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Sample records for mutant cloth-ears shows

  1. Analysis of the mouse mutant Cloth-ears shows a role for the voltage-gated sodium channel Scn8a in peripheral neural hearing loss.

    PubMed

    Mackenzie, F E; Parker, A; Parkinson, N J; Oliver, P L; Brooker, D; Underhill, P; Lukashkina, V A; Lukashkin, A N; Holmes, C; Brown, S D M

    2009-10-01

    Deafness is the most common sensory disorder in humans and the aetiology of genetic deafness is complex. Mouse mutants have been crucial in identifying genes involved in hearing. However, many deafness genes remain unidentified. Using N-ethyl N-nitrosourea (ENU) mutagenesis to generate new mouse models of deafness, we identified a novel semi-dominant mouse mutant, Cloth-ears (Clth). Cloth-ears mice show reduced acoustic startle response and mild hearing loss from approximately 30 days old. Auditory-evoked brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) analyses indicate that the peripheral neural auditory pathway is impaired in Cloth-ears mice, but that cochlear function is normal. In addition, both Clth/Clth and Clth/+ mice display paroxysmal tremor episodes with behavioural arrest. Clth/Clth mice also show a milder continuous tremor during movement and rest. Longitudinal phenotypic analysis showed that Clth/+ and Clth/Clth mice also have complex defects in behaviour, growth, neurological and motor function. Positional cloning of Cloth-ears identified a point mutation in the neuronal voltage-gated sodium channel alpha-subunit gene, Scn8a, causing an aspartic acid to valine (D981V) change six amino acids downstream of the sixth transmembrane segment of the second domain (D2S6). Complementation testing with a known Scn8a mouse mutant confirmed that this mutation is responsible for the Cloth-ears phenotype. Our findings suggest a novel role for Scn8a in peripheral neural hearing loss and paroxysmal motor dysfunction. PMID:19737145

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

    PubMed Central

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

    2014-01-01

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

  3. The Identification of Zebrafish Mutants Showing Alterations in Senescence-Associated Biomarkers

    PubMed Central

    Uchiyama, Junzo; Koshimizu, Eriko; Qi, Jie; Nanjappa, Purushothama; Imamura, Shintaro; Islam, Asiful; Neuberg, Donna; Amsterdam, Adam; Roberts, Thomas M.

    2008-01-01

    There is an interesting overlap of function in a wide range of organisms between genes that modulate the stress responses and those that regulate aging phenotypes and, in some cases, lifespan. We have therefore screened mutagenized zebrafish embryos for the altered expression of a stress biomarker, senescence-associated β-galactosidase (SA-β-gal) in our current study. We validated the use of embryonic SA-β-gal production as a screening tool by analyzing a collection of retrovirus-insertional mutants. From a pool of 306 such mutants, we identified 11 candidates that showed higher embryonic SA-β-gal activity, two of which were selected for further study. One of these mutants is null for a homologue of Drosophila spinster, a gene known to regulate lifespan in flies, whereas the other harbors a mutation in a homologue of the human telomeric repeat binding factor 2 (terf2) gene, which plays roles in telomere protection and telomere-length regulation. Although the homozygous spinster and terf2 mutants are embryonic lethal, heterozygous adult fish are viable and show an accelerated appearance of aging symptoms including lipofuscin accumulation, which is another biomarker, and shorter lifespan. We next used the same SA-β-gal assay to screen chemically mutagenized zebrafish, each of which was heterozygous for lesions in multiple genes, under the sensitizing conditions of oxidative stress. We obtained eight additional mutants from this screen that, when bred to homozygosity, showed enhanced SA-β-gal activity even in the absence of stress, and further displayed embryonic neural and muscular degenerative phenotypes. Adult fish that are heterozygous for these mutations also showed the premature expression of aging biomarkers and the accelerated onset of aging phenotypes. Our current strategy of mutant screening for a senescence-associated biomarker in zebrafish embryos may thus prove to be a useful new tool for the genetic dissection of vertebrate stress response and

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

    PubMed

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

    2014-05-01

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

  7. 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

  8. Open-gate mutants of the mammalian proteasome show enhanced ubiquitin-conjugate degradation

    PubMed Central

    Choi, Won Hoon; de Poot, Stefanie A. H.; Lee, Jung Hoon; Kim, Ji Hyeon; Han, Dong Hoon; Kim, Yun Kyung; Finley, Daniel; Lee, Min Jae

    2016-01-01

    When in the closed form, the substrate translocation channel of the proteasome core particle (CP) is blocked by the convergent N termini of α-subunits. To probe the role of channel gating in mammalian proteasomes, we deleted the N-terminal tail of α3; the resulting α3ΔN proteasomes are intact but hyperactive in the hydrolysis of fluorogenic peptide substrates and the degradation of polyubiquitinated proteins. Cells expressing the hyperactive proteasomes show markedly elevated degradation of many established proteasome substrates and resistance to oxidative stress. Multiplexed quantitative proteomics revealed ∼200 proteins with reduced levels in the mutant cells. Potentially toxic proteins such as tau exhibit reduced accumulation and aggregate formation. These data demonstrate that the CP gate is a key negative regulator of proteasome function in mammals, and that opening the CP gate may be an effective strategy to increase proteasome activity and reduce levels of toxic proteins in cells. PMID:26957043

  9. Open-gate mutants of the mammalian proteasome show enhanced ubiquitin-conjugate degradation.

    PubMed

    Choi, Won Hoon; de Poot, Stefanie A H; Lee, Jung Hoon; Kim, Ji Hyeon; Han, Dong Hoon; Kim, Yun Kyung; Finley, Daniel; Lee, Min Jae

    2016-01-01

    When in the closed form, the substrate translocation channel of the proteasome core particle (CP) is blocked by the convergent N termini of α-subunits. To probe the role of channel gating in mammalian proteasomes, we deleted the N-terminal tail of α3; the resulting α3ΔN proteasomes are intact but hyperactive in the hydrolysis of fluorogenic peptide substrates and the degradation of polyubiquitinated proteins. Cells expressing the hyperactive proteasomes show markedly elevated degradation of many established proteasome substrates and resistance to oxidative stress. Multiplexed quantitative proteomics revealed ∼ 200 proteins with reduced levels in the mutant cells. Potentially toxic proteins such as tau exhibit reduced accumulation and aggregate formation. These data demonstrate that the CP gate is a key negative regulator of proteasome function in mammals, and that opening the CP gate may be an effective strategy to increase proteasome activity and reduce levels of toxic proteins in cells. PMID:26957043

  10. Irxl1 mutant mice show reduced tendon differentiation and no patterning defects in musculoskeletal system development.

    PubMed

    Kimura, Wataru; Machii, Masashi; Xue, XiaoDong; Sultana, Nishat; Hikosaka, Keisuke; Sharkar, Mohammad T K; Uezato, Tadayoshi; Matsuda, Masashi; Koseki, Haruhiko; Miura, Naoyuki

    2011-01-01

    Irxl1 (Iroquois-related homeobox like-1) is a newly identified three amino-acid loop extension (TALE) homeobox gene, which is expressed in various mesoderm-derived tissues, particularly in the progenitors of the musculoskeletal system. To analyze the roles of Irxl1 during embryonic development, we generated mice carrying a null allele of Irxl1. Mice homozygous for the targeted allele were viable, fertile, and showed reduced tendon differentiation. Skeletal morphology and skeletal muscle weight in Irxl1-knockout mice appeared normal. Expression patterns of several marker genes for cartilage, tendon, and muscle progenitors in homozygous mutant embryos were unchanged. These results suggest that Irxl1 is required for the tendon differentiation but dispensable for the patterning of the musculoskeletal system in development. PMID:21254332

  11. Arabidopsis thaliana sku mutant seedlings show exaggerated surface-dependent alteration in root growth vector

    NASA Technical Reports Server (NTRS)

    Rutherford, R.; Masson, P. H.

    1996-01-01

    Roots of wild-type Arabidopsis thaliana seedlings in the Wassilewskija (WS) and Landsberg erecta (Ler) ecotypes often grow aslant on vertical agar surfaces. Slanted root growth always occurs to the right of the gravity vector when the root is viewed through the agar surface, and is not observed in the Columbia ecotype. Right-slanted root growth is surface-dependent and does not result directly from directional environmental stimuli or gradients in the plane of skewing. We have isolated two partially dominant mutations in WS (sku1 and sku2) that show an exaggerated right-slanting root-growth phenotype on agar surfaces. The right-slanting root-growth phenotype of wild-type and mutant roots is not the result of diagravitropism or of an alteration in root gravitropism. It is accompanied by a left-handed rotation of the root about its axis within the elongation zone, the rate of which positively correlates with the degree of right-slanted curvature. Our data suggest that the right-slanting root growth phenotype results from an endogenous structural asymmetry that expresses itself by a directional root-tip rotation.

  12. Arabidopsis thaliana sku mutant seedlings show exaggerated surface-dependent alteration in root growth vector.

    PubMed

    Rutherford, R; Masson, P H

    1996-08-01

    Roots of wild-type Arabidopsis thaliana seedlings in the Wassilewskija (WS) and Landsberg erecta (Ler) ecotypes often grow aslant on vertical agar surfaces. Slanted root growth always occurs to the right of the gravity vector when the root is viewed through the agar surface, and is not observed in the Columbia ecotype. Right-slanted root growth is surface-dependent and does not result directly from directional environmental stimuli or gradients in the plane of skewing. We have isolated two partially dominant mutations in WS (sku1 and sku2) that show an exaggerated right-slanting root-growth phenotype on agar surfaces. The right-slanting root-growth phenotype of wild-type and mutant roots is not the result of diagravitropism or of an alteration in root gravitropism. It is accompanied by a left-handed rotation of the root about its axis within the elongation zone, the rate of which positively correlates with the degree of right-slanted curvature. Our data suggest that the right-slanting root growth phenotype results from an endogenous structural asymmetry that expresses itself by a directional root-tip rotation. PMID:8756492

  13. Otx1 null mutant mice show partial segregation of sensory epithelia comparable to lamprey ears

    NASA Technical Reports Server (NTRS)

    Fritzsch, B.; Signore, M.; Simeone, A.

    2001-01-01

    We investigated the development of inner ear innervation in Otx1 null mutants, which lack a horizontal canal, between embryonic day 12 (E12) and postnatal day 7 (P7) with DiI and immunostaining for acetylated tubulin. Comparable to control animals, horizontal crista-like fibers were found to cross over the utricle in Otx1 null mice. In mutants these fibers extend toward an area near the endolymphatic duct, not to a horizontal crista. Most Otx1 null mutants had a small patch of sensory hair cells at this position. Measurement of the area of the utricular macula suggested it to be enlarged in Otx1 null mutants. We suggest that parts of the horizontal canal crista remain incorporated in the utricular sensory epithelium in Otx1 null mutants. Other parts of the horizontal crista appear to be variably segregated to form the isolated patch of hair cells identifiable by the unique fiber trajectory as representing the horizontal canal crista. Comparison with lamprey ear innervation reveals similarities in the pattern of innervation with the dorsal macula, a sensory patch of unknown function. SEM data confirm that all foramina are less constricted in Otx1 null mutants. We propose that Otx1 is not directly involved in sensory hair cell formation of the horizontal canal but affects the segregation of the horizontal canal crista from the utricle. It also affects constriction of the two main foramina in the ear, but not their initial formation. Otx1 is thus causally related to horizontal canal morphogenesis as well as morphogenesis of these foramina.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  16. Kiss1 mutant placentas show normal structure and function in the mouse

    PubMed Central

    Herreboudt, A.M.; Kyle, V.R.L.; Lawrence, J.; Doran, J.; Colledge, W.H.

    2015-01-01

    Introduction Kisspeptins, encoded by the Kiss1 gene, are a set of related neuropeptides that are required for activation of the mammalian reproductive axis at puberty and to maintain fertility. In addition, kisspeptin signaling via the G-protein coupled receptor GPR54 (KISS1R) has been suggested to regulate human placental formation and correlations have been found between altered kisspeptin levels in the maternal blood and the development of pre-eclampsia. Methods We have used Kiss1 and Gpr54 mutant mice to investigate the role of kisspeptin signaling in the structure and function of the mouse placenta. Results Expression of Kiss1 and Gpr54 was confirmed in the mouse placenta but no differences in birth weight were found in mice that had been supported by a mutant placenta during fetal development. Stereological measurements found no differences between Kiss1 mutant and wild-type placentas. Measurement of amino-acid and glucose transport across the Kiss1 mutant placentas at E15.5 days did not reveal any functional defects. Discussion These data indicate that mouse placentas can develop a normal structure and function without kisspeptin signaling and can support normal fetal development and growth. PMID:25468546

  17. Development of a mutant strain of Bacillus polymyxa showing enhanced production of 2,3-butanediol

    SciTech Connect

    Mallonee, D.H.; Speckman, R.A.

    1988-01-01

    2,3-Butanediol is a feedstock chemical of potential industrial importance. It can serve as a monomer for many polymers of consumer interest that are currently supplied by the fossil fuel industry. Bacillus polymyxa can grow on inexpensive waste products of the food-processing industry and produce this glycol. This paper describes a mutant strain of B. polymyxa which displays constitutive production of catabolic ..cap alpha..-acetolactate synthase, an enzyme in the 2,3-butanediol pathway which is normally produced only in the late log or stationary phase of growth. The mutant was obtained by treating the wild type with nitrosoguanidine and subjecting it to a penicillin counterselection procedure. One of the selected mutant strains produced four times as much of the glycol as the wild type and utilized approximately 25% of the energy source, compared with essentially complete utilization of the energy source by the wild type. Studies are under way to optimize the production of the glycol by the mutant.

  18. Spirogyra varians mutant generated by high dose gamma-irradiation shows increased antioxidant properties

    NASA Astrophysics Data System (ADS)

    Lee, Hak-Jyung; Yoon, Minchul; Sung, Nak-Yun; Choi, Jong-il

    2012-08-01

    The aim of this study was to evaluate the antioxidant properties of a Spirogyra varians mutant (Mut) produced by gamma irradiation. Methanol extracts were prepared from Spirogyra varians wild-type and Mut plants, and their antioxidant activities and total phenolic content (TPC) were determined. Antioxidant parameters, including the 2-diphenyl-1-picrylhydrazyl radical-scavenging activity and ferric-reducing/antioxidant power, were higher in the Mut extract. Moreover, the TPC level was higher (P<0.05) in the Mut methanol extract. Therefore, these results suggest that gamma irradiation-induced S. varians Mut has superior antioxidant properties.

  19. X-ray survival characteristics and genetic analysis for nine saccharomyces deletion mutants that show altered radiation sensitivity

    SciTech Connect

    Game, John C.; Williamson, Marsha S.; Baccari, Clelia

    2004-01-07

    The availability of a genome-wide set of Saccharomyces deletion mutants provides a chance to identify all the yeast genes involved in DNA repair. Using X-rays, we are screening these mutants to identify additional genes that show increased sensitivity to the lethal effects of ionizing radiation. For each mutant identified as sensitive, we are confirming that the sensitivity phenotype co-segregates with the deletion allele and are obtaining multipoint survival-versus-dose assays in at least two haploid and one homozygous diploid strains. We present data for deletion mutants involving the genes DOT1, MDM20, NAT3, SPT7, SPT20, GCN5, HFI1, DCC1 and VID21/EAF1, and discuss their potential roles in repair. Eight of these genes have a clear radiation-sensitive phenotype when deleted, but the ninth, GCN5, has at most a borderline phenotype. None of the deletions confer substantial sensitivity to ultra-violet radiation, although one or two may confer marginal sensitivity. The DOT1 gene is of interest because its only known function is to methylate one lysine residue in the core of the histone H3 protein. We find that histone H3 mutants (supplied by K. Struhl) in which this residue is replaced by other amino-acids are also X-ray sensitive, seeming to confirm that methylation of the lysine-79 residue is required for effective repair of radiation damage.

  20. Arabidopsis tic62 trol mutant lacking thylakoid-bound ferredoxin-NADP+ oxidoreductase shows distinct metabolic phenotype.

    PubMed

    Lintala, Minna; Schuck, Natalie; Thormählen, Ina; Jungfer, Andreas; Weber, Katrin L; Weber, Andreas P M; Geigenberger, Peter; Soll, Jürgen; Bölter, Bettina; Mulo, Paula

    2014-01-01

    Ferredoxin-NADP+ oxidoreductase (FNR), functioning in the last step of the photosynthetic electron transfer chain, exists both as a soluble protein in the chloroplast stroma and tightly attached to chloroplast membranes. Surface plasmon resonance assays showed that the two FNR isoforms, LFNR1 and LFNR2, are bound to the thylakoid membrane via the C-terminal domains of Tic62 and TROL proteins in a pH-dependent manner. The tic62 trol double mutants contained a reduced level of FNR, exclusively found in the soluble stroma. Although the mutant plants showed no visual phenotype or defects in the function of photosystems under any conditions studied, a low ratio of NADPH/NADP+ was detected. Since the CO₂ fixation capacity did not differ between the tic62 trol plants and wild-type, it seems that the plants are able to funnel reducing power to most crucial reactions to ensure survival and fitness of the plants. However, the activity of malate dehydrogenase was down-regulated in the mutant plants. Apparently, the plastid metabolism is able to cope with substantial changes in directing the electrons from the light reactions to stromal metabolism and thus only few differences are visible in steady-state metabolite pool sizes of the tic62 trol plants. PMID:24043709

  1. Atf3 mutant mice show reduced axon regeneration and impaired regeneration-associated gene induction after peripheral nerve injury.

    PubMed

    Gey, Manuel; Wanner, Renate; Schilling, Corinna; Pedro, Maria T; Sinske, Daniela; Knöll, Bernd

    2016-08-01

    Axon injury in the peripheral nervous system (PNS) induces a regeneration-associated gene (RAG) response. Atf3 (activating transcription factor 3) is such a RAG and ATF3's transcriptional activity might induce 'effector' RAGs (e.g. small proline rich protein 1a (Sprr1a), Galanin (Gal), growth-associated protein 43 (Gap43)) facilitating peripheral axon regeneration. We provide a first analysis of Atf3 mouse mutants in peripheral nerve regeneration. In Atf3 mutant mice, facial nerve regeneration and neurite outgrowth of adult ATF3-deficient primary dorsal root ganglia neurons was decreased. Using genome-wide transcriptomics, we identified a neuropeptide-encoding RAG cluster (vasoactive intestinal peptide (Vip), Ngf, Grp, Gal, Pacap) regulated by ATF3. Exogenous administration of neuropeptides enhanced neurite growth of Atf3 mutant mice suggesting that these molecules might be effector RAGs of ATF3's pro-regenerative function. In addition to the induction of growth-promoting molecules, we present data that ATF3 suppresses growth-inhibiting molecules such as chemokine (C-C motif) ligand 2. In summary, we show a pro-regenerative ATF3 function during PNS nerve regeneration involving transcriptional activation of a neuropeptide-encoding RAG cluster. ATF3 is a general injury-inducible factor, therefore ATF3-mediated mechanisms identified herein might apply to other cell and injury types. PMID:27581653

  2. Atf3 mutant mice show reduced axon regeneration and impaired regeneration-associated gene induction after peripheral nerve injury

    PubMed Central

    Gey, Manuel; Wanner, Renate; Schilling, Corinna; Pedro, Maria T.; Sinske, Daniela

    2016-01-01

    Axon injury in the peripheral nervous system (PNS) induces a regeneration-associated gene (RAG) response. Atf3 (activating transcription factor 3) is such a RAG and ATF3's transcriptional activity might induce ‘effector’ RAGs (e.g. small proline rich protein 1a (Sprr1a), Galanin (Gal), growth-associated protein 43 (Gap43)) facilitating peripheral axon regeneration. We provide a first analysis of Atf3 mouse mutants in peripheral nerve regeneration. In Atf3 mutant mice, facial nerve regeneration and neurite outgrowth of adult ATF3-deficient primary dorsal root ganglia neurons was decreased. Using genome-wide transcriptomics, we identified a neuropeptide-encoding RAG cluster (vasoactive intestinal peptide (Vip), Ngf, Grp, Gal, Pacap) regulated by ATF3. Exogenous administration of neuropeptides enhanced neurite growth of Atf3 mutant mice suggesting that these molecules might be effector RAGs of ATF3's pro-regenerative function. In addition to the induction of growth-promoting molecules, we present data that ATF3 suppresses growth-inhibiting molecules such as chemokine (C-C motif) ligand 2. In summary, we show a pro-regenerative ATF3 function during PNS nerve regeneration involving transcriptional activation of a neuropeptide-encoding RAG cluster. ATF3 is a general injury-inducible factor, therefore ATF3-mediated mechanisms identified herein might apply to other cell and injury types. PMID:27581653

  3. Genes identified by visible mutant phenotypes show increased bias toward one of two subgenomes of maize.

    PubMed

    Schnable, James C; Freeling, Michael

    2011-01-01

    Not all genes are created equal. Despite being supported by sequence conservation and expression data, knockout homozygotes of many genes show no visible effects, at least under laboratory conditions. We have identified a set of maize (Zea mays L.) genes which have been the subject of a disproportionate share of publications recorded at MaizeGDB. We manually anchored these "classical" maize genes to gene models in the B73 reference genome, and identified syntenic orthologs in other grass genomes. In addition to proofing the most recent version 2 maize gene models, we show that a subset of these genes, those that were identified by morphological phenotype prior to cloning, are retained at syntenic locations throughout the grasses at much higher levels than the average expressed maize gene, and are preferentially found on the maize1 subgenome even with a duplicate copy is still retained on the opposite subgenome. Maize1 is the subgenome that experienced less gene loss following the whole genome duplication in maize lineage 5-12 million years ago and genes located on this subgenome tend to be expressed at higher levels in modern maize. Links to the web based software that supported our syntenic analyses in the grasses should empower further research and support teaching involving the history of maize genetic research. Our findings exemplify the concept of "grasses as a single genetic system," where what is learned in one grass may be applied to another. PMID:21423772

  4. Mice with mutant Inf2 show impaired podocyte and slit diaphragm integrity in response to protamine-induced kidney injury.

    PubMed

    Subramanian, Balajikarthick; Sun, Hua; Yan, Paul; Charoonratana, Victoria T; Higgs, Henry N; Wang, Fang; Lai, Ka-Man V; Valenzuela, David M; Brown, Elizabeth J; Schlöndorff, Johannes S; Pollak, Martin R

    2016-08-01

    Mutations in the INF2 (inverted formin 2) gene, encoding a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause human focal segmental glomerulosclerosis (FSGS). INF2 interacts directly with certain other mammalian diaphanous formin proteins (mDia) that function as RhoA effector molecules. FSGS-causing INF2 mutations impair these interactions and disrupt the ability of INF2 to regulate Rho/Dia-mediated actin dynamics in vitro. However, the precise mechanisms by which INF2 regulates and INF2 mutations impair glomerular structure and function remain unknown. Here, we characterize an Inf2 R218Q point-mutant (knockin) mouse to help answer these questions. Knockin mice have no significant renal pathology or proteinuria at baseline despite diminished INF2 protein levels. INF2 mutant podocytes do show impaired reversal of protamine sulfate-induced foot process effacement by heparin sulfate perfusion. This is associated with persistent podocyte cytoplasmic aggregation, nephrin phosphorylation, and nephrin and podocin mislocalization, as well as impaired recovery of mDia membrane localization. These changes were partially mimicked in podocyte outgrowth cultures, in which podocytes from knockin mice show altered cellular protrusions compared to those from wild-type mice. Thus, in mice, normal INF2 function is not required for glomerular development but normal INF2 is required for regulation of the actin-based behaviors necessary for response to and/or recovery from injury. PMID:27350175

  5. An Arabidopsis Mutant Tolerant to Lethal Ultraviolet-B Levels Shows Constitutively Elevated Accumulation of Flavonoids and Other Phenolics1

    PubMed Central

    Bieza, Kim; Lois, Rodrigo

    2001-01-01

    The isolation and characterization of mutants hypersensitive to ultraviolet (UV) radiation has been a powerful tool to learn about the mechanisms that protect plants against UV-induced damage. To increase our understanding of the various mechanisms of defense against UVB radiation, we searched for mutations that would increase the level of tolerance of Arabidopsis plants to UV radiation. We describe a single gene dominant mutation (uvt1) that leads to a remarkable tolerance to UVB radiation conditions that would kill wild-type plants. Pigment analyses show a constitutive increase in accumulation of UV-absorbing compounds in uvt1 that increases the capacity of the leaves to block UVB radiation and therefore is likely to be responsible for the elevated resistance of this mutant to UVB radiation. These increases in absorption in the UV region are due, at least in part, to increases in flavonoid and sinapate accumulation. Expression of chalcone synthase (CHS) mRNA was shown to be constitutively elevated in uvt1 plants, suggesting that the increases in absorption may be a consequence of changes in gene expression. Expression of CHS in uvt1 was shown to be still inducible by UV, indicating that the uvt1 lesion may not affect the UV-mediated regulation of CHS gene expression. Our data support an important role for UV screens in the overall protection of plants to UVB radiation. The uvt1 mutant could prove to be an important tool to elucidate further the exact role of UV-absorbing pigments in UV protection as well as the relative contribution of other mechanisms to the overall tolerance of plants to UV radiation. PMID:11457961

  6. BRI1-EMS-suppressor 1 gain-of-function mutant shows higher susceptibility to necrotrophic fungal infection.

    PubMed

    Shin, Seo Youn; Chung, Hayung; Kim, Sun Young; Nam, Kyoung Hee

    2016-02-19

    Brassinosteroids (BRs) are plant-specific steroids that are involved in plant growth and defense responses. However, the exact roles of BR in plant defense are unclear. We used the bes1-D gain-of-function mutant to define the underlying relationship between plant growth and defense through BR signaling and innate immunity. In bes1-D, further downstream component BES1 transcription factor is stabilized, leading to the activation of BR signaling. Previous reports on BES1 target genes showed that approximately 10% are related to biotic stress responses. Therefore, the bes1-D PTI responses were examined. The bes1-D mutant was specifically susceptible to Alternaria brassicicola, a necrotrophic fungus, which successfully produced spore, resulting in considerable cell death. However, it was not affected by a biotrophic pathogen, Pseudomonas syringae pv. tomato (Pst) DC3000. Instead of a ROS burst, a representative initial PTI responses, higher ROS accumulation was sustained in bes1-D than in the wild type plant. PDF1.2 expression was not induced in response to fungal pathogen infection in bes1-D. These results suggest that BES1 is also involved in JA-related defense responses, especially in response to necrotrophic pathogens. PMID:26809089

  7. Mutant forms of tumour necrosis factor receptor I that occur in TNF-receptor-associated periodic syndrome retain signalling functions but show abnormal behaviour

    PubMed Central

    Todd, Ian; Radford, Paul M; Draper-Morgan, Kelly-Ann; McIntosh, Richard; Bainbridge, Susan; Dickinson, Peter; Jamhawi, Lama; Sansaridis, Marios; Huggins, Mary L; Tighe, Patrick J; Powell, Richard J

    2004-01-01

    Tumour necrosis factor (TNF)-receptor-associated periodic syndrome (TRAPS) is a hereditary autoinflammatory disorder involving autosomal-dominant missense mutations in TNF receptor superfamily 1A (TNFRSF1A) ectodomains. To elucidate the molecular effects of TRAPS-related mutations, we transfected HEK-293 cells to produce lines stably expressing high levels of either wild-type (WT) or single mutant recombinant forms of TNFRSF1A. Mutants with single amino acid substitutions in the first cysteine-rich domain (CRD1) were produced both as full-length receptor proteins and as truncated forms lacking the cytoplasmic signalling domain (Δsig). High-level expression of either WT or mutant full-length TNFRSF1A spontaneously induced apoptosis and interleukin-8 production, indicating that the mutations in CRD1 did not abrogate signalling. Consistent with this, WT and mutant full-length TNFRSF1A formed cytoplasmic aggregates that co-localized with ubiquitin and chaperones, and with the signal transducer TRADD, but not with the inhibitor, silencer of death domain (SODD). Furthermore, as expected, WT and mutant Δsig forms of TNFRSF1A did not induce apoptosis or interleukin-8 production. However, whereas the WT full-length TNFRSF1A was expressed both in the cytoplasm and on the cell surface, the mutant receptors showed strong cytoplasmic expression but reduced cell-surface expression. The WT and mutant Δsig forms of TNFRSF1A were all expressed at the cell surface, but a proportion of the mutant receptors were also retained in the cytoplasm and co-localized with BiP. Furthermore, the mutant forms of surface-expressed Δsig TNFRSF1A were defective in binding TNF-α. We conclude that TRAPS-related CRD1 mutants of TNFRSF1A possess signalling properties associated with the cytoplasmic death domain, but other behavioural features of the mutant receptors are abnormal, including intracellular trafficking and TNF binding. PMID:15312137

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

    PubMed

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

    2016-08-01

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

  9. X-ray survival characteristics and genetic analysis for nine Saccharomyces deletion mutants that show altered radiation sensitivity.

    PubMed

    Game, John C; Williamson, Marsha S; Baccari, Clelia

    2005-01-01

    The availability of a genome-wide set of Saccharomyces deletion mutants provides a chance to identify all the yeast genes involved in DNA repair. Using X rays, we are screening these mutants to identify additional genes that cause increased sensitivity to the lethal effects of ionizing radiation. For each mutant identified as sensitive, we are confirming that the sensitivity phenotype cosegregates with the deletion allele and are obtaining multipoint survival-vs.-dose assays in at least one homozygous diploid and two haploid strains. We present data for deletion mutants involving the genes DOT1, MDM20, NAT3, SPT7, SPT20, GCN5, HFI1, DCC1, and VID21/EAF1 and discuss their potential roles in repair. Eight of these genes cause a clear radiation-sensitive phenotype when deleted, but the ninth, GCN5, results in at most a borderline phenotype. None of the deletions confer substantial sensitivity to ultraviolet radiation, although one or two may confer marginal sensitivity. The DOT1 gene is of interest because its only known function is to methylate one lysine residue in the core of the histone H3 protein. We find that histone H3 mutants (supplied by K. Struhl) in which this residue is replaced by other amino acids are also X-ray sensitive, which confirms that methylation of the lysine-79 residue is required for effective repair of radiation damage. PMID:15371366

  10. Structural and functional characterization of "laboratory evolved" cytochrome P450cam mutants showing enhanced naphthalene oxygenation activity.

    PubMed

    Matsuura, Koji; Tosha, Takehiko; Yoshioka, Shiro; Takahashi, Satoshi; Ishimori, Koichiro; Morishima, Isao

    2004-10-29

    To elucidate molecular mechanisms for the enhanced oxygenation activity in the three mutants of cytochrome P450cam screened by 'laboratory evolution' [Nature 399 (1999) 670], we purified the mutants and characterized their functional and structural properties. The electronic absorption and resonance Raman spectra revealed that the structures of heme binding site of all purified mutants were quite similar to that of the wild-type enzyme, although the fraction of the inactivated form, called "P420," was increased. In the reaction with H(2)O(2), only trace amounts of the naphthalene hydroxylation product were detected by gas chromatography. We, therefore, conclude that the three mutants do not exhibit significant changes in the structural and functional properties from those of wild-type P450cam except for the stability of the axial ligand in the reduced form. The enhanced fluorescence in the whole-cell assay would reflect enhancement in the oxygenation activity below the detectable limit of the gas chromatography and/or contributions of other reactions catalyzed by the heme iron. PMID:15451425

  11. Spontaneous Chloroplast Mutants Mostly Occur by Replication Slippage and Show a Biased Pattern in the Plastome of Oenothera.

    PubMed

    Massouh, Amid; Schubert, Julia; Yaneva-Roder, Liliya; Ulbricht-Jones, Elena S; Zupok, Arkadiusz; Johnson, Marc T J; Wright, Stephen I; Pellizzer, Tommaso; Sobanski, Johanna; Bock, Ralph; Greiner, Stephan

    2016-04-01

    Spontaneous plastome mutants have been used as a research tool since the beginning of genetics. However, technical restrictions have severely limited their contributions to research in physiology and molecular biology. Here, we used full plastome sequencing to systematically characterize a collection of 51 spontaneous chloroplast mutants in Oenothera (evening primrose). Most mutants carry only a single mutation. Unexpectedly, the vast majority of mutations do not represent single nucleotide polymorphisms but are insertions/deletions originating from DNA replication slippage events. Only very few mutations appear to be caused by imprecise double-strand break repair, nucleotide misincorporation during replication, or incorrect nucleotide excision repair following oxidative damage. U-turn inversions were not detected. Replication slippage is induced at repetitive sequences that can be very small and tend to have high A/T content. Interestingly, the mutations are not distributed randomly in the genome. The underrepresentation of mutations caused by faulty double-strand break repair might explain the high structural conservation of seed plant plastomes throughout evolution. In addition to providing a fully characterized mutant collection for future research on plastid genetics, gene expression, and photosynthesis, our work identified the spectrum of spontaneous mutations in plastids and reveals that this spectrum is very different from that in the nucleus. PMID:27053421

  12. Spontaneous Chloroplast Mutants Mostly Occur by Replication Slippage and Show a Biased Pattern in the Plastome of Oenothera[OPEN

    PubMed Central

    Massouh, Amid; Schubert, Julia; Yaneva-Roder, Liliya; Ulbricht-Jones, Elena S.; Johnson, Marc T.J.; Wright, Stephen I.; Pellizzer, Tommaso; Sobanski, Johanna; Greiner, Stephan

    2016-01-01

    Spontaneous plastome mutants have been used as a research tool since the beginning of genetics. However, technical restrictions have severely limited their contributions to research in physiology and molecular biology. Here, we used full plastome sequencing to systematically characterize a collection of 51 spontaneous chloroplast mutants in Oenothera (evening primrose). Most mutants carry only a single mutation. Unexpectedly, the vast majority of mutations do not represent single nucleotide polymorphisms but are insertions/deletions originating from DNA replication slippage events. Only very few mutations appear to be caused by imprecise double-strand break repair, nucleotide misincorporation during replication, or incorrect nucleotide excision repair following oxidative damage. U-turn inversions were not detected. Replication slippage is induced at repetitive sequences that can be very small and tend to have high A/T content. Interestingly, the mutations are not distributed randomly in the genome. The underrepresentation of mutations caused by faulty double-strand break repair might explain the high structural conservation of seed plant plastomes throughout evolution. In addition to providing a fully characterized mutant collection for future research on plastid genetics, gene expression, and photosynthesis, our work identified the spectrum of spontaneous mutations in plastids and reveals that this spectrum is very different from that in the nucleus. PMID:27053421

  13. ß-amylase1 mutant Arabidopsis plants show improved drought tolerance due to reduced starch breakdown in guard cells

    PubMed Central

    Prasch, Christian Maximilian; Ott, Kirsten Verena; Bauer, Hubert; Ache, Peter; Hedrich, Rainer; Sonnewald, Uwe

    2015-01-01

    In plants, drought stress is a major growth limiting factor causing cell water loss through open stomata. In this study, guard cell-specific transcripts from drought-stressed Arabidopsis plants were analysed and a down-regulation of β-amylase 1 (BAM1) was found. In previous studies, BAM1 was shown to be involved in stomatal starch degradation under ambient conditions. Impaired starch breakdown of bam1 mutant plants was accompanied by decreased stomatal opening. Here, it is shown that drought tolerance of bam1 mutant plants is improved as compared with wild-type controls. Microarray analysis of stomata-specific transcripts from bam1 mutant plants revealed a significant down-regulation of genes encoding aquaporins, auxin- and ethylene-responsive factors, and cell-wall modifying enzymes. This expression pattern suggests that reduced water uptake and limited cell wall extension are associated with the closed state of stomata of bam1 mutant plants. Together these data suggest that regulation of stomata-specific starch turnover is important for adapting stomata opening to environmental needs and its breeding manipulation may result in drought tolerant crop plants. PMID:26139825

  14. ß-amylase1 mutant Arabidopsis plants show improved drought tolerance due to reduced starch breakdown in guard cells.

    PubMed

    Prasch, Christian Maximilian; Ott, Kirsten Verena; Bauer, Hubert; Ache, Peter; Hedrich, Rainer; Sonnewald, Uwe

    2015-09-01

    In plants, drought stress is a major growth limiting factor causing cell water loss through open stomata. In this study, guard cell-specific transcripts from drought-stressed Arabidopsis plants were analysed and a down-regulation of β-amylase 1 (BAM1) was found. In previous studies, BAM1 was shown to be involved in stomatal starch degradation under ambient conditions. Impaired starch breakdown of bam1 mutant plants was accompanied by decreased stomatal opening. Here, it is shown that drought tolerance of bam1 mutant plants is improved as compared with wild-type controls. Microarray analysis of stomata-specific transcripts from bam1 mutant plants revealed a significant down-regulation of genes encoding aquaporins, auxin- and ethylene-responsive factors, and cell-wall modifying enzymes. This expression pattern suggests that reduced water uptake and limited cell wall extension are associated with the closed state of stomata of bam1 mutant plants. Together these data suggest that regulation of stomata-specific starch turnover is important for adapting stomata opening to environmental needs and its breeding manipulation may result in drought tolerant crop plants. PMID:26139825

  15. Characterization of a Corynebacterium glutamicum dnaB mutant that shows temperature-sensitive growth and mini-cell formation.

    PubMed

    Uchida, Makoto; Hirasawa, Takashi; Wachi, Masaaki

    2014-12-01

    Corynebacterium glutamicum is known to perform a unique form of cell division called post-fission snapping division. In order to investigate the mechanism of cell division of this bacterium, we isolated temperature-sensitive mutants from C. glutamicum wild-type strain ATCC 31831, and found that one of them, M45, produced high frequencies of mini-cells with no nucleoids. Cell pairs composed of an elongated cell, with one nucleoid, connected to a mini-cell, with no nucleoids, were occasionally observed. The temperature sensitivity and mini-cell formation of M45 was complemented by a 2-kb DraI-EcoRI fragment derived from the ATCC 31831 chromosomal DNA, which carried a dnaB homolog encoding a replicative DNA helicase. DNA sequence analysis revealed that M45 carried a missense mutation in the dnaB gene, which caused a substitution of Thr364 to Ile. Microscopic observation after 4',6-diamidino-2-phenylindole staining revealed that the DNA content of single cells was decreased by culturing at the restrictive temperature, suggesting that the mutation affects chromosomal replication. These results suggest that the C. glutamicum dnaB mutant performs an asymmetric cell division even after DNA replication is inhibited, which results in the production of mini-cells. PMID:25141796

  16. Proteomic and biochemical analyses show a functional network of proteins involved in antioxidant defense of the Arabidopsis anp2anp3 double mutant.

    PubMed

    Takáč, Tomáš; Šamajová, Olga; Vadovič, Pavol; Pechan, Tibor; Košútová, Petra; Ovečka, Miroslav; Husičková, Alexandra; Komis, George; Šamaj, Jozef

    2014-12-01

    Disentanglement of functional complexity associated with plant mitogen-activated protein kinase (MAPK) signaling has benefited from transcriptomic, proteomic, phosphoproteomic, and genetic studies. Published transcriptomic analysis of a double homozygous recessive anp2anp3 mutant of two MAPK kinase kinase (MAPKKK) genes called Arabidopsis thaliana Homologues of Nucleus- and Phragmoplast-localized Kinase 2 (ANP2) and 3 (ANP3) showed the upregulation of stress-related genes. In this study, a comparative proteomic analysis of anp2anp3 mutant against its respective Wassilevskaja ecotype (Ws) wild type background is provided. Such differential proteomic analysis revealed overabundance of core enzymes such as FeSOD1, MnSOD, DHAR1, and FeSOD1-associated regulatory protein CPN20, which are involved in the detoxification of reactive oxygen species in the anp2anp3 mutant. The proteomic results were validated at the level of single protein abundance by Western blot analyses and by quantitative biochemical determination of antioxidant enzymatic activities. Finally, the functional network of proteins involved in antioxidant defense in the anp2anp3 mutant was physiologically linked with the increased resistance of mutant seedlings against paraquat treatment. PMID:25325904

  17. Transcriptomic and proteomic analyses of a pale-green durum wheat mutant shows variations in photosystem components and metabolic deficiencies under drought stress

    PubMed Central

    2014-01-01

    Background Leaf pigment content is an important trait involved in environmental interactions. In order to determine its impact on drought tolerance in wheat, we characterized a pale-green durum wheat mutant (Triticum turgidum L. var. durum) under contrasting water availability conditions. Results The pale-green mutant was investigated by comparing pigment content and gene/protein expression profiles to wild-type plants at anthesis. Under well-watered (control) conditions the mutant had lower levels of chlorophylls and carotenoids, but higher levels of xanthophyll de-epoxidation compared to wild-type. Transcriptomic analysis under control conditions showed that defense genes (encoding e.g. pathogenesis-related proteins, peroxidases and chitinases) were upregulated in the mutant, suggesting the presence of mild oxidative stress that was compensated without altering the net rate of photosynthesis. Transcriptomic analysis under terminal water stress conditions, revealed the modulation of antioxidant enzymes, photosystem components, and enzymes representing carbohydrate metabolism and the tricarboxylic acid cycle, indicating that the mutant was exposed to greater oxidative stress than the wild-type plants, but had a limited capacity to respond. We also compared the two genotypes under irrigated and rain-fed field conditions over three years, finding that the greater oxidative stress and corresponding molecular changes in the pale-green mutant were associated to a yield reduction. Conclusions This study provides insight on the effect of pigment content in the molecular response to drought. Identified genes differentially expressed under terminal water stress may be valuable for further studies addressing drought resistance in wheat. PMID:24521234

  18. The human tyrosine kinase Kit and its gatekeeper mutant T670I, show different kinetic properties: Implications for drug design.

    PubMed

    Kissova, Miroslava; Maga, Giovanni; Crespan, Emmanuele

    2016-10-01

    The tyrosine kinase Kit, a receptor for Stem Cell Factor, is involved, among others, in processes associated to cell survival, proliferation and migration. Upon physiological conditions, the activity of Kit is tightly regulated. However, primary mutations that lead to its constitutive activation are the causal oncogenic driver of gastrointestinal stromal tumours (GISTs). GISTs are known to be refractory to conventional therapies but the introduction of Imatinib, a selective inhibitor of tyrosine kinases Abl and Kit, significantly ameliorated the treatment options of GISTs patients. However, the acquisition of secondary mutations renders Kit resistant towards all available drugs. Mutation involving gatekeeper residues (such as V654a and T670I) influence both the structure and the catalytic activity of the enzyme. Therefore, detailed knowledge of the enzymatic properties of the mutant forms, in comparison with the wild type enzyme, is an important pre-requisite for the rational development of specific inhibitors. In this paper we report a thorough kinetic analysis of the reaction catalyzed by the Kit kinase and its gatekeeper mutated form T670I. Our results revealed the different mechanisms of action of these two enzymes and may open a new avenue for the future design of specific Kit inhibitors. PMID:27527414

  19. Sendai virus wild-type and mutant C proteins show a direct correlation between L polymerase binding and inhibition of viral RNA synthesis.

    PubMed

    Grogan, C C; Moyer, S A

    2001-09-15

    The Sendai virus C proteins, C', C, Y1, and Y2, are a nested set of four independently initiated carboxy-coterminal proteins encoded on the P mRNA from an alternate reading frame. Together the C proteins have been shown to inhibit viral transcription and replication in vivo and in vitro and C' binds the Sendai virus L protein, the presumed catalytic subunit of the viral RNA polymerase. To identify amino acids within the C' protein that are important for binding L, site-directed mutagenesis of the gstC' gene was used to change conserved charged amino acids to alanine, generating nine mutants. Additionally, a tenth natural mutant, gstF170S, was also constructed. Six of the gstC' mutants, primarily in the C-terminal half of C', exhibited a defect in the ability to bind L protein. The mutants were assayed for their effect on in vitro transcription and replication from the antigenomic promoter, and the data suggest in all but one case a direct correlation between the ability of C to bind L and to inhibit these steps in RNA synthesis. Further studies with two nonfusion C mutants showed that this correlation was specifically due to the C' portion, and not the gst portion, of the fusion proteins. To study their individual functions, each of the four C proteins was fused downstream of glutathione S-transferase. The gstC', gstC, gstY1, and gstY1 fusion proteins were all able to bind L protein and to inhibit viral mRNA and (+)-leader RNA synthesis, and antigenome replication in vitro. In addition, the nonfusion C, Y1, and Y2 proteins all inhibited transcription. The inhibition of (+)-leader RNA and mRNA synthesis by wt C proteins (nonfusion) showed nearly identical dose-response curves, suggesting that inhibition occurs early in RNA synthesis. PMID:11543662

  20. Myristoylation negative msbB-mutants of probiotic E. coli Nissle 1917 retain tumor specific colonization properties but show less side effects in immunocompetent mice.

    PubMed

    Stritzker, Jochen; Hill, Philip J; Gentschev, Ivaylo; Szalay, Aladar A

    2010-01-01

    Specific colonization of solid tumors by bacteria opens the way to novel approaches in both tumor diagnosis and therapy. However, even non-pathogenic bacteria induce responses by the immune system, which could be devastating for a tumor bearing patient. As such effects are caused e.g., by the lipid A moiety of the lipopolysaccharide, a msbB-mutant of the probiotic E. coli Nissle 1917 strain was investigated. Bacteria of the mutant strain did not show any growth defects in culture media when compared to wild-type E. coli Nissle 1917 but were unable to myristoylate lipid A, had less toxic effects on immunocompetent BALB/c mice, and were still able to specifically colonize tumors. Therefore, the modification of lipid A could result in bacterial strains that might be better suited for diagnosis and therapy of tumors than the corresponding wild-type strains, even if those are not considered pathogenic or are of probiotic background. PMID:21326939

  1. Myristoylation negative msbB-mutants of probiotic E. coli Nissle 1917 retain tumor specific colonization properties but show less side effects in immunocompetent mice

    PubMed Central

    Stritzker, Jochen; Hill, Philip J; Gentschev, Ivaylo

    2010-01-01

    Specific colonization of solid tumors by bacteria opens the way to novel approaches in both tumor diagnosis and therapy. However, even non-pathogenic bacteria induce responses by the immune system, which could be devastating for a tumor bearing patient. As such effects are caused e.g., by the lipid A moiety of the lipopolysaccharide, a msbB-mutant of the probiotic E. coli Nissle 1917 strain was investigated. Bacteria of the mutant strain did not show any growth defects in culture media when compared to wild-type E. coli Nissle 1917 but were unable to myristoylate lipid A, had less toxic effects on immunocompetent BALB/c mice, and were still able to specifically colonize tumors. Therefore, the modification of lipid A could result in bacterial strains that might be better suited for diagnosis and therapy of tumors than the corresponding wild-type strains, even if those are not considered pathogenic or are of probiotic background. PMID:21326939

  2. Arabidopsis AtDjA3 Null Mutant Shows Increased Sensitivity to Abscisic Acid, Salt, and Osmotic Stress in Germination and Post-germination Stages

    PubMed Central

    Salas-Muñoz, Silvia; Rodríguez-Hernández, Aída A.; Ortega-Amaro, Maria A.; Salazar-Badillo, Fatima B.; Jiménez-Bremont, Juan F.

    2016-01-01

    DnaJ proteins are essential co-chaperones involved in abiotic and biotic stress responses. Arabidopsis AtDjA3 gene encodes a molecular co-chaperone of 420 amino acids, which belongs to the J-protein family. In this study, we report the functional characterization of the AtDjA3 gene using the Arabidopsis knockout line designated j3 and the 35S::AtDjA3 overexpression lines. Loss of AtDjA3 function was associated with small seed production. In fact, j3 mutant seeds showed a reduction of 24% in seed weight compared to Col-0 seeds. Expression analysis showed that the AtDjA3 gene was modulated in response to NaCl, glucose, and abscisic acid (ABA). The j3 line had increased sensitivity to NaCl and glucose treatments in the germination and cotyledon development in comparison to parental Col-0. Furthermore, the j3 mutant line exhibited higher ABA sensitivity in comparison to parental Col-0 and 35S::AtDjA3 overexpression lines. In addition, we examined the expression of ABI3 gene, which is a central regulator in ABA signaling, in j3 mutant and 35S::AtDjA3 overexpression lines. Under 5 μM ABA treatment at 24 h, j3 mutant seedlings displayed higher ABI3 expression, whereas in 35S::AtDjA3 overexpression lines, ABI3 gene expression was repressed. Taken together, these results demonstrate that the AtDjA3 gene is involved in seed development and abiotic stress tolerance. PMID:26941772

  3. Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington's disease.

    PubMed

    Zeng, Yixuan; Guo, Wenyuan; Xu, Guangqing; Wang, Qinmei; Feng, Luyang; Long, Simei; Liang, Fengyin; Huang, Yi; Lu, Xilin; Li, Shichang; Zhou, Jiebin; Burgunder, Jean-Marc; Pang, Jiyan; Pei, Zhong

    2016-01-01

    Huntington's disease is an autosomal-dominant neurodegenerative disorder, with chorea as the most prominent manifestation. The disease is caused by abnormal expansion of CAG codon repeats in the IT15 gene, which leads to the expression of a glutamine-rich protein named mutant Huntingtin (Htt). Because of its devastating disease burden and lack of valid treatment, development of more effective therapeutics for Huntington's disease is urgently required. Xyloketal B, a natural product from mangrove fungus, has shown protective effects against toxicity in other neurodegenerative disease models such as Parkinson's and Alzheimer's diseases. To identify potential neuroprotective molecules for Huntington's disease, six derivatives of xyloketal B were screened in a Caenorhabditis elegans Huntington's disease model; all six compounds showed a protective effect. Molecular docking studies indicated that compound 1 could bind to residues GLN369 and GLN393 of the mutant Htt protein, forming a stable trimeric complex that can prevent the formation of mutant Htt aggregates. Taken together, we conclude that xyloketal derivatives could be novel drug candidates for treating Huntington's disease. Molecular target analysis is a good method to simulate the interaction between proteins and drug compounds. Further, protective candidate drugs could be designed in future using the guidance of molecular docking results. PMID:27110099

  4. Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington’s disease

    PubMed Central

    Zeng, Yixuan; Guo, Wenyuan; Xu, Guangqing; Wang, Qinmei; Feng, Luyang; Long, Simei; Liang, Fengyin; Huang, Yi; Lu, Xilin; Li, Shichang; Zhou, Jiebin; Burgunder, Jean-Marc; Pang, Jiyan; Pei, Zhong

    2016-01-01

    Huntington’s disease is an autosomal-dominant neurodegenerative disorder, with chorea as the most prominent manifestation. The disease is caused by abnormal expansion of CAG codon repeats in the IT15 gene, which leads to the expression of a glutamine-rich protein named mutant Huntingtin (Htt). Because of its devastating disease burden and lack of valid treatment, development of more effective therapeutics for Huntington’s disease is urgently required. Xyloketal B, a natural product from mangrove fungus, has shown protective effects against toxicity in other neurodegenerative disease models such as Parkinson’s and Alzheimer’s diseases. To identify potential neuroprotective molecules for Huntington’s disease, six derivatives of xyloketal B were screened in a Caenorhabditis elegans Huntington’s disease model; all six compounds showed a protective effect. Molecular docking studies indicated that compound 1 could bind to residues GLN369 and GLN393 of the mutant Htt protein, forming a stable trimeric complex that can prevent the formation of mutant Htt aggregates. Taken together, we conclude that xyloketal derivatives could be novel drug candidates for treating Huntington’s disease. Molecular target analysis is a good method to simulate the interaction between proteins and drug compounds. Further, protective candidate drugs could be designed in future using the guidance of molecular docking results. PMID:27110099

  5. Characterization and Genetic Analysis of a Novel Light-Dependent Lesion Mimic Mutant, lm3, Showing Adult-Plant Resistance to Powdery Mildew in Common Wheat

    PubMed Central

    Wang, Fang; Wu, Wenying; Wang, Dongzhi; Yang, Wenlong; Sun, Jiazhu; Liu, Dongcheng; Zhang, Aimin

    2016-01-01

    Lesion mimics (LMs) that exhibit spontaneous disease-like lesions in the absence of pathogen attack might confer enhanced plant disease resistance to a wide range of pathogens. The LM mutant, lm3 was derived from a single naturally mutated individual in the F1 population of a 3-1/Jing411 cross, backcrossed six times with 3–1 as the recurrent parent and subsequently self-pollinated twice. The leaves of young seedlings of the lm3 mutant exhibited small, discrete white lesions under natural field conditions. The lesions first appeared at the leaf tips and subsequently expanded throughout the entire leaf blade to the leaf sheath. The lesions were initiated through light intensity and day length. Histochemical staining revealed that lesion formation might reflect programmed cell death (PCD) and abnormal accumulation of reactive oxygen species (ROS). The chlorophyll content in the mutant was significantly lower than that in wildtype, and the ratio of chlorophyll a/b was increased significantly in the mutant compared with wildtype, indicating that lm3 showed impairment of the biosynthesis or degradation of chlorophyll, and that Chlorophyll b was prone to damage during lesion formation. The lm3 mutant exhibited enhanced resistance to wheat powdery mildew fungus (Blumeria graminis f. sp. tritici; Bgt) infection, which was consistent with the increased expression of seven pathogenesis-related (PR) and two wheat chemically induced (WCI) genes involved in the defense-related reaction. Genetic analysis showed that the mutation was controlled through a single partially dominant gene, which was closely linked to Xbarc203 on chromosome 3BL; this gene was delimited to a 40 Mb region between SSR3B450.37 and SSR3B492.6 using a large derived segregating population and the available Chinese Spring chromosome 3B genome sequence. Taken together, our results provide information regarding the identification of a novel wheat LM gene, which will facilitate the additional fine-mapping and

  6. Characterization and Genetic Analysis of a Novel Light-Dependent Lesion Mimic Mutant, lm3, Showing Adult-Plant Resistance to Powdery Mildew in Common Wheat.

    PubMed

    Wang, Fang; Wu, Wenying; Wang, Dongzhi; Yang, Wenlong; Sun, Jiazhu; Liu, Dongcheng; Zhang, Aimin

    2016-01-01

    Lesion mimics (LMs) that exhibit spontaneous disease-like lesions in the absence of pathogen attack might confer enhanced plant disease resistance to a wide range of pathogens. The LM mutant, lm3 was derived from a single naturally mutated individual in the F1 population of a 3-1/Jing411 cross, backcrossed six times with 3-1 as the recurrent parent and subsequently self-pollinated twice. The leaves of young seedlings of the lm3 mutant exhibited small, discrete white lesions under natural field conditions. The lesions first appeared at the leaf tips and subsequently expanded throughout the entire leaf blade to the leaf sheath. The lesions were initiated through light intensity and day length. Histochemical staining revealed that lesion formation might reflect programmed cell death (PCD) and abnormal accumulation of reactive oxygen species (ROS). The chlorophyll content in the mutant was significantly lower than that in wildtype, and the ratio of chlorophyll a/b was increased significantly in the mutant compared with wildtype, indicating that lm3 showed impairment of the biosynthesis or degradation of chlorophyll, and that Chlorophyll b was prone to damage during lesion formation. The lm3 mutant exhibited enhanced resistance to wheat powdery mildew fungus (Blumeria graminis f. sp. tritici; Bgt) infection, which was consistent with the increased expression of seven pathogenesis-related (PR) and two wheat chemically induced (WCI) genes involved in the defense-related reaction. Genetic analysis showed that the mutation was controlled through a single partially dominant gene, which was closely linked to Xbarc203 on chromosome 3BL; this gene was delimited to a 40 Mb region between SSR3B450.37 and SSR3B492.6 using a large derived segregating population and the available Chinese Spring chromosome 3B genome sequence. Taken together, our results provide information regarding the identification of a novel wheat LM gene, which will facilitate the additional fine-mapping and

  7. The cataract-associated V41M mutant of human γS-crystallin shows specific structural changes that directly enhance local surface hydrophobicity.

    PubMed

    Bharat, Somireddy Venkata; Shekhtman, Alexander; Pande, Jayanti

    2014-01-01

    The major crystallins expressed in the human lens are γS-, γC- and γD-crystallins. Several mutations in γS-crystallin are associated with hereditary cataracts, one of which involves the substitution of a highly conserved Valine at position 41 to Methionine. According to a recent report, the mutant protein, V41M, shows lower stability and increased surface hydrophobicity compared to the wild-type, and a propensity for self-aggregation. Here we address the structural differences between the two proteins, with residue-level specificity using NMR spectroscopy. Based on the structural model of the mutant protein, our results clearly show that the mutation creates a major local perturbation almost at the junction of the first and second "Greek-key" motifs in the N-terminal domain. A larger section of the second motif (residues 44-86) appears to be mainly affected. Based on the sizeable chemical shift of the imino proton of the indole side-chain of Trp46 in V41M, we suggest that the sulphur atom of Met41 is involved in an S-π interaction with Trp46. This interaction would bring the last β-strand of the first "Greek-key" motif closer to the first β-strand of the second motif. This appears to lead to a domino effect, towards both the N- and C-terminal ends, even as it decays off substantially beyond the domain interface. During this process discreet hydrophobic surface patches are created, as revealed by ANS-binding. Such changes would not affect the secondary structure or cause a major change in the tertiary structure, but can lead to self-aggregation or aberrant binding interactions of the mutant protein in vivo, and lead to lens opacity or cataract. PMID:24287181

  8. The cataract-associated V41M mutant of human γS-crystallin shows specific structural changes that directly enhance local surface hydrophobicity

    SciTech Connect

    Bharat, Somireddy Venkata; Shekhtman, Alexander; Pande, Jayanti

    2014-01-03

    Highlights: •We present NMR analysis of V41M, a cataract-causing mutant of human γS-crystallin. •Mutation alters strand–strand interactions throughout the N-terminal domain. •Mutation directly affects Trp46 due to key Met41-S–Trp46-pi interactions. •We identify the basis of the surface hydrophobicity increase and residues involved. -- Abstract: The major crystallins expressed in the human lens are γS-, γC- and γD-crystallins. Several mutations in γS-crystallin are associated with hereditary cataracts, one of which involves the substitution of a highly conserved Valine at position 41 to Methionine. According to a recent report, the mutant protein, V41M, shows lower stability and increased surface hydrophobicity compared to the wild-type, and a propensity for self-aggregation. Here we address the structural differences between the two proteins, with residue-level specificity using NMR spectroscopy. Based on the structural model of the mutant protein, our results clearly show that the mutation creates a major local perturbation almost at the junction of the first and second “Greek-key” motifs in the N-terminal domain. A larger section of the second motif (residues 44–86) appears to be mainly affected. Based on the sizeable chemical shift of the imino proton of the indole side-chain of Trp46 in V41M, we suggest that the sulphur atom of Met41 is involved in an S–π interaction with Trp46. This interaction would bring the last β-strand of the first “Greek-key” motif closer to the first β-strand of the second motif. This appears to lead to a domino effect, towards both the N- and C-terminal ends, even as it decays off substantially beyond the domain interface. During this process discreet hydrophobic surface patches are created, as revealed by ANS-binding. Such changes would not affect the secondary structure or cause a major change in the tertiary structure, but can lead to self-aggregation or aberrant binding interactions of the mutant

  9. Transcriptomic analysis of Clostridium thermocellum Populus hydrolysate-tolerant mutant strain shows increased cellular efficiency in response to Populus hydrolysate compared to the wild type strain

    PubMed Central

    2014-01-01

    Background The thermophilic, anaerobic bacterium, Clostridium thermocellum is a model organism for consolidated processing due to its efficient fermentation of cellulose. Constituents of dilute acid pretreatment hydrolysate are known to inhibit C. thermocellum and other microorganisms. To evaluate the biological impact of this type of hydrolysate, a transcriptomic analysis of growth in hydrolysate-containing medium was conducted on 17.5% v/v Populus hydrolysate-tolerant mutant (PM) and wild type (WT) strains of C. thermocellum. Results In two levels of Populus hydrolysate medium (0% and 10% v/v), the PM showed both gene specific increases and decreases of gene expression compared to the wild-type strain. The PM had increased expression of genes in energy production and conversion, and amino acid transport and metabolism in both standard and 10% v/v Populus hydrolysate media. In particular, expression of the histidine metabolism increased up to 100 fold. In contrast, the PM decreased gene expression in cell division and sporulation (standard medium only), cell defense mechanisms, cell envelope, cell motility, and cellulosome in both media. The PM downregulated inorganic ion transport and metabolism in standard medium but upregulated it in the hydrolysate media when compared to the WT. The WT differentially expressed 1072 genes in response to the hydrolysate medium which included increased transcription of cell defense mechanisms, cell motility, and cellulosome, and decreased expression in cell envelope, amino acid transport and metabolism, inorganic ion transport and metabolism, and lipid metabolism, while the PM only differentially expressed 92 genes. The PM tolerates up to 17.5% v/v Populus hydrolysate and growth in it elicited 489 genes with differential expression, which included increased expression in energy production and conversion, cellulosome production, and inorganic ion transport and metabolism and decreased expression in transcription and cell

  10. A bovine herpesvirus 1 pUL51 deletion mutant shows impaired viral growth in vitro and reduced virulence in rabbits

    PubMed Central

    Raza, Sohail; Deng, Mingliang; Shahin, Farzana; Yang, Kui; Hu, Changmin; Chen, Yingyu; Chen, Huanchun; Guo, Aizhen

    2016-01-01

    Bovine herpesvirus 1 (BoHV-1) UL51 protein (pUL51) is a tegument protein of BoHV-1 whose function is currently unknown. Here, we aimed to illustrate the specific role of pUL51 in virion morphogenesis and its importance in BoHV-1 virulence. To do so, we constructed a BoHV-1 bacterial artificial chromosome (BAC). We used recombinant BAC and transgenic techniques to delete a major part of the UL51 open reading frame. Deletion of pUL51 resulted in severe viral growth defects, as evidenced by lower single and multi-step growth kinetics, reduced plaque size, and the accumulation of non-enveloped capsids in the cytoplasm of infected cells. Using tagged BoHV-1 recombinant viruses, it was determined that the pUL51 protein completely co-localized with the cis-Golgi marker protein GM-130. Taken altogether, pUL51 was demonstrated to play a critical role in BoHV-1 growth and it is involved in viral maturation and egress. Moreover, an in vivo analysis showed that the pUL51 mutant exhibited reduced virulence in rabbits, with no clinical signs, no nasal shedding of the virus, and no detectable serum neutralizing antibodies. Therefore, we conclude that the BoHV-1 pUL51 is indispensable for efficient viral growth in vitro and is essential for virulence in vivo. PMID:26934330

  11. Lipoprotein LpqS deficient M. tuberculosis mutant is attenuated for virulence in vivo and shows protective efficacy better than BCG in guinea pigs.

    PubMed

    Sakthi, Suba; Palaniyandi, Kannan; Gupta, Umesh D; Gupta, Pushpa; Narayanan, Sujatha

    2016-02-01

    Bacterial lipoproteins are a functionally diverse class of membrane anchored proteins. Lipoproteins constitute nearly 2.5% of the Mycobacterium tuberculosis proteome. Inactivation of genes coding for individual lipoproteins results in attenuated phenotype of the mutants. LpqS is a lipoprotein highly conserved among slow growing pathogenic mycobacteria. Our previous study has shown that the lpqS gene deletion mutant of M. tuberculosis (MtbΔlpqS) poorly replicates in THP1-(human acute monocytic leukemia cell line) derived macrophagic cell line. In addition, guinea pigs, when infected with the mutant strain exhibited significantly reduced bacterial burden and pathological damage in the infected tissues in comparison with the parental strain infected group. Subsequently, we evaluated the protective efficacy of the mutant by immunization of guinea pigs through aerosol and subcutaneous routes. We observed that immunization of guinea pigs with MtbΔlpqS offered superior protection in lungs as compared to BCG. In addition, MtbΔlpqS also prevented the haematogenous spread of the disease which was evident from the significantly reduced splenic bacillary load compared to saline vaccinated animals. The gross pathological observations and the histopathological observations well corroborated the bacterial findings. We also observed that aerogenic route of immunization imparts superior protection compared to subcutaneous route of immunization. These findings well establishes the efficacy of M. tuberculosis mutant in imparting protection against pulmonary TB. PMID:26768127

  12. Translation elongation factor 1A mutants with altered actin bundling activity show reduced aminoacyl-tRNA binding and alter initiation via eIF2α phosphorylation.

    PubMed

    Perez, Winder B; Kinzy, Terri Goss

    2014-07-25

    Apart from its canonical function in translation elongation, eukaryotic translation elongation factor 1A (eEF1A) has been shown to interact with the actin cytoskeleton. Amino acid substitutions in eEF1A that reduce its ability to bind and bundle actin in vitro cause improper actin organization in vivo and reduce total translation. Initial in vivo analysis indicated the reduced translation was through initiation. The mutant strains exhibit increased levels of phosphorylated initiation factor 2α (eIF2α) dependent on the presence of the general control non-derepressible 2 (Gcn2p) protein kinase. Gcn2p causes downregulation of total protein synthesis at initiation in response to increases in deacylated tRNA levels in the cell. Increased levels of eIF2α phosphorylation are not due to a general reduction in translation elongation as eEF2 and eEF3 mutants do not exhibit this effect. Deletion of GCN2 from the eEF1A actin bundling mutant strains revealed a second defect in translation. The eEF1A actin-bundling proteins exhibit changes in their elongation activity at the level of aminoacyl-tRNA binding in vitro. These findings implicate eEF1A in a feedback mechanism for regulating translation at initiation. PMID:24936063

  13. 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

  14. Analysis of a Spontaneous Non-Motile and Avirulent Mutant Shows That FliM Is Required for Full Endoflagella Assembly in Leptospira interrogans

    PubMed Central

    Fontana, Célia; Lambert, Ambroise; Benaroudj, Nadia; Gasparini, David; Gorgette, Olivier; Cachet, Nathalie; Bomchil, Natalia; Picardeau, Mathieu

    2016-01-01

    Pathogenic Leptospira strains are responsible for leptospirosis, a worldwide emerging zoonotic disease. These spirochetes are unique amongst bacteria because of their corkscrew-like cell morphology and their periplasmic flagella. Motility is reported as an important virulence determinant, probably favoring entry and dissemination of pathogenic Leptospira in the host. However, proteins constituting the periplasmic flagella and their role in cell shape, motility and virulence remain poorly described. In this study, we characterized a spontaneous L. interrogans mutant strain lacking motility, correlated with the loss of the characteristic hook-shaped ends, and virulence in the animal model. Whole genome sequencing allowed the identification of one nucleotide deletion in the fliM gene resulting in a premature stop codon, thereby preventing the production of flagellar motor switch protein FliM. Genetic complementation restored cell morphology, motility and virulence comparable to those of wild type cells. Analyses of purified periplasmic flagella revealed a defect in flagella assembly, resulting in shortened flagella compared to the wild type strain. This also correlated with a lower amount of major filament proteins FlaA and FlaB. Altogether, these findings demonstrate that FliM is required for full and correct assembly of the flagella which is essential for motility and virulence. PMID:27044038

  15. Analysis of a Spontaneous Non-Motile and Avirulent Mutant Shows That FliM Is Required for Full Endoflagella Assembly in Leptospira interrogans.

    PubMed

    Fontana, Célia; Lambert, Ambroise; Benaroudj, Nadia; Gasparini, David; Gorgette, Olivier; Cachet, Nathalie; Bomchil, Natalia; Picardeau, Mathieu

    2016-01-01

    Pathogenic Leptospira strains are responsible for leptospirosis, a worldwide emerging zoonotic disease. These spirochetes are unique amongst bacteria because of their corkscrew-like cell morphology and their periplasmic flagella. Motility is reported as an important virulence determinant, probably favoring entry and dissemination of pathogenic Leptospira in the host. However, proteins constituting the periplasmic flagella and their role in cell shape, motility and virulence remain poorly described. In this study, we characterized a spontaneous L. interrogans mutant strain lacking motility, correlated with the loss of the characteristic hook-shaped ends, and virulence in the animal model. Whole genome sequencing allowed the identification of one nucleotide deletion in the fliM gene resulting in a premature stop codon, thereby preventing the production of flagellar motor switch protein FliM. Genetic complementation restored cell morphology, motility and virulence comparable to those of wild type cells. Analyses of purified periplasmic flagella revealed a defect in flagella assembly, resulting in shortened flagella compared to the wild type strain. This also correlated with a lower amount of major filament proteins FlaA and FlaB. Altogether, these findings demonstrate that FliM is required for full and correct assembly of the flagella which is essential for motility and virulence. PMID:27044038

  16. Gene Expressing and sRNA Sequencing Show That Gene Differentiation Associates with a Yellow Acer palmatum Mutant Leaf in Different Light Conditions

    PubMed Central

    Li, Shu-Shun; Li, Qian-Zhong; Rong, Li-Ping; Tang, Ling; Zhang, Bo

    2015-01-01

    Acer palmatum Thunb., like other maples, is a widely ornamental-use small woody tree for leaf shapes and colors. Interestingly, we found a yellow-leaves mutant “Jingling Huangfeng” turned to green when grown in shade or low-density light condition. In order to study the potential mechanism, we performed high-throughput sequencing and obtained 1,082 DEGs in leaves grown in different light conditions that result in A. palmatum significant morphological and physiological changes. A total of 989 DEGs were annotated and clustered, of which many DEGs were found associating with the photosynthesis activity and pigment synthesis. The expression of CHS and FDR gene was higher while the expression of FLS gene was lower in full-sunlight condition; this may cause more colorful substance like chalcone and anthocyanin that were produced in full-light condition, thus turning the foliage to yellow. Moreover, this is the first available miRNA collection which contains 67 miRNAs of A. palmatum, including 46 conserved miRNAs and 21 novel miRNAs. To get better understanding of which pathways these miRNAs involved, 102 Unigenes were found to be potential targets of them. These results will provide valuable genetic resources for further study on the molecular mechanisms of Acer palmatum leaf coloration. PMID:26788511

  17. Gene Expressing and sRNA Sequencing Show That Gene Differentiation Associates with a Yellow Acer palmatum Mutant Leaf in Different Light Conditions.

    PubMed

    Li, Shu-Shun; Li, Qian-Zhong; Rong, Li-Ping; Tang, Ling; Zhang, Bo

    2015-01-01

    Acer palmatum Thunb., like other maples, is a widely ornamental-use small woody tree for leaf shapes and colors. Interestingly, we found a yellow-leaves mutant "Jingling Huangfeng" turned to green when grown in shade or low-density light condition. In order to study the potential mechanism, we performed high-throughput sequencing and obtained 1,082 DEGs in leaves grown in different light conditions that result in A. palmatum significant morphological and physiological changes. A total of 989 DEGs were annotated and clustered, of which many DEGs were found associating with the photosynthesis activity and pigment synthesis. The expression of CHS and FDR gene was higher while the expression of FLS gene was lower in full-sunlight condition; this may cause more colorful substance like chalcone and anthocyanin that were produced in full-light condition, thus turning the foliage to yellow. Moreover, this is the first available miRNA collection which contains 67 miRNAs of A. palmatum, including 46 conserved miRNAs and 21 novel miRNAs. To get better understanding of which pathways these miRNAs involved, 102 Unigenes were found to be potential targets of them. These results will provide valuable genetic resources for further study on the molecular mechanisms of Acer palmatum leaf coloration. PMID:26788511

  18. Allosteric mutants show that PrfA activation is dispensable for vacuole escape but required for efficient spread and Listeria survival in vivo

    PubMed Central

    Deshayes, Caroline; Bielecka, Magdalena K; Cain, Robert J; Scortti, Mariela; de las Heras, Aitor; Pietras, Zbigniew; Luisi, Ben F; Núñez Miguel, Ricardo; Vázquez-Boland, José A

    2012-01-01

    The transcriptional regulator PrfA controls key virulence determinants of the facultative intracellular pathogen Listeria monocytogenes. PrfA-dependent gene expression is strongly induced within host cells. While the basis of this activation is unknown, the structural homology of PrfA with the cAMP receptor protein (Crp) and the finding of constitutively activated PrfA* mutants suggests it may involve ligand-induced allostery. Here, we report the identification of a solvent-accessible cavity within the PrfA N-terminal domain that may accommodate an activating ligand. The pocket occupies a similar position to the cAMP binding site in Crp but lacks the cyclic nucleotide-anchoring motif and has its entrance on the opposite side of the β-barrel. Site-directed mutations in this pocket impaired intracellular PrfA-dependent gene activation without causing extensive structural/functional alterations to PrfA. Two substitutions, L48F and Y63W, almost completely abolished intracellular virulence gene induction and thus displayed the expected phenotype for allosteric activation-deficient PrfA mutations. Neither PrfAallo substitution affected vacuole escape and initial intracellular growth of L. monocytogenes in epithelial cells and macrophages but caused defective cell-to-cell spread and strong attenuation in mice. Our data support the hypothesis that PrfA is allosterically activated during intracellular infection and identify the probable binding site for the effector ligand. They also indicate that PrfA allosteric activation is not required for early intracellular survival but is essential for full Listeria virulence and colonization of host tissues. PMID:22646689

  19. G2019S LRRK2 mutant fibroblasts from Parkinson's disease patients show increased sensitivity to neurotoxin 1-methyl-4-phenylpyridinium dependent of autophagy.

    PubMed

    Yakhine-Diop, Sokhna M S; Bravo-San Pedro, José M; Gómez-Sánchez, Rubén; Pizarro-Estrella, Elisa; Rodríguez-Arribas, Mario; Climent, Vicente; Aiastui, Ana; López de Munain, Adolfo; Fuentes, José M; González-Polo, Rosa A

    2014-10-01

    Parkinson's disease (PD) is a neurodegenerative disorder of unknown etiology. It is considered as a multifactorial disease dependent on environmental and genetic factors. Deregulation in cell degradation has been related with a significant increase in cell damage, becoming a target for studies on the PD etiology. In the present study, we have characterized the parkinsonian toxin 1-methyl-4-phenylpyridinium ion (MPP(+))-induced damage in fibroblasts from Parkinson's patients with the mutation G2019S in leucine-rich repeat kinase 2 protein (LRRK2) and control individuals without this mutation. The results reveal that MPP(+) induces mTOR-dependent autophagy in fibroblasts. Moreover, the effects of caspase-dependent cell death to MPP(+) were higher in cells with the G2019S LRRK2 mutation, which showed basal levels of autophagy due to the G2019S LRRK2 mutation (mTOR-independent). The inhibition of autophagy by 3-methyladenine (3-MA) treatment reduces these sensitivity differences between both cell types, however, the inhibition of autophagosome-lysosome fusion by bafilomycin A1 (Baf A1) increases these differences. This data confirm the importance of the combination of genetic and environmental factors in the PD etiology. Thereby, the sensitivity to the same damage may be different in function of a genetic predisposition, reason why individuals with certain mutations can develop some early-onset diseases, such as individuals with G2019S LRRK2 mutation and PD. PMID:25017139

  20. Mutants of metal binding site M1 in APP E2 show metal specific differences in binding of heparin but not of sorLA.

    PubMed

    Dienemann, Christian; Coburger, Ina; Mehmedbasic, Arnela; Andersen, Olav M; Than, Manuel E

    2015-04-21

    The amyloid precursor protein (APP) and its neurotoxic cleavage product Aβ are key players in the development of Alzheimer's disease (AD) and appear to be essential for neuronal development and cell homeostasis. Proteolytic processing of APP and its physiological function depend on its interaction with heparin and are influenced by the binding of metal ions and sorLA. We created various mutations of metal binding site M1 residing within the extracellular E2 domain of APP. Using isothermal titration calorimetry and circular dichroism spectroscopy, we analyzed the binding of Cu(2+) and Zn(2+) to APP E2 and identified two mutations that are most suited for functional studies to dissect ion specific effects of metal binding. The H313A mutation abrogates only copper-based effects, whereas the H382A mutation weakens any metal binding at M1 of APP E2. Subsequently, we tested the effect of Cu(2+) and Zn(2+) on the binding of heparin and sorLA to APP E2 using a chromatographic technique and surface plasmon resonance. We show that Zn(2+) and to a larger degree also Cu(2+) enhance the binding of heparin to APP E2, consistent with an extracellular regulation of the function of APP by both metal ions. In contrast, neither ion seemed to affect the interaction between APP E2 and sorLA. This supports an intracellular interaction between the latter two partners that would not sense extracellular variations of metal ions upon synaptic activity. PMID:25835329

  1. A Porcine Circovirus Type 2 (PCV2) Mutant with 234 Amino Acids in Capsid Protein Showed More Virulence In Vivo, Compared with Classical PCV2a/b Strain

    PubMed Central

    Guo, Longjun; Fu, Yujie; Wang, Yiping; Lu, Yuehua; Wei, Yanwu; Tang, Qinghai; Fan, Peihu; Liu, Jianbo; Zhang, Long; Zhang, Feiyan; Huang, Liping; Liu, Dan; Li, Shengbin; Wu, Hongli; Liu, Changming

    2012-01-01

    Background Porcine circovirus type 2 (PCV2) is considered to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), which has become a serious economic problem for the swine industry worldwide. The major genotypes, PCV2a and PCV2b, are highly prevalent in the pig population and are present worldwide. However, another newly emerging PCV2b genotype mutant, which has a mutation in its ORF2-encoded capsid protein, has been sporadically present in China, as well as in other countries. It is therefore important to determine the relative virulence of the newly emerging PCV2b genotype mutant, compared with the existing PCV2a and PCV2b genotypes, and to investigate whether the newly emerging mutant virus induces more severe illness. Methodology/Principal Findings Twenty healthy, 30-day-old, commercial piglets served as controls or were challenged with PCV2a, PCV2b and the newly emerging mutant virus. A series of indexes representing different parameters were adopted to evaluate virulence, including clinical signs, serological detection, viral load and distribution, changes in immune cell subsets in the peripheral blood, and evaluation of pathological lesions. The newly emerging PCV2 mutant demonstrated more severe signs compatible with PMWS, characterized by wasting, coughing, dyspnea, diarrhea, rough hair-coat and depression. Moreover, the pathological lesions and viremia, as well as the viral loads in lymph nodes, tonsils and spleen, were significantly more severe (P<0.05) for piglets challenged with the newly emerging mutant compared with those in the groups challenged with PCV2a and PCV2b. In addition, a significantly lower average daily weight gain (P<0.05) was recorded in the group challenged with the newly emerging PCV2 mutant than in the groups challenged with the prevailing PCV2a and PCV2b. Conclusions This is believed to be the first report to confirm the enhanced virulence of the newly emerging PCV2 mutant in vivo. PMID:22829951

  2. Nonchemotactic Mutants of Escherichia coli

    PubMed Central

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

    1967-01-01

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

  3. "The Show"

    ERIC Educational Resources Information Center

    Gehring, John

    2004-01-01

    For the past 16 years, the blue-collar city of Huntington, West Virginia, has rolled out the red carpet to welcome young wrestlers and their families as old friends. They have come to town chasing the same dream for a spot in what many of them call "The Show". For three days, under the lights of an arena packed with 5,000 fans, the state's best…

  4. Evaluation of histone 3 lysine 27 trimethylation (H3K27me3) and enhancer of Zest 2 (EZH2) in pediatric glial and glioneuronal tumors shows decreased H3K27me3 in H3F3A K27M mutant glioblastomas.

    PubMed

    Venneti, Sriram; Garimella, Mihir T; Sullivan, Lisa M; Martinez, Daniel; Huse, Jason T; Heguy, Adriana; Santi, Mariarita; Thompson, Craig B; Judkins, Alexander R

    2013-09-01

    H3F3A mutations are seen in ∼30% of pediatric glioblastoma (GBMs) and involve either the lysine residue at position 27 (K27M) or glycine at position 34 (G34R/V). Sixteen genes encode histone H3, each variant differing in only a few amino acids. Therefore, how mutations in a single H3 gene contribute to carcinogenesis is unknown. H3F3A K27M mutations are predicted to alter methylation of H3K27. H3K27me3 is a repressive mark critical to stem cell maintenance and is mediated by EZH2, a member of the polycomb-group (PcG) family. We evaluated H3K27me3 and EZH2 expression using immunohistochemistry in 76 pediatric brain tumors. H3K27me3 was lowered/absent in tumor cells but preserved in endothelial cells and infiltrating lymphocytes in six out of 20 GBMs. H3K27me3 showed strong immunoreactivity in all other tumor subtypes. Sequencing of GBMs showed H3F3A K27M mutations in all six cases with lowered/absent H3K27me3. EZH2 expression was high in GBMs, but absent/focal in other tumors. However, no significant differences in EZH2 expression were observed between H3F3A K27M mutant and wild type GBMs, suggesting that EZH2 mediated trimethylation of H3K27 is inhibited in GBM harboring K27M mutations. Our results indicate that H3F3A K27M mutant GBMs show decreased H3K27me3 that may be of both diagnostic and biological relevance. PMID:23414300

  5. Nebulin binding impedes mutant desmin filament assembly

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  7. Kasugamycin-dependent mutants of Escherichia coli.

    PubMed Central

    Dabbs, E R

    1978-01-01

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

  8. Arabidopsis mutants impaired in cosuppression.

    PubMed Central

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

    1998-01-01

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

  9. New therapeutic strategies for BRAF mutant colorectal cancers

    PubMed Central

    2015-01-01

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

  10. Characterization of the procera Tomato Mutant Shows Novel Functions of the SlDELLA Protein in the Control of Flower Morphology, Cell Division and Expansion, and the Auxin-Signaling Pathway during Fruit-Set and Development1[C][W

    PubMed Central

    Carrera, Esther; Ruiz-Rivero, Omar; Peres, Lazaro Eustaquio Pereira; Atares, Alejandro; Garcia-Martinez, Jose Luis

    2012-01-01

    procera (pro) is a tall tomato (Solanum lycopersicum) mutant carrying a point mutation in the GRAS region of the gene encoding SlDELLA, a repressor in the gibberellin (GA) signaling pathway. Consistent with the SlDELLA loss of function, pro plants display a GA-constitutive response phenotype, mimicking wild-type plants treated with GA3. The ovaries from both nonemasculated and emasculated pro flowers had very strong parthenocarpic capacity, associated with enhanced growth of preanthesis ovaries due to more and larger cells. pro parthenocarpy is facultative because seeded fruits were obtained by manual pollination. Most pro pistils had exserted stigmas, thus preventing self-pollination, similar to wild-type pistils treated with GA3 or auxins. However, Style2.1, a gene responsible for long styles in noncultivated tomato, may not control the enhanced style elongation of pro pistils, because its expression was not higher in pro styles and did not increase upon GA3 application. Interestingly, a high percentage of pro flowers had meristic alterations, with one additional petal, sepal, stamen, and carpel at each of the four whorls, respectively, thus unveiling a role of SlDELLA in flower organ development. Microarray analysis showed significant changes in the transcriptome of preanthesis pro ovaries compared with the wild type, indicating that the molecular mechanism underlying the parthenocarpic capacity of pro is complex and that it is mainly associated with changes in the expression of genes involved in GA and auxin pathways. Interestingly, it was found that GA activity modulates the expression of cell division and expansion genes and an auxin signaling gene (tomato AUXIN RESPONSE FACTOR7) during fruit-set. PMID:22942390

  11. Mutant fatty acid desaturase

    DOEpatents

    Shanklin, John; Cahoon, Edgar B.

    2004-02-03

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

  12. Arabidopsis mutants with a reduced seed dormancy.

    PubMed Central

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

    1996-01-01

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

  13. Connexin Mutants and Cataracts

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2001-03-01

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

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

    PubMed

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

    2016-03-10

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

  17. 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

  18. Isolation and characterization of Klebsiella pneumoniae unencapsulated mutants

    SciTech Connect

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

    1989-01-01

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

  19. Synaptic transmission deficits in Caenorhabditis elegans synaptobrevin mutants.

    PubMed

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

    1998-01-01

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

  20. Phanerochaete mutants with enhanced ligninolytic activity

    SciTech Connect

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

    1993-06-01

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

  1. Mutant p53 in cell adhesion and motility.

    PubMed

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

    2013-01-01

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

  2. ECB deacylase mutants

    DOEpatents

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

    2002-01-01

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

  3. Sphingolipid synthesis deficiency in a mutant of Bacteroides levii

    SciTech Connect

    Brumleve, B.; Lev, M.

    1986-05-01

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

  4. Escherichia coli mutants deficient in exonuclease VII.

    PubMed Central

    Chase, J W; Richardson, C C

    1977-01-01

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

  5. Isolation and characterization of Francisella novicida mutants defective in lipopolysaccharide biosynthesis.

    PubMed

    Cowley, S C; Gray, C J; Nano, F E

    2000-01-01

    In order to identify genes involved in LPS biosynthesis we isolated random mutants generated by transposon insertion in Francisella novicida. The resulting mutant bank yielded mutants with three distinct LPS phenotypes, and three representative mutants were chosen for further study. One mutant that had short O-antigen chains was sensitive to serum; this mutant and one other were more sensitive to killing by deoxycholate than control strains. The third mutant was resistant to deoxycholate killing but slightly sensitive to serum. The three mutants varied in their ability to grow in macrophages. The DNA sequences interrupted by the transposon in two of the three mutants showed similarity to known LPS biosynthetic genes at the deduced amino acid level. PMID:10612732

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2016-01-01

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

  12. Motility mutants of Dictyostelium discoideum

    PubMed Central

    1982-01-01

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

  13. Television Quiz Show Simulation

    ERIC Educational Resources Information Center

    Hill, Jonnie Lynn

    2007-01-01

    This article explores the simulation of four television quiz shows for students in China studying English as a foreign language (EFL). It discusses the adaptation and implementation of television quiz shows and how the students reacted to them.

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

    NASA Astrophysics Data System (ADS)

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

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

  15. The Wordpath Show.

    ERIC Educational Resources Information Center

    Anderton, Alice

    The Intertribal Wordpath Society is a nonprofit educational corporation formed to promote the teaching, status, awareness, and use of Oklahoma Indian languages. The Society produces "Wordpath," a weekly 30-minute public access television show about Oklahoma Indian languages and the people who are teaching and preserving them. The show aims to…

  16. Characterization of fig operon mutants of Francisella novicida U112

    PubMed Central

    Kiss, Katalin; Liu, Wei; Huntley, Jason F.; Norgard, Michael V.; Hansen, Eric J.

    2009-01-01

    Francisella species secrete a polycarboxylate siderophore that resembles rhizoferrin to acquire ferric iron. Several of the Francisella siderophore synthesis genes are contained in a Fur-regulated operon (designated fig or fsl) comprised of at least seven open reading frames (ORFs) including fur. Reverse transcriptase-PCR showed transcriptional linkage between figD and figE and between figE and figF. Mutations were constructed in four of these ORFs (figB, figC, figD, and figE) in F. novicida U112. All four of these new mutants and a F. novicida figA mutant grew at rates comparable to that of wild-type under iron-replete conditions but growth of all five mutants was stunted in iron-limiting media. When ferric rhizoferrin was added to the iron-limited media, growth of the figA, figB, figC, and figD mutants was restored to levels similar to those obtained in iron-replete media. However, this exogenously added siderophore could not rescue the figE mutant. When Chrome Azurol S assays were used to measure siderophore production, the figA, figB, and figC mutants were markedly deficient in their ability to synthesize siderophore whereas the figD and figE mutants produced siderophore at levels equivalent to the wild-type parent strain. PMID:18564336

  17. A Holographic Road Show.

    ERIC Educational Resources Information Center

    Kirkpatrick, Larry D.; Rugheimer, Mac

    1979-01-01

    Describes the viewing sessions and the holograms of a holographic road show. The traveling exhibits, believed to stimulate interest in physics, include a wide variety of holograms and demonstrate several physical principles. (GA)

  18. Resistant mechanism study of benzalkonium chloride selected Salmonella Typhimurium mutants.

    PubMed

    Guo, Wei; Cui, Shenghui; Xu, Xiao; Wang, Haoyan

    2014-02-01

    Benzalkonium chloride is one of the invaluable biocides that is extensively used in healthcare settings as well as in the food processing industry. After exposing wild-type Salmonella Typhimurium 14028s or its AcrAB inactivation mutant to gradually increasing levels of benzalkonium chloride, resistance mutants S-41, S-150, S-AB-23, S-AB-38, and S-AB-73 were selected and these mutants also showed a 2-64-fold stable minimum inhibitory concentration (MIC) increase to chloramphenicol, ciprofloxacin, nalidixic acid, and tetracycline. In S-41 and S-150, the expression of acrB was increased 2.7- and 7.6-fold, and ΔtolC or ΔacrAB mutants of S-41 and S-150 showed the same MICs to all tested antimicrobials as the equivalent Salmonella Typhimurium 14028s mutants. However, in S-AB-23, S-AB-38, and S-AB-73, the expression of acrF was increased 96-, 230-, and 267-fold, respectively, and ΔtolC or ΔacrEF mutants of S-AB-23, S-AB-38, and S-AB-73 showed the similar MICs to all tested antimicrobials as the ΔtolC mutant of Salmonella Typhimurium 14028s. Our data showed that constitutively over-expressed AcrAB working through TolC was the main resistance mechanism in ST14028s benzalkonium chloride resistance mutants. However, after AcrAB had been inactivated, benzalkonium chloride-resistant mutants could still be selected and constitutively over-expressed, AcrEF became the dominant efflux pump working through TolC and being responsible for the increasing antimicrobial resistance. These data indicated that different mechanisms existed for acrB and acrF constitutive over-expression. Since exposure to benzalkonium chloride may lead to Salmonella mutants with a decreased susceptibility to quinolones, which is currently one of the drugs of choice for the treatment of life-threatening salmonelosis, research into the pathogenesis and epidemiology of the benzalkonium chloride resistance mutants will be of increasing importance. PMID:23987991

  19. 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

  20. Show What You Know

    ERIC Educational Resources Information Center

    Eccleston, Jeff

    2007-01-01

    Big things come in small packages. This saying came to the mind of the author after he created a simple math review activity for his fourth grade students. Though simple, it has proven to be extremely advantageous in reinforcing math concepts. He uses this activity, which he calls "Show What You Know," often. This activity provides the perfect…

  1. The Ozone Show.

    ERIC Educational Resources Information Center

    Mathieu, Aaron

    2000-01-01

    Uses a talk show activity for a final assessment tool for students to debate about the ozone hole. Students are assessed on five areas: (1) cooperative learning; (2) the written component; (3) content; (4) self-evaluation; and (5) peer evaluation. (SAH)

  2. Honored Teacher Shows Commitment.

    ERIC Educational Resources Information Center

    Ratte, Kathy

    1987-01-01

    Part of the acceptance speech of the 1985 National Council for the Social Studies Teacher of the Year, this article describes the censorship experience of this honored social studies teacher. The incident involved the showing of a videotape version of the feature film entitled "The Seduction of Joe Tynan." (JDH)

  3. Talk Show Science.

    ERIC Educational Resources Information Center

    Moore, Mitzi Ruth

    1992-01-01

    Proposes having students perform skits in which they play the roles of the science concepts they are trying to understand. Provides the dialog for a skit in which hot and cold gas molecules are interviewed on a talk show to study how these properties affect wind, rain, and other weather phenomena. (MDH)

  4. Stage a Water Show

    ERIC Educational Resources Information Center

    Frasier, Debra

    2008-01-01

    In the author's book titled "The Incredible Water Show," the characters from "Miss Alaineus: A Vocabulary Disaster" used an ocean of information to stage an inventive performance about the water cycle. In this article, the author relates how she turned the story into hands-on science teaching for real-life fifth-grade students. The author also…

  5. Showing What They Know

    ERIC Educational Resources Information Center

    Cech, Scott J.

    2008-01-01

    Having students show their skills in three dimensions, known as performance-based assessment, dates back at least to Socrates. Individual schools such as Barrington High School--located just outside of Providence--have been requiring students to actively demonstrate their knowledge for years. The Rhode Island's high school graduating class became…

  6. Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants.

    PubMed

    Sung, Dong-Yul; Lee, David; Harris, Hugh; Raab, Andrea; Feldmann, Jörg; Meharg, Andrew; Kumabe, Bryan; Komives, Elizabeth A; Schroeder, Julian I

    2007-03-01

    A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5. PMID:17335514

  7. Identification of An Arsenic Tolerant Double Mutant With a Thiol-Mediated Component And Increased Arsenic Tolerance in PhyA Mutants

    SciTech Connect

    Sung, D.Y.; Lee, D.; Harris, H.; Raab, A.; Feldmann, J.; Meharg, A.; Kumabe, B.; Komives, E.A.; Schroeder, J.I.; /SLAC, SSRL /Sydney U. /Aberdeen U. /UC, San Diego

    2007-04-06

    A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.

  8. Biofilm formation-defective mutants in Pseudomonas putida.

    PubMed

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

    2016-07-01

    Out of 8000 candidates from a genetic screening for Pseudomonas putida KT2442 mutants showing defects in biofilm formation, 40 independent mutants with diminished levels of biofilm were analyzed. Most of these mutants carried insertions in genes of the lap cluster, whose products are responsible for synthesis, export and degradation of the adhesin LapA. All mutants in this class were strongly defective in biofilm formation. Mutants in the flagellar regulatory genes fleQ and flhF showed similar defects to that of the lap mutants. On the contrary, transposon insertions in the flagellar structural genes fliP and flgG, that also impair flagellar motility, had a modest defect in biofilm formation. A mutation in gacS, encoding the sensor element of the GacS/GacA two-component system, also had a moderate effect on biofilm formation. Additional insertions targeted genes involved in cell envelope function: PP3222, encoding the permease element of an ABC-type transporter and tolB, encoding the periplasmic component of the Tol-OprL system required for outer membrane stability. Our results underscore the central role of LapA, suggest cross-regulation between motility and adhesion functions and provide insights on the role of cell envelope trafficking and maintenance for biofilm development in P. putida. PMID:27190143

  9. Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities.

    PubMed

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

    2016-03-01

    The RAS/MAPK (mitogen-activated protein kinase) signalling pathway is frequently deregulated in non-small-cell lung cancer, often through KRAS activating mutations. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations. We recently showed that advanced lung tumours from Kras(G12D/+);p53-null mice frequently exhibit Kras(G12D) allelic enrichment (Kras(G12D)/Kras(wild-type) > 1) (ref. 7), implying that mutant Kras copy gains are positively selected during progression. Here we show, through a comprehensive analysis of mutant Kras homozygous and heterozygous mouse embryonic fibroblasts and lung cancer cells, that these genotypes are phenotypically distinct. In particular, Kras(G12D/G12D) cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the tricarboxylic acid cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous non-small-cell lung cancer cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of Kras(G12D) copy gain), but not in the corresponding early tumours (Kras(G12D) heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprising two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated on the basis of their relative mutant allelic content. We also provide the first, to our knowledge, in vivo evidence of metabolic rewiring during lung cancer malignant progression. PMID:26909577

  10. Mutants of Arabidopsis thaliana with altered phototropism

    NASA Technical Reports Server (NTRS)

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

    1989-01-01

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

  11. Taking in a Show.

    PubMed

    Boden, Timothy W

    2016-01-01

    Many medical practices have cut back on education and staff development expenses, especially those costs associated with conventions and conferences. But there are hard-to-value returns on your investment in these live events--beyond the obvious benefits of acquired knowledge and skills. Major vendors still exhibit their services and wares at many events, and the exhibit hall is a treasure-house of information and resources for the savvy physician or administrator. Make and stick to a purposeful plan to exploit the trade show. You can compare products, gain new insights and ideas, and even negotiate better deals with representatives anxious to realize returns on their exhibition investments. PMID:27249887

  12. Not a "reality" show.

    PubMed

    Wrong, Terence; Baumgart, Erica

    2013-01-01

    The authors of the preceding articles raise legitimate questions about patient and staff rights and the unintended consequences of allowing ABC News to film inside teaching hospitals. We explain why we regard their fears as baseless and not supported by what we heard from individuals portrayed in the filming, our decade-long experience making medical documentaries, and the full un-aired context of the scenes shown in the broadcast. The authors don't and can't know what conversations we had, what documents we reviewed, and what protections we put in place in each televised scene. Finally, we hope to correct several misleading examples cited by the authors as well as their offhand mischaracterization of our program as a "reality" show. PMID:23631336

  13. 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

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

    SciTech Connect

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

    1986-03-01

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

  15. Public medical shows.

    PubMed

    Walusinski, Olivier

    2014-01-01

    In the second half of the 19th century, Jean-Martin Charcot (1825-1893) became famous for the quality of his teaching and his innovative neurological discoveries, bringing many French and foreign students to Paris. A hunger for recognition, together with progressive and anticlerical ideals, led Charcot to invite writers, journalists, and politicians to his lessons, during which he presented the results of his work on hysteria. These events became public performances, for which physicians and patients were transformed into actors. Major newspapers ran accounts of these consultations, more like theatrical shows in some respects. The resultant enthusiasm prompted other physicians in Paris and throughout France to try and imitate them. We will compare the form and substance of Charcot's lessons with those given by Jules-Bernard Luys (1828-1897), Victor Dumontpallier (1826-1899), Ambroise-Auguste Liébault (1823-1904), Hippolyte Bernheim (1840-1919), Joseph Grasset (1849-1918), and Albert Pitres (1848-1928). We will also note their impact on contemporary cinema and theatre. PMID:25273491

  16. FTIR and EDXRF investigations of salt tolerant soybean mutants

    NASA Astrophysics Data System (ADS)

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

    2013-07-01

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

  17. Enhanced Symbiotic Performance by Rhizobium tropici Glycogen Synthase Mutants

    PubMed Central

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

    2001-01-01

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

  18. The Great Cometary Show

    NASA Astrophysics Data System (ADS)

    2007-01-01

    its high spatial and spectral resolution, it was possible to zoom into the very heart of this very massive star. In this innermost region, the observations are dominated by the extremely dense stellar wind that totally obscures the underlying central star. The AMBER observations show that this dense stellar wind is not spherically symmetric, but exhibits a clearly elongated structure. Overall, the AMBER observations confirm that the extremely high mass loss of Eta Carinae's massive central star is non-spherical and much stronger along the poles than in the equatorial plane. This is in agreement with theoretical models that predict such an enhanced polar mass-loss in the case of rapidly rotating stars. ESO PR Photo 06c/07 ESO PR Photo 06c/07 RS Ophiuchi in Outburst Several papers from this special feature focus on the later stages in a star's life. One looks at the binary system Gamma 2 Velorum, which contains the closest example of a star known as a Wolf-Rayet. A single AMBER observation allowed the astronomers to separate the spectra of the two components, offering new insights in the modeling of Wolf-Rayet stars, but made it also possible to measure the separation between the two stars. This led to a new determination of the distance of the system, showing that previous estimates were incorrect. The observations also revealed information on the region where the winds from the two stars collide. The famous binary system RS Ophiuchi, an example of a recurrent nova, was observed just 5 days after it was discovered to be in outburst on 12 February 2006, an event that has been expected for 21 years. AMBER was able to detect the extension of the expanding nova emission. These observations show a complex geometry and kinematics, far from the simple interpretation of a spherical fireball in extension. AMBER has detected a high velocity jet probably perpendicular to the orbital plane of the binary system, and allowed a precise and careful study of the wind and the shockwave

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  1. Problem-Solving Test: Tryptophan Operon Mutants

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

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

  2. Altered lipid composition in Streptococcus pneumoniae cpoA mutants

    PubMed Central

    2014-01-01

    Background Penicillin-resistance in Streptococcus pneumoniae is mainly due to alterations in genes encoding the target enzymes for beta-lactams, the penicillin-binding proteins (PBPs). However, non-PBP genes are altered in beta-lactam-resistant laboratory mutants and confer decreased susceptibility to beta-lactam antibiotics. Two piperacillin resistant laboratory mutants of Streptococcus pneumoniae R6 contain mutations in the putative glycosyltransferase gene cpoA. The CpoA gene is part of an operon including another putative glycosyltransferase gene spr0982, both of which being homologous to glycolipid synthases present in other Gram-positive bacteria. Results We now show that the cpoA mutants as well as a cpoA deletion mutant are defective in the synthesis of galactosyl-glucosyl-diacylglycerol (GalGlcDAG) in vivo consistent with the in vitro function of CpoA as α-GalGlcDAG synthase as shown previously. In addition, the proportion of phosphatidylglycerol increased relative to cardiolipin in cpoA mutants. Moreover, cpoA mutants are more susceptible to acidic stress, have an increased requirement for Mg2+ at low pH, reveal a higher resistance to lysis inducing conditions and are hypersensitive to bacitracin. Conclusions The data show that deficiency of the major glycolipid GalGlcDAG causes a pleitotropic phenotype of cpoA mutant cells consistent with severe membrane alterations. We suggest that the cpoA mutations selected with piperacillin are directed against the lytic response induced by the beta-lactam antibiotic. PMID:24443834

  3. Enhanced N2-fixing ability of a deletion mutant of arctic rhizobia with sainfoin (Onobrychis viciifolia).

    PubMed

    Jain, D K; Bordeleau, L M

    1990-12-01

    Mutagenesis provoked by exposure at elevated temperature of the cold-adapted, arctic Rhizobium strain N31 resulted in the generation of five deletion mutants, which exhibited loss of their smaller plasmid (200 kb), whereas the larger plasmid (> 500 kb) was still present in all mutants. Deletion mutants did not show differences from the wild type in the antibiotic resistance pattern, the carbohydrates and organic acids utilization, and the growth rate at low temperature. However, deletion mutants differed from the wild type and among themselves in the ex planta nitrogenase activity, the nodulation index, and the symbiotic effectiveness. The deletion mutant N31.6rif (r) showed higher nodulation index and exhibited higher nitrogenase activity and symbiotic efficiency than the other deletion mutants and the wild type. The process of deletion mutation resulted in the improvement of an arctic Rhizobium strain having an earlier and higher symbiotic nitrogen fixation efficiency than the wild type. PMID:24221111

  4. 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.

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

    PubMed

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

    2001-12-01

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

  6. Hepatitis B escape mutants in Scottish blood donors.

    PubMed

    Larralde, Osmany; Dow, Brian; Jarvis, Lisa; Davidson, Fiona; Petrik, Juraj

    2013-06-01

    Hepatitis B virus (HBV) remains as the viral infection with the highest risk of transmission by transfusion. This risk is associated with window period donations, occult HBV infection (OBI) and the emergence of escape mutants, which render blood donations false negative for hepatitis B surface antigen (HBsAg) serological testing. A retrospective study was conducted to gain insights into the molecular epidemiology of HBV escape mutants in Scottish blood donors. The criterion for selection was HBV positivity either by serology or nucleic acid testing (NAT). HBsAg detection was compared across several commercial immunoassays. The full length S gene from plasma samples was PCR amplified, cloned and expressed in HepG2 cells. Eight samples showed HBsAg discordant results, while 5 OBI samples were found. Four escape mutants, containing missense mutations in the S gene, are described here. These mutations impaired HBsAg detection both from HBV infected plasma samples and from recombinant proteins derived from its infected donors. Phylogenetic analysis showed that most of the mutants were clustered in the genotype D and were closely related to strains from Asia and the Middle East. We report here a proline substitution, outside the major hydrophilic region, that impaired HBsAg detection in vivo and in vitro, warning about the risk for the emergence of vaccine escape mutants with mutations outside the major neutralisation site. PMID:23274404

  7. Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase.

    PubMed Central

    Mat-Jan, F; Alam, K Y; Clark, D P

    1989-01-01

    Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion. PMID:2644194

  8. Analysis of a Bacillus subtilis Proteinase Mutant1

    PubMed Central

    Shoer, Ruth; Rappaport, Harry P.

    1972-01-01

    A Bacillus subtilis mutant having a phenotype manifesting reduced extracellular proteolytic activity was investigated. An extracellular protein was isolated and shown by fingerprint analysis to be a fragment of the wild-type enzyme. By using previously established molecular weights for the wild-type enzyme (2.9 × 104) and the two polypeptide chains derived from it (1.4 × 104 each), with the amino acid analysis and fingerprints of both wild-type and mutant proteins, a molecular weight of 1.57 × 104 was assigned to the mutant protein. 32P-diisopropylphosphate labeling of the mutant protein showed only 1 in 53 molecules to be functional. Thin-layer chromatography on Sephadex G-75 demonstrated that the active molecules were separable from the bulk of the isolated protein and had the same mobility as the wild-type enzyme. Fingerprints of tryptic digests of 32P-diisopropylphosphate-labeled wild-type and mutant proteins showed that the labeled peptides had identical characteristics. Images PMID:4621679

  9. Colletotrichum sublineolum genetic instability assessed by mutants resistant to chlorate.

    PubMed

    Cecília de Lima Fávaro, Léia; Luiz Araújo, Welington; Aparecida de Souza-Paccola, Ednéia; Lúcio Azevedo, João; Paccola-Meirelles, Luzia Doretto

    2007-01-01

    The fungus Colletotrichum sublineolum, causal agent of sorghum anthracnose, presents high variability, genetic instability and host specialization. The aims of the present work were to investigate the mechanisms involved in the genetic instability in this species. Mutants resistant to chlorate and unable to use nitrate (Nit mutants), were obtained spontaneously, isolated and characterized for complementation pattern, reversion frequency and RAPD profile. The results showed that chlorate-resistant mutants could be divided into six phenotypic classes that probably represented mutations in the structural nitrate reductase locus (nit1), in the structural nitrite reductase locus (nit6 and niiA of Neurospora and Aspergillus, respectively), in the specific regulator locus (nit3), in the main regulator locus (nit2), in loci that codified the cofactor containing molybdenum necessary for nitrate reductase activity (NitM), and one or more genes responsible for nitrate intake (crn). In addition, the genetic control of this metabolism in C. sublineolum seems to be similar to other fungi species such as Aspergillus, Neurospora and Fusarium. The high reversion frequency (10(-4) to 10(-5)) presented by nit1 mutants suggests that the instability in evaluated strains could be a result of transposable elements activity. The RAPD analysis enabled confirmation that the Nit mutants have a similar genetic background to original strain, and that polymorphism exists among wild-type strains, nit1 mutants and revertants of C. sublineolum. These are important aspects for the later direction of molecular analysis, where these mutants will be used as a tool to isolate the active transposable elements in the C. sublineolum genome. PMID:17158042

  10. Hydrocarbon assimilation and biosurfactant production in Pseudomonas aeruginosa mutants

    SciTech Connect

    Koch, A.K.; Fiechter, A.; Reiser, J. ); Kaeppeli, O. )

    1991-07-01

    The authors isolated transposon Tn5-GM-induced mutants of Pseudomonas aeruginosa PG201 that were unable to grow in minimal media containing hexadecane as a carbon source. Some of these mutants lacked extracellular rhamnolipids, as shown by measuring the surface and interfacial tensions of the cell culture supernatants. Furthermore, the concentrated culture media of the mutant strains were tested for the presence of rhamnolipids by thin-layer chromatography and for rhamnolipid activities, including hemolysis and growth inhibition of Bacillus subtilis. Mutant 65E12 was unable to produce extracellular rhamnolipids under any of the inhibition of Bacillus subtilis. Mutant 65E12 was unable to produce extracellular rhamnolipids under any of the conditions tested, lacked the capacity to take up {sup 14}C-labeled hexadecane, and did not grow in media containing individual alkanes with chain lengths ranging from C{sub 12} to C{sub 19}. However, growth on these alkanes and uptake of ({sup 14}C)hexadecane were restored when small amounts of purified rhamnolipids were added to the cultures. Mutant 59C7 was unable to grow in media containing hexadecane, nor was it able to take up ({sup 14}C)hexadecane uptake. The addition of small amounts of rhamnolipids restored on alkanes and ({sup 14}C)hexadecane uptake. In glucose-containing media, however, mutant 59C7 produced rhamnolipids at levels about twice as high as those of the wild-type strain. These results show that rhamnolipids play a major role in hexadecane uptake and utilization by P.aeruginosa.

  11. Phenotypes of Myopathy-Related Beta-Tropomyosin Mutants in Human and Mouse Tissue Cultures

    PubMed Central

    Abdul-Hussein, Saba; Rahl, Karin; Moslemi, Ali-Reza; Tajsharghi, Homa

    2013-01-01

    Mutations in TPM2 result in a variety of myopathies characterised by variable clinical and morphological features. We used human and mouse cultured cells to study the effects of β-TM mutants. The mutants induced a range of phenotypes in human myoblasts, which generally changed upon differentiation to myotubes. Human myotubes transfected with the E41K-β-TMEGFP mutant showed perinuclear aggregates. The G53ins-β-TMEGFP mutant tended to accumulate in myoblasts but was incorporated into filamentous structures of myotubes. The K49del-β-TMEGFP and E122K-β-TMEGFP mutants induced the formation of rod-like structures in human cells. The N202K-β-TMEGFP mutant failed to integrate into thin filaments and formed accumulations in myotubes. The accumulation of mutant β-TMEGFP in the perinuclear and peripheral areas of the cells was the striking feature in C2C12. We demonstrated that human tissue culture is a suitable system for studying the early stages of altered myofibrilogenesis and morphological changes linked to myopathy-related β-TM mutants. In addition, the histopathological phenotype associated with expression of the various mutant proteins depends on the cell type and varies with the maturation of the muscle cell. Further, the phenotype is a combinatorial effect of the specific amino acid change and the temporal expression of the mutant protein. PMID:24039757

  12. ALS-linked mutant SOD1 damages mitochondria by promoting conformational changes in Bcl-2

    PubMed Central

    Pedrini, Steve; Sau, Daniela; Guareschi, Stefania; Bogush, Marina; Brown, Robert H.; Naniche, Nicole; Kia, Azadeh; Trotti, Davide; Pasinelli, Piera

    2010-01-01

    In mutant superoxide dismutase (SOD1)-linked amyotrophic lateral sclerosis (ALS), accumulation of misfolded mutant SOD1 in spinal cord mitochondria is thought to cause mitochondrial dysfunction. Whether mutant SOD1 is toxic per se or whether it damages the mitochondria through interactions with other mitochondrial proteins is not known. We previously identified Bcl-2 as an interacting partner of mutant SOD1 specifically in spinal cord, but not in liver, mitochondria of SOD1 mice and patients. We now show that mutant SOD1 toxicity relies on this interaction. Mutant SOD1 induces mitochondrial morphological changes and compromises mitochondrial membrane integrity leading to release of Cytochrome C only in the presence of Bcl-2. In cells, mouse and human spinal cord with SOD1 mutations, the binding to mutant SOD1 triggers a conformational change in Bcl-2 that results in the uncovering of its toxic BH3 domain and conversion of Bcl-2 into a toxic protein. Bcl-2 carrying a mutagenized, non-toxic BH3 domain fails to support mutant SOD1 mitochondrial toxicity. The identification of Bcl-2 as a specific target and active partner in mutant SOD1 mitochondrial toxicity suggests new therapeutic strategies to inhibit the formation of the toxic mutant SOD1/Bcl-2 complex and to prevent mitochondrial damage in ALS. PMID:20460269

  13. Modifiers of mutant huntingtin aggregation

    PubMed Central

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

    2011-01-01

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

  14. Construction and Characterization of Moraxella catarrhalis Mutants Defective in Expression of Transferrin Receptors

    PubMed Central

    Luke, Nicole R.; Campagnari, Anthony A.

    1999-01-01

    We have previously reported the construction of an isogenic mutant defective in expression of OmpB1, the TbpB homologue, in Moraxella catarrhalis 7169. In this report, we have extended these studies by constructing and characterizing two new isogenic mutants in this clinical isolate. One mutant is defective in expression of TbpA, and the other mutant is defective in expression of both TbpA and TbpB. These isogenic mutants were confirmed by using PCR analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and sequencing. In vitro growth studies, comparing all three mutants, demonstrated that the tbpA mutant and the tbpAB mutant were severely limited in their ability to grow with human holotransferrin as the sole source of iron. In contrast, the ompB1 (tbpB) mutant was capable of utilizing iron from human transferrin, although not to the extent of the parental strain. While affinity chromatography with human holotransferrin showed that each Tbp was capable of binding independently to transferrin, solid-phase transferrin binding studies using whole cells demonstrated that the tbpA mutant exhibited binding characteristics similar to those seen with the wild-type bacteria. However, the ompB1 (tbpB) mutant exhibited a diminished capacity for binding transferrin, and no binding was detected with the double mutant. These data suggest that the M. catarrhalis TbpA is necessary for the acquisition of iron from transferrin. In contrast, TbpB is not essential but may serve as a facilitory protein that functions to optimize this process. Together these mutants are essential to provide a more thorough understanding of iron acquisition mechanisms utilized by M. catarrhalis. PMID:10531234

  15. Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse)

    PubMed Central

    Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R.; Marzban, Hassan

    2014-01-01

    The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18–19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22–23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation. PMID:24722417

  16. Purkinje cell compartmentation in the cerebellum of the lysosomal Acid phosphatase 2 mutant mouse (nax - naked-ataxia mutant mouse).

    PubMed

    Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R; Marzban, Hassan

    2014-01-01

    The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18-19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22-23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation. PMID:24722417

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

    PubMed Central

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

    1973-01-01

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

  18. Azetidine-2-carboxylic acid resistant mutants of Arabidopsis thaliana with increased salt tolerance

    SciTech Connect

    Lehle, F.R.; Murphy, M.A.; Khan, R.A. )

    1989-04-01

    Nineteen mutant Arabidopsis families resistant to the proline analog azetidine-2-carboxylic acid (ACA) were characterized in terms of NaCl tolerance and proline content. Mutants were selected from about 64,000 progeny of about 16,000 self-pollinated Columbia parents which had been mutated with ethyl methane sulfonate during seed imbibition. Selections were performed during seed germination on aseptic agar medium containing 0.2 to 0.25 mM ACA. Nineteen mutant families, 12 clearly independent, retained resistance to ACA in the M{sub 4} generation. Based on germination on 150 mM NaCl, 13 of the mutant families were more tolerant than the wild type. Two mutants of intermediate resistance to ACA were markedly more salt tolerant than the others. Four mutant families appeared to overproduce proline. Of these, only 3 showed slight increases in salt tolerance.

  19. Radish, a Drosophila mutant deficient in consolidated memory.

    PubMed Central

    Folkers, E; Drain, P; Quinn, W G

    1993-01-01

    We have characterized the behavior and genetics of the Drosophila mutant radish (rsh gene). Initial learning of radish flies in two olfactory discrimination tests is high, but subsequent memory decays rapidly at both early and late times after training. Anesthesia-resistant memory (consolidated memory) is undetectable in radish flies 3 hr after training. The mutant shows normal locomotor activity and normal sensitivity to the odor cues and electric-shock reinforcement used in the learning tests. The radish gene maps within a 180-kb interval in the 11D-E region of the X chromosome. PMID:8367473

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

    PubMed

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

    1980-03-01

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

  1. Isolation and biochemical analysis of Mucor bacilliformis monomorphic mutants.

    PubMed Central

    Ruiz-Herrera, J; Ruiz, A; Lopez-Romero, E

    1983-01-01

    Fourteen stable mutants of Mucor bacilliformis which grew yeastlike under both aerobic and anaerobic conditions were isolated after treatment of growing mycelium with N-methyl-N'-nitro-N-nitrosoguanidine. Biochemical characterization of the mutants included determination of growth in different carbon and nitrogen sources, determination of sensitivity of respiration to cyanide and salicylhydroxamate, analysis of cytochrome spectra, determination of glutamate dehydrogenases, glutamine synthase, and ornithine decarboxylase activities, and measurement of cyclic AMP levels. Data showed that all mutants were defective in some aspect of oxidative metabolism and had low levels of ornithine decarboxylase, whereas other characters were variable. It was concluded that morphological transition in M. bacilliformis is probably associated with mitochondrial functions and expression of ornithine decarboxylase, but may be independent of cyclic AMP and glutamate dehydrogenase levels. The importance of genetic studies in the analysis of dimorphism is stressed. PMID:6137477

  2. [Isolation and characterization of PAOX2 mutant in Pichia pastoris].

    PubMed

    Dai, X Y; Wang, Y X; Zhou, J; Wang, Y Q

    2000-01-01

    Spontaneous Mut+ mutants of P. pastoris AOX1-defective expression strain have been isolated, they were identified as phenotypically utilized methanol to grow as wild type. The results obtained from measuring growth curve when cultivated in medium in which methanol as a sole carbon source and detecting HSA protein on SDS-PAGE confirmed that the mutants have increased ability to utilize methanol and express foreign HSA gene product. The promoter region of AOX2 gene from the mutants has been cloned by PCR amplification, and the DNA fragment is 1022bp in size. Sequencing analysis showed that there are two point mutations at positions of -529 and -255 from the translation initiation codon respectively. The mutations improved AOX-1 defective function and facilitate the foreign gene for higher expression. PMID:11051726

  3. Developmental mechanisms underlying polydactyly in the mouse mutant Doublefoot

    PubMed Central

    Crick, Alexandra P; Babbs, Christian; Brown, Jennifer M; Morriss-Kay, Gillian M

    2003-01-01

    The pre-axial polydactylous mouse mutant Doublefoot has 6–9 digits per limb but lacks anteroposterior polarity (there is no biphalangeal digit 1). It differs from other polydactylous mutants in showing normal Shh expression, but polarizing activity (shown by mouse-chick grafting experiments) and hedgehog signalling activity (shown by expression of Ptc1) are present throughout the distal mesenchyme. The Dbf mutation has not yet been identified. Here we review current understanding of this mutant, and briefly report new results indicating (1) that limb bud expansion is concomitant with ectopic Ihh expression and with extension of the posterior high cell proliferation rate into the anterior region, and (2) that the Dbf mutation is epistatic to Shh in the limb. PMID:12587916

  4. Generation and Characterization of dickkopf3 Mutant Mice

    PubMed Central

    del Barco Barrantes, Ivan; Montero-Pedrazuela, Ana; Guadaño-Ferraz, Ana; Obregon, Maria-Jesus; Martinez de Mena, Raquel; Gailus-Durner, Valérie; Fuchs, Helmut; Franz, Tobias J.; Kalaydjiev, Svetoslav; Klempt, Martina; Hölter, Sabine; Rathkolb, Birgit; Reinhard, Claudia; Morreale de Escobar, Gabriella; Bernal, Juan; Busch, Dirk H.; Wurst, Wolfgang; Wolf, Eckhard; Schulz, Holger; Shtrom, Svetlana; Greiner, Erich; Hrabé de Angelis, Martin; Westphal, Heiner; Niehrs, Christof

    2006-01-01

    dickkopf (dkk) genes encode a small family of secreted Wnt antagonists, except for dkk3, which is divergent and whose function is poorly understood. Here, we describe the generation and characterization of dkk3 mutant mice. dkk3-deficient mice are viable and fertile. Phenotypic analysis shows no major alterations in organ morphology, physiology, and most clinical chemistry parameters. Since Dkk3 was proposed to function as thyroid hormone binding protein, we have analyzed deiodinase activities, as well as thyroid hormone levels. Mutant mice are euthyroid, and the data do not support a relationship of dkk3 with thyroid hormone metabolism. Altered phenotypes in dkk3 mutant mice were observed in the frequency of NK cells, immunoglobulin M, hemoglobin, and hematocrit levels, as well as lung ventilation. Furthermore, dkk3-deficient mice display hyperactivity. PMID:16508007

  5. How Life History Can Sway the Fixation Probability of Mutants.

    PubMed

    Li, Xiang-Yi; Kurokawa, Shun; Giaimo, Stefano; Traulsen, Arne

    2016-07-01

    In this work, we study the effects of demographic structure on evolutionary dynamics when selection acts on reproduction, survival, or both. In contrast to the previously discovered pattern that the fixation probability of a neutral mutant decreases while the population becomes younger, we show that a mutant with a constant selective advantage may have a maximum or a minimum of the fixation probability in populations with an intermediate fraction of young individuals. This highlights the importance of life history and demographic structure in studying evolutionary dynamics. We also illustrate the fundamental differences between selection on reproduction and selection on survival when age structure is present. In addition, we evaluate the relative importance of size and structure of the population in determining the fixation probability of the mutant. Our work lays the foundation for also studying density- and frequency-dependent effects in populations when demographic structures cannot be neglected. PMID:27129737

  6. Isolation of ABA hypersensitive mutants in allhexaploid breadwheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The plant hormone abscisic acid (ABA) stimulates seed dormancy during embryo maturation, inhibits germination of mature seed, and stimulates stress responses such as stomatal closure in response to drought stress. Arabidopsis mutants isolated for ABA hypersensitive (ABH) seed germination showed incr...

  7. Allele Specific p53 Mutant Reactivation

    PubMed Central

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

    2012-01-01

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

  8. Abnormal lignin in a loblolly pine mutant

    SciTech Connect

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

    1997-07-11

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

  9. Identifying representative drug resistant mutants of HIV

    PubMed Central

    2015-01-01

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

  10. The MSC phenotype of cucumber (Cucumis sativus L.) - a method for production of plant mitochondrial mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mosaic (MSC) mutants of cucumber (Cucumis sativus L.) appear after passage through cell cultures. The MSC phenotype shows paternal transmission and is associated with mitochondrial DNA rearrangements. This review describes the origins and phenotypes of independently produced MSC mutants of cuc...

  11. Mutant IDH is sufficient to initiate enchondromatosis in mice.

    PubMed

    Hirata, Makoto; Sasaki, Masato; Cairns, Rob A; Inoue, Satoshi; Puviindran, Vijitha; Li, Wanda Y; Snow, Bryan E; Jones, Lisa D; Wei, Qingxia; Sato, Shingo; Tang, Yuning J; Nadesan, Puviindran; Rockel, Jason; Whetstone, Heather; Poon, Raymond; Weng, Angela; Gross, Stefan; Straley, Kimberly; Gliser, Camelia; Xu, Yingxia; Wunder, Jay; Mak, Tak W; Alman, Benjamin A

    2015-03-01

    Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas. Somatic mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) are present in the majority of these tumor types. How these mutations cause enchondromas is unclear. Here, we identified the spectrum of IDH mutations in human enchondromas and chondrosarcomas and studied their effects in mice. A broad range of mutations was identified, including the previously unreported IDH1-R132Q mutation. These mutations harbored enzymatic activity to catalyze α-ketoglutarate to d-2-hydroxyglutarate (d-2HG). Mice expressing Idh1-R132Q in one allele in cells expressing type 2 collagen showed a disordered growth plate, with persistence of type X-expressing chondrocytes. Chondrocyte cell cultures from these animals or controls showed that there was an increase in proliferation and expression of genes characteristic of hypertrophic chondrocytes with expression of Idh1-R132Q or 2HG treatment. Col2a1-Cre;Idh1-R132Q mutant knock-in mice (mutant allele expressed in chondrocytes) did not survive after the neonatal stage. Col2a1-Cre/ERT2;Idh1-R132 mutant conditional knock-in mice, in which Cre was induced by tamoxifen after weaning, developed multiple enchondroma-like lesions. Taken together, these data show that mutant IDH or d-2HG causes persistence of chondrocytes, giving rise to rests of growth-plate cells that persist in the bone as enchondromas. PMID:25730874

  12. Mutant IDH is sufficient to initiate enchondromatosis in mice

    PubMed Central

    Hirata, Makoto; Sasaki, Masato; Cairns, Rob A.; Inoue, Satoshi; Puviindran, Vijitha; Li, Wanda Y.; Snow, Bryan E.; Jones, Lisa D.; Wei, Qingxia; Sato, Shingo; Tang, Yuning J.; Nadesan, Puviindran; Rockel, Jason; Whetstone, Heather; Poon, Raymond; Weng, Angela; Gross, Stefan; Straley, Kimberly; Gliser, Camelia; Xu, Yingxia; Wunder, Jay; Mak, Tak W.; Alman, Benjamin A.

    2015-01-01

    Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas. Somatic mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) are present in the majority of these tumor types. How these mutations cause enchondromas is unclear. Here, we identified the spectrum of IDH mutations in human enchondromas and chondrosarcomas and studied their effects in mice. A broad range of mutations was identified, including the previously unreported IDH1-R132Q mutation. These mutations harbored enzymatic activity to catalyze α-ketoglutarate to d-2-hydroxyglutarate (d-2HG). Mice expressing Idh1-R132Q in one allele in cells expressing type 2 collagen showed a disordered growth plate, with persistence of type X-expressing chondrocytes. Chondrocyte cell cultures from these animals or controls showed that there was an increase in proliferation and expression of genes characteristic of hypertrophic chondrocytes with expression of Idh1-R132Q or 2HG treatment. Col2a1-Cre;Idh1-R132Q mutant knock-in mice (mutant allele expressed in chondrocytes) did not survive after the neonatal stage. Col2a1-Cre/ERT2;Idh1-R132 mutant conditional knock-in mice, in which Cre was induced by tamoxifen after weaning, developed multiple enchondroma-like lesions. Taken together, these data show that mutant IDH or d-2HG causes persistence of chondrocytes, giving rise to rests of growth-plate cells that persist in the bone as enchondromas. PMID:25730874

  13. Tumbling chemotaxis mutants of Escherichia coli: possible gene-dependent effect of methionine starvation.

    PubMed Central

    Kondoh, H

    1980-01-01

    Some mutants defective in chemotaxis show incessant tumbling behavior and are called tumbling mutants. Previously described tumbling mutations lie in two genes, cheB and cheZ (41.5 min on Escherichia coli map). Genetic analysis of various tumbling mutants, however, revealed that two more genetic loci, cheC (43 min) and cheE (99.2 min), could also mutate to produce tumbling mutants. The genetic map around cheC was revised: his flaP flaQ flaR flbD flaA (= cheC) flaE. flbD is a new gene. When cells were starved for methionine, the tumbling mutants changed their swimming behavior depending on the che gene mutated. cheZ mutants, like wild-type bacteria, ceased tumbling shortly after removal of methionine. The tumbling of cheB or cheE mutants was depressed after prolonged methionine starvation in the presence of a constant level of an attractant. cheC tumbling mutants appeared unique in that they did not cease tumbling even when cells were deprived of methionine. By contrast, arsenate treatment of the tumbling mutants resulted in smooth swimming of the cells in every case. These results suggest that two different processes are involved in regulation of tumbling; one requiring methionine and the other requiring some phosphorylated compound. PMID:6991478

  14. Characterization of a new Arabidopsis mutant exhibiting enhanced disease resistance.

    PubMed

    Silva, H; Yoshioka, K; Dooner, H K; Klessig, D F

    1999-12-01

    In many plant-pathogen interactions, resistance is associated with the synthesis and accumulation of salicylic acid (SA) and pathogenesis-related (PR) proteins. At least two general classes of mutants with altered resistance to pathogen attack have been identified in Arabidopsis. One class exhibits increased susceptibility to pathogen infection; the other class exhibits enhanced resistance to pathogens. In an attempt to identify mutations in resistance-associated loci, we screened a population of T-DNA tagged Arabidopsis thaliana ecotype Wassilewskija (Ws) for mutants showing constitutive expression of the PR-1 gene (cep). A mutant was isolated and shown to constitutively express PR-1, PR-2, and PR-5 genes. This constitutive phenotype segregated as a single recessive trait in the Ws genetic background. The mutant also had elevated levels of SA, which are responsible for the cep phenotype. The cep mutant spontaneously formed hypersensitive response (HR)-like lesions on the leaves and cotyledons and also exhibited enhanced resistance to virulent bacterial and fungal pathogens. Genetic analyses of segregating progeny from outcrosses to other ecotypes unexpectedly revealed that alterations in more than one gene condition the constitutive expression of PR genes in the original mutant. One of the mutations, designated cpr20, maps to the lower arm of chromosome 4 and is required for the cep phenotype. Another mutation, which has been termed cpr21, maps to chromosome 1 and is often, but not always, associated with this phenotype. The recessive nature of the cep trait suggests that the CPR20 and CPR21 proteins may act as negative regulators in the disease resistance signal transduction pathway. PMID:10624014

  15. Genetic Analysis of Rough Sheath1 Developmental Mutants of Maize

    PubMed Central

    Becraft, P. W.; Freeling, M.

    1994-01-01

    Maize Rough sheath1 (Rs1) mutants are dominant and cause a proliferation of sheath-like tissue at the base of the blade and throughout the ligular region. They also cause ligule displacement, a chaotic pattern of vasculature and abnormal cellular structure of vascular bundles. The affected region of Rs1-O leaves displays genetic and morphological attributes of both sheath and auricle, suggesting an overlap of these genetic programs. The rs1 locus maps approximately 26 map units distal to opaque2 (o2) on chromosome 7S, defining a new distal-most locus on the genetic map. Three mutant alleles, Rs1-O, Rs1-1025 and Rs1-Z, all display similar phenotypes. The mutations are completely dominant and the Rs1-O phenotype is not affected by dosage of the chromosome arm carrying the rs1(+) allele, indicating that these alleles are neomorphic. Analysis of genetic mosaics showed that the Rs1-O phenotype is non-cell-autonomous, suggesting that intercellular signals convey the phenotype. Rs1 mutant phenotypes are affected by modifiers present in particular genetic backgrounds. An enhancer of Rs1-O was identified; segregation data imply a single recessive gene, ers1. Rs1 mutants were also found to enhance the expression of unlinked rs2 and Rs4 mutants, suggesting that these mutations affect similar developmental processes. We discuss the phenotypic and genetic similarities between Rs1 and Knotted1 (Kn1) mutants that led to the identification of rs1 as a kn1-like homeobox gene (unpublished data). PMID:8138166

  16. Peroxisomes in wild-type and rosy mutant Drosophila melanogaster.

    PubMed Central

    Beard, M E; Holtzman, E

    1987-01-01

    This study shows that peroxisomes are abundant in the Malpighian tubule and gut of wild-type Oregon R Drosophila melanogaster and that the peroxisomal population of the rosy-506 eye-color mutant differs from that of the wild type. Catalase activity in wild-type flies is demonstrable in bodies of appearance and centrifugal behavior comparable to the peroxisomes of vertebrate tissues. Xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.1.3.22) activity of the Malpighian tubule of wild-type flies is demonstrable cytochemically in bodies like those containing catalase. The rosy-506 mutant flies, with a deletion in the structural gene for xanthine dehydrogenase (xanthine:NAD+ oxidoreductase, EC 1.1.1.204), lack cytochemically demonstrable peroxisomal xanthine oxidase activity. In addition, peroxisomes in the rosy-506 mutants show less intense cytochemical staining for catalase than those in wild-type flies, and biochemical assays indicate that catalase in the rosy mutant is much more accessible to substrate in the absence of detergent than in the wild type. Thus, the rosy-506 mutation appears to affect peroxisomes and may mimic aspects of the defects of peroxisomes in some human metabolic disorders. Images PMID:3118368

  17. Increase in NRAS mutant allele percentage during metastatic melanoma progression.

    PubMed

    Funck-Brentano, Elisa; Hélias-Rodzewicz, Zofia; Longvert, Christine; Mokhtari, Karima; Saiag, Philippe; Emile, Jean-François

    2016-06-01

    One-fifth of cutaneous melanomas have dominant gain-of-function mutations of the NRAS oncogene. We report the first two cases of increasing NRAS mutant allele frequency in melanoma metastases and show that the chromosomal mechanism of this homozygosity is an increased polysomy of chromosome 1. We observed an increase in NRAS mutant allele percentage (NRAS-MA%) in the metastatic melanoma progression from 2 patients with melanomas harbouring a NRAS mutation (p.Q61K in case 1 and p.Q61R in case 2). In case 1, we observed a NRAS-MA% increase from 18% within the first metastatic node to 81%, 92% and 85% respectively in the three subsequent metastases: lymph node, brain and subcutaneous metastases biopsied 1, 6 and 17 months, respectively, after the initial lymph node biopsy. In case 2, we observed an increase in NRAS-MA% from 40% within the primary melanoma to 63% within the metastatic lymph node. FISH analysis showed the same results in both cases: a frequent polysomy of chromosome 1 in metastasis samples with NRAS mutant allele percentage >60%, while most cells were disomic in the samples with well-balanced heterozygous mutations. The percentage of NRAS mutant allele may increase during metastatic progression and may be associated with chromosomal instability. Further studies are needed to evaluate the prognostic impact of the NRAS homozygous status and/or polyploidy in metastatic cutaneous melanomas. PMID:26990546

  18. Regulation of Mutant p53 Protein Expression

    PubMed Central

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

    2015-01-01

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

  19. Uncaging Mutants: Moving From Menageries to Menages

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  20. 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.

  1. Southern analysis of genomic alterations in gamma-ray-induced aprt- hamster cell mutants

    SciTech Connect

    Grosovsky, A.J.; Drobetsky, E.A.; deJong, P.J.; Glickman, B.W.

    1986-06-01

    The role of genomic alterations in mutagenesis induced by ionizing radiation has been the subject of considerable speculation. By Southern blotting analysis we show here that 9 of 55 (approximately 1/6) gamma-ray-induced mutants at the adenine phosphoribosyl transferase (aprt) locus of Chinese hamster ovary (CHO) cells have a detectable genomic rearrangement. These fall into two classes: intragenic deletions and chromosomal rearrangements. In contrast, no major genomic alterations were detected among 67 spontaneous mutants, although two restriction site loss events were observed. Three gamma-ray-induced mutants were found to be intragenic deletions; all may have identical break-points. The remaining six gamma-ray-induced mutants demonstrating a genomic alteration appear to be the result of chromosomal rearrangements, possibly translocation or inversion events. None of the remaining gamma-ray-induced mutants showed any observable alteration in blotting pattern indicating a substantial role for point mutation in gamma-ray-induced mutagenesis at the aprt locus.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-02-01

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

  4. Characterization of Sugar Insensitive (sis) Mutants of Arabidopsis

    SciTech Connect

    Gibson, Susan I.

    2009-06-08

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

  5. A combinatorial strategy for treating KRAS-mutant lung cancer.

    PubMed

    Manchado, Eusebio; Weissmueller, Susann; Morris, John P; Chen, Chi-Chao; Wullenkord, Ramona; Lujambio, Amaia; de Stanchina, Elisa; Poirier, John T; Gainor, Justin F; Corcoran, Ryan B; Engelman, Jeffrey A; Rudin, Charles M; Rosen, Neal; Lowe, Scott W

    2016-06-30

    Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proved difficult to inhibit, and the effectiveness of agents that target key KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, a MEK inhibitor approved by the US Food and Drug Administration, which acts downstream of KRAS to suppress signalling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signalling rebound and adaptive drug resistance. As a consequence, genetic or pharmacological inhibition of FGFR1 in combination with trametinib enhances tumour cell death in vitro and in vivo. This compensatory response shows distinct specificities: it is dominated by FGFR1 in KRAS-mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patients’ tumours treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer. PMID:27338794

  6. Mutants of thermotaxis in Dictyostelium discoideum

    SciTech Connect

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

    1982-08-01

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

  7. Production of Extracellular Polysaccharides by CAP Mutants of Cryptococcus neoformans▿

    PubMed Central

    Grijpstra, Jan; Gerwig, Gerrit J.; Wösten, Han; Kamerling, Johannis P.; de Cock, Hans

    2009-01-01

    The human pathogen Cryptococcus neoformans causes meningoencephalitis. The polysaccharide capsule is one of the main virulence factors and consists of two distinct polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM). How capsular polysaccharides are synthesized, transported, and assembled is largely unknown. Previously, it was shown that mutations in the CAP10, CAP59, CAP60, and CAP64 genes result in an acapsular phenotype. Here, it is shown that these acapsular mutants do secrete GalXM and GXM-like polymers. GXM and GalXM antibodies specifically reacted with whole cells and the growth medium of the wild type and CAP mutants, indicating that the capsule polysaccharides adhere to the cell wall and are shed into the environment. These polysaccharides were purified from the medium, either with or without anion-exchange chromatography. Monosaccharide analysis of polysaccharide fractions by gas-liquid chromatography/mass spectrometry showed that wild-type cells secrete both GalXM and GXM. The CAP mutants, on the other hand, were shown to secrete GalXM and GXM-like polymers. Notably, the GalXM polymers were shown to contain glucuronic acid. One-dimensional 1H nuclear magnetic resonance confirmed that the CAP mutants secrete GalXM and also showed the presence of O-acetylated polymers. This is the first time it is shown that CAP mutants secrete GXM-like polymers in addition to GalXM. The small amount of this GXM-like polymer, 1 to 5% of the total amount of secreted polysaccharides, may explain the acapsular phenotype. PMID:19542308

  8. Selection and phenotypic characterization of nonhemagglutinating mutants of Porphyromonas gingivalis.

    PubMed Central

    Chandad, F; Mayrand, D; Grenier, D; Hinode, D; Mouton, C

    1996-01-01

    To further investigate the relationship between fimbriae and the hemagglutinating adhesin HA-Ag2 of Porphyromonas gingivalis, three spontaneous mutants of the type strain ATCC 33277 were selected by a hemadsorption procedure. They were characterized for hemagglutination, trypsin-like and lectin-binding activities, and hydrophobicity and for the presence of fimbriae. The presence of the 42-kDa (the fimbrilin subunit) and the 43- and 49-kDa (the HA-Ag2 components) polypeptides was investigated by immunoblotting using polyclonal and monoclonal antibodies directed to fimbriae and to the hemagglutinating adhesin HA-Ag2. Cells from two of the three mutants (M1 and M2) exhibited no or little hemagglutination activity and very low trypsin-like activity and did not show the 43- and 49-kDa polypeptides. Abnormal fimbriation in M1 was deduced from the following observations of cells grown for 18 h: absence of the 42-kDa polypeptide and of a 14-kDa polypeptide and no fimbriae visible on electron micrographs. While the cells of mutant M2, irrespective of the age of the culture, were found to lack the 43- and 49-kDa polypeptides and hemagglutination activity, the supernatants of cultures grown for 72 h had high hemagglutination and trypsin-like activities and revealed the presence of the 42-, 43-, and 49-kDa polypeptides. This suggests that M2 may be missing some molecules which anchor the components to the cell surface. Mutant M3 showed levels of activities similar to those of the parental strain but lacked the 43-kDa polypeptide. Other pleiotropic effects observed for the mutants included loss of dark pigmentation and lower hydrophobicity. The data from this study fuel an emerging consensus whereby fimbriation, hemagglutination, and proteolytic activities, as well as other functions in P. gingivalis, are intricate. PMID:8641806

  9. Rest Mutant zebrafish swim erratically and display atypical spatial preferences

    PubMed Central

    Moravec, Cara E.; Li, Edward; Maaswinkel, Hans; Kritzer, Mary F.; Weng, Wei; Sirotkin, Howard I.

    2015-01-01

    The Rest/Nrsf transcriptional repressor modulates expression of a large set of neural specific genes. Many of these target genes have well characterized roles in nervous system processes including development, plasticity and synaptogenesis. However, the impact of Rest-mediated transcriptional regulation on behavior has been understudied due in part to the embryonic lethality of the mouse knockout. To investigate the requirement for Rest in behavior, we employed the zebrafish rest mutant to explore a range of behaviors in adults and larva. Adult rest mutants of both sexes showed abnormal behaviors in a novel environment including increased vertical swimming, erratic swimming patterns and a proclivity for the tank walls. Adult males also had diminished reproductive success. At 6 days post fertilization (dpf), rest mutant larva were hypoactive, but displayed normal evoked responses to light and sound stimuli. Overall, these results provide evidence that rest dysfunction produces atypical swimming patterns and preferences in adults, and reduced locomotor activity in larvae. This study provides the first behavioral analysis of rest mutants and reveals specific behaviors that are modulated by Rest. PMID:25712696

  10. Streptomycin-resistant, asporogenous mutant of Bacillus subtilis.

    PubMed

    Campbell, K M; Chambliss, G H

    1977-12-30

    A streptomycin-resistant mutantant of Bacillus subtilis that is also asporogenous, was isolated and partially characterized. This strain, SRB15, sporulated at a frequency of about 1% compared to the wild type frequency of greater than 70%. The two phenotypes were inseparable by transformation, suggesting that this strain carries a single mutation that causes it to be both streptomycin-resistant and spore-minus. The mutation cotransduces with cysA, the closest auxotrophic marker to the "ribosomal region" of the B. subtilis chromosome, with a frequency of 68%. SRB15 showed no cross resistence to other antiobiotics tested, including the aminoglycosides kanamycin, neomycin and spectinomycin. Ribosomes obtained from the mutant were at least 200-fold more resistant in vitro to streptomycin than were wild type ribosomes in the translation of phage SPO1 RNA. The kinetics of in vitro translation of this natural message were indistinguishable for mutant and wild type ribosomes. The level of misreading, as measured by poly(U)-directed isoleucine incorporation, by mutant ribosomes was less than that by wild type ribosomes. PMID:414073

  11. Crystallographic Investigation and Selective Inhibition of Mutant Isocitrate Dehydrogenase

    PubMed Central

    2013-01-01

    Mutations in isocitrate dehydrogenase (IDH), a key enzyme in the tricarboxylic acid cycle, have recently been found in ∼75% glioma and ∼20% acute myeloid leukemia. Different from the wild-type enzyme, mutant IDH1 catalyzes the reduction of α-ketoglutaric acid to d-2-hydroxyglutaric acid. Strong evidence has shown mutant IDH1 represents a novel target for this type of cancer. We found two 1-hydroxypyridin-2-one compounds that are potent inhibitors of R132H and R132C IDH1 mutants with Ki values as low as 120 nM. These compounds exhibit >60-fold selectivity against wild-type IDH1 and can inhibit the production of d-2-hydroxyglutaric acid in IDH1 mutated cells, representing novel chemical probes for cancer biology studies. We also report the first inhibitor-bound crystal structures of IDH1(R132H), showing these inhibitors have H-bond, electrostatic, and hydrophobic interactions with the mutant enzyme. Comparison with the substrate-bound IDH1 structures revealed the structural basis for the high enzyme selectivity of these compounds. PMID:23795241

  12. Crystallographic Investigation and Selective Inhibition of Mutant Isocitrate Dehydrogenase.

    PubMed

    Zheng, Baisong; Yao, Yuan; Liu, Zhen; Deng, Lisheng; Anglin, Justin L; Jiang, Hong; Prasad, B V Venkataram; Song, Yongcheng

    2013-06-13

    Mutations in isocitrate dehydrogenase (IDH), a key enzyme in the tricarboxylic acid cycle, have recently been found in ~75% glioma and ~20% acute myeloid leukemia. Different from the wild-type enzyme, mutant IDH1 catalyzes the reduction of α-ketoglutaric acid to D-2-hydroxyglutaric acid. Strong evidence has shown mutant IDH1 represents a novel target for this type of cancer. We found two 1-hydroxypyridin-2-one compounds that are potent inhibitors of R132H and R132C IDH1 mutants with Ki values as low as 120 nM. These compounds exhibit >60-fold selectivity against wild-type IDH1 and can inhibit the production of D-2-hydroxyglutaric acid in IDH1 mutated cells, representing novel chemical probes for cancer biology studies. We also report the first inhibitor-bound crystal structures of IDH1(R132H), showing these inhibitors have H-bond, electrostatic and hydrophobic interactions with the mutant enzyme. Comparison with the substrate-bound IDH1 structures revealed the structural basis for the high enzyme selectivity of these compounds. PMID:23795241

  13. Lactobacillus crispatus and its nonaggregating mutant in human colonization trials.

    PubMed

    Cesena, C; Morelli, L; Alander, M; Siljander, T; Tuomola, E; Salminen, S; Mattila-Sandholm, T; Vilpponen-Salmela, T; von Wright, A

    2001-05-01

    A wild-type Lactobacillus crispatus, showing a cell aggregation phenotype and its spontaneous nonaggregating mutant were compared for their in vitro adhesion properties to human ileal mucus and to a cultured human colonic cell line (Caco2) and for their in vivo colonization and adhesion potential with colonoscopy patients as volunteers in feeding trials. The wild-type strain adhered better to mucus or to Caco2 cells than did the mutant. Altogether, three human trials with the wild type and two with the mutant strain were performed. In two of the trials, the wild type could be recovered from either fecal samples or biopsies taken from the colon, while the mutant strain could not be demonstrated in either of the trials where it was used. The L. crispatus colonies recovered from the trials were often mixed, and several enterococci and lactobacillus strains coaggregating with L. crispatus wild type could be isolated. The results indicate that the surface-mediated properties, such as aggregation, of lactobacilli can have a role in adhesion and colonization. PMID:11384025

  14. Insulator dysfunction and oncogene activation in IDH mutant gliomas.

    PubMed

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

    2016-01-01

    Gain-of-function IDH mutations are initiating events that define major clinical and prognostic classes of gliomas. Mutant IDH protein produces a new onco-metabolite, 2-hydroxyglutarate, which interferes with iron-dependent hydroxylases, including the TET family of 5'-methylcytosine hydroxylases. TET enzymes catalyse a key step in the removal of DNA methylation. IDH mutant gliomas thus manifest a CpG island methylator phenotype (G-CIMP), although the functional importance of this altered epigenetic state remains unclear. Here we show that human IDH mutant gliomas exhibit hypermethylation at cohesin and CCCTC-binding factor (CTCF)-binding sites, compromising binding of this methylation-sensitive insulator protein. Reduced CTCF binding is associated with loss of insulation between topological domains and aberrant gene activation. We specifically demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to interact aberrantly with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Treatment of IDH mutant gliomaspheres with a demethylating agent partially restores insulator function and downregulates PDGFRA. Conversely, CRISPR-mediated disruption of the CTCF motif in IDH wild-type gliomaspheres upregulates PDGFRA and increases proliferation. Our study suggests that IDH mutations promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression. PMID:26700815

  15. Insulator dysfunction and oncogene activation in IDH mutant gliomas

    PubMed Central

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

    2015-01-01

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

  16. Rapid Mutation of Spirulina platensis by a New Mutagenesis System of Atmospheric and Room Temperature Plasmas (ARTP) and Generation of a Mutant Library with Diverse Phenotypes

    PubMed Central

    Zhang, Chong; Tan, Yinyee; Jiang, Peixia; Ge, Nan; Heping Li; Xing, Xinhui

    2013-01-01

    In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9th subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae. PMID:24319517

  17. Rapid mutation of Spirulina platensis by a new mutagenesis system of atmospheric and room temperature plasmas (ARTP) and generation of a mutant library with diverse phenotypes.

    PubMed

    Fang, Mingyue; Jin, Lihua; Zhang, Chong; Tan, Yinyee; Jiang, Peixia; Ge, Nan; Heping Li; Xing, Xinhui

    2013-01-01

    In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9(th) subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae. PMID:24319517

  18. Incomplete flagellar structures in Escherichia coli mutants.

    PubMed Central

    Suzuki, T; Komeda, Y

    1981-01-01

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

  19. Swimming activity in dystonia musculorum mutant mice.

    PubMed

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

    1993-07-01

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

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

    PubMed Central

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

    1988-01-01

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

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

    PubMed

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

    2001-01-01

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

  2. Mutant Huntingtin Downregulates Myelin Regulatory Factor-Mediated Myelin Gene Expression and Affects Mature Oligodendrocytes

    PubMed Central

    Huang, Brenda; Wei, Wenjie; Wang, Guohao; Gaertig, Marta A.; Feng, Yue; Wang, Wei; Li, Xiao-Jiang; Li, Shihua

    2015-01-01

    SUMMARY Growing evidence indicates that non-neuronal mutant huntingtin toxicity plays an important role in Huntington’s disease (HD); however, whether and how mutant huntingtin affects oligodendrocytes, which are vitally important for neural function and axonal integrity, remain unclear. We first verified the presence of mutant huntingtin in oligodendrocytes in HD140Q knock-in mice. We then established transgenic mice (PLP-150Q) that selectively express mutant huntingtin in oligodendrocytes. PLP-150Q mice show progressive neurological symptoms and early death, as well as age-dependent demyelination and reduced expression of myelin genes that are downstream of myelin regulatory factor (MYRF or MRF), a transcriptional regulator that specifically activates and maintains the expression of myelin genes in mature oligodendrocytes. Consistently, mutant huntingtin binds abnormally to MYRF and affects its transcription activity. Our findings suggest that dysfunction of mature oligodendrocytes is involved in HD pathogenesis and may also make a good therapeutic target. PMID:25789755

  3. Mutant huntingtin downregulates myelin regulatory factor-mediated myelin gene expression and affects mature oligodendrocytes.

    PubMed

    Huang, Brenda; Wei, WenJie; Wang, Guohao; Gaertig, Marta A; Feng, Yue; Wang, Wei; Li, Xiao-Jiang; Li, Shihua

    2015-03-18

    Growing evidence indicates that non-neuronal mutant huntingtin toxicity plays an important role in Huntington's disease (HD); however, whether and how mutant huntingtin affects oligodendrocytes, which are vitally important for neural function and axonal integrity, remains unclear. We first verified the presence of mutant huntingtin in oligodendrocytes in HD140Q knockin mice. We then established transgenic mice (PLP-150Q) that selectively express mutant huntingtin in oligodendrocytes. PLP-150Q mice show progressive neurological symptoms and early death, as well as age-dependent demyelination and reduced expression of myelin genes that are downstream of myelin regulatory factor (MYRF or MRF), a transcriptional regulator that specifically activates and maintains the expression of myelin genes in mature oligodendrocytes. Consistently, mutant huntingtin binds abnormally to MYRF and affects its transcription activity. Our findings suggest that dysfunction of mature oligodendrocytes is involved in HD pathogenesis and may also make a good therapeutic target. PMID:25789755

  4. Pharmacodynamic assessment based on mutant prevention concentrations of fluoroquinolones to prevent the emergence of resistant mutants of Streptococcus pneumoniae.

    PubMed

    Homma, Tomoyuki; Hori, Toshihiko; Sugimori, Giichi; Yamano, Yoshinori

    2007-11-01

    The objective of this study was to investigate the relationship between pharmacokinetic and pharmacodynamic parameters, on the basis of the mutant prevention concentration (MPC) concept, and the emergence of resistant mutants of Streptococcus pneumoniae to fluoroquinolone antibacterials. Some clinical isolates with various MIC and MPC values of moxifloxacin and levofloxacin were exposed under conditions simulating the time-concentration curves observed when moxifloxacin (400 or 80 mg, once a day) or levofloxacin (200 mg, twice a day) was orally administered by using an in vitro pharmacodynamic model. The decrease in susceptibility was evaluated by altering the population analysis profiles after moxifloxacin or levofloxacin treatment for 72 h. When the area under the concentration-time curve from 0 to 24 h (AUC(0-24))/MPC and peak concentration (C(max))/MPC were above 13.41 and 1.20, respectively, complete eradication occurred and no decrease in susceptibility was observed. On the other hand, when AUC(0-24)/MPC and C(max)/MPC were below 0.84 and 0.08, respectively, the susceptibility decreased. However, the time inside the mutant selective window and the time above the MPC did not show any correlation with the decrease in susceptibility. These results suggest that AUC(0-24)/MPC and C(max)/MPC are important parameters for predicting the emergence of resistant mutants and that higher values indicate greater effectiveness. PMID:17664314

  5. Replication and transformation functions of in vitro-generated simian virus 40 large T antigen mutants.

    PubMed Central

    Rutila, J E; Imperiale, M J; Brockman, W W

    1986-01-01

    We used sodium bisulfite mutagenesis to introduce point mutations within the early region of the simian virus 40 genome. Seventeen mutants which contained amino acid changes in the amino-terminal half of the large T antigen coding sequence were assayed for their ability to replicate viral DNA and to induce transformation in the established rodent cell line Rat-3. The mutants fell into four basic classes with respect to these two biological functions. Five mutants had wild-type replication and transformation activities, six were totally defective, three were replication deficient and transformation competent, and two were replication competent and transformation deficient. Within these classes were mutants which displayed intermediate phenotypes, such as four mutants which were not totally deficient in viral replication or cellular transformation but instead showed reduced large T antigen function relative to wild type. Three large T mutants displayed transforming activity that was greater than that of wild type and are called supertransforming mutants. Of the most interest are mutants differentially defective in replication and transformation activities. These results both support and extend previous findings that two important biological functions of large T antigen can be genetically separated. Images PMID:3009866

  6. An Avirulent Mutant of Rabies Virus Is Unable To Infect Motoneurons In Vivo and In Vitro

    PubMed Central

    Coulon, Patrice; Ternaux, Jean-Pierre; Flamand, Anne; Tuffereau, Christine

    1998-01-01

    An antigenic double mutant of rabies virus (challenge virus standard [CVS] strain) was selected by successive use of two neutralizing antiglycoprotein monoclonal antibodies, both specific for antigenic site III. This mutant differed from the original virus strain by two amino acid substitutions in the ectodomain of the glycoprotein. The lysine in position 330 and the arginine in position 333 were replaced by asparagine and methionine, respectively. This double mutant was not pathogenic for adult mice. When injected intramuscularly into the forelimbs of adult mice, this virus could not penetrate the nervous system, either by the motor or by the sensory route, while respective single mutants infected motoneurons in the spinal cord and sensory neurons in the dorsal root ganglia. In vitro experiments showed that the double mutant was able to infect BHK cells, neuroblastoma cells, and freshly prepared embryonic motoneurons, albeit with a lower efficiency than the CVS strain. Upon further incubation at 37°C, the motoneurons became resistant to infection by the mutant while remaining permissive to CVS infection. These results suggest that rabies virus uses different types of receptors: a molecule which is ubiquitously expressed at the surface of continuous cell lines and which is recognized by both CVS and the double mutant and a neuron-specific molecule which is not recognized by the double mutant. PMID:9420224

  7. 3-hydroxypyruvate substitutes for pyridoxine in serC mutants of Escherichia coli K-12.

    PubMed Central

    Shimizu, S; Dempsey, W B

    1978-01-01

    Escherichia coli K-12 mutants with serC genotype required pyridoxine and serine for normal growth, as do E. coli B mutants of this type. Mutants of the K-12 strain, however, reverted easily to pyridoxine independence without regaining activity in the 3-phosphoserine oxoglutarate transaminase coded for by the serC gene. Both these revertants and the parental type synthesized pyridoxine in normal amounts when 3-hydroxypyruvate was used as a supplement, although neither of these mutants could use this compound to satisfy their serine requirement. Since serine alone was inadequate to provide the nutritional requirement of serC mutants, these mutants must have been unable to synthesize 3-hydroxypyruvate from serine. We suggest that 3-phosphoserine oxoglutarate transaminase in normal E. coli serves as a catalyst for transaminating small amounts of serine to 3-hydroxypyruvate, which is then used in pyridoxine biosynthesis. In serC mutants, this activity is blocked, and these mutants then show a double requirement for serine and pyridoxine. PMID:350858

  8. Analysis of proteomic changes in colored mutants of Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

    PubMed

    Barbachano-Torres, Alejandra; Castelblanco-Matiz, Lina M; Ramos-Valdivia, Ana C; Cerda-García-Rojas, Carlos M; Salgado, Luis M; Flores-Ortiz, César M; Ponce-Noyola, Teresa

    2014-06-01

    The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin as its most prevalent xanthophyll derivative. Comparisons between the protein profiles of mutant lines of this yeast can provide insight into the carotenogenic pathway. Differently colored mutants (red, orange, pink, yellow, and white) were obtained from this yeast species, and their protein profiles were determined using two-dimensional polyacrylamide gel electrophoresis (2DE). Individual proteins differentially expressed were identified using mass spectrometry. The red mutants hyperproduced total carotenoids (mainly astaxanthin), while in white and orange mutants, mutagenesis affected the phytoene dehydrogenase activity as indicated by the accumulation of phytoene. Inactivation of astaxanthin synthase after the mutagenic treatment was evident in β-carotene accumulating mutants. Differences in the proteomic profiles of wild-type X. dendrorhous and its colored mutants were demonstrated using 2DE. Of the total number of spots detected in each gel (297-417), 128 proteins were present in all strains. The red mutant showed the greatest number of matches with respect to the wild type (305 spots), while the white and yellow mutants, which had reduced concentrations of total carotenoids, presented the highest correlation coefficient (0.6) between each other. A number of differentially expressed proteins were sequenced, indicating that tricarboxylic acid cycle and stress response proteins are closely related to the carotenogenic process. PMID:24676883

  9. Initial characterization of a pea mutant with light-independent photomorphogenesis.

    PubMed

    Frances, S; White, M J; Edgerton, M D; Jones, A M; Elliott, R C; Thompson, W F

    1992-12-01

    We have identified a mutant of pea cultivar Alaska that has many of the characteristics normally associated with light-grown seedlings even when grown in complete darkness. We have designated this mutant lip1, for light independent photomorphogenesis. Etiolated wild-type pea seedlings are white to slightly yellow in color and have a distinct morphology characterized by elongated epicotyls and buds containing unexpanded leaves with small, undifferentiated cells. In contrast, mutant seedlings grown under the same conditions are yellow in color and have short epicotyls and expanded leaves showing clear cellular differentiation. Transmission electron microscopy revealed partially developed, agranal plastids in the dark-grown mutant, unlike wild-type seedlings that contain etioplasts with prolamellar bodies. The mutant also exhibits a much shorter lag period for chlorophyll accumulation when etiolated seedlings are transferred from darkness to white light. The dark-grown mutant has 10-fold less spectrally detectable phytochrome, which can be attributed to a 10-fold reduction in the level of the PHYA polypeptide. Cab, Fed1, and RbcS transcripts are present in dark-grown mutant seedlings at levels comparable to those produced in light-grown material. The levels of these transcripts show a normal decrease when green plants grown for 15 days in a light/dark cycle are transferred to continuous darkness. However, transcript levels remain high during dark treatment of seedlings grown for 9 days in continuous light, indicating that the dark adaptation response in this mutant is developmentally plastic. The lip1 mutant has several features in common with the deetiolated Arabidopsis mutants det1, det2, and cop1. However, there are also several important differences, including varying effects on phytochrome levels, organ-specific gene expression, plastid development, and response to dark adaptation. PMID:1467651

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    Tao, Ya-Xiong; Huang, Hui

    2014-01-01

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

  12. Initial characterization of a pea mutant with light-independent photomorphogenesis.

    PubMed Central

    Frances, S; White, M J; Edgerton, M D; Jones, A M; Elliott, R C; Thompson, W F

    1992-01-01

    We have identified a mutant of pea cultivar Alaska that has many of the characteristics normally associated with light-grown seedlings even when grown in complete darkness. We have designated this mutant lip1, for light independent photomorphogenesis. Etiolated wild-type pea seedlings are white to slightly yellow in color and have a distinct morphology characterized by elongated epicotyls and buds containing unexpanded leaves with small, undifferentiated cells. In contrast, mutant seedlings grown under the same conditions are yellow in color and have short epicotyls and expanded leaves showing clear cellular differentiation. Transmission electron microscopy revealed partially developed, agranal plastids in the dark-grown mutant, unlike wild-type seedlings that contain etioplasts with prolamellar bodies. The mutant also exhibits a much shorter lag period for chlorophyll accumulation when etiolated seedlings are transferred from darkness to white light. The dark-grown mutant has 10-fold less spectrally detectable phytochrome, which can be attributed to a 10-fold reduction in the level of the PHYA polypeptide. Cab, Fed1, and RbcS transcripts are present in dark-grown mutant seedlings at levels comparable to those produced in light-grown material. The levels of these transcripts show a normal decrease when green plants grown for 15 days in a light/dark cycle are transferred to continuous darkness. However, transcript levels remain high during dark treatment of seedlings grown for 9 days in continuous light, indicating that the dark adaptation response in this mutant is developmentally plastic. The lip1 mutant has several features in common with the deetiolated Arabidopsis mutants det1, det2, and cop1. However, there are also several important differences, including varying effects on phytochrome levels, organ-specific gene expression, plastid development, and response to dark adaptation. PMID:1467651

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

    PubMed

    Bhalla, S C

    1976-12-01

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

  14. 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

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

    PubMed

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

    2002-01-01

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

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

    SciTech Connect

    Datta, T.; Doermer, P.

    1987-12-01

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

  17. Phosphoglucomutase Mutants of Escherichia coli K-12

    PubMed Central

    Adhya, Sankar; Schwartz, Maxime

    1971-01-01

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

  18. Cooperative Interaction Within RNA Virus Mutant Spectra.

    PubMed

    Shirogane, Yuta; Watanabe, Shumpei; Yanagi, Yusuke

    2016-01-01

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

  19. Mutant p53: one name, many proteins

    PubMed Central

    Freed-Pastor, William A.; Prives, Carol

    2012-01-01

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

  20. Mapping Pathological Phenotypes in Reelin Mutant Mice

    PubMed Central

    Michetti, Caterina; Romano, Emilia; Altabella, Luisa; Caruso, Angela; Castelluccio, Paolo; Bedse, Gaurav; Gaetani, Silvana; Canese, Rossella; Laviola, Giovanni; Scattoni, Maria Luisa

    2014-01-01

    Autism Spectrum Disorders (ASD) are neurodevelopmental disorders with multifactorial origin characterized by social communication deficits and the presence of repetitive behaviors/interests. Several studies showed an association between the reelin gene mutation and increased risk of ASD and a reduced reelin expression in some brain regions of ASD subjects, suggesting a role for reelin deficiency in ASD etiology. Reelin is a large extracellular matrix glycoprotein playing important roles during development of the central nervous system. To deeply investigate the role of reelin dysfunction as vulnerability factor in ASD, we assessed the behavioral, neurochemical, and brain morphological features of reeler male mice. We recently reported a genotype-dependent deviation in the ultrasonic vocal repertoire and a general delay in motor development of reeler pups. We now report that adult male heterozygous (Het) reeler mice did not show social behavior and communication deficits during male–female social interactions. Wildtype and Het mice showed a typical light/dark locomotor activity profile, with a peak during the central interval of the dark phase. However, when faced with a mild stressful stimulus (a saline injection) only Het mice showed an over response to stress. In addition to the behavioral studies, we conducted high performance liquid chromatography and magnetic resonance imaging and spectroscopy to investigate whether reelin mutation influences brain monoamine and metabolites levels in regions involved in ASD. Low levels of dopamine in cortex and high levels of glutamate and taurine in hippocampus were detected in Het mice, in line with clinical data collected on ASD children. Altogether, our data detected subtle but relevant neurochemical abnormalities in reeler mice supporting this mutant line, particularly male subjects, as a valid experimental model to estimate the contribution played by reelin deficiency in the global ASD neurobehavioral phenotype. PMID

  1. VIEW SHOWING WEST ELEVATION, EAST SIDE OF MEYER AVENUE. SHOWS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    VIEW SHOWING WEST ELEVATION, EAST SIDE OF MEYER AVENUE. SHOWS 499-501, MUNOZ HOUSE (AZ-73-37) ON FAR RIGHT - Antonio Bustamente House, 485-489 South Meyer Avenue & 186 West Kennedy Street, Tucson, Pima County, AZ

  2. 15. Detail showing lower chord pinconnected to vertical member, showing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    15. Detail showing lower chord pin-connected to vertical member, showing floor beam riveted to extension of vertical member below pin-connection, and showing brackets supporting cantilevered sidewalk. View to southwest. - Selby Avenue Bridge, Spanning Short Line Railways track at Selby Avenue between Hamline & Snelling Avenues, Saint Paul, Ramsey County, MN

  3. A physiological, biochemical and proteomic characterization of Saccharomyces cerevisiae trk1,2 transport mutants grown under limiting potassium conditions.

    PubMed

    Gelis, Samuel; González-Fernández, Raquel; Herrera, Rito; Jorrín, Jesús; Ramos, José

    2015-06-01

    Saccharomyces cerevisiae mutants lacking both isoforms of the main plasma membrane potassium transporter display impaired potassium transport and defective growth at limiting concentrations of the cation. Moreover, they are hyperpolarized and have a lower intracellular pH than wild-type. In order to unravel global physiological processes altered in trk1,2 mutants, we have established conditions at which both wild-type and mutants can grow at different rates. Using a combination of physiological, biochemical and proteomic approaches, we show that during growth at suboptimal potassium concentrations, double trk1,2 mutants accumulate less potassium and reach lower yields. In contrast, the mutants maintain increased viability in the stationary phase and retain more potassium. Moreover, the mutants show increased expression of stress-related proteins such as catalase T, thioredoxin peroxidase or hexokinase 2, suggesting that they are better adapted to the additional stress factors associated with entry into stationary growth phase. PMID:25777080

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

    PubMed

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

    2016-01-10

    To breed industrially useful strains of a slow-growing, yellow pigment producing strain of Monascus sp., protoplasts of Monascus purpureus yellow mutant (ade(-)) and rapid-growing M. purpureus white mutant (prototroph) were fused and fusants were selected on minimal medium (MM). Preliminary conventional protoplast fusion of the two strains was performed and the result showed that only white colonies were detected on MM. It was not able to differentiate the fusants from the white parental prototroph. To solve this problem, the white parental prototroph was thus pretreated with 20mM iodoacetamide (IOA) for cytoplasm inactivation and subsequently taken into protoplast fusion with slow-growing Monascus yellow mutant. Under this development technique, only the fusants, with viable cytoplasm from Monascus yellow mutant (ade(-)), could thus grow on MM, whereas neither IOA pretreated white parental prototroph nor yellow auxotroph (ade(-)) could survive. Fifty-three fusants isolated from yellow colonies obtained through this developed technique were subsequently inoculated on complete medium (MY agar). Fifteen distinguished yellow colonies from their parental yellow mutant were then selected for biochemical, morphological and fermentative properties in cassava starch and soybean flour (SS) broth. Finally, three most stable fusants (F7, F10 and F43) were then selected and compared in rice solid culture. Enhancement of yellow pigment production over the parental yellow auxotroph was found in F7 and F10, while enhanced glucoamylase activity was found in F43. The formation of fusants was further confirmed by monacolin K content, which was intermediate between the two parents (monacolin K-producing yellow auxotroph and non-monacolin K producing white prototroph). PMID:26562446

  5. Survival, growth, and localization of epiphytic fitness mutants of pseudomonas syringae on leaves

    SciTech Connect

    Beattie, G.A.; Lindow, S.E. )

    1994-10-01

    Among 82 epiphytic fitness mutants of a Pseudomonas syringae pv. syringae strain that were characterized in a previous study, 4 mutants were particularly intolerant of the stresses associated with dry leaf surfaces. These four mutants each exhibited distinctive behaviors when inoculated into and into plant leaves. For example, while non showed measurable growth on dry potato leaf surfaces, they grew to different population sizes in the intercellular space of bean leaves and on dry bean leaf surfaces, and one mutant appeared incapable of growth in both environments although it grew well on moist bean leaves. The presence of the parental strain did not influence the survival of the mutants immediately following exposure of leaves to dry, high-light incubation conditions, suggesting that the reduced survival of the mutants did not result from an inability to produce extracellular factors in planta. On moist bean leaves that were colonized by either a mutant or the wild type, the proportion of the total epiphytic population that was located in sizes protected from a surface sterilant was smaller for the mutants than for the wild type, indicating that the mutants were reduced in their ability to locate, multiply in, and/or survive in such protected sites. This reduced ability was only one of possible several factors contributing to the reduced epiphytic fitness of each mutant. Their reduced fitness was not specific to the host plant bean, since they also exhibited reduced fitness on the nonhost plant potato; the functions altered in these strains are thus of interest for their contribution to the general fitness of bacterial epiphytes. 52 refs., 6 figs., 1 tab.

  6. [Evaluation of penicillin expandase mutants and complex substrate inhibition characteristics at high concentrations of penicillin G].

    PubMed

    Wu, Linjun; Fan, Keqiang; Ji, Junjie; Yang, Keqian

    2015-12-01

    Penicillin expandase, also known as deacetoxycephalosporin C synthase (DAOCS), is an essential enzyme involved in cephalosporin C biosynthesis. To evaluate the catalytic behaviors of penicillin expandase under high penicillin G concentration and to identify mutants suitable for industrial applications, the specific activities of wild-type DAOCS and several mutants with increased activities toward penicillin G were determined by HPLC under high penicillin G concentrations. Their specific activity profiles were compared with theoretical predictions by different catalytic dynamics models. We evaluated the specific activities of wild-type DAOCS and previous reported high-activity mutants H4, H5, H6 and H7 at concentrations ranging from 5.6 to 500 mmol/L penicillin G. The specific activities of wild-type DAOCS and mutant H4 increased as penicillin G concentration increased, but decreased when concentrations of substrate go above 200 mmol/L. Other mutants H5, H6 and H7 showed more complex behaviors under high concentration of penicillin G. Among all tested enzymes, mutant H6 showed the highest activity when concentration of penicillin G is above 100 mmol/L. Our results revealed that the substrate inhibition to wild-type DAOCS' by penicillin G is noncompetitive. Other DAOCS mutants showed more complex trends in their specific activities at high concentration of penicillin G (>100 mmol/L), indicating more complex substrate inhibition mechanism might exist. The substrate inhibition and activity of DAOCS mutants at high penicillin G concentration provide important insight to help select proper mutants for industrial application. PMID:27093832

  7. Increased sensitivity to salt stress in tocopherol-deficient Arabidopsis mutants growing in a hydroponic system

    PubMed Central

    Ellouzi, Hasna; Hamed, Karim Ben; Cela, Jana; Müller, Maren; Abdelly, Chedly; Munné-Bosch, Sergi

    2013-01-01

    Recent studies suggest that tocopherols could play physiological roles in salt tolerance but the mechanisms are still unknown. In this study, we analyzed changes in growth, mineral and oxidative status in vte1 and vte4 Arabidopsis thaliana mutants exposed to salt stress. vte1 and vte4 mutants lack α-tocopherol, but only the vte1 mutant is additionally deficient in γ-tocopherol. Results showed that a deficiency in vitamin E leads to reduced growth and increased oxidative stress in hydroponically-grown plants. This effect was observed at early stages, not only in rosettes but also in roots. The vte1 mutant was more sensitive to salt-induced oxidative stress than the wild type and the vte4 mutant. Salt sensitivity was associated with (i) high contents of Na+, (ii) reduced efficiency of PSII photochemistry (Fv/Fm ratio) and (iii) more pronounced oxidative stress as indicated by increased hydrogen peroxide and malondialdeyde levels. The vte 4 mutant, which accumulates γ- instead of α-tocopherol showed an intermediate sensitivity to salt stress between the wild type and the vte1 mutant. Contents of abscisic acid, jasmonic acid and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid were higher in the vte1 mutant than the vte4 mutant and wild type. It is concluded that vitamin E-deficient plants show an increased sensitivity to salt stress both in rosettes and roots, therefore indicating the positive role of tocopherols in stress tolerance, not only by minimizing oxidative stress, but also controlling Na+/K+ homeostasis and hormonal balance. PMID:23299430

  8. Increased sensitivity to salt stress in tocopherol-deficient Arabidopsis mutants growing in a hydroponic system.

    PubMed

    Ellouzi, Hasna; Hamed, Karim Ben; Cela, Jana; Müller, Maren; Abdelly, Chedly; Munné-Bosch, Sergi

    2013-02-01

    Recent studies suggest that tocopherols could play physiological roles in salt tolerance but the mechanisms are still unknown. In this study, we analyzed changes in growth, mineral and oxidative status in vte1 and vte4 Arabidopsis thaliana mutants exposed to salt stress. vte1 and vte4 mutants lack α-tocopherol, but only the vte1 mutant is additionally deficient in γ-tocopherol. Results showed that a deficiency in vitamin E leads to reduced growth and increased oxidative stress in hydroponically-grown plants. This effect was observed at early stages, not only in rosettes but also in roots. The vte1 mutant was more sensitive to salt-induced oxidative stress than the wild type and the vte4 mutant. Salt sensitivity was associated with (i) high contents of Na(+), (ii) reduced efficiency of PSII photochemistry (Fv/Fm ratio) and (iii) more pronounced oxidative stress as indicated by increased hydrogen peroxide and malondialdeyde levels. The vte 4 mutant, which accumulates γ- instead of α-tocopherol showed an intermediate sensitivity to salt stress between the wild type and the vte1 mutant. Contents of abscisic acid, jasmonic acid and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid were higher in the vte1 mutant than the vte4 mutant and wild type. It is concluded that vitamin E-deficient plants show an increased sensitivity to salt stress both in rosettes and roots, therefore indicating the positive role of tocopherols in stress tolerance, not only by minimizing oxidative stress, but also controlling Na(+)/K(+) homeostasis and hormonal balance. PMID:23299430

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

    SciTech Connect

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

    1990-03-01

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

  10. Characterization of novel sorghum brown midrib mutants from an EMS-mutagenized population

    DOE PAGESBeta

    Sattler, Scott E.; Saballos, Ana; Xin, Zhanguo; Funnell-Harris, Deanna L.; Vermerris, Wilfred; Pedersen, Jeffrey F.

    2014-09-02

    Reducing lignin concentration in lignocellulosic biomass can increase forage digestibility for ruminant livestock and saccharification yields of biomass for bioenergy. In sorghum (Sorghum bicolor (L.) Moench) and several other C4 grasses, brown midrib (bmr) mutants have been shown to reduce lignin concentration. Putative bmr mutants isolated from an EMS-mutagenized population were characterized and classified based on their leaf midrib phenotype and allelism tests with the previously described sorghum bmr mutants bmr2, bmr6, and bmr12. These tests resulted in the identification of additional alleles of bmr2, bmr6,and bmr12, and, in addition, six bmr mutants were identified that were not allelic tomore » these previously described loci. Further allelism testing among these six bmr mutants showed that they represented four novel bmr loci. Based on this study, the number of bmr loci uncovered in sorghum has doubled. The impact of these lines on agronomic traits and lignocellulosic composition was assessed in a 2-yr field study. Most of the identified bmr lines showed reduced lignin concentration of their biomass relative to wild-type (WT). Effects of the six new bmr mutants on enzymatic saccharification of lignocellulosic materials were determined, but the amount of glucose released from the stover was similar to WT in all cases. Like bmr2, bmr6, and bmr12, these mutants may affect monolignol biosynthesis and may be useful for bioenergy and forage improvement when stacked together or in combination with the three previously described bmr alleles.« less

  11. Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia.

    PubMed

    Liu, Chao; Gates, Keith P; Fang, Longhou; Amar, Marcelo J; Schneider, Dina A; Geng, Honglian; Huang, Wei; Kim, Jungsu; Pattison, Jennifer; Zhang, Jian; Witztum, Joseph L; Remaley, Alan T; Dong, P Duc; Miller, Yury I

    2015-08-01

    Apolipoprotein C-II (APOC2) is an obligatory activator of lipoprotein lipase. Human patients with APOC2 deficiency display severe hypertriglyceridemia while consuming a normal diet, often manifesting xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia is also an important risk factor for development of cardiovascular disease. Animal models to study hypertriglyceridemia are limited, with no Apoc2-knockout mouse reported. To develop a genetic model of hypertriglyceridemia, we generated an apoc2 mutant zebrafish characterized by the loss of Apoc2 function. apoc2 mutants show decreased plasma lipase activity and display chylomicronemia and severe hypertriglyceridemia, which closely resemble the phenotype observed in human patients with APOC2 deficiency. The hypertriglyceridemia in apoc2 mutants is rescued by injection of plasma from wild-type zebrafish or by injection of a human APOC2 mimetic peptide. Consistent with a previous report of a transient apoc2 knockdown, apoc2 mutant larvae have a minor delay in yolk consumption and angiogenesis. Furthermore, apoc2 mutants fed a normal diet accumulate lipid and lipid-laden macrophages in the vasculature, which resemble early events in the development of human atherosclerotic lesions. In addition, apoc2 mutant embryos show ectopic overgrowth of pancreas. Taken together, our data suggest that the apoc2 mutant zebrafish is a robust and versatile animal model to study hypertriglyceridemia and the mechanisms involved in the pathogenesis of associated human diseases. PMID:26044956

  12. Characterization of Novel Sorghum brown midrib Mutants from an EMS-Mutagenized Population

    PubMed Central

    Sattler, Scott E.; Saballos, Ana; Xin, Zhanguo; Funnell-Harris, Deanna L.; Vermerris, Wilfred; Pedersen, Jeffrey F.

    2014-01-01

    Reducing lignin concentration in lignocellulosic biomass can increase forage digestibility for ruminant livestock and saccharification yields of biomass for bioenergy. In sorghum (Sorghum bicolor (L.) Moench) and several other C4 grasses, brown midrib (bmr) mutants have been shown to reduce lignin concentration. Putative bmr mutants isolated from an EMS-mutagenized population were characterized and classified based on their leaf midrib phenotype and allelism tests with the previously described sorghum bmr mutants bmr2, bmr6, and bmr12. These tests resulted in the identification of additional alleles of bmr2, bmr6, and bmr12, and, in addition, six bmr mutants were identified that were not allelic to these previously described loci. Further allelism testing among these six bmr mutants showed that they represented four novel bmr loci. Based on this study, the number of bmr loci uncovered in sorghum has doubled. The impact of these lines on agronomic traits and lignocellulosic composition was assessed in a 2-yr field study. Overall, most of the identified bmr lines showed reduced lignin concentration of their biomass relative to wild-type (WT). Effects of the six new bmr mutants on enzymatic saccharification of lignocellulosic materials were determined, but the amount of glucose released from the stover was similar to WT in all cases. Like bmr2, bmr6, and bmr12, these mutants may affect monolignol biosynthesis and may be useful for bioenergy and forage improvement when stacked together or in combination with the three previously described bmr alleles. PMID:25187038

  13. Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia

    PubMed Central

    Liu, Chao; Gates, Keith P.; Fang, Longhou; Amar, Marcelo J.; Schneider, Dina A.; Geng, Honglian; Huang, Wei; Kim, Jungsu; Pattison, Jennifer; Zhang, Jian; Witztum, Joseph L.; Remaley, Alan T.; Dong, P. Duc; Miller, Yury I.

    2015-01-01

    ABSTRACT Apolipoprotein C-II (APOC2) is an obligatory activator of lipoprotein lipase. Human patients with APOC2 deficiency display severe hypertriglyceridemia while consuming a normal diet, often manifesting xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia is also an important risk factor for development of cardiovascular disease. Animal models to study hypertriglyceridemia are limited, with no Apoc2-knockout mouse reported. To develop a genetic model of hypertriglyceridemia, we generated an apoc2 mutant zebrafish characterized by the loss of Apoc2 function. apoc2 mutants show decreased plasma lipase activity and display chylomicronemia and severe hypertriglyceridemia, which closely resemble the phenotype observed in human patients with APOC2 deficiency. The hypertriglyceridemia in apoc2 mutants is rescued by injection of plasma from wild-type zebrafish or by injection of a human APOC2 mimetic peptide. Consistent with a previous report of a transient apoc2 knockdown, apoc2 mutant larvae have a minor delay in yolk consumption and angiogenesis. Furthermore, apoc2 mutants fed a normal diet accumulate lipid and lipid-laden macrophages in the vasculature, which resemble early events in the development of human atherosclerotic lesions. In addition, apoc2 mutant embryos show ectopic overgrowth of pancreas. Taken together, our data suggest that the apoc2 mutant zebrafish is a robust and versatile animal model to study hypertriglyceridemia and the mechanisms involved in the pathogenesis of associated human diseases. PMID:26044956

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

    PubMed

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

    2004-06-01

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

  15. 28. MAP SHOWING LOCATION OF ARVFS FACILITY AS BUILT. SHOWS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    28. MAP SHOWING LOCATION OF ARVFS FACILITY AS BUILT. SHOWS LINCOLN BOULEVARD, BIG LOST RIVER, AND NAVAL REACTORS FACILITY. F.C. TORKELSON DRAWING NUMBER 842-ARVFS-101-2. DATED OCTOBER 12, 1965. INEL INDEX CODE NUMBER: 075 0101 851 151969. - Idaho National Engineering Laboratory, Advanced Reentry Vehicle Fusing System, Scoville, Butte County, ID

  16. 8. Detail showing concrete abutment, showing substructure of bridge, specifically ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    8. Detail showing concrete abutment, showing substructure of bridge, specifically west side of arch and substructure. - Presumpscot Falls Bridge, Spanning Presumptscot River at Allen Avenue extension, 0.75 mile west of U.S. Interstate 95, Falmouth, Cumberland County, ME

  17. Arabidopsis AMY1 expressions and early flowering mutant phenotype.

    PubMed

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

    2009-02-28

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

  18. Regulation of apoptosis of rbf mutant cells during Drosophila development

    PubMed Central

    Tanaka-Matakatsu, Miho; Xu, Jinhua; Cheng, Leping; Du, Wei

    2008-01-01

    Inactivation of the retinoblastoma gene Rb leads to defects in cell proliferation, differentiation, or apoptosis, depending on specific cell or tissue types. To gain insights into the genes that can modulate the consequences of Rb inactivation, we carried out a genetic screen in Drosophila to identify mutations that affected apoptosis induced by inactivation of the Retinoblastoma-family protein (rbf) and identified a mutation that blocked apoptosis induced by rbf. We found this mutation to be a new allele of head involution defective (hid) and showed that hid expression is deregulated in rbf mutant cells in larval imaginal discs. We identified an enhancer that regulates hid expression in response to developmental cues as well as to radiation and demonstrated that this hid enhancer is directly repressed by RBF through an E2F binding site. These observations indicate that apoptosis of rbf mutant cells is mediated by an upregulation of hid. Finally, we showed that bantam, a miRNA that regulates hid translation, is expressed in the interommatidial cells in the larval eye discs and modulates the survival of rbf mutant cells. PMID:19100727

  19. Mutant SOD1 Forms Ion Channel: Implications for ALS Pathophysiology

    PubMed Central

    Allen, Michael J.; Lacroix, Jérome J.; Ramachandran, Srinivasan; Capone, Ricardo; Whitlock, Jenny L.; Ghadge, Ghanashyam D.; Arnsdorf, Morton F.; Roos, Raymond P.; Lal, Ratnesh

    2011-01-01

    Point mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) impart a gain-of-function to this protein that underlies 20-25% of all familial amyotrophic lateral sclerosis (FALS) cases. However, the specific mechanism of mutant SOD1 toxicity has remained elusive. Using the complementary techniques of atomic force microscopy (AFM), electrophysiology, and cell and molecular biology, here we examine the structure and activity of A4VSOD1, a mutant SOD1. AFM of A4VSOD1 reconstituted in lipid membrane shows discrete tetrameric pore-like structure with outer and inner diameters 12.2 and 3.0 nm respectively. Electrophysiological recordings show distinct ionic conductances across bilayer for A4VSOD1 and none for wild-type SOD1. Mouse neuroblastoma cells exposed to A4VSOD1 undergo membrane depolarization and increases in intracellular calcium. These results provide compelling new evidence that a mutant SOD1 is capable of disrupting cellular homeostasis via an unregulated ion channel mechanism. Such a “toxic channel” mechanism presents a new therapeutic direction for ALS research. PMID:21930207

  20. Virulence of Burkholderia mallei Quorum-Sensing Mutants

    PubMed Central

    Majerczyk, Charlotte; Kinman, Loren; Han, Tony; Bunt, Richard

    2013-01-01

    Many Proteobacteria use acyl-homoserine lactone-mediated quorum-sensing (QS) to activate specific sets of genes as a function of cell density. QS often controls the virulence of pathogenic species, and in fact a previous study indicated that QS was important for Burkholderia mallei mouse lung infections. To gain in-depth information on the role of QS in B. mallei virulence, we constructed and characterized a mutant of B. mallei strain GB8 that was unable to make acyl-homoserine lactones. The QS mutant showed virulence equal to that of its wild-type parent in an aerosol mouse infection model, and growth in macrophages was indistinguishable from that of the parent strain. Furthermore, we assessed the role of QS in B. mallei ATCC 23344 by constructing and characterizing a mutant strain producing AiiA, a lactonase enzyme that degrades acyl-homoserine lactones. Although acyl-homoserine lactone levels in cultures of this strain are very low, it showed full virulence. Contrary to the previous report, we conclude that QS is not required for acute B. mallei infections of mice. QS may be involved in some stage of chronic infections in the natural host of horses, or the QS genes may be remnants of the QS network in B. pseudomallei from which this host-adapted pathogen evolved. PMID:23429539

  1. Generating amphioxus Hedgehog knockout mutants and phenotype analysis.

    PubMed

    Hui, Wang; Guang, Li; Yiquan, Wang

    2015-10-01

    The amphioxus is a promising animal model for evolutionary-developmental studies due to its key position on the animal phylogenetic tree. In the present study, we reported a genetically modified amphioxus strain on the Hedgehog (Hh) gene locus using the TALEN method. The result showed that our TALEN pair injection could bring about 34% mutations in the amphioxus Hh coding region. Further analysis on the F(0) gametic DNA revealed that the mutations had entered into gametes. So, we paired one F(0) male carrying an 8 bp deletion with a wild-type (WT) female, and carefully nursed the F(1) embryos up to adulthood. We then screened F(1) individually via analyzing their genomic DNA from a tiny tail tip, and obtained eight heterozygous mutants from the F(1) offspring. Moreover, our observation on the F(2) embryos generated by mating F(1) mutants also revealed that about 25% of early larvae developed aberrantly with head and tail curving ventrally, agenesis of the mesoblastic tissue under their anterior notochord, and no mouth opening. With the larva growth, deformities (such as twist of head and tail, mouth absent, ventrally localized endostyle and gill slits) became more severe, and eventually those malformed larvae died due to no food intake. Genetic analysis showed that all these deformed embryos were homozygous mutants and the ratio of Hh hetorozygotes vs WT agreed with Mondel's law. WT amphioxus larvae are asymmetric with the mouth on the left and gill slits on the right side. However, the homozygous mutant larvae became left-right symmetric with the gill slits on the ventral side, indicating a conserved role of Hedgehog signaling in establishing the left-right embryonic axis. PMID:26496756

  2. Distinct cellular properties of oncogenic KIT receptor tyrosine kinase mutants enable alternative courses of cancer cell inhibition.

    PubMed

    Shi, Xiarong; Sousa, Leiliane P; Mandel-Bausch, Elizabeth M; Tome, Francisco; Reshetnyak, Andrey V; Hadari, Yaron; Schlessinger, Joseph; Lax, Irit

    2016-08-16

    Large genomic sequencing analysis as part of precision medicine efforts revealed numerous activating mutations in receptor tyrosine kinases, including KIT. Unfortunately, a single approach is not effective for inhibiting cancer cells or treating cancers driven by all known oncogenic KIT mutants. Here, we show that each of the six major KIT oncogenic mutants exhibits different enzymatic, cellular, and dynamic properties and responds distinctly to different KIT inhibitors. One class of KIT mutants responded well to anti-KIT antibody treatment alone or in combination with a low dose of tyrosine kinase inhibitors (TKIs). A second class of KIT mutants, including a mutant resistant to imatinib treatment, responded well to a combination of TKI with anti-KIT antibodies or to anti-KIT toxin conjugates, respectively. We conclude that the preferred choice of precision medicine treatments for cancers driven by activated KIT and other RTKs may rely on clear understanding of the dynamic properties of oncogenic mutants. PMID:27482095

  3. Identification of novel attenuated Salmonella Enteritidis mutants.

    PubMed

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

    2008-06-01

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

  4. Inositol-Requiring Mutants of SACCHAROMYCES CEREVISIAE

    PubMed Central

    Culbertson, Michael R.; Henry, Susan A.

    1975-01-01

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

  5. Escherichia coli mutants deficient in deoxyuridine triphosphatase.

    PubMed Central

    Hochhauser, S J; Weiss, B

    1978-01-01

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

  6. Characterization of a cytochalasin D-resistant mutant of Entamoeba histolytica.

    PubMed

    de la Garza, M; Gallegos, B; Meza, I

    1989-01-01

    Characterization of a cytochalasin D-resistant mutant of the human parasite Entamoeba histolytica capable of growing at 10 microM cytochalasin is described. The mutant cells also show resistance to 5 mM colchicine and 100 microM cytochalasin B, drugs proved deleterious for wild type trophozoites. The mutants show increased osmotic fragility and electric mobility but reduced phagocytic activity, and agglutination by Concanavalin A. On the other hand pinocytic activity remains unaltered when compared with the wild type cells. Polymerized actin, seen by staining with phalloidin, often appears polarized to one end of the trophozoites and forms few of the endocytic invaginations found in wild type amebas. An altered distribution of part of the actin could explain the differences in surface properties and motility observed in the mutant amebas. PMID:2557444

  7. hydra Mutants of Arabidopsis Are Defective in Sterol Profiles and Auxin and Ethylene Signaling

    PubMed Central

    Souter, Martin; Topping, Jennifer; Pullen, Margaret; Friml, Jiri; Palme, Klaus; Hackett, Rachel; Grierson, Don; Lindsey, Keith

    2002-01-01

    The hydra mutants of Arabidopsis are characterized by a pleiotropic phenotype that shows defective embryonic and seedling cell patterning, morphogenesis, and root growth. We demonstrate that the HYDRA1 gene encodes a Δ8-Δ7 sterol isomerase, whereas HYDRA2 encodes a sterol C14 reductase, previously identified as the FACKEL gene product. Seedlings mutant for each gene are similarly defective in the concentrations of the three major Arabidopsis sterols. Promoter::reporter gene analysis showed misexpression of the auxin-regulated DR5 and ACS1 promoters and of the epidermal cell file–specific GL2 promoter in the mutants. The mutants exhibit enhanced responses to auxin. The phenotypes can be rescued partially by inhibition of auxin and ethylene signaling but not by exogenous sterols or brassinosteroids. We propose a model in which correct sterol profiles are required for regulated auxin and ethylene signaling through effects on membrane function. PMID:12034894

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

    PubMed Central

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

    2016-01-01

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

  9. Transcriptomic comparison of Drosophila snRNP biogenesis mutants reveals mutant-specific changes in pre-mRNA processing: implications for spinal muscular atrophy.

    PubMed

    Garcia, Eric L; Wen, Ying; Praveen, Kavita; Matera, A Gregory

    2016-08-01

    Survival motor neuron (SMN) functions in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) that catalyze pre-mRNA splicing. Here, we used disruptions in Smn and two additional snRNP biogenesis genes, Phax and Ars2, to classify RNA processing differences as snRNP-dependent or gene-specific in Drosophila Phax and Smn mutants exhibited comparable reductions in snRNAs, and comparison of their transcriptomes uncovered shared sets of RNA processing changes. In contrast, Ars2 mutants displayed only small decreases in snRNA levels, and RNA processing changes in these mutants were generally distinct from those identified in Phax and Smn animals. Instead, RNA processing changes in Ars2 mutants support the known interaction of Ars2 protein with the cap-binding complex, as splicing changes showed a clear bias toward the first intron. Bypassing disruptions in snRNP biogenesis, direct knockdown of spliceosomal proteins caused similar changes in the splicing of snRNP-dependent events. However, these snRNP-dependent events were largely unaltered in three Smn mutants expressing missense mutations that were originally identified in human spinal muscular atrophy (SMA) patients. Hence, findings here clarify the contributions of Phax, Smn, and Ars2 to snRNP biogenesis in Drosophila, and loss-of-function mutants for these proteins reveal differences that help disentangle cause and effect in SMA model flies. PMID:27268418

  10. Planning a Successful Tech Show

    ERIC Educational Resources Information Center

    Nikirk, Martin

    2011-01-01

    Tech shows are a great way to introduce prospective students, parents, and local business and industry to a technology and engineering or career and technical education program. In addition to showcasing instructional programs, a tech show allows students to demonstrate their professionalism and skills, practice public presentations, and interact…

  11. Hey Teacher, Your Personality's Showing!

    ERIC Educational Resources Information Center

    Paulsen, James R.

    1977-01-01

    A study of 30 fourth, fifth, and sixth grade teachers and 300 of their students showed that a teacher's age, sex, and years of experience did not relate to students' mathematics achievement, but that more effective teachers showed greater "freedom from defensive behavior" than did less effective teachers. (DT)

  12. What Do Blood Tests Show?

    MedlinePlus

    ... shows the ranges for blood glucose levels after 8 to 12 hours of fasting (not eating). It shows the normal range and the abnormal ranges that are a sign of prediabetes or diabetes. Plasma Glucose Results (mg/dL)* Diagnosis 70 to 99 ...

  13. Oncolytic reovirus preferentially induces apoptosis in KRAS mutant colorectal cancer cells, and synergizes with irinotecan

    PubMed Central

    Maitra, Radhashree; Seetharam, Raviraja; Tesfa, Lydia; Augustine, Titto A.; Klampfer, Lidija; Coffey, Matthew C.; Mariadason, John M.; Goel, Sanjay

    2014-01-01

    Reovirus is a double stranded RNA virus, with an intrinsic preference for replication in KRAS mutant cells. As 45% of human colorectal cancers (CRC) harbor KRAS mutations, we sought to investigate its efficacy in KRAS mutant CRC cells, and examine its impact in combination with the topoisimerase-1 inhibitor, irinotecan. Reovirus efficacy was examined in the KRAS mutant HCT116, and the isogenic KRAS WT Hke3 cell line, and in the non-malignant rat intestinal epithelial cell line. Apoptosis was determined by flow cytometry and TUNEL staining. Combination treatment with reovirus and irintoecan was investigated in 15 CRC cell lines, including the HCT116 p21 isogenic cell lines. Reovirus preferentially induced apoptosis in KRAS mutant HCT116 cells compared to its isogenic KRAS WT derivative, and in KRAS mutant IEC cells. Reovirus showed a greater degree of caspase 3 activation with PARP 1 cleavage, and preferential inhibition of p21 protein expression in KRAS mutant cells. Reovirus synergistically induced growth inhibition when combined with irinotecan. This synergy was lost upon p21 gene knock out. Reovirus preferentially induces apoptosis in KRAS mutant colon cancer cells. Reovirus and irinotecan combination therapy is synergistic, p21 mediated, and represents a novel potential treatment for patients with CRC. PMID:24798549

  14. Oncolytic reovirus preferentially induces apoptosis in KRAS mutant colorectal cancer cells, and synergizes with irinotecan.

    PubMed

    Maitra, Radhashree; Seetharam, Raviraja; Tesfa, Lydia; Augustine, Titto A; Klampfer, Lidija; Coffey, Matthew C; Mariadason, John M; Goel, Sanjay

    2014-05-15

    Reovirus is a double stranded RNA virus, with an intrinsic preference for replication in KRAS mutant cells. As 45% of human colorectal cancers (CRC) harbor KRAS mutations, we sought to investigate its efficacy in KRAS mutant CRC cells, and examine its impact in combination with the topoisimerase-1 inhibitor, irinotecan. Reovirus efficacy was examined in the KRAS mutant HCT116, and the isogenic KRAS WT Hke3 cell line, and in the non-malignant rat intestinal epithelial cell line. Apoptosis was determined by flow cytometry and TUNEL staining. Combination treatment with reovirus and irintoecan was investigated in 15 CRC cell lines, including the HCT116 p21 isogenic cell lines. Reovirus preferentially induced apoptosis in KRAS mutant HCT116 cells compared to its isogenic KRAS WT derivative, and in KRAS mutant IEC cells. Reovirus showed a greater degree of caspase 3 activation with PARP 1 cleavage, and preferential inhibition of p21 protein expression in KRAS mutant cells. Reovirus synergistically induced growth inhibition when combined with irinotecan. This synergy was lost upon p21 gene knock out. Reovirus preferentially induces apoptosis in KRAS mutant colon cancer cells. Reovirus and irinotecan combination therapy is synergistic, p21 mediated, and represents a novel potential treatment for patients with CRC. PMID:24798549

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

    PubMed

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

    2014-06-01

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

  16. Analysis of Induced Pluripotent Stem Cells from a BRCA1 Mutant Family

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    Uprichard, S L; Knipe, D M

    1996-01-01

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

  20. A naturally occurring deletion mutant of figwort mosaic virus (caulimovirus) is generated by RNA splicing.

    PubMed

    Scholthof, H B; Wu, F C; Richins, R D; Shepherd, R J

    1991-09-01

    A naturally occurring deletion mutant is observed in plants infected with figwort mosaic virus (FMV), a caulimovirus. The encapsidated mutant genome is formed spontaneously in association with two different strains of FMV in four host plant species. The mutant also appears when cloned wild-type viral DNA is used as the inoculum. The deletion mutant alone is not infectious and it appears unable to replicate after its formation, even in the presence of wild-type virus. The gene for chloramphenicol acetyltransferase was inserted at different positions in the deletion mutant genome, and subsequent transient assays showed that gene expression of the mutant occurs despite the deletion. Sequence analyses of the mutant genome revealed a deletion of 1237-bp segment encompassing a major portion of the coat protein gene and the 5' end of the downstream reverse transcriptase gene. This deletion is associated with consensus signals for RNA splicing including the conserved 5' and 3' splice sites plus surrounding sequences, putative branch point(s) for lariat formation, and an extremely high adenosine content (41%) of the removed fragment. This suggests that splicing of the FMV full-length transcript has occurred prior to reverse transcription and this accounts for the presence and accumulation of encapsidated DNAs with the same deletion. PMID:1871973

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

    PubMed Central

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

    2009-01-01

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

  2. Pla2g16 phospholipase mediates gain-of-function activities of mutant p53.

    PubMed

    Xiong, Shunbin; Tu, Huolin; Kollareddy, Madhusudhan; Pant, Vinod; Li, Qin; Zhang, Yun; Jackson, James G; Suh, Young-Ah; Elizondo-Fraire, Ana C; Yang, Peirong; Chau, Gilda; Tashakori, Mehrnoosh; Wasylishen, Amanda R; Ju, Zhenlin; Solomon, Hilla; Rotter, Varda; Liu, Bin; El-Naggar, Adel K; Donehower, Lawrence A; Martinez, Luis Alfonso; Lozano, Guillermina

    2014-07-29

    p53(R172H/+) mice inherit a p53 mutation found in Li-Fraumeni syndrome and develop metastatic tumors at much higher frequency than p53(+/-) mice. To explore the mutant p53 metastatic phenotype, we used expression arrays to compare primary osteosarcomas from p53(R172H/+) mice with metastasis to osteosarcomas from p53(+/-) mice lacking metastasis. For this study, 213 genes were differentially expressed with a P value <0.05. Of particular interest, Pla2g16, which encodes a phospholipase that catalyzes phosphatidic acid into lysophosphatidic acid and free fatty acid (both implicated in metastasis), was increased in p53(R172H/+) osteosarcomas. Functional analyses showed that Pla2g16 knockdown decreased migration and invasion in mutant p53-expressing cells, and vice versa: overexpression of Pla2g16 increased the invasion of p53-null cells. Furthermore, Pla2g16 levels were increased upon expression of mutant p53 in both mouse and human osteosarcoma cell lines, indicating that Pla2g16 is a downstream target of the mutant p53 protein. ChIP analysis revealed that several mutant p53 proteins bind the Pla2g16 promoter at E26 transformation-specific (ETS) binding motifs and knockdown of ETS2 suppressed mutant p53 induction of Pla2g16. Thus, our study identifies a phospholipase as a transcriptional target of mutant p53 that is required for metastasis. PMID:25024203

  3. Uncoupling of longevity and paraquat resistance in mutants of the nematode Caenorhabditis elegans.

    PubMed

    Fujii, Michihiko; Tanaka, Nanae; Miki, Kensuke; Hossain, Mohammad Nazir; Endoh, Morio; Ayusawa, Dai

    2005-10-01

    To analyze the relationship between resistance to oxidative stress and longevity, we isolated three novel paraquat-resistant mutants, mev-5, mev-6, and mev-7, from the nematode Caenorhabditis elegans. They all showed the Dyf (defective in dye filling) phenotype, but not always resistance to heat or UV. Life-span extension was observed only in the mev-5 mutant at 26 degrees C. These results indicate that longevity is uncoupled with the phenotype of paraquat resistance. PMID:16244463

  4. Comparative macromolecular composition of filaments and rods of a Bacillus megaterium thermoconditional morphological mutant.

    PubMed

    Yan, L P; Hitchins, A D

    1980-10-01

    Filaments of a thermosensitive Bacillus megaterium mutant showed an altered macromolecular composition compared with salt-cured mutant cells and parental cells. Filaments contained more peptidoglycan, polyglucose, poly-beta-hydroxy-butyrate, and deoxyribonucleic acid per unit of protein. The ribonucleic acid-to-protein ratio of filaments was similar to that of rods or salt-cured cells. Filament formation seemed to be due to defective protein or ribonucleic acid metabolism. PMID:6158510

  5. Structure of mutant human oncogene protein determined

    SciTech Connect

    Baum, R.

    1989-01-16

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

  6. Amphid defective mutant of Caenorhabditis elegans.

    PubMed

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

    1994-01-01

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

  7. High Persister Mutants in Mycobacterium tuberculosis.

    PubMed

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

    2016-01-01

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

  8. High Persister Mutants in Mycobacterium tuberculosis

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2011-02-01

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

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

    PubMed Central

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

    2011-01-01

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

  11. Satellite Movie Shows Erika Dissipate

    NASA Video Gallery

    This animation of visible and infrared imagery from NOAA's GOES-West satellite from Aug. 27 to 29 shows Tropical Storm Erika move through the Eastern Caribbean Sea and dissipate near eastern Cuba. ...

  12. Caenorhabditis elegans Mutants Resistant to Attachment of Yersinia Biofilms

    PubMed Central

    Darby, Creg; Chakraborti, Amrita; Politz, Samuel M.; Daniels, Calvin C.; Tan, Li; Drace, Kevin

    2007-01-01

    The detailed composition and structure of the Caenorhabditis elegans surface are unknown. Previous genetic studies used antibody or lectin binding to identify srf genes that play roles in surface determination. Infection by Microbacterium nematophilum identified bus (bacterially unswollen) genes that also affect surface characteristics. We report that biofilms produced by Yersinia pestis and Y. pseudotuberculosis, which bind the C. elegans surface predominantly on the head, can be used to identify additional surface-determining genes. A screen for C. elegans mutants with a biofilm absent on the head (Bah) phenotype identified three novel genes: bah-1, bah-2, and bah-3. The bah-1 and bah-2 mutants have slightly fragile cuticles but are neither Srf nor Bus, suggesting that they are specific for surface components involved in biofilm attachment. A bah-3 mutant has normal cuticle integrity, but shows a stage-specific Srf phenotype. The screen produced alleles of five known surface genes: srf-2, srf-3, bus-4, bus-12, and bus-17. For the X-linked bus-17, a paternal effect was observed in biofilm assays. PMID:17339204

  13. Functional Analysis of Jasmonates in Rice through Mutant Approaches

    PubMed Central

    Dhakarey, Rohit; Kodackattumannil Peethambaran, Preshobha; Riemann, Michael

    2016-01-01

    Jasmonic acid, one of the major plant hormones, is, unlike other hormones, a lipid-derived compound that is synthesized from the fatty acid linolenic acid. It has been studied intensively in many plant species including Arabidopsis thaliana, in which most of the enzymes participating in its biosynthesis were characterized. In the past 15 years, mutants and transgenic plants affected in the jasmonate pathway became available in rice and facilitate studies on the functions of this hormone in an important crop. Those functions are partially conserved compared to other plant species, and include roles in fertility, response to mechanical wounding and defense against herbivores. However, new and surprising functions have also been uncovered by mutant approaches, such as a close link between light perception and the jasmonate pathway. This was not only useful to show a phenomenon that is unique to rice but also helped to establish this role in plant species where such links are less obvious. This review aims to provide an overview of currently available rice mutants and transgenic plants in the jasmonate pathway and highlights some selected roles of jasmonate in this species, such as photomorphogenesis, and abiotic and biotic stress. PMID:27135235

  14. Functional verification of a porcine myostatin propeptide mutant.

    PubMed

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

    2015-10-01

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

  15. Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase

    PubMed Central

    Bendikov-Bar, Inna; Maor, Gali; Filocamo, Mirella; Horowitz, Mia

    2013-01-01

    Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase β-glucocerebrosidase (GCase). The disease has a broad spectrum of phenotypes, which were divided into three different Types; Type 1 GD is not associated with primary neurological disease while Types 2 and 3 are associated with central nervous system disease. GCase molecules are synthesized on endoplasmic reticulum (ER)-bound polyribosomes, translocated into the ER and following modifications and correct folding, shuttle to the lysosomes. Mutant GCase molecules, which fail to fold correctly, undergo ER associated degradation (ERAD) in the proteasomes, the degree of which is one of the factors that determine GD severity. Several pharmacological chaperones have already been shown to assist correct folding of mutant GCase molecules in the ER, thus facilitating their trafficking to the lysosomes. Ambroxol, a known expectorant, is one such chaperone. Here we show that ambroxol increases both the lysosomal fraction and the enzymatic activity of several mutant GCase variants in skin fibroblasts derived from Type 1 and Type 2 GD patients. PMID:23158495

  16. Distinct Phyllosphere Bacterial Communities on Arabidopsis Wax Mutant Leaves

    PubMed Central

    Reisberg, Eva E.; Hildebrandt, Ulrich; Riederer, Markus; Hentschel, Ute

    2013-01-01

    The phyllosphere of plants is inhabited by diverse microorganisms, however, the factors shaping their community composition are not fully elucidated. The plant cuticle represents the initial contact surface between microorganisms and the plant. We thus aimed to investigate whether mutations in the cuticular wax biosynthesis would affect the diversity of the phyllosphere microbiota. A set of four Arabidopsis thaliana eceriferum mutants (cer1, cer6, cer9, cer16) and their respective wild type (Landsberg erecta) were subjected to an outdoor growth period and analysed towards this purpose. The chemical distinctness of the mutant wax phenotypes was confirmed by gas chromatographic measurements. Next generation amplicon pyrosequencing of the bacterial communities showed distinct community patterns. This observation was supported by denaturing gradient gel electrophoresis experiments. Microbial community analyses revealed bacterial phylotypes that were ubiquitously present on all plant lines (termed “core” community) while others were positively or negatively affected by the wax mutant phenotype (termed “plant line-specific“ community). We conclude from this study that plant cuticular wax composition can affect the community composition of phyllosphere bacteria. PMID:24223831

  17. Functional Analysis of Jasmonates in Rice through Mutant Approaches.

    PubMed

    Dhakarey, Rohit; Kodackattumannil Peethambaran, Preshobha; Riemann, Michael

    2016-01-01

    Jasmonic acid, one of the major plant hormones, is, unlike other hormones, a lipid-derived compound that is synthesized from the fatty acid linolenic acid. It has been studied intensively in many plant species including Arabidopsis thaliana, in which most of the enzymes participating in its biosynthesis were characterized. In the past 15 years, mutants and transgenic plants affected in the jasmonate pathway became available in rice and facilitate studies on the functions of this hormone in an important crop. Those functions are partially conserved compared to other plant species, and include roles in fertility, response to mechanical wounding and defense against herbivores. However, new and surprising functions have also been uncovered by mutant approaches, such as a close link between light perception and the jasmonate pathway. This was not only useful to show a phenomenon that is unique to rice but also helped to establish this role in plant species where such links are less obvious. This review aims to provide an overview of currently available rice mutants and transgenic plants in the jasmonate pathway and highlights some selected roles of jasmonate in this species, such as photomorphogenesis, and abiotic and biotic stress. PMID:27135235

  18. A mutant of barley lacking NADH-hydroxypyruvate reductase

    SciTech Connect

    Blackwell, R.; Lea, P. )

    1989-04-01

    A mutant of barley, LaPr 88/29, deficient in peroxisomal NADH-hydroxypyruvate reductase (HPR) activity has been identified. Compared to the wild type the activities of NADH-HPR and NADPH-HPR were severely reduced but the mutant was still capable of fixing CO{sub 2} at rates equivalent to 75% of that of the wild type in air. Although lacking an enzyme in the main photorespiratory pathway, there appeared to be little disruption to photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{sup 14}C) serine were similar in both mutant and wild type. LaPr 88/29 has been used to show that NADH-glyoxylate reductase (GR) and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-HPR activity is due to the NADH-HPR enzyme. Immunological studies, using antibodies raised against spinach HPR, have shown that the NADH-dependent enzyme protein is absent in LaPr 88/29 but there appears to be enhanced synthesis of the NADPH-dependent enzyme protein.

  19. Crystal Structure of a Thermally Stable Rhodopsin Mutant

    PubMed Central

    Standfuss, Jörg; Xie, Guifu; Edwards, Patricia C.; Burghammer, Manfred; Oprian, Daniel D.; Schertler, Gebhard F. X.

    2007-01-01

    We determined the structure of the rhodopsin mutant N2C/D282C expressed in mammalian cells; the first structure of a recombinantly produced G protein-coupled receptor (GPCR). The mutant was designed to form a disulfide bond between the N-terminus and loop E3 which allows handling of opsin in detergent solution and increases thermal stability of rhodopsin by 10°C. It furthermore allowed us to crystallize a fully deglycosylated rhodopsin (N2C/N15D/D282C). N15 mutations are normally misfolding and cause retinitis pigmentosa in humans. Microcrystallographic techniques and a 5μm x-ray beam were used to collect data along a single needle measuring 5x5x90μm3. The disulfide introduces only minor changes but fixes the N-terminal cap over the β-sheet lid covering the ligand binding site, a likely explanation for the increased stability. This work allows structural investigation of rhodopsin mutants and shows the problems encountered during structure determination of GPCRs and other mammalian membrane proteins. PMID:17825322

  20. Paracoccus denitrificans cytochrome c1 gene replacement mutants.

    PubMed Central

    Gerhus, E; Steinrücke, P; Ludwig, B

    1990-01-01

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

  1. Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase.

    PubMed

    Bendikov-Bar, Inna; Maor, Gali; Filocamo, Mirella; Horowitz, Mia

    2013-02-01

    Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase β-glucocerebrosidase (GCase). The disease has a broad spectrum of phenotypes, which were divided into three different Types; Type 1 GD is not associated with primary neurological disease while Types 2 and 3 are associated with central nervous system disease. GCase molecules are synthesized on endoplasmic reticulum (ER)-bound polyribosomes, translocated into the ER and following modifications and correct folding, shuttle to the lysosomes. Mutant GCase molecules, which fail to fold correctly, undergo ER associated degradation (ERAD) in the proteasomes, the degree of which is one of the factors that determine GD severity. Several pharmacological chaperones have already been shown to assist correct folding of mutant GCase molecules in the ER, thus facilitating their trafficking to the lysosomes. Ambroxol, a known expectorant, is one such chaperone. Here we show that ambroxol increases both the lysosomal fraction and the enzymatic activity of several mutant GCase variants in skin fibroblasts derived from Type 1 and Type 2 GD patients. PMID:23158495

  2. Neurospora crassa mutants deficient in asparagine synthetase.

    PubMed Central

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

    1983-01-01

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

  3. Evolutionary Mutant Models for Human Disease

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2015-06-01

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

  5. Comparative Analysis of Mutant Tyrosine Kinase Chemical Rescue†

    PubMed Central

    Muratore, Kathryn E.; Seeliger, Markus A.; Wang, Zhihong; Fomina, Dina; Neiswinger, Johnathan; Havranek, James J.; Baker, David; Kuriyan, John; Cole, Philip A.

    2009-01-01

    Protein tyrosine kinases are critical cell signaling enzymes. These enzymes have a highly conserved Arg residue in their catalytic loop which is present two residues or four residues downstream from an absolutely conserved Asp catalytic base. Prior studies on protein tyrosine kinases Csk and Src revealed the potential for chemical rescue of catalytically-deficient mutant kinases (Arg to Ala mutations) by small diamino compounds, particularly imidazole, however the potency and efficiency of rescue was greater for Src. This current study further examines the structural and kinetic basis of rescue for mutant Src as compared to mutant Abl tyrosine kinase. An X-ray crystal structure of R388A Src revealed the surprising finding that a histidine residue of the N-terminus of a symmetry-related kinase inserts into the active site of the adjacent Src and mimics the hydrogen bonding pattern seen in wild-type protein tyrosine kinases. Abl R367A shows potent and efficient rescue more comparable to Src, even though its catalytic loop is more like that of Csk. Various enzyme redesigns of the active sites indicate that the degree and specificity of rescue is somewhat flexible, but the overall properties of the enzymes and rescue agents play an overarching role. The newly discovered rescue agent 2-aminoimidazole is about as efficient as imidazole in rescuing R/A Src and Abl. Rate vs. pH studies with these imidazole analogs suggest that the protonated imidazolium is the preferred form for chemical rescue, consistent with structural models. The efficient rescue seen with mutant Abl points to the potential of this approach to be used effectively to analyze Abl phosphorylation pathways in cells. PMID:19260709

  6. Prion propagation in cells expressing PrP glycosylation mutants.

    PubMed

    Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

    2011-04-01

    Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection. PMID:21248032

  7. Characterization of a Lignified Secondary Phloem Fibre‐deficient Mutant of Jute (Corchorus capsularis)

    PubMed Central

    SENGUPTA, GARGI; PALIT, P.

    2004-01-01

    • Background and Aims High lignin content of lignocellulose jute fibre does not favour its utilization in making finer fabrics and other value‐added products. To aid the development of low‐lignin jute fibre, this study aimed to identify a phloem fibre mutant with reduced lignin. • Methods An x‐ray‐induced mutant line (CMU) of jute (Corchorus capsularis) was morphologically evaluated and the accession (CMU 013) with the most undulated phenotype was compared with its normal parent (JRC 212) for its growth, secondary fibre development and lignification of the fibre cell wall. • Key Results The normal and mutant plants showed similar leaf photosynthetic rates. The mutant grew more slowly, had shorter internodes and yielded much less fibre after retting. The fibre of the mutant contained 50 % less lignin but comparatively more cellulose than that of the normal type. Differentiation of primary and secondary vascular tissues throughout the CMU 013 stem was regular but it did not have secondary phloem fibre bundles as in JRC 212. Instead, a few thin‐walled, less lignified fibre cells formed uni‐ or biseriate radial rows within the phloem wedges of the middle stem. The lower and earliest developed part of the mutant stem had no lignified fibre cells. This developmental deficiency in lignification of fibre cells was correlated to a similar deficiency in phenylalanine ammonia lyase activity, but not peroxidase activity, in the bark tissue along the stem axis. In spite of severe reduction in lignin synthesis in the phloem cells this mutant functioned normally and bred true. • Conclusions In view of the observations made, the mutant is designated as deficient lignified phloem fibre (dlpf). This mutant may be utilized to engineer low‐lignin jute fibre strains and may also serve as a model to study the positional information that coordinates secondary wall thickening of fibre cells. PMID:14707004

  8. National Orange Show Photovoltaic Demonstration

    SciTech Connect

    Dan Jimenez Sheri Raborn, CPA; Tom Baker

    2008-03-31

    National Orange Show Photovoltaic Demonstration created a 400KW Photovoltaic self-generation plant at the National Orange Show Events Center (NOS). The NOS owns a 120-acre state fairground where it operates an events center and produces an annual citrus fair known as the Orange Show. The NOS governing board wanted to employ cost-saving programs for annual energy expenses. It is hoped the Photovoltaic program will result in overall savings for the NOS, help reduce the State's energy demands as relating to electrical power consumption, improve quality of life within the affected grid area as well as increase the energy efficiency of buildings at our venue. In addition, the potential to reduce operational expenses would have a tremendous effect on the ability of the NOS to service its community.

  9. Creating Slide Show Book Reports.

    ERIC Educational Resources Information Center

    Taylor, Harriet G.; Stuhlmann, Janice M.

    1995-01-01

    Describes the use of "Kid Pix 2" software by fourth grade students to develop slide-show book reports. Highlights include collaboration with education majors from Louisiana State University, changes in attitudes of the education major students and elementary students, and problems with navigation and disk space. (LRW)

  10. Producing Talent and Variety Shows.

    ERIC Educational Resources Information Center

    Szabo, Chuck

    1995-01-01

    Identifies key aspects of producing talent shows and outlines helpful hints for avoiding pitfalls and ensuring a smooth production. Presents suggestions concerning publicity, scheduling, and support personnel. Describes types of acts along with special needs and problems specific to each act. Includes a list of resources. (MJP)

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-04-01

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

  13. A Temporarily Red Light-Insensitive Mutant of Tomato Lacks a Light-Stable, B-Like Phytochrome.

    PubMed Central

    Van Tuinen, A.; Kerckhoffs, LHJ.; Nagatani, A.; Kendrick, R. E.; Koornneef, M.

    1995-01-01

    We have selected four recessive mutants in tomato (Lycopersicon esculentum Mill.) that, under continuous red light (R), have long hypocotyls and small cotyledons compared to wild type (WT), a phenotype typical of phytochrome B (phyB) mutants of other species. These mutants, which are allelic, are only insensitive to R during the first 2 days upon transition from darkness to R, and therefore we propose the gene symbol tri (temporarily red light insensitive). White light-grown mutant plants have a more elongated growth habit than that of the WT. An immunochemically and spectrophotometrically detectable phyB-like polypeptide detectable in the WT is absent or below detection limits in the tri1 mutant. In contrast to the absence of an elongation growth response to far-red light (FR) given at the end of the daily photoperiod (EODFR) in all phyB-deficient mutants so far characterized, the tri1 mutant responds to EODFR treatment. The tri1 mutant also shows a strong response to supplementary daytime far-red light. We propose that the phyB-like phytochrome deficient in the tri mutants plays a major role during de-etiolation and that other light-stable phytochromes can regulate the EODFR and shade-avoidance responses in tomato. PMID:12228517

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

    PubMed Central

    Almond, B D; Dean, D H

    1993-01-01

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

  15. Mutant p53 cooperates with ETS and selectively up-regulates human MDR1 not MRP1.

    PubMed

    Sampath, J; Sun, D; Kidd, V J; Grenet, J; Gandhi, A; Shapiro, L H; Wang, Q; Zambetti, G P; Schuetz, J D

    2001-10-19

    The most frequently expressed drug resistance genes, MDR1 and MRP1, occur in human tumors with mutant p53. However, it was unknown if mutant p53 transcriptionally regulated both MDR1 and MRP1. We demonstrated that mutant p53 did not activate either the MRP1 promoter or the endogenous gene. In contrast, mutant p53 strongly up-regulated the MDR1 promoter and expression of the endogenous MDR1 gene. Notably, cells that expressed either a transcriptionally inactive mutant p53 or the empty vector showed no endogenous MDR1 up-regulation. Transcriptional activation of the MDR1 promoter by mutant p53 required an Ets binding site, and mutant p53 and Ets-1 synergistically activated MDR1 transcription. Biochemical analysis revealed that Ets-1 interacted exclusively with mutant p53s in vivo but not with wild-type p53. These findings are the first to demonstrate the induction of endogenous MDR1 by mutant p53 and provide insight into the mechanism. PMID:11483599

  16. [Mutations of Cx26 gene in patients with NSHL and intracellular distribution of two mutants].

    PubMed

    Li, Jing-Zhi; Hu, Yi-Qiao; Wang, Shu-Hui; Cheng, Hong-Sheng; Pan, Qian; Xia, Kun; Hu, Zheng-Mao; Feng, Yong

    2009-07-01

    To analyze the frequencies and characteristics of Cx26 gene mutations in Chinese patients with nonsyndromic hearing loss (NSHL) and investigate the intracellular localization of two mutants, 139 unrelated familial cases with non-syndromic hearing loss were screened for mutation in Cx26 gene by direct sequencing. Two mutants, p.F115C and p.V37I, were structured into pEGFP vectors and transfected into Hela cells to detect their expression and fluorescent localization in cells. Cx26 variations were detected in 31 patients, with a detection rate of 22.3%. The 10 variations included 6 types of mutations and 4 types of polymorphisms. A novel variation p.F115C was found. The fluorescent localization assay of the two mutants p.F115C and p.V37I showed no difference from the wild-type, indicating that both mutants did not impair the formation of the gap junctions. PMID:19586875

  17. The phenotype of Arabidopsis thaliana det1 mutants suggest a role for cytokinins in greening

    SciTech Connect

    Chory, J.; Aguilar, N.; Peto, C.A.

    1990-01-01

    When grown in the absence of light, the det1 mutants of Arabidopsis thaliana develop characteristics of light-grown plants by morphological, cellular, and molecular criteria. Further, in light-grown plants, mutations in the DET1 gene affect cell-type-specific expression of light-regulated genes and the chloroplast developmental program. Here we show that the addition of exogenously added cytokinins (either 2-isopentenyl adenine, kinetin, or benzyladenine) to the growth medium of dark-germinated wild-type seedlings results in seedlings that resemble det1 mutants, instead of having the normal etiolated morphology. Like det1 mutants, these dark-grown seedlings now contain chloroplasts and have high levels of expression of genes that are normally light''-regulated. These results suggest an important role for cytokinins during greening of Arabidopsis, and may implicate cytokinin levels or an increased sensitivity to cytokinins as explanations for some of the observed phenotypes of det1 mutants.

  18. Accumulation of infectious mutants in stocks during the propagation of fiber-modified recombinant adenoviruses

    SciTech Connect

    Ugai, Hideyo; Inabe, Kumiko; Yamasaki, Takahito; Murata, Takehide; Obata, Yuichi; Hamada, Hirofumi; Yokoyama, Kazunari K. . E-mail: kazu@brc.riken.jp

    2005-11-25

    In infected cells, replication errors during viral proliferation generate mutations in adenoviruses (Ads), and the mutant Ads proliferate and evolve in the intracellular environment. Genetically fiber-modified recombinant Ads (rAd variants) were generated, by modification of the fiber gene, for therapeutic applications in host cells that lack or express reduced levels of the Coxsackievirus and adenovirus receptor. To assess the genetic modifications of rAd variants that might induce the instability of Ad virions, we examined the frequencies of mutants that accumulated in propagated stocks. Seven of 41 lines of Ad variants generated mutants in the stocks and all mutants were infectious. Moreover, all the mutations occurred in the modified region that had been added at the 3' end of the fiber gene. Our results show that some genetic modifications at the carboxyl terminus of Ad fiber protein lead to the instability of Ad virions.

  19. Ethanol production using engineered mutant E. coli

    DOEpatents

    Ingram, Lonnie O.; Clark, David P.

    1991-01-01

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

  20. Phenotypic mutant library: potential for gene discovery

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  1. Rapid Antibiotic Resistance Evolution of GASP Mutants

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  2. Yeast mutants overproducing iso-cytochromes c

    SciTech Connect

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

    1980-01-01

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

  3. Genotyping-by-sequencing of glossy mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. Quantitative genetics and utilization of mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  5. Bacteriophages show promise as antimicrobial agents.

    PubMed

    Alisky, J; Iczkowski, K; Rapoport, A; Troitsky, N

    1998-01-01

    The emergence of antibiotic-resistant bacteria has prompted interest in alternatives to conventional drugs. One possible option is to use bacteriophages (phage) as antimicrobial agents. We have conducted a literature review of all Medline citations from 1966-1996 that dealt with the therapeutic use of phage. There were 27 papers from Poland, the Soviet Union, Britain and the U.S.A. The Polish and Soviets administered phage orally, topically or systemically to treat a wide variety of antibiotic-resistant pathogens in both adults and children. Infections included suppurative wound infections, gastroenteritis, sepsis, osteomyelitis, dermatitis, empyemas and pneumonia; pathogens included Staphylococcus, Streptococcus, Klebsiella, Escherichia, Proteus, Pseudomonas, Shigella and Salmonella spp. Overall, the Polish and Soviets reported success rates of 80-95% for phage therapy, with rare, reversible gastrointestinal or allergic side effects. However, efficacy of phage was determined almost exclusively by qualitative clinical assessment of patients, and details of dosages and clinical criteria were very sketchy. There were also six British reports describing controlled trials of phage in animal models (mice, guinea pigs and livestock), measuring survival rates and other objective criteria. All of the British studies raised phage against specific pathogens then used to create experimental infections. Demonstrable efficacy against Escherichia, Acinetobacter, Pseudomonas and Staphylococcus spp. was noted in these model systems. Two U.S. papers dealt with improving the bioavailability of phage. Phage is sequestered in the spleen and removed from circulation. This can be overcome by serial passage of phage through mice to isolate mutants that resist sequestration. In conclusion, bacteriophages may show promise for treating antibiotic resistant pathogens. To facilitate further progress, directions for future research are discussed and a directory of authors from the reviewed

  6. Magic Carpet Shows Its Colors

    NASA Technical Reports Server (NTRS)

    2004-01-01

    The upper left image in this display is from the panoramic camera on the Mars Exploration Rover Spirit, showing the 'Magic Carpet' region near the rover at Gusev Crater, Mars, on Sol 7, the seventh martian day of its journey (Jan. 10, 2004). The lower image, also from the panoramic camera, is a monochrome (single filter) image of a rock in the 'Magic Carpet' area. Note that colored portions of the rock correlate with extracted spectra shown in the plot to the side. Four different types of materials are shown: the rock itself, the soil in front of the rock, some brighter soil on top of the rock, and some dust that has collected in small recesses on the rock face ('spots'). Each color on the spectra matches a line on the graph, showing how the panoramic camera's different colored filters are used to broadly assess the varying mineral compositions of martian rocks and soils.

  7. Isolation of transposon mutants and characterization of genes involved in biofilm formation by Pseudomonas fluorescens TC222.

    PubMed

    Nian, Hongjuan; Zhang, Jie; Song, Fuping; Fan, Liqiang; Huang, Dafang

    2007-09-01

    Biofilm formation mutants are often found to have defective or altered motility. The motility phenotype was exploited to identify Pseudomonas fluorescens biofilm formation mutants. Fourteen motility mutants were obtained from P. fluorescens isolate TC222 and eight stable mutants were studied further. The eight transposon insertion mutants showed altered ability to form biofilm compared with the parent. Five Tn5-inserted genes from these mutants were cloned and sequenced. Genetic analysis showed that two insertions were located in genes affecting multiple cell surface characteristics, including lipopolysaccharide (rfbD) and polar flagella (fliR). Three genes encoding for a putative Mig-14 family protein (epsB), a probable bacteriophage signal peptide protein (bspA) and a soluble pyridine nucleotide transhydrogenase (pyrA) were reported for the first time to be involved in biofilm formation. Complementation experiments of rfbD and epsB genes proved that biofilm formation of the corresponding mutants could be restored. Further semi-quantitative reverse transcription-PCR analysis showed that both rfbD and epsB can express their transcripts much higher in the complemented strains than that in wild-type strains. The transcripts of both genes in their mutants could not be detected. PMID:17453174

  8. Isolation of mutants of Aspergillus awamori with enhanced production of extracellular xylanase and β-xylosidase.

    PubMed

    Smith, D C; Wood, T M

    1991-05-01

    Plate screening tests were designed for the selection and isolation of mutant strains of the fungus Aspergillus awamori CMI 142717 showing over-production and constitutive synthesis of xylanase and β-xylosidase. Following mutation by N-methyl-N-nitro-N-nitrosoguanidine, nitrous acid and UV (254 nm), two generations of mutants were isolated and cultured in shake fiasks containing glucose, ball-milled oat straw or oat speit xylan as carbon source. Growth of a number of selected mutants in shake flask culture on medium containing oat spelt xylan produced the highest titres of xylanase and β-xylosidase. Thus, xylanase producton by mutant AANTG43 was 132 U/ml when the Somogyl-Nelson (alkaline copper) method of measuring reducing sugar released was used, or 1160 U/ml using the dinitrosalicylic acid method of reducing sugar analysis. These values were 8-fold higher than those produced by the wild type. A 20-fold improvement in β-xylosidase production was produced by mutant AANO19 (3.51 U/ml). The titres for these two enzyme activities are the highest recorded so far in the literature. Mutant AANTG43 also produced high levels of xylanase (49.8 U/ml) in submerged culture in a fermenter and showed a substantial improvement in the overall productivity of enzyme compared to the wild type strain. PMID:24425022

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

    PubMed Central

    Frustaci, J M; O'Brian, M R

    1992-01-01

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

  10. Comparative proteomic study of Arabidopsis mutants mpk4 and mpk6.

    PubMed

    Takáč, Tomáš; Vadovič, Pavol; Pechan, Tibor; Luptovčiak, Ivan; Šamajová, Olga; Šamaj, Jozef

    2016-01-01

    Arabidopsis MPK4 and MPK6 are implicated in different signalling pathways responding to diverse external stimuli. This was recently correlated with transcriptomic profiles of Arabidopsis mpk4 and mpk6 mutants, and thus it should be reflected also on the level of constitutive proteomes. Therefore, we performed a shot gun comparative proteomic analysis of Arabidopsis mpk4 and mpk6 mutant roots. We have used bioinformatic tools and propose several new proteins as putative MPK4 and MPK6 phosphorylation targets. Among these proteins in the mpk6 mutant were important modulators of development such as CDC48A and phospholipase D alpha 1. In the case of the mpk4 mutant transcriptional reprogramming might be mediated by phosphorylation and change in the abundance of mRNA decapping complex VCS. Further comparison of mpk4 and mpk6 root differential proteomes showed differences in the composition and regulation of defense related proteins. The mpk4 mutant showed altered abundances of antioxidant proteins. The examination of catalase activity in response to oxidative stress revealed that this enzyme might be preferentially regulated by MPK4. Finally, we proposed developmentally important proteins as either directly or indirectly regulated by MPK4 and MPK6. These proteins contribute to known phenotypic defects in the mpk4 and mpk6 mutants. PMID:27324189

  11. Comparative proteomic study of Arabidopsis mutants mpk4 and mpk6

    PubMed Central

    Takáč, Tomáš; Vadovič, Pavol; Pechan, Tibor; Luptovčiak, Ivan; Šamajová, Olga; Šamaj, Jozef

    2016-01-01

    Arabidopsis MPK4 and MPK6 are implicated in different signalling pathways responding to diverse external stimuli. This was recently correlated with transcriptomic profiles of Arabidopsis mpk4 and mpk6 mutants, and thus it should be reflected also on the level of constitutive proteomes. Therefore, we performed a shot gun comparative proteomic analysis of Arabidopsis mpk4 and mpk6 mutant roots. We have used bioinformatic tools and propose several new proteins as putative MPK4 and MPK6 phosphorylation targets. Among these proteins in the mpk6 mutant were important modulators of development such as CDC48A and phospholipase D alpha 1. In the case of the mpk4 mutant transcriptional reprogramming might be mediated by phosphorylation and change in the abundance of mRNA decapping complex VCS. Further comparison of mpk4 and mpk6 root differential proteomes showed differences in the composition and regulation of defense related proteins. The mpk4 mutant showed altered abundances of antioxidant proteins. The examination of catalase activity in response to oxidative stress revealed that this enzyme might be preferentially regulated by MPK4. Finally, we proposed developmentally important proteins as either directly or indirectly regulated by MPK4 and MPK6. These proteins contribute to known phenotypic defects in the mpk4 and mpk6 mutants. PMID:27324189

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

    PubMed

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

    2012-11-30

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

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

    PubMed

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

    2016-06-01

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

  14. Butterfly wing pattern mutants: developmental heterochrony and co-ordinately regulated phenotypes.

    PubMed

    Koch, P B; Lorenz, U; Brakefield, P M; ffrench-Constant, R H

    2000-11-01

    Butterfly wings are colored late in development, when pigments are synthesized in specialized wing scale cells in a fixed developmental succession. In this succession, colored pigments are deposited first and the remaining areas are later melanized black or brown. Here we studied the developmental changes underlying two wing pattern mutants, firstly melanic mutants of the swallowtail Papilio glaucus, in which the yellow background is turned black, and secondly a Spotty mutant of the satyrid Bicyclus anynana, which carries two additional eyespots. Despite the very different pattern changes in these two mutants, they are both associated with changes in rates of scale development and correspondingly, the final color pattern. In the melanic swallowtail, background scales originally destined to become yellow (normally developing early and synthesizing papiliochrome) show delayed development, fail to make papiliochrome, and subsequently melanize at the same time as scales in the wild-type black pattern. In the B. anynana eyespot, scale maturation begins with the central white focus, then progresses to the surrounding gold ring and later finishes with melanization of the black center. Mutants showing additional eyespots display accelerated rates of scale development (corresponding to new eyespots) in wing cells not normally occupied by eyespots. Thus by either delaying or accelerating rates of scale development, the final color, or position, of a wing pattern element can be changed. We propose that this heterochrony of scale development is a basic mechanism of color pattern formation on which developmental mutants act to change lepidopteran color patterns. PMID:11180804

  15. Isolation of a novel mutant gene for soil-surface rooting in rice (Oryza sativa L.)

    PubMed Central

    2013-01-01

    Background Root system architecture is an important trait affecting the uptake of nutrients and water by crops. Shallower root systems preferentially take up nutrients from the topsoil and help avoid unfavorable environments in deeper soil layers. We have found a soil-surface rooting mutant from an M2 population that was regenerated from seed calli of a japonica rice cultivar, Nipponbare. In this study, we examined the genetic and physiological characteristics of this mutant. Results The primary roots of the mutant showed no gravitropic response from the seedling stage on, whereas the gravitropic response of the shoots was normal. Segregation analyses by using an F2 population derived from a cross between the soil-surface rooting mutant and wild-type Nipponbare indicated that the trait was controlled by a single recessive gene, designated as sor1. Fine mapping by using an F2 population derived from a cross between the mutant and an indica rice cultivar, Kasalath, revealed that sor1 was located within a 136-kb region between the simple sequence repeat markers RM16254 and 2935-6 on the terminal region of the short arm of chromosome 4, where 13 putative open reading frames (ORFs) were found. We sequenced these ORFs and detected a 33-bp deletion in one of them, Os04g0101800. Transgenic plants of the mutant transformed with the genomic fragment carrying the Os04g0101800 sequence from Nipponbare showed normal gravitropic responses and no soil-surface rooting. Conclusion These results suggest that sor1, a rice mutant causing soil-surface rooting and altered root gravitropic response, is allelic to Os04g0101800, and that a 33-bp deletion in the coding region of this gene causes the mutant phenotypes. PMID:24280269

  16. A proteome map of a quadruple photoreceptor mutant sustains its severe photosynthetic deficient phenotype.

    PubMed

    Fox, Ana Romina; Barberini, Maria Laura; Ploschuk, Edmundo Leonardo; Muschietti, Jorge Prometeo; Mazzella, Maria Agustina

    2015-08-01

    Light is the environmental factor that most affects plant growth and development through its impact on photomorphogenesis and photosynthesis. A quadruple photoreceptor mutant lacking four of the most important photoreceptors in plants, phytochromes A and B (phyA, phyB) and cryptochromes 1 and 2 (cry1, cry2), is severely affected in terms of growth and development. Previous studies have suggested that in addition to a photomorphogenic disorder, the phyA phyB cry1 cry2 quadruple mutant might have severe alterations in photosynthetic ability. Here, we investigated the photosynthetic processes altered in the quadruple mutant and performed a proteomic profiling approach to identify some of the proteins involved. The phyA phyB cry1 cry2 quadruple mutant showed reduced leaf area and total chlorophyll content. Photosynthetic rates at high irradiances were reduced approximately 65% compared to the wild type (WT). Light-saturated photosynthesis and the response of net CO2 exchange to low and high internal CO2 concentrations suggest that the levels or activity of the components of the Calvin cycle and electron transport might be reduced in the quadruple mutant. Most of the under-expressed proteins in the phyA phyB cry1 cry2 quadruple mutant consistently showed a chloroplastic localization, whereas components of the Calvin cycle and light reaction centers were overrepresented. Additionally, Rubisco expression was reduced threefold in the phyA phyB cry1 cry2 quadruple mutant. Together, these results highlight the importance of the phytochrome and cryptochrome families in proper autotrophy establishment in plants. They also suggest that an overall limitation in the chlorophyll levels, expression of Rubisco, and enzymes of the Calvin Cycle and electron transport that affect ribulose-1,5-biphosphate (RuBP) regeneration reduced photosynthetic capacity in the phyA phyB cry1 cry2 quadruple mutant. PMID:26264966

  17. A Small Molecule Agonist THIQ as a Novel Pharmacoperone for Intracellularly Retained Melanocortin-4 Receptor Mutants

    PubMed Central

    Huang, Hui; Tao, Ya-Xiong

    2014-01-01

    Although mutations in the melanocortin-4 receptor (MC4R) gene cause severe early-onset obesity, we still do not have effective approaches to correct the defects of these mutations. Several antagonists have been identified as pharmacoperones of the MC4R whereas no agonist of the MC4R has been reported. In the present study, we investigated the effect of a small molecule agonist of the MC4R, THIQ, on the cell surface expression and signaling of ten intracellularly retained MC4R mutants using different cell lines. We showed that THIQ increased the cell surface expression of three mutants (N62S, C84R, and C271Y) and two of them (N62S and C84R) had increased signaling in HEK293 cells. Interestingly, THIQ increased the signaling of two other mutants (P78L and P260Q) without increasing their cell surface expression in HEK293 cells. In neuronal cells, THIQ exhibited a more potent effect, correcting the cell surface expression and signaling of seven mutants (N62S, I69R, P78L, C84R, W174C, P260Q, and C271Y). Other mutants were not rescued by THIQ. We also showed that THIQ did not rescue MC4R mutants defective in ligand binding or signaling or one intracellularly retained mutant of the melanocortin-3 receptor. In summary, we demonstrated that a small molecule agonist acted as a pharmacoperone of the MC4R rescuing the cell surface expression and signaling of some intracellularly retained MC4R mutants. PMID:25076858

  18. ENVITEC shows off air technologies

    SciTech Connect

    McIlvaine, R.W.

    1995-08-01

    The ENVITEC International Trade Fair for Environmental Protection and Waste Management Technologies, held in June in Duesseldorf, Germany, is the largest air pollution exhibition in the world and may be the largest environmental technology show overall. Visitors saw thousands of environmental solutions from 1,318 companies representing 29 countries and occupying roughly 43,000 square meters of exhibit space. Many innovations were displayed under the category, ``thermal treatment of air pollutants.`` New technologies include the following: regenerative thermal oxidizers; wet systems for removing pollutants; biological scrubbers;electrostatic precipitators; selective adsorption systems; activated-coke adsorbers; optimization of scrubber systems; and air pollution monitors.

  19. Selection and Characterization of Phage-Resistant Mutant Strains of Listeria monocytogenes Reveal Host Genes Linked to Phage Adsorption

    PubMed Central

    Denes, Thomas; den Bakker, Henk C.; Tokman, Jeffrey I.; Guldimann, Claudia

    2015-01-01

    Listeria-infecting phages are readily isolated from Listeria-containing environments, yet little is known about the selective forces they exert on their host. Here, we identified that two virulent phages, LP-048 and LP-125, adsorb to the surface of Listeria monocytogenes strain 10403S through different mechanisms. We isolated and sequenced, using whole-genome sequencing, 69 spontaneous mutant strains of 10403S that were resistant to either one or both phages. Mutations from 56 phage-resistant mutant strains with only a single mutation mapped to 10 genes representing five loci on the 10403S chromosome. An additional 12 mutant strains showed two mutations, and one mutant strain showed three mutations. Two of the loci, containing seven of the genes, accumulated the majority (n = 64) of the mutations. A representative mutant strain for each of the 10 genes was shown to resist phage infection through mechanisms of adsorption inhibition. Complementation of mutant strains with the associated wild-type allele was able to rescue phage susceptibility for 6 out of the 10 representative mutant strains. Wheat germ agglutinin, which specifically binds to N-acetylglucosamine, bound to 10403S and mutant strains resistant to LP-048 but did not bind to mutant strains resistant to only LP-125. We conclude that mutant strains resistant to only LP-125 lack terminal N-acetylglucosamine in their wall teichoic acid (WTA), whereas mutant strains resistant to both phages have disruptive mutations in their rhamnose biosynthesis operon but still possess N-acetylglucosamine in their WTA. PMID:25888172

  20. Selection and Characterization of Phage-Resistant Mutant Strains of Listeria monocytogenes Reveal Host Genes Linked to Phage Adsorption.

    PubMed

    Denes, Thomas; den Bakker, Henk C; Tokman, Jeffrey I; Guldimann, Claudia; Wiedmann, Martin

    2015-07-01

    Listeria-infecting phages are readily isolated from Listeria-containing environments, yet little is known about the selective forces they exert on their host. Here, we identified that two virulent phages, LP-048 and LP-125, adsorb to the surface of Listeria monocytogenes strain 10403S through different mechanisms. We isolated and sequenced, using whole-genome sequencing, 69 spontaneous mutant strains of 10403S that were resistant to either one or both phages. Mutations from 56 phage-resistant mutant strains with only a single mutation mapped to 10 genes representing five loci on the 10403S chromosome. An additional 12 mutant strains showed two mutations, and one mutant strain showed three mutations. Two of the loci, containing seven of the genes, accumulated the majority (n = 64) of the mutations. A representative mutant strain for each of the 10 genes was shown to resist phage infection through mechanisms of adsorption inhibition. Complementation of mutant strains with the associated wild-type allele was able to rescue phage susceptibility for 6 out of the 10 representative mutant strains. Wheat germ agglutinin, which specifically binds to N-acetylglucosamine, bound to 10403S and mutant strains resistant to LP-048 but did not bind to mutant strains resistant to only LP-125. We conclude that mutant strains resistant to only LP-125 lack terminal N-acetylglucosamine in their wall teichoic acid (WTA), whereas mutant strains resistant to both phages have disruptive mutations in their rhamnose biosynthesis operon but still possess N-acetylglucosamine in their WTA. PMID:25888172

  1. Cadmium trapping in an epithelial sodium channel pore mutant.

    PubMed

    Takeda, Armelle-Natsuo; Gautschi, Ivan; van Bemmelen, Miguel X; Schild, Laurent

    2007-11-01

    The putative selectivity filter of the epithelial sodium channel (ENaC) comprises a three-residue sequence G/SXS, but it remains uncertain whether the backbone atoms of this sequence or whether their side chains are lining the pore. It has been reported that the S589C mutation in the selectivity filter of alphaENaC renders the channel sensitive to block by externally applied Cd2+; this was interpreted as evidence for Cd2+ coordination with the thiol group of the side chain of alpha589C, pointing toward the pore lumen. Because the alphaS589C mutation alters the monovalent to divalent cation selectivity ratio of ENaC and because internally applied Cd2+ blocks wild-type ENaC with high affinity, we hypothesized that the inhibition of alphaS589C ENaC by Cd2+ results rather from the coordination of this cation with native cysteine residues located in the internal pore of ENaC. We show here that Cd2+ inhibits not only ENaC alphaS589C and alphaS589D but also alphaS589N mutants and that Ca2+ weakly interacts with the S589D mutant. The block of alphaS589C, -D, and -N mutants is characterized by a slow on-rate, is nearly irreversible, is voltage-dependent, and can be prevented by amiloride. The C546S mutation in the second transmembrane helix of gamma subunit in the background of the ENaC alphaS589C, -D, or -N mutants reduces the sensitivity to block by Cd2+ and renders the block rapidly reversible. We conclude therefore that the block by Cd2+ of the alphaS589C, -D, and -N mutants results from the trapping of Cd2+ ions in the internal pore of the channel and involves Cys-546 in the second transmembrane helix of the gammaENaC subunit. PMID:17804416

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-01-01

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

  4. ShowMe3D

    Energy Science and Technology Software Center (ESTSC)

    2012-01-05

    ShowMe3D is a data visualization graphical user interface specifically designed for use with hyperspectral image obtained from the Hyperspectral Confocal Microscope. The program allows the user to select and display any single image from a three dimensional hyperspectral image stack. By moving a slider control, the user can easily move between images of the stack. The user can zoom into any region of the image. The user can select any pixel or region from themore » displayed image and display the fluorescence spectrum associated with that pixel or region. The user can define up to 3 spectral filters to apply to the hyperspectral image and view the image as it would appear from a filter-based confocal microscope. The user can also obtain statistics such as intensity average and variance from selected regions.« less

  5. ShowMe3D

    SciTech Connect

    Sinclair, Michael B

    2012-01-05

    ShowMe3D is a data visualization graphical user interface specifically designed for use with hyperspectral image obtained from the Hyperspectral Confocal Microscope. The program allows the user to select and display any single image from a three dimensional hyperspectral image stack. By moving a slider control, the user can easily move between images of the stack. The user can zoom into any region of the image. The user can select any pixel or region from the displayed image and display the fluorescence spectrum associated with that pixel or region. The user can define up to 3 spectral filters to apply to the hyperspectral image and view the image as it would appear from a filter-based confocal microscope. The user can also obtain statistics such as intensity average and variance from selected regions.

  6. Pleiotropic phenotypes of the salt-tolerant and cytosine hypomethylated leafless inflorescence, evergreen dwarf and irregular leaf lamina mutants of Catharanthus roseus possessing Mendelian inheritance.

    PubMed

    Kumari, Renu; Sharma, Vishakha; Sharma, Vinay; Kumar, Sushil

    2013-12-01

    In Catharanthus roseus, three morphological cum salt-tolerant chemically induced mutants of Mendelian inheritance and their wild-type parent cv Nirmal were characterized for overall cytosine methylation at DNA repeats, expression of 119 protein coding and seven miRNA-coding genes and 50 quantitative traits. The mutants, named after their principal morphological feature(s), were leafless inflorescence (lli), evergreen dwarf (egd) and irregular leaf lamina (ill). The Southern-blot analysis of MspI digested DNAs of mutants probed with centromeric and 5S and 18S rDNA probes indicated that, in comparison to wild type, the mutants were extensively demethylated at cytosine sites. Among the 126 genes investigated for transcriptional expression, 85 were upregulated and 41 were downregulated in mutants. All of the five genes known to be stress responsive had increased expression in mutants. Several miRNA genes showed either increased or decreased expression in mutants. The C. roseus counterparts of CMT3, DRM2 and RDR2 were downregulated in mutants. Among the cell, organ and plant size, photosynthesis and metabolism related traits studied, 28 traits were similarly affected in mutants as compared to wild type. Each of the mutants also expressed some traits distinctively. The egd mutant possessed superior photosynthesis and water retention abilities. Biomass was hyperaccumulated in roots, stems, leaves and seeds of the lli mutant. The ill mutant was richest in the pharmaceutical alkaloids catharanthine, vindoline, vincristine and vinblastine. The nature of mutations, origins of mutant phenotypes and evolutionary importance of these mutants are discussed. PMID:24371160

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

    PubMed

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

    2003-08-01

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

  8. Pea Plants Show Risk Sensitivity.

    PubMed

    Dener, Efrat; Kacelnik, Alex; Shemesh, Hagai

    2016-07-11

    Sensitivity to variability in resources has been documented in humans, primates, birds, and social insects, but the fit between empirical results and the predictions of risk sensitivity theory (RST), which aims to explain this sensitivity in adaptive terms, is weak [1]. RST predicts that agents should switch between risk proneness and risk aversion depending on state and circumstances, especially according to the richness of the least variable option [2]. Unrealistic assumptions about agents' information processing mechanisms and poor knowledge of the extent to which variability imposes specific selection in nature are strong candidates to explain the gap between theory and data. RST's rationale also applies to plants, where it has not hitherto been tested. Given the differences between animals' and plants' information processing mechanisms, such tests should help unravel the conflicts between theory and data. Measuring root growth allocation by split-root pea plants, we show that they favor variability when mean nutrient levels are low and the opposite when they are high, supporting the most widespread RST prediction. However, the combination of non-linear effects of nitrogen availability at local and systemic levels may explain some of these effects as a consequence of mechanisms not necessarily evolved to cope with variance [3, 4]. This resembles animal examples in which properties of perception and learning cause risk sensitivity even though they are not risk adaptations [5]. PMID:27374342

  9. Casimir experiments showing saturation effects

    SciTech Connect

    Sernelius, Bo E.

    2009-10-15

    We address several different Casimir experiments where theory and experiment disagree. First out is the classical Casimir force measurement between two metal half spaces; here both in the form of the torsion pendulum experiment by Lamoreaux and in the form of the Casimir pressure measurement between a gold sphere and a gold plate as performed by Decca et al.; theory predicts a large negative thermal correction, absent in the high precision experiments. The third experiment is the measurement of the Casimir force between a metal plate and a laser irradiated semiconductor membrane as performed by Chen et al.; the change in force with laser intensity is larger than predicted by theory. The fourth experiment is the measurement of the Casimir force between an atom and a wall in the form of the measurement by Obrecht et al. of the change in oscillation frequency of a {sup 87}Rb Bose-Einstein condensate trapped to a fused silica wall; the change is smaller than predicted by theory. We show that saturation effects can explain the discrepancies between theory and experiment observed in all these cases.

  10. Glutamate Racemase Mutants of Bacillus anthracis

    PubMed Central

    Oh, So-Young; Richter, Stefan G.; Missiakas, Dominique M.

    2015-01-01

    ABSTRACT d-Glutamate is an essential component of bacterial peptidoglycan and a building block of the poly-γ-d-glutamic acid (PDGA) capsule of Bacillus anthracis, the causative agent of anthrax. Earlier work suggested that two glutamate racemases, encoded by racE1 and racE2, are each essential for growth of B. anthracis, supplying d-glutamic acid for the synthesis of peptidoglycan and PDGA capsule. Earlier work could not explain, however, why two enzymes that catalyze the same reaction may be needed for bacterial growth. Here, we report that deletion of racE1 or racE2 did not prevent growth of B. anthracis Sterne (pXO1+ pXO2−), the noncapsulating vaccine strain, or of B. anthracis Ames (pXO1+ pXO2+), a fully virulent, capsulating isolate. While mutants with deletions in racE1 and racE2 were not viable, racE2 deletion delayed vegetative growth of B. anthracis following spore germination and caused aberrant cell shapes, phenotypes that were partially restored by exogenous d-glutamate. Deletion of racE1 or racE2 from B. anthracis Ames did not affect the production or stereochemical composition of the PDGA capsule. A model is presented whereby B. anthracis, similar to Bacillus subtilis, utilizes two functionally redundant racemase enzymes to synthesize d-glutamic acid for peptidoglycan synthesis. IMPORTANCE Glutamate racemases, enzymes that convert l-glutamate to d-glutamate, are targeted for antibiotic development. Glutamate racemase inhibitors may be useful for the treatment of bacterial infections such as anthrax, where the causative agent, B. anthracis, requires d-glutamate for the synthesis of peptidoglycan and poly-γ-d-glutamic acid (PDGA) capsule. Here we show that B. anthracis possesses two glutamate racemase genes that can be deleted without abolishing either bacterial growth or PDGA synthesis. These data indicate that drug candidates must inhibit both glutamate racemases, RacE1 and RacE2, in order to block B. anthracis growth and achieve therapeutic

  11. Disruption of Olfactory Receptor Neuron Patterning in Scutoid mutant Drosophila

    PubMed Central

    Tom, W.; de Bruyne, M.; Haehnel, M.; Carlson, J. R.; Ray, A.

    2010-01-01

    Olfactory neurons show an extreme diversity of cell types with each cell usually expressing one member from a large family of 60 Odorant receptor (Or)genes in Drosophila. Little is known about the developmental processes and transcription factors that generate this stereotyped pattern of cellular diversity. Here we investigate the molecular and cellular basis of defects in olfactory system function in an unusual dominant mutant, Scutoid. We show that the defects map to olfactory neurons innervating a specific morphological class of sensilla on the antenna, large basiconics. Molecular analysis indicates defects in neurons expressing specific classes of receptor genes that map to large basiconic sensilla. Previous studies have shown that in Scutoid mutants the coding region of the transcriptional repressor snail is translocated near the no-ocelli promoter, leading to misexpression of snail in the developing eye-antenna disc. We show that ectopic expression of snail in developing olfactory neurons leads to severe defects in neurons of the antennal large basiconics supporting the model that the dominant olfactory phenotype in Scutoid is caused by misexpression of snail. PMID:20875862

  12. The Arabidopsis elch mutant reveals functions of an ESCRT component in cytokinesis.

    PubMed

    Spitzer, Christoph; Schellmann, Swen; Sabovljevic, Aneta; Shahriari, Mojgan; Keshavaiah, Channa; Bechtold, Nicole; Herzog, Michel; Müller, Stefan; Hanisch, Franz-Georg; Hülskamp, Martin

    2006-12-01

    Recently, an alternative route to the proteasomal protein-degradation pathway was discovered that specifically targets transmembrane proteins marked with a single ubiquitin to the endosomal multivesicular body (MVB) and, subsequently, to the vacuole (yeast) or lysosome (animals), where they are degraded by proteases. Vps23p/TSG101 is a key component of the ESCRT I-III machinery in yeast and animals that recognizes mono-ubiquitylated proteins and sorts them into the MVB. Here, we report that the Arabidopsis ELCH (ELC) gene encodes a Vps23p/TSG101 homolog, and that homologs of all known ESCRT I-III components are present in the Arabidopsis genome. As with its animal and yeast counterparts, ELC binds ubiquitin and localizes to endosomes. Gel-filtration experiments indicate that ELC is a component of a high-molecular-weight complex. Yeast two-hybrid and immunoprecipitation assays showed that ELC interacts with Arabidopsis homologs of the ESCRT I complex. The elc mutant shows multiple nuclei in various cell types, indicating a role in cytokinesis. Double-mutant analysis with kaktus shows that increased ploidy levels do not influence the cytokinesis effect of elc mutants, suggesting that ELC is only important during the first endoreduplication cycle. Double mutants with tubulin folding cofactor a mutants show a synergistic phenotype, suggesting that ELC regulates cytokinesis through the microtubule cytoskeleton. PMID:17090720

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

    PubMed

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

    2016-01-01

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

  14. Identification and characterization of Photorhabdus temperata mutants altered in hemolysis and virulence.

    PubMed

    Chapman, Christine; Tisa, Louis S

    2016-08-01

    Photorhabdus temperata is a symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora and an insect pathogen. This bacterium produces a wide variety of virulence factors and hemolytic activity. The goal of this study was to identify hemolysin-defective mutants and test their virulence. A genetic approach was used to identify mutants with altered hemolytic activity by screening a library of 10 000 P. temperata transposon mutants. Three classes of mutants were identified: (i) defective (no hemolytic activity), (ii) delayed (delayed initiation of hemolytic activity), and (iii) early (early initiation of hemolytic activity). The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis and motility. The hemolysin-defective mutants, P10A-C11, P10A-H12, and P79-B5, had inserts in genes involved in RNA turnover (RNase II and 5'-pentaphospho-5'-adenosine pyrophosphohydrolase) and showed reduced virulence and production of extracellular factors. These data support the role of RNA turnover in insect pathogenesis and other physiological functions. PMID:27300499

  15. Enhanced dimerization drives ligand-independent activity of mutant epidermal growth factor receptor in lung cancer

    PubMed Central

    Valley, Christopher C.; Arndt-Jovin, Donna J.; Karedla, Narain; Steinkamp, Mara P.; Chizhik, Alexey I.; Hlavacek, William S.; Wilson, Bridget S.; Lidke, Keith A.; Lidke, Diane S.

    2015-01-01

    Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with tumorigenesis. In particular, a number of EGFR mutants that demonstrate ligand-independent signaling are common in non–small cell lung cancer (NSCLC), including kinase domain mutations L858R (also called L834R) and exon 19 deletions (e.g., ΔL747-P753insS), which collectively make up nearly 90% of mutations in NSCLC. The molecular mechanisms by which these mutations confer constitutive activity remain unresolved. Using multiple subdiffraction-limit imaging modalities, we reveal the altered receptor structure and interaction kinetics of NSCLC-associated EGFR mutants. We applied two-color single quantum dot tracking to quantify receptor dimerization kinetics on living cells and show that, in contrast to wild-type EGFR, mutants are capable of forming stable, ligand-independent dimers. Two-color superresolution localization microscopy confirmed ligand-independent aggregation of EGFR mutants. Live-cell Förster resonance energy transfer measurements revealed that the L858R kinase mutation alters ectodomain structure such that unliganded mutant EGFR adopts an extended, dimerization-competent conformation. Finally, mutation of the putative dimerization arm confirmed a critical role for ectodomain engagement in ligand-independent signaling. These data support a model in which dysregulated activity of NSCLC-associated kinase mutants is driven by coordinated interactions involving both the kinase and extracellular domains that lead to enhanced dimerization. PMID:26337388

  16. Rapid Purification and Characterization of Mutant Origin Recognition Complexes in Saccharomyces cerevisiae

    PubMed Central

    Kawakami, Hironori; Ohashi, Eiji; Tsurimoto, Toshiki; Katayama, Tsutomu

    2016-01-01

    Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as several canonical protein fractionations. Here, we present a streamlined method to construct mutant overproducers, followed by purification of mutant ORCs. Use of mammalian cells co-transfected with conveniently mutagenized plasmids bearing a His tag excludes many of the construction and fractionation steps. Transfection is highly efficient. All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex. Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells. The method is applicable to wild-type and at least three mutant ORCs, and the resultant purified complexes show expected biochemical activities. The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC. PMID:27148210

  17. Rapid Purification and Characterization of Mutant Origin Recognition Complexes in Saccharomyces cerevisiae.

    PubMed

    Kawakami, Hironori; Ohashi, Eiji; Tsurimoto, Toshiki; Katayama, Tsutomu

    2016-01-01

    Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as several canonical protein fractionations. Here, we present a streamlined method to construct mutant overproducers, followed by purification of mutant ORCs. Use of mammalian cells co-transfected with conveniently mutagenized plasmids bearing a His tag excludes many of the construction and fractionation steps. Transfection is highly efficient. All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex. Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells. The method is applicable to wild-type and at least three mutant ORCs, and the resultant purified complexes show expected biochemical activities. The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC. PMID:27148210

  18. Rescuing cystic fibrosis transmembrane conductance regulator (CFTR)-processing mutants by transcomplementation

    PubMed Central

    Cormet-Boyaka, Estelle; Jablonsky, Michael; Naren, Anjaparavanda P.; Jackson, Patricia L.; Muccio, Donald D.; Kirk, Kevin L.

    2004-01-01

    Most cases of cystic fibrosis (CF) are caused by mutations that block the biosynthetic maturation of the CF gene product, the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR-processing mutants fail to escape the endoplasmic reticulum and are rapidly degraded. Current efforts to induce the maturation of CFTR mutants target components of the biosynthetic pathway (e.g., chaperones) rather than CFTR per se. Such methods are inherently nonspecific. Here we show that the most common CF-causing mutant (ΔF508-CFTR) can form mature, functional chloride channels that reach the cell surface when coexpressed with several other CFTR-processing mutants or with amino fragments of the wild-type CFTR protein. This transcomplementation effect required a specific match between the region flanking the disease-causing mutation and the complementing fragment; e.g., amino fragments complemented ΔF508-CFTR but not H1085R (a carboxy-processing mutant), whereas a carboxy fragment complemented H1085R but not ΔF508-CFTR. Transcomplementing fragments did not affect CFTR interactions with Hsc70, a chaperone previously implicated in CFTR biosynthesis. Instead, they may promote CFTR maturation by blocking nonproductive interactions between domains within the same or neighboring CFTR polypeptides that prevent normal processing. These findings indicate that it may be possible to develop CF therapies (e.g., mini-cDNA constructs for gene therapy) that are tailored to specific disease-causing mutants of CFTR. PMID:15141088

  19. Analysis of crystal structure of Arabidopsis MPK6 and generation of its mutants with higher activity.

    PubMed

    Wang, Bo; Qin, Xinghua; Wu, Juan; Deng, Hongying; Li, Yuan; Yang, Hailian; Chen, Zhongzhou; Liu, Guoqin; Ren, Dongtao

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades, which are the highly conserved signalling modules in eukaryotic organisms, have been shown to play important roles in regulating growth, development, and stress responses. The structures of various MAPKs from yeast and animal have been solved, and structure-based mutants were generated for their function analyses, however, the structures of plant MAPKs remain unsolved. Here, we report the crystal structure of Arabidopsis MPK6 at a 3.0 Å resolution. Although MPK6 is topologically similar to ERK2 and p38, the structures of the glycine-rich loop, MAPK insert, substrate binding sites, and L16 loop in MPK6 show notable differences from those of ERK2 and p38. Based on the structural comparison, we constructed MPK6 mutants and analyzed their kinase activity both in vitro and in planta. MPK6(F364L) and MPK6(F368L) mutants, in which Phe364 and Phe368 in the L16 loop were changed to Leu, respectively, acquired higher intrinsic kinase activity and retained the normal MAPKK activation property. The expression of MPK6 mutants with basal activity is sufficient to induce camalexin biosynthesis; however, to induce ethylene and leaf senescence, the expression of MPK6 mutants with higher activity is required. The results suggest that these mutants can be used to analyze the specific biological functions of MPK6. PMID:27160427

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

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

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

    PubMed

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

    2016-01-01

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

  2. Reduced Infectivity in Cattle for an Outer Membrane Protein Mutant of Anaplasma marginale

    PubMed Central

    Brayton, Kelly A.; Magunda, Forgivemore; Munderloh, Ulrike G.; Kelley, Karen L.; Barbet, Anthony F.

    2015-01-01

    Anaplasma marginale is the causative agent of anaplasmosis in cattle. Transposon mutagenesis of this pathogen using the Himar1 system resulted in the isolation of an omp10 operon insertional mutant referred to as the omp10::himar1 mutant. The work presented here evaluated if this mutant had morphological and/or growth rate defects compared to wild-type A. marginale. Results showed that the morphology, developmental cycle, and growth in tick and mammalian cell cultures are similar for the mutant and the wild type. Tick transmission experiments established that tick infection levels with the mutant were similar to those with wild-type A. marginale and that infected ticks successfully infected cattle. However, this mutant exhibited reduced infectivity and growth in cattle. The possibility of transforming A. marginale by transposon mutagenesis coupled with in vitro and in vivo assessment of altered phenotypes can aid in the identification of genes associated with virulence. The isolation of deliberately attenuated organisms that can be evaluated in their natural biological system is an important advance for the rational design of vaccines against this species. PMID:25595772

  3. Isolation of Chlamydomonas reinhardtii mutants with altered mitochondrial respiration by chlorophyll fluorescence measurement.

    PubMed

    Massoz, Simon; Larosa, Véronique; Horrion, Bastien; Matagne, René F; Remacle, Claire; Cardol, Pierre

    2015-12-10

    The unicellular green alga Chlamydomonas reinhardtii is a model organism for studying energetic metabolism. Most mitochondrial respiratory-deficient mutants characterized to date have been isolated on the basis of their reduced ability to grow in heterotrophic conditions. Mitochondrial deficiencies are usually partly compensated by adjustment of photosynthetic activity and more particularly by transition to state 2. In this work, we explored the opportunity to select mutants impaired in respiration and/or altered in dark metabolism by measuring maximum photosynthetic efficiency by chlorophyll fluorescence analyses (FV/FM). Out of about 2900 hygromycin-resistant insertional mutants generated from wild type or from a mutant strain deficient in state transitions (stt7 strain), 22 were found to grow slowly in heterotrophic conditions and 8 of them also showed a lower FV/FM value. Several disrupted coding sequences were identified, including genes coding for three different subunits of respiratory-chain complex I (NUO9, NUOA9, NUOP4) or for isocitrate lyase (ICL1). Overall, the comparison of respiratory mutants obtained in wild-type or stt7 genetic backgrounds indicated that the FV/FM value can be used to isolate mutants severely impaired in dark metabolism. PMID:26022424

  4. Quorum sensing control of Type VI secretion factors restricts the proliferation of quorum-sensing mutants.

    PubMed

    Majerczyk, Charlotte; Schneider, Emily; Greenberg, E Peter

    2016-01-01

    Burkholderia thailandensis uses acyl-homoserine lactone-mediated quorum sensing systems to regulate hundreds of genes. Here we show that cell-cell contact-dependent type VI secretion (T6S) toxin-immunity systems are among those activated by quorum sensing in B. thailandensis. We also demonstrate that T6S is required to constrain proliferation of quorum sensing mutants in colony cocultures of a BtaR1 quorum-sensing signal receptor mutant and its parent. However, the BtaR1 mutant is not constrained by and outcompetes its parent in broth coculture, presumably because no cell contact occurs and there is a metabolic cost associated with quorum sensing gene activation. The increased fitness of the wild type over the BtaR1 mutant during agar surface growth is dependent on an intact T6SS-1 apparatus. Thus, quorum sensing activates B. thailandensis T6SS-1 growth inhibition and this control serves to police and constrain quorum-sensing mutants. This work defines a novel role for T6SSs in intraspecies mutant control. PMID:27183270

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

    SciTech Connect

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

    2013-12-15

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

  6. Light-Induced Acclimation of the Arabidopsis chlorina1 Mutant to Singlet Oxygen[C][W

    PubMed Central

    Ramel, Fanny; Ksas, Brigitte; Akkari, Elsy; Mialoundama, Alexis S.; Monnet, Fabien; Krieger-Liszkay, Anja; Ravanat, Jean-Luc; Mueller, Martin J.; Bouvier, Florence; Havaux, Michel

    2013-01-01

    Singlet oxygen (1O2) is a reactive oxygen species that can function as a stress signal in plant leaves leading to programmed cell death. In microalgae, 1O2-induced transcriptomic changes result in acclimation to 1O2. Here, using a chlorophyll b–less Arabidopsis thaliana mutant (chlorina1 [ch1]), we show that this phenomenon can also occur in vascular plants. The ch1 mutant is highly photosensitive due to a selective increase in the release of 1O2 by photosystem II. Under photooxidative stress conditions, the gene expression profile of ch1 mutant leaves very much resembled the gene responses to 1O2 reported in the Arabidopsis mutant flu. Preexposure of ch1 plants to moderately elevated light intensities eliminated photooxidative damage without suppressing 1O2 formation, indicating acclimation to 1O2. Substantial differences in gene expression were observed between acclimation and high-light stress: A number of transcription factors were selectively induced by acclimation, and contrasting effects were observed for the jasmonate pathway. Jasmonate biosynthesis was strongly induced in ch1 mutant plants under high-light stress and was noticeably repressed under acclimation conditions, suggesting the involvement of this hormone in 1O2-induced cell death. This was confirmed by the decreased tolerance to photooxidative damage of jasmonate-treated ch1 plants and by the increased tolerance of the jasmonate-deficient mutant delayed-dehiscence2. PMID:23590883

  7. Directed mutagenesis in Candida albicans: one-step gene disruption to isolate ura3 mutants

    SciTech Connect

    Kelly, R.; Miller, S.M.; Kurtz, M.B.; Kirsch, D.R.

    1987-01-01

    A method for introducing specific mutations into the diploid Candida albicans by one-step gene disruption and subsequent UV-induced recombination was developed. The cloned C. albicans URA3 gene was disrupted with the C. albicans ADE2 gene, and the linearized DNA was used for transformation of two ade2 mutants, SGY-129 and A81-Pu. Both an insertional inactivation of the URA3 gene and a disruption which results in a 4.0-kilobase deletion were made. Southern hybridization analyses demonstrated that the URA3 gene was disrupted on one of the chromosomal homologs in 15 of the 18 transformants analyzed. These analyses also revealed restriction site dimorphism of EcoRI at the URA3 locus which provides a unique marker to distinguish between chromosomal homologs. This enabled us to show that either homolog could be disrupted and that disrupted transformants of SGY-129 contained more than two copies of the URA3 locus. The A81-Pu transformants heterozygous for the ura3 mutations were rendered homozygous and Ura- by UV-induced recombination. The homozygosity of a deletion mutant and an insertion mutant was confirmed by Southern hybridization. Both mutants were transformed to Ura+ with plasmids containing the URA3 gene and in addition, were resistant to 5-fluoro-orotic acid, a characteristic of Saccharomyces cerevisiae ura3 mutants as well as of orotidine-5'-phosphate decarboxylase mutants of other organisms.

  8. A Mutant Library Approach to Identify Improved Meningococcal Factor H Binding Protein Vaccine Antigens

    PubMed Central

    Konar, Monica; Rossi, Raffaella; Walter, Helen; Pajon, Rolando; Beernink, Peter T.

    2015-01-01

    Factor H binding protein (FHbp) is a virulence factor used by meningococci to evade the host complement system. FHbp elicits bactericidal antibodies in humans and is part of two recently licensed vaccines. Using human complement Factor H (FH) transgenic mice, we previously showed that binding of FH decreased the protective antibody responses to FHbp vaccination. Therefore, in the present study we devised a library-based method to identify mutant FHbp antigens with very low binding of FH. Using an FHbp sequence variant in one of the two licensed vaccines, we displayed an error-prone PCR mutant FHbp library on the surface of Escherichia coli. We used fluorescence-activated cell sorting to isolate FHbp mutants with very low binding of human FH and preserved binding of control anti-FHbp monoclonal antibodies. We sequenced the gene encoding FHbp from selected clones and introduced the mutations into a soluble FHbp construct. Using this approach, we identified several new mutant FHbp vaccine antigens that had very low binding of FH as measured by ELISA and surface plasmon resonance. The new mutant FHbp antigens elicited protective antibody responses in human FH transgenic mice that were up to 20-fold higher than those elicited by the wild-type FHbp antigen. This approach offers the potential to discover mutant antigens that might not be predictable even with protein structural information and potentially can be applied to other microbial vaccine antigens that bind host proteins. PMID:26057742

  9. [Genetical analysis and characterization of a new mutant, black tremor appearing in the Syrian hamster].

    PubMed

    Mizutani, M; Katsuie, Y; Umezawa, H; Kuramasu, S

    1986-04-01

    A black coat-color mutant with tremor was discovered in babies of 61 generations of an inbred strain APG of Syrian hamster which had been maintained in the Nippon Institute for Biological Science, Laboratory Animal Research Station. The genetical analysis by matings between four inbred strains which had different genes in the E and B loci and four mutant strains which were introduced the mutant gene into the four inbred strains and characterization were carried out on the mutant. The results obtained are summarized as follows: 1) The mutation occurred in a different locus with E and B loci. 2) The mutant was controlled by an autosomal recessive gene designated as "bt", and it was thought that both tremor and black coat-color were the pleiotropic effect of bt gene. 3) At least one E gene in the E locus was necessary for the appearance of black coat color. Therefore, the coat-color remained cream in ee (cream) hamsters showing only trembling. 4) The degree of blackness of the coat-color of EE hamsters differed from Ee ones. The former was darker than the latter. 5) The mutant may be a useful animal model for studying abnormal myelogenesis and biosynthesis of melanin. PMID:3732409

  10. Bacillus subtilis mutant with temperature-sensitive net synthesis of phosphatidylethanolamine.

    PubMed Central

    Lindgren, V; Holmgren, E; Rutberg, L

    1977-01-01

    Bacillus subtilis mutants with temperature-sensitive growth on complex media were screened for defects in phospholipid metabolism. One mutant was isolated that showed temperature-sensitive net synthesis of phosphatidylethanolamine. The mutant did not accumulate phosphatidylserine at the nonpermissive temperature. In the presence of hydroxylamine, wild-type B. subtilis accumulated phosphatidylserine at both 32 and 45 degrees C, whereas the mutant did only at 32 degrees C. In vitro phosphatidylethanolamine synthesis with bacterial membranes is no more temperature sensitive with mutant membranes than with wild-type membranes. The mutation probably affects the synthesis indirectly, possibly by altering a membrane protein. The mutant bacteria grew at the nonpermissive temperature, 45 degrees C, in a phosphate buffer-based minimal medium, although net synthesis of phosphatidylethanolamine was also temperature sensitive in this medium. One mutation caused both temperature-sensitive growth on complex media and temperature-sensitive net synthesis of phosphatidylethanolamine. The mutation is linked to aroD by transformation. PMID:410792

  11. Genetic analysis and characterization of a Caulobacter crescentus mutant defective in membrane biogenesis.

    PubMed Central

    Hodgson, D; Shaw, P; Letts, V; Henry, S; Shapiro, L

    1984-01-01

    A mutant of Caulobacter crescentus has been isolated which has an auxotrophic requirement for unsaturated fatty acids or biotin for growth on medium containing glucose as the carbon source. This mutant exhibits a pleiotropic phenotype which includes (i) the auxotrophic requirement, (ii) cell death in cultures attempting to grow on glucose in the absence of fatty acids or biotin, and (iii) a major change in the outer membrane protein composition before cell death. This genetic lesion did not appear to affect directly a fatty acid biosynthetic reaction because fatty acid and phospholipid syntheses were found to continue in the absence of supplement. Oleic acid repressed fatty acid biosynthesis and induced fatty acid degradation in the wild-type parent, AE5000 . The mutant strain, AE6000 , was altered in both of these regulatory functions. The AE6000 mutant also showed specific inhibition of the synthesis of outer membrane and flagellar proteins. Total phospholipid, DNA, RNA, and protein syntheses were unaffected. The multiple phenotypes of the AE6000 mutant were found to cosegregate and to map between hclA and lacA on the C. crescentus chromosome. The defect in this mutant appears to be associated with a regulatory function in membrane biogenesis and provides evidence for a direct coordination of membrane protein synthesis and lipid metabolism in C. crescentus. Images PMID:6202671

  12. Analysis of crystal structure of Arabidopsis MPK6 and generation of its mutants with higher activity

    PubMed Central

    Wang, Bo; Qin, Xinghua; Wu, Juan; Deng, Hongying; Li, Yuan; Yang, Hailian; Chen, Zhongzhou; Liu, Guoqin; Ren, Dongtao

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades, which are the highly conserved signalling modules in eukaryotic organisms, have been shown to play important roles in regulating growth, development, and stress responses. The structures of various MAPKs from yeast and animal have been solved, and structure-based mutants were generated for their function analyses, however, the structures of plant MAPKs remain unsolved. Here, we report the crystal structure of Arabidopsis MPK6 at a 3.0 Å resolution. Although MPK6 is topologically similar to ERK2 and p38, the structures of the glycine-rich loop, MAPK insert, substrate binding sites, and L16 loop in MPK6 show notable differences from those of ERK2 and p38. Based on the structural comparison, we constructed MPK6 mutants and analyzed their kinase activity both in vitro and in planta. MPK6F364L and MPK6F368L mutants, in which Phe364 and Phe368 in the L16 loop were changed to Leu, respectively, acquired higher intrinsic kinase activity and retained the normal MAPKK activation property. The expression of MPK6 mutants with basal activity is sufficient to induce camalexin biosynthesis; however, to induce ethylene and leaf senescence, the expression of MPK6 mutants with higher activity is required. The results suggest that these mutants can be used to analyze the specific biological functions of MPK6. PMID:27160427

  13. Quorum sensing control of Type VI secretion factors restricts the proliferation of quorum-sensing mutants

    PubMed Central

    Majerczyk, Charlotte; Schneider, Emily; Greenberg, E Peter

    2016-01-01

    Burkholderia thailandensis uses acyl-homoserine lactone-mediated quorum sensing systems to regulate hundreds of genes. Here we show that cell-cell contact-dependent type VI secretion (T6S) toxin-immunity systems are among those activated by quorum sensing in B. thailandensis. We also demonstrate that T6S is required to constrain proliferation of quorum sensing mutants in colony cocultures of a BtaR1 quorum-sensing signal receptor mutant and its parent. However, the BtaR1 mutant is not constrained by and outcompetes its parent in broth coculture, presumably because no cell contact occurs and there is a metabolic cost associated with quorum sensing gene activation. The increased fitness of the wild type over the BtaR1 mutant during agar surface growth is dependent on an intact T6SS-1 apparatus. Thus, quorum sensing activates B. thailandensis T6SS-1 growth inhibition and this control serves to police and constrain quorum-sensing mutants. This work defines a novel role for T6SSs in intraspecies mutant control. DOI: http://dx.doi.org/10.7554/eLife.14712.001 PMID:27183270

  14. A Mutant Library Approach to Identify Improved Meningococcal Factor H Binding Protein Vaccine Antigens.

    PubMed

    Konar, Monica; Rossi, Raffaella; Walter, Helen; Pajon, Rolando; Beernink, Peter T

    2015-01-01

    Factor H binding protein (FHbp) is a virulence factor used by meningococci to evade the host complement system. FHbp elicits bactericidal antibodies in humans and is part of two recently licensed vaccines. Using human complement Factor H (FH) transgenic mice, we previously showed that binding of FH decreased the protective antibody responses to FHbp vaccination. Therefore, in the present study we devised a library-based method to identify mutant FHbp antigens with very low binding of FH. Using an FHbp sequence variant in one of the two licensed vaccines, we displayed an error-prone PCR mutant FHbp library on the surface of Escherichia coli. We used fluorescence-activated cell sorting to isolate FHbp mutants with very low binding of human FH and preserved binding of control anti-FHbp monoclonal antibodies. We sequenced the gene encoding FHbp from selected clones and introduced the mutations into a soluble FHbp construct. Using this approach, we identified several new mutant FHbp vaccine antigens that had very low binding of FH as measured by ELISA and surface plasmon resonance. The new mutant FHbp antigens elicited protective antibody responses in human FH transgenic mice that were up to 20-fold higher than those elicited by the wild-type FHbp antigen. This approach offers the potential to discover mutant antigens that might not be predictable even with protein structural information and potentially can be applied to other microbial vaccine antigens that bind host proteins. PMID:26057742

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

    PubMed Central

    Banerjee, M

    1989-01-01

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

  16. Deficits in memory and motor performance in synaptotagmin IV mutant mice

    PubMed Central

    Ferguson, Gregory D.; Anagnostaras, Stephan G.; Silva, Alcino J.; Herschman, Harvey R.

    2000-01-01

    Synaptotagmin (Syt) IV is a synaptic vesicle protein. Syt IV expression is induced in the rat hippocampus after systemic kainic acid treatment. To examine the functional role of this protein in vivo, we derived Syt IV null [Syt IV(−/−)] mutant mice. Studies with the rotorod revealed that the Syt IV mutants have impaired motor coordination, a result consistent with constitutive Syt IV expression in the cerebellum. Because Syt IV is thought to modulate synaptic function, we also have examined Syt IV mutant mice in learning and memory tests. Our studies show that the Syt IV mutation disrupts contextual fear conditioning, a learning task sensitive to hippocampal and amygdala lesions. In contrast, cued fear conditioning is normal in the Syt IV mutants, suggesting that this mutation did not disrupt amygdala function. Conditioned taste aversion, which also depends on the amygdala, is normal in the Syt IV mutants. Consistent with the idea that the Syt IV mutation preferentially affects hippocampal function, Syt IV mutant mice also display impaired social transmission of food preference. These studies demonstrate that Syt IV is critical for brain function and suggest that the Syt IV mutation affects hippocampal-dependent learning and memory, as well as motor coordination. PMID:10792055

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

    PubMed Central

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

    2015-01-01

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

  18. Characterization of factor IIIGLc in catabolite repression-resistant (crr) mutants of Salmonella typhimurium.

    PubMed Central

    Scholte, B J; Schuitema, A R; Postma, P W

    1982-01-01

    crr mutants of Salmonella typhimurium are thought to be defective in the regulation of adenylate cyclase and a number of transport systems by the phosphoenolpyruvate-dependent sugar phosphotransferase system, crr mutants are also defective in the enzymatic activity of factor IIIGlc (IIIGlc), a protein component of the phosphotransferase system involved in glucose transport. Therefore, it has been proposed that IIIGlc is the primary effector of phosphotransferase system-mediated regulation of cell metabolism. We characterized crr mutants with respect to the presence and function of IIIGlc by using an immunochemical approach. All of the crr mutants tested had low (0 to 30%) levels of IIIGlc compared with wild-type cells, as determined by rocket immunoelectrophoresis. The IIIGlc isolated from one crr mutant was investigated in more detail and showed abnormal aggregation behavior, which indicated a structural change in the protein. These results supported the hypothesis that a crr mutation directly affects IIIGlc, probably by altering the structural gene of IIIGlc. Several crr strains which appeared to be devoid of IIIGlc in immunoprecipitation assays were still capable of in vitro phosphorylation and transport of methyl alpha-glucoside. This phosphorylation activity was sensitive to specific anti-IIIGlc serum. Moreover, the membranes of crr mutants, as well as those of wild-type cells, contained a protein that reacted strongly with our anti-IIIGlc serum. We propose that S. typhimurium contains a membrane-bound form of IIIGlc which may be involved in phosphotransferase system activity. Images PMID:7035434

  19. Trichomonas vaginalis lipophosphoglycan mutants have reduced adherence and cytotoxicity to human ectocervical cells.

    PubMed

    Bastida-Corcuera, Felix D; Okumura, Cheryl Y; Colocoussi, Angie; Johnson, Patricia J

    2005-11-01

    The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite. PMID

  20. Trichomonas vaginalis Lipophosphoglycan Mutants Have Reduced Adherence and Cytotoxicity to Human Ectocervical Cells

    PubMed Central

    Bastida-Corcuera, Felix D.; Okumura, Cheryl Y.; Colocoussi, Angie; Johnson, Patricia J.

    2005-01-01

    The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite. PMID

  1. Lysyl-tRNA Synthetase Is a Target for Mutant SOD1 Toxicity in Mitochondria*

    PubMed Central

    Kawamata, Hibiki; Magrané, Jordi; Kunst, Catherine; King, Michael P.; Manfredi, Giovanni

    2008-01-01

    Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting the motor neurons. The majority of familial forms of ALS are caused by mutations in the Cu,Zn-superoxide dismutase (SOD1). In mutant SOD1 spinal cord motor neurons, mitochondria develop abnormal morphology, bioenergetic defects, and degeneration. However, the mechanisms of mitochondrial toxicity are still unclear. One possibility is that mutant SOD1 establishes aberrant interactions with nuclear-encoded mitochondrial proteins, which can interfere with their normal trafficking from the cytosol to mitochondria. Lysyl-tRNA synthetase (KARS), an enzyme required for protein translation that was shown to interact with mutant SOD1 in yeast, is a good candidate as a target for interaction with mutant SOD1 at the mitochondrion in mammals because of its dual cytosolic and mitochondrial localization. Here, we show that in mammalian cells mutant SOD1 interacts preferentially with the mitochondrial form of KARS (mitoKARS). KARS-SOD1 interactions occur also in the mitochondria of the nervous system in transgenic mice. In the presence of mutant SOD1, mitoKARS displays a high propensity to misfold and aggregate prior to its import into mitochondria, becoming a target for proteasome degradation. Impaired mitoKARS import correlates with decreased mitochondrial protein synthesis. Ultimately, the abnormal interactions between mutant SOD1 and mitoKARS result in mitochondrial morphological abnormalities and cell toxicity. mitoKARS is the first described member of a group of mitochondrial proteins whose interaction with mutant SOD1 contributes to mitochondrial dysfunction in ALS. PMID:18715867

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

    PubMed

    Iskandar, Kartini; Rezlan, Majidah; Pervaiz, Shazib

    2014-10-01

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

  3. Homology-dependent repair is involved in 45S rDNA loss in plant CAF-1 mutants

    PubMed Central

    Muchová, Veronika; Amiard, Simon; Mozgová, Iva; Dvořáčková, Martina; Gallego, Maria E; White, Charles; Fajkus, Jiří

    2015-01-01

    Arabidopsis thaliana mutants in FAS1 and FAS2 subunits of chromatin assembly factor 1 (CAF1) show progressive loss of 45S rDNA copies and telomeres. We hypothesized that homology-dependent DNA damage repair (HDR) may contribute to the loss of these repeats in fas mutants. To test this, we generated double mutants by crossing fas mutants with knock-out mutants in RAD51B, one of the Rad51 paralogs of A. thaliana. Our results show that the absence of RAD51B decreases the rate of rDNA loss, confirming the implication of RAD51B-dependent recombination in rDNA loss in the CAF1 mutants. Interestingly, this effect is not observed for telomeric repeat loss, which thus differs from that acting in rDNA loss. Involvement of DNA damage repair in rDNA dynamics in fas mutants is further supported by accumulation of double-stranded breaks (measured as γ-H2AX foci) in 45S rDNA. Occurrence of the foci is not specific for S-phase, and is ATM-independent. While the foci in fas mutants occur both in the transcribed (intranucleolar) and non-transcribed (nucleoplasmic) fraction of rDNA, double fas rad51b mutants show a specific increase in the number of the intranucleolar foci. These results suggest that the repair of double-stranded breaks present in the transcribed rDNA region is RAD51B dependent and that this contributes to rDNA repeat loss in fas mutants, presumably via the single-stranded annealing recombination pathway. Our results also highlight the importance of proper chromatin assembly in the maintenance of genome stability. PMID:25359579

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

    PubMed Central

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

    2015-01-01

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

  5. Intact Interval Timing in Circadian CLOCK Mutants

    PubMed Central

    Cordes, Sara; Gallistel, C. R.

    2008-01-01

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

  6. Oxygen sensitivity of an Escherichia coli mutant.

    PubMed

    Adler, H; Mural, R; Suttle, B

    1992-04-01

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

  7. Oxygen sensitivity of an Escherichia coli mutant.

    PubMed Central

    Adler, H; Mural, R; Suttle, B

    1992-01-01

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

  8. Survival of rifampin-resistant mutants of Pseudomonas fluorescens and Pseudomonas putida in soil systems.

    PubMed Central

    Compeau, G; Al-Achi, B J; Platsouka, E; Levy, S B

    1988-01-01

    The fate of spontaneous chromosomal rifampin-resistant (Rifr) mutants of Pseudomonas putida and Pseudomonas fluorescens in sterile and live organic soil from which they were isolated was studied. In sterile native-soil assays, a Rifr mutant of P. putida showed no decrease in competitive fitness when compared with the wild-type parent. However, mutants of P. fluorescens were of two general categories. Group 1 showed no difference from the wild type in terms of growth rate, competitive fitness, and membrane protein composition. Group 2 showed a slower growth rate in both minimal and enriched media and an altered membrane protein profile. These mutants also demonstrated decreased competitive fitness compared with the wild-type strain. In live soil, the Rifr P. putida strain persisted throughout the 38-day test period with a decay rate of 0.7 log10 CFU/g of soil per 10 days. A group 1 Rifr P. fluorescens mutant maintained its inoculated titer for 7 to 10 days and then decayed at a rate of 0.2 to 0.4 log10 CFU/g of soil per 10 days. A group 2 Rifr P. fluorescens mutant remained at its titer for 1 to 5 days before decaying at a two- to threefold-faster rate. These findings indicate that rifampin resistance may not be an innocuous mutation in some pseudomonads and that marked strains should be compared with wild-type parents before being used as monitors of parental strain survival. Colonization of sterile soil with either the wild-type or mutant strain precluded normal colonization of the second added strain.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:3144244

  9. Mutants with enhanced nitrogenase activity in hydroponic Azospirillum brasilense-wheat associations.

    PubMed

    Pereg Gerk, L; Gilchrist, K; Kennedy, I R

    2000-05-01

    The effect of a mutation affecting flocculation, differentiation into cyst-like forms, and root colonization on nitrogenase expression by Azospirillum brasilense is described. The gene flcA of strain Sp7 restored these phenotypes in spontaneous mutants of both strains Sp7 and Sp245. Employing both constitutive pLA-lacZ and nifH-lacZ reporter fusions expressed in situ, the colony morphology, colonization pattern, and potential for nitrogenase activity of spontaneous mutants and flcA Tn5-induced mutants were established. The results of this study show that the ability of Sp7 and Sp245 mutant strains to remain in a vegetative form improved their ability to express nitrogenase activity in association with wheat in a hydroponic system. Restoring the cyst formation and colonization pattern to the spontaneous mutant Sp7-S reduced nitrogenase activity rates in association with plants to that of the wild-type Sp7. Although Tn5-induced flcA mutants showed higher potentials for nitrogenase expression than Sp7, their potentials were lower than that of Sp7-S, indicating that other factors in this strain contribute to its exceptional nitrogenase activity rates on plants. The lack of lateral flagella is not one of these factors, as Sp7-PM23, a spontaneous mutant impaired in swarming and lateral-flagellum production but not in flocculation, showed wild-type nitrogenase activity and expression. The results also suggest factors of importance in evolving an effective symbiosis between Azospirillum and wheat, such as increasing the availability of microaerobic niches along the root, increased supply of carbon sources by the plant, and the retention of the bacterial cells in vegetative form for faster metabolism. PMID:10788397

  10. Recombination-deficient mutant of Streptococcus faecalis

    SciTech Connect

    Yagi, Y.; Clewell, D.B.

    1980-08-01

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

  11. Mimas Showing False Colors #1

    NASA Technical Reports Server (NTRS)

    2005-01-01

    False color images of Saturn's moon, Mimas, reveal variation in either the composition or texture across its surface.

    During its approach to Mimas on Aug. 2, 2005, the Cassini spacecraft narrow-angle camera obtained multi-spectral views of the moon from a range of 228,000 kilometers (142,500 miles).

    The image at the left is a narrow angle clear-filter image, which was separately processed to enhance the contrast in brightness and sharpness of visible features. The image at the right is a color composite of narrow-angle ultraviolet, green, infrared and clear filter images, which have been specially processed to accentuate subtle changes in the spectral properties of Mimas' surface materials. To create this view, three color images (ultraviolet, green and infrared) were combined into a single black and white picture that isolates and maps regional color differences. This 'color map' was then superimposed over the clear-filter image at the left.

    The combination of color map and brightness image shows how the color differences across the Mimas surface materials are tied to geological features. Shades of blue and violet in the image at the right are used to identify surface materials that are bluer in color and have a weaker infrared brightness than average Mimas materials, which are represented by green.

    Herschel crater, a 140-kilometer-wide (88-mile) impact feature with a prominent central peak, is visible in the upper right of each image. The unusual bluer materials are seen to broadly surround Herschel crater. However, the bluer material is not uniformly distributed in and around the crater. Instead, it appears to be concentrated on the outside of the crater and more to the west than to the north or south. The origin of the color differences is not yet understood. It may represent ejecta material that was excavated from inside Mimas when the Herschel impact occurred. The bluer color of these materials may be caused by subtle differences in

  12. Sleep restores behavioral plasticity to Drosophila mutants

    PubMed Central

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

    2015-01-01

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

  13. Mutant Sodium Channel for Tumor Therapy

    PubMed Central

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

    2009-01-01

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

  14. Induction of sarcomas by mutant IDH2

    PubMed Central

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

    2013-01-01

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

  15. Ultrastructure of the outer membrane of Salmonella typhimurium bacteriocin-resistant mutants deficient in the 33K protein.

    PubMed Central

    Lounatmaa, K

    1979-01-01

    Outer membrane mutants of Salmonella typhimurium deficient in one, two, or three of the 33,000-dalton (33K), 34K, and 36K outer membrane proteins (7) were studied by using thin sectioning and freeze-fracturing electron microscopy techniques. The outer concave fracture face of all mutants deficient in the 33K protein had numerous particleless patches. In contrast to all previously examined 34K to 36K-deficient mutants, the 33K-deficient mutants showed marked heterogeneity in the size and distribution of such "empty" patches between cells of a culture. One mutant was deficient in both the 33K and the 34K to 36K "porin" protein complex; its outer membrane had very large particleless smooth areas. It is concluded that the 33K protein on one hand and the porin on the other are both able to form intramembraneous particles. Images PMID:378982

  16. Registration of two allelic erect leaf mutants of sorghum

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  17. Targeting Oncogenic Mutant p53 for Cancer Therapy

    PubMed Central

    Parrales, Alejandro; Iwakuma, Tomoo

    2015-01-01

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

  18. PARK2 patient neuroprogenitors show increased mitochondrial sensitivity to copper.

    PubMed

    Aboud, Asad A; Tidball, Andrew M; Kumar, Kevin K; Neely, M Diana; Han, Bingying; Ess, Kevin C; Hong, Charles C; Erikson, Keith M; Hedera, Peter; Bowman, Aaron B

    2015-01-01

    Poorly-defined interactions between environmental and genetic risk factors underlie Parkinson's disease (PD) etiology. Here we tested the hypothesis that human stem cell derived forebrain neuroprogenitors from patients with known familial risk for early onset PD will exhibit enhanced sensitivity to PD environmental risk factors compared to healthy control subjects without a family history of PD. Two male siblings (SM and PM) with biallelic loss-of-function mutations in PARK2 were identified. Human induced pluripotent stem cells (hiPSCs) from SM, PM, and four control subjects with no known family histories of PD or related neurodegenerative diseases were utilized. We tested the hypothesis that hiPSC-derived neuroprogenitors from patients with PARK2 mutations would show heightened cell death, mitochondrial dysfunction, and reactive oxygen species generation compared to control cells as a result of exposure to heavy metals (PD environmental risk factors). We report that PARK2 mutant neuroprogenitors showed increased cytotoxicity with copper (Cu) and cadmium (Cd) exposure but not manganese (Mn) or methyl mercury (MeHg) relative to control neuroprogenitors. PARK2 mutant neuroprogenitors also showed a substantial increase in mitochondrial fragmentation, initial ROS generation, and loss of mitochondrial membrane potential following Cu exposure. Our data substantiate Cu exposure as an environmental risk factor for PD. Furthermore, we report a shift in the lowest observable effect level (LOEL) for greater sensitivity to Cu-dependent mitochondrial dysfunction in patients SM and PM relative to controls, correlating with their increased genetic risk for PD. PMID:25315681

  19. 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

  20. A glutathione reductase mutant of yeast accumulates high levels of oxidized glutathione and requires thioredoxin for growth.

    PubMed Central

    Muller, E G

    1996-01-01

    A glutathione reductase null mutant of Saccharomyces cerevisiae was isolated in a synthetic lethal genetic screen for mutations which confer a requirement for thioredoxin. Yeast mutants that lack glutathione reductase (glr1 delta) accumulate high levels of oxidized glutathione and have a twofold increase in total glutathione. The disulfide form of glutathione increases 200-fold and represents 63% of the total glutathione in a glr1 delta mutant compared with only 6% in wild type. High levels of oxidized glutathione are also observed in a trx1 delta, trx2 delta double mutant (22% of total), in a glr1 delta, trx1 delta double mutant (71% of total), and in a glr1 delta, trx2 delta double mutant (69% of total). Despite the exceptionally high ratio of oxidized/reduced glutathione, the glr1 delta mutant grows with a normal cell cycle. However, either one of the two thioredoxins is essential for growth. Cells lacking both thioredoxins and glutathione reductase are not viable under aerobic conditions and grow poorly anaerobically. In addition, the glr1 delta mutant shows increased sensitivity to the thiol oxidant diamide. The sensitivity to diamide was suppressed by deletion of the TRX2 gene. The genetic analysis of thioredoxin and glutathione reductase in yeast runs counter to previous studies in Escherichia coli and for the first time links thioredoxin with the redox state of glutathione in vivo. Images PMID:8930901

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

    PubMed

    Mandal, Aninda; Datta, Animesh K

    2014-01-01

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

  2. The emergence of cooperation from a single cooperative mutant

    NASA Astrophysics Data System (ADS)

    Cremer, Jonas; Melbinger, Anna; Frey, Erwin

    2012-02-01

    Population structure is one central condition which promotes the stability of cooperation: If cooperators more likely interact with other cooperators (positive assortment), they keep most of their benefit for themselves and are less exploited by non-cooperators. However, positive assortment can only act successfully if cooperation is already well established in the population such that cooperative individuals can successfully assort. But how can cooperation emerge when starting with a single cooperative mutant? Here we study this issue for a generic situation of microbial systems where microbes regularly form new colonies and show strong population growth. We show how and when the dynamical interplay between colony formation, population growth and evolution within colonies can provoke the emergence of cooperation. In particular, the probability for a single cooperative mutant to succeed is robustly large when colony-formation is fast or comparable to the time-scale of growth within colonies; growth supports cooperation.[4pt] [1] A. Melbinger, J. Cremer, and E. Frey, Evolutionary game theory in growing populations, Phys. Rev. Lett. 105, 178101 (2010)[0pt] [2] J. Cremer, A. Melbinger, and E. Frey, Evolutionary and population dynamics: a coupled approach, arXiv:1108.2604

  3. Microscale insights into pneumococcal antibiotic mutant selection windows

    PubMed Central

    Sorg, Robin A.; Veening, Jan-Willem

    2015-01-01

    The human pathogen Streptococcus pneumoniae shows alarming rates of antibiotic resistance emergence. The basic requirements for de novo resistance emergence are poorly understood in the pneumococcus. Here we systematically analyse the impact of antibiotics on S. pneumoniae at concentrations that inhibit wild type cells, that is, within the mutant selection window. We identify discrete growth-inhibition profiles for bacteriostatic and bactericidal compounds, providing a predictive framework for distinction between the two classifications. Cells treated with bacteriostatic agents show continued gene expression activity, and real-time mutation assays link this activity to the development of genotypic resistance. Time-lapse microscopy reveals that antibiotic-susceptible pneumococci display remarkable growth and death bistability patterns in response to many antibiotics. We furthermore capture the rise of subpopulations with decreased susceptibility towards cell wall synthesis inhibitors (heteroresisters). We show that this phenomenon is epigenetically inherited, and that heteroresistance potentiates the accumulation of genotypic resistance. PMID:26514094

  4. Microscale insights into pneumococcal antibiotic mutant selection windows.

    PubMed

    Sorg, Robin A; Veening, Jan-Willem

    2015-01-01

    The human pathogen Streptococcus pneumoniae shows alarming rates of antibiotic resistance emergence. The basic requirements for de novo resistance emergence are poorly understood in the pneumococcus. Here we systematically analyse the impact of antibiotics on S. pneumoniae at concentrations that inhibit wild type cells, that is, within the mutant selection window. We identify discrete growth-inhibition profiles for bacteriostatic and bactericidal compounds, providing a predictive framework for distinction between the two classifications. Cells treated with bacteriostatic agents show continued gene expression activity, and real-time mutation assays link this activity to the development of genotypic resistance. Time-lapse microscopy reveals that antibiotic-susceptible pneumococci display remarkable growth and death bistability patterns in response to many antibiotics. We furthermore capture the rise of subpopulations with decreased susceptibility towards cell wall synthesis inhibitors (heteroresisters). We show that this phenomenon is epigenetically inherited, and that heteroresistance potentiates the accumulation of genotypic resistance. PMID:26514094

  5. Regulation of chloroplast biogenesis: the immutans mutant of Arabidopsis

    SciTech Connect

    Rodermel, Steven

    2015-11-16

    The immutans (im) variegation mutant of Arabidopsis is an ideal model to gain insight into factors that control chloroplast biogenesis. im defines the gene for PTOX, a plastoquinol terminal oxidase that participates in control of thylakoid redox. Here, we report that the im defect can be suppressed during the late stages of plant development by gigantea (gi2), which defines the gene for GIGANTEA (GI), a central component of the circadian clock that plays a poorly-understood role in diverse plant developmental processes. imgi2 mutants are late-flowering and display other well-known phenotypes associated with gi2, such as starch accumulation and resistance to oxidative stress. We show that the restoration of chloroplast biogenesis in imgi2 is caused by a developmental-specific de-repression of cytokinin signaling that involves crosstalk with signaling pathways mediated by gibberellin (GA) and SPINDLY (SPY), a GA response inhibitor. Suppression of the plastid defect in imgi2 is likely caused by a relaxation of excitation pressures in developing plastids by factors contributed by gi2, including enhanced rates of photosynthesis and increased resistance to oxidative stress. Interestingly, the suppression phenotype of imgi can be mimicked by crossing im with the starch accumulation mutant, sex1, perhaps because sex1 utilizes pathways similar to gi. We conclude that our studies provide a direct genetic linkage between GIGANTEA and chloroplast biogenesis, and we construct a model of interactions between signaling pathways mediated by gi, GA, SPY, cytokinins, and sex1 that are required for chloroplast biogenesis.

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

    PubMed

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

    2012-01-27

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

  7. Identification of Mutants of Arabidopsis Defective in Acclimation of Photosynthesis to the Light Environment1

    PubMed Central

    Walters, Robin G.; Shephard, Freya; Rogers, Jennifer J.M.; Rolfe, Stephen A.; Horton, Peter

    2003-01-01

    In common with many other higher plant species, Arabidopsis undergoes photosynthetic acclimation, altering the composition of the photosynthetic apparatus in response to fluctuations in its growth environment. The changes in photosynthetic function that result from acclimation can be detected in a noninvasive manner by monitoring chlorophyll (Chl) fluorescence. This technique has been used to develop a screen that enables the rapid identification of plants defective at ACCLIMATION OF PHOTOSYNTHESIS TO THE ENVIRONMENT (APE) loci. The application of this screen to a population of T-DNA-transformed Arabidopsis has successfully led to the identification of a number of mutant lines with altered Chl fluorescence characteristics. Analysis of photosynthesis and pigment composition in leaves from three such mutants showed that they had altered acclimation responses to the growth light environment, each having a distinct acclimation-defective phenotype, demonstrating that screening for mutants using Chl fluorescence is a viable strategy for the investigation of acclimation. Sequencing of the genomic DNA flanking the T-DNA elements showed that in the ape1 mutant, a gene was disrupted that encodes a protein of unknown function but that appears to be specific to photosynthetic organisms, whereas the ape2 mutant carries an insertion in the region of the TPT gene encoding the chloroplast inner envelope triose phosphate/phosphate translocator. PMID:12586872

  8. Enantiomer-specific and paracrine leukemogenicity of mutant IDH metabolite 2-hydroxyglutarate.

    PubMed

    Chaturvedi, A; Araujo Cruz, M M; Jyotsana, N; Sharma, A; Goparaju, R; Schwarzer, A; Görlich, K; Schottmann, R; Struys, E A; Jansen, E E; Rohde, C; Müller-Tidow, C; Geffers, R; Göhring, G; Ganser, A; Thol, F; Heuser, M

    2016-08-01

    Canonical mutations in IDH1 and IDH2 produce high levels of the R-enantiomer of 2-hydroxyglutarate (R-2HG), which is a competitive inhibitor of α-ketoglutarate (αKG)-dependent enzymes and a putative oncometabolite. Mutant IDH1 collaborates with HoxA9 to induce monocytic leukemia in vivo. We used two mouse models and a patient-derived acute myeloid leukemia xenotransplantation (PDX) model to evaluate the in vivo transforming potential of R-2HG, S-2HG and αKG independent of the mutant IDH1 protein. We show that R-2HG, but not S-2HG or αKG, is an oncometabolite in vivo that does not require the mutant IDH1 protein to induce hyperleukocytosis and to accelerate the onset of murine and human leukemia. Thus, circulating R-2HG acts in a paracrine manner and can drive the expansion of many different leukemic and preleukemic clones that may express wild-type IDH1, and therefore can be a driver of clonal evolution and diversity. In addition, we show that the mutant IDH1 protein is a stronger oncogene than R-2HG alone when comparable intracellular R-2HG levels are achieved. We therefore propose R-2HG-independent oncogenic functions of mutant IDH1 that may need to be targeted in addition to R-2HG production to exploit the full therapeutic potential of IDH1 inhibition. PMID:27063596

  9. Light-stimulated cotyledon expansion in the blu3 and hy4 mutants of Arabidopsis thaliana.

    PubMed

    Blum, D E; Neff, M M; Van Volkenburgh, E

    1994-08-01

    Cotyledon expansion in response to blue light was compared for wild-type Arabidopsis thaliana (L.) Heynh. and the mutants blu3 and hy4, which show reduced inhibition of hypocotyl growth in blue light. White, blue, and red light stimulated cotyledon expansion in both intact and excised cotyledons of wild-type seedlings (ecotypes No-0, WS, Co-0, La-er). Cotyledons on intact blu3 and hy4 seedlings did not grow as well as those on the wild type in response to blue light, but pretreatment of blu3 seedlings with low fluence rates of red light increased their responsiveness to blue light. Excision of cotyledons alleviated the mutant phenotype so that both mutant and wild-type cotyledons grew equally well in blue light. The loss of the mutant cotyledon phenotype upon excision indicates that the blu3 and hy4 lesions affect cotyledon expansion indirectly via a whole-plant response to light. Furthermore, the ability of excised, mutant cotyledons to grow normally in blue light shows that this growth response to blue light is mediated by a photosystem other than the ones impaired by the blu3 and hy4 lesions. PMID:7972499

  10. On the origin of a slowly reversible fluorescence decay component in the Arabidopsis npq4 mutant.

    PubMed

    Dall'Osto, Luca; Cazzaniga, Stefano; Wada, Masamitsu; Bassi, Roberto

    2014-04-19

    Over-excitation of photosynthetic apparatus causing photoinhibition is counteracted by non-photochemical quenching (NPQ) of chlorophyll fluorescence, dissipating excess absorbed energy into heat. The PsbS protein plays a key role in this process, thus making the PsbS-less npq4 mutant unable to carry out qE, the major and most rapid component of NPQ. It was proposed that npq4 does perform qE-type quenching, although at lower rate than WT Arabidopsis. Here, we investigated the kinetics of NPQ in PsbS-depleted mutants of Arabidopsis. We show that red light was less effective than white light in decreasing maximal fluorescence in npq4 mutants. Also, the kinetics of fluorescence dark recovery included a decay component, qM, exhibiting the same amplitude and half-life in both WT and npq4 mutants. This component was uncoupler-sensitive and unaffected by photosystem II repair or mitochondrial ATP synthesis inhibitors. Targeted reverse genetic analysis showed that traits affecting composition of the photosynthetic apparatus, carotenoid biosynthesis and state transitions did not affect qM. This was depleted in the npq4phot2 mutant which is impaired in chloroplast photorelocation, implying that fluorescence decay, previously described as a quenching component in npq4 is, in fact, the result of decreased photon absorption caused by chloroplast relocation rather than a change in the activity of quenching reactions. PMID:24591708

  11. On the origin of a slowly reversible fluorescence decay component in the Arabidopsis npq4 mutant

    PubMed Central

    Dall'Osto, Luca; Cazzaniga, Stefano; Wada, Masamitsu; Bassi, Roberto

    2014-01-01

    Over-excitation of photosynthetic apparatus causing photoinhibition is counteracted by non-photochemical quenching (NPQ) of chlorophyll fluorescence, dissipating excess absorbed energy into heat. The PsbS protein plays a key role in this process, thus making the PsbS-less npq4 mutant unable to carry out qE, the major and most rapid component of NPQ. It was proposed that npq4 does perform qE-type quenching, although at lower rate than WT Arabidopsis. Here, we investigated the kinetics of NPQ in PsbS-depleted mutants of Arabidopsis. We show that red light was less effective than white light in decreasing maximal fluorescence in npq4 mutants. Also, the kinetics of fluorescence dark recovery included a decay component, qM, exhibiting the same amplitude and half-life in both WT and npq4 mutants. This component was uncoupler-sensitive and unaffected by photosystem II repair or mitochondrial ATP synthesis inhibitors. Targeted reverse genetic analysis showed that traits affecting composition of the photosynthetic apparatus, carotenoid biosynthesis and state transitions did not affect qM. This was depleted in the npq4phot2 mutant which is impaired in chloroplast photorelocation, implying that fluorescence decay, previously described as a quenching component in npq4 is, in fact, the result of decreased photon absorption caused by chloroplast relocation rather than a change in the activity of quenching reactions. PMID:24591708

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

    PubMed Central

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

    2015-01-01

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

  13. Ultrastructure of the ocellar visual system in normal and mutant Drosophila melanogaster.

    PubMed

    Stark, W S; Sapp, R; Carlson, S D

    1989-05-01

    unaffected. Mutants best known for their compound eye defects were examined. The norpA mutant loses its ocellar rhabdomeres with age but has normal feed forward and feed back synapses. This normal synaptology prevails despite the electrophysiological defects in norpA ocelli reported earlier. The rdgABS12 mutant has poorly formed ocellar receptors which show some degeneration with age but synapses survive. The trp. rdgBKS222 and rgdAPC47 mutants are essentially normal with respect to structure and survival of ocellar receptors and synapses. PMID:2500507

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

    SciTech Connect

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

    1991-04-15

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

  15. Poliovirus: Generation and Characterization of Mutants

    PubMed Central

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

    2016-01-01

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

  16. Molecular analysis of ethylene-insensitive mutants in arabidopsis

    SciTech Connect

    Meyerowitz, E.

    1991-01-01

    The subject of this study is the biochemical basis of ethylene reception. The Arabidopsis thaliana ETR gene codes for the ethylene receptor or is involved in transduction of the ethylene-generated signal. We have cloned an etr mutation which results in a decrease in the ethylene response of the plant, with a decrease in ethylene binding of about five-fold. Two genes have been found in the cloned region which confer resistance. By sequence analysis, the first protein contains three distinct regions: a transmembrane region, a serine/threonine protein kinase region, and a control region similar to the RAS-binding region of yeast adenylate cyclase. The second protein contains a zinc-finger; since sequence of the first protein shows no mutant-dependent changes, and transition metals have been implicated in ethylene binding, this protein could be the ETR gene product. However, no mutant dependent differences have been found in this protein, either. The mutation could be upstream of the coding region of either gene and involve regulatory elements, so we are continuing to sequence. (MHB)

  17. Identification of Drosophila Mutants Affecting Defense to an Entomopathogenic Fungus

    PubMed Central

    Lu, Hsiao-Ling; Wang, Jonathan B.; Brown, Markus A.; Euerle, Christopher; St. Leger, Raymond J.

    2015-01-01

    Fungi cause the majority of insect disease. However, to date attempts to model host–fungal interactions with Drosophila have focused on opportunistic human pathogens. Here, we performed a screen of 2,613 mutant Drosophila lines to identify host genes affecting susceptibility to the natural insect pathogen Metarhizium anisopliae (Ma549). Overall, 241 (9.22%) mutant lines had altered resistance to Ma549. Life spans ranged from 3.0 to 6.2 days, with females being more susceptible than males in all lines. Speed of kill correlated with within-host growth and onset of sporulation, but total spore production is decoupled from host genotypes. Results showed that mutations affected the ability of Drosophila to restrain rather than tolerate infections and suggested trade-offs between antifungal and antibacterial genes affecting cuticle and gut structural barriers. Approximately, 13% of mutations where in genes previously associated with host pathogen interactions. These encoded fast-acting immune responses including coagulation, phagocytosis, encapsulation and melanization but not the slow-response induction of anti-fungal peptides. The non-immune genes impact a wide variety of biological functions, including behavioral traits. Many have human orthologs already implicated in human disorders; while others were mutations in protein and non-protein coding genes for which disease resistance was the first biological annotation. PMID:26202798

  18. Iron deficiency anemia's effect on bone formation in zebrafish mutant.

    PubMed

    Bo, Lin; Liu, Zhichun; Zhong, Yingbin; Huang, Jian; Chen, Bin; Wang, Han; Xu, Youjia

    2016-07-01

    Iron is one of the essential elements of life. Iron metabolism is related to bone metabolism. Previous studies have confirmed that iron overload is a risk factor for osteoporosis. But the correlation between iron deficiency and bone metabolism remains unclear. Ferroportin 1 is identified as a cellular iron exporter and required for normal iron cycling. In zebrafish, the mutant of ferroportin 1 gene (fpn1), weh(tp85c) exhibited the defective iron transport, leading to developing severe hypochromic anemia. We used weh(tp85c) as a model for investigating iron deficiency and bone metabolism. In this study, we examined the morphology of the developing cartilage and vertebrae of the Weh(tp85) compared to the wild type siblings by staining the larvae with alcian blue for cartilage and alizarin red for the bone. In addition, we evaluated the expression patterns of the marker genes of bone development and cell signaling in bone formation. Our results showed that weh(tp85c) mutant larvae exhibited the defects in bone formation, revealing by decreases in the number of calcified vertebrae along with decreased expression of osteoblast novel genes: alpl, runx2a and col1a1a and BMPs signaling genes in osteoblast differentiation: bmp2a and bmp2b. Our data suggest that iron deficiency anemia affects bone formation, potentially through the BMPs signaling pathway in zebrafish. PMID:27184405

  19. 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

  20. The zebrafish merovingian mutant reveals a role for pH regulation in hair cell toxicity and function.

    PubMed

    Stawicki, Tamara M; Owens, Kelly N; Linbo, Tor; Reinhart, Katherine E; Rubel, Edwin W; Raible, David W

    2014-07-01

    Control of the extracellular environment of inner ear hair cells by ionic transporters is crucial for hair cell function. In addition to inner ear hair cells, aquatic vertebrates have hair cells on the surface of their body in the lateral line system. The ionic environment of these cells also appears to be regulated, although the mechanisms of this regulation are less understood than those of the mammalian inner ear. We identified the merovingian mutant through genetic screening in zebrafish for genes involved in drug-induced hair cell death. Mutants show complete resistance to neomycin-induced hair cell death and partial resistance to cisplatin-induced hair cell death. This resistance is probably due to impaired drug uptake as a result of reduced mechanotransduction ability, suggesting that the mutants have defects in hair cell function independent of drug treatment. Through genetic mapping we found that merovingian mutants contain a mutation in the transcription factor gcm2. This gene is important for the production of ionocytes, which are cells crucial for whole body pH regulation in fish. We found that merovingian mutants showed an acidified extracellular environment in the vicinity of both inner ear and lateral line hair cells. We believe that this acidified extracellular environment is responsible for the defects seen in hair cells of merovingian mutants, and that these mutants would serve as a valuable model for further study of the role of pH in hair cell function. PMID:24973752

  1. Characterization and fine mapping of a light-dependent leaf lesion mimic mutant 1 in rice.

    PubMed

    Wang, Jing; Ye, Bangquan; Yin, Junjie; Yuan, Can; Zhou, Xiaogang; Li, Weitao; He, Min; Wang, Jichun; Chen, Weilan; Qin, Peng; Ma, Bintian; Wang, Yuping; Li, Shigui; Chen, Xuewei

    2015-12-01

    Plants that spontaneously produce lesion mimics or spots, without any signs of obvious adversity, such as pesticide and mechanical damage, or pathogen infection, are so-called lesion mimic mutants (lmms). In rice, many lmms exhibit enhanced resistance to pathogens, which provides a unique opportunity to uncover the molecular mechanism underlying lmms. We isolated a rice light-dependent leaf lesion mimic mutant 1 (llm1). Lesion spots appeared in the leaves of the llm1 mutant at the tillering stage. Furthermore, the mutant llm1 had similar agronomic traits to wild type rice. Trypan blue and diamiobenzidine staining analyses revealed that the lesion spot formation on the llm1 mutant was due to programmed cell death and reactive oxygen species. The chloroplasts were severely damaged in the llm1 mutant, suggesting that chloroplast damage was associated with the formation of lesion spots in llm1. More importantly, llm1 exhibited enhanced resistance to bacterial blight pathogens within increased expression of pathogenesis related genes (PRs). Using a map-based cloning approach, we delimited the LLM1 locus to a 121-kb interval between two simple sequence repeat markers, RM17470 and RM17473, on chromosome 4. We sequenced the candidate genes on the interval and found that a base mutation had substituted adenine phosphate for thymine in the last exon of LOC_Os04g52130, which led to an amino acid change (Asp(388) to Val) in the llm1 mutant. Our investigation showed that the putative coproporphyrinogen III oxidase (CPOX) encoded by LOC_Os04g52130 was produced by LLM1 and that amino acid Asp(388) was essential for CPOX function. Our study provides the basis for further investigations into the mechanism underlying lesion mimic initiation associated with LLM1. PMID:26410574

  2. Tyrosyl-DNA phosphodiesterase I catalytic mutants reveal an alternative nucleophile that can catalyze substrate cleavage.

    PubMed

    Comeaux, Evan Q; Cuya, Selma M; Kojima, Kyoko; Jafari, Nauzanene; Wanzeck, Keith C; Mobley, James A; Bjornsti, Mary-Ann; van Waardenburg, Robert C A M

    2015-03-01

    Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3'-DNA adducts, such as the 3'-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (His(nuc)) that attacks DNA adducts to form a covalent 3'-phosphohistidyl intermediate and a general acid/base His (His(gab)), which resolves the Tdp1-DNA linkage. A His(nuc) to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of His(gab) to Arg. However, here we report that expression of the yeast His(nuc)Ala (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 His(gab) mutants, including H432N and the SCAN1-related H432R. Moreover, the His(nuc)Ala mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the His(nuc)Phe mutant was catalytically inactive and suppressed His(gab) mutant-induced toxicity. These data suggest that the activity of another nucleophile when His(nuc) is replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to His(nuc), can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate. PMID:25609251

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

    PubMed

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

    2015-12-01

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

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

    PubMed Central

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

    2010-01-01

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

  5. Arabidopsis thaliana siRNA biogenesis mutants have the lower frequency of homologous recombination.

    PubMed

    Yao, Youli; Bilichak, Andriy; Golubov, Andrey; Kovalchuk, Igor

    2016-07-01

    Small interfering RNAs (siRNAs) are involved in the regulation of plant development and response to stress. We have previously shown that mutants impaired in Dicer-like 2 (DCL2), DCL3 and DCL4, RDR2, RDR6 and NPRD1 are partially impaired in their response to stress and dcl2 and dcl3 plants are also impaired in transgenerational response to stress, including changes in homologous recombination frequency (HRF). Here, we have analyzed genome stability of dcl2, dcl3, dcl4, dcl2 dcl3, dcl2 dcl3 dcl4 and rdr6 mutants by measuring the non-induced and the stress-induced recombination frequency. We found that all mutants had the lower spontaneous HRF. The analysis of strand breaks showed that all tested Arabidopsis mutants had a higher level of spontaneous strand breaks, suggesting that the lower HRF is not due to the unusually low level of breaks. Exposure to methyl methane sulfonate (MMS) resulted in an increase in the level of strand breaks in wild-type plants and a decrease in mutants. All mutants had the higher methylation of cytosines at CpG sites under non-induced conditions. Exposure to MMS resulted in a decrease in methylation level in wild-type plants and an increase in methylation in all dcl mutants. The expression of several DNA repair genes was altered in dcl4 plants under non-induced and induced conditions. Our data suggest that siRNA biogenesis may be essential for the maintenance of the genome stability and stress response in Arabidopsis. PMID:26901311

  6. Phenotype Sequencing: Identifying the Genes That Cause a Phenotype Directly from Pooled Sequencing of Independent Mutants

    PubMed Central

    Harper, Marc A.; Chen, Zugen; Toy, Traci; Machado, Iara M. P.; Nelson, Stanley F.; Liao, James C.; Lee, Christopher J.

    2011-01-01

    Random mutagenesis and phenotype screening provide a powerful method for dissecting microbial functions, but their results can be laborious to analyze experimentally. Each mutant strain may contain 50–100 random mutations, necessitating extensive functional experiments to determine which one causes the selected phenotype. To solve this problem, we propose a “Phenotype Sequencing” approach in which genes causing the phenotype can be identified directly from sequencing of multiple independent mutants. We developed a new computational analysis method showing that 1. causal genes can be identified with high probability from even a modest number of mutant genomes; 2. costs can be cut many-fold compared with a conventional genome sequencing approach via an optimized strategy of library-pooling (multiple strains per library) and tag-pooling (multiple tagged libraries per sequencing lane). We have performed extensive validation experiments on a set of E. coli mutants with increased isobutanol biofuel tolerance. We generated a range of sequencing experiments varying from 3 to 32 mutant strains, with pooling on 1 to 3 sequencing lanes. Our statistical analysis of these data (4099 mutations from 32 mutant genomes) successfully identified 3 genes (acrB, marC, acrA) that have been independently validated as causing this experimental phenotype. It must be emphasized that our approach reduces mutant sequencing costs enormously. Whereas a conventional genome sequencing experiment would have cost $7,200 in reagents alone, our Phenotype Sequencing design yielded the same information value for only $1200. In fact, our smallest experiments reliably identified acrB and marC at a cost of only $110–$340. PMID:21364744

  7. Comparison between NOx Evolution Mechanisms of Wild-Type and nr1 Mutant Soybean Leaves 1

    PubMed Central

    Klepper, Lowell

    1990-01-01

    The nr1 soybean (Glycine max [L.] Merr.) mutant does not contain the two constitutive nitrate reductases, one of which is responsible for enzymic conversion of nitrite to NOx (NO + NO2). It was tested for possible nonenzymic NOx formation and evolution because of known chemical reactions between NO2− and plant metabolites and the instability of nitrous acid. It did not evolve NOx during the in vivo NR assay, but intact leaves did evolve small amounts of NOx under dark, anaerobic conditions. Experiments were conducted to compare NO3− reduction, NO2− accumulation, and the NOx evolution processes of the wild type (cv Williams) and the nr1 mutant. In vivo NR assays showed that wild-type leaves had three times more NO3− reducing capacity than the nr1 mutant. NOx evolution from intact, anerobic nr1 leaves was approximately 10 to 20% that from wild-type leaves. Nitrite content of the nr1 mutant leaves was usually higher than wild type due to low NOx evolution. Lag times and threshold NO2− concentrations for NOx evolution were similar for the two genotypes. While only 1 to 2% of NOx from wild type is NO2, the nr1 mutant evolved 15 to 30% NO2. The kinetic patterns of NOx evolution with time weré completely different for the mutant and wild type. Comparisons of light and heat treatments also gave very different results. It is generally accepted that the NOx evolution by wild type is primarily an enzymic conversion of NO2− to NO. However, this report concludes that NOx evolution by the nr1 mutant was due to nonenzymic, chemical reactions between plant metabolites and accumulated NO2− and/or decomposition of nitrous acid. Nonenzymic NOx evolution probably also occurs in wild type to a degree but could be easily masked by high rates of the enzymic process. PMID:16667445

  8. In vivo pathogenic role of mutant SOD1 localized in the mitochondrial intermembrane space.

    PubMed

    Igoudjil, Anissa; Magrané, Jordi; Fischer, Lindsey R; Kim, Hyun Jeong; Hervias, Isabel; Dumont, Magali; Cortez, Czrina; Glass, Jonathan D; Starkov, Anatoly A; Manfredi, Giovanni

    2011-11-01

    Mutations in Cu,Zn superoxide dismutase (SOD1) are associated with familial amyotrophic lateral sclerosis (ALS). Mutant SOD1 causes a complex array of pathological events, through toxic gain of function mechanisms, leading to selective motor neuron degeneration. Mitochondrial dysfunction is among the well established toxic effects of mutant SOD1, but its mechanisms are just starting to be elucidated. A portion of mutant SOD1 is localized in mitochondria, where it accumulates mostly on the outer membrane and inside the intermembrane space (IMS). Evidence in cultured cells suggests that mutant SOD1 in the IMS causes mitochondrial dysfunction and compromises cell viability. Therefore, to test its pathogenic role in vivo we generated transgenic mice expressing G93A mutant or wild-type (WT) human SOD1 targeted selectively to the mitochondrial IMS (mito-SOD1). We show that mito-SOD1 is correctly localized in the IMS, where it oligomerizes and acquires enzymatic activity. Mito-G93ASOD1 mice, but not mito-WTSOD1 mice, develop a progressive disease characterized by body weight loss, muscle weakness, brain atrophy, and motor impairment, which is more severe in females. These symptoms are associated with reduced spinal motor neuron counts and impaired mitochondrial bioenergetics, characterized by decreased cytochrome oxidase activity and defective calcium handling. However, there is no evidence of muscle denervation, a cardinal pathological feature of ALS. Together, our findings indicate that mutant SOD1 in the mitochondrial IMS causes mitochondrial dysfunction and neurodegeneration, but per se it is not sufficient to cause a full-fledged ALS phenotype, which requires the participation of mutant SOD1 localized in other cellular compartments. PMID:22049426

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

    PubMed

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

    2012-01-01

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

  10. Auxin physiology of the tomato mutant diageotropica

    NASA Technical Reports Server (NTRS)

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

    1989-01-01

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

  11. Auxin physiology of the tomato mutant diageotropical

    SciTech Connect

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

    1989-11-01

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

  12. Method for rapid isolation of sensitive mutants

    DOEpatents

    Freyer, James P.

    1997-01-01

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

  13. Method for rapid isolation of sensitive mutants

    DOEpatents

    Freyer, J.P.

    1997-07-29

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

  14. Isolation of mouse cell proteoglycan mutants

    SciTech Connect

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

    1986-05-01

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

  15. Too Many Mutants with Multiple Mutations

    PubMed Central

    Drake, John W.

    2007-01-01

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

  16. Neurobehavioral Mutants Identified in an ENU Mutagenesis Project

    SciTech Connect

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

    2007-01-01

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

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

    PubMed Central

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

    1994-01-01

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

  18. 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

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

    PubMed

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

    2002-06-01

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

  20. 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

  1. Structure of a His170Tyr mutant of thermostable pNPPase from Geobacillus stearothermophilus

    PubMed Central

    Shen, Tiantian; Guo, Zheng; Ji, Chaoneng

    2014-01-01

    Using directed evolution based on random mutagenesis and heat-treated selection, a thermostable His170Tyr mutant of Geobacillus stearothermophilus thermostable p-nitrophenylphosphatase (TpNPPase) was obtained. The temperature at which the His170Tyr mutant lost 50% of its activity (T 1/2) was found to be 4.40 K higher than that of wild-type TpNPPase, and the melting temperature of the His170Tyr mutant increased by 2.39 K. The crystal structure of the His170Tyr mutant was then determined at 2.0 Å resolution in the presence of a sodium ion and a sulfate ion in the active site. The cap domain of chain B shows a half-closed conformation. The hydrophobic side chain of the mutated residue, the hydroxyphenyl group, forms a hydrophobic contact with the methyl group of Ala166. This hydrophobic interaction was found using the Protein Interactions Calculator (PIC) web server with an interaction distance of 4.6 Å, and might be a key factor in the thermostabilization of the His170Tyr mutant. This study potentially offers a molecular basis for both investigation of the catalytic mechanism and thermostable protein engineering. PMID:24915075

  2. Ospapst1, a useful mutant for identifying seed purity and authenticity in hybrid rice.

    PubMed

    Lv, Qundan; Xu, Jiming; Wu, Ping

    2013-07-01

    The stability and completeness of male sterility is still a challenge in some male sterile rice lines, especially those of photoperiod/thermo-sensitive genic male sterility (P/TGMS). Leaf color marker is a widely practiced approach to reduce the impact of self-pollinated seeds of male sterile lines. The papst1 is a leaf color mutant. The newly emerged leaves of papst1 are chlorosis and have an impaired photosynthesis. But the other agronomic traits, such as germination rate, duration of maturation and seed weight, are not changed. The papst1/PAPST1 F₁ showed the wild-type leaf phenotype. The papst1/PAPST1 F₂ progenies displayed an approximately 3:1 segregation ratio of WT phenotype:mutant phenotype (72: 28, χ(2) = 0.48, p > 0.05), suggesting that papst1 mutant phenotype is caused by a single repressive gene. Map-based cloning and sequencing analysis revealed that a point mutation was occurred in Os01 g16040 (OsPAPST1). Given these results, the Ospapst1 mutant is a useful mutant for identifying seed purity and authenticity in hybrid rice. PMID:23656867

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

    PubMed Central

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

    1998-01-01

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

  4. Mutant NRASQ61 shares signaling similarities across various cancer types – potential implications for future therapies

    PubMed Central

    Vujic, Igor; Posch, Christian; Sanlorenzo, Martina; Yen, Adam J.; Tsumura, Aaron; Kwong, Andrew; Feichtenschlager, Valentin; Lai, Kevin; Arneson, Douglas V.; Rappersberger, Klemens; Ortiz-Urda, Susana M.

    2014-01-01

    Oncogenic mutations in the Neuroblastoma Rat Sarcoma oncogene (NRAS) are frequent in melanoma, but are also found in several other cancer types, such as lung cancer, neuroblastoma and colon cancer. We designed our study to analyze changes in NRAS mutant tumor cells derived from malignancies other than melanoma. A variety of small molecule inhibitors as well as their combinations was tested in order to find beneficial inhibitory modalities in NRASQ61 mutant lung cancer and neuroblastoma cell lines. Signaling changes after incubation with inhibitors were studied and compared to those found in NRAS mutant melanoma. All cell lines were most sensitive to inhibition in the MAPK pathway with the MEK inhibitor trametinib. MEK/AKT and MEK/CDK4,6 inhibitor combinations did not show any beneficial effects in vitro. However, we observed strong synergism combining MEK and PI3K/mTOR inhibitors in all cell lines. Our study provides evidence that NRAS mutant cancers share signaling similarities across different malignancies. We demonstrate that dual pathway inhibition of the MAPK and PI3K/AKT/mTOR pathway synergistically reduces cell viability in NRAS mutant cancers regardless of their tissue origin. Our results suggest that such inhibitor combinations may be a potential treatment option for non-melanoma tumors harboring activating NRAS mutations. PMID:25277205

  5. 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

  6. Swimming Behaviour and Otolith Characteristics of wildtype and mutant Zebrafish (AIE) under diminished Gravity Conditions

    NASA Astrophysics Data System (ADS)

    Weigele, J.; Anken, R.; Hilbig, R.

    During microgravity humans often suffer from sensorimotor disorders e g motion sickness a kinetosis Using fish as vertebrate model systems we could previously provide ample evidence that the individually different susceptibility to such disorders is based on an individually differently pronounced asymmetric mineralisation calcification of inner ear stones otoliths In the course of a preliminary study we subjected mutant zebrafish Danio rerio due to malformation of the inner ear - see below - this mutant was termed Asymmetric Inner Ear AIE to diminished gravity conditions during parabolic aircraft flight PF As compared to wildtype WT animals the mutants showed a pronounced kinetotic behaviour The gross-morphology of the inner ears of AIE and WT animals strikingly differed In WT specimens the saccular otoliths were located at the periphery of the inner ear whereas the utricular stones were positioned mediad as it is usually the case in teleosts in most AIE animals dissected however the respective otoliths were positioned in an opposite arrangement Moreover the mutants sported transparent otoliths whereas the otoliths of WT specimens had an opaque appearance This finding clearly indicates that mutant otoliths differed from wildtype ones in their lattice structure i e the calcium carbonate polymorph and thus the compostion of the proteinacious matrix which is a template for calcium carbonate deposition In the course of the present study the PF experiment is scheduled to be carried out in March 2006 we intend to statistically verify

  7. Transcriptional profiling of human smooth muscle cells infected with gingipain and fimbriae mutants of Porphyromonas gingivalis.

    PubMed

    Zhang, Boxi; Sirsjö, Allan; Khalaf, Hazem; Bengtsson, Torbjörn

    2016-01-01

    Porphyromonas gingivalis (P. gingivalis) is considered to be involved in the development of atherosclerosis. However, the role of different virulence factors produced by P. gingivalis in this process is still uncertain. The aim of this study was to investigate the transcriptional profiling of human aortic smooth muscle cells (AoSMCs) infected with wild type, gingipain mutants or fimbriae mutants of P. gingivalis. AoSMCs were exposed to wild type (W50 and 381), gingipain mutants (E8 and K1A), or fimbriae mutants (DPG-3 and KRX-178) of P. gingivalis. We observed that wild type P. gingivalis changes the expression of a considerable larger number of genes in AoSMCs compare to gingipain and fimbriae mutants, respectively. The results from pathway analysis revealed that the common differentially expressed genes for AoSMCs infected by 3 different wild type P. gingivalis strains were enriched in pathways of cancer, cytokine-cytokine receptor interaction, regulation of the actin cytoskeleton, focal adhesion, and MAPK signaling pathway. Disease ontology analysis showed that various strains of P. gingivalis were associated with different disease profilings. Our results suggest that gingipains and fimbriae, especially arginine-specific gingipain, produced by P. gingivalis play important roles in the association between periodontitis and other inflammatory diseases, including atherosclerosis. PMID:26907358

  8. High-level production of extracellular lipase by Yarrowia lipolytica mutants from methyl oleate.

    PubMed

    Darvishi, Farshad; Destain, Jacqueline; Nahvi, Iraj; Thonart, Philippe; Zarkesh-Esfahani, Hamid

    2011-10-01

    The yeast Yarrowia lipolytica degrades efficiently low-cost hydrophobic substrates for the production of various added-value products such as lipases. To obtain yeast strains producing high levels of extracellular lipase, Y. lipolytica DSM3286 was subjected to mutation using ethyl methanesulfonate (EMS) and ultraviolet (UV) light. Twenty mutants were selected out of 1600 mutants of Y. lipolytica treated with EMS and UV based on lipase production ability on selective medium. A new industrial medium containing methyl oleate was optimized for lipase production. In the 20 L bioreactor containing new industrial medium, one UV mutant (U6) produced 356 U/mL of lipase after 24h, which is about 10.5-fold higher than that produced by the wild type strain. The properties of the mutant lipase were the same as those of the wild type: molecular weight 38 kDa, optimum temperature 37°C and optimum pH 7. Furthermore, the nucleotide sequences of extracellular lipase gene (LIP2) in wild type and mutant strains were determined. Only two silent substitutions at 362 and 385 positions were observed in the ORF region of LIP2. Two single substitutions and two duplications of the T nucleotide were also detected in the promoter region. LIP2 sequence comparison of the Y. lipolytica DSM3286 and U6 strains shows good targets to effective DNA recombinant for extracellular lipase of Y. lipolytica. PMID:21324386

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

    PubMed

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

    2013-09-01

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

  10. Misassembled mutant DeltaF508 CFTR in the distal secretory pathway alters cellular lipid trafficking.

    PubMed

    Gentzsch, Martina; Choudhury, Amit; Chang, Xiu-bao; Pagano, Richard E; Riordan, John R

    2007-02-01

    Most patients with cystic fibrosis (CF) have a single codon deletion (DeltaF508) in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that impairs assembly of the multidomain glycoprotein. The mutant protein escapes endoplasmic reticulum (ER) quality control at low temperature, but is rapidly cleared from the distal secretory pathway and degraded in lysosomes. CF cells accumulate free cholesterol similar to Niemann-Pick disease type C cells. We show that this lipid alteration is caused by the presence of misassembled mutant CFTR proteins, including DeltaF508, in the distal secretory pathway rather than the absence of functional CFTR. By contrast, cholesterol distribution is not changed by either D572N CFTR, which does not mature even at low temperature, or G551D, which is processed normally but is inactive. On expression of the DeltaF508 mutant, cholesterol and glycosphingolipids accumulate in punctate endosomal structures and cholesterol esters are reduced, indicating a block in the translocation of cholesterol to the ER for esterification. This is overcome by Rab9 overexpression, resulting in clearance of accumulating intracellular cholesterol. Similar but less pronounced alterations in intracellular cholesterol distribution are observed on expression of a temperature-rescued mutant variant of the related ATP-binding cassette (ABC) protein multidrug resistance-associated protein 1 (MRP1). Thus, on escape from ER quality control, misassembled mutants of CFTR and MRP1 impair lipid homeostasis in endocytic compartments. PMID:17213331

  11. A new method for screening and isolation of hypersecretion mutants in Aspergillus niger.

    PubMed

    Weenink, Xavier O; Punt, Peter J; van den Hondel, Cees A M J J; Ram, Arthur F J

    2006-02-01

    Although filamentous fungi have a unique property of secreting a large amount of homologous extracellular proteins, the use of filamentous fungi as hosts for the production of heterologous proteins is limited because of the low production levels that are generally reached. Here, we report a general screening method for the isolation of mutants with increased protein production levels. The screening method makes use of an Aspergillus niger strain that lacks the two major amylolytic enzymes, glucoamylase (GlaA) and acid amylase (AamA). The double-mutant strain grows poorly on starch and its growth is restored after reintroducing the catalytic part of the glucoamylase gene (GlaA512). We show that the fusion of a heterologous protein, a laccase from Pleurotus ostreatus (Pox2), to the catalytic part of glucoamylase (GlaA512-Pox2) severely hampers efficient production of the glucoamylase protein, resulting in a slow-growth phenotype on starch. Laccase-hypersecreting mutants were obtained by isolating mutants that displayed improved growth on starch plates. The mutant with the highest growth rate on starch displayed the highest laccase activity, indicating that increased glucoamylase protein levels are correlated with higher laccase production levels. In principle, our method can be applied to any low-produced heterologous protein that is secreted as a fusion with the glucoamylase protein. PMID:16021486

  12. Characterization of a Temperature-sensitive Mutant of Bacillus subtilis Defective in Deoxyribonucleic Acid Replication

    PubMed Central

    Mendelson, Neil H.; Gross, Julian D.

    1967-01-01

    In this paper we present a preliminary characterization of a temperature-sensitive mutant of Bacillus subtilis which appears to be defective in deoxyribonucleic acid (DNA) replication at high temperature. When log-phase cells of the mutant were transferred from 30 to 45 C, protein synthesis and ribonucleic acid synthesis continued more or less normally for several hours, whereas DNA synthesis continued at a normal rate for only 20 to 30 min and then was drastically reduced. The amount of DNA synthesized prior to this reduction corresponded approximately to the amount of DNA synthesized under conditions of protein synthesis inhibition by the parent or mutant strain. After 1 hr of growth at high temperature, cells of the mutant showed a pronounced drop in viable count. After 30 or 60 min of growth at high temperature, DNA synthesis could be restored by lowering the temperature. A longer period of growth at 45 C led to a loss of reversibility of DNA synthesis. Spores of the mutant synthesized no DNA when germinated at high temperature, although an outgrowing cell appeared. When spores were germinated at low temperature until DNA synthesis began, and then were transferred to high temperature, macromolecular synthesis continued as the log-phase transfer experiments described above. Images PMID:4964484

  13. 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

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

    PubMed

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

    2016-05-28

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

  15. Kinase inhibitor profiling reveals unexpected opportunities to inhibit disease-associated mutant kinases

    PubMed Central

    Duong-Ly, Krisna C.; Devarajan, Karthik; Liang, Shuguang; Horiuchi, Kurumi Y.; Wang, Yuren; Ma, Haiching; Peterson, Jeffrey R.

    2016-01-01

    Summary Small-molecule kinase inhibitors have typically been designed to inhibit wild-type kinases rather than the mutant forms that frequently arise in diseases such as cancer. Mutations can have serious clinical implications by increasing kinase catalytic activity or conferring therapeutic resistance. To identify opportunities to repurpose inhibitors against disease-associated mutant kinases, we conducted a large-scale functional screen of 183 known kinase inhibitors against 76 recombinant, mutant kinases. The results revealed lead compounds with activity against clinically important mutant kinases including ALK, LRRK2, RET, and EGFR as well as unexpected opportunities for repurposing FDA-approved kinase inhibitors as leads for additional indications. Furthermore, using T674I PDGFRα as an example, we show how single-dose screening data can provide predictive structure-activity data to guide subsequent inhibitor optimization. This study provides a resource for the development of inhibitors against numerous disease-associated mutant kinases and illustrates the potential of unbiased profiling as an approach to compound-centric inhibitor development. PMID:26776524

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

    PubMed Central

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

    2016-01-01

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

  17. Transcriptional profiling of human smooth muscle cells infected with gingipain and fimbriae mutants of Porphyromonas gingivalis

    PubMed Central

    Zhang, Boxi; Sirsjö, Allan; Khalaf, Hazem; Bengtsson, Torbjörn

    2016-01-01

    Porphyromonas gingivalis (P. gingivalis) is considered to be involved in the development of atherosclerosis. However, the role of different virulence factors produced by P. gingivalis in this process is still uncertain. The aim of this study was to investigate the transcriptional profiling of human aortic smooth muscle cells (AoSMCs) infected with wild type, gingipain mutants or fimbriae mutants of P. gingivalis. AoSMCs were exposed to wild type (W50 and 381), gingipain mutants (E8 and K1A), or fimbriae mutants (DPG-3 and KRX-178) of P. gingivalis. We observed that wild type P. gingivalis changes the expression of a considerable larger number of genes in AoSMCs compare to gingipain and fimbriae mutants, respectively. The results from pathway analysis revealed that the common differentially expressed genes for AoSMCs infected by 3 different wild type P. gingivalis strains were enriched in pathways of cancer, cytokine-cytokine receptor interaction, regulation of the actin cytoskeleton, focal adhesion, and MAPK signaling pathway. Disease ontology analysis showed that various strains of P. gingivalis were associated with different disease profilings. Our results suggest that gingipains and fimbriae, especially arginine-specific gingipain, produced by P. gingivalis play important roles in the association between periodontitis and other inflammatory diseases, including atherosclerosis. PMID:26907358

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

    PubMed Central

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

    2015-01-01

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

  19. Characterization of singlet oxygen-accumulating mutants isolated in a screen for altered oxidative stress response in Chlamydomonas reinhardtii

    PubMed Central

    2010-01-01

    Background When photosynthetic organisms are exposed to harsh environmental conditions such as high light intensities or cold stress, the production of reactive oxygen species like singlet oxygen is stimulated in the chloroplast. In Chlamydomonas reinhardtii singlet oxygen was shown to act as a specific signal inducing the expression of the nuclear glutathione peroxidase gene GPXH/GPX5 during high light stress, but little is known about the cellular mechanisms involved in this response. To investigate components affecting singlet oxygen signaling in C. reinhardtii, a mutant screen was performed. Results Mutants with altered GPXH response were isolated from UV-mutagenized cells containing a GPXH-arylsulfatase reporter gene construct. Out of 5500 clones tested, no mutant deficient in GPXH induction was isolated, whereas several clones showed constitutive high GPXH expression under normal light conditions. Many of these GPXH overexpressor (gox) mutants exhibited higher resistance to oxidative stress conditions whereas others were sensitive to high light intensities. Interestingly, most gox mutants produced increased singlet oxygen levels correlating with high GPXH expression. Furthermore, different patterns of altered photoprotective parameters like non-photochemical quenching, carotenoid contents and α-tocopherol levels were detected in the various gox mutants. Conclusions Screening for mutants with altered GPXH expression resulted in the isolation of many gox mutants with increased singlet oxygen production, showing the relevance of controlling the production of this ROS in photosynthetic organisms. Phenotypic characterization of these gox mutants indicated that the mutations might lead to either stimulated triplet chlorophyll and singlet oxygen formation or reduced detoxification of singlet oxygen in the chloroplast. Furthermore, changes in multiple protection mechanisms might be responsible for high singlet oxygen formation and GPXH expression, which could either

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

    PubMed Central

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

    1997-01-01

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

  1. Mutations Affecting Sexual Conjugation and Related Processes in SACCHAROMYCES CEREVISIAE. II. Genetic Analysis of Nonmating Mutants

    PubMed Central

    Mackay, Vivian; Manney, Thomas R.

    1974-01-01

    Rare diploids formed by sterile mutants have been studied by tetrad analysis. Sixteen classes of mutants representing at least five distinct genetic loci have been defined. One group of mutations, isolated only in α, maps at the mating-type locus, while none of the others shows any linkage to mating type. Some of the mutations are nonspecific for mating type, while others act only on a or α. In addition, mutations were found that prevent sporulation when heterozygous in diploids. These appear to be mutations of the mating-type alleles. PMID:4595644

  2. ERβ decreases the invasiveness of triple-negative breast cancer cells by regulating mutant p53 oncogenic function

    PubMed Central

    Bado, Igor; Nikolos, Fotis; Rajapaksa, Gayani; Gustafsson, Jan-Åke; Thomas, Christoforos

    2016-01-01

    Most (80%) of the triple-negative breast cancers (TNBCs) express mutant p53 proteins that acquire oncogenic activities including promoting metastasis. We previously showed that wild-type ERβ (ERβ1) impedes epithelial to mesenchymal transition (EMT) and decreases the invasiveness of TNBC cells. In the present study we searched for signaling pathways that ERβ1 uses to inhibit EMT and invasion in TNBC cells. We show that ERβ1 binds to and opposes the transcriptional activity of mutant p53 at the promoters of genes that regulate metastasis. p63 that transcriptionally cooperates with mutant p53 also binds to ERβ1. Downregulation of p63 represses the epithelial phenotype of ERβ1-expressing cells and alters the expression of mutant p53 target genes. These results describe a novel mechanism through which ERβ1 can disturb oncogenic signals to inhibit aggressiveness in TNBCs. PMID:26871946

  3. Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus

    PubMed Central

    del Toro, Daniel; Alberch, Jordi; Lázaro-Diéguez, Francisco; Martín-Ibáñez, Raquel; Xifró, Xavier; Egea, Gustavo

    2009-01-01

    Huntingtin regulates post-Golgi trafficking of secreted proteins. Here, we studied the mechanism by which mutant huntingtin impairs this process. Colocalization studies and Western blot analysis of isolated Golgi membranes showed a reduction of huntingtin in the Golgi apparatus of cells expressing mutant huntingtin. These findings correlated with a decrease in the levels of optineurin and Rab8 in the Golgi apparatus that can be reverted by overexpression of full-length wild-type huntingtin. In addition, immunoprecipitation studies showed reduced interaction between mutant huntingtin and optineurin/Rab8. Cells expressing mutant huntingtin produced both an accumulation of clathrin adaptor complex 1 at the Golgi and an increase of clathrin-coated vesicles in the vicinity of Golgi cisternae as revealed by electron microscopy. Furthermore, inverse fluorescence recovery after photobleaching analysis for lysosomal-associated membrane protein-1 and mannose-6-phosphate receptor showed that the optineurin/Rab8-dependent post-Golgi trafficking to lysosomes was impaired in cells expressing mutant huntingtin or reducing huntingtin levels by small interfering RNA. Accordingly, these cells showed a lower content of cathepsin D in lysosomes, which led to an overall reduction of lysosomal activity. Together, our results indicate that mutant huntingtin perturbs post-Golgi trafficking to lysosomal compartments by delocalizing the optineurin/Rab8 complex, which, in turn, affects the lysosomal function. PMID:19144827

  4. Metformin and trametinib have synergistic effects on cell viability and tumor growth in NRAS mutant cancer

    PubMed Central

    Vujic, Igor; Sanlorenzo, Martina; Posch, Christian; Esteve-Puig, Rosaura; Yen, Adam J.; Kwong, Andrew; Tsumura, Aaron; Murphy, Ryan; Rappersberger, Klemens; Ortiz-Urda, Susana

    2015-01-01

    Attempts to directly block the mutant neuroblastoma rat sarcoma oncogene (NRAS) protein, a driving mutation in many cancer types, have been unsuccessful. Current treatments focus on inhibition of different components of NRAS' two main downstream cascades: PI3K/AKT/mTOR and MAPK. Here we test a novel dual therapy combination of metformin and trametinib on a panel of 16 NRAS mutant cell lines, including melanoma cells, melanoma cells with acquired trametinib resistance, lung cancer and neuroblastoma cells. We show that both of the main downstream cascades of NRAS can be blocked by this combination: metformin indirectly inhibits the PI3K/AKT/mTOR pathway and trametinib directly impedes the MAPK pathway. This dual therapy synergistically reduced cell viability in vitro and xenograft tumor growth in vivo. We conclude that metformin and trametinib combinations are effective in preclinical models and may be a possible option for treatment of NRAS mutant cancers. PMID:25504439

  5. Does the mutant mouse lurcher have deficits in spatially oriented behaviours?

    PubMed

    Lalonde, R; Lamarre, Y; Smith, A M

    1988-07-01

    Lurcher mutant mice, in comparison to normal mice, had directional deficits in the Morris milk tank test and in a water-maze spatial alternation task. The lurcher mutants also showed an initial lack of spontaneous alternation and did not alternate in one behavioral condition when the inter-trial interval was lengthened. Lurcher mice were slower to learn a simple left/right position response to escape a T-maze by swimming although their motor coordination was good. Paradoxically, no deficit was observed in learning to select a left or right position for food in the same T-maze although the goal-directed locomotion was very ataxic. Overall, the lurcher mutants have difficulty in guiding themselves in the water toward a visible goal. PMID:3416189

  6. Hypothalamic overexpression of mutant huntingtin causes dysregulation of brown adipose tissue

    PubMed Central

    Soylu-Kucharz, Rana; Adlesic, Natalie; Baldo, Barbara; Kirik, Deniz; Petersén, Åsa

    2015-01-01

    Expression of mutant huntingtin (htt) protein has been shown to cause metabolic imbalance in animal models of Huntington disease (HD). The pathways involved are not fully understood but dysfunction of both the hypothalamus and brown adipose tissue (BAT) has been implicated. Here we show that targeted expression of mutant HTT in the hypothalamus leads to loss of the A13 dopaminergic cell group located in the zona incerta and reduced mRNA expression of neuropeptide Y1 receptor in the hypothalamus. Furthermore, this is accompanied by downregulation of uncoupling protein 1 expression and PPARγ coactivator-1 alpha in BAT and a rapid body weight gain. Taken together, our data might provide a mechanistic link between expression of mutant HTT, reduced activity of a hypothalamic dopaminergic pathway and dysfunction of BAT and in part explain the development of an obese phenotype in HD mouse models. PMID:26419281

  7. A Stable Mutant Predisposes Antibody Domains to Amyloid Formation through Specific Non-Native Interactions.

    PubMed

    Nokwe, Cardine N; Hora, Manuel; Zacharias, Martin; Yagi, Hisashi; Peschek, Jirka; Reif, Bernd; Goto, Yuji; Buchner, Johannes

    2016-03-27

    The aggregation of mostly antibody light chain variable (VL) domains into amyloid fibrils in various tissues is the main cause of death in systemic amyloid light chain amyloidosis. Point mutations within the domain are important to shift the VL into the fibrillar pathway, but why and how only some site-specific mutations achieve this still remains elusive. We show here that both destabilizing and surprisingly stable mutants readily predispose an amyloid-resistant VL domain to amyloid formation. The decreased thermodynamic stability of the destabilizing mutant results in the accumulation of non-native intermediates that readily populate the amyloid state. Interestingly, the stable mutants establish site-specific non-native interactions with especially nearby serine/threonine residues that unexpectedly do not affect the folding behavior of the VL domain but rather readily induce and stabilize the fibril structure, a previously unrecognized mechanism. These findings provide a new concept for the molecular mechanism of amyloid fibril formation. PMID:26827727

  8. Phenotype of a Temperature-Sensitive, Respiration-Deficient (cyt) Mutant of Yeast

    PubMed Central

    Miyake, Setsuko; Iwamoto, Yoshihisa; Nagao, Minako; Sugimura, Takashi; Ohsumi, Masako

    1972-01-01

    A temperature-sensitive respiration-deficient mutant of yeast lacks hemoproteins and accumulates coproporphyrin III when cultivated at elevated temperatures. Cells grown at 20 C respired normally and contained cytochromes a, b, and c. Cells grown at 35 C showed respiration-deficient mutant characters; they did not respire, lacked cytochromes, and accumulated coproporphyrin III. Addition of protoporphyrin IX or protohemin IX to the culture medium restored the respiratory activity of this mutant during growth at 35 C. The activities of various enzymes, including succinate-2,6-dichlorophenol indophenol (DCPIP), reduced nicotinamide adenine dinucleotide (NADH2)-DCPIP, succinate-cytochrome c, and NADH2-cytochrome c oxidoreductase, and cytochrome oxidase, and the cytochrome c content of cells cultured in various conditions were determined. Changes in the number and structure of mitochondria were associated with changes in respiratory activity. Images PMID:4333381

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

    PubMed Central

    Davis, L; Engebrecht, J

    1998-01-01

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

  10. Acid-Soluble Nucleotides in an Asporogenous Mutant of Bacillus subtilis

    PubMed Central

    Chow, C. T.; Takahashi, I.

    1972-01-01

    An asporogenous mutant of Bacillus subtilis Sp−H12-3, which is considered to have a block at stage 0, showed general growth characteristics similar to those of sporulating cultures. However, a sudden increase in the total amount of acid-soluble nucleotides observed at t2 in sporulating bacteria was completely absent in this mutant. In sporulating cells, a marked increase in two nucleotides which were identified to be uridine diphosphate (UDP)-galactose and UDP-N-acetylglucosamine was noted, whereas UDP-glucose appeared to be accumulated in the mutant cells at t2. No unusual nucleotides were found in the strains of B. subtilis examined. The possible role of these UDP derivatives in early stages of sporulation in B. subtilis is discussed. PMID:4622128

  11. Isolation and characterization of high-temperature-induced Dauer formation mutants in Caenorhabditis elegans.

    PubMed Central

    Ailion, Michael; Thomas, James H

    2003-01-01

    Dauer formation in Caenorhabditis elegans is regulated by at least three signaling pathways, including an insulin receptor-signaling pathway. These pathways were defined by mutants that form dauers constitutively (Daf-c) at 25 degrees. Screens for Daf-c mutants at 25 degrees have probably been saturated, but failed to identify all the components involved in regulating dauer formation. Here we screen for Daf-c mutants at 27 degrees, a more strongly dauer-inducing condition. Mutations identified include novel classes of alleles for three known genes and alleles defining at least seven new genes, hid-1-hid-7. Many of the genes appear to act in the insulin branch of the dauer pathway, including pdk-1, akt-1, aex-6, and hid-1. We also molecularly identify hid-1 and show that it encodes a novel highly conserved putative transmembrane protein expressed in neurons. PMID:14504222

  12. Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

    SciTech Connect

    Brown, Steven D; Guss, Adam M; Karpinets, Tatiana V; Parks, Jerry M; Smolin, Nikolai; Yang, Shihui; Land, Miriam L; Klingeman, Dawn Marie; Bhandiwad, Ashwini; Rodriguez, Jr., Miguel; Raman, Babu; Shao, Xiongjun; Mielenz, Jonathan R; Smith, Jeremy C; Keller, Martin; Lynd, Lee R

    2011-01-01

    Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

  13. Multiple Chemosensory Defects in Daf-11 and Daf-21 Mutants of Caenorhabditis Elegans

    PubMed Central

    Vowels, J. J.; Thomas, J. H.

    1994-01-01

    Phenotypic analysis of the daf-11 and daf-21 mutants of Caenorhabditis elegans suggests that they have defects in components shared by processes analogous to vertebrate taste and olfaction. daf-11 and daf-21 mutations were previously shown to cause inappropriate response to the dauer-inducing pheromone. By mutational analysis and by disabling specific chemosensory sensilla with a laser, we show that neurons in the amphid sensilla are required for this pheromone response. Using behavioral assays, we find that daf-11 and daf-21 mutants are not defective in avoidance of certain non-volatile repellents, but are defective in taxis to non-volatile attractants. In addition, both mutants are defective in taxis to volatile attractants detected primarily by the amphid neuron AWC, but respond normally to volatile attractants detected primarily by AWA. We propose that daf-11 and daf-21 mediate sensory transduction for both volatile and non-volatile compounds in specific amphid neurons. PMID:7828815

  14. Limited photosynthetic electron flow but no CO2 fixation in Chlamydomonas mutants lacking photosystem I.

    PubMed

    Cournac, L; Redding, K; Bennoun, P; Peltier, G

    1997-10-13

    By measuring O2 and CO2 exchange in mutants of the green alga Chlamydomonas reinhardtii in which genes encoding the reaction center of photosystem I (psaA or psaB) have been deleted, we found that a photosystem II-dependent electron flow using O2 as the final acceptor can be sustained in the light. However, in contrast with recent reports using other Chlamydomonas mutants (B4 and F8), we show here that CO2 fixation does not occur in the absence of photosystem I. By deleting the psaA gene in both B4 and F8 strains, we conclude that the ability of these mutants to fix CO2 in the light is due to the presence of residual amounts of photosystem I. PMID:9369234

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

    PubMed

    Davis, L; Engebrecht, J

    1998-05-01

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

  16. Identification of differentially expressed genes in sorghum (Sorghum bicolor) brown midrib mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sorghum (Sorghum bicolor L.), with a high biomass yield and excellent tolerance to drought and low nutrition, has been recommended as one of the most competitive bioenergy crops. Brown midrib (bmr) mutant sorghum with reduced lignin content showed a high potential for the improvement of bioethanol ...

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

    PubMed

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

    2010-05-01

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

  18. A lipopolysaccharide (LPS)-resistant mutant isolated from a macrophagelike cell line, J774.1, exhibits an altered activated-macrophage phenotype in response to LPS.

    PubMed

    Amano, F; Akamatsu, Y

    1991-06-01

    A bacterial lipopolysaccharide (LPS)-resistant mutant was isolated from murine macrophagelike cell line J774.1. The mutant showed selective resistance to LPS and lipid A and was almost 10(5)- to 10(6)-fold more resistant than the parent; it grew even in the presence of 1 mg of Escherichia coli O55:B5 LPS per liter, whereas the parent did not grow with less than 10 ng of LPS per milliliter. We next examined the mutant for activation of various functions of macrophages on LPS treatment. This LPS-resistant mutant secreted interleukin-1 and tumor necrosis factor almost as effectively as the parent did. The mutant cells also changed transiently from a round to a spread form; however, they became round again afterwards. The mutant cells secreted less arachidonic acid in response to LPS. These results also suggest that this LPS-resistant mutant responds to LPS and shows activation of some macrophage functions. However, this mutant did not exhibit elevation of O2- generation or H2O2 generation after LPS treatment. Also, treatment of the mutant cells with murine recombinant gamma interferon was partly able to correct the defect in O(2-)-generating activity in response to LPS, suggesting that this defect is probably due to some of the LPS signal pathways. This implies that there is some correlation between O2- metabolism in LPS-activated macrophages and decreases in cell growth and viability. PMID:1645329

  19. Isolation and characterization of a catabolite repression-insensitive mutant of a methanol yeast, Candida boidinii A5, producing alcohol oxidase in glucose-containing medium

    SciTech Connect

    Sakai, Y.; Sawai, T.; Tani, Y.

    1987-08-01

    Mutants exhibiting alcohol oxidase activity when grown on glucose in the presence of methanol were found among 2-deoxyglucose-resistant mutants derived from a methanol yeast, Candida boidinii A5. One of these mutants, strain ADU-15, showed the highest alcohol oxidase activity in glucose-containing medium. The growth characteristics and also the induction and degradation of alcohol oxidase were compared with the parent strain and mutant strain ADU-15. In the parent strain, initiation of alcohol oxidase synthesis was delayed by the addition of 0.5% glucose to the methanol medium, whereas it was not delayed in mutant strain ADU-15. This showed that alcohol oxidase underwent repression by glucose. On the other hand, degradation of alcohol oxidase after transfer of the cells from methanol to glucose medium (catabolite inactivation) was observed to proceed at similar rates in parent and mutant strains. The results of immunochemical titration experiments suggests that catabolite inactivation of alcohol oxidase is coupled with a quantitative change in the enzyme. Mutant strain ADU-15 was proved to be a catabolite repression-insensitive mutant and to produce alcohol oxidase in the presence of glucose. However, it was not an overproducer of alcohol oxidase and, in both the parent and mutant strains, alcohol oxidase was completely repressed by ethanol.

  20. Genetic dissection of leukemia-associated IDH1 and IDH2 mutants and D-2-hydroxyglutarate in Drosophila

    PubMed Central

    Reitman, Zachary J.; Sinenko, Sergey A.; Spana, Eric P.

    2015-01-01

    Gain-of-function mutations in nicotinamide adenine dinucleotide phosphate–dependent isocitrate dehydrogenase (IDH)1 and IDH2 frequently arise in human leukemias and other cancers and produce high levels of D-2-hydroxyglutarate (D-2HG). We expressed the R195H mutant of Drosophila Idh (CG7176), which is equivalent to the human cancer-associated IDH1-R132H mutant, in fly tissues using the UAS-Gal4 binary expression system. Idh-R195H caused a >25-fold elevation of D-2HG when expressed ubiquitously in flies. Expression of mutant Idh in larval blood cells (hemocytes) resulted in higher numbers of circulating blood cells. Mutant Idh expression in fly neurons resulted in neurologic and wing-expansion defects, and these phenotypes were rescued by genetic modulation of superoxide dismutase 2, p53, and apoptotic caspase cascade mediators. Idh-R163Q, which is homologous to the common leukemia-associated IDH2-R140Q mutant, resulted in moderately elevated D-2HG and milder phenotypes. We identified the fly homolog of D-2-hydroxyglutaric acid dehydrogenase (CG3835), which metabolizes D-2HG, and showed that coexpression of this enzyme with mutant Idh abolishes mutant Idh-associated phenotypes. These results provide a flexible model system to interrogate a cancer-related genetic and metabolic pathway and offer insights into the impact of IDH mutation and D-2HG on metazoan tissues. PMID:25398939

  1. Platelet storage pool deficiency associated with inherited abnormalities of the inner ear in the mouse pigment mutants muted and mocha.

    PubMed

    Swank, R T; Reddington, M; Howlett, O; Novak, E K

    1991-10-15

    Several inherited human syndromes have combined platelet, auditory, and/or pigment abnormalities. In the mouse the pallid pigment mutant has abnormalities of the otoliths of the inner ear together with a bleeding abnormality caused by platelet storage pool deficiency (SPD). To determine if this association is common, two other mouse pigment mutants, muted and mocha, which are known to have inner ear abnormalities, were examined for hematologic abnormalities. Both mutants had prolonged bleeding times accompanied by abnormalities of dense granules as determined by whole mount electron microscopy of platelets and by labeling platelets with mepacrine. When mutant platelets were treated with collagen, there was minimal secretion of adenosine triphosphate and aggregation was reduced. Lysosomal enzyme secretion in response to thrombin treatment was partially reduced in muted platelets and markedly reduced in mocha platelets. Similar reductions in constitutive lysosomal enzyme secretion from kidney proximal tubule cells were noted in the two mutants. These studies show that several mutations that cause pigment dilution and platelet SPD are associated with abnormalities of the inner ear. Also, these mutants, like previously described mouse pigment mutants, are models for human Hermansky-Pudlak syndrome and provide additional examples of single genes that simultaneously affect melanosomes, lysosomes, and platelet dense granules. PMID:1912584

  2. 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

  3. 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.

  4. Characterization of Pseudomonas aeruginosa mutants with altered piliation.

    PubMed Central

    Johnson, K; Lory, S

    1987-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  6. Production of l-Phenylalanine from Starch by Analog-Resistant Mutants of Bacillus polymyxa†

    PubMed Central

    Shetty, Kalidas; Crawford, Don L.; Pometto, Anthony L.

    1986-01-01

    p-Fluorophenylalanine-resistant mutants of starch-degrading Bacillus polymyxa ATCC 842, generated by ethyl methanesulfonate mutagenesis followed by incubation with caffeine, overproduced small amounts of l-phenylalanine (l-phe) from starch. A β-2-thienylalanine-resistant mutant (BTR-7) derived from p-fluorophenylalanine mutant (C-4000 FPR-4) and resistant to both p-fluorophenylalanine and β-2-thienylalanine produced 0.5 g of l-phe and 0.15 g of l-tyrosine per liter from 10 g of starch per liter when growing in a minimal medium. trans-Cinnamic acid (CA) was also excreted by both mutants, indicating the possibility of l-phenylalanine ammonia-lyase-induced deamination of l-phe to CA. The amount of l-phe-derived CA detected in BTR-7 was less compared with mutant C-4000 FPR-4. CA production was induced in the parent only when l-phe was used as a sole nitrogen source. Time of CA production in the two mutants could be delayed by addition of other nitrogen sources, an indication of possible l-phenylalanine ammonia-lyase inhibition or repression. The presence of l-phenylalanine ammonia-lyase in B. polymyxa mutant C-4000 FPR-4 was confirmed by assays of cell-free extracts from cells grown in starch minimal medium containing l-phe as the sole nitrogen source. Preliminary studies of the regulation of deoxy-d-arabino-heptulosonate-7-phosphate synthase and prephenate dehydratase in the wild-type strain showed that deoxy-d-arabino-heptulosonate-7-phosphate synthase was subject to feedback inhibition by l-phe, l-tyrosine, and l-tryptophan. Inhibition by each amino acid was to a similar extent singly or in combination at a 0.5 mM level of each amino acid. Prephenate dehydratase was feedback inhibited by l-phe, but not by l-tyrosine or l-tryptophan or both. In the double analog-resistant mutant BTR-7, deoxy-d-arabino-heptulosonate-7-phosphate synthase had specific activity similar to that in the wild type, and the enzyme was still subject to feedback inhibition. However

  7. TagSmart: analysis and visualization for yeast mutant fitness data measured by tag microarrays

    PubMed Central

    Kim, Chulyun; Kim, Sangkyum; Dorer, Russell; Xie, Dan; Han, Jiawei; Zhong, Sheng

    2007-01-01

    Background A nearly complete collection of gene-deletion mutants (96% of annotated open reading frames) of the yeast Saccharomyces cerevisiae has been systematically constructed. Tag microarrays are widely used to measure the fitness of each mutant in a mutant mixture. The tag array experiments can have a complex experimental design, such as time course measurements and drug treatment with multiple dosages. Results TagSmart is a web application for analysis and visualization of Saccharomyces cerevisiae mutant fitness data measured by tag microarrays. It implements a robust statistical approach to assess the concentration differences among S. cerevisiae mutant strains. It also provides an interactive environment for data analysis and visualization. TagSmart has the following advantages over previously described analysis procedures: 1) it is user-friendly software rather than merely a description of analytical procedure; 2) It can handle complicated experimental designs, such as multiple time points and treatment with multiple dosages; 3) it has higher sensitivity and specificity; 4) It allows users to mask out "bad" tags in the analysis. Two biological tests were performed to illustrate the performance of TagSmart. First, we generated titration mixtures of mutant strains, in which the relative concentration of each strain was controlled. We used tag microarrays to measure the numbers of tag copies in each titration mixture. The data was analyzed with TagSmart and the result showed high precision and recall. Second, TagSmart was applied to a dataset in which heterozygous deletion strain mixture pools were treated with a new drug, Cincreasin. TagSmart identified 53 mutant strains as sensitive to Cincreasin treatment. We individually tested each identified mutant, and found 52 out of the 53 predicted mutants were indeed sensitive to Cincreasin. Conclusion TagSmart is provided "as is" to analyze tag array data produced by Affymetrix and Agilent arrays. TagSmart web

  8. Characterization and fine mapping of a novel rice narrow leaf mutant nal9.

    PubMed

    Li, Wei; Wu, Chao; Hu, Guocheng; Xing, Li; Qian, Wenjing; Si, Huamin; Sun, Zongxiu; Wang, Xingchun; Fu, Yaping; Liu, Wenzhen

    2013-11-01

    A narrow leaf mutant was isolated from transgenic rice (Oryza sativa L.) lines carrying a T-DNA insertion. The mutant is characterized by narrow leaves during its whole growth period, and was named nal9 (narrow leaf 9). The mutant also has other phenotypes, such as light green leaves at the seedling stage, reduced plant height, a small panicle and increased tillering. Genetic analysis revealed that the mutation is controlled by a single recessive gene. A hygromycin resistance assay showed that the mutation was not caused by T-DNA insertion, so a map-based cloning strategy was employed to isolate the nal9 gene. The mutant individuals from the F₂ generations of a cross between the nal9 mutant and Longtepu were used for mapping. With 24 F₂ mutants, the nal9 gene was preliminarily mapped near the marker RM156 on the chromosome 3. New INDEL markers were then designed based on the sequence differences between japonica and indica at the region near RM156. The nal9 gene was finally located in a 69.3 kb region between the markers V239B and V239G within BAC OJ1212_C05 by chromosome walking. Sequence and expression analysis showed that an ATP-dependent Clp protease proteolytic subunit gene (ClpP) was most likely to be the nal9 gene. Furthermore, the nal9 mutation was rescued by transformation of the ClpP cDNA driven by the 35S promoter. Accordingly, the ClpP gene was identified as the NAL9 gene. Our results provide a basis for functional studies of NAL9 in future work. PMID:23945310

  9. Stability of Iowa mutant and wild type Aβ-peptide aggregates

    SciTech Connect

    Alred, Erik J.; Scheele, Emily G.; Berhanu, Workalemahu M.; Hansmann, Ulrich H. E.

    2014-11-07

    Recent experiments indicate a connection between the structure of amyloid aggregates and their cytotoxicity as related to neurodegenerative diseases. Of particular interest is the Iowa Mutant, which causes early-onset of Alzheimer's disease. While wild-type Amyloid β-peptides form only parallel beta-sheet aggregates, the mutant also forms meta-stable antiparallel beta sheets. Since these structural variations may cause the difference in the pathological effects of the two Aβ-peptides, we have studied in silico the relative stability of the wild type and Iowa mutant in both parallel and antiparallel forms. We compare regular molecular dynamics simulations with such where the viscosity of the samples is reduced, which, we show, leads to higher sampling efficiency. By analyzing and comparing these four sets of all-atom molecular dynamics simulations, we probe the role of the various factors that could lead to the structural differences. Our analysis indicates that the parallel forms of both wild type and Iowa mutant aggregates are stable, while the antiparallel aggregates are meta-stable for the Iowa mutant and not stable for the wild type. The differences result from the direct alignment of hydrophobic interactions in the in-register parallel oligomers, making them more stable than the antiparallel aggregates. The slightly higher thermodynamic stability of the Iowa mutant fibril-like oligomers in its parallel organization over that in antiparallel form is supported by previous experimental measurements showing slow inter-conversion of antiparallel aggregates into parallel ones. Knowledge of the mechanism that selects between parallel and antiparallel conformations and determines their relative stability may open new avenues for the development of therapies targeting familial forms of early-onset Alzheimer's disease.

  10. cor Gene Expression in Barley Mutants Affected in Chloroplast Development and Photosynthetic Electron Transport1

    PubMed Central

    Dal Bosco, Cristina; Busconi, Marco; Govoni, Chiara; Baldi, Paolo; Stanca, A. Michele; Crosatti, Cristina; Bassi, Roberto; Cattivelli, Luigi

    2003-01-01

    The expression of several barley (Hordeum vulgare) cold-regulated (cor) genes during cold acclimation was blocked in the albino mutant an, implying a chloroplast control on mRNAs accumulation. By using albino and xantha mutants ordered according to the step in chloroplast biogenesis affected, we show that the cold-dependent accumulation of cor14b, tmc-ap3, and blt14 mRNAs depends on plastid developmental stage. Plants acquire the ability to fully express cor genes only after the development of primary thylakoid membranes in their chloroplasts. To investigate the chloroplast-dependent mechanism involved in cor gene expression, the activity of a 643-bp cor14b promoter fragment was assayed in wild-type and albino mutant an leaf explants using transient β-glucuronidase reporter expression assay. Deletion analysis identified a 27-bp region between nucleotides −274 and −247 with respect to the transcription start point, encompassing a boundary of some element that contributes to the cold-induced expression of cor14b. However, cor14b promoter was equally active in green and in albino an leaves, suggesting that chloroplast controls cor14b expression by posttranscriptional mechanisms. Barley mutants lacking either photosystem I or II reaction center complexes were then used to evaluate the effects of redox state of electron transport chain components on COR14b accumulation. In the mutants analyzed, the amount of COR14b protein, but not the steady-state level of the corresponding mRNA, was dependent on the redox state of the electron transport chain. Treatments of the vir-zb63 mutant with electron transport chain inhibitors showed that oxidized plastoquinone promotes COR14b accumulation, thus suggesting a molecular relationship between plastoquinone/plastoquinol pool and COR14b. PMID:12586903

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

    NASA Astrophysics Data System (ADS)

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

    2013-10-01

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

  12. Photophysiology of the Elongated Internode (ein) Mutant of Brassica rapa: ein Mutant Lacks a Detectable Phytochrome B-Like Polypeptide.

    PubMed

    Devlin, P F; Rood, S B; Somers, D E; Quail, P H; Whitelam, G C

    1992-11-01

    Several phytochrome-controlled processes have been examined in etiolated and light-grown seedlings of a normal genotype and the elongated internode (ein/ein) mutant of rapid-cycling Brassica rapa. Although etiolated ein seedlings displayed normal sensitivity to prolonged far-red light with respect to inhibition of hypocotyl elongation, expansion of cotyledons, and synthesis of anthocyanin, they displayed reduced sensitivity to prolonged red light for all three of these deetiolation responses. In contrast to normal seedlings, light-grown ein seedlings did not show a growth promotion in response to end-of-day far-red irradiation. Additionally, whereas the first internode of light-grown normal seedlings showed a marked increase in elongation in response to reduced ratio of red to far-red light, ein seedlings showed only a small elongation response. When blots of protein extracts from etiolated and light-treated ein and normal seedlings were probed with monoclonal antibody to phytochrome A, an immunostaining band at about 120 kD was observed for both extracts. The immunostaining intensity of this band was substantially reduced for extracts of light-treated normal and ein seedlings. A mixture of three monoclonal antibodies directed against phytochrome B from Arabidopsis thaliana immunostained a band at about 120 kD for extracts of etiolated and light-treated normal seedlings. This band was undetectable in extracts of ein seedlings. We propose that ein is a photoreceptor mutant that is deficient in a light-stable phytochrome B-like species. PMID:16653143

  13. Optimized cell transplantation using adult rag2 mutant zebrafish

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Kuraku, Shigehiro; Meyer, Axel

    2008-12-01

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

  15. Computational approaches for predicting mutant protein stability.

    PubMed

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

    2016-05-01

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

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

    PubMed

    Ladygin, V G

    2003-01-01

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

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

    SciTech Connect

    Susan M. Wick

    2004-07-23

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

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

    PubMed Central

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

    1988-01-01

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

  19. Enriching the Environment of [alpha]CaMKII[superscript T286A] Mutant Mice Reveals that LTD Occurs in Memory Processing but Must be Subsequently Reversed by LTP

    ERIC Educational Resources Information Center

    Soto, Florentina; Giese, K. Peter; Edwards, Frances A.; Parsley, Stephanie L.; Pilgram, Sara M.

    2007-01-01

    [alpha]CaMKII[superscript T286A] mutant mice lack long-term potentiation (LTP) in the hippocampal CA1 region and are impaired in spatial learning. In situ hybridization confirms that the mutant mice show the same developmental expression of [alpha]CaMKII as their wild-type littermates. A simple hypothesis would suggest that if LTP is a substrate…

  20. Superior triacylglycerol (TAG) accumulation in starchless mutants of Scenedesmus obliquus: (II) evaluation of TAG yield and productivity in controlled photobioreactors

    PubMed Central

    2014-01-01

    Background Many microalgae accumulate carbohydrates simultaneously with triacylglycerol (TAG) upon nitrogen starvation, and these products compete for photosynthetic products and metabolites from the central carbon metabolism. As shown for starchless mutants of the non-oleaginous model alga Chlamydomonas reinhardtii, reduced carbohydrate synthesis can enhance TAG production. However, these mutants still have a lower TAG productivity than wild-type oleaginous microalgae. Recently, several starchless mutants of the oleaginous microalga Scenedesmus obliquus were obtained which showed improved TAG content and productivity. Results The most promising mutant, slm1, is compared in detail to wild-type S. obliquus in controlled photobioreactors. In the slm1 mutant, the maximum TAG content increased to 57 ± 0.2% of dry weight versus 45 ± 1% in the wild type. In the wild type, TAG and starch were accumulated simultaneously during initial nitrogen starvation, and starch was subsequently degraded and likely converted into TAG. The starchless mutant did not produce starch and the liberated photosynthetic capacity was directed towards TAG synthesis. This increased the maximum yield of TAG on light by 51%, from 0.144 ± 0.004 in the wild type to 0.217 ± 0.011 g TAG/mol photon in the slm1 mutant. No differences in photosynthetic efficiency between the slm1 mutant and the wild type were observed, indicating that the mutation specifically altered carbon partitioning while leaving the photosynthetic capacity unaffected. Conclusions The yield of TAG on light can be improved by 51% by using the slm1 starchless mutant of S. obliquus, and a similar improvement seems realistic for the areal productivity in outdoor cultivation. The photosynthetic performance is not negatively affected in the slm1 and the main difference with the wild type is an improved carbon partitioning towards TAG. PMID:24883102

  1. Synthesis of outer membrane proteins in cpxA cpxB mutants of Escherichia coli K-12.

    PubMed Central

    McEwen, J; Sambucetti, L; Silverman, P M

    1983-01-01

    Two major proteins, the murein lipoprotein and the OmpF matrix porin, are deficient in the outer membrane of cpxA cpxB mutants of Escherichia coli K-12. We present evidence that the cpx mutations prevent or retard the translocation of these proteins to the outer membrane. The mutations had no effect on the rate of lipoprotein synthesis. Mutant cells labeled for 5 min with radioactive arginine accumulated as much lipoprotein as otherwise isogenic cpxA+ cpxB+ cells. This lipoprotein accumulated as such; no material synthesized in mutant cells and reactive with antilipoprotein antibodies had the electrophoretic mobility of prolipoprotein. Hence, the initial stages of prolipoprotein insertion into the inner membrane leading to its cleavage to lipoprotein appeared normal. However, after a long labeling interval, mutant cells were deficient in free lipoprotein and lacked lipoprotein covalently bound to peptidoglycan, suggesting that little if any of the lipoprotein synthesized in mutant cells reaches the outer membrane. Immunoreactive OmpF protein could also be detected in extracts of mutant cells labeled for 5 min, but the amount that accumulated was severalfold less in mutant cells than in cpxA+ cpxB+ cells. Analysis of beta-galactosidase synthesis from ompF-lacZ fusion genes showed this difference to be the result of a reduced rate of ompF transcription in mutant cells. Even so, little or none of the ompF protein synthesized in mutant cells was incorporated into the outer membrane. Images PMID:6339479

  2. Inhibition mechanism exploration of investigational drug TAK-441 as inhibitor against Vismodegib-resistant Smoothened mutant.

    PubMed

    Ishii, Tsuyoshi; Shimizu, Yuji; Nakashima, Kosuke; Kondo, Shigeru; Ogawa, Kazumasa; Sasaki, Satoshi; Matsui, Hideki

    2014-01-15

    Hedgehog signaling is a driving force in medulloblastoma and basal cell carcinoma (BCC), making it an attractive therapeutic target. Vismodegib recently received FDA approval for the treatment of inoperable BCC, but a drug-resistant Smoothened (Smo) mutant (D473H) was identified in a clinical study. TAK-441 is a pyrrolo[3,2-c]pyridine-4-one derivative that potently inhibits Hh signal transduction and is currently under investigation in clinical trials. We demonstrated that TAK-441 inhibits reporter activity in D473H-transfected cells with an IC50 of 79nM, while Vismodegib showed an IC50=7100nM. In order to investigate the mode of inhibition, we evaluated the Smo inhibitors with three different binding assays, such as [(3)H]-TAK-441 membrane binding assay, affinity selection-MS detection assay, and bodipy-cylopamine whole cell assay. In three different assays, Vismodegib and cyclopamine showed lower affinity for the D473H mutant in comparison with wild-type Smo. On the other hand, TAK-441 showed almost equal binding affinity for the D473H mutant compared with wild-type Smo in the binding assays, although TAK-441 binds to the same binding site as two other well-known inhibitors. These in vitro findings suggest that TAK-441 has the potential for clinical use in cancers that are dependent on Hedgehog signaling, including wild-type tumors and Vismodegib-resistant D473H mutants. PMID:24291104

  3. Functional determinants of ras interference 1 mutants required for their inhbitory activity on endocytosis

    SciTech Connect

    Galvis, Adriana; Giambini, Hugo; Villasana, Zoilmar; Barbieri, M. Alejandro

    2009-03-10

    In this study, we initiated experiments to address the structure-function relationship of Rin1. A total of ten substitute mutations were created, and their effects on Rin1 function were examined. Of the ten mutants, four of them (P541A, E574A, Y577F, T580A) were defective in Rab5 binding, while two other Rin1 mutants (D537A, Y561F) partially interacted with Rab5. Mutations in several other residues (Y506F, Y523F, T572A, Y578F) resulted in partial loss of Rab5 function. Biochemical studies showed that six of them (D537A, P541A, Y561F, E574A, Y577F, T580A) were unable to activate Rab5 in an in vitro assay. In addition, Rin1: D537A and Rin1: Y561F mutants showed dominant inhibition of Rab5 function. Consistent with the biochemical studies, we observed that these two Rin1 mutants have lost their ability to stimulate the endocytosis of EGF, form enlarged Rab5-positive endosomes, or support in vitro endosome fusion. Based on these data, our results showed that mutations in the Vps9 domain of Rin1 lead to a loss-of-function phenotype, indicating a specific structure-function relationship between Rab5 and Rin1.

  4. A Saccharomyces cerevisiae genome-wide mutant screen for altered sensitivity to K1 killer toxin.

    PubMed Central

    Pagé, Nicolas; Gérard-Vincent, Manon; Ménard, Patrice; Beaulieu, Maude; Azuma, Masayuki; Dijkgraaf, Gerrit J P; Li, Huijuan; Marcoux, José; Nguyen, Thuy; Dowse, Tim; Sdicu, Anne-Marie; Bussey, Howard

    2003-01-01

    Using the set of Saccharomyces cerevisiae mutants individually deleted for 5718 yeast genes, we screened for altered sensitivity to the antifungal protein, K1 killer toxin, that binds to a cell wall beta-glucan receptor and subsequently forms lethal pores in the plasma membrane. Mutations in 268 genes, including 42 in genes of unknown function, had a phenotype, often mild, with 186 showing resistance and 82 hypersensitivity compared to wild type. Only 15 of these genes were previously known to cause a toxin phenotype when mutated. Mutants for 144 genes were analyzed for alkali-soluble beta-glucan levels; 63 showed alterations. Further, mutants for 118 genes with altered toxin sensitivity were screened for SDS, hygromycin B, and calcofluor white sensitivity as indicators of cell surface defects; 88 showed some additional defect. There is a markedly nonrandom functional distribution of the mutants. Many genes affect specific areas of cellular activity, including cell wall glucan and mannoprotein synthesis, secretory pathway trafficking, lipid and sterol biosynthesis, and cell surface signal transduction, and offer new insights into these processes and their integration. PMID:12663529

  5. Phospholipase D δ knock-out mutants are tolerant to severe drought stress

    PubMed Central

    Distéfano, Ayelen M; Valiñas, Matías A; Scuffi, Denise; Lamattina, Lorenzo; ten Have, Arjen; García-Mata, Carlos; Laxalt, Ana M

    2015-01-01

    Phospholipase D (PLD) is involved in different plant processes, ranging from responses to abiotic and biotic stress to plant development. Phospholipase Dδ (PLDδ) is activated in dehydration and salt stress, producing the lipid second messenger phosphatidic acid. In this work we show that pldδ Arabidopsis mutants were more tolerant to severe drought than wild-type plants. PLDδ has been shown to be required for ABA regulation of stomatal closure of isolated epidermal peels. However, there was no significant difference in stomatal conductance at the whole plant level between wild-type and pldδ mutants. Since PLD hydrolyses structural phospholipids, then we looked at membrane integrity. Ion leakage measurements showed that during dehydration of leaf discs pldδ mutant has less membrane degradation compared to the wild-type. We further analyzed the mutants and showed that pldδ have higher mRNA levels of RAB18 and RD29A compared to wild-type plants under normal growth conditions. Transient expression of AtPLDδ in Nicotiana benthamiana plants induced a wilting phenotype. These findings suggest that, in wt plants PLDδ disrupt membranes in severe drought stress and, in the absence of the protein (PLDδ knock-out) might drought-prime the plants, making them more tolerant to severe drought stress. The results are discussed in relation to PLDδ role in guard cell signaling and drought tolerance. PMID:26340512

  6. Kinetic properties and thermal stabilities of mutant forms of mitochondrial aspartate aminotransferase.

    PubMed

    Azzariti, A; Vacca, R A; Giannattasio, S; Merafina, R S; Marra, E; Doonan, S

    1998-07-28

    Kinetic properties and thermal stabilities of the precursor form of mitochondrial aspartate aminotransferase, the mature form lacking 9 amino acids from the N-terminus, and forms of the mature protein in which cysteine-166 had been mutated to serine or alanine were compared with those of the mature enzyme. The precursor and the cysteine mutants showed moderately impaired catalytic properties consistent with decreased ability to undergo transition from the open to the closed conformation which is an integral part of the mechanism of action of the enzyme. The deletion mutant had a kcat only 2% of that of the mature enzyme but also much reduced Km values for both substrates. In addition it showed enhanced reactivity of cysteine-166 with 5,5'-dithiobis(2-nitrobenzoate), which is characteristic of the closed form of the enzyme, with no enhancement of reactivity in the presence of substrates. This is taken to show that the deletion mutant adopts a conformation that is significantly different from that of the mature enzyme particularly in respect of the small domain. The deletion mutant was found to be more resistant to thermal inactivation over a range of temperatures than were the other forms of the enzyme consistent with its having a more tightly packed small domain. PMID:9675237

  7. Phospholipase D δ knock-out mutants are tolerant to severe drought stress.

    PubMed

    Distéfano, Ayelen M; Valiñas, Matías A; Scuffi, Denise; Lamattina, Lorenzo; Ten Have, Arjen; García-Mata, Carlos; Laxalt, Ana M

    2015-01-01

    Phospholipase D (PLD) is involved in different plant processes, ranging from responses to abiotic and biotic stress to plant development. Phospholipase Dδ (PLDδ) is activated in dehydration and salt stress, producing the lipid second messenger phosphatidic acid. In this work we show that pldδ Arabidopsis mutants were more tolerant to severe drought than wild-type plants. PLDδ has been shown to be required for ABA regulation of stomatal closure of isolated epidermal peels. However, there was no significant difference in stomatal conductance at the whole plant level between wild-type and pldδ mutants. Since PLD hydrolyses structural phospholipids, then we looked at membrane integrity. Ion leakage measurements showed that during dehydration of leaf discs pldδ mutant has less membrane degradation compared to the wild-type. We further analyzed the mutants and showed that pldδ have higher mRNA levels of RAB18 and RD29A compared to wild-type plants under normal growth conditions. Transient expression of AtPLDδ in Nicotiana benthamiana plants induced a wilting phenotype. These findings suggest that, in wt plants PLDδ disrupt membranes in severe drought stress and, in the absence of the protein (PLDδ knock-out) might drought-prime the plants, making them more tolerant to severe drought stress. The results are discussed in relation to PLDδ role in guard cell signaling and drought tolerance. PMID:26340512

  8. Differential sensitivity of Arabidopsis siRNA biogenesis mutants to genotoxic stress.

    PubMed

    Yao, Youli; Bilichak, Andriy; Golubov, Andrey; Blevins, Todd; Kovalchuk, Igor

    2010-12-01

    Plant response to stress has been linked to different RNA-silencing processes and epigenetic mechanisms. Our recent results showed that Arabidopsis thaliana Dicer-like (DCL) mutants were impaired in transgenerational changes, recombination frequency and stress tolerance. We also found that transgenerational changes were dependent on changes in DNA methylation. Here, we hypothesized that plants deficient in the production of small RNAs would show an impaired abiotic stress response. To test this, we exposed A. thaliana dcl2, dcl3, dcl4, dcl2 dcl3 (d2d3), dcl2 dcl4 (d2d4), dcl2 dcl3 dcl4 (d2d3d4), nrpd1a, rdr2 and rdr6 mutants to methyl methane sulfonate (MMS). We found dcl4 and rdr6 to be more sensitive and dcl2, dcl3, d2d3 and rdr2 plants more resistant to MMS, as shown by fresh weight, root length and survival rate. The in vitro repair assay showed the lower ability of dcl2 and dcl3 to repair UV-damaged DNA. To summarize, we found that whereas mutants impaired in transactivating siRNA biogenesis were more sensitive to MMS, mutants impaired in natural antisense siRNA and heterochromatic siRNA biogeneses were more tolerant. Our data suggest that plant response to MMS is in part regulated through biogenesis of various siRNAs. PMID:20953786

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

    PubMed Central

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

    1992-01-01

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

  10. 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

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

    PubMed

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

    2013-10-25

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

  12. Evidence that the LRRK2 ROC domain Parkinson's disease-associated mutants A1442P and R1441C exhibit increased intracellular degradation.

    PubMed

    Greene, Izabella D; Mastaglia, Francis; Meloni, Bruno P; West, Kristin A; Chieng, Joanne; Mitchell, Chris J; Gai, Wei-Ping; Boulos, Sherif

    2014-04-01

    Mutations in the leucine-rich repeat kinase 2 (lrrk2) gene are the leading genetic cause of Parkinson's disease (PD). In characterizing the novel ROC domain mutant A1442P, we compared its steady-state protein levels, propensity to aggregate, and toxicity with the pathogenic R1441C mutant and wild-type (WT) LRRK2. Mutant (R1441C and A1442P) and WT LRRK2 fused to green fluorescent protein (GFP) and FLAG were transiently expressed in HEK293 cells using plasmid constructs. Western analysis and fluorescence microscopy consistently demonstrated lower mutant LRRK2 protein levels compared with WT. A time-course expression study using flow cytometry showed that WT LRRK2 expression increased initially but then plateaued by 72 hr. Conversely, R1441C and A1442P mutant expression attained 85% and 74% of WT levels at 24 hr but fell to 68% and 55% of WT levels by 72 hr, respectively. We found that proteasome inhibition markedly increased mutant LRRK2 to levels approaching those of WT. Taken together, our findings reveal increased intracellular degradation for both mutants. Furthermore, the impact of mutant and WT LRRK2 expression on HEK293 cell viability was assessed under normative and oxidative (hydrogen peroxide) conditions and found not to differ. Expression of WT and mutant LRRK2 protein gave rise to intracellular aggregates of similar appearance and cellular localization. In summary, we provide evidence that the novel A1442P mutant and the previously investigated R1441C pathogenic mutant exhibit increased intracellular degradation, a property reportedly demonstrated for the pathogenic LRRK2 kinase domain mutant I2020T. PMID:24375786

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

    PubMed

    Swerdlow, Sarah J; Schaaper, Roel M

    2014-12-01

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

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

    PubMed Central

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

    1998-01-01

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

  15. Growth, seed development and genetic analysis in wild type and Def mutant of Pisum sativum L

    PubMed Central

    2011-01-01

    Background The def mutant pea (Pisum sativum L) showed non-abscission of seeds from the funicule. Here we present data on seed development and growth pattern and their relationship in predicting this particular trait in wild type and mutant lines as well as the inheritance pattern of the def allele in F2 and F3 populations. Findings Pod length and seed fresh weight increase with fruit maturity and this may affect the abscission event in pea seeds. However, the seed position in either the distal and proximal ends of the pod did not show any difference. The growth factors of seed fresh weight (FW), width of funicles (WFN), seed width (SW) and seed height (SH) were highly correlated and their relationships were determined in both wild type and def mutant peas. The coefficient of determination R2 values for the relationship between WFN and FW, SW and SH and their various interactions were higher for the def dwarf type. Stepwise multiple regression analysis showed that variation of WFN was associated with SH and SW. Pearson's chi square analysis revealed that the inheritance and segregation of the Def locus in 3:1 ratio was significant in two F2 populations. Structural analysis of the F3 population was used to confirm the inheritance status of the Def locus in F2 heterozygote plants. Conclusions This study investigated the inheritance of the presence or absence of the Def allele, controlling the presence of an abscission zone (AZ) or an abscission-less zone (ALZ) forming in wild type and mutant lines respectively. The single major gene (Def) controlling this phenotype was monogenic and def mutants were characterized and controlled by the homozygous recessive def allele that showed no palisade layers in the hilum region of the seed coat. PMID:22078070

  16. Carboxypeptidase S- and carboxypeptidase Y-deficient mutants of Saccharomyces cerevisiae.

    PubMed

    Wolf, D H; Ehmann, C

    1981-08-01

    A new carboxypeptidase (carboxypeptidase S) was found in a Saccharomyces cerevisiae strain lacking carboxypeptidase Y (D. H. Wolf and U. Weiser, Eur. J. Biochem. 73:553-556, 1977). Mutants devoid of carboxypeptidase S activity were isolated from a mutant strain that was also deficient in carboxypeptidase Y. Four mutants were analyzed in detail and fell into one complementation group. The defect segregated 2:2 in meiotic tetrads. Gene dosage experiments indicated that the mutation might reside in the structural gene of carboxypeptidase S. The absence of both enzymes, carboxypeptidases Y and S, did not affect mitotic growth. Ascopore formation was only slightly affected by the absence of both carboxypeptidases. Protein degradation under conditions of nutrient deprivation and under sporulation conditions showed no obvious alteration in the absence of carboxypeptidases Y and S. When a proteinase B mutation, which led to the absence of proteinase B activity and resulted in the partial reduction of sporulation, was introduced into a mutant lacking both carboxypeptidases, the ability of diploid cells to sporulate was nearly completely lost. Mutants lacking both carboxypeptidases were unable to grow on the dipeptide benzyloxycarbonylglycyl-l-leucine as a sole nitrogen source, which indicates an additional function for carboxypeptidases Y and S in supplying nutrients from exogenous peptides. Catabolite inactivation of fructose-1,6-bisphosphatase, cytoplasmic malate dehydrogenase, and phosphoenolpyruvate carboxykinase and inactivation of nicotin-amide adenine dinucleotide phosphate-dependent, glutamate dehydrogenase, events which have been proposed to involve proteolysis in vivo, were not dependent on the presence of carboxypeptidase Y and S. In a mutant lacking both carboxypeptidases, four new proteolytic enzymes with carboxypeptidase activity were detected. PMID:7021530

  17. Phenotypic and expression analysis of a novel spontaneous myosin VI null mutant mouse.

    PubMed

    Mochizuki, Eiji; Okumura, Kazuhiro; Ishikawa, Masashi; Yoshimoto, Sachi; Yamaguchi, Junya; Seki, Yuta; Wada, Kenta; Yokohama, Michinari; Ushiki, Tatsuo; Tokano, Hisashi; Ishii, Rie; Shitara, Hiroshi; Taya, Choji; Kitamura, Ken; Yonekawa, Hiromichi; Kikkawa, Yoshiaki

    2010-01-01

    In humans, hearing is a major factor in quality of life. Mouse models are important tools for the discovery of genes responsible for genetic hearing loss, often enabling analysis of the processes that regulate the onset of deafness in humans. Thus far, at least 400 deafness mutants have been discovered in laboratory mouse populations and used in the study of deafness. Here we report the discovery of a new spontaneous recessive Rinshoken shaker/waltzer (rsv) mutant derived from our in-house C57BL/6J stock, which exhibits circling and/or head-tossing behaviour and complete lack of auditory brain response to any sound pressure. The hearing and balance phenotypes are associated with structural defects, in particular, disorganisation and fusion of stereocilia in the inner ear hair cells. Two sets of intersubspecific N(2) mice were generated for the positional cloning of the rsv mutation. The mutant locus was mapped to a 4.8-Mb region of chromosome 9, which contains myosin VI (Myo6), a gene responsible for deafness in humans and Snell's waltzer mutation in mice. The rsv mutant showed reduced expressions of Myo6 mRNA and MYO6 protein in the inner ear. Moreover, no immunoreactivity was observed in the cochlear and vestibular hair cells in the rsv mutant mice. We sequenced the genomic region (30,154 bp) of Myo6, including all coding exons, a non-coding exon, UTRs and the Myo6 promoter; however, no mutation was discovered in these regions. We therefore speculate that loss of MYO6 expression might cause shaker/waltzer behaviour and deafness in the rsv mutant; also, loss of MYO6 expression might be the result of mutations in an unidentified regulatory region(s) of the gene. PMID:20224170

  18. Differences in relative amounts of two novel mutant HEXA transcripts in a juvenile TSD Druze patient

    SciTech Connect

    Drucker, L.; Navon, R. |

    1994-09-01

    An Israeli-Druze patient with juvenile Tay-Sachs disease, born to first cousins, was found to be a compound heterozygote for two novel mutant HEXA alleles. SSCP analysis of the parents` genomic DNA revealed alterations in both exons 5 and 8. Direct sequencing showed a novel missense mutation T{sup 835}{r_arrow}C (Ser{sup 279}{r_arrow}Pro) in exon 8, of maternal origin. The mutant allele of paternal origin carried a novel double mutation in exon 5, (i) a C{sup 496} deletion, resulting in a frameshift and eventually a stop codon, (ii) a C{sup 496}{r_arrow}G transition which is a silent mutation. Both these latter mutations occur in the same codon. New restriction sites for ScrFI were introduced into the two mutant alleles, enabling rapid screening for their presence. In order to detect differences of the relative levels of the transcripts originating from the two mutant alleles, we applied allele-specific transcripts polymerase chain reaction (AST-PCR) to the RNA extractions prepared from the heterozygous parents (each carry a normal and mutant allele). In order to distinguish between the transcripts originating from the normal allele and those originating from each of the mutant alleles, the transcripts were digested by ScrFI. A severe depletion of the mRNA coded by the allele carrying the mutation in exon 5 was found. The phenomena corresponds with citations in the literature in cases of stop mutations. The allele carrying the transversion in exon 8, contrary to our expectations, also had a distinctly lower level of transcripts. The AST-PCR approach offers a molecular tool to study allele-specific gene expression in heterozygous individuals.

  19. Doravirine Suppresses Common Nonnucleoside Reverse Transcriptase Inhibitor-Associated Mutants at Clinically Relevant Concentrations.

    PubMed

    Feng, Meizhen; Sachs, Nancy A; Xu, Min; Grobler, Jay; Blair, Wade; Hazuda, Daria J; Miller, Michael D; Lai, Ming-Tain

    2016-04-01

    Doravirine (DOR), which is currently in a phase 3 clinical trial, is a novel human immunodeficiency type 1 virus (HIV-1) nonnucleoside reverse transcriptase inhibitor (NNRTI). DOR exhibits potent antiviral activity against wild-type virus and K103N, Y181C, and K103N/Y181C mutant viruses, with 50% inhibitory concentrations (IC50s) of 12, 21, 31, and 33 nM, respectively, when measured in 100% normal human serum (NHS). To assess the potential for DOR to suppress NNRTI-associated and rilpivirine (RPV)-specific mutants at concentrations achieved in the clinic setting, inhibitory quotients (IQs) were calculated by determining the ratio of the clinical trough concentration over the antiviral IC50for each virus with DOR and RPV and efavirenz (EFV). DOR displayed IQs of 39, 27, and 25 against the K103N, Y181C, and K103N/Y181C mutants, respectively. In contrast, RPV exhibited IQs of 4.6, 1.4, and 0.8, and EFV showed IQs of 2.5, 60, and 1.9 against these viruses, respectively. DOR also displayed higher IQs than those of RPV and EFV against other prevalent NNRTI-associated mutants, with the exception of Y188L. Both DOR and EFV exhibited higher IQs than RPV when analyzed with RPV-associated mutants. Resistance selections were conducted with K103N, Y181C, G190A, and K103N/Y181C mutants at clinically relevant concentrations of DOR, RPV, and EFV. No viral breakthrough was observed with DOR, whereas breakthrough viruses were readily detected with RPV and EFV against Y181C and K103N viruses, respectively. These data suggest that DOR should impose a higher barrier to the development of resistance than RPV and EFV at the concentrations achieved in the clinic setting. PMID:26833152

  20. Characterization of Escherichia coli d-Cycloserine Transport and Resistant Mutants

    PubMed Central

    Baisa, Gary; Stabo, Nicholas J.

    2013-01-01

    d-Cycloserine (DCS) is a broad-spectrum antibiotic that inhibits d-alanine ligase and alanine racemase activity. When Escherichia coli K-12 or CFT073 is grown in minimal glucose or glycerol medium, CycA transports DCS into the cell. E. coli K-12 cycA and CFT073 cycA mutant strains display increased DCS resistance when grown in minimal medium. However, the cycA mutants exhibit no change in DCS sensitivity compared to their parental strains when grown in LB (CFT073 and K-12) or human urine (CFT073 only). These data suggest that cycA does not participate in DCS sensitivity when strains are grown in a non-minimal medium. The small RNA GvcB acts as a negative regulator of E. coli K-12 cycA expression when grown in LB. Three E. coli K-12 gcvB mutant strains failed to demonstrate a change in DCS sensitivity when grown in LB. This further suggests a limited role for cycA in DCS sensitivity. To aid in the identification of E. coli genes involved in DCS sensitivity when grown on complex media, the Keio K-12 mutant collection was screened for DCS-resistant strains. dadA, pnp, ubiE, ubiF, ubiG, ubiH, and ubiX mutant strains showed elevated DCS resistance. The phenotypes associated with these mutants were used to further define three previously characterized E. coli DCS-resistant strains (χ316, χ444, and χ453) isolated by Curtiss and colleagues (R. Curtiss, III, L. J. Charamella, C. M. Berg, and P. E. Harris, J. Bacteriol. 90:1238–1250, 1965). A dadA mutation was identified in both χ444 and χ453. In addition, results are presented that indicate for the first time that DCS can antagonize d-amino acid dehydrogenase (DadA) activity. PMID:23316042

  1. Development of a forward genetic screen to isolate oil mutants in the green microalga Chlamydomonas reinhardtii

    PubMed Central

    2013-01-01

    Background Oils produced by microalgae are precursors to biodiesel. To achieve a profitable production of biodiesel from microalgae, identification of factors governing oil synthesis and turnover is desirable. The green microalga Chlamydomonas reinhardtii is amenable to genetic analyses and has recently emerged as a model to study oil metabolism. However, a detailed method to isolate various types of oil mutants that is adapted to Chlamydomonas has not been reported. Results We describe here a forward genetic approach to isolate mutants altered in oil synthesis and turnover from C. reinhardtii. It consists of a three-step screening procedure: a primary screen by flow cytometry of Nile red stained transformants grown in 96-deep-well plates under three sequential conditions (presence of nitrogen, then absence of nitrogen, followed by oil remobilization); a confirmation step using Nile red stained biological triplicates; and a validation step consisting of the quantification by thin layer chromatography of oil content of selected strains. Thirty-one mutants were isolated by screening 1,800 transformants generated by random insertional mutagenesis (1.7%). Five showed increased oil accumulation under the nitrogen-replete condition and 13 had altered oil content under nitrogen-depletion. All mutants were affected in oil remobilization. Conclusion This study demonstrates that various types of oil mutants can be isolated in Chlamydomonas based on the method set-up here, including mutants accumulating oil under optimal biomass growth. The strategy conceived and the protocol set-up should be applicable to other microalgal species such as Nannochloropsis and Chlorella, thus serving as a useful tool in Chlamydomonas oil research and algal biotechnology. PMID:24295516

  2. Effects of pseudophosphorylation mutants on the structural dynamics of smooth muscle myosin regulatory light chain.

    PubMed

    Espinoza-Fonseca, L Michel; Colson, Brett A; Thomas, David D

    2014-10-01

    We have performed 50 independent molecular dynamics (MD) simulations to determine the effect of pseudophosphorylation mutants on the structural dynamics of smooth muscle myosin (SMM) regulatory light chain (RLC). We previously showed that the N-terminal phosphorylation domain of RLC simultaneously populates two structural states in equilibrium, closed and open, and that phosphorylation at S19 induces a modest shift toward the open state, which is sufficient to activate smooth muscle. However, it remains unknown why pseudophosphorylation mutants poorly mimic phosphorylation-induced activation of SMM. We performed MD simulations of unphosphorylated, phosphorylated, and three pseudophosphorylated RLC mutants: S19E, T18D/S19D and T18E/S19E. We found that the S19E mutation does not shift the equilibrium toward the open state, indicating that simple charge replacement at position S19 does not mimic the activating effect of phosphorylation, providing a structural explanation for previously published functional data. In contrast, mutants T18D/S19D and T18E/S19E shift the equilibrium toward the open structure and partially activate in vitro motility, further supporting the model that an increase in the mol fraction of the open state is coupled to SMM motility. Structural analyses of the doubly-charged pseudophosphorylation mutants suggest that alterations in an interdomain salt bridge between residues R4 and D100 results in impaired signal transmission from RLC to the catalytic domain of SMM, which explains the low ATPase activity of these mutants. Our results demonstrate that phosphorylation produces a unique structural balance in the RLC. These observations have important implications for our understanding of the structural aspects of activation and force potentiation in smooth and striated muscle. PMID:25091814

  3. Genetic analysis of calcium spiking responses in nodulation mutants of Medicago truncatula

    PubMed Central

    Wais, Rebecca J.; Galera, Christine; Oldroyd, Giles; Catoira, Romy; Penmetsa, R. Varma; Cook, Douglas; Gough, Clare; Dénarié, Jean; Long, Sharon R.

    2000-01-01

    The symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti results in the formation of nitrogen-fixing nodules on the roots of the host plant. The early stages of nodule formation are induced by bacteria via lipochitooligosaccharide signals known as Nod factors (NFs). These NFs are structurally specific for bacterium–host pairs and are sufficient to cause a range of early responses involved in the host de