metabolic enzymes of the monoaminergic pathway were not affected. We demonstrate that the defect in response inhibition is a consequence of noradrenaline imbalance in the superficial layers of the superior colliculus caused by retinotopic map duplication. Our results suggest that structural abnormalities in the superior colliculus can cause defective response inhibition, a key feature of attention-deficit disorders. PMID:24647754

  18. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses

    PubMed Central

    Shorter, Shayla K.; Schnell, Frederick J.; McMaster, Sean R.; Pinelli, David F.; Andargachew, Rakieb; Evavold, Brian D.

    2016-01-01

    T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant. PMID:26915099

  19. Association of constitutive hyperphosphorylation of Hsf1p with a defective ethanol stress response in Saccharomyces cerevisiae sake yeast strains.

    PubMed

    Noguchi, Chiemi; Watanabe, Daisuke; Zhou, Yan; Akao, Takeshi; Shimoi, Hitoshi

    2012-01-01

    Modern sake yeast strains, which produce high concentrations of ethanol, are unexpectedly sensitive to environmental stress during sake brewing. To reveal the underlying mechanism, we investigated a well-characterized yeast stress response mediated by a heat shock element (HSE) and heat shock transcription factor Hsf1p in Saccharomyces cerevisiae sake yeast. The HSE-lacZ activity of sake yeast during sake fermentation and under acute ethanol stress was severely impaired compared to that of laboratory yeast. Moreover, the Hsf1p of modern sake yeast was highly and constitutively hyperphosphorylated, irrespective of the extracellular stress. Since HSF1 allele replacement did not significantly affect the HSE-mediated ethanol stress response or Hsf1p phosphorylation patterns in either sake or laboratory yeast, the regulatory machinery of Hsf1p is presumed to function differently between these types of yeast. To identify phosphatases whose loss affected the control of Hsf1p, we screened a series of phosphatase gene deletion mutants in a laboratory strain background. Among the 29 mutants, a Δppt1 mutant exhibited constitutive hyperphosphorylation of Hsf1p, similarly to the modern sake yeast strains, which lack the entire PPT1 gene locus. We confirmed that the expression of laboratory yeast-derived functional PPT1 recovered the HSE-mediated stress response of sake yeast. In addition, deletion of PPT1 in laboratory yeast resulted in enhanced fermentation ability. Taken together, these data demonstrate that hyperphosphorylation of Hsf1p caused by loss of the PPT1 gene at least partly accounts for the defective stress response and high ethanol productivity of modern sake yeast strains.

  20. Anti-H-Y responses of H-2b mutant mice.

    PubMed

    Simpson, E; Gordon, R D; Chandler, P R; Bailey, D

    1978-10-01

    Two strains of H-2b mutant mice, H-2ba and H-2bf, in which the mutational event took place at H-2K, make anti-H-Y cytotoxic T cell responses which are H-2-restricted, Db-associated and indistinguishable in target cell specificity from those of H-2b mice. Thus, alteration of the H-2K molecule affects neither the Ir gene controlling the response, nor the associative antigen. On the other hand, one H-2Db mutant strain, H-2bo, although it makes a good anti-H-Y cytotoxic response, shows target cell specificity restricted to its own Dbo antigen(s), and neither H-2b, H-2ba or H-2bf anti-H-Y cytotoxic cells kill H-2bo male target cells. Thus, the alteration of the H-2Db molecule does not affect the Ir gene of H-2b mice, but it does alter the H-2Db-associative antigen.

  1. Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis.

    PubMed

    Kishino, Hideyuki; Eguchi, Hiroki; Takagi, Keiko; Horiuchi, Hiroyuki; Fukuda, Ryouichi; Ohta, Akinori

    2014-03-01

    A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipid methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of (13)C-labeled diC8PC ((methyl-(13)C)3-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-(13)C)3-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast.

  2. CEK2, a Novel MAPK from Candida albicans Complement the Mating Defect of fus3/kss1 Mutant.

    PubMed

    Chen, Jing; Wang, Qing; Chen, Jiang-Ye

    2000-01-01

    A novel MAPK(mitogen-activated protein kinase) gene, CEK2(Candida albicans extracellular signal-regulated kinase 2), was isolated by screening the Candida albicans library based on oligonucleotide probe hybridization and degenerated PCR. The CEK2 gene is 1 119 bp in length, and coding for a 373 aa protein. The CEK2 shares 56% homology with CEK1 from Candida albicans, 55 % homology with FUS3 from S. cerevisiae. From the deduced amino acid sequence, the Cek2 protein contains a conserved ATP binding site and a Ser/Thr kinase activity signal and a conserved TEY sequence was located at L(12) region. In vitro kinase activity assay showed Cek2 could phosphorylate MBP(myelin bovine protein) but not histone H1. CEK2 gene could complement the fus3/kss1 mutant and underwent mating signal induction by a tester strain, but CEK1 gene could not complement with the fus3/kss1 mutant. CEK2 is therefore a FUS3 homolog in Candida albicans. PMID:12075459

  3. Gata3 Hypomorphic Mutant Mice Rescued with a Yeast Artificial Chromosome Transgene Suffer a Glomerular Mesangial Cell Defect.

    PubMed

    Moriguchi, Takashi; Yu, Lei; Otsuki, Akihito; Ainoya, Keiko; Lim, Kim-Chew; Yamamoto, Masayuki; Engel, James Douglas

    2016-09-01

    GATA3 is a zinc finger transcription factor that plays a crucial role in embryonic kidney development, while its precise functions in the adult kidney remain largely unexplored. Here, we demonstrate that GATA3 is specifically expressed in glomerular mesangial cells and plays a critical role in the maintenance of renal glomerular function. Newly generated Gata3 hypomorphic mutant mice exhibited neonatal lethality associated with severe renal hypoplasia. Normal kidney size was restored by breeding the hypomorphic mutant with a rescuing transgenic mouse line bearing a 662-kb Gata3 yeast artificial chromosome (YAC), and these animals (termed G3YR mice) survived to adulthood. However, most of the G3YR mice showed degenerative changes in glomerular mesangial cells, which deteriorated progressively during postnatal development. Consequently, the G3YR adult mice suffered severe renal failure. We found that the 662-kb Gata3 YAC transgene recapitulated Gata3 expression in the renal tubules but failed to direct sufficient GATA3 activity to mesangial cells. Renal glomeruli of the G3YR mice had significantly reduced amounts of platelet-derived growth factor receptor (PDGFR), which is known to participate in the development and maintenance of glomerular mesangial cells. These results demonstrate a critical role for GATA3 in the maintenance of mesangial cells and its absolute requirement for prevention of glomerular disease. PMID:27296697

  4. Mutagenesis of the potato ADPglucose pyrophosphorylase and characterization of an allosteric mutant defective in 3-phosphoglycerate activation

    SciTech Connect

    Greene, T.W.; Chantler, S.E.; Kahn, M.L.

    1996-02-20

    ADPglucose pyrophosphorylase (glucose-1-phosphate adenylytransferase; AD P:{alpha}-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27) catalyzes a key regulatory step in {alpha}-glucan synthesis in bacteria and higher plants. We have previously shown that the expression of the cDNA sequences of the potato tuber large (LS) and small (SS) subunits yielded a functional heterotetrameric enzyme capable of complementing a mutation in the single AGP (glgC) structural gene of Escherichia coli. This heterologous complementation provides a powerful genetic approach to obtain biochemical information on the specific roles of LS and SS in enzyme function. By mutagenizing the LS cDNA with hydroxylamine and then coexpressing with wild-type SS in an E. coli glgC{sup {minus}} strain, >350 mutant colonies were identified that were impaired in glycogen production. One mutant exhibited enzymatic and antigen levels comparable to the wild-type recombinant enzyme but required 45-fold greater levels of the activator 3-phosphoglycerate for maximum activity. Sequence analysis identified a single nucleotide change that resulted in the change of Pro-52 to Leu. This heterologous genetic system provides and efficient means to identify residues important for catalysis and allosteric functioning and should lead to novel approaches to increase plant productivity. 31 refs., 4 figs., 1 tab.

  5. Clustered Charge-to-Alanine Mutagenesis of the Vaccinia Virus H5 Gene: Isolation of a Dominant, Temperature-Sensitive Mutant with a Profound Defect in Morphogenesis

    PubMed Central

    DeMasi, Joseph; Traktman, Paula

    2000-01-01

    The vaccinia virus H5 gene encodes a 22.3-kDa phosphoprotein that is expressed during both the early and late phases of viral gene expression. It is a major component of virosomes and has been implicated in viral transcription and, as a substrate of the B1 kinase, may participate in genome replication. To enable a genetic analysis of the role of H5 during the viral life cycle, we used clustered charge-to-alanine mutagenesis in an attempt to create a temperature-sensitive (ts) virus with a lesion in the H5 gene. Five mutant viruses were isolated, with one of them, tsH5-4, having a strong ts phenotype as assayed by plaque formation and measurements of 24-h viral yield. Surprisingly, no defects in genome replication or viral gene expression were detected at the nonpermissive temperature. By electron microscopy, we observed a profound defect in the early stages of virion morphogenesis, with arrest occurring prior to the formation of crescent membranes or immature particles. Nonfunctional, “curdled” virosomes were detected in tsH5-4 infections at the nonpermissive temperature. These structures appeared to revert to functional virosomes after a temperature shift to permissive conditions. We suggest an essential role for H5 in normal virosome formation and the initiation of virion morphogenesis. By constructing recombinant genomes containing two H5 alleles, wild type and H5-4, we determined that H5-4 exerted a dominant phenotype. tsH5-4 is the first example of a dominant ts mutant isolated and characterized in vaccinia virus. PMID:10666270

  6. Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

    PubMed Central

    Goode, Alice; Butler, Kevin; Long, Jed; Cavey, James; Scott, Daniel; Shaw, Barry; Sollenberger, Jill; Gell, Christopher; Johansen, Terje; Oldham, Neil J.; Searle, Mark S.; Layfield, Robert

    2016-01-01

    ABSTRACT Growing evidence implicates impairment of autophagy as a candidate pathogenic mechanism in the spectrum of neurodegenerative disorders which includes amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS-FTLD). SQSTM1, which encodes the autophagy receptor SQSTM1/p62, is genetically associated with ALS-FTLD, although to date autophagy-relevant functional defects in disease-associated variants have not been described. A key protein-protein interaction in autophagy is the recognition of a lipid-anchored form of LC3 (LC3-II) within the phagophore membrane by SQSTM1, mediated through its LC3-interacting region (LIR), and notably some ALS-FTLD mutations map to this region. Here we show that although representing a conservative substitution and predicted to be benign, the ALS-associated L341V mutation of SQSTM1 is defective in recognition of LC3B. We place our observations on a firm quantitative footing by showing the L341V-mutant LIR is associated with a ∼3-fold reduction in LC3B binding affinity and using protein NMR we rationalize the structural basis for the effect. This functional deficit is realized in motor neuron-like cells, with the L341V mutant EGFP-mCherry-SQSTM1 less readily incorporated into acidic autophagic vesicles than the wild type. Our data supports a model in which the L341V mutation limits the critical step of SQSTM1 recruitment to the phagophore. The oligomeric nature of SQSTM1, which presents multiple LIRs to template growth of the phagophore, potentially gives rise to avidity effects which amplify the relatively modest impact of any single mutation on LC3B binding. Over the lifetime of a neuron, impaired autophagy could expose a vulnerability, which ultimately tips the balance from cell survival toward cell death. PMID:27158844

  7. Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD.

    PubMed

    Goode, Alice; Butler, Kevin; Long, Jed; Cavey, James; Scott, Daniel; Shaw, Barry; Sollenberger, Jill; Gell, Christopher; Johansen, Terje; Oldham, Neil J; Searle, Mark S; Layfield, Robert

    2016-07-01

    Growing evidence implicates impairment of autophagy as a candidate pathogenic mechanism in the spectrum of neurodegenerative disorders which includes amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS-FTLD). SQSTM1, which encodes the autophagy receptor SQSTM1/p62, is genetically associated with ALS-FTLD, although to date autophagy-relevant functional defects in disease-associated variants have not been described. A key protein-protein interaction in autophagy is the recognition of a lipid-anchored form of LC3 (LC3-II) within the phagophore membrane by SQSTM1, mediated through its LC3-interacting region (LIR), and notably some ALS-FTLD mutations map to this region. Here we show that although representing a conservative substitution and predicted to be benign, the ALS-associated L341V mutation of SQSTM1 is defective in recognition of LC3B. We place our observations on a firm quantitative footing by showing the L341V-mutant LIR is associated with a ∼3-fold reduction in LC3B binding affinity and using protein NMR we rationalize the structural basis for the effect. This functional deficit is realized in motor neuron-like cells, with the L341V mutant EGFP-mCherry-SQSTM1 less readily incorporated into acidic autophagic vesicles than the wild type. Our data supports a model in which the L341V mutation limits the critical step of SQSTM1 recruitment to the phagophore. The oligomeric nature of SQSTM1, which presents multiple LIRs to template growth of the phagophore, potentially gives rise to avidity effects which amplify the relatively modest impact of any single mutation on LC3B binding. Over the lifetime of a neuron, impaired autophagy could expose a vulnerability, which ultimately tips the balance from cell survival toward cell death.

  8. Assembly defects of desmin disease mutants carrying deletions in the alpha-helical rod domain are rescued by wild type protein.

    PubMed

    Bär, Harald; Mücke, Norbert; Katus, Hugo A; Aebi, Ueli; Herrmann, Harald

    2007-04-01

    Most mutations of desmin that cause severe autosomal dominant forms of myofibrillar myopathy are point mutations and locate in the central alpha-helical coiled-coil rod domain. Recently, two in-frame deletions of one and three amino acids, respectively, in the alpha-helix have been described and discussed to drastically interfere with the architecture of the desmin dimer and possibly also the formation of tetramers and higher order complexes [Kaminska, A., Strelkov, S.V., Goudeau, B., Olive, M., Dagvadorj, A., Fidzianska, A., Simon-Casteras, M., Shatunov, A., Dalakas, M.C., Ferrer, I., Kwiecinski, H., Vicart, P., Goldfarb, L.G., 2004. Small deletions disturb desmin architecture leading to breakdown of muscle cells and development of skeletal or cardioskeletal myopathy. Hum. Genet. 114, 306-313.]. Therefore, it was proposed that they may poison intermediate filament (IF) assembly. We have now recombinantly synthesized both mutant proteins and subjected them to comprehensive in vitro assembly experiments. While exhibiting assembly defects when analyzed on their own, both one-to-one mixtures of the respective mutant protein with wild type desmin facilitated proper filament formation. Transient transfection studies complemented this fundamental finding by demonstrating that wild type desmin is also rescuing these assembly defects in vivo. In summary, our findings strongly question the previous hypothesis that it is assembly incompetence due to molecular rearrangements caused by the mutations, which triggers the development of disease. As an alternative, we propose that these mutations cause subtle age-dependent structural alterations of desmin IFs that eventually lead to disease. PMID:17188893

  9. Specificity of inhibitors of serine palmitoyltransferase (SPT), a key enzyme in sphingolipid biosynthesis, in intact cells. A novel evaluation system using an SPT-defective mammalian cell mutant.

    PubMed

    Hanada, K; Nishijima, M; Fujita, T; Kobayashi, S

    2000-05-15

    In the present study, we demonstrate a model cell system for evaluating the specificity of inhibitors of serine palmitoyltransferase (SPT), the enzyme that catalyzes the first step of sphingolipid biosynthesis. The LY-B strain is a Chinese hamster ovary (CHO) cell mutant defective in SPT, and the LY-B/cLCB1 strain is a genetically corrected revertant of the mutant. Although LY-B cells grew only slightly in sphingolipid-deficient medium, their growth was restored to the level of LY-B/cLCB1 cells under sphingosine-supplied conditions, indicating that, in CHO cells, the growth inhibition caused by SPT inactivation was rescued almost fully by the metabolic complementation of sphingolipids. Cultivation of LY-B/cLCB1 cells in sphingolipid-deficient medium in the presence of 10 microM sphingofungin B and ISP-1 (myriocin, thermozymocidin), potent inhibitors of SPT activity, caused severe growth inhibition with approximately 95% inhibition of de novo sphingolipid synthesis. The growth inhibition by sphingofungin B and ISP-1 was rescued substantially by exogenous sphingosine, whereas the cytotoxicity of two other types of SPT inhibitor, L-cycloserine and beta-chloro-L-alanine, was hardly rescued. Similar cytotoxic patterns of these inhibitors also were observed on the growth of SPT-defective LY-B cells cultured under sphingosine-supplied conditions. The SPT inhibitors did not affect metabolic conversion of exogenous [(3)H]sphingosine to complex sphingolipids. Thus, the cytotoxicity of sphingofungin B and ISP-1, but not L-cycloserine or beta-chloro-L-alanine, is due largely to inhibition of sphingolipid synthesis by inhibiting the SPT activity.

  10. The DSL domain in mutant JAG1 ligand is essential for the severity of the liver defect in Alagille syndrome.

    PubMed

    Yuan, Z R; Okaniwa, M; Nagata, I; Tazawa, Y; Ito, M; Kawarazaki, H; Inomata, Y; Okano, S; Yoshida, T; Kobayashi, N; Kohsaka, T

    2001-05-01

    Alagille syndrome (AGS) is a congenital multi-system anomaly mainly characterized by paucity of intrahepatic bile ducts caused by haploinsufficiency of the Jagged 1 gene (JAG1). To explore the relationship between genotype and phenotype, we analyzed the JAG1 gene in 25 Japanese AGS families at the genomic DNA level and identified 15 point mutations and one large deletion. Analysis of the genotype and phenotype strongly indicated that the Delta/Serrate/Lag-2 (DSL) domain in JAG1 protein played an essential role in determining the severity of the liver disorder. In four sporadic cases, missing an entire DSL domain in mutant JAG1 resulted in progressive liver failure and all 4 patients needed a liver transplant at a very young age. This correlation was further confirmed by statistical analysis (chi2=9.143, p<0.001). Our finding demonstrated that the DSL domain in JAG1 appears to be essential for normal liver development and function.

  11. Analysis of the Legionella pneumophila fliI gene: intracellular growth of a defined mutant defective for flagellum biosynthesis.

    PubMed Central

    Merriam, J J; Mathur, R; Maxfield-Boumil, R; Isberg, R R

    1997-01-01

    Using a PCR-based strategy and degenerate oligonucleotides, we isolated a Legionella pneumophila gene that showed high sequence similarity to members of the fliI gene family. An insertion mutation that disrupted the fliI open reading frame was recombined onto the L. pneumophila chromosome and analyzed for its effects on production of flagella and intracellular growth. The mutation resulted in loss of surface-localized flagellin protein but had no effect on the ability of the bacteria to grow within cultured cells. Therefore, in spite of the fact that some aflagellar mutations render L. pneumophila unable to grow within macrophages, the isolation of this defined mutant confirms that production of flagella is not required for intracellular growth. PMID:9169800

  12. Characterization of three putative Lon proteases of Thermus thermophilus HB27 and use of their defective mutants as hosts for production of heterologous proteins.

    PubMed

    Maehara, Tomoko; Hoshino, Takayuki; Nakamura, Akira

    2008-03-01

    In the genome of a thermophilic bacterium, Thermus thermophilus HB27, three genes, TTC0418, TTC0746 and TTC1975, were annotated as ATP-dependent protease La (Lon). Sequence comparisons indicated that TTC0418 and TTC0746 showed significant similarities to bacterial LonA-type proteases, such as Escherichia coli Lon protease, especially in regions corresponding to domains for ATP-binding and hydrolysis, and for proteolysis, but TTC1975 exhibited a similarity only at the C-terminal proteolytic domain. The enzymatic analyses, using purified recombinant proteins produced by E. coli, revealed that TTC0418 and TTC0746 exhibited peptidase and protease activities against two synthetic peptides and casein, respectively, in an ATP-dependent manner, and at the same time, both the enzymes had significant ATPase activities in the presence of substrates. On the other hand, TTC1975 possessed a protease activity against casein, but addition of ATP did not enhance this activity. Moreover, a T. thermophilus mutant deficient in both TTC0418 and TTC0746 showed a similar growth characteristic to an E. coli lon mutant, i.e., a growth defect lag after a nutritional downshift. These results indicate that TTC0418 and TTC0746 are actually members of bacterial LonA-type proteases with different substrate specificities, whereas TTC1975 should not be classified as a Lon protease. Finally, the effects of mutations deficient in these proteases were assessed on production of several heterologous gene products from Pyrococcus horikoshii and Geobacillus stearothermophilus. It was shown that TTC0746 mutation was more effective in improving production than the other two mutations, especially for production of P. horikoshii alpha-mannosidase and G. stearothermophilus alpha-amylase, indicating that the TTC0746 mutant of T. thermophilus HB27 may be useful for production of heterologous proteins from thermophiles and hyperthermophiles. PMID:18157502

  13. Alanine racemase mutants of Mycobacterium tuberculosis require D-alanine for growth and are defective for survival in macrophages and mice.

    PubMed

    Awasthy, Disha; Bharath, Sowmya; Subbulakshmi, Venkita; Sharma, Umender

    2012-02-01

    Alanine racemase (Alr) is an essential enzyme in most bacteria; however, some species (e.g. Listeria monocytogenes) can utilize d-amino acid transaminase (Dat) to generate d-alanine, which renders Alr non-essential. In addition to the conflicting reports on gene knockout of alr in Mycobacterium smegmatis, a recent study concluded that depletion of Alr does not affect the growth of M. smegmatis. In order to get an unambiguous answer on the essentiality of Alr in Mycobacterium tuberculosis and validate it as a drug target in vitro and in vivo, we have inactivated the alr gene of M. tuberculosis and found that it was not possible to generate an alr knockout in the absence of a complementing gene copy or d-alanine in the growth medium. The growth kinetics of the alr mutant revealed that M. tuberculosis requires very low amounts of d-alanine (5-10 µg ml(-1)) for optimum growth. Survival kinetics of the mutant in the absence of d-alanine indicated that depletion of this amino acid results in rapid loss of viability. The alr mutant was found to be defective for growth in macrophages. Analysis of phenotype in mice suggested that non-availability of d-alanine in mice leads to clearance of bacteria followed by stabilization of bacterial number in lungs and spleen. Additionally, reversal of d-cycloserine inhibition in the presence of d-alanine in M. tuberculosis suggested that Alr is the primary target of d-cycloserine. Thus, Alr of M. tuberculosis is a valid drug target and inhibition of Alr alone should result in loss of viability in vitro and in vivo.

  14. Cellular Responses during Morphological Transformation in Azospirillum brasilense and Its flcA Knockout Mutant

    PubMed Central

    Coumans, Joëlle V. F.; Poljak, Anne; Raftery, Mark J.; Pereg, Lily

    2014-01-01

    FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA− strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome. PMID:25502569

  15. Cellular responses during morphological transformation in Azospirillum brasilense and Its flcA knockout mutant.

    PubMed

    Hou, Xingsheng; McMillan, Mary; Coumans, Joëlle V F; Poljak, Anne; Raftery, Mark J; Pereg, Lily

    2014-01-01

    FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA- strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.

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

    PubMed Central

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

    2016-01-01

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

  17. Staphylococcus aureus avirulent mutant vaccine induces humoral and cellular immune responses on pregnant heifers.

    PubMed

    Pellegrino, M; Rodriguez, N; Vivas, A; Giraudo, J; Bogni, C

    2016-06-17

    Bovine mastitis produces economic losses, attributable to the decrease in milk production, reduced milk quality, costs of treatment and replacement of animals. A successful prophylactic vaccine against Staphylococcus aureus should elicit both humoral and cellular immune responses. In a previous report we evaluated the effectiveness of a live vaccine to protect heifers against challenge with a virulent strain. In the present study the immunological response of heifers after combined immunization schedule was investigated. In a first experimental trial, heifers were vaccinated with 3 subcutaneous doses of avirulent mutant S. aureus RC122 before calving and one intramammary dose (IMD) after calving. Antibodies concentration in blood, bactericidal effect of serum from vaccinated animals and lymphocyte proliferation was determined. The levels of total IgG, IgG1 and IgG2 in colostrum and the lymphocyte proliferation index were significantly higher in vaccinated respect to non-vaccinated group throughout the experiment. The second trial, where animals were inoculated with different vaccination schedules, was carried out to determine the effect of the IMD on the level of antibodies in blood and milk, cytokines (IL-13 and IFN-γ) concentration and milk's SCC and bacteriology. The bacterial growth of the S. aureus strains was totally inhibited at 1-3×10(6) and 1-3×10(3)cfu/ml, when the strains were mixed with pooled serum diluted 1/40. The results shown that IMD has not a significant effect on the features determinate. In conclusion, a vaccination schedule involving three SC doses before calving would be enough to stimulate antibodies production in milk without an IMD. Furthermore, the results showed a bactericidal effect of serum from vaccinated animals and this provides further evidence about serum functionality. Immune responses, humoral (antigen-specific antibodies and Th2 type cytokines) and cellular (T-lymphocyte proliferation responses and Th1 type cytokines), were

  18. An altered hydrotropic response (ahr1) mutant of Arabidopsis recovers root hydrotropism with cytokinin

    PubMed Central

    Saucedo, Manuel; Ponce, Georgina; Campos, María Eugenia; Eapen, Delfeena; García, Edith; Luján, Rosario; Sánchez, Yoloxóchitl; Cassab, Gladys I.

    2012-01-01

    Roots are highly plastic and can acclimate to heterogeneous and stressful conditions. However, there is little knowledge of the effect of moisture gradients on the mechanisms controlling root growth orientation and branching, and how this mechanism may help plants to avoid drought responses. The aim of this study was to isolate mutants of Arabidopsis thaliana with altered hydrotropic responses. Here, altered hydrotropic response 1 (ahr1), a semi-dominant allele segregating as a single gene mutation, was characterized. ahr1 directed the growth of its primary root towards the source of higher water availability and developed an extensive root system over time. This phenotype was intensified in the presence of abscisic acid and was not observed if ahr1 seedlings were grown in a water stress medium without a water potential gradient. In normal growth conditions, primary root growth and root branching of ahr1 were indistinguishable from those of the wild type (wt). The altered hydrotropic growth of ahr1 roots was confirmed when the water-rich source was placed at an angle of 45° from the gravity vector. In this system, roots of ahr1 seedlings grew downward and did not display hydrotropism; however, in the presence of cytokinins, they exhibited hydrotropism like those of the wt, indicating that cytokinins play a critical role in root hydrotropism. The ahr1 mutant represents a valuable genetic resource for the study of the effects of cytokinins in the differential growth of hydrotropism and control of lateral root formation during the hydrotropic response. PMID:22442413

  19. The Primary Folding Defect and Rescue of ΔF508 CFTR Emerge during Translation of the Mutant Domain

    PubMed Central

    Schmidt, Andre; Richardson, John; Charitou, Paraskevi; Thomas, Philip J.; Braakman, Ineke

    2010-01-01

    In the vast majority of cystic fibrosis (CF) patients, deletion of residue F508 from CFTR is the cause of disease. F508 resides in the first nucleotide binding domain (NBD1) and its absence leads to CFTR misfolding and degradation. We show here that the primary folding defect arises during synthesis, as soon as NBD1 is translated. Introduction of either the I539T or G550E suppressor mutation in NBD1 partially rescues ΔF508 CFTR to the cell surface, but only I539T repaired ΔF508 NBD1. We demonstrated rescue of folding and stability of NBD1 from full-length ΔF508 CFTR expressed in cells to isolated purified domain. The co-translational rescue of ΔF508 NBD1 misfolding in CFTR by I539T advocates this domain as the most important drug target for cystic fibrosis. PMID:21152102

  20. Transcriptome Comparative Profiling of Barley eibi1 Mutant Reveals Pleiotropic Effects of HvABCG31 Gene on Cuticle Biogenesis and Stress Responsive Pathways

    PubMed Central

    Yang, Zujun; Zhang, Tao; Lang, Tao; Li, Guangrong; Chen, Guoxiong; Nevo, Eviatar

    2013-01-01

    Wild barley eibi1 mutant with HvABCG31 gene mutation has low capacity to retain leaf water, a phenotype associated with reduced cutin deposition and a thin cuticle. To better understand how such a mutant plant survives, we performed a genome-wide gene expression analysis. The leaf transcriptomes between the near-isogenic lines eibi1 and the wild type were compared using the 22-k Barley1 Affymetrix microarray. We found that the pleiotropic effect of the single gene HvABCG31 mutation was linked to the co-regulation of metabolic processes and stress-related system. The cuticle development involved cytochrome P450 family members and fatty acid metabolism pathways were significantly up-regulated by the HvABCG31 mutation, which might be anticipated to reduce the levels of cutin monomers or wax and display conspicuous cuticle defects. The candidate genes for responses to stress were induced by eibi1 mutant through activating the jasmonate pathway. The down-regulation of co-expressed enzyme genes responsible for DNA methylation and histone deacetylation also suggested that HvABCG31 mutation may affect the epigenetic regulation for barley development. Comparison of transcriptomic profiling of barley under biotic and abiotic stresses revealed that the functions of HvABCG31 gene to high-water loss rate might be different from other osmotic stresses of gene mutations in barley. The transcriptional profiling of the HvABCG31 mutation provided candidate genes for further investigation of the physiological and developmental changes caused by the mutant. PMID:24129180

  1. Different mating-type-regulated genes affect the DNA repair defects of Saccharomyces RAD51, RAD52 and RAD55 mutants.

    PubMed

    Valencia-Burton, Maria; Oki, Masaya; Johnson, Jean; Seier, Tracey A; Kamakaka, Rohinton; Haber, James E

    2006-09-01

    Saccharomyces cerevisiae cells expressing both a- and alpha-mating-type (MAT) genes (termed mating-type heterozygosity) exhibit higher rates of spontaneous recombination and greater radiation resistance than cells expressing only MATa or MATalpha. MAT heterozygosity suppresses recombination defects of four mutations involved in homologous recombination: complete deletions of RAD55 or RAD57, an ATPase-defective Rad51 mutation (rad51-K191R), and a C-terminal truncation of Rad52, rad52-Delta327. We investigated the genetic basis of MAT-dependent suppression of these mutants by deleting genes whose expression is controlled by the Mata1-Matalpha2 repressor and scoring resistance to both campothecin (CPT) and phleomycin. Haploid rad55Delta strains became more damage resistant after deleting genes required for nonhomologous end-joining (NHEJ), a process that is repressed in MATa/MATalpha cells. Surprisingly, NHEJ mutations do not suppress CPT sensitivity of rad51-K191R or rad52-Delta327. However, rad51-K191R is uniquely suppressed by deleting the RME1 gene encoding a repressor of meiosis or its coregulator SIN4; this effect is independent of the meiosis-specific homolog, Dmc1. Sensitivity of rad52-Delta327 to CPT was unexpectedly increased by the MATa/MATalpha-repressed gene YGL193C, emphasizing the complex ways in which MAT regulates homologous recombination. The rad52-Delta327 mutation is suppressed by deleting the prolyl isomerase Fpr3, which is not MAT regulated. rad55Delta is also suppressed by deletion of PST2 and/or YBR052C (RFS1, rad55 suppressor), two members of a three-gene family of flavodoxin-fold proteins that associate in a nonrandom fashion with chromatin. All three recombination-defective mutations are made more sensitive by deletions of Rad6 and of the histone deacetylases Rpd3 and Ume6, although these mutations are not themselves CPT or phleomycin sensitive.

  2. Heat shock response in tomato brassinosteroid mutants indicates that thermotolerance is independent of brassinosteroid homeostasis.

    PubMed

    Mazorra, Luis Miguel; Holton, Nicholas; Bishop, Gerard J; Núñez, Miriam

    2011-12-01

    Brassinosteroids (BRs) are plant steroid hormones and, when applied exogenously, they induce physiological responses, including tolerance to heat shock (HS). How endogenous BR content and altered perception of BRs influence thermal tolerance is poorly understood. BR-induced thermotolerance in tomato seedlings with altered BR homeostasis was examined by assessing the survival, ion leakage and lipid peroxidation of seedlings from a BR-deficient mutant (extreme dwarf d(x)), a partially BR-insensitive mutant curl3(-abs) allele (curl3 altered brassinolide sensitivity) and a line overexpressing the Dwarf, BR-biosynthesis gene (35SD). We confirmed that treatment with 1 μM of epi-brassinolide (EBL) induces thermotolerance of wild type seedlings following a HS regime at 45 °C. The curl3(-abs) seedlings had the highest basal tolerance to heat, whereas the EBL-induced thermal tolerance of d(x) seedlings was greatest and responded to lower EBL concentrations. The d(x) and 35SD seedlings had similar thermal tolerance; however, they showed increased signs of oxidative stress. EBL reduced the induction of lipid peroxidation of seedlings after recovery from heat. Highest oxidative stress and peroxidase (POX) activity (EC 1.11.1.7) was in BR-deficient d(x) mutant seedlings. EBL was able of inducing POX activity but not other antioxidant enzymes; however, effects of HS on POX activity of seedlings were absent or less marked. Taking together, results indicate that thermal tolerance is independent of endogenous BR content, but HS-mediated oxidative stress depends on BR levels. PMID:22078379

  3. Large-Scale Molecular Simulations on the Mechanical Response and Failure Behavior of a defective Graphene: Cases of 5–8–5 Defects

    PubMed Central

    Wang, Shuaiwei; Yang, Baocheng; Yuan, Jinyun; Si, Yubing; Chen, Houyang

    2015-01-01

    Understanding the effect of defects on mechanical responses and failure behaviors of a graphene membrane is important for its applications. As examples, in this paper, a family of graphene with various 5–8–5 defects are designed and their mechanical responses are investigated by employing molecular dynamics simulations. The dependence of fracture strength and strain as well as Young’s moduli on the nearest neighbor distance and defect types is examined. By introducing the 5–8–5 defects into graphene, the fracture strength and strain become smaller. However, the Young’s moduli of DL (Linear arrangement of repeat unit 5–8–5 defect along zigzag-direction of graphene), DS (a Slope angle between repeat unit 5–8–5 defect and zigzag direction of graphene) and DZ (Zigzag-like 5–8–5 defects) defects in the zigzag direction become larger than those in the pristine graphene in the same direction. A maximum increase of 11.8% of Young’s modulus is obtained. Furthermore, the brittle cracking mechanism is proposed for the graphene with 5–8–5 defects. The present work may provide insights in controlling the mechanical properties by preparing defects in the graphene, and give a full picture for the applications of graphene with defects in flexible electronics and nanodevices. PMID:26449655

  4. Activated mutant of Galpha(12) enhances the hyperosmotic stress response of NIH3T3 cells.

    PubMed

    Dermott, J M; Wadsworth, S J; van Rossum, G D; Dhanasekaran, N

    2001-01-01

    Heterotrimeric G protein G12 stimulates diverse physiological responses including the activities of Na+/H+ exchangers and Jun kinases. We have observed that the expression of the constitutively activated, GTPase-deficient mutant of Galpha(12) (Galpha(12)QL) accelerates the hyperosmotic response of NIH3T3 cells as monitored by the hyperosmotic stress-stimulated activity of JNK1. The accelerated response appears to be partly due to the increased basal activity of JNK since cell lines-such as NIH3T3 cells expressing JNK1-in which JNK activity is elevated, show a similar response. NIH3T3 cells expressing Galpha(12)QL also display heightened sensitivity to hyperosmotic stress. This is in contrast to JNK1-NIH3T3 cells that failed to enhance sensitivity although they do exhibit an accelerated hyperosmotic response. Reasoning that the increased sensitivity seen in Galpha(12)QL cells is due to a signaling component other than JNK, the effect of dimethyamiloride, an inhibitor of Na+/H+ exchanger in this response, was assessed. Treatment of vector control NIH3T3 cells with 50 microM dimethylamiloride potently inhibited their hyperosmotic response whereas the response was only partially inhibited in Galpha(12)QL-NIH3T3 cells. These results, for the first time, identify that NHEs are upstream of the JNK module in the hyperosmotic stress-signaling pathway and that Galpha(12) can enhance this response by modulating either or both of these components namely, JNKs and NHEs in NIH3T3 cells. PMID:11180393

  5. A no hydrotropic response root mutant that responds positively to gravitropism in Arabidopsis.

    PubMed

    Eapen, Delfeena; Barroso, María Luisa; Campos, María Eugenia; Ponce, Georgina; Corkidi, Gabriel; Dubrovsky, Joseph G; Cassab, Gladys I

    2003-02-01

    For most plants survival depends upon the capacity of root tips to sense and move towards water and other nutrients in the soil. Because land plants cannot escape environmental stress they use developmental solutions to remodel themselves in order to better adapt to the new conditions. The primary site for perception of underground signals is the root cap (RC). Plant roots have positive hydrotropic response and modify their growth direction in search of water. Using a screening system with a water potential gradient, we isolated a no hydrotropic response (nhr) semi-dominant mutant of Arabidopsis that continued to grow downwardly into the medium with the lowest water potential contrary to the positive hydrotropic and negative gravitropic response seen in wild type-roots. The lack of hydrotropic response of nhr1 roots was confirmed in a system with a gradient in air moisture. The root gravitropic response of nhr1 seedlings was significantly faster in comparison with those of wild type. The frequency of the waving pattern in nhr1 roots was increased compared to those of wild type. nhr1 seedlings had abnormal root cap morphogenesis and reduced root growth sensitivity to abscisic acid (ABA) and the polar auxin transport inhibitor N-(1-naphtyl)phtalamic acid (NPA). These results showed that hydrotropism is amenable to genetic analysis and that an ABA signaling pathway participates in sensing water potential gradients through the root cap.

  6. Cystic fibrosis–adapted Pseudomonas aeruginosa quorum sensing lasR mutants cause hyperinflammatory responses

    PubMed Central

    LaFayette, Shantelle L.; Houle, Daniel; Beaudoin, Trevor; Wojewodka, Gabriella; Radzioch, Danuta; Hoffman, Lucas R.; Burns, Jane L.; Dandekar, Ajai A.; Smalley, Nicole E.; Chandler, Josephine R.; Zlosnik, James E.; Speert, David P.; Bernier, Joanie; Matouk, Elias; Brochiero, Emmanuelle; Rousseau, Simon; Nguyen, Dao

    2015-01-01

    Cystic fibrosis lung disease is characterized by chronic airway infections with the opportunistic pathogen Pseudomonas aeruginosa and severe neutrophilic pulmonary inflammation. P. aeruginosa undergoes extensive genetic adaptation to the cystic fibrosis (CF) lung environment, and adaptive mutations in the quorum sensing regulator gene lasR commonly arise. We sought to define how mutations in lasR alter host-pathogen relationships. We demonstrate that lasR mutants induce exaggerated host inflammatory responses in respiratory epithelial cells, with increased accumulation of proinflammatory cytokines and neutrophil recruitment due to the loss of bacterial protease–dependent cytokine degradation. In subacute pulmonary infections, lasR mutant–infected mice show greater neutrophilic inflammation and immunopathology compared with wild-type infections. Finally, we observed that CF patients infected with lasR mutants have increased plasma interleukin-8 (IL-8), a marker of inflammation. These findings suggest that bacterial adaptive changes may worsen pulmonary inflammation and directly contribute to the pathogenesis and progression of chronic lung disease in CF patients. PMID:26457326

  7. SPI1 defective mutants of Salmonella enterica induce cross-protective immunity in chickens against challenge with serovars Typhimurium and Enteritidis.

    PubMed

    Matulova, Marta; Havlickova, Hana; Sisak, Frantisek; Babak, Vladimir; Rychlik, Ivan

    2013-06-28

    In this study we were interested in the serovar cross-protection potential of Salmonella Pathogenicity Island 1 (SPI1) attenuated vaccine strains of Salmonella enterica serovars Enteritidis and Typhimurium and immune response of vaccinated and naive chickens to Salmonella infection. The immune response was characterized by real time PCR quantifying transcripts of interleukins IL1β, IL17, IL22, interferon gamma (IFNγ), inducible NO synthase (iNOS), immunoglobulins IgM, IgA, IgY and Ig light chain, and six genes of acute phase response including avidin, serum amyloid A, extracellular fatty acid-binding protein (Ex-FABP), immune responsive gene 1, chemokine AH221 and trappin-6. Vaccination with SPI1 mutants of both serovars protected chickens against Salmonella infection, independent of the serovar used for the challenge and the time post infection. However, expressions of all interleukins, iNOS and Ex-FABP showed that protection against homologous serovars was significantly higher than against heterologous serovars after intravenous challenge at 4 days post infection. The vaccination with a mixture of S. Enteritidis and S. Typhimurium SPI1 mutants induced an intermediate protection against challenge with both serovars, i.e. the mixed vaccine provided an additional protective effect when compared with the chickens vaccinated with a vaccine formed by only a single Salmonella serovar. PMID:23684831

  8. VPX mutants of HIV-2 are infectious in established cell lines but display a severe defect in peripheral blood lymphocytes.

    PubMed Central

    Guyader, M; Emerman, M; Montagnier, L; Peden, K

    1989-01-01

    Nucleotide sequence comparison between HIV-1, HIV-2 and SIV has revealed the presence of an open reading frame (ORF) in the central region of the genomes of HIV-2 and SIV that has no counterpart in HIV-1. This new ORF, called vpx, is highly conserved between HIV-2ROD and SIVmac. Using anti-peptide sera to the predicted protein and site-directed mutagenesis, we show that mutations in the vpx ORF eliminate the synthesis of a 16 kd protein in HIV-2 infected cells, confirming that this protein is the product of this gene. Full-length clones of HIV-2 containing these mutations are infectious in two permanent T lymphocytic cell lines and two monocytic cell lines. In contrast, we show that loss of VPX function results in a severe defect in the productive infection of human peripheral blood lymphocytes both in the amount of reverse transcriptase activity produced and in core protein expression. These findings suggest that the VPX protein plays an important role in the in vivo life cycle of the HIV-2/SIV viruses. Images PMID:2743977

  9. Escherichia coli K-12 Suppressor-free Mutants Lacking Early Glycosyltransferases and Late Acyltransferases: minimal lipopolysaccharide structure and induction of envelope stress response.

    PubMed

    Klein, Gracjana; Lindner, Buko; Brabetz, Werner; Brade, Helmut; Raina, Satish

    2009-06-01

    To elucidate the minimal lipopolysaccharide (LPS) structure needed for the viability of Escherichia coli, suppressor-free strains lacking either the 3-deoxy-d-manno-oct-2-ulosonic acid transferase waaA gene or derivatives of the heptosyltransferase I waaC deletion with lack of one or all late acyltransferases (lpxL/M/P) and/or various outer membrane biogenesis factors were constructed. Delta(waaC lpxL lpxM lpxP) and waaA mutants exhibited highly attenuated growth, whereas simultaneous deletion of waaC and surA was lethal. Analyses of LPS of suppressor-free waaA mutants grown at 21 degrees C, besides showing accumulation of free lipid IV(A) precursor, also revealed the presence of its pentaacylated and hexaacylated derivatives, indicating in vivo late acylation can occur without Kdo. In contrast, LPS of Delta(waaC lpxL lpxM lpxP) strains showed primarily Kdo(2)-lipid IV(A), indicating that these minimal LPS structures are sufficient to support growth of E. coli under slow-growth conditions at 21/23 degrees C. These lipid IV(A) derivatives could be modified biosynthetically by phosphoethanolamine, but not by 4-amino-4-deoxy-l-arabinose, indicating export defects of such minimal LPS. DeltawaaA and Delta(waaC lpxL lpxM lpxP) exhibited cell-division defects with a decrease in the levels of FtsZ and OMP-folding factor PpiD. These mutations led to strong constitutive additive induction of envelope responsive CpxR/A and sigma(E) signal transduction pathways. Delta(lpxL lpxM lpxP) mutant, with intact waaC, synthesized tetraacylated lipid A and constitutively incorporated a third Kdo in growth medium inducing synthesis of P-EtN and l-Ara4N. Overexpression of msbA restored growth of Delta(lpxL lpxM lpxP) under fast-growing conditions, but only partially that of the Delta(waaC lpxL lpxM lpxP) mutant. This suppression could be alleviated by overexpression of certain mutant msbA alleles or the single-copy chromosomal MsbA-498V variant in the vicinity of Walker-box II.

  10. Identification of a Maize Locus that Modulates the Hypersensitive Defense Response, Using Mutant-Assisted Gene Identification and Characterization (MAGIC)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The hypersensitive response (HR) is the most visible and arguably the most important defense response in plants, although the details of how it is controlled and executed remain patchy. In this paper a novel genetic technique called MAGIC (Mutant-Assisted Gene Identification and Characterization) i...

  11. Shank2 mutant mice display a hypersecretory response to cholera toxin.

    PubMed

    Jung, Eun Suk; Park, Joonhee; Gee, Heon Yung; Jung, Jinsei; Noh, Shin Hye; Lee, Jung-Soo; Richter, Wito; Namkung, Wan; Lee, Min Goo

    2014-04-15

    Shank2 is a PDZ (PSD-95/discs large/ZO-1)-based adaptor that has been suggested to regulate membrane transporting proteins in the brain and epithelial tissues. Here, we report that Shank2 mutant (Shank2(-/-)) mice exhibit aberrant fluid and ion transport in the intestine. Molecular characterization using epithelial tissues from Shank2(+/+) and Shank2(-/-) mice revealed that a long spliceoform of Shank2 (Shank2E) is predominantly expressed in the pancreatic, renal and intestinal epithelia. In functional assays, deletion of Shank2 increased the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent short-circuit currents by 84% (P < 0.05) and 101% (P < 0.05) in the mouse colon and rectum, respectively. Disruption of the CFTR-Shank2-phosphodiesterase 4D protein complex appeared to be mostly responsible for the changes in CFTR activities. Notably, Shank2 deletion profoundly increased cholera toxin-induced fluid accumulation in the mouse intestine (∼90%, P < 0.01). Analyses with chemical inhibitors confirmed that the hyperactivation of CFTR channel function is responsible for the increased response to cholera toxin. These results suggest that Shank2 is a key molecule that participates in epithelial homeostasis, in particular to prevent overt secretory responses caused by epithelial pathogens. PMID:24445315

  12. Mutants of Arabidopsis thaliana with Decreased Amplitude in Their Phototropic Response 1

    PubMed Central

    Khurana, Jitendra P.; Ren, Zhangling; Steinitz, Benjamin; Parks, Brian; Best, Thérèse R.; Poff, Kenneth L.

    1989-01-01

    Two mutants of Arabidopsis thaliana have been identified with decreased phototropism to 450-nanometer light. Fluence-response relationships for these strains (ZR8 and ZR19) to single and multiple flashes of light show thresholds, curve shapes, and fluence for maximum curvature in `first positive' phototropism which are the same as those of the wild type. Similarly, there is no alteration from the wild type in the kinetics of curvature or in the optimum dark period separating sequential flashes in a multiple flash regimen. In addition, in both strains, gravitropism is decreased compared to the wild type by an amount which is comparable to the decrease in phototropism. Based on reciprocal backcrosses, it appears that the alteration is due to a recessive nuclear mutation. It is suggested that ZR8 and ZR19 represent alterations in some step analogous to an amplifier, downstream of the photoreceptor pigment, and common to both phototropism and gravitropism. PMID:11537461

  13. Identification of rice ethylene-response mutants and characterization of MHZ7/OsEIN2 in distinct ethylene response and yield trait regulation.

    PubMed

    Ma, Biao; He, Si-Jie; Duan, Kai-Xuan; Yin, Cui-Cui; Chen, Hui; Yang, Chao; Xiong, Qing; Song, Qing-Xin; Lu, Xiang; Chen, Hao-Wei; Zhang, Wan-Ke; Lu, Tie-Gang; Chen, Shou-Yi; Zhang, Jin-Song

    2013-11-01

    Ethylene plays essential roles in adaptive growth of rice plants in water-saturating environment; however, ethylene signaling pathway in rice is largely unclear. In this study, we report identification and characterization of ethylene-response mutants based on the specific ethylene-response phenotypes of etiolated rice seedlings, including ethylene-inhibited root growth and ethylene-promoted coleoptile elongation, which is different from the ethylene triple-response phenotype in Arabidopsis. We establish an efficient system for screening and a set of rice mutants have been identified. Genetic analysis reveals that these mutants form eight complementation groups. All the mutants show insensitivity or reduced sensitivity to ethylene in root growth but exhibit differential responses in coleoptile growth. One mutant group mhz7 has insensitivity to ethylene in both root and coleoptile growth. We identified the corresponding gene by a map-based cloning method. MHZ7 encodes a membrane protein homologous to EIN2, a central component of ethylene signaling in Arabidopsis. Upon ethylene treatment, etiolated MHZ7-overexpressing seedlings exhibit enhanced coleoptile elongation, increased mesocotyl growth and extremely twisted short roots, featuring enhanced ethylene-response phenotypes in rice. Grain length was promoted in MHZ7-transgenic plants and 1000-grain weight was reduced in mhz7 mutants. Leaf senescent process was also affected by MHZ7 expression. Manipulation of ethylene signaling may improve adaptive growth and yield-related traits in rice.

  14. Members of a Novel Family of Mammalian Protein Kinases Complement the DNA-Negative Phenotype of a Vaccinia Virus ts Mutant Defective in the B1 Kinase

    PubMed Central

    Boyle, Kathleen A.; Traktman, Paula

    2004-01-01

    Temperature-sensitive (ts) mutants of vaccinia virus defective in the B1 kinase demonstrate a conditionally lethal defect in DNA synthesis. B1 is the prototypic member of a new family of protein kinases (vaccinia virus-related kinases, or VRK) that possess distinctive B1-like sequence features within their catalytic motifs (R. J. Nichols and P. Traktman, J. Biol. Chem., in press). Given the striking sequence similarity between B1 and the VRK enzymes, we proposed that they might share overlapping substrate specificity. We therefore sought to determine whether the human and mouse VRK1 enzymes (hVRK1 and mVRK1, respectively) could complement a B1 deficiency in vivo. Recombinant ts2 viruses expressing hVRK1, mVRK1, or wild-type B1 were able to synthesize viral DNA at high temperature, but those expressing the more distantly related human casein kinase 1α2 could not. Complementation required the enzymatic activity of hVRK1, since a catalytically inactive allele of hVRK1 was unable to confer a temperature-insensitive phenotype. Interestingly, rescue of viral DNA synthesis was not coupled to the ability to phosphorylate H5, the only virus-encoded protein shown to be a B1 substrate in vivo. Expression of hVRK1 during nonpermissive ts2 infections restored virus production and plaque formation, whereas expression of mVRK1 resulted in an intermediate level of rescue. Taken together, these observations indicate that enzymatically active cellular VRK1 kinases can perform the function(s) of B1 required for genome replication, most likely due to overlapping specificity for cellular and/or viral substrates. PMID:14747564

  15. Cell-type-specific growth restriction of vesicular stomatitis virus polR mutants is linked to defective viral polymerase function.

    PubMed

    Ostertag, Derek; Hoblitzell-Ostertag, Traci M; Perrault, Jacques

    2007-01-01

    Vesicular stomatitis virus polR mutants synthesize defective RNA replication products in vitro and display growth restriction in some cultured cells (J. L. Chuang, R. L. Jackson, and J. Perrault, Virology 229:57-67, 1997). We show here that a recombinant virus carrying the polR N protein mutation (R179H) yielded approximately 100-fold- and approximately 40-fold-lower amounts of infectious virus than the wild type in mouse L-929 and rat 3Y1 cells, respectively, but only approximately 3-fold less in hamster BHK cells. Virus genome accumulation was inhibited 6- to 10-fold in restricting cells, but transcription was not affected. No defect in encapsidation of replication products was detected, but virus protein accumulation was reduced two- to threefold in both restricting and nonrestricting cells. polR virus particles released from the latter were 5- to 10-fold less infectious than the wild type but showed no difference in protein composition. Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha) was enhanced approximately 3-fold in polR versus wild-type virus-infected L-929 cells, but neither inhibition of host gene transcription nor inhibition of double-stranded RNA (dsRNA)-activated protein kinase showed significant effects on restriction. Conditioned medium studies revealed no evidence for secretion of antiviral factors from restricting cells. We conclude that the block in polR growth is due to the combined effect of reduced genome replication and lower infectivity of released virus particles and may be due to overproduction of dsRNA. An accompanying paper (D. Ostertag, T. M. Hoblitzell-Ostertag, and J. Perrault, J. Virol. 81:503-513, 2007) provides compelling evidence for the role of dsRNA in this unique restriction phenomenon.

  16. Staphylococcus aureus avirulent mutant vaccine induces humoral and cellular immune responses on pregnant heifers.

    PubMed

    Pellegrino, M; Rodriguez, N; Vivas, A; Giraudo, J; Bogni, C

    2016-06-17

    Bovine mastitis produces economic losses, attributable to the decrease in milk production, reduced milk quality, costs of treatment and replacement of animals. A successful prophylactic vaccine against Staphylococcus aureus should elicit both humoral and cellular immune responses. In a previous report we evaluated the effectiveness of a live vaccine to protect heifers against challenge with a virulent strain. In the present study the immunological response of heifers after combined immunization schedule was investigated. In a first experimental trial, heifers were vaccinated with 3 subcutaneous doses of avirulent mutant S. aureus RC122 before calving and one intramammary dose (IMD) after calving. Antibodies concentration in blood, bactericidal effect of serum from vaccinated animals and lymphocyte proliferation was determined. The levels of total IgG, IgG1 and IgG2 in colostrum and the lymphocyte proliferation index were significantly higher in vaccinated respect to non-vaccinated group throughout the experiment. The second trial, where animals were inoculated with different vaccination schedules, was carried out to determine the effect of the IMD on the level of antibodies in blood and milk, cytokines (IL-13 and IFN-γ) concentration and milk's SCC and bacteriology. The bacterial growth of the S. aureus strains was totally inhibited at 1-3×10(6) and 1-3×10(3)cfu/ml, when the strains were mixed with pooled serum diluted 1/40. The results shown that IMD has not a significant effect on the features determinate. In conclusion, a vaccination schedule involving three SC doses before calving would be enough to stimulate antibodies production in milk without an IMD. Furthermore, the results showed a bactericidal effect of serum from vaccinated animals and this provides further evidence about serum functionality. Immune responses, humoral (antigen-specific antibodies and Th2 type cytokines) and cellular (T-lymphocyte proliferation responses and Th1 type cytokines), were

  17. A study of eukaryotic response mechanisms to atmospheric pressure cold plasma by using Saccharomyces cerevisiae single gene mutants

    SciTech Connect

    Feng Hongqing; Wang Ruixue; Sun Peng; Wu Haiyan; Liu Qi; Li Fangting; Fang Jing; Zhang Jue; Zhu Weidong

    2010-09-27

    The mechanisms of eukaryotic cell response to cold plasma are studied. A series of single gene mutants of eukaryotic model organism Saccharomyces cerevisiae are used to compare their sensitivity to plasma treatment with the wild type. We examined 12 mutants in the oxidative stress pathway and the cell cycle pathway, in which 8 are found to be hypersensitive to plasma processing. The mutated genes' roles in the two pathways are analyzed to understand the biological response mechanisms of plasma treatment. The results demonstrate that genes from both pathways are needed for the eukaryotic cells to survive the complex plasma treatment.

  18. Anisotropic compressive response of Stone-Thrower-Wales defects in graphene: A molecular dynamics study

    NASA Astrophysics Data System (ADS)

    Rajasekaran, G.; Parashar, Avinash

    2016-09-01

    The mechanical properties of graphene sheet can be tailored with the help of topological defects. In this research article, the effects of Stone-Thrower-Wales (STW) defects on the mechanical properties of graphene sheet was investigated with the help of molecular dynamics based simulations. Authors has made an attempt to analyse the stress field developed in and around the vicinity of defect due to bond reorientation and further systematic evaluation has been carried out to study the effect of these stress fields against the applied axial compressive load. The results obtained with the pristine graphene were made to compare with the available open literature and the results were reported to be in good agreement with theoretical and experimental data. It was predicted that graphene with STW defect cannot able to bear compressive strength in zigzag direction, whereas on the other hand it was predicted that graphene sheet containing STW defect can bear higher compressive load in armchair direction, which shows an anisotropic response of STW defects in graphene. From the obtained results it can be observed that orientation of STW defects and the loading direction plays an important role to alter the strength of graphene under axial compression.

  19. Correlation Between Bulk Material Defects and Spectroscopic Response in Cadmium Zinc Telluride Detectors

    NASA Technical Reports Server (NTRS)

    Parker, Bradford H.; Stahle, C. M.; Barthelmy, S. D.; Parsons, A. M.; Tueller, J.; VanSant, J. T.; Munoz, B. F.; Snodgrass, S. J.; Mullinix, R. E.

    1999-01-01

    One of the critical challenges for large area cadmium zinc telluride (CdZnTe) detector arrays is obtaining material capable of uniform imaging and spectroscopic response. Two complementary nondestructive techniques for characterizing bulk CdZnTe have been developed to identify material with a uniform response. The first technique, infrared transmission imaging, allows for rapid visualization of bulk defects. The second technique, x-ray spectral mapping, provides a map of the material spectroscopic response when it is configured as a planar detector. The two techniques have been used to develop a correlation between bulk defect type and detector performance. The correlation allows for the use of infrared imaging to rapidly develop wafer mining maps. The mining of material free of detrimental defects has the potential to dramatically increase the yield and quality of large area CdZnTe detector arrays.

  20. Classical ethylene insensitive mutants of the Arabidopsis EIN2 orthologue lack the expected 'hypernodulation' response in Lotus japonicus.

    PubMed

    Chan, Pick Kuen; Biswas, Bandana; Gresshoff, Peter M

    2013-04-01

    Three independent ethylene insensitive mutants were selected from an EMS- mutagenized population of Lotus japonicus MG-20 (Miyakojima). The mutants, called 'Enigma', were mutated in the LjEIN2a gene from Lotus chromosome 1, sharing significant homology with Arabidopsis EIN2 (ethylene-insensitive2). All three alleles showed classical ethylene insensitivity phenotypes (e.g., Triple Response), but lacked the increased nodulation phenotype commonly associated with ethylene insensitivity. Indeed, all showed a marginal reduction in nodule number per plant, a phenotype that is enigmatic to sickle, an ethylene-insensitive EIN2 mutant in Medicago truncatula. In contrast to wild type, but similar to an ETR1-1 ethylene ethylene-insensitive transgenic of L. japonicus, enigma mutants formed nodules in between the protoxylem poles, demonstrating the influence of ethylene on radial positioning. Suppression of nodule numbers by nitrate and colonisation by mycorrhizal fungi in the enigma-1 mutant were indistinguishable from the wild-type MG-20. However, reflecting endogenous ethylene feedback, the enigma-1 mutant released more than twice the wild-type amount of ethylene. enigma-1 had a moderate reduction in growth, greater root mass (and lateral root formation), delayed flowering and ripening, smaller pods and seeds. Expression analysis of ethylene-regulated genes, such as ETR1, NRL1 (neverripe-like 1), and EIL3 in shoots and roots of enigma-1 and MG-20 illustrated that the ethylene-insensitive mutation strongly affected transcriptional responses in the root. These mutants open the possibility that EIN2 in L. japonicus, a determinate nodulating legume, acts in a more complex fashion possibly through the presence of a duplicated copy of LjEIN2.

  1. The Timing of Senescence and Response to Pathogens Is Altered in the Ascorbate-Deficient Arabidopsis Mutant vitamin c-11

    PubMed Central

    Barth, Carina; Moeder, Wolfgang; Klessig, Daniel F.; Conklin, Patricia L.

    2004-01-01

    The ozone-sensitive Arabidopsis mutant vitamin c-1 (vtc1) is deficient in l-ascorbic acid (AsA) due to a mutation in GDP-Man pyrophosphorylase (Conklin et al., 1999), an enzyme involved in the AsA biosynthetic pathway (Smirnoff et al., 2001). In this study, the physiology of this AsA deficiency was initially investigated in response to biotic (virulent pathogens) stress and subsequently with regards to the onset of senescence. Infection with either virulent Pseudomonas syringae or Peronospora parasitica resulted in largely reduced bacterial and hyphal growth in the vtc1 mutant in comparison to the wild type. When vitamin c-2 (vtc2), another AsA-deficient mutant, was challenged with P. parasitica, growth of the fungus was also reduced, indicating that the two AsA-deficient mutants are more resistant to these pathogens. Induction of pathogenesis-related proteins PR-1 and PR-5 is significantly higher in vtc1 than in the wild type when challenged with virulent P. syringae. In addition, the vtc1 mutant exhibits elevated levels of some senescence-associated gene (SAG) transcripts as well as heightened salicylic acid levels. Presumably, therefore, low AsA is causing vtc1 to enter at least some stage(s) of senescence prematurely with an accompanying increase in salicylic acid levels that results in a faster induction of defense responses. PMID:15064386

  2. Systems Analysis of Adaptive Responses to MAP Kinase Pathway Blockade in BRAF Mutant Melanoma

    PubMed Central

    Capaldo, Brian J.; Roller, Devin; Axelrod, Mark J.; Koeppel, Alex F.; Petricoin, Emanuel F.; Slingluff, Craig L.; Weber, Michael J.; Mackey, Aaron J.; Gioeli, Daniel; Bekiranov, Stefan

    2015-01-01

    Fifty percent of cutaneous melanomas are driven by activated BRAFV600E, but tumors treated with RAF inhibitors, even when they respond dramatically, rapidly adapt and develop resistance. Thus, there is a pressing need to identify the major mechanisms of intrinsic and adaptive resistance and develop drug combinations that target these resistance mechanisms. In a combinatorial drug screen on a panel of 12 treatment-naïve BRAFV600E mutant melanoma cell lines of varying levels of resistance to mitogen-activated protein kinase (MAPK) pathway inhibition, we identified the combination of PLX4720, a targeted inhibitor of mutated BRaf, and lapatinib, an inhibitor of the ErbB family of receptor tyrosine kinases, as synergistically cytotoxic in the subset of cell lines that displayed the most resistance to PLX4720. To identify potential mechanisms of resistance to PLX4720 treatment and synergy with lapatinib treatment, we performed a multi-platform functional genomics analysis to profile the genome as well as the transcriptional and proteomic responses of these cell lines to treatment with PLX4720. We found modest levels of resistance correlated with the zygosity of the BRAF V600E allele and receptor tyrosine kinase (RTK) mutational status. Layered over base-line resistance was substantial upregulation of many ErbB pathway genes in response to BRaf inhibition, thus generating the vulnerability to combination with lapatinib. The transcriptional responses of ErbB pathway genes are associated with a number of transcription factors, including ETS2 and its associated cofactors that represent a convergent regulatory mechanism conferring synergistic drug susceptibility in the context of diverse mutational landscapes. PMID:26405815

  3. Lens ER-stress response during cataract development in Mip-mutant mice.

    PubMed

    Zhou, Yuefang; Bennett, Thomas M; Shiels, Alan

    2016-08-01

    Major intrinsic protein (MIP) is a functional water-channel (AQP0) that also plays a key role in establishing lens fiber cell architecture. Genetic variants of MIP have been associated with inherited and age-related forms of cataract; however, the underlying pathogenic mechanisms are unclear. Here we have used lens transcriptome profiling by microarray-hybridization and qPCR to identify pathogenic changes during cataract development in Mip-mutant (Lop/+) mice. In postnatal Lop/+ lenses (P7) 99 genes were up-regulated and 75 were down-regulated (>2-fold, p=<0.05) when compared with wild-type. A pathway analysis of up-regulated genes in the Lop/+ lens (P7) was consistent with endoplasmic reticulum (ER)-stress and activation of the unfolded protein response (UPR). The most up-regulated UPR genes (>4-fold) in the Lop/+ lens included Chac1>Ddit3>Atf3>Trib3>Xbp1 and the most down-regulated genes (>5-fold) included two anti-oxidant genes, Hspb1 and Hmox1. Lop/+ lenses were further characterized by abundant TUNEL-positive nuclei within central degenerating fiber cells, glutathione depletion, free-radical overproduction, and calpain hyper-activation. These data suggest that Lop/+ lenses undergo proteotoxic ER-stress induced cell-death resulting from prolonged activation of the Eif2ak3/Perk-Atf4-Ddit3-Chac1 branch of the UPR coupled with severe oxidative-stress.

  4. A novel AKT1 mutant amplifies an adaptive melanoma response to BRAF inhibition

    PubMed Central

    Shi, Hubing; Hong, Aayoung; Kong, Xiangju; Koya, Richard C.; Song, Chunying; Moriceau, Gatien; Hugo, Willy; Yu, Clarissa C.; Ng, Charles; Chodon, Thinle; Scolyer, Richard A.; Kefford, Richard F.; Ribas, Antoni; Long, Georgina V.; Lo, Roger S.

    2013-01-01

    BRAF inhibitor (BRAFi) therapy leads to remarkable anti-melanoma responses, but the initial tumor shrinkage is commonly incomplete, providing a nidus for subsequent disease progression. Adaptive signaling may underlie early BRAFi resistance and influence the selection pattern for genetic variants causing late, acquired resistance. We show here that BRAFi (or BRAFi+MEKi) therapy in patients frequently led to rebound p-AKT levels in their melanomas early on treatment. In cell lines, BRAFi treatment led to rebound levels of RTKs (including PDGFRβ), PIP3, pleckstrin homology domain (PHD) recruitment, and p-AKT. PTEN expression limited this BRAFi-elicited PI3K-AKT signaling, which could be rescued by introduction of a mutant AKT1 (Q79K) kown to confer acquired BRAFi resistance. Functionally, AKT1 Q79K conferred BRAFi resistance via amplifying BRAFi-elicited PI3K-AKT signaling. Additionally, MAPK pathway inhibition enhanced clonogenic growth dependency on PI3K or AKT. Thus, adaptive or genetic upregulation of AKT critically participates in melanoma survival during BRAFi therapy. PMID:24265152

  5. A positively gravitropic mutant mirrors the wild-type protonemal response in the moss Ceratodon purpureus

    NASA Technical Reports Server (NTRS)

    Wagner, T. A.; Cove, D. J.; Sack, F. D.

    1997-01-01

    Wild-type Ceratodon purpureus (Hedw.) Brid. protonemata grow up in the dark by negative gravitropism. When upright wild-type protonemata are reoriented 90 degrees, they temporarily grow down soon after reorientation ("initial reversal") and also prior to cytokinesis ("mitotic reversal"). A positively gravitropic mutant designated wrong- way response (wwr-1) has been isolated by screening ultraviolet light-mutagenized Ceratodon protonemata. Protonemata of wwr-l reoriented from the vertical to the horizontal grow down with kinetics comparable to those of the wild-type. Protonemata of wwr-1 also show initial and mitotic reversals where they temporarily grow up. Thus, the direction of gravitropism, initial reversal, and mitotic reversal are coordinated though each are opposite in wwr-1 compared to the wild-type. Normal plastid zonation is still maintained in dark-grown wwr-1 apical cells, but the plastids are more numerous and plastid sedimentation is more pronounced. In addition, wwr-1 apical cells are wider and the tips greener than in the wild-type. These data suggest that a functional WWR gene product is not necessary for the establishment of some gravitropic polarity, for gravitropism, or for the coordination of the reversals. Thus, the WWR protein may normally transduce information about cell orientation.

  6. A positively gravitropic mutant mirrors the wild-type protonemal response in the moss Ceratodon purpureus.

    PubMed

    Wagner, T A; Cove, D J; Sack, F D

    1997-06-01

    Wild-type Ceratodon purpureus (Hedw.) Brid. protonemata grow up in the dark by negative gravitropism. When upright wild-type protonemata are reoriented 90 degrees, they temporarily grow down soon after reorientation ("initial reversal") and also prior to cytokinesis ("mitotic reversal"). A positively gravitropic mutant designated wrong- way response (wwr-1) has been isolated by screening ultraviolet light-mutagenized Ceratodon protonemata. Protonemata of wwr-l reoriented from the vertical to the horizontal grow down with kinetics comparable to those of the wild-type. Protonemata of wwr-1 also show initial and mitotic reversals where they temporarily grow up. Thus, the direction of gravitropism, initial reversal, and mitotic reversal are coordinated though each are opposite in wwr-1 compared to the wild-type. Normal plastid zonation is still maintained in dark-grown wwr-1 apical cells, but the plastids are more numerous and plastid sedimentation is more pronounced. In addition, wwr-1 apical cells are wider and the tips greener than in the wild-type. These data suggest that a functional WWR gene product is not necessary for the establishment of some gravitropic polarity, for gravitropism, or for the coordination of the reversals. Thus, the WWR protein may normally transduce information about cell orientation. PMID:11541791

  7. Further phenotypic characterization of pso mutants of Saccharomyces cerevisiae with respect to DNA repair and response to oxidative stress.

    PubMed

    Pungartnik, Cristina; Picada, Jaqueline; Brendel, Martin; Henriques, João A P

    2002-03-31

    The sensitivity responses of seven pso mutants of Saccharomyces cerevisiae towards the mutagens N-nitrosodiethylamine (NDEA), 1,2:7,8-diepoxyoctane (DEO), and 8-hydroxyquinoline (8HQ) further substantiated their allocation into two distinct groups: genes PSO1 (allelic to REV3), PSO2 (SNM1), PSO4 (PRP19), and PSO5 (RAD16) constitute one group in that they are involved in repair of damaged DNA or in RNA processing whereas genes PSO6 (ERG3) and PSO7 (COX11) are related to metabolic steps protecting from oxidative stress and thus form a second group, not responsible for DNA repair. PSO3 has not yet been molecularly characterized but its pleiotropic phenotype would allow its integration into either group. The first three PSO genes of the DNA repair group and PSO3, apart from being sensitive to photo-activated psoralens, have another common phenotype: they are also involved in error-prone DNA repair. While all mutants of the DNA repair group and pso3 were sensitive to DEO and NDEA the pso6 mutant revealed WT or near WT resistance to these mutagens. As expected, the repair-proficient pso7-1 and cox11-Delta mutant alleles conferred high sensitivity to NDEA, a chemical known to be metabolized via redox cycling that yields hydroxylamine radicals and reactive oxygen species. All pso mutants exhibited some sensitivity to 8HQ and again pso7-1 and cox11-Delta conferred the highest sensitivity to this drug. Double mutant snm1-Delta cox11-Delta exhibited additivity of 8HQ and NDEA sensitivities of the single mutants, indicating that two different repair/recovery systems are involved in survival. DEO sensitivity of the double mutant was equal or less than that of the single snm1-Delta mutant. In order to determine if there was oxidative damage to nucleotide bases by these drugs we employed an established bacterial test with and without metabolic activation. After S9-mix biotransformation, NDEA and to a lesser extent 8HQ, lead to significantly higher mutagenesis in an Escherichia

  8. Integrated Transcriptomic and Metabolomic Characterization of the Low-Carbon Response Using an ndhR Mutant of Synechocystis sp. PCC 6803.

    PubMed

    Klähn, Stephan; Orf, Isabel; Schwarz, Doreen; Matthiessen, Jasper K F; Kopka, Joachim; Hess, Wolfgang R; Hagemann, Martin

    2015-11-01

    The acquisition and assimilation of inorganic carbon (Ci) represents the largest flux of inorganic matter in photosynthetic organisms; hence, this process is tightly regulated. We examined the Ci-dependent transcriptional and metabolic regulation in wild-type Synechocystis sp. PCC 6803 compared with a mutant defective in the main transcriptional repressor for Ci acquisition genes, the NAD(P)H dehydrogenase transcriptional regulator NdhR. The analysis revealed that many protein-coding transcripts that are normally repressed in the presence of high CO2 (HC) concentrations were strongly expressed in ∆ndhR, whereas other messenger RNAs were strongly down-regulated in mutant cells, suggesting a potential activating role for NdhR. A conserved NdhR-binding motif was identified in the promoters of derepressed genes. Interestingly, the expression of some NdhR-regulated genes remained further inducible under low-CO2 conditions, indicating the involvement of additional NdhR-independent Ci-regulatory mechanisms. Intriguingly, we also observed that the abundance of 52 antisense RNAs and 34 potential noncoding RNAs was affected by Ci supply, although most of these molecules were not regulated through NdhR. Thus, antisense and noncoding RNAs could contribute to NdhR-independent carbon regulation. In contrast to the transcriptome, the metabolome in ∆ndhR cells was similar to that of wild-type cells under HC conditions. This observation and the delayed metabolic responses to the low-CO2 shift in ∆ndhR, specifically the lack of transient increases in the photorespiratory pathway intermediates 2-phosphoglycolate, glycolate, and glycine, suggest that the deregulation of gene expression in the ΔndhR mutant successfully preacclimates cyanobacterial cells to lowered Ci supply under HC conditions. PMID:25630438

  9. Integrated Transcriptomic and Metabolomic Characterization of the Low-Carbon Response Using an ndhR Mutant of Synechocystis sp. PCC 6803.

    PubMed

    Klähn, Stephan; Orf, Isabel; Schwarz, Doreen; Matthiessen, Jasper K F; Kopka, Joachim; Hess, Wolfgang R; Hagemann, Martin

    2015-11-01

    The acquisition and assimilation of inorganic carbon (Ci) represents the largest flux of inorganic matter in photosynthetic organisms; hence, this process is tightly regulated. We examined the Ci-dependent transcriptional and metabolic regulation in wild-type Synechocystis sp. PCC 6803 compared with a mutant defective in the main transcriptional repressor for Ci acquisition genes, the NAD(P)H dehydrogenase transcriptional regulator NdhR. The analysis revealed that many protein-coding transcripts that are normally repressed in the presence of high CO2 (HC) concentrations were strongly expressed in ∆ndhR, whereas other messenger RNAs were strongly down-regulated in mutant cells, suggesting a potential activating role for NdhR. A conserved NdhR-binding motif was identified in the promoters of derepressed genes. Interestingly, the expression of some NdhR-regulated genes remained further inducible under low-CO2 conditions, indicating the involvement of additional NdhR-independent Ci-regulatory mechanisms. Intriguingly, we also observed that the abundance of 52 antisense RNAs and 34 potential noncoding RNAs was affected by Ci supply, although most of these molecules were not regulated through NdhR. Thus, antisense and noncoding RNAs could contribute to NdhR-independent carbon regulation. In contrast to the transcriptome, the metabolome in ∆ndhR cells was similar to that of wild-type cells under HC conditions. This observation and the delayed metabolic responses to the low-CO2 shift in ∆ndhR, specifically the lack of transient increases in the photorespiratory pathway intermediates 2-phosphoglycolate, glycolate, and glycine, suggest that the deregulation of gene expression in the ΔndhR mutant successfully preacclimates cyanobacterial cells to lowered Ci supply under HC conditions.

  10. 49 CFR 210.7 - Responsibility for noise defective railroad equipment.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 4 2014-10-01 2014-10-01 false Responsibility for noise defective railroad equipment. 210.7 Section 210.7 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION RAILROAD NOISE EMISSION...

  11. 49 CFR 210.7 - Responsibility for noise defective railroad equipment.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 4 2011-10-01 2011-10-01 false Responsibility for noise defective railroad equipment. 210.7 Section 210.7 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION RAILROAD NOISE EMISSION...

  12. 49 CFR 210.7 - Responsibility for noise defective railroad equipment.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 4 2013-10-01 2013-10-01 false Responsibility for noise defective railroad equipment. 210.7 Section 210.7 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION RAILROAD NOISE EMISSION...

  13. 49 CFR 210.7 - Responsibility for noise defective railroad equipment.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 4 2012-10-01 2012-10-01 false Responsibility for noise defective railroad equipment. 210.7 Section 210.7 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION RAILROAD NOISE EMISSION...

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

    PubMed

    Chaumeil, Myriam M; Radoul, Marina; Najac, Chloé; Eriksson, Pia; Viswanath, Pavithra; Blough, Michael D; Chesnelong, Charles; Luchman, H Artee; Cairncross, J Gregory; Ronen, Sabrina M

    2016-01-01

    Metabolic imaging of brain tumors using (13)C Magnetic Resonance Spectroscopy (MRS) of hyperpolarized [1-(13)C] 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-(13)C] lactate produced from [1-(13)C] pyruvate, and response to therapy has been associated with a drop in hyperpolarized [1-(13)C] 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 (13)C MRS of hyperpolarized [1-(13)C] 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-(13)C] lactate compared to GBM, consistent with their metabolic reprogramming. Furthermore, hyperpolarized [1-(13)C] 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. PMID:27437179

  15. Defective regulation of the immune response to tetanus toxoid in Hashimoto's disease.

    PubMed Central

    Fawcett, J; Hutton, C; Mclachlan, S M; Clark, F; Rees Smith, B

    1984-01-01

    The humoral immune response to tetanus toxoid has been studied in patients with Hashimoto's disease. Although the magnitude of the response was similar to that observed in normal subjects, the Hashimoto patients demonstrated an inability to regulate their levels of tetanus toxoid antibody. This apparent defect in the control of antibody synthesis may be an important factor in both the initiation and perpetuation of autoimmune thyroid disease. PMID:6378767

  16. Association between Interferon Response and Protective Efficacy of NS1-Truncated Mutants as Influenza Vaccine Candidates in Chickens

    PubMed Central

    Jang, Hyesun; Ngunjiri, John M.; Lee, Chang-Won

    2016-01-01

    Influenza virus mutants that encode C-terminally truncated NS1 proteins (NS1-truncated mutants) are attractive candidates for avian live attenuated influenza vaccine (LAIV) development because they are both attenuated and immunogenic in chickens. We previously showed that a high protective efficacy of NS1-truncated LAIV in chickens corresponds with induction of high levels of type I interferon (IFN) responses in chicken embryonic fibroblast cells. In this study, we investigated the relationship between induction of IFN and IFN-stimulated gene responses in vivo and the immunogenicity and protective efficacy of NS1-truncated LAIV. Our data demonstrates that accelerated antibody induction and protective efficacy of NS1-truncated LAIV correlates well with upregulation of IFN-stimulated genes. Further, through oral administration of recombinant chicken IFN alpha in drinking water, we provide direct evidence that type I IFN can promote rapid induction of adaptive immune responses and protective efficacy of influenza vaccine in chickens. PMID:27257989

  17. Association between Interferon Response and Protective Efficacy of NS1-Truncated Mutants as Influenza Vaccine Candidates in Chickens.

    PubMed

    Jang, Hyesun; Ngunjiri, John M; Lee, Chang-Won

    2016-01-01

    Influenza virus mutants that encode C-terminally truncated NS1 proteins (NS1-truncated mutants) are attractive candidates for avian live attenuated influenza vaccine (LAIV) development because they are both attenuated and immunogenic in chickens. We previously showed that a high protective efficacy of NS1-truncated LAIV in chickens corresponds with induction of high levels of type I interferon (IFN) responses in chicken embryonic fibroblast cells. In this study, we investigated the relationship between induction of IFN and IFN-stimulated gene responses in vivo and the immunogenicity and protective efficacy of NS1-truncated LAIV. Our data demonstrates that accelerated antibody induction and protective efficacy of NS1-truncated LAIV correlates well with upregulation of IFN-stimulated genes. Further, through oral administration of recombinant chicken IFN alpha in drinking water, we provide direct evidence that type I IFN can promote rapid induction of adaptive immune responses and protective efficacy of influenza vaccine in chickens.

  18. Anomalous satellite inductive peaks in alternating current response of defective carbon nanotubes

    SciTech Connect

    Hirai, Daisuke; Watanabe, Satoshi; Yamamoto, Takahiro

    2014-05-07

    AC response of defective metallic carbon nanotubes is investigated from first principles. We found that capacitive peaks appear at electron scattering states. Moreover, we show that satellite inductive peaks are seen adjacent to a main capacitive peak, which is in contrast to the conductance spectra having no satellite features. The appearance of satellite inductive peaks seems to depend on the scattering states. Our analysis with a simple resonant scattering model reveals that the origin of the satellite inductive peaks can be understood by just one parameter, i.e., the lifetime of electrons at a defect state.

  19. Defect-Induced Optoelectronic Response in Single-layer Group-VI Transition-Metal Dichalcogenides

    NASA Astrophysics Data System (ADS)

    Chow, Philippe K.

    The ever-evolving symbiosis between mankind and nanoelectronics-driven technology pushes the limits of its constituent materials, largely due to the dominance of undesirable hetero-interfacial physiochemical behavior at the few-nanometer length scale, which dominates over bulk material characteristics. Driven by such instabilities, research into two-dimensional (2D) van der Waals-layered materials (e.g. graphene, transition metal dichalcogenides (TMDCs), boron nitride), which have characteristically inert surface chemistry, has virtually exploded over the past few years. The discovery of an indirect- to direct-gap conversion in semiconducting group-VI TMDCs (e.g. MoS2) upon thinning to a single atomic layer provided the critical link between metallic and insulating 2D materials. While proof-of-concept demonstrations of single-layer TMDC-based devices for visible-range photodetection, light-emission and solar energy conversion have showed promising results, the exciting qualities are downplayed by poorly-understood defectinduced photocarrier traps, limiting the best-achieved external quantum efficiencies to approximately ~1%. This thesis explores the behavior of defects in atomically-thin TMDC layers in response to optical stimuli using a combination of steady-state photoluminescence, reflectance and Raman spectroscopy at room-temperature. By systematically varying the defect density using plasma-irradiation techniques, an unprecedented room-temperature defect-induced monolayer PL feature was discovered. High-resolution transmission electron microscopy correlated the defect-induced PL with plasma-generation of sulfur vacancy defects while reflectance measurements indicate defect-induced sub-bandgap light absorption. Excitation intensity-dependent PL measurements and exciton rate modeling further help elucidate the origin of the defect-induced PL response and highlights the role of non-radiative recombination on exciton conversion processes. The results in this

  20. Photic entrainment of Period mutant mice is predicted from their phase response curves

    PubMed Central

    Pendergast, Julie S.; Friday, Rio C.; Yamazaki, Shin

    2010-01-01

    A fundamental property of circadian clocks is that they entrain to environmental cues. The circadian genes, Period1 and Period2, are involved in entrainment of the mammalian circadian system. To investigate the roles of the Period genes in photic entrainment, we constructed phase response curves (PRC) to light pulses for C57BL/6J wild-type, Per1−/−, Per2−/−, and Per3−/− mice and tested whether the PRCs accurately predict entrainment to non-24 light-dark cycles (T-cycles) and constant light (LL). The PRCs of wild-type and Per3−/− mice are similar in shape and amplitude and have relatively large delay zones and small advance zones, resulting in successful entrainment to T26, but not T21, with similar phase angles. Per1−/− mice have a high-amplitude PRC, resulting in entrainment to a broad range of T-cycles. Per2−/− mice also entrain to a wide range of T-cycles because the advance portion of their PRC is larger than wild-types. Period aftereffects following entrainment to T-cycles were similar among all genotypes. We found that the ratio of the advance portion to the delay portion of the PRC accurately predicts the lengthening of the period of the activity rhythm in LL. Wild-type, Per1−/−, and Per3−/− mice had larger delay zones than advance zones and lengthened (>24hrs) periods in LL, while Per2−/− mice had delay and advance zones that were equal in size and no period lengthening in LL. Together, these results demonstrate that PRCs are powerful tools for predicting and understanding photic entrainment of circadian mutant mice. PMID:20826680

  1. Defective carbon nanotube-silicon heterojunctions for photodetector and chemical sensor with improved responses

    NASA Astrophysics Data System (ADS)

    Choi, Jungwook; Kim, Jongbaeg

    2015-11-01

    The direct growth and integration of defect-modulated carbon nanotubes (CNTs) on n-type silicon (Si) microstructures for high performance photodetectors and chemiresistive sensors is presented. By devising a Si microspring that is strained by the growth force of the CNTs, a vertical load from the restoring force of the microspring is perpendicularly applied against the growth direction of the CNTs. This vertical load induces the formation of defective structures in the CNTs while the CNT-Si heterojunctions are fabricated. Under the illumination of UV light, photogenerated carriers from both the CNTs and the Si can be separated at the CNT-Si heterojunctions and at the defects in the CNTs before recombination, which contributes to a high photoresponsivity of 262.3 mA W-1 and an external quantum efficiency of 91.4%. Moreover, the adsorption of chemical species can be promoted by increasing the defects in the CNTs, thereby improving the sensing responsiveness toward ethanol and NO2 vapors. Our simple and facile growth of defect-adjusted CNTs on conductive Si microstructures would be beneficial to the scalable, high throughput manufacturing of heterojunctioned devices with tunable properties and functionalities of the CNTs.

  2. Mutant human torsinA, responsible for early-onset dystonia, dominantly suppresses GTPCH expression, dopamine levels and locomotion in Drosophila melanogaster

    PubMed Central

    Wakabayashi-Ito, Noriko; Ajjuri, Rami R.; Henderson, Benjamin W.; Doherty, Olugbenga M.; Breakefield, Xandra O.; O'Donnell, Janis M.; Ito, Naoto

    2015-01-01

    Dystonia represents the third most common movement disorder in humans with over 20 genetic loci identified. TOR1A (DYT1), the gene responsible for the most common primary hereditary dystonia, encodes torsinA, an AAA ATPase family protein. Most cases of DYT1 dystonia are caused by a 3 bp (ΔGAG) deletion that results in the loss of a glutamic acid residue (ΔE302/303) in the carboxyl terminal region of torsinA. This torsinAΔE mutant protein has been speculated to act in a dominant-negative manner to decrease activity of wild type torsinA. Drosophila melanogaster has a single torsin-related gene, dtorsin. Null mutants of dtorsin exhibited locomotion defects in third instar larvae. Levels of dopamine and GTP cyclohydrolase (GTPCH) proteins were severely reduced in dtorsin-null brains. Further, the locomotion defect was rescued by the expression of human torsinA or feeding with dopamine. Here, we demonstrate that human torsinAΔE dominantly inhibited locomotion in larvae and adults when expressed in neurons using a pan-neuronal promoter Elav. Dopamine and tetrahydrobiopterin (BH4) levels were significantly reduced in larval brains and the expression level of GTPCH protein was severely impaired in adult and larval brains. When human torsinA and torsinAΔE were co-expressed in neurons in dtorsin-null larvae and adults, the locomotion rates and the expression levels of GTPCH protein were severely reduced. These results support the hypothesis that torsinAΔE inhibits wild type torsinA activity. Similarly, neuronal expression of a Drosophila DtorsinΔE equivalent mutation dominantly inhibited larval locomotion and GTPCH protein expression. These results indicate that both torsinAΔE and DtorsinΔE act in a dominant-negative manner. We also demonstrate that Dtorsin regulates GTPCH expression at the post-transcriptional level. This Drosophila model of DYT1 dystonia provides an important tool for studying the differences in the molecular function between the wild type and the

  3. Response of BRAF mutant melanoma to BRAF inhibition is mediated by a network of transcriptional regulators of glycolysis

    PubMed Central

    Parmenter, Tiffany J.; Kleinschmidt, Margarete; Kinross, Kathryn M.; Bond, Simon T.; Li, Jason; Kaadige, Mohan R.; Rao, Aparna; Sheppard, Karen E.; Hugo, Willy; Pupo, Gulietta M.; Pearson, Richard B.; McGee, Sean L.; Long, Georgina V.; Scolyer, Richard A.; Rizos, Helen; Lo, Roger S.; Cullinane, Carleen; Ayer, Donald E.; Ribas, Antoni; Johnstone, Ricky W.; Hicks, Rodney J.; McArthur, Grant A.

    2014-01-01

    Deregulated glucose metabolism fulfils the energetic and biosynthetic requirements for tumour growth driven by oncogenes. Because inhibition of oncogenic BRAF causes profound reductions in glucose uptake and a strong clinical benefit in BRAF mutant melanoma, we examined the role of energy metabolism in responses to BRAF inhibition. We observed pronounced and consistent decreases in glycolytic activity in BRAF mutant melanoma cells. Moreover, we identified a network of BRAF-regulated transcription factors that control glycolysis in melanoma cells. Remarkably, this network of transcription factors, including HIF1α, c-Myc and MondoA, drives glycolysis downstream of BRAFV600, is critical for responses to BRAF inhibition and is modulated by BRAF inhibition in clinical melanoma specimens. Furthermore, we show that concurrent inhibition of BRAF and glycolysis induces cell death in BRAF inhibitor-resistant melanoma cells. Thus, we provide a proof of principle for treatment of melanoma with combinations of BRAF inhibitors and glycolysis inhibitors. PMID:24469106

  4. Diabetic pdx1-mutant zebrafish show conserved responses to nutrient overload and anti-glycemic treatment.

    PubMed

    Kimmel, Robin A; Dobler, Stefan; Schmitner, Nicole; Walsen, Tanja; Freudenblum, Julia; Meyer, Dirk

    2015-01-01

    Diabetes mellitus is characterized by disrupted glucose homeostasis due to loss or dysfunction of insulin-producing beta cells. In this work, we characterize pancreatic islet development and function in zebrafish mutant for pdx1, a gene which in humans is linked to genetic forms of diabetes and is associated with increased susceptibility to Type 2 diabetes. Pdx1 mutant zebrafish have the key diabetic features of reduced beta cells, decreased insulin and elevated glucose. The hyperglycemia responds to pharmacologic anti-diabetic treatment and, as often seen in mammalian diabetes models, beta cells of pdx1 mutants show sensitivity to nutrient overload. This unique genetic model of diabetes provides a new tool for elucidating the mechanisms behind hyperglycemic pathologies and will allow the testing of novel therapeutic interventions in a model organism that is amenable to high-throughput approaches. PMID:26384018

  5. Diabetic pdx1-mutant zebrafish show conserved responses to nutrient overload and anti-glycemic treatment

    PubMed Central

    Kimmel, Robin A.; Dobler, Stefan; Schmitner, Nicole; Walsen, Tanja; Freudenblum, Julia; Meyer, Dirk

    2015-01-01

    Diabetes mellitus is characterized by disrupted glucose homeostasis due to loss or dysfunction of insulin-producing beta cells. In this work, we characterize pancreatic islet development and function in zebrafish mutant for pdx1, a gene which in humans is linked to genetic forms of diabetes and is associated with increased susceptibility to Type 2 diabetes. Pdx1 mutant zebrafish have the key diabetic features of reduced beta cells, decreased insulin and elevated glucose. The hyperglycemia responds to pharmacologic anti-diabetic treatment and, as often seen in mammalian diabetes models, beta cells of pdx1 mutants show sensitivity to nutrient overload. This unique genetic model of diabetes provides a new tool for elucidating the mechanisms behind hyperglycemic pathologies and will allow the testing of novel therapeutic interventions in a model organism that is amenable to high-throughput approaches. PMID:26384018

  6. Survival and SOS response induction in ultraviolet B irradiated Escherichia coli cells with defective repair mechanisms.

    PubMed

    Prada Medina, Cesar Augusto; Aristizabal Tessmer, Elke Tatjana; Quintero Ruiz, Nathalia; Serment-Guerrero, Jorge; Fuentes, Jorge Luis

    2016-06-01

    Purpose In this paper, the contribution of different genes involved in DNA repair for both survival and SOS induction in Escherichia coli mutants exposed to ultraviolet B radiation (UVB, [wavelength range 280-315 nm]) was evaluated. Materials and methods E. coli strains defective in uvrA, oxyR, recO, recN, recJ, exoX, recB, recD or xonA genes were used to determine cell survival. All strains also had the genetic sulA::lacZ fusion, which allowed for the quantification of SOS induction through the SOS Chromotest. Results Five gene products were particularly important for survival, as follows: UvrA > RecB > RecO > RecJ > XonA. Strains defective in uvrA and recJ genes showed elevated SOS induction compared with the wild type, which remained stable for up to 240 min after UVB-irradiation. In addition, E. coli strains carrying the recO or recN mutation showed no SOS induction. Conclusions The nucleotide excision and DNA recombination pathways were equally used to repair UVB-induced DNA damage in E. coli cells. The sulA gene was not turned off in strains defective in UvrA and RecJ. RecO protein was essential for processing DNA damage prior to SOS induction. In this study, the roles of DNA repair proteins and their contributions to the mechanisms that induce SOS genes in E. coli are proposed. PMID:26967458

  7. Survival and SOS response induction in ultraviolet B irradiated Escherichia coli cells with defective repair mechanisms.

    PubMed

    Prada Medina, Cesar Augusto; Aristizabal Tessmer, Elke Tatjana; Quintero Ruiz, Nathalia; Serment-Guerrero, Jorge; Fuentes, Jorge Luis

    2016-06-01

    Purpose In this paper, the contribution of different genes involved in DNA repair for both survival and SOS induction in Escherichia coli mutants exposed to ultraviolet B radiation (UVB, [wavelength range 280-315 nm]) was evaluated. Materials and methods E. coli strains defective in uvrA, oxyR, recO, recN, recJ, exoX, recB, recD or xonA genes were used to determine cell survival. All strains also had the genetic sulA::lacZ fusion, which allowed for the quantification of SOS induction through the SOS Chromotest. Results Five gene products were particularly important for survival, as follows: UvrA > RecB > RecO > RecJ > XonA. Strains defective in uvrA and recJ genes showed elevated SOS induction compared with the wild type, which remained stable for up to 240 min after UVB-irradiation. In addition, E. coli strains carrying the recO or recN mutation showed no SOS induction. Conclusions The nucleotide excision and DNA recombination pathways were equally used to repair UVB-induced DNA damage in E. coli cells. The sulA gene was not turned off in strains defective in UvrA and RecJ. RecO protein was essential for processing DNA damage prior to SOS induction. In this study, the roles of DNA repair proteins and their contributions to the mechanisms that induce SOS genes in E. coli are proposed.

  8. Abiraterone Treatment in Castration-Resistant Prostate Cancer Selects for Progesterone Responsive Mutant Androgen Receptors

    PubMed Central

    Chen, Eddy J.; Sowalsky, Adam G.; Gao, Shuai; Cai, Changmeng; Voznesensky, Olga; Schaefer, Rachel; Loda, Massimo; True, Lawrence D.; Ye, Huihui; Troncoso, Patricia; Lis, Rosina L.; Kantoff, Philip W.; Montgomery, Robert B.; Nelson, Peter S.; Bubley, Glenn J.; Balk, Steven P.; Taplin, Mary-Ellen

    2014-01-01

    Purpose The CYP17A1 inhibitor abiraterone markedly reduces androgen precursors and is thereby effective in castration-resistant prostate cancer (CRPC). However, abiraterone increases progesterone, which can activate certain mutant androgen receptors (ARs) identified previously in flutamide-resistant tumors. Therefore, we sought to determine if CYP17A1 inhibitor treatment selects for progesterone activated mutant ARs. Experimental Design AR was examined by targeted sequencing in metastatic tumor biopsies from 18 CRPC patients who were progressing on a CYP17A1 inhibitor (17 on abiraterone, 1 on ketoconazole), alone or in combination with dutasteride, and by whole exome sequencing in residual tumor in one patient treated with neoadjuvant leuprolide plus abiraterone. Results The progesterone-activated T878A mutant AR was present at high allele frequency in 3 of the 18 CRPC cases. It was also present in one focus of resistant tumor in the neoadjuvant treated patient, but not in a second clonally related resistant focus which instead had lost one copy of PTEN and both copies of CHD1. The T878A mutation appeared to be less common in the subset of CRPC patients treated with abiraterone plus dutasteride, and transfection studies showed that dutasteride was a more potent direct antagonist of the T878A versus the wildtype AR. Conclusions These findings indicate that selection for tumor cells expressing progesterone-activated mutant ARs is a mechanism of resistance to CYP17A1 inhibition. PMID:25320358

  9. Clinical response to chemotherapy in oesophageal adenocarcinoma patients is linked to defects in mitochondria.

    PubMed

    Aichler, Michaela; Elsner, Mareike; Ludyga, Natalie; Feuchtinger, Annette; Zangen, Verena; Maier, Stefan K; Balluff, Benjamin; Schöne, Cédrik; Hierber, Ludwig; Braselmann, Herbert; Meding, Stephan; Rauser, Sandra; Zischka, Hans; Aubele, Michaela; Schmitt, Manfred; Feith, Marcus; Hauck, Stefanie M; Ueffing, Marius; Langer, Rupert; Kuster, Bernhard; Zitzelsberger, Horst; Höfler, Heinz; Walch, Axel K

    2013-08-01

    Chemotherapeutic drugs kill cancer cells, but it is unclear why this happens in responding patients but not in non-responders. Proteomic profiles of patients with oesophageal adenocarcinoma may be helpful in predicting response and selecting more effective treatment strategies. In this study, pretherapeutic oesophageal adenocarcinoma biopsies were analysed for proteomic changes associated with response to chemotherapy by MALDI imaging mass spectrometry. Resulting candidate proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and investigated for functional relevance in vitro. Clinical impact was validated in pretherapeutic biopsies from an independent patient cohort. Studies on the incidence of these defects in other solid tumours were included. We discovered that clinical response to cisplatin correlated with pre-existing defects in the mitochondrial respiratory chain complexes of cancer cells, caused by loss of specific cytochrome c oxidase (COX) subunits. Knockdown of a COX protein altered chemosensitivity in vitro, increasing the propensity of cancer cells to undergo cell death following cisplatin treatment. In an independent validation, patients with reduced COX protein expression prior to treatment exhibited favourable clinical outcomes to chemotherapy, whereas tumours with unchanged COX expression were chemoresistant. In conclusion, previously undiscovered pre-existing defects in mitochondrial respiratory complexes cause cancer cells to become chemosensitive: mitochondrial defects lower the cells' threshold for undergoing cell death in response to cisplatin. By contrast, cancer cells with intact mitochondrial respiratory complexes are chemoresistant and have a high threshold for cisplatin-induced cell death. This connection between mitochondrial respiration and chemosensitivity is relevant to anticancer therapeutics that target the mitochondrial electron transport chain.

  10. Growth and sporulation defects in Bacillus subtilis mutants with a single rrn operon can be suppressed by amplification of the rrn operon.

    PubMed

    Yano, Koichi; Masuda, Kenta; Akanuma, Genki; Wada, Tetsuya; Matsumoto, Takashi; Shiwa, Yuh; Ishige, Taichiro; Yoshikawa, Hirofumi; Niki, Hironori; Inaoka, Takashi; Kawamura, Fujio

    2016-01-01

    The genome of Bacillus subtilis strain 168 encodes ten rRNA (rrn) operons. We previously reported that strains with only a single rrn operon had a decreased growth and sporulation frequency. We report here the isolation and characterization of suppressor mutants from seven strains that each have a single rrn operon (rrnO, A, J, I, E, D or B). The suppressor mutants for strain RIK656 with a single rrnO operon had a higher frequency of larger colonies. These suppressor mutants had not only increased growth rates, but also increased sporulation frequencies and ribosome levels compared to the parental mutant strain RIK656. Quantitative PCR analyses showed that all these suppressor mutants had an increased number of copies of the rrnO operon. Suppressor mutants were also isolated from the six other strains with single rrn operons (rrnA, J, I, E, D or B). Next generation and capillary sequencing showed that all of the suppressor mutants had tandem repeats of the chromosomal locus containing the remaining rrn operon (amplicon). These amplicons varied in size from approximately 9 to 179 kb. The amplifications were likely to be initiated by illegitimate recombination between non- or micro-homologous sequences, followed by unequal crossing-over during DNA replication. These results are consistent with our previous report that rrn operon copy number has a major role in cellular processes such as cell growth and sporulation.

  11. Alterations of cellular physiology in Escherichia coli in response to oxidative phosphorylation impaired by defective F1-ATPase.

    PubMed

    Noda, Sakiko; Takezawa, Yuji; Mizutani, Tomohiko; Asakura, Tomoaki; Nishiumi, Eiichiro; Onoe, Kazunori; Wada, Masaru; Tomita, Fusao; Matsushita, Kazunobu; Yokota, Atsushi

    2006-10-01

    The physiological changes in an F1-ATPase-defective mutant of Escherichia coli W1485 growing in a glucose-limited chemostat included a decreased growth yield (60%) and increased specific rates of both glucose consumption (168%) and respiration (171%). Flux analysis revealed that the mutant showed approximately twice as much flow in glycolysis but only an 18% increase in the tricarboxylic acid (TCA) cycle, owing to the excretion of acetate, where most of the increased glycolytic flux was directed. Genetic and biochemical analyses of the mutant revealed the downregulation of many TCA cycle enzymes, including citrate synthase, and the upregulation of the pyruvate dehydrogenase complex in both transcription and enzyme activities. These changes seemed to contribute to acetate excretion in the mutant. No transcriptional changes were observed in the glycolytic enzymes, despite the enhanced glycolysis. The most significant alterations were found in the respiratory-chain components. The total activity of NADH dehydrogenases (NDHs) and terminal oxidases increased about twofold in the mutant, which accounted for its higher respiration rate. These changes arose primarily from the increased (3.7-fold) enzyme activity of NDH-2 and an increased amount of cytochrome bd in the mutant. Transcriptional upregulation appeared to be involved in these phenomena. As NDH-2 cannot generate an electrochemical gradient of protons and as cytochrome bd is inferior to cytochrome bo3 in this ability, the mutant was able to recycle NADH at a higher rate than the parent and avoid generating an excess proton-motive force. We discuss the physiological benefits of the alterations in the mutant.

  12. Skin microbiome imbalance in patients with STAT1/STAT3 defects impairs innate host defense responses

    PubMed Central

    Smeekens, Sanne P.; Huttenhower, Curtis; Riza, Anca; van de Veerdonk, Frank; Zeeuwen, Patrick L.J.M.; Schalkwijk, Joost; van der Meer, Jos W.M.; Xavier, Ramnik J.; Netea, Mihai G.; Gevers, Dirk

    2014-01-01

    Background Chronic mucocutaneous candidiasis (CMC) and hyper IgE syndrome (HIES) are primary immunodeficiencies mainly caused by mutations in STAT1 and STAT3, respectively. CMC and HIES patients have an increased risk for skin and mucosal infections with fungal pathogens and Staphylococcus aureus. However, it is unknown whether the genetic defects in these patients also affect the skin and mucosal microbiome, which in turn may influence host defense mechanisms. Methods The skin and oral microbiome of CMC and HIES patients was compared to that of healthy controls at five body sites using 16S rRNA sequencing. The influence of skin colonizers on the immune response was investigated using in-vitro experiments. Results The microbiome of CMC and HIES patients contained more Gram-negative bacteria, especially Acinetobacter spp, and less of the normal Corynebacterium spp., compared to healthy controls. Exposure of human primary leukocytes to Acinetobacter suppressed the cytokine response to C. albicans and S. aureus, while the normal Corynebacteria did not suppress cytokine responses. Discussion These results demonstrate that central mediators of immune responses like STAT1 and STAT3 not only directly influence immune responses, but also result in changes of the skin microbiome that in turn can amplify the defective immune response against fungal and microbial pathogens. PMID:23796786

  13. Mechanical touch responses of Arabidopsis TCH1-3 mutant roots on inclined hard-agar surface

    NASA Astrophysics Data System (ADS)

    Zha, Guodong; Wang, Bochu; Liu, Junyu; Yan, Jie; Zhu, Liqing; Yang, Xingyan

    2016-01-01

    The gravity-induced mechanical touch stimulus can affect plant root architecture. Mechanical touch responses of plant roots are an important aspect of plant root growth and development. Previous studies have reported that Arabidopsis TCH1-3 genes are involved in mechano-related events, how-ever, the physiological functions of TCH1-3 genes in Arabidopsis root mechanoresponses remain unclear. In the present study, we applied an inclined hard agar plate method to produce mechanical touch stimulus, and provided evidence that altered mechanical environment could influence root growth. Furthermore, tch1-3 Arabidopsis mutants were investigated on inclined agar surfaces to explore the functions of TCH1-3 genes on Arabidopsis root mechanoresponses. The results showed that two tch2 mutants, cml24-2 and cml24-4, exhibited significantly reduced root length, biased skewing, and decreased density of lateral root. In addition, primary root length and density of lateral root of tch3 (cml12-2) was significantly decreased on inclined agar surfaces. This study indicates that the tch2 and tch3 mutants are hypersensitive to mechanical touch stimulus, and TCH2 (CML24-2 and CML24-4) and TCH3 (CML12-2) genes may participate in the mechanical touch response of Arabidopsis roots.

  14. Functional analyses of cotton (Gossypium hirsutum L.) immature fiber (im) mutant infer that fiber cell wall development is associated with stress responses

    PubMed Central

    2013-01-01

    Background Cotton fiber maturity is an important factor for determining the commercial value of cotton. How fiber cell wall development affects fiber maturity is not well understood. A comparison of fiber cross-sections showed that an immature fiber (im) mutant had lower fiber maturity than its near isogenic wild type, Texas marker-1 (TM-1). The availability of the im mutant and TM-1 provides a unique way to determine molecular mechanisms regulating cotton fiber maturity. Results Transcriptome analysis showed that the differentially expressed genes (DEGs) in the im mutant fibers grown under normal stress conditions were similar to those in wild type cotton fibers grown under severe stress conditions. The majority of these DEGs in the im mutant were related to stress responses and cellular respiration. Stress is known to reduce the activity of a classical respiration pathway responsible for energy production and reactive oxygen species (ROS) accumulation. Both energy productions and ROS levels in the im mutant fibers are expected to be reduced if the im mutant is associated with stress responses. In accord with the prediction, the transcriptome profiles of the im mutant showed the same alteration of transcriptional regulation that happened in energy deprived plants in which expressions of genes associated with cell growth processes were reduced whereas expressions of genes associated with recycling and transporting processes were elevated. We confirmed that ROS production in developing fibers from the im mutant was lower than that from the wild type. The lower production of ROS in the im mutant fibers might result from the elevated levels of alternative respiration induced by stress. Conclusion The low degree of fiber cell wall thickness of the im mutant fibers is associated with deregulation of the genes involved in stress responses and cellular respiration. The reduction of ROS levels and up-regulation of the genes involved in alternative respirations suggest that

  15. Identification of sonic hedgehog as a candidate gene responsible for the polydactylous mouse mutant Sasquatch.

    PubMed

    Sharpe, J; Lettice, L; Hecksher-Sorensen, J; Fox, M; Hill, R; Krumlauf, R

    1999-01-28

    The mouse mutants of the hemimelia-luxate group (lx, lu, lst, Dh, Xt, and the more recently identified Hx, Xpl and Rim4; [1] [2] [3] [4] [5]) have in common preaxial polydactyly and longbone abnormalities. Associated with the duplication of digits are changes in the regulation of development of the anterior limb bud resulting in ectopic expression of signalling components such as Sonic hedgehog (Shh) and fibroblast growth factor-4 (Fgf4), but little is known about the molecular causes of this misregulation. We generated, by a transgene insertion event, a new member of this group of mutants, Sasquatch (Ssq), which disrupted aspects of both anteroposterior (AP) and dorsoventral (DV) patterning. The mutant displayed preaxial polydactyly in the hindlimbs of heterozygous embryos, and in both hindlimbs and forelimbs of homozygotes. The Shh, Fgf4, Fgf8, Hoxd12 and Hoxd13 genes were all ectopically expressed in the anterior region of affected limb buds. The insertion site was found to lie close to the Shh locus. Furthermore, expression from the transgene reporter has come under the control of a regulatory element that directs a pattern mirroring the endogenous expression pattern of Shh in limbs. In abnormal limbs, both Shh and the reporter were ectopically induced in the anterior region, whereas in normal limbs the reporter and Shh were restricted to the zone of polarising activity (ZPA). These data strongly suggest that Ssq is caused by direct interference with the cis regulation of the Shh gene.

  16. α-Mangostin, a Natural Agent, Enhances the Response of NRAS Mutant Melanoma to Retinoic Acid

    PubMed Central

    Xia, Yun; Chen, Jing; Gong, Chongwen; Chen, Hongxiang; Sun, Jiaming

    2016-01-01

    Background The identification and use of novel compounds alone or in combination hold promise for the fight against NRAS mutant melanoma. Material/Methods We screened a kinase-specific inhibitor library through combining it with α-Mangostin in NRAS mutant melanoma cell line, and verified the enhancing effect of α-Mangostin through inhibition of the tumorigenesis pathway. Results Within the kinase inhibitors, retinoic acid showed a significant synergistic effect with α-Mangostin. α-Mangostin also can reverse the drug resistance of retinoic acid in RARa siRNA-transduced sk-mel-2 cells. Colony assay, TUNEL staining, and the expressions of several apoptosis-related genes revealed that α-Mangostin enhanced the effect of retinoic acid-induced apoptosis. The combination treatment resulted in marked induction of ROS generation and inhibition of the AKT/S6 pathway. Conclusions These results indicate that the combination of these novel natural agents with retinoid acid may be clinically effective in NRAS mutant melanoma. PMID:27104669

  17. AarF Domain Containing Kinase 3 (ADCK3) Mutant Cells Display Signs of Oxidative Stress, Defects in Mitochondrial Homeostasis and Lysosomal Accumulation

    PubMed Central

    Cullen, Jason K.; Abdul Murad, Norazian; Yeo, Abrey; McKenzie, Matthew; Ward, Micheal; Chong, Kok Leong; Schieber, Nicole L.; Parton, Robert G.; Lim, Yi Chieh; Wolvetang, Ernst; Maghzal, Ghassan J.; Stocker, Roland; Lavin, Martin F.

    2016-01-01

    Autosomal recessive ataxias are a clinically diverse group of syndromes that in some cases are caused by mutations in genes with roles in the DNA damage response, transcriptional regulation or mitochondrial function. One of these ataxias, known as Autosomal Recessive Cerebellar Ataxia Type-2 (ARCA-2, also known as SCAR9/COQ10D4; OMIM: #612016), arises due to mutations in the ADCK3 gene. The product of this gene (ADCK3) is an atypical kinase that is thought to play a regulatory role in coenzyme Q10 (CoQ10) biosynthesis. Although much work has been performed on the S. cerevisiae orthologue of ADCK3, the cellular and biochemical role of its mammalian counterpart, and why mutations in this gene lead to human disease is poorly understood. Here, we demonstrate that ADCK3 localises to mitochondrial cristae and is targeted to this organelle via the presence of an N-terminal localisation signal. Consistent with a role in CoQ10 biosynthesis, ADCK3 deficiency decreased cellular CoQ10 content. In addition, endogenous ADCK3 was found to associate in vitro with recombinant Coq3, Coq5, Coq7 and Coq9, components of the CoQ10 biosynthetic machinery. Furthermore, cell lines derived from ARCA-2 patients display signs of oxidative stress, defects in mitochondrial homeostasis and increases in lysosomal content. Together, these data shed light on the possible molecular role of ADCK3 and provide insight into the cellular pathways affected in ARCA-2 patients. PMID:26866375

  18. Defects in CVD Diamond Films from Their Response as Nuclear Detectors

    NASA Astrophysics Data System (ADS)

    Marinelli, Marco; Milani, Enrico; Tucciarone, Aldo; Rinati, Gianluca Verona

    CVD diamond films can be used to realize nuclear detectors with outstanding working capability in harsh environments. Since efficient particle detection requires high drift lengths of the carriers produced by the ionizing particle, the presence of defects severely limits the performance of these detectors. This is a major issue because the fabrication technology of CVD diamond is much less advanced than that of more conventional materials like silicon. The different kinds of defects in CVD diamond and their influence on the detector response are discussed. The connections between the microscopic structure of CVD diamond and the priming (or pumping) effect, which is widely used to increase CVD diamond detector performance, are elucidated. The analysis of the response of CVD diamond-based detectors is used to extract qualitative and quantitative information on the properties of defects limiting the free movement of charge carriers in the detector (e.g., carrier type for which the traps are active, activation energies, geometrical distribution in the film, etc.). Milani-begin

  19. phoP, SPI1, SPI2 and aroA mutants of Salmonella Enteritidis induce a different immune response in chickens.

    PubMed

    Elsheimer-Matulova, Marta; Varmuzova, Karolina; Kyrova, Kamila; Havlickova, Hana; Sisak, Frantisek; Rahman, Masudur; Rychlik, Ivan

    2015-01-01

    Poultry is the most frequent reservoir of non-typhoid Salmonella enterica for humans. Understanding the interactions between chickens and S. enterica is therefore important for vaccine design and subsequent decrease in the incidence of human salmonellosis. In this study we therefore characterized the interactions between chickens and phoP, aroA, SPI1 and SPI2 mutants of S. Enteritidis. First we tested the response of HD11 chicken macrophage-like cell line to S. Enteritidis infection monitoring the transcription of 36 genes related to immune response. All the mutants and the wild type strain induced inflammatory signaling in the HD11 cell line though the response to SPI1 mutant infection was different from the rest of the mutants. When newly hatched chickens were inoculated, the phoP as well as the SPI1 mutant did not induce an expression of any of the tested genes in the cecum. Despite this, such chickens were protected against challenge with wild-type S. Enteritidis. On the other hand, inoculation of chickens with the aroA or SPI2 mutant induced expression of 27 and 18 genes, respectively, including genes encoding immunoglobulins. Challenge of chickens inoculated with these two mutants resulted in repeated induction of 11 and 13 tested genes, respectively, including the genes encoding immunoglobulins. In conclusion, SPI1 and phoP mutants induced protective immunity without inducing an inflammatory response and antibody production. Inoculation of chickens with the SPI2 and aroA mutants also led to protective immunity but was associated with inflammation and antibody production. The differences in interaction between the mutants and chicken host can be used for a more detailed understanding of the chicken immune system.

  20. Influenza virus-specific RNA and protein syntheses in cells infected with temperature-sensitive mutants defective in the genome segment encoding nonstructural proteins.

    PubMed

    Wolstenholme, A J; Barrett, T; Nichol, S T; Mahy, B W

    1980-07-01

    Virus-specific protein and RNA syntheses have been analyzed in chicken embryo fibroblast cells infected with two group IV temperature-sensitive (ts) mutants of influenza A (fowl plague) virus in which the ts lesion maps in RNA segment 8 (J. W. Almond, D. McGeoch, and R. D. Barry, Virology 92:416-427, 1979), known to code to code for two nonstructural proteins, NS1 and NS2. Both mutants induced the synthesis of similar amounts of all the early virus-specific proteins (P1, P2, P3, NP, and NS1) at temperatures that were either permissive (34 degrees C) or nonpermissive (40.5 degrees C) for replication. However, the synthesis of M protein, which normally accumulates late in infection, was greatly reduced in ts mutant-infected cells at 40.5 degrees C compared to 34 degrees C. The NS2 protein was not detected at either temperature in cells infected with one mutant (mN3), and was detected only at the permissive temperature in cells infected with mutant ts47. There was no overall reduction in polyadenylated (A+) complementary RNA, which functions as mRNA, in cells infected with these mutants at 40.5 degrees C compared to 34 degrees C, nor was there any evidence of selective accumulation of this type of RNA within the nucleus at the nonpermissive temperature. No significant differences in ts mutant virion RNA transcriptase activity were detected by assays in vitro at 31 and 40.5 degrees C compared to wild-type virus. Virus-specific non-polyadenylated (A-) complementary RNA, which is believed to act as the template for new virion RNA production, accumulated normally in cells at both 34 and 40.5 degrees C, but at 40.5 degrees C accumulation of new virion RNA was reduced by greater than 90% when compared to accumulation at 34 degrees C.

  1. Isolation of the GA-response mutant sly1 as a suppressor of ABI1-1 in Arabidopsis thaliana.

    PubMed Central

    Steber, C M; Cooney, S E; McCourt, P

    1998-01-01

    Seed dormancy and germination in higher plants are partially controlled by the plant hormones abscisic acid (ABA) and gibberellic acid (GA). ABA establishes dormancy during embryo maturation, whereas GA breaks dormancy and induces germination. Previous attempts to identify GA response genes were confounded because GA mutants are not expected to germinate and, unlike GA auxotrophs, should fail to be rescued by exogenous GA. Here, we describe a screen for suppressors of the ABA-insensitive mutant ABI1-1 that enriches for GA auxotrophs and GA-insensitive mutants. The vast majority (76%) of the suppressors of ABI1-1 strongly resemble GA auxotrophs in that they are severely dwarfed and have dark green foliage and flowers with underdeveloped petals and stamen. Three isolates were alleles of the GA auxotroph ga1. The remaining severe dwarves were not rescued by GA and belong to a single complementation group that we designate sly1 (Sleepy 1). The alleles of sly1 identified are the first recessive GA-insensitive mutations to reflect the full spectrum of GA-associated phenotypes, including the failure to germinate in the absence of the ABI1-1 lesion. Thus, we postulate that SLY1 is a key factor in GA reception. PMID:9611170

  2. A vaccine based on a mutant transferrin binding protein B of Haemophilus parasuis induces a strong T-helper 2 response and bacterial clearance after experimental infection.

    PubMed

    Martínez-Martínez, Sonia; Frandoloso, Rafael; Rodríguez-Ferri, Elías-Fernando; García-Iglesias, María-José; Pérez-Martínez, Claudia; Álvarez-Estrada, Álvaro; Gutiérrez-Martín, César-Bernardo

    2016-10-15

    This study aimed to characterize the type of immune response induced by an experimental vaccine based on a mutant Haemophilus parasuis transferrin binding protein (Tbp) B (Y167A) defective in its ability to bind porcine transferrin. Clinical and pathological signs, bacterial clearance, antibody response and the cytokine profile in alveolar macrophages and spleen after the vaccination and challenge of twenty-two colostrum-deprived pigs with 10(8) CFU of H. parasuis were analysed. Pigs vaccinated with Y167A were compared to those vaccinated with native TbpB (nTbpB), those treated with a commercial bacterin (CB) against Glässer's disease, those unvaccinated challenged (CH) and those unvaccinated unchallenged (UNCH) pigs. The rectal temperatures of Y167A pigs resembled those of UNCH pigs and were significantly lower than those of the nTbpB, CB and CH animals. A major reduction in pathological changes of the challenged pigs was observed in the Y167A group. H. parasuis was cleared from 88.9% of the samples from Y167A pigs versus 60.0% and 55.6% from those of the CB and nTbpB groups, respectively. The antibody response elicited by Y167A by ELISA was notably higher than that observed for nTbpB and CB pigs and was capable of preventing the expression and secretion of IL-8. The expression of IL-4 and IL-5, which were associated with the specific antibody levels, suggests that the main mechanism of protection conferred by Y167A vaccine is based on a strong T-helper 2 response. PMID:27590421

  3. Relationships of RNA polymerase II genetic interactors to transcription start site usage defects and growth in Saccharomyces cerevisiae.

    PubMed

    Jin, Huiyan; Kaplan, Craig D

    2014-11-06

    Transcription initiation by RNA Polymerase II (Pol II) is an essential step in gene expression and regulation in all organisms. Initiation requires a great number of factors, and defects in this process can be apparent in the form of altered transcription start site (TSS) selection in Saccharomyces cerevisiae (Baker's yeast). It has been shown previously that TSS selection in S. cerevisiae is altered in Pol II catalytic mutants defective in a conserved active site feature known as the trigger loop. Pol II trigger loop mutants show growth phenotypes in vivo that correlate with biochemical defects in vitro and exhibit wide-ranging genetic interactions. We assessed how Pol II mutant growth phenotypes and TSS selection in vivo are modified by Pol II genetic interactors to estimate the relationship between altered TSS selection in vivo and organismal fitness of Pol II mutants. We examined whether the magnitude of TSS selection defects could be correlated with Pol II mutant-transcription factor double mutant phenotypes. We observed broad genetic interactions among Pol II trigger loop mutants and General Transcription Factor (GTF) alleles, with reduced-activity Pol II mutants especially sensitive to defects in TFIIB. However, Pol II mutant growth defects could be uncoupled from TSS selection defects in some Pol II allele-GTF allele double mutants, whereas a number of other Pol II genetic interactors did not influence ADH1 start site selection alone or in combination with Pol II mutants. Initiation defects are likely only partially responsible for Pol II allele growth phenotypes, with some Pol II genetic interactors able to exacerbate Pol II mutant growth defects while leaving initiation at a model TSS selection promoter unaffected.

  4. The Tumor Suppressor Prostate Apoptosis Response-4 (Par-4) is Regulated by Mutant IDH1 and Kills Glioma Stem Cells

    PubMed Central

    Liu, Yinxing; Gilbert, Misty R.; Kyprianou, Natasha; Rangnekar, Vivek M.; Horbinski, Craig

    2014-01-01

    Prostate apoptosis response-4 (Par-4) is an endogenous tumor suppressor that selectively induces apoptosis in a variety of cancers. Although it has been the subject of intensive research in other cancers, less is known about its significance in gliomas, including whether it is regulated by key driver mutations, has therapeutic potential against glioma stem cells (GSCs), and/or is a prognostic marker. We found that patient-derived gliomas with mutant isocitrate dehydrogenase 1 have markedly lower Par-4 expression (P < 0.0001), which was validated by The Cancer Genome Atlas dataset (P = 2.0 E-13). The metabolic product of mutant IDH1, D-2-hydroxyglutarate (2-HG), can suppress Par-4 transcription in vitro via inhibition of promoter activity as well as enhanced mRNA degradation, but interestingly not by direct DNA promoter hypermethylation. The Selective for Apoptosis induction in Cancer cells (SAC) domain within Par-4 is highly active against glioma cells, including orthotopic xenografts of patient-derived primary GSCs (P < 0.0001). Among high-grade gliomas that are IDH1 wild-type, those that express more Par-4 have significantly longer median survival (18.4 versus 8.0 months, P = 0.002), a finding confirmed in two external GBM cohorts. Together, these data suggest that Par-4 is a significant component of the mutant IDH1 phenotype, that the activity of 2-HG is complex and can extend beyond direct DNA hypermethylation, and that Par-4 is a promising therapeutic strategy against GSCs. Furthermore, not every effect of mutant IDH1 necessarily contributes to the overall favorable prognosis seen in such tumors; inhibition of Par-4 may be one such effect. PMID:25135281

  5. Bobber: a cervical ataxic mutant in the domestic turkey responsive to sunlight.

    PubMed

    Harper, J A; Bernier, P E; Savage, T F; West, D J

    1988-01-01

    Bobber is a genetic disorder in the domestic turkey that is usually expressed between two and four weeks of age. The condition is permanent and is characterized by ventrocaudad bending of the neck accompanied by a lateral pendulumlike motion of the head between the legs. Expressivity of the defect is variable and may be exhibited in some turkeys as a stargazing posture or a rapid clockwise twirling motion. When suspended by the legs in a head-down orientation, afflicted turkeys exhibit an inward turning of the neck and head toward the breast as opposed to an outward turning in normal turkeys. The disorder is inherited as a sex-linked recessive trait. The symbol bo is used for the gene. The defect can be corrected by exposure to intense light in the visible spectrum. PMID:3392387

  6. Isolation of mammalian cell mutants that are X-ray sensitive, impaired in DNA double-strand break repair and defective for V(D)J recombination.

    PubMed

    Lee, S E; Pulaski, C R; He, D M; Benjamin, D M; Voss, M; Um, J; Hendrickson, E A

    1995-05-01

    The Chinese hamster lung V79-4 cell line was infected with a Moloney murine leukemia retrovirus and the infected cells were subsequently screened for mutants that were sensitive to X-rays using a toothpicking/96-well replica plating technique. Four independent mutants that were sensitive to X-irradiation (sxi-1 to sxi-4) were isolated from 9000 retrovirally infected colonies. A pulse-field gel electrophoresis (PFGE) assay demonstrated that all of the sxi mutants were impaired in DNA double-strand break (DSB) repair, thus providing a molecular explanation for the observed X-ray sensitivity. Interestingly, additional PFGE experiments demonstrated that for any given X-ray dose all of the mutants incurred more DNA DSBs than the parental V79-4 cell line indicating there may be some inherent fragility to sxi chromosomes. Cross-sensitivity to other DNA-damaging agents including bleomycin, mitomycin C and methyl methanesulfonate indicated that sxi-2, sxi-3 and sxi-4 appear to be specifically hypersensitive to genotoxic agents that cause DNA DSBs, whereas sxi-1 appeared to be hypersensitive to multiple types of DNA lesions. Lastly, in preliminary experiments all of the sxi mutants demonstrated an inability to carry out V(D)J recombination, a somatic DNA rearrangement process required for the assembly of lymphoid antigen receptor genes. Thus, the sxi cell lines have interesting phenotypes which should make them valuable tools for unraveling the mechanism(s) of DNA DSB repair and recombination in mammalian cells. PMID:7537861

  7. Pregnancy Hyperglycemia in Prolactin Receptor Mutant, but Not Prolactin Mutant, Mice and Feeding-Responsive Regulation of Placental Lactogen Genes Implies Placental Control of Maternal Glucose Homeostasis1

    PubMed Central

    Rawn, Saara M.; Huang, Carol; Hughes, Martha; Shaykhutdinov, Rustem; Vogel, Hans J.; Cross, James C.

    2015-01-01

    Pregnancy is often viewed as a conflict between the fetus and mother over metabolic resources. Insulin resistance occurs in mothers during pregnancy but does not normally lead to diabetes because of an increase in the number of the mother's pancreatic beta cells. In mice, this increase is dependent on prolactin (Prl) receptor signaling but the source of the ligand has been unclear. Pituitary-derived Prl is produced during the first half of pregnancy in mice but the placenta produces Prl-like hormones from implantation to term. Twenty-two separate mouse genes encode the placenta Prl-related hormones, making it challenging to assess their roles in knockout models. However, because at least four of them are thought to signal through the Prl receptor, we analyzed Prlr mutant mice and compared their phenotypes with those of Prl mutants. We found that whereas Prlr mutants develop hyperglycemia during gestation, Prl mutants do not. Serum metabolome analysis showed that Prlr mutants showed other changes consistent with diabetes. Despite the metabolic changes, fetal growth was normal in Prlr mutants. Of the four placenta-specific, Prl-related hormones that have been shown to interact with the Prlr, their gene expression localizes to different endocrine cell types. The Prl3d1 gene is expressed by trophoblast giant cells both in the labyrinth layer, sitting on the arterial side where maternal blood is highest in oxygen and nutrients, and in the junctional zone as maternal blood leaves the placenta. Expression increases during the night, though the increase in the labyrinth is circadian whereas it occurs only after feeding in the junctional zone. These data suggest that the placenta has a sophisticated endocrine system that regulates maternal glucose metabolism during pregnancy. PMID:26269505

  8. Pregnancy Hyperglycemia in Prolactin Receptor Mutant, but Not Prolactin Mutant, Mice and Feeding-Responsive Regulation of Placental Lactogen Genes Implies Placental Control of Maternal Glucose Homeostasis.

    PubMed

    Rawn, Saara M; Huang, Carol; Hughes, Martha; Shaykhutdinov, Rustem; Vogel, Hans J; Cross, James C

    2015-09-01

    Pregnancy is often viewed as a conflict between the fetus and mother over metabolic resources. Insulin resistance occurs in mothers during pregnancy but does not normally lead to diabetes because of an increase in the number of the mother's pancreatic beta cells. In mice, this increase is dependent on prolactin (Prl) receptor signaling but the source of the ligand has been unclear. Pituitary-derived Prl is produced during the first half of pregnancy in mice but the placenta produces Prl-like hormones from implantation to term. Twenty-two separate mouse genes encode the placenta Prl-related hormones, making it challenging to assess their roles in knockout models. However, because at least four of them are thought to signal through the Prl receptor, we analyzed Prlr mutant mice and compared their phenotypes with those of Prl mutants. We found that whereas Prlr mutants develop hyperglycemia during gestation, Prl mutants do not. Serum metabolome analysis showed that Prlr mutants showed other changes consistent with diabetes. Despite the metabolic changes, fetal growth was normal in Prlr mutants. Of the four placenta-specific, Prl-related hormones that have been shown to interact with the Prlr, their gene expression localizes to different endocrine cell types. The Prl3d1 gene is expressed by trophoblast giant cells both in the labyrinth layer, sitting on the arterial side where maternal blood is highest in oxygen and nutrients, and in the junctional zone as maternal blood leaves the placenta. Expression increases during the night, though the increase in the labyrinth is circadian whereas it occurs only after feeding in the junctional zone. These data suggest that the placenta has a sophisticated endocrine system that regulates maternal glucose metabolism during pregnancy.

  9. Water Stress Responses of Tomato Mutants Impaired in Hormone Biosynthesis Reveal Abscisic Acid, Jasmonic Acid and Salicylic Acid Interactions.

    PubMed

    Muñoz-Espinoza, Valeria A; López-Climent, María F; Casaretto, José A; Gómez-Cadenas, Aurelio

    2015-01-01

    To investigate the putative crosstalk between JA and ABA in Solanum lycopersicum plants in response to drought, suppressor of prosystemin-mediated responses2 (spr2, JA-deficient) and flacca (flc, ABA-deficient) mutants together with the naphthalene/salicylate hydroxylase (NahG) transgenic (SA-deficient) line were used. Hormone profiling and gene expression of key enzymes in ABA, JA and SA biosynthesis were analyzed during early stages of drought. ABA accumulation was comparable in spr2 and wild type (WT) plants whereas expression of 9-cis-epoxycarotenoid dioxygenase 1 (NCED1) and NCED2 was different, implying a compensation mechanism between NCED genes and an organ-specific regulation of NCED1 expression. JA levels and 12-oxo-phytodienoic acid reductase 3 (OPR3) expression in flc plants suggest that ABA regulates the induction of the OPR3 gene in roots. By contrast, ABA treatment to flc plants leads to a reduction of JA and SA contents. Furthermore, different pattern of SA accumulation (and expression of isochorismate synthase and phenylalanine ammonia lyase 1) was observed between WT seedlings and mutants, suggesting that SA plays an important role on the early response of tomato plants to drought and also that JA and ABA modulate its biosynthesis. Finally, hormone profiling in spr2 and NahG plants indicate a crosstalk between JA and SA that could enhance tolerance of tomato to water stress. PMID:26635826

  10. Water Stress Responses of Tomato Mutants Impaired in Hormone Biosynthesis Reveal Abscisic Acid, Jasmonic Acid and Salicylic Acid Interactions

    PubMed Central

    Muñoz-Espinoza, Valeria A.; López-Climent, María F.; Casaretto, José A.; Gómez-Cadenas, Aurelio

    2015-01-01

    To investigate the putative crosstalk between JA and ABA in Solanum lycopersicum plants in response to drought, suppressor of prosystemin-mediated responses2 (spr2, JA-deficient) and flacca (flc, ABA-deficient) mutants together with the naphthalene/salicylate hydroxylase (NahG) transgenic (SA-deficient) line were used. Hormone profiling and gene expression of key enzymes in ABA, JA and SA biosynthesis were analyzed during early stages of drought. ABA accumulation was comparable in spr2 and wild type (WT) plants whereas expression of 9-cis-epoxycarotenoid dioxygenase 1 (NCED1) and NCED2 was different, implying a compensation mechanism between NCED genes and an organ-specific regulation of NCED1 expression. JA levels and 12-oxo-phytodienoic acid reductase 3 (OPR3) expression in flc plants suggest that ABA regulates the induction of the OPR3 gene in roots. By contrast, ABA treatment to flc plants leads to a reduction of JA and SA contents. Furthermore, different pattern of SA accumulation (and expression of isochorismate synthase and phenylalanine ammonia lyase 1) was observed between WT seedlings and mutants, suggesting that SA plays an important role on the early response of tomato plants to drought and also that JA and ABA modulate its biosynthesis. Finally, hormone profiling in spr2 and NahG plants indicate a crosstalk between JA and SA that could enhance tolerance of tomato to water stress. PMID:26635826

  11. Expression of gibberellin 3 beta-hydroxylase gene in a gravi-response mutant, weeping Japanese flowering cherry

    NASA Technical Reports Server (NTRS)

    Sugano, Mami; Nakagawa, Yuriko; Nyunoya, Hiroshi; Nakamura, Teruko

    2004-01-01

    Expressions of the gibberellin biosynthesis gene were investigated in a normal upright type and a gravi-response mutant, a weeping type of Japanese flowering cherry (Prunus spachiana), that is unable to support its own weight and elongates downward. A segment of the gibberellin 3 beta-hydroxylase cDNA of Prunus spachiana (Ps3ox), which is responsible for active gibberellin synthesis, was amplified by using real-time RT-PCR. The content of Ps3ox mRNA in the weeping type was much greater than that in the upright type, while the endogenous gibberellin level was much higher in the elongating zone of the weeping type. These results suggest that the amount and distribution of synthesized gibberellin regulate secondary xylem formation, and the unbalanced distribution of gibberellin affects the gravi-response of the Prunus tree.

  12. Influence of surface defects in ZnO thin films on its biosensing response characteristic

    SciTech Connect

    Saha, Shibu; Gupta, Vinay

    2011-09-15

    Highly c-axis oriented zinc oxide (ZnO) thin films deposited by rf magnetron sputtering under varying processing pressure (20-50 mT) in a reactive gas mixture of argon and oxygen were studied for biosensing application. The as-deposited ZnO thin films were in a state of compressive stress having defects related to interstitial Zn and antisite oxygen. Glucose oxidase has been chosen as the model enzyme in the present study and was immobilized on the surface of ZnO thin films deposited on indium tin oxide coated Corning Glass substrate. The studies reveal a correlation between the biosensing characteristic and the presence of defects in the ZnO films. The ZnO films deposited under high pressure (50 mT) are found to be more sensitive for biosensing application due to availability of more surface area for effective immobilization of biomolecules and exhibits a suitable microenvironment with good electron transfer characteristic. The obtained results highlight the importance of desired microstate besides availability of suitable native defects in the ZnO thin film for exhibiting enhanced biosensing response.

  13. Anesthetic-resistant spontaneous mutant of Drosophila melanogaster: intensified response to /sup 60/Cobalt radiation damage

    SciTech Connect

    Gamo, S.; Nakashima-Tanaka, E.; Megumi, T.; Ueda, I.

    1985-02-25

    Accumulating evidence suggests that the extent of acute damage by ionizing irradiation is closely related to the state of membrane orderliness. Decreased orderliness apparently protects organisms from ionizing irradiation. Because anesthetics decrease membrane orderliness, anesthesia is expected to affect damages caused by ionizing irradiation. The present study compared the effects of /sup 60/Co irradiation on Drosophila melanogaster between an anesthetic-resistant spontaneous mutant and an anesthetic-sensitive strain. An anesthetic-resistant mutant strain, Eth-29, of Drosophila melanogaster has previously been established. Eth-29 is resistant to diethyl-ether, chloroform and halothane. The anesthetic-resistant strain was found to be radiosensitive when evaluated by survival at the eighth day after irradiation or by dyskinesia (knock-down) at the second day. The results indicate that anesthetic resistance may be related to an increase in orderliness. The findings in reciprocal crosses between Eth-29 and the control strain indicate that the mechanism of survival is different from that of knock-down. Presumably, knock-down is the direct sequela of irradiation, and the present result suggests that membrane damage may be involved in inducing knock-down. 18 references, 3 figures.

  14. Direct demonstration of Ca2+ binding defects in sarco-endoplasmic reticulum Ca2+ ATPase mutants overexpressed in COS-1 cells transfected with adenovirus vectors.

    PubMed

    Strock, C; Cavagna, M; Peiffer, W E; Sumbilla, C; Lewis, D; Inesi, G

    1998-06-12

    Single mutations of specific amino acids within the membrane-bound region of the sarco-endoplasmic reticulum Ca2+ (SERCA)-1 ATPase interfere with Ca2+ inhibition of ATPase phosphorylation by Pi (1), suggesting that these residues may be involved in complexation of two Ca2+ that are known to bind to the enzyme. However, direct measurements of Ca2+ binding in the absence of ATP have been limited by the low quantities of available mutant protein. We have improved the transfection efficiency by means of recombinant adenovirus vectors, yielding sufficient expression of wild type and mutant SERCA-1 ATPase for measurements of Ca2+ binding to the microsomal fraction of the transfected cells. We find that in the presence of 20 microM Ca2+ and in the absence of ATP, the Glu771 --> Gln, Thr799 --> Ala, Asp800 --> Asn, and Glu908 --> Ala mutants exhibit negligible binding, indicating that the oxygen functions of Glu771, Thr799, Asp800, and Glu908 are involved in interactions whose single disruption causes major changes in the highly cooperative "duplex" binding. Total loss of Ca2+ binding is accompanied by loss of Ca2+ inhibition of the Pi reaction. We also find that, at pH 7.0, the Glu309 --> Gln and the Asn796 --> Ala mutants bind approximately half as much Ca2+ as the wild type ATPase and do not interfere with Ca2+ inhibition of the Pi reaction. At pH 6.2, the Glu309 --> Gln mutant does not bind any Ca2+, and its phosphorylation by Pi is not inhibited by Ca2+. On the contrary, the Asn796 --> Ala mutant retains the behavior displayed at pH 7.0. This suggests that in the Glu309 --> Gln mutant, ionization of acidic functions in other amino acids (e.g. Glu771 and Asp800) occurs as the pH is shifted, thereby rendering Ca2+ binding possible. In the Asn796 --> Ala mutant, on the other hand, the Glu309 carboxylic function allows binding of inhibitory Ca2+ even at pH 6.2. In all cases mutational interference with the inhibition of the Pi reaction by Ca2+ can be overcome by raising

  15. Isolation of a fission yeast mutant that is sensitive to valproic acid and defective in the gene encoding Ric1, a putative component of Ypt/Rab-specific GEF for Ryh1 GTPase.

    PubMed

    Ma, Yan; Sugiura, Reiko; Zhang, Lili; Zhou, Xin; Takeuchi, Mai; He, Yi; Kuno, Takayoshi

    2010-09-01

    Valproic acid (VPA) causes various therapeutic and biological effects, but the exact mechanisms underlying these effects, however, remain elusive. To gain insights into the molecular mechanisms of VPA action, we performed in fission yeast a genetic screen for mutants that show VPA hypersensitivity and have identified several membrane-trafficking mutants including vas1-1/vps45 and vas2-1/aps1. Here, we describe the isolation and characterization of vas3-1/ric1-v3, a mutant allele of the ric1 (+) gene encoding a fission yeast homolog of the budding yeast Ric1p, a component of Ypt/Rab-specific guanyl-nucleotide exchange factor (GEF). The Rab GTPase Ryh1 knockout (Deltaryh1) cells and Deltaric1 cells exhibited similar phenotypes. The double knockout Deltaric1Deltaryh1 cells did not display synthetic growth defects. These results are consistent with the notion that Ric1 may be a component of the GEF complex for Ryh1. Overexpression of wild-type Ryh1 and the constitutively active Ryh1Q70L only partially suppressed the phenotypes of ric1-v3 and Deltaric1 cells, and they failed to localize to the Golgi/endosomes in ric1-v3 and Deltaric1 cells. Furthermore, we isolated vps15 (+) gene, encoding a serine/threonine protein kinase, as a dosage-dependent suppressor of the temperature-sensitive phenotype of ric1-v3 mutant, but not that of Deltaric1 cells. Our results showed that the ric1-v3 mutant allele has some residual functional activity and suggest that Vps15 plays a role in the regulation of Ric1 function. In conclusion, Ric1 is a putative component of GEF for Ryh1 and might be regulated by Vps15. Further studies are needed to reveal the mechanism underlying the regulation.

  16. Altered poly(ADP-ribose) metabolism impairs cellular responses to genotoxic stress in a hypomorphic mutant of poly(ADP-ribose) glycohydrolase

    SciTech Connect

    Gao Hong; Coyle, Donna L.; Meyer-Ficca, Mirella L.; Meyer, Ralph G.; Jacobson, Elaine L.; Wang, Zhao-Qi; Jacobson, Myron K. . E-mail: mjacobson@pharmacy.arizona.edu

    2007-03-10

    Genotoxic stress activates nuclear poly(ADP-ribose) (PAR) metabolism leading to PAR synthesis catalyzed by DNA damage activated poly(ADP-ribose) polymerases (PARPs) and rapid PAR turnover by action of nuclear poly(ADP-ribose) glycohydrolase (PARG). The involvement of PARP-1 and PARP-2 in responses to DNA damage has been well studied but the involvement of nuclear PARG is less well understood. To gain insights into the function of nuclear PARG in DNA damage responses, we have quantitatively studied PAR metabolism in cells derived from a hypomorphic mutant mouse model in which exons 2 and 3 of the PARG gene have been deleted (PARG-{delta}2,3 cells), resulting in a nuclear PARG containing a catalytic domain but lacking the N-terminal region (A domain) of the protein. Following DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we found that the activity of both PARG and PARPs in intact cells is increased in PARG-{delta}2,3 cells. The increased PARG activity leads to decreased PARP-1 automodification with resulting increased PARP activity. The degree of PARG activation is greater than PARP, resulting in decreased PAR accumulation. Following MNNG treatment, PARG-{delta}2,3 cells show reduced formation of XRCC1 foci, delayed H2AX phosphorylation, decreased DNA break intermediates during repair, and increased cell death. Our results show that a precise coordination of PARPs and PARG activities is important for normal cellular responses to DNA damage and that this coordination is defective in the absence of the PARG A domain.

  17. The content of mutant EGFR DNA correlates with response to EGFR-TKIs in lung adenocarcinoma patients with common EGFR mutations

    PubMed Central

    Hung, Ming-Szu; Lung, Jr-Hau; Lin, Yu-Ching; Fang, Yu-Hung; Hsieh, Meng-Jer; Tsai, Ying-Huang

    2016-01-01

    Abstract This study aimed to elucidate the association of the content of mutant epidermal growth factor receptor (EGFR) deoxyribonucleic acid (DNA) with the treatment response to EGFR-tyrosine kinase inhibitor (TKI) and survival in patients with lung cancer. This retrospective cohort study included 77 lung adenocarcinoma patients with common EGFR mutations from December 2012 to February 2015. The content of mutant EGFR DNA in lung cancer tissues was determined using an Amplification Refractory Mutation System. The association of the amount of mutant EGFR DNA with treatment response, the clinical variables, and the progression-free survival (PFS) after EGFR-TKI therapy were evaluated. Using the amount of mutant EGR DNA above 4.77% as the cut-off value, the sensitivity to predict EGFR-TKI responder is 82.0% and the specificity is 75.0% (area under the curve [AUC]: 0.734, P = 0.003). The high content of mutant EGFR DNA is an independent factor associated with the response to EGFR-TKIs (odds ratio: 13.07, 95% confidence interval [CI]: 3.23–52.11, P = 0.0003). A significantly longer PFS was observed in the group with the high content of mutant EGFR DNA (26.3 months, 95% CI: 12.2–26.3) compared with the low content of mutant EGFR DNA groups (12.3 months, 95% CI: 5.7–14.8, P = 0.0155). A better predictive value of the content of mutant EGFR DNA was noted in patients with exon 19 deletions (AUC: 0.892, P < 0.0001) than exon 21 L858R mutations (AUC: 0.675, P = 0.0856). Our results show that the content of mutant EGFR DNA is associated with the clinical response to EGFR-TKIs, especially in patients with exon 19 deletions mutation. PMID:27368002

  18. The content of mutant EGFR DNA correlates with response to EGFR-TKIs in lung adenocarcinoma patients with common EGFR mutations.

    PubMed

    Hung, Ming-Szu; Lung, Jr-Hau; Lin, Yu-Ching; Fang, Yu-Hung; Hsieh, Meng-Jer; Tsai, Ying-Huang

    2016-06-01

    This study aimed to elucidate the association of the content of mutant epidermal growth factor receptor (EGFR) deoxyribonucleic acid (DNA) with the treatment response to EGFR-tyrosine kinase inhibitor (TKI) and survival in patients with lung cancer.This retrospective cohort study included 77 lung adenocarcinoma patients with common EGFR mutations from December 2012 to February 2015. The content of mutant EGFR DNA in lung cancer tissues was determined using an Amplification Refractory Mutation System. The association of the amount of mutant EGFR DNA with treatment response, the clinical variables, and the progression-free survival (PFS) after EGFR-TKI therapy were evaluated.Using the amount of mutant EGR DNA above 4.77% as the cut-off value, the sensitivity to predict EGFR-TKI responder is 82.0% and the specificity is 75.0% (area under the curve [AUC]: 0.734, P = 0.003). The high content of mutant EGFR DNA is an independent factor associated with the response to EGFR-TKIs (odds ratio: 13.07, 95% confidence interval [CI]: 3.23-52.11, P = 0.0003). A significantly longer PFS was observed in the group with the high content of mutant EGFR DNA (26.3 months, 95% CI: 12.2-26.3) compared with the low content of mutant EGFR DNA groups (12.3 months, 95% CI: 5.7-14.8, P = 0.0155). A better predictive value of the content of mutant EGFR DNA was noted in patients with exon 19 deletions (AUC: 0.892, P < 0.0001) than exon 21 L858R mutations (AUC: 0.675, P = 0.0856).Our results show that the content of mutant EGFR DNA is associated with the clinical response to EGFR-TKIs, especially in patients with exon 19 deletions mutation. PMID:27368002

  19. Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

    PubMed

    Gao, Rui; Liu, Yongping; Silva-Fernandes, Anabela; Fang, Xiang; Paulucci-Holthauzen, Adriana; Chatterjee, Arpita; Zhang, Hang L; Matsuura, Tohru; Choudhary, Sanjeev; Ashizawa, Tetsuo; Koeppen, Arnulf H; Maciel, Patricia; Hazra, Tapas K; Sarkar, Partha S

    2015-01-01

    Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-δ pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible

  20. Helicobacter pylori Mutants Defective in RuvC Holliday Junction Resolvase Display Reduced Macrophage Survival and Spontaneous Clearance from the Murine Gastric Mucosa

    PubMed Central

    Loughlin, Michael F.; Barnard, Faye M.; Jenkins, David; Sharples, Gary J.; Jenks, Peter J.

    2003-01-01

    Homologous recombination contributes to the extraordinary genetic diversity of Helicobacter pylori and may be critical for surface antigen expression and adaptation to environmental challenges within the stomach. We generated isogenic, nonpolar H. pylori ruvC mutants to investigate the function of RuvC, a Holliday junction endonuclease that resolves recombinant joints into nicked duplex products. Inactivation of ruvC reduced the frequency of homologous recombination of H. pylori between 17- and 45-fold and increased sensitivity to DNA-damaging agents and the antimicrobial agents levofloxacin and metronidazole. The H. pylori ruvC mutants were more susceptible to oxidative stress and exhibited reduced survival within macrophages. Experiments with the H. pylori SS1 mouse model revealed that the 50% infective dose of the ruvC mutant was approximately 100-fold higher than that of the wild-type SS1 strain. Although the ruvC mutant was able to establish colonization with bacterial loads that were initially similar to those of the parental SS1 strain, infection was spontaneously cleared from the murine gastric mucosa over periods that varied from 36 to 67 days. These results demonstrate that, in this infection model, RuvC is essential for continued survival of H. pylori in vivo and raises the possibility that inactivation of ruvC might be of value in an attenuated vaccine strain. PMID:12654822

  1. Isolation and characterization of mutants defective in the localization of LCIB, an essential factor for the carbon-concentrating mechanism in Chlamydomonas reinhardtii.

    PubMed

    Yamano, Takashi; Asada, Atsuko; Sato, Emi; Fukuzawa, Hideya

    2014-09-01

    The unicellular green alga Chlamydomonas reinhardtii acclimates to low-CO2 (LC) conditions by actively transporting inorganic carbon (Ci) into the cell, resulting in an increase in photosynthetic efficiency. This mechanism is called the carbon-concentrating mechanism (CCM), and soluble protein LCIB is essential for the CCM. LCIB is localized in the vicinity of pyrenoid, a prominent structure in the chloroplast, under LC conditions in the light. In contrast, in the dark or in high-CO2 conditions, where the CCM is inactive, LCIB diffuses away from the pyrenoid. Although the functional importance of LCIB for the CCM has been shown, the significance and mechanism of the change in suborganellar localization of LCIB remain to be elucidated. In this study, we screened 13,000 DNA-tagged mutants and isolated twelve aberrant LCIB localization (abl) mutants under LC conditions. abl-1 and abl-3 with dispersed and speckled localization of LCIB in the chloroplast showed significant decreases in Ci affinity, Ci accumulation, and CO2 fixation. Ten abl mutants (abl-1, abl-3, abl-4, abl-5, abl-6, abl-7, abl-8, abl-9, abl-11, and abl-12) showed not only aberrant LCIB localization but also reduced pyrenoid sizes. Moreover, three abl mutants (abl-10, abl-11, and abl-12) showed the increased numbers of pyrenoids per cell. These results suggested that the specific LCIB localization could be related to pyrenoid development.

  2. Genomic Instability and DNA Damage Responses in Progeria Arising from Defective Maturation of Prelamin A

    PubMed Central

    Musich, Phillip R.; Zou, Yue

    2009-01-01

    Progeria syndromes have in common a premature aging phenotype and increased genome instability. The susceptibility to DNA damage arises from a compromised repair system, either in the repair proteins themselves or in the DNA damage response pathways. The most severe progerias stem from mutations affecting lamin A production, a filamentous protein of the nuclear lamina. Hutchinson-Gilford progeria syndrome (HGPS) patients are heterozygous for a LMNA gene mutation while Restrictive Dermopathy (RD) individuals have a homozygous deficiency in the processing protease Zmpste24. These mutations generate the mutant lamin A proteins progerin and FC-lamina A, respectively, which cause nuclear deformations and chromatin perturbations. Genome instability is observed even though genome maintenance and repair genes appear normal. The unresolved question is what features of the DNA damage response pathways are deficient in HGPS and RD cells. Here we review and discuss recent findings which resolve some mechanistic details of how the accumulation of progerin/FC-lamin A proteins may disrupt DNA damage response pathways in HGPS and RD cells. As the mutant lamin proteins accumulate they sequester replication and repair factors, leading to stalled replication forks which collapse into DNA double-strand beaks (DSBs). In a reaction unique to HGPS and RD cells these accessible DSB termini bind Xeroderma pigmentosum group A (XPA) protein which excludes normal binding by DNA DSB repair proteins. The bound XPA also signals activation of ATM and ATR, arresting cell cycle progression, leading to arrested growth. In addition, the effective sequestration of XPA at these DSB damage sites makes HGPS and RD cells more sensitive to ultraviolet light and other mutagens normally repaired by the nucleotide excision repair pathway of which XPA is a necessary and specific component. PMID:19851476

  3. Structure-relaxation mechanism for the response of T4 lysozyme cavity mutants to hydrostatic pressure

    PubMed Central

    Lerch, Michael T.; López, Carlos J.; Yang, Zhongyu; Kreitman, Margaux J.; Horwitz, Joseph; Hubbell, Wayne L.

    2015-01-01

    Application of hydrostatic pressure shifts protein conformational equilibria in a direction to reduce the volume of the system. A current view is that the volume reduction is dominated by elimination of voids or cavities in the protein interior via cavity hydration, although an alternative mechanism wherein cavities are filled with protein side chains resulting from a structure relaxation has been suggested [López CJ, Yang Z, Altenbach C, Hubbell WL (2013) Proc Natl Acad Sci USA 110(46):E4306–E4315]. In the present study, mechanisms for elimination of cavities under high pressure are investigated in the L99A cavity mutant of T4 lysozyme and derivatives thereof using site-directed spin labeling, pressure-resolved double electron–electron resonance, and high-pressure circular dichroism spectroscopy. In the L99A mutant, the ground state is in equilibrium with an excited state of only ∼3% of the population in which the cavity is filled by a protein side chain [Bouvignies et al. (2011) Nature 477(7362):111–114]. The results of the present study show that in L99A the native ground state is the dominant conformation to pressures of 3 kbar, with cavity hydration apparently taking place in the range of 2–3 kbar. However, in the presence of additional mutations that lower the free energy of the excited state, pressure strongly populates the excited state, thereby eliminating the cavity with a native side chain rather than solvent. Thus, both cavity hydration and structure relaxation are mechanisms for cavity elimination under pressure, and which is dominant is determined by details of the energy landscape. PMID:25918400

  4. A defect in the p53 response pathway induced by de novo purine synthesis inhibition.

    PubMed

    Bronder, Julie L; Moran, Richard G

    2003-12-01

    p53 is believed to sense cellular ribonucleotide depletion in the absence of DNA strand breaks and to respond by imposition of a p21-dependent G1 cell cycle arrest. We now report that the p53-dependent G1 checkpoint is blocked in human carcinoma cell lines after inhibition of de novo purine synthesis by folate analogs inhibitory to glycinamide ribonucleotide formyltransferase (GART). p53 accumulated in HCT116, MCF7, or A549 carcinoma cells upon GART inhibition, but, surprisingly, transcription of several p53 targets, including p21cip1/waf1, was impaired. The mechanism of this defect was examined. The p53 accumulating in these cells was nuclear but was not phosphorylated at serines 6, 15, and 20, nor was it acetylated at lysines 373 or 382. The DDATHF-stabilized p53 bound to the p21 promoter in vitro and in vivo but did not activate histone acetylation over the p53 binding sites in the p21 promoter that is an integral part of the transcriptional response mediated by the DNA damage pathway. We concluded that the robust initial response of the p53 pathway to GART inhibitors is not transcriptionally propagated to target genes due to a defect in p53 post-translational modifications and a failure to open chromatin structure despite promoter binding of this unmodified p53. PMID:14517211

  5. An Enhanced Immune Response of Mclk1+/− Mutant Mice Is Associated with Partial Protection from Fibrosis, Cancer and the Development of Biomarkers of Aging

    PubMed Central

    Wang, Dantong; Wang, Ying; Argyriou, Catherine; Carrière, Audrey; Malo, Danielle; Hekimi, Siegfried

    2012-01-01

    The immune response is essential for survival by destroying microorganisms and pre-cancerous cells. However, inflammation, one aspect of this response, can result in short- and long-term deleterious side-effects. Mclk1+/− mutant mice can be long-lived despite displaying a hair-trigger inflammatory response and chronically activated macrophages as a result of high mitochondrial ROS generation. Here we ask whether this phenotype is beneficial or simply tolerated. We used models of infection by Salmonella serovars and found that Mclk1+/− mutants mount a stronger immune response, control bacterial proliferation better, and are resistant to cell and tissue damage resulting from the response, including fibrosis and types of oxidative damage that are considered to be biomarkers of aging. Moreover, these same types of tissue damage were found to be low in untreated 23 months-old mutants. We also examined the initiation of tumour growth after transplantation of mouse LLC1 carcinoma cells into Mclk1+/− mutants, as well as during spontaneous tumorigenesis in Mclk1+/− Trp53+/− double mutants. Tumour latency was increased by the Mclk1+/− genotype in both models. Furthermore, we used the transplantation model to show that splenic CD8+ T lymphocytes from Mclk1+/− graft recipients show enhanced cytotoxicity against LLC1 cells in vitro. Mclk1+/− mutants thus display an association of an enhanced immune response with partial protection from age-dependent processes and from pathologies similar to those that are found with increased frequency during the aging process. This suggests that the immune phenotype of these mutants might contribute to their longevity. We discuss how these findings suggest a broader view of how the immune response might impact the aging process. PMID:23166727

  6. The symbiotic defect of Rhizobium meliloti exopolysaccharide mutants is suppressed by lpsZ sup + , a gene involved in lipopolysaccharide biosynthesis

    SciTech Connect

    Williams, M.N.V.; Klein, S.; Signer, E.R. ); Hollingsworth, R.I. )

    1990-05-01

    exo mutants of Rhizobium meliloti SU47, which fail to secrete acidic extracellular polysaccharide (EPS), induce Fix{sup {minus}} nodules on alfalfa. However, mutants of R. meliloti Rm41 carrying the same exo lesions induce normal Fix{sup +} nodules. The authors show that such induction is due to a gene from strain Rm41, which they call lpsZ{sup +}, that is missing in strain SU47. lpsZ{sup +} does not restore EPS production but instead alters the composition an structure of lipopolysaccharide. In both SU47 and Rm41, either lpsZ{sup +} or exo{sup +} is sufficient for normal nodulation. This suggests that in R. meliloti EPS and lipopolysaccharide can perform the same function in nodule development.

  7. Staphylococcus aureus β-Toxin Mutants Are Defective in Biofilm Ligase and Sphingomyelinase Activity, and Causation of Infective Endocarditis and Sepsis.

    PubMed

    Herrera, Alfa; Vu, Bao G; Stach, Christopher S; Merriman, Joseph A; Horswill, Alexander R; Salgado-Pabón, Wilmara; Schlievert, Patrick M

    2016-05-01

    β-Toxin is an important virulence factor of Staphylococcus aureus, contributing to colonization and development of disease [Salgado-Pabon, W., et al. (2014) J. Infect. Dis. 210, 784-792; Huseby, M. J., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14407-14412; Katayama, Y., et al. (2013) J. Bacteriol. 195, 1194-1203]. This cytotoxin has two distinct mechanisms of action: sphingomyelinase activity and DNA biofilm ligase activity. However, the distinct mechanism that is most important for its role in infective endocarditis is unknown. We characterized the active site of β-toxin DNA biofilm ligase activity by examining deficiencies in site-directed mutants through in vitro DNA precipitation and biofilm formation assays. Possible conformational changes in mutant structure compared to that of wild-type toxin were assessed preliminarily by trypsin digestion analysis, retention of sphingomyelinase activity, and predicted structures based on the native toxin structure. We addressed the contribution of each mechanism of action to producing infective endocarditis and sepsis in vivo in a rabbit model. The H289N β-toxin mutant, lacking sphingomyelinase activity, exhibited lower sepsis lethality and infective endocarditis vegetation formation compared to those of the wild-type toxin. β-Toxin mutants with disrupted biofilm ligase activity did not exhibit decreased sepsis lethality but were deficient in infective endocarditis vegetation formation compared to the wild-type protein. Our study begins to characterize the DNA biofilm ligase active site of β-toxin and suggests β-toxin functions importantly in infective endocarditis through both of its mechanisms of action. PMID:27015018

  8. Staphylococcus aureus β-Toxin Mutants Are Defective in Biofilm Ligase and Sphingomyelinase Activity, and Causation of Infective Endocarditis and Sepsis.

    PubMed

    Herrera, Alfa; Vu, Bao G; Stach, Christopher S; Merriman, Joseph A; Horswill, Alexander R; Salgado-Pabón, Wilmara; Schlievert, Patrick M

    2016-05-01

    β-Toxin is an important virulence factor of Staphylococcus aureus, contributing to colonization and development of disease [Salgado-Pabon, W., et al. (2014) J. Infect. Dis. 210, 784-792; Huseby, M. J., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14407-14412; Katayama, Y., et al. (2013) J. Bacteriol. 195, 1194-1203]. This cytotoxin has two distinct mechanisms of action: sphingomyelinase activity and DNA biofilm ligase activity. However, the distinct mechanism that is most important for its role in infective endocarditis is unknown. We characterized the active site of β-toxin DNA biofilm ligase activity by examining deficiencies in site-directed mutants through in vitro DNA precipitation and biofilm formation assays. Possible conformational changes in mutant structure compared to that of wild-type toxin were assessed preliminarily by trypsin digestion analysis, retention of sphingomyelinase activity, and predicted structures based on the native toxin structure. We addressed the contribution of each mechanism of action to producing infective endocarditis and sepsis in vivo in a rabbit model. The H289N β-toxin mutant, lacking sphingomyelinase activity, exhibited lower sepsis lethality and infective endocarditis vegetation formation compared to those of the wild-type toxin. β-Toxin mutants with disrupted biofilm ligase activity did not exhibit decreased sepsis lethality but were deficient in infective endocarditis vegetation formation compared to the wild-type protein. Our study begins to characterize the DNA biofilm ligase active site of β-toxin and suggests β-toxin functions importantly in infective endocarditis through both of its mechanisms of action.

  9. Mutations in the unc-52 gene responsible for body wall muscle defects in adult Caenorhabditis elegans are located in alternatively spliced exons

    SciTech Connect

    Rogalski, T.M.; Gilchrist, E.J.; Mullen, G.P.

    1995-01-01

    The unc-52 gene in Caenorhabditis elegans produces several large proteins that function in the basement membrane underlying muscle cells. Mutations in this gene result in defects in myofilament assembly and in the attachment of the myofilament lattice to the muscle cell membrane. The st549 and ut111 alleles of unc-52 produce a lethal (Pat) terminal phenotype whereas the e444, e669, e998, e1012 and e1421 mutations result in viable, paralyzed animals. We have identified the sequence alterations responsible for these mutant phenotypes. The st549 allele has a premature stop codon in exon 7 that should result in the complete elimination of unc-52 gene function, and the ut111 allele has a Tc1 transposon inserted into the second exon of the gene. The five remaining mutations are clustered in a small interval containing three adjacent, alternatively spliced exons (16, 17 and 18). These mutations affect some, but not all of the unc-52-encoded proteins. Thirteen intragenic revertants of the e669, e998, e1012 and e1421 alleles have also been sequenced. The majority of these carry the original mutation plus a G to A transition in the conserved splice acceptor site of the affected exon. This result suggests that reversion of the mutant phenotype in these strains may be the result of exon-skipping. 38 refs., 6 figs., 2 tabs.

  10. Partial suppression of the respiratory defect of qrs1/her2 glutamyl-tRNA amidotransferase mutants by overexpression of the mitochondrial pentatricopeptide Msc6p.

    PubMed

    Moda, Bruno S; Ferreira-Júnior, José Ribamar; Barros, Mario H

    2016-08-01

    Recently, a large body of evidences indicates the existence in the mitochondrial matrix of foci that contain different proteins involved in mitochondrial RNA metabolism. Some of these proteins have a pentatricopeptide repeat motif that constitutes their RNA-binding structures. Here we report that MSC6, a mitochondrial pentatricopeptide protein of unknown function, is a multi copy suppressor of mutations in QRS1/HER2 a component of the trimeric complex that catalyzes the transamidation of glutamyl-tRNAQ to glutaminyl-tRNAQ. This is an essential step in mitochondrial translation because of the lack of a specific mitochondrial aminoacyl glutaminyl-tRNA synthetase. MSC6 over-expression did not abolish translation of an aberrant variant form of Cox2p detected in QRS1/HER2 mutants, arguing against a suppression mechanism that bypasses Qrs1p function. A slight decrement of the mitochondrial translation capacity as well as diminished growth on respiratory carbon sources media for respiratory activity was observed in the msc6 null mutant. Additionally, the msc6 null mutant did not display any impairment in RNA transcription, processing or turnover. We concluded that Msc6p is a mitochondrial matrix protein and further studies are required to indicate the specific function of Msc6p in mitochondrial translation.

  11. [Salmonella typhi vaccination response study reveals defective antibody production selective IgA deficiency patient].

    PubMed

    Pleguezuelo, Daniel E; Gianelli, Carla

    2015-01-01

    Selective IgA deficiency (SIgAD) is the most prevalent immunodeficiency worldwide, progressing to common variable immunodeficiency only in few reported cases. We report the case of a Spanish female aged 22 and diagnosed of selective IgA deficiency, a long history of bronchitis, several episodes of pneumonia, bilateral bronchiectasis, normal IgG, IgM, IgG subclasses, and detectable pre-vaccination IgG antibodies against tetanus toxoid and Streptococcus pneumoniae. She was evaluated in our clinic in order to rule out common variable immunodeficiency. We observed good antibody response to tetanus toxoid, absence of circulating switched memory B cells, decreased response to pneumococcal polysaccharide antigens and a lack of response to Salmonella typhi vaccine. Most SIgAD patients presents with upper respiratory tract infections or mild diarrhea. Those with lower tract infections, pneumonia or untreatable diarrhea should follow B-cell subpopulations' study and antibody response to vaccines. Absence of response to Salmonella typhi vaccine allowed us to expose the defective antibody production.

  12. MuLV IN Mutants Responsive to HDAC Inhibitors Enhance Transcription from Unintegrated Retroviral DNA

    PubMed Central

    Schneider, William M.; Wu, Dai-tze; Amin, Vaibhav; Aiyer, Sriram; Roth, Monica J.

    2012-01-01

    For Moloney murine leukemia virus (M-MuLV), sustained viral infections require expression from an integrated provirus. For many applications, non-integrating retroviral vectors have been utilized to avoid the unwanted effects of integration, however, the level of expression from unintegrated DNA is significantly less than that of integrated provirus. We find that unintegrated DNA expression can be increased in the presence of HDAC inhibitors, such as TSA, when applied in combination with integrase (IN) mutations. These mutants include an active site mutation as well as catalytically active INs bearing mutations of K376 in the MuLV C-terminal domain of IN. MuLV IN K376 is homologous to K266 in HIV-1 IN, a known substrate for acetylation. The MuLV IN protein is acetylated by p300 in vitro, however, the effect of HDAC inhibitors on gene expression from unintegrated DNA is not dependent on the acetylation state of MuLV IN K376. PMID:22365328

  13. Cox4i2, Ifit2, and Prdm11 Mutant Mice: Effective Selection of Genes Predisposing to an Altered Airway Inflammatory Response from a Large Compendium of Mutant Mouse Lines.

    PubMed

    Horsch, Marion; Aguilar-Pimentel, Juan Antonio; Bönisch, Clemens; Côme, Christophe; Kolster-Fog, Cathrine; Jensen, Klaus T; Lund, Anders H; Lee, Icksoo; Grossman, Lawrence I; Sinkler, Christopher; Hüttemann, Maik; Bohn, Erwin; Fuchs, Helmut; Ollert, Markus; Gailus-Durner, Valérie; de Angelis, Martin Hrabĕ; Beckers, Johannes

    2015-01-01

    We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype-envirotype interactions for other diseases.

  14. Cox4i2, Ifit2, and Prdm11 Mutant Mice: Effective Selection of Genes Predisposing to an Altered Airway Inflammatory Response from a Large Compendium of Mutant Mouse Lines

    PubMed Central

    Bönisch, Clemens; Côme, Christophe; Kolster-Fog, Cathrine; Jensen, Klaus T.; Lund, Anders H.; Lee, Icksoo; Grossman, Lawrence I.; Sinkler, Christopher; Hüttemann, Maik; Bohn, Erwin; Fuchs, Helmut; Ollert, Markus; Gailus-Durner, Valérie; Hrabĕ de Angelis, Martin; Beckers, Johannes

    2015-01-01

    We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype – envirotype interactions for other diseases. PMID:26263558

  15. The tomato mutant ars1 (altered response to salt stress 1) identifies an R1-type MYB transcription factor involved in stomatal closure under salt acclimation.

    PubMed

    Campos, Juan F; Cara, Beatriz; Pérez-Martín, Fernando; Pineda, Benito; Egea, Isabel; Flores, Francisco B; Fernandez-Garcia, Nieves; Capel, Juan; Moreno, Vicente; Angosto, Trinidad; Lozano, Rafael; Bolarin, Maria C

    2016-06-01

    A screening under salt stress conditions of a T-DNA mutant collection of tomato (Solanum lycopersicum L.) led to the identification of the altered response to salt stress 1 (ars1) mutant, which showed a salt-sensitive phenotype. Genetic analysis of the ars1 mutation revealed that a single T-DNA insertion in the ARS1 gene was responsible of the mutant phenotype. ARS1 coded for an R1-MYB type transcription factor and its expression was induced by salinity in leaves. The mutant reduced fruit yield under salt acclimation while in the absence of stress the disruption of ARS1 did not affect this agronomic trait. The stomatal behaviour of ars1 mutant leaves induced higher Na(+) accumulation via the transpiration stream, as the decreases of stomatal conductance and transpiration rate induced by salt stress were markedly lower in the mutant plants. Moreover, the mutation affected stomatal closure in a response mediated by abscisic acid (ABA). The characterization of tomato transgenic lines silencing and overexpressing ARS1 corroborates the role of the gene in regulating the water loss via transpiration under salinity. Together, our results show that ARS1 tomato gene contributes to reduce transpirational water loss under salt stress. Finally, this gene could be interesting for tomato molecular breeding, because its manipulation could lead to improved stress tolerance without yield penalty under optimal culture conditions. PMID:26578112

  16. Different epitope structures select distinct mutant forms of an antibody variable region for expression during the immune response

    PubMed Central

    1991-01-01

    Antibody variable (V) regions that initially differ from one another by only single amino acid residues at VH-D and D-JH segment junctions (termed canonical V regions) can be elicited in strain A/J mice by three different haptens. Among such V regions an amino acid substitution due to somatic mutation is recurrently observed at VH CDR2 position 58, regardless of which of these haptens is used for immunization. This substitution confers upon a canonical V region a generic increase in affinity for all the haptens. Conversely, the type of amino acid substitution at VH position 59 resulting from somatic mutation that is recurrently observed among such V regions changes with the eliciting hapten, in a manner that correlates directly with the cognate affinity increases (or decreases) for hapten conferred by the observed substitutions. This small subregion of VH CDR2 therefore plays a major role in determining both affinity and specificity for antigen. The data confirm that affinity for antigen is of pivotal importance in determining the degree of selection of different mutant forms of a V region. Moreover, during an immune response a sufficiently diverse mutant repertoire can be generated from a single canonical V region to allow adaptation to increase affinity for three different epitopes. PMID:1705280

  17. Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis.

    PubMed Central

    Nonet, M; Scafe, C; Sexton, J; Young, R

    1987-01-01

    We have isolated a yeast conditional mutant which rapidly ceases synthesis of mRNA when subjected to the nonpermissive temperature. This mutant (rpb1-1) was constructed by replacing the wild-type chromosomal copy of the gene encoding the largest subunit of RNA polymerase II with one mutagenized in vitro. The rapid cessation of mRNA synthesis in vivo and the lack of RNA polymerase II activity in crude extracts indicate that the mutant possesses a functionally defective, rather than an assembly-defective, RNA polymerase II. The shutdown in mRNA synthesis in the rpb1-1 mutant has pleiotropic effects on the synthesis of other RNAs and on the heat shock response. This mutant provides direct evidence that the RPB1 protein has a functional role in mRNA synthesis. Images PMID:3299050

  18. Short term variability in FEV1 and bronchodilator responsiveness in patients with obstructive ventilatory defects.

    PubMed Central

    Tweeddale, P M; Alexander, F; McHardy, G J

    1987-01-01

    Short term variability in FEV1 and responsiveness to inhaled bronchodilator were measured in 150 patients with obstructive ventilatory defects. The range of initial FEV1 was 0.5-4.71 and the natural variability over a 20 minute period when expressed in absolute terms was similar over the entire range, and differed insignificantly from that found in normal subjects. The increase in FEV1 and vital capacity (VC) required to exclude natural variability with 95% confidence in these patients was 160 ml and 330 ml respectively. Natural variability when expressed in percentage terms was negatively correlated with the level of FEV1 recorded. The analysis of changes in FEV1 and VC after administration of bronchodilator used absolute and percentage criteria for response. The number of responders differed considerably according to the criterion used. In those defined by the absolute criterion as responders there was no evidence that size of response was related to level of FEV1. Percentage criteria have traditionally been used to identify responses to bronchodilator that may be clinically useful, while absolute criteria, although statistically valid, have not been favoured. Reappraisal of the criteria used and their limitations and implications is required. PMID:3438892

  19. Immune responses and protection against infection and abortion in cattle experimentally vaccinated with mutant strains of Brucella abortus.

    PubMed

    Cheville, N F; Stevens, M G; Jensen, A E; Tatum, F M; Halling, S M

    1993-10-01

    Twenty-four 10-month-old Polled Hereford heifers were inoculated SC with live cells of one of the following strains of Brucella abortus: S19 delta 31K (n = 4), S19 delta SOD (n = 4), RB51 (n = 4), and strain 19 (n = 6); controls (n = 6) were given saline solution. Heifers given the deletion mutants S19 delta 31K and S19 delta SOD, and those given strain 19 developed antibody responses to B abortus and cutaneous reactions to brucellin. Heifers given strain RB51 did not develop antibodies that reacted in the standard tube agglutination test, but sera reacted in tests, using an antibody dot-blot assay containing RB51 antigen. The S19 delta 31K and S19 delta SOD strains of B abortus isolated from lymph node tissue after vaccination did not differ genetically from the master stock strain. All heifers were bred naturally at 16 to 17 months of age, and were challenge-exposed intraconjunctivally with virulent B abortus strain 2308 during the fifth month of pregnancy. All vaccinated heifers were protected (ie, none aborted and none had B abortus isolated from their tissues after parturition). Calves born from vaccinated dams were free of B abortus. Antibody responses in heifers after challenge exposure were an indicator of immunity. All 5 control heifers (nonvaccinated) developed serum antibodies after challenge exposure; 3 aborted, and 1 delivered a small, weak calf at 8.5 months of gestation. Thus live mutant strains of B abortus can induce protective immunity when given at 10 months of age, and strain RB51 is a strong candidate for further testing.

  20. Parkin is a lipid-responsive regulator of fat uptake in mice and mutant human cells

    PubMed Central

    Kim, Kye-Young; Stevens, Mark V.; Akter, M. Hasina; Rusk, Sarah E.; Huang, Robert J.; Cohen, Alexandra; Noguchi, Audrey; Springer, Danielle; Bocharov, Alexander V.; Eggerman, Tomas L.; Suen, Der-Fen; Youle, Richard J.; Amar, Marcelo; Remaley, Alan T.; Sack, Michael N.

    2011-01-01

    It has long been hypothesized that abnormalities in lipid biology contribute to degenerative brain diseases. Consistent with this, emerging epidemiologic evidence links lipid alterations with Parkinson disease (PD), and disruption of lipid metabolism has been found to predispose to α-synuclein toxicity. We therefore investigated whether Parkin, an E3 ubiquitin ligase found to be defective in patients with early onset PD, regulates systemic lipid metabolism. We perturbed lipid levels by exposing Parkin+/+ and Parkin–/– mice to a high-fat and -cholesterol diet (HFD). Parkin–/– mice resisted weight gain, steatohepatitis, and insulin resistance. In wild-type mice, the HFD markedly increased hepatic Parkin levels in parallel with lipid transport proteins, including CD36, Sr-B1, and FABP. These lipid transport proteins were not induced in Parkin–/– mice. The role of Parkin in fat uptake was confirmed by increased oleate accumulation in hepatocytes overexpressing Parkin and decreased uptake in Parkin–/– mouse embryonic fibroblasts and patient cells harboring complex heterozygous mutations in the Parkin-encoding gene PARK2. Parkin conferred this effect, in part, via ubiquitin-mediated stabilization of the lipid transporter CD36. Reconstitution of Parkin restored hepatic fat uptake and CD36 levels in Parkin–/– mice, and Parkin augmented fat accumulation during adipocyte differentiation. These results demonstrate that Parkin is regulated in a lipid-dependent manner and modulates systemic fat uptake via ubiquitin ligase–dependent effects. Whether this metabolic regulation contributes to premature Parkinsonism warrants investigation. PMID:21865652

  1. Admittance spectroscopy in kesterite solar cells: Defect signal or circuit response

    NASA Astrophysics Data System (ADS)

    Paul Weiss, Thomas; Redinger, Alex; Luckas, Jennifer; Mousel, Marina; Siebentritt, Susanne

    2013-05-01

    Unlike Cu(In,Ga)Se2 based solar cells, Cu2ZnSn(S,Se)4 solar cells show a strong increase in series resistance with decreasing temperature. In this study we deduce the series resistance from temperature dependent current-voltage measurements on a 5.5% efficient Cu2ZnSnSe4 solar cell. By applying a simple circuit model an increasing series resistance with decreasing temperature alone results in a capacitance step within the C-f profile. We show that this step needs to be distinguished from a step caused by a defect state or a carrier freeze-out. Consequently, the deduced activation energy is strongly distorted by the circuit response.

  2. Membrane-Associated Proteins of a Lipopolysaccharide-Deficient Mutant of Neisseria meningitidis Activate the Inflammatory Response through Toll-Like Receptor 2

    PubMed Central

    Ingalls, Robin R.; Lien, Egil; Golenbock, Douglas T.

    2001-01-01

    The recent isolation of a lipopolysaccharide (LPS)-deficient mutant of Neisseria meningitidis has allowed us to explore the roles of other gram-negative cell wall components in the host response to infection. The experiments in this study were designed to examine the ability of this mutant strain to activate cells. Although it was clearly less potent than the parental strain, we found the LPS-deficient mutant to be a capable inducer of the inflammatory response in monocytic cells, inducing a response similar to that seen with Staphylococcus aureus. Cellular activation by the LPS mutant was related to expression of CD14, a high-affinity receptor for LPS and other microbial products, as well as Toll-like receptor 2, a member of the Toll family of receptors recently implicated in host responses to gram-positive bacteria. In contrast to the parental strain, the synthetic LPS antagonist E5564 did not inhibit the LPS-deficient mutant. We conclude that even in the absence of LPS, the gram-negative cell wall remains a potent inflammatory stimulant, utilizing signaling pathways independent of those involved in LPS signaling. PMID:11254578

  3. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects.

    PubMed

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A

    2016-09-19

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes. PMID:27257060

  4. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects

    PubMed Central

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A.

    2016-01-01

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes. PMID:27257060

  5. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects.

    PubMed

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A

    2016-09-19

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes.

  6. Increased valinomycin production in mutants of Streptomyces sp. M10 defective in bafilomycin biosynthesis and branched-chain α-keto acid dehydrogenase complex expression.

    PubMed

    Lee, Dong Wan; Ng, Bee Gek; Kim, Beom Seok

    2015-11-01

    Streptomyces sp. M10 is a valinomycin-producing bacterial strain that shows potent bioactivity against Botrytis blight of cucumber plants. During studies to increase the yield of valinomycin (a cyclododecadepsipeptide) in strain M10, additional antifungal metabolites, including bafilomycin derivatives (macrolide antibiotics), were identified. To examine the effect of bafilomycin biosynthesis on valinomycin production, the bafilomycin biosynthetic gene cluster was cloned from the genome of strain M10, as were two branched-chain α-keto acid dehydrogenase (BCDH) gene clusters related to precursor supply for bafilomycin biosynthesis. A null mutant (M10bafm) of one bafilomycin biosynthetic gene (bafV) failed to produce bafilomycin, but resulted in a 1.2- to 1.5-fold increase in the amount of valinomycin produced. In another null mutant (M10bkdFm) of a gene encoding a subunit of the BCDH complex (bkdF), bafilomycin production was completely abolished and valinomycin production increased fourfold relative to that in the wild-type M10 strain. The higher valinomycin yield was likely the result of redistribution of the metabolic flux from bafilomycin to valinomycin biosynthesis, because the two antibiotics share a common precursor, 2-ketoisovaleric acid, a deamination product of valine. The results show that directing precursor flux toward active ingredient biosynthesis could be used as a prospective tool to increase the competence of biofungicides.

  7. The disease-linked Glu-26-Lys mutant version of Coronin 1A exhibits pleiotropic and pathway-specific signaling defects

    PubMed Central

    Ojeda, Virginia; Robles-Valero, Javier; Barreira, María; Bustelo, Xosé R.

    2015-01-01

    Coronin 1A (Coro1A) is involved in cytoskeletal and signaling events, including the regulation of Rac1 GTPase– and myosin II–dependent pathways. Mutations that generate truncated or unstable Coro1A proteins cause immunodeficiencies in both humans and rodents. However, in the case of the peripheral T-cell–deficient (Ptcd) mouse strain, the immunodeficiency is caused by a Glu-26-Lys mutation that targets a surface-exposed residue unlikely to affect the intramolecular architecture and stability of the protein. Here we report that this mutation induces pleiotropic effects in Coro1A protein, including the exacerbation of Coro1A-dependent actin-binding and -bundling activities; the formation of large meshworks of Coro1AE26K-decorated filaments endowed with unusual organizational, functional, and staining properties; and the elimination of Coro1A functions associated with both Rac1 and myosin II signaling. By contrast, it does not affect the ability of Coro1A to stimulate the nuclear factor of activated T-cells (NF-AT). Coro1AE26K is not a dominant-negative mutant, indicating that its pathological effects are derived from the inability to rescue the complete loss of the wild-type counterpart in cells. These results indicate that Coro1AE26K behaves as either a recessive gain-of-function or loss-of-function mutant protein, depending on signaling context and presence of the wild-type counterpart in cells. PMID:26108624

  8. An ion-current mutant of Paramecium tetraurelia with defects in the primary structure and post-translational N-methylation of calmodulin

    SciTech Connect

    Wallen-Friedman, M.A.

    1988-01-01

    My work on pantophobiac A{sup 2} (pntA{sup 2}), a behavioral mutant of Paramecium tetraurelia, suggest that the Ca{sup ++}-binding protein calmodulin (CaM), and post-translation N-methylation of CaM, are important for Ca{sup ++}-related ion-current function. Calmodulin from wild-type Paramecium has two sites of lysine-N-methylation. Both of these sites are almost fully methylated in vivo; thus wild-type calmodulin is a poor substrate for N-methylation in vitro. In contrast, pntA/{sup 2} CaM can be heavily N-methylated in vitro, suggesting that the mutant calmodulin is under-methylated in vivo. Amino-acid composition analysis showed that CaM lysine 115 is undermethylated in pntA{sup 2}. Once pntA{sup 2} CaM is N-methylated, the (methyl-{sup 3}H) group does not turn over in either wild-type or pntA{sup 2} cytoplasmic fractions. The methylating enzymes in pntA{sup 2} high-speed supernatant fractions are active, but may be less robust than those of the wild type, suggesting a possible control of these enzymes by CaM.

  9. A genetic screen for mutants defective in IAA1-LUC degradation in Arabidopsis thaliana reveals an important requirement for TOPOISOMERASE6B in auxin physiology

    PubMed Central

    Gilkerson, Jonathan; Callis, Judy

    2014-01-01

    Many plant growth and developmental processes are modulated by the hormone auxin. Auxin-modulated proteolysis of Aux/IAAs, a family of transcriptional repressors, represents a major mode of auxin action. Auxin facilitates the interaction of Aux/IAAs with TIR1/AFB F-box proteins, promoting their ubiquitination by the SCFTIR1/AFB ubiquitin E3 ligase leading to subsequent degradation by the 26S proteasome. To identify new genes regulating Aux/IAA proteolysis in Arabidopsis thaliana, we took a genetic approach, identifying individuals with altered degradation of an IAA1-luciferase fusion protein (IAA1-LUC). A mutant with 2-fold slower IAA1-LUC degradation rate compared with wild-type was isolated. Positional cloning identified the mutant as an allele of TOPOISOMERASE6B, named top6b-7. TOP6B encodes a subunit of a plant and archea-specific enzyme regulating endoreduplication, DNA damage repair and transcription in plants. T-DNA insertion alleles (top6b-8 and top6b-9) were also analyzed. top6b-7 seedlings are less sensitive to exogenous auxin than wild-type siblings in primary root growth assays, and experiments with DR5:GUS. Additionally, top6b-7 seedlings have a 40% reduction in the amount of endogenous IAA. These data suggest that increased IAA1-LUC half-life in top6b-7 probably results from a combination of both lower endogenous IAA levels and reduced sensitivity to auxin. PMID:25482814

  10. Evolved Osmotolerant Escherichia coli Mutants Frequently Exhibit Defective N-Acetylglucosamine Catabolism and Point Mutations in Cell Shape-Regulating Protein MreB

    PubMed Central

    Winkler, James D.; Garcia, Carlos; Olson, Michelle; Callaway, Emily

    2014-01-01

    Biocatalyst robustness toward stresses imposed during fermentation is important for efficient bio-based production. Osmotic stress, imposed by high osmolyte concentrations or dense populations, can significantly impact growth and productivity. In order to better understand the osmotic stress tolerance phenotype, we evolved sexual (capable of in situ DNA exchange) and asexual Escherichia coli strains under sodium chloride (NaCl) stress. All isolates had significantly improved growth under selection and could grow in up to 0.80 M (47 g/liter) NaCl, a concentration that completely inhibits the growth of the unevolved parental strains. Whole genome resequencing revealed frequent mutations in genes controlling N-acetylglucosamine catabolism (nagC, nagA), cell shape (mrdA, mreB), osmoprotectant uptake (proV), and motility (fimA). Possible epistatic interactions between nagC, nagA, fimA, and proV deletions were also detected when reconstructed as defined mutations. Biofilm formation under osmotic stress was found to be decreased in most mutant isolates, coupled with perturbations in indole secretion. Transcriptional analysis also revealed significant changes in ompACGL porin expression and increased transcription of sulfonate uptake systems in the evolved mutants. These findings expand our current knowledge of the osmotic stress phenotype and will be useful for the rational engineering of osmotic tolerance into industrial strains in the future. PMID:24727267

  11. Elements That Regulate the DNA Damage Response of Proteins Defective in Cockayne Syndrome.

    PubMed

    Iyama, Teruaki; Wilson, David M

    2016-01-16

    Cockayne syndrome (CS) is a premature aging disorder characterized by developmental defects, multisystem progressive degeneration and sensitivity to ultraviolet light. CS is divided into two primary complementation groups, A and B, with the CSA and CSB proteins presumably functioning in DNA repair and transcription. Using laser microirradiation and confocal microscopy, we characterized the nature and regulation of the CS protein response to oxidative DNA damage, double-strand breaks (DSBs), angelicin monoadducts and trioxsalen interstrand crosslinks (ICLs). Our data indicate that CSB recruitment is influenced by the type of DNA damage and is most rapid and robust as follows: ICLs>DSBs>monoadducts>oxidative lesions. Transcription inhibition reduced accumulation of CSB at sites of monoadducts and ICLs, but it did not affect recruitment to (although slightly affected retention at) oxidative damage. Inhibition of histone deacetylation altered the dynamics of CSB assembly, suggesting a role for chromatin status in the response to DNA damage, whereas the proteasome inhibitor MG132 had no effect. The C-terminus of CSB and, in particular, its ubiquitin-binding domain were critical to recruitment, while the N-terminus and a functional ATPase domain played a minor role at best in facilitating protein accumulation. Although the absence of CSA had no effect on CSB recruitment, CSA itself localized at sites of ICLs, DSBs and monoadducts but not at oxidative lesions. Our results reveal molecular components of the CS protein response and point to a major involvement of complex lesions in the pathology of CS.

  12. Heart failure after transvenous closure of atrial septal defect associated with atrial standstill and thiamine-responsive megaloblastic anemia.

    PubMed

    Doğan, Vehbi; Senocak, Filiz; Orün, Utku Arman; Ceylan, Ozben

    2013-10-01

    Despite advances in device closure for atrial septal defect, post-closure heart failure remains a clinical problem in adult patients but is seen only rarely in children. An eight-year-old boy, who had been followed by a local pediatrician with the diagnosis of diabetes mellitus and congenital heart disease, was consulted to us for cardiac re-evaluation. Electrocardiography demonstrated absent P waves, and echocardiography revealed enlargement of the right ventricle and both atria and secundum atrial septal defect. With the diagnosis of atrial standstill, secundum atrial septal defect and thiamine-responsive megaloblastic anemia, acute heart failure developed after transvenous closure of the atrial septal defect, which improved dramatically with thiamine and supportive treatment. PMID:24164997

  13. Abscisic acid-responsive guard cell metabolomes of Arabidopsis wild-type and gpa1 G-protein mutants.

    PubMed

    Jin, Xiaofen; Wang, Rui-Sheng; Zhu, Mengmeng; Jeon, Byeong Wook; Albert, Reka; Chen, Sixue; Assmann, Sarah M

    2013-12-01

    Individual metabolites have been implicated in abscisic acid (ABA) signaling in guard cells, but a metabolite profile of this specialized cell type is lacking. We used liquid chromatography-multiple reaction monitoring mass spectrometry for targeted analysis of 85 signaling-related metabolites in Arabidopsis thaliana guard cell protoplasts over a time course of ABA treatment. The analysis utilized ∼ 350 million guard cell protoplasts from ∼ 30,000 plants of the Arabidopsis Columbia accession (Col) wild type and the heterotrimeric G-protein α subunit mutant, gpa1, which has ABA-hyposensitive stomata. These metabolomes revealed coordinated regulation of signaling metabolites in unrelated biochemical pathways. Metabolites clustered into different temporal modules in Col versus gpa1, with fewer metabolites showing ABA-altered profiles in gpa1. Ca(2+)-mobilizing agents sphingosine-1-phosphate and cyclic adenosine diphosphate ribose exhibited weaker ABA-stimulated increases in gpa1. Hormone metabolites were responsive to ABA, with generally greater responsiveness in Col than in gpa1. Most hormones also showed different ABA responses in guard cell versus mesophyll cell metabolomes. These findings suggest that ABA functions upstream to regulate other hormones, and are also consistent with G proteins modulating multiple hormonal signaling pathways. In particular, indole-3-acetic acid levels declined after ABA treatment in Col but not gpa1 guard cells. Consistent with this observation, the auxin antagonist α-(phenyl ethyl-2-one)-indole-3-acetic acid enhanced ABA-regulated stomatal movement and restored partial ABA sensitivity to gpa1.

  14. Immune activation and response to pembrolizumab in POLE-mutant endometrial cancer

    PubMed Central

    Mehnert, Janice M.; Panda, Anshuman; Zhong, Hua; Hirshfield, Kim; Damare, Sherri; Lane, Katherine; Sokol, Levi; Stein, Mark N.; Rodriguez-Rodriquez, Lorna; Kaufman, Howard L.; Ali, Siraj; Ross, Jeffrey S.; Pavlick, Dean C.; Bhanot, Gyan; White, Eileen P.; DiPaola, Robert S.; Lovell, Ann; Cheng, Jonathan

    2016-01-01

    Antibodies that target the immune checkpoint receptor programmed cell death protein 1 (PD-1) have resulted in prolonged and beneficial responses toward a variety of human cancers. However, anti–PD-1 therapy in some patients provides no benefit and/or results in adverse side effects. The factors that determine whether patients will be drug sensitive or resistant are not fully understood; therefore, genomic assessment of exceptional responders can provide important insight into patient response. Here, we identified a patient with endometrial cancer who had an exceptional response to the anti–PD-1 antibody pembrolizumab. Clinical grade targeted genomic profiling of a pretreatment tumor sample from this individual identified a mutation in DNA polymerase epsilon (POLE) that associated with an ultramutator phenotype. Analysis of The Cancer Genome Atlas (TCGA) revealed that the presence of POLE mutation associates with high mutational burden and elevated expression of several immune checkpoint genes. Together, these data suggest that cancers harboring POLE mutations are good candidates for immune checkpoint inhibitor therapy. PMID:27159395

  15. Loss-of-Function Mutants and Overexpression Lines of the Arabidopsis Cyclin CYCA1;2/TARDY ASYNCHRONOUS MEIOSIS Exhibit Different Defects in Prophase-I Meiocytes but Produce the Same Meiotic Products

    PubMed Central

    Wang, Yixing; Yang, Ming

    2014-01-01

    In Arabidopsis, loss-of-function mutations in the A-type cyclin CYCA1;2/TARDY ASYNCHRONOUS MEIOSIS (TAM) gene lead to the production of abnormal meiotic products including triads and dyads. Here we report that overexpression of TAM by the ASK1:TAM transgene also led to the production of triads and dyads in meiosis, as well as shriveled seeds, in a dominant fashion. However, the partial loss-of-function mutant tam-1, an ASK1:TAM line, and the wild type differed in dynamic changes in chromosome thread thickness from zygotene to diplotene. We also found that the pericentromeric heterochromatin regions in male meiocytes in tam-1 and tam-2 (a null allele) frequently formed a tight cluster at the pachytene and diplotene stages, in contrast to the infrequent occurrences of such clusters in the wild type and the ASK1:TAM line. Immunolocalization studies of the chromosome axial component ASY1 revealed that ASY1 was highly expressed at the appropriate male meiotic stages but not localized to the chromosomes in tam-2. The level of ASY1, however, was greatly reduced in another ASK1:TAM line with much overexpressed TAM. Our results indicate that the reduction and increase in the activity of TAM differentially affect chromosomal morphology and the action of ASY1 in prophase I. Based on these results, we propose that either the different meiotic defects or a common defect such as missing ASY1 on the chromosomal axes triggers a hitherto uncharacterized cell cycle checkpoint in the male meiocytes in the tam mutants and ASK1:TAM lines, leading to the production of the same abnormal meiotic products. PMID:25402453

  16. Loss-of-function mutants and overexpression lines of the Arabidopsis cyclin CYCA1;2/Tardy Asynchronous Meiosis exhibit different defects in prophase-i meiocytes but produce the same meiotic products.

    PubMed

    Wang, Yixing; Yang, Ming

    2014-01-01

    In Arabidopsis, loss-of-function mutations in the A-type cyclin CYCA1;2/Tardy Asynchronous Meiosis (TAM) gene lead to the production of abnormal meiotic products including triads and dyads. Here we report that overexpression of TAM by the ASK1:TAM transgene also led to the production of triads and dyads in meiosis, as well as shriveled seeds, in a dominant fashion. However, the partial loss-of-function mutant tam-1, an ASK1:TAM line, and the wild type differed in dynamic changes in chromosome thread thickness from zygotene to diplotene. We also found that the pericentromeric heterochromatin regions in male meiocytes in tam-1 and tam-2 (a null allele) frequently formed a tight cluster at the pachytene and diplotene stages, in contrast to the infrequent occurrences of such clusters in the wild type and the ASK1:TAM line. Immunolocalization studies of the chromosome axial component ASY1 revealed that ASY1 was highly expressed at the appropriate male meiotic stages but not localized to the chromosomes in tam-2. The level of ASY1, however, was greatly reduced in another ASK1:TAM line with much overexpressed TAM. Our results indicate that the reduction and increase in the activity of TAM differentially affect chromosomal morphology and the action of ASY1 in prophase I. Based on these results, we propose that either the different meiotic defects or a common defect such as missing ASY1 on the chromosomal axes triggers a hitherto uncharacterized cell cycle checkpoint in the male meiocytes in the tam mutants and ASK1:TAM lines, leading to the production of the same abnormal meiotic products. PMID:25402453

  17. Effects of natural and synthetic auxins on the gravitropic growth habit of roots in two auxin-resistant mutants of Arabidopsis, axr1 and axr4: evidence for defects in the auxin influx mechanism of axr4

    NASA Technical Reports Server (NTRS)

    Yamamoto, M.; Yamamoto, K. T.

    1999-01-01

    The partially agravitropic growth habit of roots of an auxin-resistant mutant of Arabidopsis thaliana, axr4, was restored by the addition of 30-300 nM 1-naphthaleneacetic acid (NAA) to the growth medium. Neither indole 3-acetic acid (IAA) nor 2,4-dichlorophenoxyacetic acid (2,4-D) showed such an effect. Growth of axr4 roots was resistant to IAA and 2,4-D, but not at all to NAA. The differential effects of the three auxins suggest that the defects of axr4 result from a lower auxin influx into its cells. The partially agravitropic growth habit of axr1 roots, which was less severe than that of axr4 roots, was only slightly affected by the three auxins in the growth medium at concentrations up to 300 nM; growth of axr1 roots was resistant to all three of the auxins. These results suggest that the lesion of axrl mutants is different from that of axr4.

  18. Isolation and characterization of S. cerevisiae mutants defective in somatostatin expression: cloning and functional role of a yeast gene encoding an aspartyl protease in precursor processing at monobasic cleavage sites.

    PubMed

    Bourbonnais, Y; Ash, J; Daigle, M; Thomas, D Y

    1993-01-01

    The peptide somatostatin exists as two different molecular species. In addition to the most common form, somatostatin-14, there is also a fourteen amino acid N-terminally extended form of the tetradecapeptide, somatostatin-28. Both peptides are synthesized as larger precursors containing paired basic and monobasic amino acids at their processing sites, which upon cleavage generate either somatostatin-14 or -28, respectively. In some species of fish two distinct, but homologous, precursors (prosomatostatin-I and -II) give rise to somatostatin-14 and -28, respectively. Whereas anglerfish prosomatostatin-II was previously shown to release exclusively somatostatin-28, the yeast Saccharomyces cerevisiae proteolytically matures the homologous prosomatostatin-I precursor to somatostatin-28 and -14 as well as to a lysine-extended form of somatostatin-14. The Kex2 endoprotease appears to be essential for the formation of lysine somatostatin-14 and is involved either directly or indirectly in the release of mature somatostatin-14. The isolation of yeast mutants defective in somatostatin-28 expression (sex mutant) allowed the cloning of a non-essential gene, which encodes an aspartyl protease, whose disruption severely affects the cleavage of mature somatostatin-28 from both somatostatin precursors. We conclude that two distinct endoproteases, which demonstrate some cross specificity in vivo, are involved in the proteolytic maturation of prosomatostatin at mono- and dibasic processing sites in yeast. PMID:8094050

  19. Isolation and characterization of S. cerevisiae mutants defective in somatostatin expression: cloning and functional role of a yeast gene encoding an aspartyl protease in precursor processing at monobasic cleavage sites.

    PubMed Central

    Bourbonnais, Y; Ash, J; Daigle, M; Thomas, D Y

    1993-01-01

    The peptide somatostatin exists as two different molecular species. In addition to the most common form, somatostatin-14, there is also a fourteen amino acid N-terminally extended form of the tetradecapeptide, somatostatin-28. Both peptides are synthesized as larger precursors containing paired basic and monobasic amino acids at their processing sites, which upon cleavage generate either somatostatin-14 or -28, respectively. In some species of fish two distinct, but homologous, precursors (prosomatostatin-I and -II) give rise to somatostatin-14 and -28, respectively. Whereas anglerfish prosomatostatin-II was previously shown to release exclusively somatostatin-28, the yeast Saccharomyces cerevisiae proteolytically matures the homologous prosomatostatin-I precursor to somatostatin-28 and -14 as well as to a lysine-extended form of somatostatin-14. The Kex2 endoprotease appears to be essential for the formation of lysine somatostatin-14 and is involved either directly or indirectly in the release of mature somatostatin-14. The isolation of yeast mutants defective in somatostatin-28 expression (sex mutant) allowed the cloning of a non-essential gene, which encodes an aspartyl protease, whose disruption severely affects the cleavage of mature somatostatin-28 from both somatostatin precursors. We conclude that two distinct endoproteases, which demonstrate some cross specificity in vivo, are involved in the proteolytic maturation of prosomatostatin at mono- and dibasic processing sites in yeast. Images PMID:8094050

  20. The Rice brassinosteroid-deficient dwarf2 Mutant, Defective in the Rice Homolog of Arabidopsis DIMINUTO/DWARF1, Is Rescued by the Endogenously Accumulated Alternative Bioactive Brassinosteroid, Dolichosterone

    PubMed Central

    Hong, Zhi; Ueguchi-Tanaka, Miyako; Fujioka, Shozo; Takatsuto, Suguru; Yoshida, Shigeo; Hasegawa, Yasuko; Ashikari, Motoyuki; Kitano, Hidemi; Matsuoka, Makoto

    2005-01-01

    We have identified a rice (Oryza sativa) brassinosteroid (BR)-deficient mutant, BR-deficient dwarf2 (brd2). The brd2 locus contains a single base deletion in the coding region of Dim/dwf1, a homolog of Arabidopsis thaliana DIMINUTO/DWARF1 (DIM/DWF1). Introduction of the wild-type Dim/dwf1 gene into brd2 restored the normal phenotype. Overproduction and repression of Dim/dwf1 resulted in contrasting phenotypes, with repressors mimicking the brd2 phenotype and overproducers having large stature with increased numbers of flowers and seeds. Although brd2 contains low levels of common 6-oxo-type BRs, the severity of the brd2 phenotype is much milder than brd1 mutants and most similar to d2 and d11, which show a semidwarf phenotype at the young seedling stage. Quantitative analysis suggested that in brd2, the 24-methylene BR biosynthesis pathway is activated and the uncommon BR, dolichosterone (DS), is produced. DS enhances the rice lamina joint bending angle, rescues the brd1 dwarf phenotype, and inhibits root elongation, indicating that DS is a bioactive BR in rice. Based on these observations, we discuss an alternative BR biosynthetic pathway that produces DS when Dim/dwf1 is defective. PMID:15994910

  1. p53-Dependent DNA damage response sensitive to editing-defective tRNA synthetase in zebrafish.

    PubMed

    Song, Youngzee; Shi, Yi; Carland, Tristan M; Lian, Shanshan; Sasaki, Tomoyuki; Schork, Nicholas J; Head, Steven R; Kishi, Shuji; Schimmel, Paul

    2016-07-26

    Brain and heart pathologies are caused by editing defects of transfer RNA (tRNA) synthetases, which preserve genetic code fidelity by removing incorrect amino acids misattached to tRNAs. To extend understanding of the broader impact of synthetase editing reactions on organismal homeostasis, and based on effects in bacteria ostensibly from small amounts of mistranslation of components of the replication apparatus, we investigated the sensitivity to editing of the vertebrate genome. We show here that in zebrafish embryos, transient overexpression of editing-defective valyl-tRNA synthetase (ValRS(ED)) activated DNA break-responsive H2AX and p53-responsive downstream proteins, such as cyclin-dependent kinase (CDK) inhibitor p21, which promotes cell-cycle arrest at DNA damage checkpoints, and Gadd45 and p53R2, with pivotal roles in DNA repair. In contrast, the response of these proteins to expression of ValRS(ED) was abolished in p53-deficient fish. The p53-activated downstream signaling events correlated with suppression of abnormal morphological changes caused by the editing defect and, in adults, reversed a shortened life span (followed for 2 y). Conversely, with normal editing activities, p53-deficient fish have a normal life span and few morphological changes. Whole-fish deep sequencing showed genomic mutations associated with the editing defect. We suggest that the sensitivity of p53 to expression of an editing-defective tRNA synthetase has a critical role in promoting genome integrity and organismal homeostasis.

  2. Reversible and Irreversible Responses of Defect-Engineered Graphene-Based Electrolyte-Gated pH Sensors.

    PubMed

    Kwon, Sun Sang; Yi, Jaeseok; Lee, Won Woo; Shin, Jae Hyeok; Kim, Su Han; Cho, Seunghee H; Nam, SungWoo; Park, Won Il

    2016-01-13

    We have studied the role of defects in electrolyte-gated graphene mesh (GM) field-effect transistors (FETs) by introducing engineered edge defects in graphene (Gr) channels. Compared with Gr-FETs, GM-FETs were characterized as having large increments of Dirac point shift (∼30-100 mV/pH) that even sometimes exceeded the Nernst limit (59 mV/pH) by means of electrostatic gating of H(+) ions. This feature was attributed to the defect-mediated chemisorptions of H(+) ions to the graphene edge, as supported by Raman measurements and observed cycling characteristics of the GM FETs. Although the H(+) ion binding to the defects increased the device response to pH change, this binding was found to be irreversible. However, the irreversible component showed relatively fast decay, almost disappearing after 5 cycles of exposure to solutions of decreasing pH value from 8.25 to 6.55. Similar behavior could be found in the Gr-FET, but the irreversible component of the response was much smaller. Finally, after complete passivation of the defects, both Gr-FETs and GM-FETs exhibited only reversible response to pH change, with similar magnitude in the range of 6-8 mV/pH. PMID:26692009

  3. Evidence for Oxidative Stress and Defective Antioxidant Response in Guinea Pigs with Tuberculosis

    PubMed Central

    Palanisamy, Gopinath S.; Kirk, Natalie M.; Ackart, David F.; Shanley, Crystal A.; Orme, Ian M.; Basaraba, Randall J.

    2011-01-01

    The development of granulomatous inflammation with caseous necrosis is an important but poorly understood manifestation of tuberculosis in humans and some animal models. In this study we measured the byproducts of oxidative stress in granulomatous lesions as well as the systemic antioxidant capacity of BCG vaccinated and non-vaccinated guinea pigs experimentally infected with Mycobacterium tuberculosis. In non-vaccinated guinea pigs, oxidative stress was evident within 2 weeks of infection as measured by a decrease in the serum total antioxidant capacity and blood glutathione levels accompanied by an increase in malondialdehyde, a byproduct of lipid peroxidation, within lesions. Despite a decrease in total and reduced blood glutathione concentrations, there was an increase in lesion glutathione by immunohistochemistry in response to localized oxidative stress. In addition there was an increase in the expression of the host transcription factor nuclear erythroid 2 p45-related factor 2 (Nrf2), which regulates several protein and non-proteins antioxidants, including glutathione. Despite the increase in cytoplasmic expression of Nrf2, immunohistochemical staining revealed a defect in Nrf2 nuclear translocation within granulomatous lesions as well as a decrease in the expression of the Nrf2-regulated antioxidant protein NQO1. Treating M. tuberculosis–infected guinea pigs with the antioxidant drug N-acetyl cysteine (NAC) partially restored blood glutathione concentrations and the serum total antioxidant capacity. Treatment with NAC also decreased spleen bacterial counts, as well as decreased the lung and spleen lesion burden and the severity of lesion necrosis. These data suggest that the progressive oxidative stress during experimental tuberculosis in guinea pigs is due in part to a defect in host antioxidant defenses, which, we show here, can be partially restored with antioxidant treatment. These data suggest that the therapeutic strategies that reduce oxidant

  4. Mitotic UV irradiation induces a DNA replication-licensing defect that potentiates G1 arrest response.

    PubMed

    Morino, Masayuki; Nukina, Kohei; Sakaguchi, Hiroki; Maeda, Takeshi; Takahara, Michiyo; Shiomi, Yasushi; Nishitani, Hideo

    2015-01-01

    Cdt1 begins to accumulate in M phase and has a key role in establishing replication licensing at the end of mitosis or in early G1 phase. Treatments that damage the DNA of cells, such as UV irradiation, induce Cdt1 degradation through PCNA-dependent CRL4-Cdt2 ubiquitin ligase. How Cdt1 degradation is linked to cell cycle progression, however, remains unclear. In G1 phase, when licensing is established, UV irradiation leads to Cdt1 degradation, but has little effect on the licensing state. In M phase, however, UV irradiation does not induce Cdt1 degradation. When mitotic UV-irradiated cells were released into G1 phase, Cdt1 was degraded before licensing was established. Thus, these cells exhibited both defective licensing and G1 cell cycle arrest. The frequency of G1 arrest increased in cells expressing extra copies of Cdt2, and thus in cells in which Cdt1 degradation was enhanced, whereas the frequency of G1 arrest was reduced in cell expressing an extra copy of Cdt1. The G1 arrest response of cells irradiated in mitosis was important for cell survival by preventing the induction of apoptosis. Based on these observations, we propose that mammalian cells have a DNA replication-licensing checkpoint response to DNA damage induced during mitosis.

  5. Revertant analysis of a temperature-sensitive mutant of Newcastle disease virus with defective glycoproteins: implication of the fusion glycoprotein in cell killing and isolation of a neuraminidase-deficient hemagglutinating virus.

    PubMed Central

    Smith, G W; Hightower, L E

    1982-01-01

    Biological and molecular properties of a temperature-sensitive mutant (C1) of Newcastle disease virus and its revertants were analyzed. C1 exhibited three temperature-sensitive alterations (plaque formation, virion assembly, and cytopathogenicity) and several defects which were also present at the permissive temperature. C1 virions contained low amounts of hemagglutinin-neuraminidase glycopeptides and consequently were deficient in hemagglutinating and neuraminidase activities. These virions also contained defective fusion glycoproteins which rendered them poorly hemolytic and slow to penetrate cultured chicken embryo cells. The biological activities of the membrane glycoproteins were recovered sequentially in a series of plaque-forming revertants. The coreversion of hemolysis, membrane-penetrating activities, and cytopathogenicity in the first-step revertant (S1) suggested that fusion glycoproteins were major contributors to cellular destruction. This revertant also provided evidence of a role for fusion glycoproteins in virion assembly. From S1 we isolated a large-plaque-forming revertant (L1) that assembled wild-type amounts of biologically active hemagglutinin-neuraminidase glycoproteins into virions. Although it was normal for hemagglutination, L1 had less than 3% of the neuraminidase activity of the wild type, demonstrating that these two activities can be uncoupled genetically. The neuraminidase deficiency of L1 did not impair its virulence in ovo or its reproduction in cultured cells. PMID:6896347

  6. Rapid selection of escape mutants by the first CD8 T cell responses in acute HIV-1 infection

    SciTech Connect

    Korber, Bette Tina Marie

    2008-01-01

    The recent failure of a vaccine that primes T cell responses to control primary HIV-1 infection has raised doubts about the role of CD8+ T cells in early HIV-1 infection. We studied four patients who were identified shortly after HIV-1 infection and before seroconversion. In each patient there was very rapid selection of multiple HIV-1 escape mutants in the transmitted virus by CD8 T cells, including examples of complete fixation of non-synonymous substitutions within 2 weeks. Sequencing by single genome amplification suggested that the high rate of virus replication in acute infection gave a selective advantage to virus molecules that contained simultaneous and gained sequential T cell escape mutations. These observations show that whilst early HIV-1 specific CD8 T cells can act against virus, rapid escape means that these T cell responses are unlikely to benefit the patient and may in part explain why current HIV-1 T cell vaccines may not be protective.

  7. Characterization of the endemic equilibrium and response to mutant injection in a multi-strain disease model.

    PubMed

    Aquino, Tomás; Bolster, Diogo; Nunes, Ana

    2015-03-01

    We explore a model of an antigenically diverse infection whose otherwise identical strains compete through cross-immunity. We assume that individuals may produce upon infection different numbers of antibody types, each of which matches the antigenic configuration of a particular epitope, and that one matching antibody type grants total immunity against a challenging strain. In order to reduce the number of equations involved in the analytic description of the dynamics, we follow the strategy proposed by Kryazhimskiy et al. (2007) and apply a low-order closure reminiscent of a pair approximation. Using this approximation, we go beyond the numerical studies of Kryazhimskiy et al. (2007) and explore the analytic properties of the ensuing model in the absence of mutation. We characterize its endemic equilibrium, comparing with the results of agent based simulations of the full model to assess the performance of the closure assumption. We show that a particular choice of immune response leads to a degenerate endemic equilibrium, where different strain prevalences may exist, breaking the symmetry of the model. Finally we study the behavior of the system under the injection of mutant strains. We find that the build up of diversity from a single founding strain is extremely unlikely for different choices of the population׳s immune response. PMID:25496729

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

    PubMed

    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.

  9. Heat Stress Phenotypes of Arabidopsis Mutants Implicate Multiple Signaling Pathways in the Acquisition of Thermotolerance1[w

    PubMed Central

    Larkindale, Jane; Hall, Jennifer D.; Knight, Marc R.; Vierling, Elizabeth

    2005-01-01

    To investigate the importance of different processes to heat stress tolerance, 45 Arabidopsis (Arabidopsis thaliana) mutants and one transgenic line were tested for basal and acquired thermotolerance at different stages of growth. Plants tested were defective in signaling pathways (abscisic acid, salicylic acid, ethylene, and oxidative burst signaling) and in reactive oxygen metabolism (ascorbic acid or glutathione production, catalase) or had previously been found to have temperature-related phenotypes (e.g. fatty acid desaturase mutants, uvh6). Mutants were assessed for thermotolerance defects in seed germination, hypocotyl elongation, root growth, and seedling survival. To assess oxidative damage and alterations in the heat shock response, thiobarbituric acid reactive substances, heat shock protein 101, and small heat shock protein levels were determined. Fifteen mutants showed significant phenotypes. Abscisic acid (ABA) signaling mutants (abi1 and abi2) and the UV-sensitive mutant, uvh6, showed the strongest defects in acquired thermotolerance of root growth and seedling survival. Mutations in nicotinamide adenine dinucleotide phosphate oxidase homolog genes (atrbohB and D), ABA biosynthesis mutants (aba1, aba2, and aba3), and NahG transgenic lines (salicylic acid deficient) showed weaker defects. Ethylene signaling mutants (ein2 and etr1) and reactive oxygen metabolism mutants (vtc1, vtc2, npq1, and cad2) were more defective in basal than acquired thermotolerance, especially under high light. All mutants accumulated wild-type levels of heat shock protein 101 and small heat shock proteins. These data indicate that, separate from heat shock protein induction, ABA, active oxygen species, and salicylic acid pathways are involved in acquired thermotolerance and that UVH6 plays a significant role in temperature responses in addition to its role in UV stress. PMID:15923322

  10. The zebrafish early arrest mutants.

    PubMed

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

    1996-12-01

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

  11. Polymorphic human (CTAT)n microsatellite provides a conserved linkage marker for mouse mutants causing cleft palate, vestibular defects, obesity and ataxia

    SciTech Connect

    Griffith, A.J.; Burgess, D.L.; Kohrman, D.

    1994-09-01

    The Twirler mutation (Tw) causing cleft palate {plus_minus} cleft lip, vestibular defects and obesity is located within 0.5 cM of an ataxia locus (ax) on mouse chromosome 18. We identified a transgene-induced insertional mutation with vestibular and craniofacial defects that appears to be a new allele of Twirler. Mouse DNA flanking the transgene insertion site was isolated from a cosmid library. An evolutionarily conserved, zoo blot positive cosmid subclone was used to probe a human {lambda} genomic library. From the sequence of a highly homologous human {lambda} clone, we designed STS primers and screened a human P1 library. DNA from two positive P1 clones was hybridized with simple sequence probes, and a (CTAT){sub 12} repeat was detected. Analysis of 62 CEPH parents with primers flanking the repeat identified six alleles containing 9 to 14 copies of the repeat, at frequencies of 0.17, 0.17, 0.17, 0.27, 0.15 and 0.07, respectively. The observed heterozygosity was 49/62 with a calculated PIC value of 0.76. This polymorphic microsatellite marker, designated Umi3, was mapped to the predicted conserved human linkage group by analysis of somatic cell hybrid panels. The anticipated short distance between Umi3 and the disease genes will facilitate detection of linkage in small families. We would like to type appropriate human pedigrees with Umi3 in order to identify patients with inherited disorders homologous to the mouse mutations Twirler and ataxia.

  12. The effects of additional flora on the response of salmonella mutants lodged in the gastrointestinal tract.

    PubMed

    Wheeler, L A; Carter, J H; Ingelfinger, J A; Soderberg, F B; Goldman, P

    1977-02-01

    A histidine auxotroph of Salmonella typhimurium, strain TA1538, will lodge for several months in the gastrointestinal tract of otherwise germ-free rats and of rats additionally associated with bacteria characteristic of the normal flora such as Lactobacillus plantarum and Bacteroides vulgatus. In the presence of the additional flora, the concentration of strain TA1538 is diminished in the stomach but not in the lower gastrointestinal tract or in the feces. Following the ingestion of 2-nitrofluorene, there is an increase in the concentration of revertants in the feces which reflects that observed in the colon and cecum. A dose-response relationship can be demonstrated between the amount of 2-nitrofluorene ingested and the concentration of revertants in the feces. A given dose of 2-nitrofluorene, however, produces fewer revertants in the feces of rats with the additional flora than in the feces of rats associated only with strain TA1538. It is not clear whether the decreased number of revertants in the feces in the presence of the additional flora is a result of metabolic transformations of 2-nitrofluorene by B. vulgatus, which can be demonstrated in vitro, or a result of the displacement of strain TA1538 from the stomach. The rat associated with strain TA1538, or other Ames tester strains, may be useful for detecting carcinogens as mutagens within the gastrointestinal tract and for determining the influence of various constituents of the bacterial flora on the concentration of mutagenic compounds. PMID:318921

  13. Mutant MHC class II epitopes drive therapeutic immune responses to cancer

    PubMed Central

    Kreiter, Sebastian; Vormehr, Mathias; van de Roemer, Niels; Diken, Mustafa; Löwer, Martin; Diekmann, Jan; Boegel, Sebastian; Schrörs, Barbara; Vascotto, Fulvia; Castle, John C.; Tadmor, Arbel D.; Schoenberger, Stephen P.; Huber, Christoph; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient’s tumour possesses a unique set of mutations (‘the mutanome’) that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient’s individual tumour-specific mutations1. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4+ T cells. Vaccination with such CD4+ immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4+ T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo

  14. Fitness of Escherichia coli mutants with reduced susceptibility to tigecycline

    PubMed Central

    Linkevicius, Marius; Anderssen, Jytte Mark; Sandegren, Linus; Andersson, Dan I.

    2016-01-01

    Objectives The objective of this study was to determine the fitness of Escherichia coli mutants with reduced susceptibility to tigecycline after exposure to adverse conditions in vitro and in vivo. Methods Survival in response to low pH, bile salts, oxidative stress and human serum was examined for E. coli mutants with reduced susceptibility to tigecycline due to single mutations that caused increased efflux (marR, lon) or impaired LPS (rfaC, rfaE, lpcA). An in vitro competition assay was used to determine growth fitness defects. Competitive fitness was assessed using mouse infection models. MICs, exponential growth rates and expression levels of efflux-related genes were measured for genetically reconstructed double and triple mutants. Results The LPS mutants were 48–85-fold more susceptible to bile salts compared with the ERN mutants and the WT. As shown by in vitro competitions, the fitness reduction was 0.3%–13% for ERN mutants and ∼24% for LPS mutants. During in vivo survival experiments, LPS mutants were outcompeted by the WT strain in the thigh infection model. Constructed double ERN and LPS mutants showed additive and synergistic increases in tigecycline MICs. Conclusions Generally, reduced susceptibility to tigecycline caused a decrease in fitness under stressful in vitro and in vivo conditions with ERN mutants being fitter than LPS mutants. When combined, ERN mutations caused a synergistic increase in the MIC of tigecycline. These findings could explain why clinical resistance to tigecycline in E. coli is mainly associated with up-regulation of the AcrAB efflux system. PMID:26851608

  15. Normal or defective immune response to Hepatitis B vaccine in patients with diabetes and celiac disease

    PubMed Central

    Zanoni, Giovanna; Contreas, Giovanna; Valletta, Enrico; Gabrielli, Oretta; Mengoli, Carlo; Veneri, Dino

    2014-01-01

    A defective production of protective levels of antibodies to Hepatitis B (HB) vaccine is reported to occur in 4–10% of healthy subjects and a correlation with the presence of specific human leukocyte antigen (HLA) molecules, including DQ2, which also confers genetic predisposition to celiac disease (CD) and type I diabetes mellitus (T1DM), has been suggested. The aim of this study was to analyze the serological response to HB vaccine and measles-containing vaccines in 69 diabetic patients (T1DM), 42 patients with celiac disease (CD) and 79 healthy control subjects (CT). The median interval between the third dose of HB vaccine and serum collection was 6.8, 3.5, and 4.7 years for T1DM, CD and CT groups, respectively. 50/69 (72%) T1DM patients, 32/42 (76%) CD patients and 61/79 (77%) CT subjects showed protective anti-HBs antibodies after vaccination, with no statistically significant difference. On the contrary, a lower statistically significant difference was found in the mean HBsAb level of T1DM subjects when compared with the other two groups. No correlation between HLA DQ2 expression in T1DM and vaccine response was detected. The comparison of serological response to measles after vaccination also showed no statistically significant differences in the three groups. Contrasting results between these data and those reported in the literature might be due to differences in the time intervals between vaccination and testing. Prospective studies in pathological and healthy groups with the same age at HBV vaccination and with the same time interval for blood sample collection to determine antibody titers are necessary in order to provide more conclusive data. PMID:25483516

  16. Normal or defective immune response to Hepatitis B vaccine in patients with diabetes and celiac disease.

    PubMed

    Zanoni, Giovanna; Contreas, Giovanna; Valletta, Enrico; Gabrielli, Oretta; Mengoli, Carlo; Veneri, Dino

    2015-01-01

    A defective production of protective levels of antibodies to Hepatitis B (HB) vaccine is reported to occur in 4-10% of healthy subjects and a correlation with the presence of specific human leukocyte antigen (HLA) molecules, including DQ2, which also confers genetic predisposition to celiac disease (CD) and type I diabetes mellitus (T1DM), has been suggested.   The aim of this study was to analyze the serological response to HB vaccine and measles-containing vaccines in 69 diabetic patients (T1DM), 42 patients with celiac disease (CD) and 79 healthy control subjects (CT). The median interval between the third dose of HB vaccine and serum collection was 6.8, 3.5, and 4.7 years for T1DM, CD and CT groups, respectively. 50/69 (72%) T1DM patients, 32/42 (76%) CD patients and 61/79 (77%) CT subjects showed protective anti-HBs antibodies after vaccination, with no statistically significant difference. On the contrary, a lower statistically significant difference was found in the mean HBsAb level of T1DM subjects when compared with the other two groups. No correlation between HLA DQ2 expression in T1DM and vaccine response was detected. The comparison of serological response to measles after vaccination also showed no statistically significant differences in the three groups. Contrasting results between these data and those reported in the literature might be due to differences in the time intervals between vaccination and testing. Prospective studies in pathological and healthy groups with the same age at HBV vaccination and with the same time interval for blood sample collection to determine antibody titers are necessary in order to provide more conclusive data.

  17. Method for vibration response simulation and sensor placement optimization of a machine tool spindle system with a bearing defect.

    PubMed

    Cao, Hongrui; Niu, Linkai; He, Zhengjia

    2012-01-01

    Bearing defects are one of the most important mechanical sources for vibration and noise generation in machine tool spindles. In this study, an integrated finite element (FE) model is proposed to predict the vibration responses of a spindle bearing system with localized bearing defects and then the sensor placement for better detection of bearing faults is optimized. A nonlinear bearing model is developed based on Jones' bearing theory, while the drawbar, shaft and housing are modeled as Timoshenko's beam. The bearing model is then integrated into the FE model of drawbar/shaft/housing by assembling equations of motion. The Newmark time integration method is used to solve the vibration responses numerically. The FE model of the spindle-bearing system was verified by conducting dynamic tests. Then, the localized bearing defects were modeled and vibration responses generated by the outer ring defect were simulated as an illustration. The optimization scheme of the sensor placement was carried out on the test spindle. The results proved that, the optimal sensor placement depends on the vibration modes under different boundary conditions and the transfer path between the excitation and the response.

  18. Method for Vibration Response Simulation and Sensor Placement Optimization of a Machine Tool Spindle System with a Bearing Defect

    PubMed Central

    Cao, Hongrui; Niu, Linkai; He, Zhengjia

    2012-01-01

    Bearing defects are one of the most important mechanical sources for vibration and noise generation in machine tool spindles. In this study, an integrated finite element (FE) model is proposed to predict the vibration responses of a spindle bearing system with localized bearing defects and then the sensor placement for better detection of bearing faults is optimized. A nonlinear bearing model is developed based on Jones' bearing theory, while the drawbar, shaft and housing are modeled as Timoshenko's beam. The bearing model is then integrated into the FE model of drawbar/shaft/housing by assembling equations of motion. The Newmark time integration method is used to solve the vibration responses numerically. The FE model of the spindle-bearing system was verified by conducting dynamic tests. Then, the localized bearing defects were modeled and vibration responses generated by the outer ring defect were simulated as an illustration. The optimization scheme of the sensor placement was carried out on the test spindle. The results proved that, the optimal sensor placement depends on the vibration modes under different boundary conditions and the transfer path between the excitation and the response. PMID:23012514

  19. Response of the pearly eye melon fly Bactrocera cucurbitae(Coquillett)(Diptera:Tephritidae) mutant to host-associated visual cues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report on a pearly eye mutant (PEM) line generated from a single male Bactrocera cucurbitae collected in Kapoho, Hawaii. Crossing experiments with colony wild-type flies indicate that the locus controlling this trait is autosomal and the mutant allele is recessive. Experiments with females to ass...

  20. Auditory development in progressive motor neuronopathy mouse mutants.

    PubMed

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

    2009-11-01

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

  1. Auditory development in progressive motor neuronopathy mouse mutants.

    PubMed

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

    2009-11-01

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

  2. Angiogenic response to bioactive glass promotes bone healing in an irradiated calvarial defect.

    PubMed

    Leu, Ann; Stieger, Susanne M; Dayton, Paul; Ferrara, Katherine W; Leach, J Kent

    2009-04-01

    Localized radiation is an effective treatment modality for carcinomas, yet the associated reduction of the host vasculature significantly inhibits the tissue's regenerative capacity. Low concentrations of bioactive glass (BG) possess angiogenic potential, and we hypothesized that localized BG presentation would increase neovascularization and promote healing in an irradiated bone defect. An isolated calvarial region of Sprague-Dawley rats was irradiated 2 weeks before surgery. Bilateral critical-sized defects were created and immediately filled with a BG-loaded collagen sponge or an empty sponge as an internal control. Histological analysis of calvaria collected after 2 weeks demonstrated greater neovascularization within the defect in the presence of BG than with collagen alone. Noninvasive ultrasound imaging at 4 weeks detected less contrast agent in the brain below BG-treated defects than in the nearby untreated defects and images of treated defects acquired at 2 weeks. The reduced ability to detect contrast agent in BG-treated defects suggested greater attenuation of ultrasound signal due to early bone formation. Micro-computed tomography imaging at 12 weeks demonstrated significantly greater bone volume fraction within BG-treated defects than in controls. These results suggest that neovascularization induced by localized BG delivery promotes bone regeneration in this highly compromised model of bone healing and may offer an alternative approach to costly growth factors and their potential side-effects. PMID:18795867

  3. Smad-binding defective mutant of transforming growth factor beta type I receptor enhances tumorigenesis but suppresses metastasis of breast cancer cell lines.

    PubMed

    Tian, Fang; Byfield, Stacey DaCosta; Parks, W Tony; Stuelten, Christina H; Nemani, Deepa; Zhang, Ying E; Roberts, Anita B

    2004-07-01

    The role of transforming growth factor beta (TGF-beta) in carcinogenesis is complex, with tumor suppressor and pro-oncogenic activities depending on the particular tumor cell and its stage in malignant progression. We previously have demonstrated in breast cancer cell lines that Smad2/3 signaling played a dominant role in mediating tumor suppressor effects on well-differentiated breast cancer cell lines grown as xenografts and prometastatic effects on a more invasive, metastatic cell line. Our present data based on selective interference with activation of endogenous Smad2 and Smad3 by stable expression of a mutant form of the TGF-beta type I receptor (RImL45) unable to bind Smad2/3 but with a functional kinase again show that reduction in Smad2/3 signaling by expression of RImL45 enhanced the malignancy of xenografted tumors of the well-differentiated MCF10A-derived tumor cell line MCF10CA1h, resulting in formation of larger tumors with a higher proliferative index and more malignant histologic features. In contrast, expression of RImL45 in the more aggressive MCF10CA1a cell line strongly suppressed formation of lung metastases following tail vein injection. These results suggest a causal, dominant role for the endogenous Smad2/3 signaling pathway in the tumor suppressor and prometastatic activities of TGF-beta in these cells. Using an in vitro assay, we further show that non-Smad signaling pathways, including p38 and c-Jun NH(2)-terminal kinase, cooperate with TGF-beta/Smads in enhancing migration of metastatic MCF10CA1a cells, but that, although necessary for migration, these other pathways are not sufficient for metastasis.

  4. Identification of eight new mutations in familial neurogenic diabetes insipidus supports the concept that defective folding of the mutant provasopressin-neurophysin causes the disease

    SciTech Connect

    Rittig, S.; Siggaard, C.; Pedersen, E.B.

    1994-09-01

    Familial neurogenic diabetes insipidus (FNDI) is an autosomal dominant disorder with a uniform phenotype characterized by polyuria, polydipsia and a severe deficiency of arginine vasopressin (AVP). These abnormalities develop postnatally and appear to be due to progressive degeneration of AVP producing neurons. Previous studies in 8 FNDI kindreds have identified 5 different mutations in the gene that codes for the AVP-neurophysin (NP) precursor, AVP-NP. Four kindreds had the same missense mutation in the part of exon 1 that codes for the C-terminal amino acid of the signal peptide (SP). The other 4 had different missense mutations or a codon deletion in exon 2 which codes for the highly conserved part of NP. In the present study, the AVP-NP genes from 8 other kindreds with FNDI were sequenced bidirectionally using sequence and single-stranded DNA amplified by PCR with biotinylated primers flanking each of the 3 exons. We find that each of the 8 kindreds has a different, previously unreported mutation in either the SP coding part of exon 1, in exon 2 or in the variable, NP-coding part of exon 3. Combining these 8 new mutations with the 5 described previously reveals a distribution pattern that corresponds closely to the domains involved in the mutually interactive processes of AVP binding, folding and dimerization of NP. Based on these findings and the clinical features of FNDI, we postulate that the precursors produced by the mutant alleles are cytotoxic because they do not fold or dimerize properly for subsequent packaging and processing.

  5. Myeloid decidual dendritic cells and immunoregulation of pregnancy: defective responsiveness to Coxiella burnetii and Brucella abortus

    PubMed Central

    Gorvel, Laurent; Ben Amara, Amira; Ka, Mignane B.; Textoris, Julien; Gorvel, Jean-Pierre; Mege, Jean-Louis

    2014-01-01

    Dendritic cells (DCs) are a component of the placental immune system, but their role in pregnancy is still poorly understood. Decidual DCs (dDCs) were selected from at-term pregnancy on the basis of CD14 and CD11c expression. A phenotypic analysis revealed that dDCs are characterized by the expression of monocyte-derived DC (moDCs) markers and specific markers such as HLA-G and its ligand ILT4. As demonstrated by whole-genome microarray, dDCs expressed a specific gene program markedly distinct from that of moDCs; it included estrogen- and progesterone-regulated genes and genes encoding immunoregulatory cytokines, which is consistent with the context of foeto-maternal tolerance. A functional analysis of dDCs showed that they were unable to mature in response to bacterial ligands such as lipopolysaccharide or peptidoglycan, as assessed by the expression of HLA-DR, CD80, CD83, and CD86. When dDCs were incubated with bacteria known for their placenta tropism, Coxiella burnetii and Brucella abortus, they were also unable to mature and to produce inflammatory cytokines. It is likely that the defective maturation of dDCs and their inability to produce inflammatory cytokines is related to the spontaneous release of IL-10 by these cells. Taken together, these results suggest that dDCs exhibit an immunoregulatory program, which may favor the pathogenicity of C. burnetii or B. abortus. PMID:25566514

  6. Symmetry, strain, defects, and the nonlinear optical response of crystalline BaTiO3/silicon

    NASA Astrophysics Data System (ADS)

    Kormondy, Kristy; Abel, Stefan; Popoff, Youri; Sousa, Marilyne; Caimi, Daniele; Siegwart, Heinz; Marchiori, Chiara; Rossell, Marta; Demkov, Alex; Fompeyrine, Jean

    Recent progress has been made towards exploiting the linear electro-optic or Pockels effect in ferroelectric BaTiO3 (BTO) for novel integrated silicon photonics devices. In such structures, the crystalline symmetry and domain structure of BTO determine which electro-optic tensor elements are accessible under application of an external electric field. For epitaxial thin films of BTO on Si (001), the role of defects in strain relaxation can lead to very different crystalline symmetry even for films of identical thickness. Indeed, through geometric phase analysis of high-resolution scanning transmission electron microscopy images, we map changes of the in-plane and out-of-plane lattice parameters across two 80-nm-thick BTO films. A corresponding 20% difference in the effective electro-optic response was measured by analyzing induced rotation of the polarization of a laser beam (λ = 1550 nm) transmitted through lithographically defined electrodes. Understanding, controlling, and modelling the role of BTO symmetry in nonlinear optics is of fundamental importance for the development of a hybrid BTO/Si photonics platform.. Work supported by the NSF (IRES-1358111), AFOSR (FA9550-12-10494), and European Commission (FP7-ICT-2013-11-619456-SITOGA).

  7. Low sodium diet corrects the defect in lymphocyte beta-adrenergic responsiveness in hypertensive subjects.

    PubMed Central

    Feldman, R D; Lawton, W J; McArdle, W L

    1987-01-01

    To determine the role of dietary sodium intake in the reduction in beta-adrenergic sensitivity in hypertension, lymphocyte beta-receptors from 8 borderline hypertensive and 16 normotensive subjects were studied after 5 d on a high sodium diet (400 meq/d) and also following a low sodium diet (10 meq/d). During the high sodium diet, lymphocyte beta-receptor-stimulated adenylate cyclase activity, expressed as the relative increase over basal levels stimulated by the beta-agonist isoproterenol, was significantly (P less than 0.025) decreased in hypertensive (24 +/- 5%, mean +/- SE) compared with normotensive (42 +/- 4%) subjects. Neither beta-receptor density nor the proportion of nonsequestered beta-receptors differed between groups. A low sodium diet significantly increased beta-receptor-stimulated adenylate cyclase activity in hypertensives (low sodium, 51 +/- 7%; high sodium, 24 +/- 5%, P less than 0.025) to a level not different than that of normotensives (46 +/- 5%). Thus, reduced lymphocyte beta-receptor responsiveness in hypertensive subjects is not due to beta-receptor sequestration and is corrected on a low sodium diet. Dietary sodium may be an important factor in the beta-receptor defect in early hypertension. Images PMID:3025262

  8. Constitutive expression of the tzs gene from Agrobacterium tumefaciens virG mutant strains is responsible for improved transgenic plant regeneration in cotton meristem transformation.

    PubMed

    Ye, Xudong; Chen, Yurong; Wan, Yuechun; Hong, Yun-Jeong; Ruebelt, Martin C; Gilbertson, Larry A

    2016-03-01

    KEY MESSAGE : virG mutant strains of a nopaline type of Agrobacterium tumefaciens increase the transformation frequency in cotton meristem transformation. Constitutive cytokinin expression from the tzs gene in the virG mutant strains is responsible for the improvement. Strains of Agrobacterium tumefaciens were tested for their ability to improve cotton meristem transformation frequency. Two disarmed A. tumefaciens nopaline strains with either a virGN54D constitutively active mutation or virGI77V hypersensitive induction mutation significantly increased the transformation frequency in a cotton meristem transformation system. The virG mutant strains resulted in greener explants after three days of co-culture in the presence of light, which could be attributed to a cytokinin effect of the mutants. A tzs knockout strain of virGI77V mutant showed more elongated, less green explants and decreased cotton transformation frequency, as compared to a wild type parental strain, suggesting that expression of the tzs gene is required for transformation frequency improvement in cotton meristem transformation. In vitro cytokinin levels in culture media were tenfold higher in the virGN54D strain, and approximately 30-fold higher in the virGI77V strain, in the absence of acetosyringone induction, compared to the wild type strain. The cytokinin level in the virGN54D strain is further increased upon acetosyringone induction, while the cytokinin level in the virGI77V mutant is decreased by induction, suggesting that different tzs gene expression regulation mechanisms are present in the two virG mutant strains. Based on these data, we suggest that the increased cytokinin levels play a major role in increasing Agrobacterium attachment and stimulating localized division of the attached plant cells. PMID:26650837

  9. Virus growth and antibody responses following respiratory tract infection of ferrets and mice with WT and P/V mutants of the paramyxovirus Simian Virus 5

    PubMed Central

    Capraro, Gerald A.; Johnson, John B.; Kock, Nancy D.; Parks, Griffith D.

    2008-01-01

    P/V gene substitutions convert the non-cytopathic paramyxovirus Simian Virus 5 (SV5), which is a poor inducer of host cell responses in human tissue culture cells, into a mutant (P/V-CPI–) that induces high levels of apoptosis, interferon (IFN)-beta, and proinflammatory cytokines. However, the effect of SV5-P/V gene mutations on virus growth and adaptive immune responses in animals has not been determined. Here, we used two distinct animal model systems to test the hypothesis that SV5-P/V mutants which are more potent activators of innate responses in tissue culture will also elicit higher antiviral antibody responses. In mouse cells, in vitro studies identified a panel of SV5-P/V mutants that ranged in their ability to limit IFN responses. Intranasal infection of mice with these WT and P/V mutant viruses elicited equivalent anti-SV5 IgG responses at all doses tested, and viral titers recovered from the respiratory tract were indistinguishable. In primary cultures of ferret lung fibroblasts, WT rSV5 and P/V-CPI– viruses had phenotypes similar to those established in human cell lines, including differential induction of IFN secretion, IFN signaling and apoptosis. Intranasal infection of ferrets with a low dose of WT rSV5 elicited ~500 fold higher anti-SV5 serum IgG responses compared to the P/V-CPI– mutant, and this correlated with overall higher viral titers for the WT virus in tracheal tissues. There was a dose-dependent increase in antibody response to infection of ferrets with P/V-CPI–, but not with WT rSV5. Together our data indicate that WT rSV5 and P/V mutants can elicit distinct innate and adaptive immunity phenotypes in the ferret animal model system, but not in the mouse system. We present a model for the effect of P/V gene substitutions on SV5 growth and immune responses in vivo. PMID:18456301

  10. DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe.

    PubMed Central

    al-Khodairy, F; Carr, A M

    1992-01-01

    We have tested mutants corresponding to 20 DNA repair genes of the fission yeast Schizosaccharomyces pombe for their ability to arrest in G2 after DNA damage. Of the mutants tested, four are profoundly defective in this damage dependent G2 arrest. In addition, these four mutants are highly sensitive to a transient inhibition of DNA synthesis by hydroxyurea. This suggests that the pathway responsible for the recognition of DNA damage and the subsequent mitotic arrest, shares many functions with the mechanism that controls the dependency of mitosis on the completion of S phase. The phenotype of these checkpoint rad mutants in wee mutant backgrounds indicate that the G2 arrest response is mediated either through, or in parallel with, the activity of the cdc2 gene product. Images PMID:1563350

  11. EFFECT OF TRANSLOCATION DEFECTIVE REVERSE TRANSCRIPTASE INHIBITORS ON THE ACTIVITY OF N348I, A CONNECTION SUBDOMAIN DRUG RESISTANT HIV-1 REVERSE TRANSCRIPTASE MUTANT

    PubMed Central

    MICHAILIDIS, E.; SINGH, K.; RYAN, E.M.; HACHIYA, A.; ONG, Y.T.; KIRBY, K.A.; MARCHAND, B.; KODAMA, E.N.; MITSUYA, H.; PARNIAK, M.A.; SARAFIANOS, S.G.

    2013-01-01

    4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a highly potent inhibitor of HIV-1 reverse transcriptase (RT). We have previously shown that its exceptional antiviral activity stems from a unique mechanism of action that is based primarily on blocking translocation of RT; therefore we named EFdA a Translocation Defective RT Inhibitor (TDRTI). The N348I mutation at the connection subdomain (CS) of HIV-1 RT confers clinically significant resistance to both nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). In this study we tested EFdA-triphosphate (TP) together with a related compound, ENdA-TP (4′-ethynyl-2-amino-2′-deoxyadenosine triphosphate) against HIV-1 RTs that carry clinically relevant drug resistance mutations: N348I, D67N/K70R/L210Q/T215F, D67N/K70R/L210Q/T215F/N348I, and A62V/V75I/F77L/F116Y/Q151M. We demonstrate that these enzymes remain susceptible to TDRTIs. Similar to WT RT, the N348I RT is inhibited by EFdA mainly at the point of incorporation through decreased translocation. In addition, the N348I substitution decreases the RNase H cleavage of DNA terminated with EFdA-MP (T/PEFdA-MP). Moreover, N348I RT unblocks EFdA-terminated primers with similar efficiency as the WT enzyme, and further enhances EFdA unblocking in the background of AZT-resistance mutations. This study provides biochemical insights into the mechanism of inhibition of N348I RT by TDRTIs and highlights the excellent efficacy of this class of inhibitors against WT and drug-resistant HIV-1 RTs. PMID:23273211

  12. Effect of translocation defective reverse transcriptase inhibitors on the activity of N348I, a connection subdomain drug resistant HIV-1 reverse transcriptase mutant.

    PubMed

    Michailidis, E; Singh, K; Ryan, E M; Hachiya, A; Ong, Y T; Kirby, K A; Marchand, B; Kodama, E N; Mitsuya, H; Parniak, M A; Sarafianos, S G

    2012-01-01

    4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a highly potent inhibitor of HIV-1 reverse transcriptase (RT). We have previously shown that its exceptional antiviral activity stems from a unique mechanism of action that is based primarily on blocking translocation of RT; therefore we named EFdA a Translocation Defective RT Inhibitor (TDRTI). The N348I mutation at the connection subdomain (CS) of HIV-1 RT confers clinically significant resistance to both nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). In this study we tested EFdA-triphosphate (TP) together with a related compound, ENdA-TP (4'-ethynyl-2-amino-2'-deoxdyadenosine triphosphate) against HIV-1 RTs that carry clinically relevant drug resistance mutations: N348I, D67N/K70R/L210Q/T215F, D67N/K70R/L210Q/T215F/N348I, and A62V/V5I/F77L/F116Y/Q151M. We demonstrate that these enzymes remain susceptible to TDRTIs. Similar to WT RT, the N348I RT is inhibited by EFdA mainly at the point of incorporation through decreased translocation. In addition, the N348I substitution decreases the RNase H cleavage of DNA terminated with EFdA-MP (T/P(EFdA-MP)). Moreover, N348I RT unblocks EFdA-terminated primers with similar efficiency as the WT enzyme, and further enhances EFdA unblocking in the background of AZT-resistance mutations. This study provides biochemical insights into the mechanism of inhibition of N348I RT by TDRTIs and highlights the excellent efficacy of this class of inhibitors against WT and drug-resistant HIV-1 RTs. PMID:23273211

  13. Mutant MCP-1 Protein Delivery from Layer-by-Layer Coatings on Orthopaedic Implants to Modulate Inflammatory Response

    PubMed Central

    Keeney, Michael; Waters, Heather; Barcay, Katie; Jiang, Xinyi; Yao, Zhenyu; Pajarinen, Jukka; Egashira, Kensuke; Goodman, Stuart; Yang, Fan

    2013-01-01

    Total joint replacement (TJR) is a common and effective surgical procedure for hip or knee joint reconstruction. However, the production of wear particles is inevitable for all TJRs, which activates macrophages and initiates an inflammatory cascade often resulting in bone loss, prosthetic loosening and eventual TJR failure. Macrophage Chemoattractant Protein-1 (MCP-1) is one of the most potent cytokines responsible for macrophage cell recruitment, and previous studies suggest that mutant MCP-1 proteins such as 7ND may be used as a decoy drug to block the receptor and reduce inflammatory cell recruitment. Here we report the development of a biodegradable, layer-by-layer (LBL) coating platform that allows efficient loading and controlled release of 7ND proteins from the surface of orthopaedic implants using as few as 14 layers. Scanning electron microscopy and fluorescence imaging confirmed effective coating using the LBL procedure on titanium rods. 7ND protein loading concentration and release kinetics can be modulated by varying the polyelectrolytes of choice, the polymer chemistry, the pH of the polyelectrolyte solution, and the degradation rate of the LBL assembly. The released 7ND from LBL coating retained its bioactivity and effectively reduced macrophage migration towards MCP-1. Finally, the LBL coating remained intact following a femoral rod implantation procedure as determined by immunostaining of the 7ND coating. The LBL platform reported herein may be applied for in situ controlled release of 7ND protein from orthopaedic implants, to reduce wear particle-induced inflammatory responses in an effort to prolong the lifetime of implants. PMID:24075408

  14. Transcriptomic profiling of Arabidopsis thaliana mutant pad2.1 in response to combined cold and osmotic stress.

    PubMed

    Kumar, Deepak; Datta, Riddhi; Hazra, Saptarshi; Sultana, Asma; Mukhopadhyay, Ria; Chattopadhyay, Sharmila

    2015-01-01

    The contribution of glutathione (GSH) in stress tolerance, defense response and antioxidant signaling is an established fact. In this study transcriptome analysis of pad2.1, an Arabidopsis thaliana mutant, after combined osmotic and cold stress treatment has been performed to explore the intricate position of GSH in the stress and defense signaling network in planta. Microarray data revealed the differential regulation of about 1674 genes in pad2.1 amongst which 973 and 701 were significantly up- and down-regulated respectively. Gene enrichment, functional pathway analysis by DAVID and MapMan analysis identified various stress and defense related genes viz. members of heat shock protein family, peptidyl prolyl isomerase (PPIase), thioredoxin peroxidase (TPX2), glutathione-S-transferase (GST), NBS-LRR type resistance protein etc. as down-regulated. The expression pattern of the above mentioned stress and defense related genes and APETALA were also validated by comparative proteomic analysis of combined stress treated Col-0 and pad2.1. Functional annotation noted down-regulation of UDP-glycosyl transferase, 4-coumarate CoA ligase 8, cinnamyl alcohol dehydrogenase 4 (CAD4), ACC synthase and ACC oxidase which are the important enzymes of phenylpropanoid, lignin and ethylene (ET) biosynthetic pathway respectively. Since the only difference between Col-0 (Wild type) and pad2.1 is the content of GSH, so, this study suggested that in addition to its association with specific stress responsive genes and proteins, GSH provides tolerance to plants by its involvement with phenylpropanoid, lignin and ET biosynthesis under stress conditions. PMID:25822199

  15. Structural and biophysical analysis of sero-specific immune responses using epitope grafted Dengue ED3 mutants.

    PubMed

    Kulkarni, Manjiri R; Islam, Monirul M; Numoto, Nobutaka; Elahi, Montasir; Mahib, Mamunur R; Ito, Nobutoshi; Kuroda, Yutaka

    2015-10-01

    Dengue fever is a re-emerging tropical disease and its severe form is caused by cross-reactivity between its four serotypes (DEN1, DEN2, DEN3 and DEN4). The third domain of the viral envelope protein (ED3) contains the two major putative epitopes and is a highly suitable model protein for examining the molecular determinants of a virus' sero-specificity. Here we examine d the sero-specificity and cross-reactivity of the immune response against DEN3 and DEN4 ED3 using six epitope grafted ED3 variants where the surface-exposed epitope residues from DEN3 ED3 were switched to those of DEN4 ED3 and vice versa. We prepared anti-DEN3 and anti-DEN4 ED3 serum by immunizing Swiss albino mice and measured their reactivities against all six grafted mutants. As expected, both sera exhibited strong reactivity against its own serotype's ED3, and little cross-reactivity against their counterpart serotype's ED3s. E2 played a major role in the sero-specificity of anti-DEN3 serum, whereas E1 was important for DEN4 ED3's sero-specificity. Next, the reactivity patterns corroborated our working hypothesis that sero-specificity could be transferred by grafting the surface exposed epitope residues from one serotype to the other. To analyze the above results from a structural viewpoint, we determined the crystal structure of a DEN4 ED3 variant, where E2 was grafted from DEN3 ED3, at 2.78Å resolution and modeled the structures of the five remaining grafted variants by assuming that the overall backbone remained unchanged. The examination of the electrostatic and molecular surfaces of the variants suggested some further rationale for the sero-specificity of the immune responses.

  16. Radionuclide analysis of right and left ventricular response to exercise in patients with atrial and ventricular septal defects

    SciTech Connect

    Peter, C.A.; Bowyer, K.; Jones, R.H.

    1983-03-01

    In patients with ventricular or atrial septal defect, the ventricle which is chronically volume overloaded might not appropriately respond to increased demand for an augmentation in output and thereby might limit total cardiac function. In this study we simultaneously measured right and left ventricular response to exercise in 10 normal individuals, 10 patients with ventricular septal defect (VSD), and 10 patients with atrial septal defect (ASD). The normal subjects increased both right and left ventricular ejection fraction, end-diastolic volume, and stroke volume to achieve a higher cardiac output during exercise. Patients with VSD failed to increase right ventricular ejection fraction, but increased right ventricular end-diastolic volume and stroke volume. Left ventricular end-diastolic volume did not increase in these patients but ejection fraction, stroke volume, and forward left ventricular output achieved during exercise were comparable to the response observed in healthy subjects. In the patients with ASD, no rest-to-exercise change occurred in either right ventricular ejection fraction, end-diastolic volume, or stroke volume. In addition, left ventricular end-diastolic volume failed to increase, and despite an increase in ejection fraction, left ventricular stroke volume remained unchanged from rest to exercise. Therefore, cardiac output was augmented only by the heart rate increase in these patients. Right ventricular function appeared to be the major determinant of total cardiac output during exercise in patients with cardiac septal defects and left-to-right shunt.

  17. p53-Dependent DNA damage response sensitive to editing-defective tRNA synthetase in zebrafish.

    PubMed

    Song, Youngzee; Shi, Yi; Carland, Tristan M; Lian, Shanshan; Sasaki, Tomoyuki; Schork, Nicholas J; Head, Steven R; Kishi, Shuji; Schimmel, Paul

    2016-07-26

    Brain and heart pathologies are caused by editing defects of transfer RNA (tRNA) synthetases, which preserve genetic code fidelity by removing incorrect amino acids misattached to tRNAs. To extend understanding of the broader impact of synthetase editing reactions on organismal homeostasis, and based on effects in bacteria ostensibly from small amounts of mistranslation of components of the replication apparatus, we investigated the sensitivity to editing of the vertebrate genome. We show here that in zebrafish embryos, transient overexpression of editing-defective valyl-tRNA synthetase (ValRS(ED)) activated DNA break-responsive H2AX and p53-responsive downstream proteins, such as cyclin-dependent kinase (CDK) inhibitor p21, which promotes cell-cycle arrest at DNA damage checkpoints, and Gadd45 and p53R2, with pivotal roles in DNA repair. In contrast, the response of these proteins to expression of ValRS(ED) was abolished in p53-deficient fish. The p53-activated downstream signaling events correlated with suppression of abnormal morphological changes caused by the editing defect and, in adults, reversed a shortened life span (followed for 2 y). Conversely, with normal editing activities, p53-deficient fish have a normal life span and few morphological changes. Whole-fish deep sequencing showed genomic mutations associated with the editing defect. We suggest that the sensitivity of p53 to expression of an editing-defective tRNA synthetase has a critical role in promoting genome integrity and organismal homeostasis. PMID:27402763

  18. Isolation of New Gravitropic Mutants under Hypergravity Conditions

    PubMed Central

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

    2016-01-01

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

  19. The Transcriptional Response of Lactobacillus sanfranciscensis DSM 20451T and Its tcyB Mutant Lacking a Functional Cystine Transporter to Diamide Stress

    PubMed Central

    Stetina, Mandy; Behr, Jürgen

    2014-01-01

    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 20451T (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. PMID:24795368

  20. Molecular heterogeneity assessment by next-generation sequencing and response to gefitinib of EGFR mutant advanced lung adenocarcinoma.

    PubMed

    Bria, Emilio; Pilotto, Sara; Amato, Eliana; Fassan, Matteo; Novello, Silvia; Peretti, Umberto; Vavalà, Tiziana; Kinspergher, Stefania; Righi, Luisella; Santo, Antonio; Brunelli, Matteo; Corbo, Vincenzo; Giglioli, Eliana; Sperduti, Isabella; Milella, Michele; Chilosi, Marco; Scarpa, Aldo; Tortora, Giampaolo

    2015-05-20

    Cancer molecular heterogeneity might explain the variable response of EGFR mutant lung adenocarcinomas to tyrosine kinase inhibitors (TKIs). We assessed the mutational status of 22 cancer genes by next-generation sequencing (NGS) in poor, intermediate or good responders to first-line gefitinib. Clinical outcome was correlated with Additional Coexisting Mutations (ACMs) and the EGFR Proportion of Mutated Alleles (PMA). Thirteen ACMs were found in 10/17 patients: TP53 (n=6), KRAS (n=2), CTNNB1 (n=2), PIK3CA, SMAD4 and MET (n=1 each). TP53 mutations were exclusive of poor/intermediate responders (66.7% versus 0, p=0.009). Presence of ACMs significantly affected both PFS (median 3.0 versus 12.3 months, p=0.03) and survival (3.6 months versus not reached, p=0.03). TP53 mutation was the strongest negative modifier (median PFS 4.0 versus 14.0 months). Higher EGFR PMA was present in good versus poor/intermediate responders. Median PFS and survival were longer in patients with EGFR PMA ≥0.36 (12.0 versus 4.0 months, p=0.31; not reached versus 18.0 months, p=0.59). Patients with an EGFR PMA ≥0.36 and no ACMs fared significantly better (p=0.03), with a trend towards increased survival (p=0.06). Our exploratory data suggest that a quantitative (PMA) and qualitative (ACMs) molecular heterogeneity assessment using NGS might be useful for a better selection of patients.

  1. Molecular heterogeneity assessment by next-generation sequencing and response to gefitinib of EGFR mutant advanced lung adenocarcinoma

    PubMed Central

    Amato, Eliana; Fassan, Matteo; Novello, Silvia; Peretti, Umberto; Vavalà, Tiziana; Kinspergher, Stefania; Righi, Luisella; Santo, Antonio; Brunelli, Matteo; Corbo, Vincenzo; Giglioli, Eliana; Sperduti, Isabella; Milella, Michele; Chilosi, Marco; Scarpa, Aldo; Tortora, Giampaolo

    2015-01-01

    Cancer molecular heterogeneity might explain the variable response of EGFR mutant lung adenocarcinomas to tyrosine kinase inhibitors (TKIs). We assessed the mutational status of 22 cancer genes by next-generation sequencing (NGS) in poor, intermediate or good responders to first-line gefitinib. Clinical outcome was correlated with Additional Coexisting Mutations (ACMs) and the EGFR Proportion of Mutated Alleles (PMA). Thirteen ACMs were found in 10/17 patients: TP53 (n=6), KRAS (n=2), CTNNB1 (n=2), PIK3CA, SMAD4 and MET (n=1 each). TP53 mutations were exclusive of poor/intermediate responders (66.7% versus 0, p=0.009). Presence of ACMs significantly affected both PFS (median 3.0 versus 12.3 months, p=0.03) and survival (3.6 months versus not reached, p=0.03). TP53 mutation was the strongest negative modifier (median PFS 4.0 versus 14.0 months). Higher EGFR PMA was present in good versus poor/intermediate responders. Median PFS and survival were longer in patients with EGFR PMA ≥0.36 (12.0 versus 4.0 months, p=0.31; not reached versus 18.0 months, p=0.59). Patients with an EGFR PMA ≥0.36 and no ACMs fared significantly better (p=0.03), with a trend towards increased survival (p=0.06). Our exploratory data suggest that a quantitative (PMA) and qualitative (ACMs) molecular heterogeneity assessment using NGS might be useful for a better selection of patients. PMID:25904052

  2. Desensitization contributes to the synaptic response of gain-of-function mutants of the muscle nicotinic receptor.

    PubMed

    Elenes, Sergio; Ni, Ying; Cymes, Gisela D; Grosman, Claudio

    2006-11-01

    Although the muscle nicotinic receptor (AChR) desensitizes almost completely in the steady presence of high concentrations of acetylcholine (ACh), it is well established that AChRs do not accumulate in desensitized states under normal physiological conditions of neurotransmitter release and clearance. Quantitative considerations in the framework of plausible kinetic schemes, however, lead us to predict that mutations that speed up channel opening, slow down channel closure, and/or slow down the dissociation of neurotransmitter (i.e., gain-of-function mutations) increase the extent to which AChRs desensitize upon ACh removal. In this paper, we confirm this prediction by applying high-frequency trains of brief ( approximately 1 ms) ACh pulses to outside-out membrane patches expressing either lab-engineered or naturally occurring (disease-causing) gain-of-function mutants. Entry into desensitization was evident in our experiments as a frequency-dependent depression in the peak value of succesive macroscopic current responses, in a manner that is remarkably consistent with the theoretical expectation. We conclude that the comparatively small depression of the macroscopic currents observed upon repetitive stimulation of the wild-type AChR is due, not to desensitization being exceedingly slow but, rather, to the particular balance between gating, entry into desensitization, and ACh dissociation rate constants. Disruption of this fine balance by, for example, mutations can lead to enhanced desensitization even if the kinetics of entry into, and recovery from, desensitization themselves are not affected. It follows that accounting for the (usually overlooked) desensitization phenomenon is essential for the correct interpretation of mutagenesis-driven structure-function relationships and for the understanding of pathological synaptic transmission at the vertebrate neuromuscular junction.

  3. Role of trichomes in defense against herbivores: comparison of herbivore response to woolly and hairless trichome mutants in tomato (Solanum lycopersicum).

    PubMed

    Tian, Donglan; Tooker, John; Peiffer, Michelle; Chung, Seung Ho; Felton, Gary W

    2012-10-01

    Trichomes contribute to plant resistance against herbivory by physical and chemical deterrents. To better understand their role in plant defense, we systemically studied trichome morphology, chemical composition and the response of the insect herbivores Helicoverpa zea and Leptinotarsa decemlineata (Colorado potato beetle = CPB) on the tomato hairless (hl), hairy (woolly) mutants and wild-type Rutgers (RU) and Alisa Craig (AC) plants. Hairless mutants showed reduced number of twisted glandular trichomes (types I, IV, VI and VII) on leaf and stem compared to wild-type Rutgers (RU), while woolly mutants showed high density of non-glandular trichomes (types II, III and V) but only on the leaf. In both mutants, trichome numbers were increased by methyl jasmonate (MeJA), but the types of trichomes present were not affected by MeJA treatment. Glandular trichomes contained high levels of monoterpenes and sesquiterpenes. A similar pattern of transcript accumulation was observed for monoterpene MTS1 (=TPS5) and sesquiterpene synthase SST1 (=TPS9) genes in trichomes. While high density of non-glandular trichome on leaves negatively influenced CPB feeding behavior and growth, it stimulated H. zea growth. High glandular trichome density impaired H. zea growth, but had no effect on CPB. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that glandular trichomes highly express protein inhibitors (PIN2), polyphenol oxidase (PPOF) and hydroperoxide lyase (HPL) when compared to non-glandular trichomes. The SlCycB2 gene, which participates in woolly trichome formation, was highly expressed in the woolly mutant trichomes. PIN2 in trichomes was highly induced by insect feeding in both mutant and wild-type plants. Thus, both the densities of trichomes and the chemical defenses residing in the trichomes are inducible. PMID:22552638

  4. Identification of genes potentially involved in solute stress response in Sphingomonas wittichii RW1 by transposon mutant recovery

    PubMed Central

    Coronado, Edith; Roggo, Clémence; van der Meer, Jan R.

    2014-01-01

    The term water stress refers to the effects of low water availability on microbial growth and physiology. Water availability has been proposed as a major constraint for the use of microorganisms in contaminated sites with the purpose of bioremediation. Sphingomonas wittichii RW1 is a bacterium capable of degrading the xenobiotic compounds dibenzofuran and dibenzo-p-dioxin, and has potential to be used for targeted bioremediation. The aim of the current work was to identify genes implicated in water stress in RW1 by means of transposon mutagenesis and mutant growth experiments. Conditions of low water potential were mimicked by adding NaCl to the growth media. Three different mutant selection or separation method were tested which, however recovered different mutants. Recovered transposon mutants with poorer growth under salt-induced water stress carried insertions in genes involved in proline and glutamate biosynthesis, and further in a gene putatively involved in aromatic compound catabolism. Transposon mutants growing poorer on medium with lowered water potential also included ones that had insertions in genes involved in more general functions such as transcriptional regulation, elongation factor, cell division protein, RNA polymerase β or an aconitase. PMID:25408691

  5. A Forward Genetic Screen and Whole Genome Sequencing Identify Deflagellation Defective Mutants in Chlamydomonas, Including Assignment of ADF1 as a TRP Channel

    PubMed Central

    Hilton, Laura K.; Meili, Fabian; Buckoll, Paul D.; Rodriguez-Pike, Julie C.; Choutka, Courtney P.; Kirschner, Jaime A.; Warner, Freda; Lethan, Mette; Garces, Fabian A.; Qi, Jingnan; Quarmby, Lynne M.

    2016-01-01

    With rare exception, ciliated cells entering mitosis lose their cilia, thereby freeing basal bodies to serve as centrosomes in the formation of high-fidelity mitotic spindles. Cilia can be lost by shedding or disassembly, but either way, it appears that the final release may be via a coordinated severing of the nine axonemal outer doublet microtubules linking the basal body to the ciliary transition zone. Little is known about the mechanism or regulation of this important process. The stress-induced deflagellation response of Chlamydomonas provides a basis to identifying key players in axonemal severing. In an earlier screen we uncovered multiple alleles for each of three deflagellation genes, ADF1, FA1, and FA2. Products of the two FA genes localize to the site of axonemal severing and encode a scaffolding protein and a member of the NIMA-related family of ciliary-cell cycle kinases. The identity of the ADF1 gene remained elusive. Here, we report a new screen using a mutagenesis that yields point mutations in Chlamydomonas, an enhanced screening methodology, and whole genome sequencing. We isolated numerous new alleles of the three known genes, and one or two alleles each of at least four new genes. We identify ADF1 as a TRP ion channel, which we suggest may reside at the flagellar transition zone. PMID:27520959

  6. Insights into Metabolic Mechanisms Underlying Folate-Responsive Neural Tube Defects: A Minireview

    PubMed Central

    Beaudin, Anna E.; Stover, Patrick J.

    2015-01-01

    Neural tube defects (NTDs), including anencephaly and spina bifida, arise from the failure of neurulation during early embryonic development. Neural tube defects are common birth defects with a heterogenous and multifactorial etiology with interacting genetic and environmental risk factors. Although the mechanisms resulting in failure of neural tube closure are unknown, up to 70% of NTDs can be prevented by maternal folic acid supplementation. However, the metabolic mechanisms underlying the association between folic acid and NTD pathogenesis have not been identified. This review summarizes our current understanding of the mechanisms by which impairments in folate metabolism might ultimately lead to failure of neural tube closure, with an emphasis on untangling the relative contributions of nutritional deficiency and genetic risk factors to NTD pathogenesis. PMID:19180567

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

    PubMed Central

    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

  8. Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions.

    PubMed Central

    Noel, K D; Sanchez, A; Fernandez, L; Leemans, J; Cevallos, M A

    1984-01-01

    Rhizobium phaseoli CFN42 DNA was mutated by random insertion of Tn5 from suicide plasmid pJB4JI to obtain independently arising strains that were defective in symbiosis with Phaseolus vulgaris but grew normally outside the plant. When these mutants were incubated with the plant, one did not initiate visible nodule tissue (Nod-), seven led to slow nodule development (Ndv), and two led to superficially normal early nodule development but lacked symbiotic nitrogenase activity (Sna-). The Nod- mutant lacked the large transmissible indigenous plasmid pCFN42d that has homology to Klebsiella pneumoniae nitrogenase (nif) genes. The other mutants had normal plasmid content. In the two Sna- mutants and one Ndv mutant, Tn5 had inserted into plasmid pCFN42d outside the region of nif homology. The insertions of the other Ndv mutants were apparently in the chromosome. They were not in plasmids detected on agarose gels, and, in contrast to insertions on indigenous plasmids, they were transmitted in crosses to wild-type strain CFN42 at the same frequency as auxotrophic markers and with the same enhancement of transmission by conjugation plasmid R68.45. In these Ndv mutants the Tn5 insertions were the same as or very closely linked to mutations causing the Ndv phenotype. However, in two mutants with Tn5 insertions on plasmid pCFN42d, an additional mutation on the same plasmid, rather than Tn5, was responsible for the Sna- or Ndv phenotype. When plasmid pJB4JI was transferred to two other R. phaseoli strains, analysis of symbiotic mutants was complicated by Tn5-containing deleted forms of pJB4JI that were stably maintained. Images PMID:6325385

  9. Histomorphometric analysis of tissue responses to bioactive glass implants in critical defects in rat calvaria.

    PubMed

    Cardoso, Ana Karina M V; Barbosa, Aryon de Almeida; Miguel, Fúlvio Borges; Marcantonio, Elcio; Farina, Marcos; Soares, Glória Dulce de Almeida; Rosa, Fabiana Paim

    2006-01-01

    The aim of this study was to evaluate the osteogenic behavior of two chemically similar bioactive glass products (Biogranand Perioglas) implanted in critical bone defects in rat calvaria. Thirty-six tr