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Sample records for response mutants defective

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

    PubMed

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

    2009-08-01

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

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

    PubMed Central

    2013-01-01

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

  3. Epididymis response partly compensates for spermatozoa oxidative defects in snGPx4 and GPx5 double mutant mice.

    PubMed

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

    2012-01-01

    We report here that spermatozoa of mice lacking both the sperm nucleus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H(2)O(2)-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice.

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

    PubMed Central

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

    2012-01-01

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

  5. Differential Activation of Cellular DNA Damage Responses by Replication-Defective and Replication-Competent Adenovirus Mutants

    PubMed Central

    Prakash, Anand; Jayaram, Sumithra

    2012-01-01

    Adenovirus (Ad) mutants that lack early region 4 (E4) activate the phosphorylation of cellular DNA damage response proteins. In wild-type Ad type 5 (Ad5) infections, E1b and E4 proteins target the cellular DNA repair protein Mre11 for redistribution and degradation, thereby interfering with its ability to activate phosphorylation cascades important during DNA repair. The characteristics of Ad infection that activate cellular DNA repair processes are not yet well understood. We investigated the activation of DNA damage responses by a replication-defective Ad vector (AdRSVβgal) that lacks E1 and fails to produce the immediate-early E1a protein. E1a is important for activating early gene expression from the other viral early transcription units, including E4. AdRSVβgal can deliver its genome to the cell, but it is subsequently deficient for viral early gene expression and DNA replication. We studied the ability of AdRSVβgal-infected cells to induce cellular DNA damage responses. AdRSVβgal infection does activate formation of foci containing the Mdc1 protein. However, AdRSVβgal fails to activate phosphorylation of the damage response proteins Nbs1 and Chk1. We found that viral DNA replication is important for Nbs1 phosphorylation, suggesting that this step in the viral life cycle may provide an important trigger for activating at least some DNA repair proteins. PMID:23015708

  6. Characterization of Halobacterium halobium mutants defective in taxis.

    PubMed Central

    Sundberg, S A; Alam, M; Lebert, M; Spudich, J L; Oesterhelt, D; Hazelbauer, G L

    1990-01-01

    Mutant derivatives of Halobacterium halobium previously isolated by using a procedure that selected for defective phototactic response to white light were examined for an array of phenotypic characteristics related to phototaxis and chemotaxis. The properties tested were unstimulated swimming behavior, behaviorial responses to temporal gradients of light and spatial gradients of chemoattractants, content of photoreceptor pigments, methylation of methyl-accepting taxis proteins, and transient increases in rate of release of volatile methyl groups induced by tactic stimulation. Several distinct phenotypes were identified, corresponding to a mutant missing photoreceptors, a mutant defective in the methyltransferase, a mutant altered in control of the methylesterase, and mutants apparently defective in intracellular signaling. All except the photoreceptor mutant were defective in both chemotaxis and phototaxis. Images PMID:2332402

  7. Characterization of Halobacterium halobium mutants defective in taxis.

    PubMed

    Sundberg, S A; Alam, M; Lebert, M; Spudich, J L; Oesterhelt, D; Hazelbauer, G L

    1990-05-01

    Mutant derivatives of Halobacterium halobium previously isolated by using a procedure that selected for defective phototactic response to white light were examined for an array of phenotypic characteristics related to phototaxis and chemotaxis. The properties tested were unstimulated swimming behavior, behaviorial responses to temporal gradients of light and spatial gradients of chemoattractants, content of photoreceptor pigments, methylation of methyl-accepting taxis proteins, and transient increases in rate of release of volatile methyl groups induced by tactic stimulation. Several distinct phenotypes were identified, corresponding to a mutant missing photoreceptors, a mutant defective in the methyltransferase, a mutant altered in control of the methylesterase, and mutants apparently defective in intracellular signaling. All except the photoreceptor mutant were defective in both chemotaxis and phototaxis.

  8. Mutagenic and Recombinagenic Responses to Defective DNA Polymerase δ Are Facilitated by the Rev1 Protein in pol3-t Mutants of Saccharomyces cerevisiae

    PubMed Central

    Mito, Erica; Mokhnatkin, Janet V.; Steele, Molly C.; Buettner, Victoria L.; Sommer, Steve S.; Manthey, Glenn M.; Bailis, Adam M.

    2008-01-01

    Defective DNA replication can result in substantial increases in the level of genome instability. In the yeast Saccharomyces cerevisiae, the pol3-t allele confers a defect in the catalytic subunit of replicative DNA polymerase δ that results in increased rates of mutagenesis, recombination, and chromosome loss, perhaps by increasing the rate of replicative polymerase failure. The translesion polymerases Pol η, Pol ζ, and Rev1 are part of a suite of factors in yeast that can act at sites of replicative polymerase failure. While mutants defective in the translesion polymerases alone displayed few defects, loss of Rev1 was found to suppress the increased rates of spontaneous mutation, recombination, and chromosome loss observed in pol3-t mutants. These results suggest that Rev1 may be involved in facilitating mutagenic and recombinagenic responses to the failure of Pol δ. Genome stability, therefore, may reflect a dynamic relationship between primary and auxiliary DNA polymerases. PMID:18711219

  9. Identification of residues responsible for the defective virulence gene regulator Mga produced by a natural mutant of Streptococcus pyogenes.

    PubMed

    Vahling, Cheryl M; McIver, Kevin S

    2005-09-01

    Mga is a transcriptional regulator in the pathogen Streptococcus pyogenes that positively activates several important virulence genes involved in colonization and immune evasion in the human host. A naturally occurring mutant of Mga that is defective in its ability to activate transcription has been identified in the serotype M50 strain B514-Sm. Sequence alignment of the defective M50 Mga with the fully functional Mga from serotypes M4 and M49 revealed only three amino acid changes that might result in a defective protein. Electrophoretic mobility shift assays using purified M50 and M4 maltose binding protein-Mga found that both exhibited DNA-binding activity towards regulated promoters. Thus, the significance of each residue for the functionality of M50 Mga was explored through introduction of "gain-of-function" mutations based on M4 Mga. Transcriptional studies of the mutant alleles under both constitutive (PrpsL) and autoactivated (Pmga4) promoters illustrated that an arginine-to-methionine change at position 461 of M50 Mga protein fully restored activation of downstream genes. Western blot analyses of steady-state Mga levels suggest that the M461 residue may play a role in overall conformation and protein stability of Mga. However, despite the conservation of the M461 protein among all other Mga proteins, it does not appear to be necessary for activity in a divergent M6 Mga. These studies highlight the potential differences that exist between divergent Mga proteins in this important human pathogen.

  10. Identification of Residues Responsible for the Defective Virulence Gene Regulator Mga Produced by a Natural Mutant of Streptococcus pyogenes

    PubMed Central

    Vahling, Cheryl M.; McIver, Kevin S.

    2005-01-01

    Mga is a transcriptional regulator in the pathogen Streptococcus pyogenes that positively activates several important virulence genes involved in colonization and immune evasion in the human host. A naturally occurring mutant of Mga that is defective in its ability to activate transcription has been identified in the serotype M50 strain B514-Sm. Sequence alignment of the defective M50 Mga with the fully functional Mga from serotypes M4 and M49 revealed only three amino acid changes that might result in a defective protein. Electrophoretic mobility shift assays using purified M50 and M4 maltose binding protein-Mga found that both exhibited DNA-binding activity towards regulated promoters. Thus, the significance of each residue for the functionality of M50 Mga was explored through introduction of “gain-of-function” mutations based on M4 Mga. Transcriptional studies of the mutant alleles under both constitutive (PrpsL) and autoactivated (Pmga4) promoters illustrated that an arginine-to-methionine change at position 461 of M50 Mga protein fully restored activation of downstream genes. Western blot analyses of steady-state Mga levels suggest that the M461 residue may play a role in overall conformation and protein stability of Mga. However, despite the conservation of the M461 protein among all other Mga proteins, it does not appear to be necessary for activity in a divergent M6 Mga. These studies highlight the potential differences that exist between divergent Mga proteins in this important human pathogen. PMID:16109937

  11. Induction of the Pho Regulon Suppresses the Growth Defect of an Escherichia coli sgrS Mutant, Connecting Phosphate Metabolism to the Glucose-Phosphate Stress Response

    PubMed Central

    Richards, Gregory R.

    2012-01-01

    Some bacteria experience stress when glucose-6-phosphate or analogues like α-methyl glucoside-6-phosphate (αMG6P) accumulate in the cell. In Escherichia coli, the small SgrS RNA is vital to recovery from glucose-phosphate stress; the growth of sgrS mutants is strongly inhibited by αMG. SgrS helps to restore growth in part through inhibiting translation of the ptsG mRNA, which encodes the major glucose transporter EIICBGlc. While the regulatory mechanism of SgrS has been characterized, little is known about how glucose-phosphate stress connects to other aspects of cell physiology. In the present study, we discovered that mutation of pitA, which encodes the low-affinity transporter of inorganic phosphate, partially suppresses the αMG growth defect of an sgrS mutant. Induction of the stress response was also reduced in the sgrS pitA mutant compared to its sgrS parent. Microarray analysis suggested that expression of phosphate (Pho) regulon genes is increased in the sgrS pitA mutant compared to the sgrS parent. Consistent with this, we found increased PhoA (alkaline phosphatase) activity in the sgrS pitA mutant compared to the sgrS strain. Further, direct induction of the Pho regulon (in a pitA+ background) also resulted in partial suppression of the sgrS growth defect. The suppression was reversed when Pho induction was prevented by mutation of phoB, which encodes the Pho transcriptional activator. Deletion of individual Pho structural genes in suppressed strains did not identify a single gene responsible for suppression. Altogether, this work describes one of the first studies of glucose-phosphate stress physiology and suggests a novel connection of carbon and phosphate metabolism. PMID:22427626

  12. 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.

  13. Isolation of a Defective Prion Mutant from Natural Scrapie

    PubMed Central

    Migliore, Sergio; Cosseddu, Gian Mario; Pirisinu, Laura; Riccardi, Geraldina; Nonno, Romolo

    2016-01-01

    It is widely known that prion strains can mutate in response to modification of the replication environment and we have recently reported that prion mutations can occur in vitro during amplification of vole-adapted prions by Protein Misfolding Cyclic Amplification on bank vole substrate (bvPMCA). Here we exploited the high efficiency of prion replication by bvPMCA to study the in vitro propagation of natural scrapie isolates. Although in vitro vole-adapted PrPSc conformers were usually similar to the sheep counterpart, we repeatedly isolated a PrPSc mutant exclusively when starting from extremely diluted seeds of a single sheep isolate. The mutant and faithful PrPSc conformers showed to be efficiently autocatalytic in vitro and were characterized by different PrP protease resistant cores, spanning aa ∼155–231 and ∼80–231 respectively, and by different conformational stabilities. The two conformers could thus be seen as different bona fide PrPSc types, putatively accounting for prion populations with different biological properties. Indeed, once inoculated in bank vole the faithful conformer was competent for in vivo replication while the mutant was unable to infect voles, de facto behaving like a defective prion mutant. Overall, our findings confirm that prions can adapt and evolve in the new replication environments and that the starting population size can affect their evolutionary landscape, at least in vitro. Furthermore, we report the first example of “authentic” defective prion mutant, composed of brain-derived PrPC and originating from a natural scrapie isolate. Our results clearly indicate that the defective mutant lacks of some structural characteristics, that presumably involve the central region ∼90–155, critical for infectivity but not for in vitro replication. Finally, we propose a molecular mechanism able to account for the discordant in vitro and in vivo behavior, suggesting possible new paths for investigating the molecular bases of

  14. Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1delta mutant of Saccharomyces cerevisiae.

    PubMed Central

    Popolo, L; Gilardelli, D; Bonfante, P; Vai, M

    1997-01-01

    The GGP1/GAS1 gene codes for a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae. The ggp1delta mutant shows morphogenetic defects which suggest changes in the cell wall matrix. In this work, we have investigated cell wall glucan levels and the increase of chitin in ggp1delta mutant cells. In these cells, the level of alkali-insoluble 1,6-beta-D-glucan was found to be 50% of that of wild-type cells and was responsible for the observed decrease in the total alkali-insoluble glucan. Moreover, the ratio of alkali-soluble to alkali-insoluble glucan almost doubled, suggesting a change in glucan solubility. The increase of chitin in ggp1delta cells was found to be essential since the chs3delta ggp1delta mutations determined a severe reduction in the growth rate and in cell viability. Electron microscopy analysis showed the loss of the typical structure of yeast cell walls. Furthermore, in the chs3delta ggp1delta cells, the level of alkali-insoluble glucan was 57% of that of wild-type cells and the alkali-soluble/alkali-insoluble glucan ratio was doubled. We tested the effect of inhibition of chitin synthesis also by a different approach. The ggp1delta cells were treated with nikkomycin Z, a well-known inhibitor of chitin synthesis, and showed a hypersensitivity to this drug. In addition, studies of genetic interactions with genes related to the construction of the cell wall indicate a synthetic lethal effect of the ggp1delta kre6delta and the ggp1delta pkc1delta combined mutations. Our data point to an involvement of the GGP1 gene product in the cross-links between cell wall glucans (1,3-beta-D-glucans with 1,6-beta-D-glucans and with chitin). Chitin is essential to compensate for the defects due to the lack of Ggp1p. Moreover, the activities of Ggp1p and Chs3p are essential to the formation of the organized structure of the cell wall in vegetative cells. PMID:8990299

  15. Isolation and characterization of an olfactory mutant in Drosophila with a chemically specific defect.

    PubMed Central

    Helfand, S L; Carlson, J R

    1989-01-01

    A Drosophila mutant was isolated and shown to exhibit defective response to the chemical odorant benzaldehyde in two distinctly different behavioral assays. The defect exhibited chemical specificity: response to three other chemicals was normal. The mutant also showed abnormalities in pigmentation and fertility. Genetic mapping and complementation analysis provide evidence that the olfactory, pigmentation, and fertility defects arise as a result of a lesion at the pentagon locus. The specificity of the olfactory defect suggests the possibility that the mutation may define a molecule required in reception, transduction, or processing of a specific subset of chemical information in the olfactory system. Images PMID:2495539

  16. 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

  17. Bacteriophage SPO1 development: defects in a gene 31 mutant.

    PubMed Central

    Sarachu, A N; Añón, M C; Grau, O

    1978-01-01

    SPO1 temperature-sensitive mutant ts14-1, located in cistron 31, has a DD (DNA synthesis-delayed) phenotype at 37 degrees C and produces progeny in a stretched program. At 44 degrees C it behaves as a DO (DNA synthesis-defective) mutant and shuts off the viral RNA synthesis about 10 min after infection. The thermal sensitivity of this mutant is due to the inactivity of gp-31 (the product of gene 31) at 44 degrees C. However, gp-31 is synthesized at that temperature and partly recovers its activity at 37 degrees C. Only 5 min at the permissive temperature is enough to trigger the continuation of the phage program and to produce progeny. The partial defect at 37 degrees C and the expansion of the middle program together with the pleiotropic defects at the nonpermissive temperature could be suitable for the study of the controls involved in bacteriophage development. PMID:100606

  18. Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

    PubMed Central

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

    2009-01-01

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

  19. 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.

  20. Rhizobium japonicum mutants defective in symbiotic nitrogen fixation.

    PubMed Central

    Noel, K D; Stacey, G; Tandon, S R; Silver, L E; Brill, W J

    1982-01-01

    Rhizobium japonicum strains 3I1b110 and 61A76 were mutagenized to obtain 25 independently derived mutants that produced soybean nodules defective in nitrogen fixation, as assayed by acetylene reduction. The proteins of both the bacterial and the plant portions of the nodules were analyzed by two-dimensional polyacrylamide gel electrophoresis. All of the mutants had lower-than-normal levels of the nitrogenase components, and all but four contained a prominent bacteroid protein not observed in wild-type bacteroids. Experiments with bacteria grown ex planta suggested that this protein was derepressed by the absence of ammonia. Nitrogenase component II of one mutant was altered in isoelectric point. The soluble plant fraction of the nodules of seven mutants had very low levels of heme, yet the nodules of five of these seven mutants contained the polypeptide of leghemoglobin. Thus, the synthesis of the globin may not be coupled to the content of available heme in soybean nodules. The nodules of the other two of these seven mutants lacked not only leghemoglobin but most of the other normal plant and bacteroid proteins. Ultrastructural examination of nodules formed by these two mutants indicated normal ramification of infection threads but suggested a problem in subsequent survival of the bacteria and their release from the infection threads. Images PMID:6956566

  1. A Rice Mutant Defective in Si Uptake1

    PubMed Central

    Ma, Jian Feng; Tamai, Kazunori; Ichii, Masahiko; Wu, Guo Feng

    2002-01-01

    Rice (Oryza sativa) accumulates silicon (Si) in the tops to levels up to 10.0% of shoot dry weight, but the mechanism responsible for high Si uptake by rice roots is not understood. We isolated a rice mutant (GR1) that is defective in active Si uptake by screening M2 seeds (64,000) of rice cv Oochikara that were treated with 10−3 m sodium azide for 6 h at 25oC. There were no phenotypic differences between wild type (WT) and GR1 except that the leaf blade of GR1 remained droopy when Si was supplied. Uptake experiments showed that Si uptake by GR1 was significantly lower than that by WT at both low and high Si concentrations. However, there was no difference in the uptake of other nutrients such as phosphorus and potassium. Si concentration in the xylem sap of WT was 33-fold that of the external solution, but that of GR1 was 3-fold higher than the external solution at 0.15 mm Si. Si uptake by WT was inhibited by metabolic inhibitors including NaCN and 2,4-dinitrophenol and by low temperature, whereas Si uptake by GR1 was not inhibited by these agents. These results suggest that an active transport system for Si uptake is disrupted in GR1. Analysis of F2 populations between GR1 and WT showed that roots with high Si uptake and roots with low Si uptake segregated at a 3:1 ratio, suggesting that GR1 is a recessive mutant of Si uptake. PMID:12481095

  2. 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

  3. 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.

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

    PubMed

    McCammon, M T

    1996-09-01

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

  5. 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

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

    PubMed

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

    2005-01-01

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

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

    PubMed

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

    2015-12-01

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

  8. Temperature Sensitivity of Neural Tube Defects in Zoep Mutants

    PubMed Central

    Ma, Phyo; Swartz, Morgan R.; Kindt, Lexy M.; Kangas, Ashley M.

    2015-01-01

    Abstract Neural tube defects (NTD) occur when the flat neural plate epithelium fails to fold into the neural tube, the precursor to the brain and spinal cord. Squint (Sqt/Ndr1), a Nodal ligand, and One-eyed pinhead (Oep), a component of the Nodal receptor, are required for anterior neural tube closure in zebrafish. The NTD in sqt and Zoep mutants are incompletely penetrant. The penetrance of several defects in sqt mutants increases upon heat or cold shock. In this project, undergraduate students tested whether temperature influences the Zoep open neural tube phenotype. Single pairs of adults were spawned at 28.5°C, the normal temperature for zebrafish, and one half of the resulting embryos were moved to 34°C at different developmental time points. Analysis of variance indicated temperature and clutch/genetic background significantly contributed to the penetrance of the open neural tube phenotype. Heat shock affected the embryos only at or before the midblastula stage. Many factors, including temperature changes in the mother, nutrition, and genetic background, contribute to NTD in humans. Thus, sqt and Zoep mutants may serve as valuable models for studying the interactions between genetics and the environment during neurulation. PMID:26366681

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

    PubMed

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

    2014-05-01

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

  10. Defective Kernel Mutants of Maize II. Morphological and Embryo Culture Studies.

    PubMed

    Sheridan, W F; Neuffer, M G

    1980-08-01

    This report presents the initial results of our study of the immature kernel stage of 150 defective kernel maize mutants. They are single gene, recessive mutants that map throughout the genome, defective in both endosperm and embryo development and, for the most part, lethal (Neuffer and Sheridan 1980). All can be distinguished on immature ears, and 85% of them reveal a mutant phenotype within 11 to 17 days post-pollination. Most have immature kernels that are smaller and lighter in color than their normal counterparts. Forty of the mutants suffer from their defects early in kernel development and are blocked in embryogenesis before their primordia differentiate, or, if primordia are formed, they are unable to germinate when cultured as immature embryos or tested at maturity; a few begin embryo degeneration prior to the time that mutant kernels became visually distinguishable. The others express the associated lesion later in kernel development and form at least one leaf primordium by the time kernels are distinguishable and will germinate when cultured or tested at maturity. In most cases, on a fresh weight basis, the mutants have embryos that are more severely defective than the endosperm; their embryos usually are no more than one-half to two-thirds the size, and lag behind by one or two developmental stages. in comparison with embryos in normal kernels from the same ear. One hundred and two mutants were examined by culturing embryos on basal and enriched media; 21 simply enlarged or completely failed to grow on any of the media tested; and 81 produced shoots and roots on at least one medium. Many grew equally well on basal and enriched media; 16 grew at a faster rate on basal medium and 23 displayed a superior growth on enriched medium. Among the latter group, 10 may be auxotrophs. One of these mutants and another mutant isolated by E. H. Coe are proline-requiring mutants, allelic to pro-1. Considering their diversity of expression as evidenced by their

  11. Defective Kernel Mutants of Maize II. Morphological and Embryo Culture Studies

    PubMed Central

    Sheridan, William F.; Neuffer, M. G.

    1980-01-01

    This report presents the initial results of our study of the immature kernel stage of 150 defective kernel maize mutants. They are single gene, recessive mutants that map throughout the genome, defective in both endosperm and embryo development and, for the most part, lethal (Neuffer and Sheridan 1980). All can be distinguished on immature ears, and 85% of them reveal a mutant phenotype within 11 to 17 days post-pollination. Most have immature kernels that are smaller and lighter in color than their normal counterparts. Forty of the mutants suffer from their defects early in kernel development and are blocked in embryogenesis before their primordia differentiate, or, if primordia are formed, they are unable to germinate when cultured as immature embryos or tested at maturity; a few begin embryo degeneration prior to the time that mutant kernels became visually distinguishable. The others express the associated lesion later in kernel development and form at least one leaf primordium by the time kernels are distinguishable and will germinate when cultured or tested at maturity. In most cases, on a fresh weight basis, the mutants have embryos that are more severely defective than the endosperm; their embryos usually are no more than one-half to two-thirds the size, and lag behind by one or two developmental stages. in comparison with embryos in normal kernels from the same ear. One hundred and two mutants were examined by culturing embryos on basal and enriched media; 21 simply enlarged or completely failed to grow on any of the media tested; and 81 produced shoots and roots on at least one medium. Many grew equally well on basal and enriched media; 16 grew at a faster rate on basal medium and 23 displayed a superior growth on enriched medium. Among the latter group, 10 may be auxotrophs. One of these mutants and another mutant isolated by E. H. Coe are proline-requiring mutants, allelic to pro-1. Considering their diversity of expression as evidenced by their

  12. Aspergillus nidulans mutants defective in stc gene cluster regulation.

    PubMed Central

    Butchko, R A; Adams, T H; Keller, N P

    1999-01-01

    The genes involved in the biosynthesis of sterigmatocystin (ST), a toxic secondary metabolite produced by Aspergillus nidulans and an aflatoxin (AF) precursor in other Aspergillus spp., are clustered on chromosome IV of A. nidulans. The sterigmatocystin gene cluster (stc gene cluster) is regulated by the pathway-specific transcription factor aflR. The function of aflR appears to be conserved between ST- and AF-producing aspergilli, as are most of the other genes in the cluster. We describe a novel screen for detecting mutants defective in stc gene cluster activity by use of a genetic block early in the ST biosynthetic pathway that results in the accumulation of the first stable intermediate, norsolorinic acid (NOR), an orange-colored compound visible with the unaided eye. We have mutagenized this NOR-accumulating strain and have isolated 176 Nor(-) mutants, 83 of which appear to be wild type in growth and development. Sixty of these 83 mutations are linked to the stc gene cluster and are likely defects in aflR or known stc biosynthetic genes. Of the 23 mutations not linked to the stc gene cluster, 3 prevent accumulation of NOR due to the loss of aflR expression. PMID:10511551

  13. 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

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

    SciTech Connect

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

    1990-11-01

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

  15. Reduced heme levels underlie the exponential growth defect of the Shewanella oneidensis hfq mutant.

    PubMed

    Brennan, Christopher M; Mazzucca, Nicholas Q; Mezoian, Taylor; Hunt, Taylor M; Keane, Meaghan L; Leonard, Jessica N; Scola, Shelby E; Beer, Emma N; Perdue, Sarah; Pellock, Brett J

    2014-01-01

    The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA) function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA), the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.

  16. Reduced Heme Levels Underlie the Exponential Growth Defect of the Shewanella oneidensis hfq Mutant

    PubMed Central

    Mezoian, Taylor; Hunt, Taylor M.; Keane, Meaghan L.; Leonard, Jessica N.; Scola, Shelby E.; Beer, Emma N.; Perdue, Sarah; Pellock, Brett J.

    2014-01-01

    The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA) function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA), the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step. PMID:25356668

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

    PubMed

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

    2016-06-01

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

  18. Characterization of axonal transport defects in Drosophila Huntingtin mutants.

    PubMed

    Weiss, Kurt R; Littleton, J Troy

    Polyglutamine (polyQ) expansion within Huntingtin (Htt) causes the fatal neurodegenerative disorder Huntington's Disease (HD). Although Htt is ubiquitously expressed and conserved from Drosophila to humans, its normal biological function is still being elucidated. Here we characterize a role for the Drosophila Htt homolog (dHtt) in fast axonal transport (FAT). Generation and expression of transgenic dHtt-mRFP and human Htt-mRFP fusion proteins in Drosophila revealed co-localization with mitochondria and synaptic vesicles undergoing FAT. However, Htt was not ubiquitously associated with the transport machinery, as it was excluded from dense-core vesicles and APLIP1 containing vesicles. Quantification of cargo movement in dHtt deficient axons revealed that mitochondria and synaptic vesicles show a decrease in the distance and duration of transport, and an increase in the number of pauses. In addition, the ratio of retrograde to anterograde flux was increased in mutant animals. Dense-core vesicles did not display similar defects in processivity, but did show altered retrograde to anterograde flux along axons. Given the co-localization with mitochondria and synaptic vesicles, but not dense-core vesicles, the data suggest dHtt likely acts locally at cargo interaction sites to regulate processivity. An increase in dynein heavy chain expression was also observed in dHtt mutants, suggesting that the altered flux observed for all cargo may represent secondary transport changes occurring independent of dHtt's primary function. Expression of dHtt in a milton (HAP1) mutant background revealed that the protein does not require mitochondria or HAP1 to localize along axons, suggesting Htt has an independent mechanism for coupling with motors to regulate their processivity during axonal transport.

  19. Mutations in rpoBC suppress the defects of a Sinorhizobium meliloti relA mutant.

    PubMed

    Wells, Derek H; Long, Sharon R

    2003-09-01

    The nitrogen-fixing symbiosis between Sinorhizobium meliloti and Medicago sativa requires complex physiological adaptation by both partners. One method by which bacteria coordinately control physiological adaptation is the stringent response, which is triggered by the presence of the nucleotide guanosine tetraphosphate (ppGpp). ppGpp, produced by the RelA enzyme, is thought to bind to and alter the ability of RNA polymerase (RNAP) to initiate and elongate transcription and affect the affinity of the core enzyme for various sigma factors. An S. meliloti relA mutant which cannot produce ppGpp was previously shown to be defective in the ability to form nodules. This mutant also overproduces a symbiotically necessary exopolysaccharide called succinoglycan. The work presented here encompasses the analysis of suppressor mutants, isolated from host plants, that suppress the symbiotic defects of the relA mutant. All suppressor mutations are extragenic and map to either rpoB or rpoC, which encode the beta and beta' subunits of RNAP. Phenotypic, structural, and gene expression analyses reveal that suppressor mutants can be divided into two classes; one is specific in its effect on stringent response-regulated genes and shares striking similarity with suppressor mutants of Escherichia coli strains that lack ppGpp, and another reduces transcription of all genes tested in comparison to that in the relA parent strain. Our findings indicate that the ability to successfully establish symbiosis is tightly coupled with the bacteria's ability to undergo global physiological adjustment via the stringent response.

  20. A rhizobium leguminosarum mutant defective in symbiotic iron acquisition

    SciTech Connect

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

    1990-02-01

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

  1. Molecular defect of isovaleryl-CoA dehydrogenase in the skunk mutant of silkworm, Bombyx mori.

    PubMed

    Urano, Kei; Daimon, Takaaki; Banno, Yutaka; Mita, Kazuei; Terada, Tohru; Shimizu, Kentaro; Katsuma, Susumu; Shimada, Toru

    2010-11-01

    The isovaleric acid-emanating silkworm mutant skunk (sku) was first studied over 30 years ago because of its unusual odour and prepupal lethality. Here, we report the identification and characterization of the gene responsible for the sku mutant. Because of its specific features and symptoms similar to human isovaleryl-CoA dehydrogenase (IVD) deficiency, also known as isovaleric acidaemia, IVD dysfunction in silkworms was predicted to be responsible for the phenotype of the sku mutant. Linkage analysis revealed that the silkworm IVD gene (BmIVD) was closely linked to the odorous phenotype as expected, and a single amino acid substitution (G376V) was found in BmIVD of the sku mutant. To investigate the effect of the G376V substitution on BmIVD function, wild-type and sku-type recombinants were constructed with a baculovirus expression system and the subsequent enzyme activity of sku-type BmIVD was shown to be significantly reduced compared with that of wild-type BmIVD. Molecular modelling suggested that this reduction in the enzyme activity may be due to negative effects of G376V mutation on FAD-binding or on monomer-monomer interactions. These observations strongly suggest that BmIVD is responsible for the sku locus and that the molecular defect in BmIVD causes the characteristic smell and prepupal lethality of the sku mutant. To our knowledge, this is, aside from humans, the first characterization of IVD deficiency in metazoa. Considering that IVD acts in the third step of leucine degradation and the sku mutant accumulates branched-chain amino acids in haemolymph, this mutant may be useful in the investigation of unique branched-chain amino acid catabolism in insects.

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

    PubMed

    Konstantinidou, Nina; Morrissey, John Patrick

    2015-12-01

    Pathogenicity of Candida albicans is linked with its developmental stages, notably the capacity switch from yeast-like to hyphal growth, and to form biofilms on surfaces. To better understand the cellular processes involved in C. albicans development, a collection of 63 C. albicans protein kinase mutants was screened for biofilm formation in a microtitre plate assay. Thirty-eight mutants displayed some degree of biofilm impairment, with 20 categorised as poor biofilm formers. All the poor biofilm formers were also defective in the switch from yeast to hyphae, establishing it as a primary defect. Five genes, VPS15, IME2, PKH3, PGA43 and CEX1, encode proteins not previously reported to influence hyphal development or biofilm formation. Network analysis established that individual components of some processes, most interestingly MAP kinase pathways, are not required for biofilm formation, most likely indicating functional redundancy. Mutants were also screened for their response to bacterial supernatants and it was found that Pseudomonas aeruginosa supernatants inhibited biofilm formation in all mutants, regardless of the presence of homoserine lactones (HSLs). In contrast, Candida morphology was only affected by supernatant containing HSLs. This confirms the distinct HSL-dependent inhibition of filamentation and the HSL-independent impairment of biofilm development by P. aeruginosa. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Cog2 Null Mutant CHO Cells Show Defective Sphingomyelin Synthesis*

    PubMed Central

    Spessott, Waldo; Uliana, Andrea; Maccioni, Hugo J. F.

    2010-01-01

    The COG (conserved oligomeric Golgi complex) is a Golgi-associated tethering complex involved in retrograde trafficking of multiple Golgi enzymes. COG deficiencies lead to misorganization of the Golgi, defective trafficking of glycosylation enzymes, and abnormal N-, O- and ceramide-linked oligosaccharides. Here, we show that in Cog2 null mutant ldlC cells, the content of sphingomyelin (SM) is reduced to ∼25% of WT cells. Sphingomyelin synthase (SMS) activity is essentially normal in ldlC cells, but in contrast with the typical Golgi localization in WT cells, in ldlC cells, transfected SMS1 localizes to vesicular structures scattered throughout the cytoplasm, which show almost no signal of co-transfected ceramide transfer protein (CERT). Cog2 transfection restores SM formation and the typical SMS1 Golgi localization phenotype. Adding exogenous N-6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl-4-d-erythro-sphingosine (C6-NBD-ceramide) to ldlC cell cultures results in normal SM formation. Endogenous ceramide levels were 3-fold higher in ldlC cells than in WT cells, indicating that Golgi misorganization caused by Cog2 deficiency affects the delivery of ceramide to sites of SM synthesis by SMS1. Considering the importance of SM as a structural component of membranes, this finding is also worth of consideration in relation to a possible contribution to the clinical phenotype of patients suffering congenital disorders of glycosylation type II. PMID:21047787

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

    NASA Technical Reports Server (NTRS)

    Charlang, G.; Williams, N. P.

    1977-01-01

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

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

    NASA Technical Reports Server (NTRS)

    Charlang, G.; Williams, N. P.

    1977-01-01

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

  6. A Mutant of Mycobacterium smegmatis Defective in Dipeptide Transport

    PubMed Central

    Bhatt, Achal; Green, Renee; Coles, Roswell; Condon, Michael; Connell, Nancy D.

    1998-01-01

    A mutant of Mycobacterium smegmatis unable to use the dipeptide carnosine (β-alanyl-l-histidine) as a sole carbon or nitrogen source was isolated. Carnosinase activity and the ability to grow on β-Ala and/or l-His were similar in the mutant and the wild type. However, the mutant showed significant impairment in the uptake of carnosine. This study is the first description of a peptide utilization mutant of a mycobacterium. PMID:9852030

  7. A Rhizobium leguminosarum mutant defective in symbiotic iron acquisition.

    PubMed Central

    Nadler, K D; Johnston, A W; Chen, J W; John, T R

    1990-01-01

    Iron acquisition by symbiotic Rhizobium spp. is essential for nitrogen fixation in the legume root nodule symbiosis. Rhizobium leguminosarum 116, an ineffective mutant strain with a defect in iron acquisition, was isolated after nitrosoguanidine mutagenesis of the effective strain 1062. The pop-1 mutation in strain 116 imparted to it a complex phenotype, characteristic of iron deficiency: the accumulation of porphyrins (precursors of hemes) so that colonies emitted a characteristic pinkish-red fluorescence when excited by UV light, reduced levels of cytochromes b and c, and wild-type growth on high-iron media but low or no growth in low-iron broth and on solid media supplemented with the iron scavenger dipyridyl. Several iron(III)-solubilizing agents, such as citrate, hydroxyquinoline, and dihydroxybenzoate, stimulated growth of 116 on low-iron solid medium; anthranilic acid, the R. leguminosarum siderophore, inhibited low-iron growth of 116. The initial rate of 55Fe uptake by suspensions of iron-starved 116 cells was 10-fold less than that of iron-starved wild-type cells. Electron microscopic observations revealed no morphological abnormalities in the small, white nodules induced by 116. Nodule cortical cells were filled with vesicles containing apparently normal bacteroids. No premature degeneration of bacteroids or of plant cell organelles was evident. We mapped pop-1 by R plasmid-mediated conjugation and recombination to the ade-27-rib-2 region of the R. leguminosarum chromosome. No segregation of pop-1 and the symbiotic defect was observed among the recombinants from these crosses. Cosmid pKN1, a pLAFR1 derivative containing a 24-kilobase-pair fragment of R. leguminosarum DNA, conferred on 116 the ability to grow on dipyridyl medium and to fix nitrogen symbiotically. These results indicate that the insert cloned in pKN1 encodes an element of the iron acquisition system of R. leguminosarum that is essential for symbiotic nitrogen fixation. Images FIG. 3A-3B FIG

  8. Chloroplast dysfunction causes multiple defects in cell cycle progression in the Arabidopsis crumpled leaf mutant.

    PubMed

    Hudik, Elodie; Yoshioka, Yasushi; Domenichini, Séverine; Bourge, Mickaël; Soubigout-Taconnat, Ludivine; Mazubert, Christelle; Yi, Dalong; Bujaldon, Sandrine; Hayashi, Hiroyuki; De Veylder, Lieven; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

    2014-09-01

    The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants.

  9. Responses of a triple mutant defective in three iron deficiency-induced Basic Helix-Loop-Helix genes of the subgroup Ib(2) to iron deficiency and salicylic acid.

    PubMed

    Maurer, Felix; Naranjo Arcos, Maria Augusta; Bauer, Petra

    2014-01-01

    Plants are sessile organisms that adapt to external stress by inducing molecular and physiological responses that serve to better cope with the adverse growth condition. Upon low supply of the micronutrient iron, plants actively increase the acquisition of soil iron into the root and its mobilization from internal stores. The subgroup Ib(2) BHLH genes function as regulators in this response, however their concrete functions are not fully understood. Here, we analyzed a triple loss of function mutant of BHLH39, BHLH100 and BHLH101 (3xbhlh mutant). We found that this mutant did not have any iron uptake phenotype if iron was provided. However, under iron deficiency the mutant displayed a more severe leaf chlorosis than the wild type. Microarray-based transcriptome analysis revealed that this mutant phenotype resulted in the mis-regulation of 198 genes, out of which only 15% were associated with iron deficiency regulation itself. A detailed analysis revealed potential targets of the bHLH transcription factors as well as genes reflecting an exaggerated iron deficiency response phenotype. Since the BHLH genes of this subgroup have been brought into the context of the plant hormone salicylic acid, we investigated whether the 3xbhlh mutant might have been affected by this plant signaling molecule. Although a very high number of genes responded to SA, also in a differential manner between mutant and wild type, we did not find any indication for an association of the BHLH gene functions in SA responses upon iron deficiency. In summary, our study indicates that the bHLH subgroup Ib(2) transcription factors do not only act in iron acquisition into roots but in other aspects of the adaptation to iron deficiency in roots and leaves.

  10. Dendritic reduction in Passover, a Drosophila mutant with a defective giant fiber neuronal pathway.

    PubMed

    Baird, D H; Koto, M; Wyman, R J

    1993-07-01

    The jump response to a light-off startle stimulus in Drosophila melanogaster occurs when the Giant Fiber (GF), a neuron descending from the brain to the thorax, drives the jump (tergotrochanteral) muscle motorneuron (TTMn). Nonjumping mutants have been isolated in which this response is disrupted. Flies bearing the X-chromosome mutation Passover (Pas) fail to jump in response to a light-off stimulus, and electrical stimulation of the GF in the brain no longer elicits the normal response in the TTM. We have used retrograde HRP labelling to examine the TTMn motorneuron in wild-type flies and in a variety of newly identified Pas alleles. In wild type the medial branch (MB) of the TTMn has an extensive region of apposition with the GF. In Pas alleles, there is a general reduction in anterior-posterior (A-P) extent of the medial branch but not of the posterior branch. Nevertheless, Pas alleles usually leave the TTMn close enough to the GF so that contact would not be precluded. In flies carrying a particular deficiency of Pas, Df(1) 16-3-22, including Pas/Df(1) 16-3-22 heterozygotes, there can be extensive growth of the medial-branch including a contralateral projection; these heterozygotes have more than the normal amount of overlap between the GF and the TTMn. This phenotype, originally ascribed to Pas mutants, is associated with Df(1) 16-3-22, but not with other deletions of the Pas gene. The driving of the TTMn by the GF is defective in mutant genotypes with extensive medial branches as well as in mutants where GF-TTMn contact is reduced. The fact that the TTMn grows into its normal synaptic region in mutant genotypes, but the GF pathway functions abnormally suggests that pathfinding by the TTMn is not impaired. It is more likely that the Pas mutation disrupts cell recognition, synaptogenesis, or synaptic function in the TTMn or its presynaptic partners.

  11. Defective Kernel Mutants of Maize. I. Genetic and Lethality Studies

    PubMed Central

    Neuffer, M. G.; Sheridan, William F.

    1980-01-01

    A planting of 3,919 M1 kernels from normal ears crossed by EMS-treated pollen produced 3,461 M1 plants and 3,172 selfed ears. These plants yielded 2,477 (72%) total heritable changes; the selfed ears yielded 2,457 (78%) recessive mutants, including 855 (27%) recessive kernel mutants and 8 (0.23%) viable dominant mutants. The ratio of recessive to dominant mutants was 201:1. The average mutation frequency for four known loci was three per 3,172 genomes analyzed. The estimated total number of loci mutated was 535 and the estimated number of kernel mutant loci mutated was 285. Among the 855 kernel mutants, 432 had a nonviable embryo, and 59 germinated but had a lethal seedling. A sample of 194 of the latter two types was tested for heritability, lethality, chromosome arm location and endosperm-embryo interaction between mutant and nonmutant tissues in special hyper-hypoploid combinations produced by manipulation of B-A translocations. The selected 194 mutants were characterized and catalogued according to endosperm phenotype and investigated to determine their effects on the morphology and development of the associated embryo. The possibility of rescuing some of the lethal mutants by covering the mutant embryo with a normal endosperm was investigated. Ninety of these 194 mutants were located on 17 of the 18 chromosome arms tested. Nineteen of the located mutants were examined to determine the effect of having a normal embryo in the same kernel with a mutant endosperm, and vice versa, as compared to the expression observed in kernels with both embryo and endosperm in a mutant condition. In the first situation, for three of the 19 mutants, the mutant endosperm was less extreme (the embryo helped); for seven cases, the mutant endosperm was more extreme (the embryo hindered); and for nine cases, there was no change. In the reverse situation, for four cases the normal endosperm helped the mutant embryo; for 14 cases there was no change and one case was inconclusive. PMID

  12. Defective kernel mutants of maize. I. Genetic and lethality studies.

    PubMed

    Neuffer, M G; Sheridan, W F

    1980-08-01

    A planting of 3,919 M(1) kernels from normal ears crossed by EMS-treated pollen produced 3,461 M(1) plants and 3,172 selfed ears. These plants yielded 2,477 (72%) total heritable changes; the selfed ears yielded 2,457 (78%) recessive mutants, including 855 (27%) recessive kernel mutants and 8 (0.23%) viable dominant mutants. The ratio of recessive to dominant mutants was 201:1. The average mutation frequency for four known loci was three per 3,172 genomes analyzed. The estimated total number of loci mutated was 535 and the estimated number of kernel mutant loci mutated was 285. Among the 855 kernel mutants, 432 had a nonviable embryo, and 59 germinated but had a lethal seedling. A sample of 194 of the latter two types was tested for heritability, lethality, chromosome arm location and endosperm-embryo interaction between mutant and nonmutant tissues in special hyper-hypoploid combinations produced by manipulation of B-A translocations. The selected 194 mutants were characterized and catalogued according to endosperm phenotype and investigated to determine their effects on the morphology and development of the associated embryo. The possibility of rescuing some of the lethal mutants by covering the mutant embryo with a normal endosperm was investigated. Ninety of these 194 mutants were located on 17 of the 18 chromosome arms tested. Nineteen of the located mutants were examined to determine the effect of having a normal embryo in the same kernel with a mutant endosperm, and vice versa, as compared to the expression observed in kernels with both embryo and endosperm in a mutant condition. In the first situation, for three of the 19 mutants, the mutant endosperm was less extreme (the embryo helped); for seven cases, the mutant endosperm was more extreme (the embryo hindered); and for nine cases, there was no change. In the reverse situation, for four cases the normal endosperm helped the mutant embryo; for 14 cases there was no change and one case was inconclusive.

  13. Multiple classes of yeast mutants are defective in vacuole partitioning yet target vacuole proteins correctly.

    PubMed Central

    Wang, Y X; Zhao, H; Harding, T M; Gomes de Mesquita, D S; Woldringh, C L; Klionsky, D J; Munn, A L; Weisman, L S

    1996-01-01

    In Saccharomyces cerevisiae the vacuoles are partitioned from mother cells to daughter cells in a cell-cycle-coordinated process. The molecular basis of this event remains obscure. To date, few yeast mutants had been identified that are defective in vacuole partitioning (vac), and most such mutants are also defective in vacuole protein sorting (vps) from the Golgi to the vacuole. Both the vps mutants and previously identified non-vps vac mutants display an altered vacuolar morphology. Here, we report a new method to monitor vacuole inheritance and the isolation of six new non-vps vac mutants. They define five complementation groups (VAC8-VAC12). Unlike mutants identified previously, three of the complementation groups exhibit normal vacuolar morphology. Zygote studies revealed that these vac mutants are also defective in intervacuole communication. Although at least four pathways of protein delivery to the vacuole are known, only the Vps pathway seems to significantly overlap with vacuole partitioning. Mutants defective in both vacuole partitioning and endocytosis or vacuole partitioning and autophagy were not observed. However, one of the new vac mutants was additionally defective in direct protein transport from the cytoplasm to the vacuole. Images PMID:8885233

  14. Cloning of spiramycin biosynthetic genes and their use in constructing Streptomyces ambofaciens mutants defective in spiramycin biosynthesis.

    PubMed Central

    Richardson, M A; Kuhstoss, S; Huber, M L; Ford, L; Godfrey, O; Turner, J R; Rao, R N

    1990-01-01

    Several cosmid clones from Streptomyces ambofaciens containing the spiramycin resistance gene srmB were introduced into S. fradiae PM73, a mutant defective in tylosin synthesis, resulting in tylosin synthesis. The DNA responsible for this complementation was localized to a 10.5-kilobase EcoRI fragment. A 32-kilobase DNA segment which included the srmB spiramycin resistance gene and DNA which complemented the defect in strain PM73 were mutagenized in vivo with Tn10 carrying the gene for Nmr (which is expressed in Streptomyces spp.) or in vitro by insertional mutagenesis with a drug resistance gene (Nmr) cassette. When these mutagenized DNA segments were crossed into the S. ambofaciens chromosome, three mutant classes blocked in spiramycin synthesis were obtained. One mutant accumulated two precursors of spiramycin, platenolide I and platenolide II. Two mutants, when cofermented with the platenolide-accumulating mutant, produced spiramycin. Tylactone supplementation of these two mutants resulted in the synthesis of a group of compounds exhibiting antibiotic activity. Two other mutants failed to coferment with any of the other mutants or to respond to tylactone supplementation. Images PMID:2193916

  15. What cardiovascular defect does my prenatal mouse mutant have, and why?

    PubMed

    Conway, Simon J; Kruzynska-Frejtag, Agnieszka; Kneer, Paige L; Machnicki, Michal; Koushik, Srinagesh V

    2003-01-01

    Since the advent of mouse targeted mutations, gene traps, an escalating use of a variety of complex transgenic manipulations, and large-scale chemical mutagenesis projects yielding many mutants with cardiovascular defects, it has become increasingly evident that defects within the heart and vascular system are largely responsible for the observed in utero lethality of the embryo and early fetus. If a transgenically altered embryo survives implantation but fails to be born, it usually indicates that there is some form of lethal cardiovascular defect present. A number of embryonic organ and body systems, including the central nervous system, gut, lungs, urogenital system, and musculoskeletal system appear to have little or no survival value in utero (Copp, 1995). Cardiovascular abnormalities include the failure to establish an adequate yolk-sac vascular circulation, which results in early lethality (E8.5-10.5); poor cardiac function (E9.0-birth); failure to undergo correct looping and chamber formation of the primitive heart tube (E9.0-11.0); improper septation, including division of the common ventricle and atria and the establishment of a divided outflow tract (E11.0-13.0); inadequate establishment of the cardiac conduction system (E12.0-birth); and the failure of the in utero cardiovascular system to adapt to adult life (birth) and close the interatrial and aorta-pulmonary trunk shunts that are required for normal fetal life. Importantly, the developmental timing of lethality is usually a good indicator of both the type of the cardiovascular defect present and may also suggest the possible underlying cause/s. The purpose of this review is both to review the literature and to provide a beginner's guide for analysing cardiovascular defects in mouse mutants.

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

    SciTech Connect

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

    1984-11-01

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

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

    PubMed Central

    Winkelman, J W; Clark, D P

    1984-01-01

    We devised a positive selection procedure for bacterial mutants incapable of producing acid from sugars by fermentation. The method relied on the production of elemental bromine from a mixture of bromide and bromate under acidic conditions. When wild-type Escherichia coli cells were plated on media containing a fermentable sugar and an equimolar mixture of bromide and bromate, most of the cells were killed but a variety of mutants unable to produce acid from the sugar survived. Among these mutants were those defective in (i) sugar uptake, (ii) the glycolytic pathway, and (iii) the excretion. There were also novel mutants with some presumed regulatory defects affecting fermentation. PMID:6094484

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

    PubMed

    Winkelman, J W; Clark, D P

    1984-11-01

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

  19. Defective telomere elongation and hematopoiesis from telomerase-mutant aplastic anemia iPSCs

    PubMed Central

    Winkler, Thomas; Hong, So Gun; Decker, Jake E.; Morgan, Mary J.; Wu, Chuanfeng; Hughes, William M.; Yang, Yanqin; Wangsa, Danny; Padilla-Nash, Hesed M.; Ried, Thomas; Young, Neal S.; Dunbar, Cynthia E.; Calado, Rodrigo T.

    2013-01-01

    Critically short telomeres activate p53-mediated apoptosis, resulting in organ failure and leading to malignant transformation. Mutations in genes responsible for telomere maintenance are linked to a number of human diseases. We derived induced pluripotent stem cells (iPSCs) from 4 patients with aplastic anemia or hypocellular bone marrow carrying heterozygous mutations in the telomerase reverse transcriptase (TERT) or the telomerase RNA component (TERC) telomerase genes. Both mutant and control iPSCs upregulated TERT and TERC expression compared with parental fibroblasts, but mutant iPSCs elongated telomeres at a lower rate compared with healthy iPSCs, and the deficit correlated with the mutations’ impact on telomerase activity. There was no evidence for alternative lengthening of telomere (ALT) pathway activation. Elongation varied among iPSC clones derived from the same patient and among clones from siblings harboring identical mutations. Clonal heterogeneity was linked to genetic and environmental factors, but was not influenced by residual expression of reprogramming transgenes. Hypoxia increased telomere extension in both mutant and normal iPSCs. Additionally, telomerase-mutant iPSCs showed defective hematopoietic differentiation in vitro, mirroring the clinical phenotype observed in patients and demonstrating that human telomere diseases can be modeled utilizing iPSCs. Our data support the necessity of studying multiple clones when using iPSCs to model disease. PMID:23585473

  20. Defective telomere elongation and hematopoiesis from telomerase-mutant aplastic anemia iPSCs.

    PubMed

    Winkler, Thomas; Hong, So Gun; Decker, Jake E; Morgan, Mary J; Wu, Chuanfeng; Hughes, William M; Yang, Yanqin; Wangsa, Danny; Padilla-Nash, Hesed M; Ried, Thomas; Young, Neal S; Dunbar, Cynthia E; Calado, Rodrigo T

    2013-05-01

    Critically short telomeres activate p53-mediated apoptosis, resulting in organ failure and leading to malignant transformation. Mutations in genes responsible for telomere maintenance are linked to a number of human diseases. We derived induced pluripotent stem cells (iPSCs) from 4 patients with aplastic anemia or hypocellular bone marrow carrying heterozygous mutations in the telomerase reverse transcriptase (TERT) or the telomerase RNA component (TERC) telomerase genes. Both mutant and control iPSCs upregulated TERT and TERC expression compared with parental fibroblasts, but mutant iPSCs elongated telomeres at a lower rate compared with healthy iPSCs, and the deficit correlated with the mutations' impact on telomerase activity. There was no evidence for alternative lengthening of telomere (ALT) pathway activation. Elongation varied among iPSC clones derived from the same patient and among clones from siblings harboring identical mutations. Clonal heterogeneity was linked to genetic and environmental factors, but was not influenced by residual expression of reprogramming transgenes. Hypoxia increased telomere extension in both mutant and normal iPSCs. Additionally, telomerase-mutant iPSCs showed defective hematopoietic differentiation in vitro, mirroring the clinical phenotype observed in patients and demonstrating that human telomere diseases can be modeled utilizing iPSCs. Our data support the necessity of studying multiple clones when using iPSCs to model disease.

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

    PubMed

    Harris, Muriel J

    2009-04-01

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

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

    PubMed

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

    2016-09-01

    The widespread use of nanosilver in various antibacterial, antifungal, and antiviral products warrants the studies of the toxicity pathways of nanosilver-enabled materials toward microbes and viruses. We profiled the toxicity mechanisms of uncoated, casein-coated, and polyvinylpyrrolidone-coated silver nanoparticles (AgNPs) using Saccharomyces cerevisiae wild-type (wt) and its 9 single-gene deletion mutants defective in oxidative stress (OS) defense, cell wall/membrane integrity, and endocytosis. The 48-h growth inhibition assay in organic-rich growth medium and 24-h cell viability assay in deionized (DI) water were applied whereas AgNO3, H2O2, and SDS served as positive controls. Both coated AgNPs (primary size 8-12nm) were significantly more toxic than the uncoated (~85nm) AgNPs. All studied AgNPs were ~30 times more toxic if exposed to yeast cells in DI water than in the rich growth medium: the IC50 based on nominal concentration of AgNPs in the growth inhibition test ranged from 77 to 576mg Ag/L and in the cell viability test from 2.7 to 18.7mg Ag/L, respectively. Confocal microscopy showed that wt but not endocytosis mutant (end3Δ) internalized AgNPs. Comparison of toxicity patterns of wt and mutant strains defective in OS defense and membrane integrity revealed that the toxicity of the studied AgNPs to S. cerevisiae was not caused by the OS or cell wall/membrane permeabilization. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Saccharomyces cerevisiae mutant with a partial defect in the synthesis of CDP-diacylglycerol and altered regulation of phospholipid biosynthesis.

    PubMed Central

    Klig, L S; Homann, M J; Kohlwein, S D; Kelley, M J; Henry, S A; Carman, G M

    1988-01-01

    A Saccharomyces cerevisiae mutant (cdg1 mutation) was isolated on the basis of an inositol excretion phenotype and exhibited pleiotropic deficiencies in phospholipid biosynthesis. Genetic analysis of the mutant confirmed that the cdg1 mutation represents a new genetic locus and that a defect in a single gene was responsible for the Cdg1 phenotype. CDP-diacylglycerol synthase activity in mutant haploid cells was 25% of the wild-type derepressed level. Biochemical and immunoblot analyses revealed that the defect in CDP-diacylglycerol synthase activity in the cdg1 mutant was due to a reduced level of the CDP-diacylglycerol synthase Mr-56,000 subunit rather than to an alteration in the enzymological properties of the enzyme. This defect resulted in a reduced rate of CDP-diacylglycerol synthesis, an elevated phosphatidate content, and alterations in overall phospholipid synthesis. Unlike wild-type cells, CDP-diacylglycerol synthase was not regulated in response to water-soluble phospholipid precursors. The cdg1 lesion also caused constitutive expression of inositol-1-phosphate synthase and elevated phosphatidylserine synthase. Phosphatidylinositol synthase was not affected in the cdg1 mutant. Images PMID:2832385

  4. Mutants of Myxococcus xanthus dsp defective in fibril binding.

    PubMed Central

    Chang, B Y; Dworkin, M

    1996-01-01

    The dsp mutant of Myxococcus xanthus lacks extracellular fibrils and as a result is unable to undergo cohesion, group motility, or development (J. W. Arnold and L. J. Shimkets, J. Bacteriol. 170:5765-5770, 1983; J. W. Arnold and L. J. Shimkets, J. Bacteriol. 170:5771-5777, 1983; R. M. Behmlander and M. Dworkin, J. Bacteriol. 173:7810-7821, 1991; L. J. Shimkets, J. Bacteriol. 166:837-841, 1986; L. J. Shimkets, J. Bacteriol. 166:842-848, 1986). However, cohesion and development can be phenotypically restored by the addition of isolated fibrils (R. M. Behmlander, Ph.D. thesis, University of Minnesota, Minneapolis, 1994; B.-Y. Chang and M. Dworkin, J. Bacteriol. 176:7190-7196, 1994). As part of our attempts to examine the interaction of fibrils and cells of M. xanthus, we have isolated a series of secondary mutants of M. xanthus dsp in which cohesion, unlike that of the parent strain, could not be rescued by the addition of isolated fibrils. Cells of M. xanthus dsp were mutagenized either by ethyl methanesulfonate or by Tn5 insertions. Mutagenized cultures were enriched by selection of those cells that could not be rescued, i.e., that failed to cohere in the presence of isolated fibrils. Seven mutants of M. xanthus dsp, designated fbd mutants, were isolated from 6,983 colonies; these represent putative fibril receptor-minus mutants. The fbd mutants, like the parent dsp mutant, still lacked fibrils, but displayed a number of unexpected properties. They regained group motility and the ability to aggregate but not the ability to form mature fruiting bodies. In addition, they partially regained the ability to form myxospores. The fbd mutant was backcrossed into the dsp mutant by Mx4 transduction. Three independently isolated transconjugants showed essentially the same properties as the fbd mutants--loss of fibril rescue of cohesion, partial restoration of myxospore morphogenesis, and restoration of group motility. These results suggest that the physical presence of fibrils

  5. Mutants of Saccharomycopsis lipolytica defective in lysine catabolism.

    PubMed Central

    Gaillardin, C; Fournier, P; Sylvestre, G; Heslot, H

    1976-01-01

    Wild-type strains of Saccharomycopsis lipolytica are able to use lysine as a carbon or a nitrogen source, but not as a unique source for both. Mutants were selected that could not use lysine either as a nitrogen or as a carbon source. Some of them, however, utilized N-6-acetyllysine or 5-aminovaleric acid. Many of the mutants appeared to be blocked in both utilizations, suggesting a unique pathway for lysine degradation (either as a carbon or as a nitrogen source). Genetic characterization of these mutants was achieved by complementation and recombination tests. PMID:1245461

  6. Incision and postincision steps of pyrimidine dimer removal in excision-defective mutants of Saccharomyces cerevisiae

    SciTech Connect

    Wilcox, D.R.; Prakash, L.

    1981-11-01

    cdc9, a temperature-sensitive mutant defective in polynucleotide deoxyribonucleic acid (DNA) ligase activity, accumulates low-molecular-weight DNA fragments (as measured by sedimentation of DNA in alkaline sucrose gradients) at the nonpermissive temperature after irradiation with ultraviolet light. This phenotype of cdc9 is a sensitive indicator of successful incision during excision repair of dimers. In strains containing excision-defective mutations in any of nine genes in combination with the cdc9 mutation, the absence of low-molecular-weight DNA at the nonpermissive temperature after ultraviolet treatment suggests that these mutants are incision defective, whereas the presence of low-molecular-weight DNA indicates that the mutants are defective in a step after incision. With rad1, rad2, rad3, rad4, and rad10 mutants, the molecular weight of the DNA remained unchanged after ultraviolet irradiation and incubation at the restrictive temperature, despite the presence of the cdc9 mutation; these mutants are therefore incision defective. Low-molecular-weight DNA was observed in rad14 cdc9 and rad16 cdc9 strains. With the rad16 strain, the accumulation of low-molecular-weight DNA correlated with the amount of excision taking place, whereas in the rad14 mutant strain, no evidence of dimer removal was obtained. Therefore, rad14 is likely to be defective in a step after incision.

  7. Anti-oncogenic activity of signalling-defective epidermal growth factor receptor mutants.

    PubMed Central

    Redemann, N; Holzmann, B; von Rüden, T; Wagner, E F; Schlessinger, J; Ullrich, A

    1992-01-01

    Overexpression and autocrine activation of the epidermal growth factor receptor (EGF-R) cause transformation of cultured cells and correlate with tumor progression in cancer patients. Dimerization and transphosphorylation are crucial events in the process by which receptors with tyrosine kinase activity generate normal and transforming cellular signals. Interruption of this process by inactive receptor mutants offers the potential to inhibit ligand-induced cellular responses. Using recombinant retroviruses, we have examined the effects of signalling-incompetent EGF-R mutants on the growth-promoting and transforming potential of ligand-activated, overexpressed wild-type EGF-R and the v-erbB oncogene product. Expression of a soluble extracellular EGF-R domain had little if any effect on the growth and transformation of NIH 3T3 cells by either tyrosine kinase. However, both a kinase-negative EGF-R point mutant (HERK721A) and an EGF-R lacking 533 C-terminal amino acids efficiently inhibited wild-type EGF-R-mediated, de novo DNA synthesis and cell transformation in a dose-dependent manner. Furthermore, coexpression with the v-erbBES4 oncogene product in NIH 3T3 cells resulted in transphosphorylation of the HERK721A mutant receptor and reduced soft-agar colony growth but had no effect in a focus formation assay. These results demonstrate that signalling-defective receptor tyrosine kinase mutants differentially interfere with oncogenic signals generated by either overexpressed EGF-R or the retroviral v-erbBES4 oncogene product. Images PMID:1346334

  8. The pineal gland in wild-type and two zebrafish mutants with retinal defects.

    PubMed

    Allwardt, B A; Dowling, J E

    2001-06-01

    Light and transmission electron microscopy were used to characterize the ultrastructural features of the pineal glands of wild-type and two mutant zebrafish strains that have retinal defects. Particular attention was given to the pineal photoreceptors. Photoreceptors in the pineal gland appear quite similar to retinal cone photoreceptors, having many of the same structural characteristics including outer segment disk membranes often confluent with the plasma membrane, calycal processes surrounding the outer segments, and classic connecting cilia. The pineal photoreceptor terminals differ from photoreceptor terminals in the retina in that they have short synaptic ribbons and make dyad synapses which may or may not be invaginated. Pineal photoreceptors in two zebrafish mutants with abnormal retinal photoreceptors were also studied. Pineal photoreceptors in the niezerka (nie) mutant degenerate, as they do in the retina, indicating that pineal and retinal photoreceptors share at least some genes. However, the synaptic terminals of no optokinetic response c (nrc) pineal photoreceptors are normal, suggesting that this mutation is specific to the retina.

  9. fused-somites-like mutants exhibit defects in trunk vessel patterning.

    PubMed

    Shaw, Kenna M; Castranova, Daniel A; Pham, Van N; Kamei, Makoto; Kidd, Kameha R; Lo, Brigid D; Torres-Vasquez, Jesus; Ruby, Alexander; Weinstein, Brant M

    2006-07-01

    We identified four mutants in two distinct loci exhibiting similar trunk vascular patterning defects in an F3 genetic screen for zebrafish vascular mutants. Initial vasculogenesis is not affected in these mutants, with proper specification and differentiation of endothelial cells. However, all four display severe defects in the growth and patterning of angiogenic vessels in the trunk, with ectopic branching and disoriented migration of intersegmental vessels. The four mutants are allelic to previously characterized mutants at the fused-somites (fss) and beamter (bea) loci, and they exhibit comparable defects in trunk somite boundary formation. The fss locus has been shown to correspond to tbx24; we show here that bea mutants are defective in the zebrafish dlC gene. Somitic expression of known vascular guidance factors efnb2a, sema3a1, and sema3a2 is aberrantly patterned in fss and bea mutants, suggesting that the vascular phenotype is due to loss of proper guidance cues provided by these factors. Published 2006 Wiley-Liss, Inc.

  10. 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

  11. Susceptibility of lipopolysaccharide-defective mutants of Pseudomonas aeruginosa strain PAO to dyes, detergents, and antibiotics.

    PubMed Central

    Kropinski, A M; Chan, L; Milazzo, F H

    1978-01-01

    Lipopolysaccharide-defective mutants of Pseudomonas aeruginosa strain PAO have been isolated on the basis of their resistance to lipopolysaccharide-specific bacteriophages. These mutants have been differentiated by their agglutination in NaCl and acriflavine, phage sensitivity, and chemical analysis of the lipopolysaccharides. The susceptibility of the wild-type strain and four mutants to a series of twenty-six agents, including dyes, detergents, antibiotics, and lysozyme, was examined. The roughest mutant (AK-43) exhibited increased susceptibility to sodium deoxycholate, hexadecylpyridinium chloride, benzalkonium chloride, ampicillin, penicillin G, erythromycin, colymycin, and polymyxin B. The role of cell envelope fractions in antibiotic resistance in P. aeruginosa is discussed. PMID:122525

  12. Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants.

    PubMed Central

    Manos, M M; Gluzman, Y

    1985-01-01

    To study the role of the biochemical and physiological activities of simian virus 40 (SV40) large T antigen in the lytic and transformation processes, we have analyzed DNA replication-defective, transformation-competent T-antigen mutants. Here we describe two such mutants, C8/SV40 and T22/SV40, and also summarize the properties of all of the mutants in this collection. C8/SV40 and T22/SV40 were isolated from C8 and T22 cells (simian cell lines transformed with UV-irradiated SV40). Early regions encoding the defective T antigens were cloned into a plasmid vector to generate pC8 and pT22. The mutations responsible for the defects in viral DNA replication were localized by marker rescue, and subsequent DNA sequencing revealed missense and one nonsense mutation. The T22 mutation predicts a change of histidine to glutamine at residue 203. C8 has two mutations, one predicts lysine224 to glutamamic acid and the other changes the codon for glutamic acid660 to a stop codon; therefore, C8 T antigen lacks the 49 carboxy-terminal amino acids. pC8A and pC8B were constructed to contain the C8 mutations separately. Plasmids pT22, pC8, pC8A, and pC8B were able to transform primary rodent cell cultures. T22 T antigen is defective in binding to the SV40 origin. C8B (49-amino-acid truncation) is a host-range mutant defective in a late function in CV-1 but not BSC cells. Analysis of T antigens in mutant SV40-transformed mouse cells suggests that the replicative function of T antigen is important in generating SV40 DNA rearrangements that allow the expression of "100K" variant T antigens in the transformants. Images PMID:2981330

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

    SciTech Connect

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

    1989-07-01

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

  14. Temperature-sensitive yeast mutants defective in mitochondrial inheritance.

    PubMed

    McConnell, S J; Stewart, L C; Talin, A; Yaffe, M P

    1990-09-01

    The distribution of mitochondria to daughter cells is an essential feature of mitotic cell growth, yet the molecular mechanisms facilitating this mitochondrial inheritance are unknown. We have isolated mutants of Saccharomyces cerevisiae that are temperature-sensitive for the transfer of mitochondria into a growing bud. Two of these mutants contain single, recessive, nuclear mutations, mdm1 and mdm2, that cause temperature-sensitive growth and aberrant mitochondrial distribution at the nonpermissive temperature. The absence of mitochondria from the buds of mutant cells was confirmed by indirect immunofluorescence microscopy and by transmission electron microscopy. The mdm1 lesion also retards nuclear division and prevents the transfer of nuclei into the buds. Cells containing the mdm2 mutation grown at the nonpermissive temperature sequentially form multiple buds, each receiving a nucleus but no mitochondria. Neither mdm1 or mdm2 affects the transfer of vacuolar material into the buds or causes apparent changes in the tubulin- or actin-based cytoskeletons. The mdm1 and mdm2 mutations are cell-cycle specific, displaying an execution point in late G1 or early S phase.

  15. Isolation of Rhodospirillum centenum Mutants Defective in Phototactic Colony Motility by Transposon Mutagenesis

    PubMed Central

    Jiang, Ze-Yu; Rushing, Brenda G.; Bai, Yong; Gest, Howard; Bauer, Carl E.

    1998-01-01

    The purple photosynthetic bacterium Rhodospirillum centenum is capable of forming swarm colonies that rapidly migrate toward or away from light, depending on the wavelength of excitation. To identify components specific for photoperception, we conducted mini-Tn5-mediated mutagenesis and screened approximately 23,000 transposition events for mutants that failed to respond to either continuous illumination or to a step down in light intensity. A majority of the ca. 250 mutants identified lost the ability to form motile swarm cells on an agar surface. These cells appeared to contain defects in the synthesis or assembly of surface-induced lateral flagella. Another large fraction of mutants that were unresponsive to light were shown to be defective in the formation of a functional photosynthetic apparatus. Several photosensory mutants also were obtained with defects in the perception and transmission of light signals. Twelve mutants in this class were shown to contain disruptions in a chemotaxis operon, and five mutants contained disruptions of components unique to photoperception. It was shown that screening for photosensory defective R. centenum swarm colonies is an effective method for genetic dissection of the mechanism of light sensing in eubacteria. PMID:9495765

  16. Proteus mirabilis mutants defective in swarmer cell differentiation and multicellular behavior.

    PubMed Central

    Belas, R; Erskine, D; Flaherty, D

    1991-01-01

    Proteus mirabilis is a dimorphic bacterium which exists in liquid cultures as a 1.5- to 2.0-microns motile swimmer cell possessing 6 to 10 peritrichous flagella. When swimmer cells are placed on a surface, they differentiate by a combination of events that ultimately produce a swarmer cell. Unlike the swimmer cell, the polyploid swarmer cell is 60 to 80 microns long and possesses hundreds to thousands of surface-induced flagella. These features, combined with multicellular behavior, allow the swarmer cells to move over a surface in a process called swarming. Transposon Tn5 was used to produce P. mirabilis mutants defective in wild-type swarming motility. Two general classes of mutants were found to be defective in swarming. The first class was composed of null mutants that were completely devoid of swarming motility. The majority of nonswarming mutations were the result of defects in the synthesis of flagella or in the ability to rotate the flagella. The remaining nonswarming mutants produced flagella but were defective in surface-induced elongation. Strains in the second general class of mutants, which made up more than 65% of all defects in swarming were motile but were defective in the control and coordination of multicellular swarming. Analysis of consolidation zones produced by such crippled mutants suggested that this pleiotropic phenotype was caused by a defect in the regulation of multicellular behavior. A possible mechanism controlling the cyclic process of differentiation and dediferentiation involved in the swarming behavior of P. mirabilis is discussed. Images PMID:1917860

  17. Isolation and characterization of prophage mutants of the defective Bacillus subtilis bacteriophage PBSX.

    PubMed Central

    Thurm, P; Garro, A J

    1975-01-01

    Bacillus subtilis mutants with lesions in PBSX prophage genes have been isolated. One of these appears to be a regulatory mutant and is defective for mitomycin C-induced derepression of PBSX; the others are defective for phage capsid formation. All of the PBSX structural proteins are synthesized during induction of the capsid defective mutants; however, several of these proteins exhibit abnormal serological reactivity with anti-PBSX antiserum. The two head proteins X4 and X7 are not immunoprecipitable in a mutant which fails to assemble phage head structures. In the tail mutant, proteins X5 and X6 are not immunoprecipitable, tails are not assembled, and a possible tail protein precursor remains uncleaved. The noninducible mutant does not synthesize any PBSX structural proteins after exposure to mitomycin C. The mutation is specific for PBSX since ø105 and SPO2 lysogens of the mutant are inducible. All of the known PBSX-specific mutations were shown to be clustered between argC and metC on the host chromosome. In addition, the metC marker was shown to be present in multiple copies in cells induced for PBSX replication. This suggests that the derepressed prophage replicates while still integrated and that replication extends into the adjacent regions of the host chromosome. Images PMID:805847

  18. C. elegans feeding defective mutants have shorter body lengths and increased autophagy.

    PubMed

    Mörck, Catarina; Pilon, Marc

    2006-08-03

    Mutations that cause feeding defects in the nematode C. elegans are known to increase life span. Here we show that feeding defective mutants also have a second general trait in common, namely that they are small. Our measurements of the body lengths of a variety of feeding defective mutants, or of a variety of double mutants affecting other pathways that regulate body length in C. elegans, i.e. the DBL-1/TGFbeta, TAX-6/calcineurin and the SMA-1/betaH-spectrin pathways, indicate that food uptake acts as a separate pathway regulating body length. In early stages, before eating begins, feeding defective worms have no defect in body length or, in some cases, have only slightly smaller body length compared to wild-type. A significant difference in body length is first noticeable at later larval stages, a difference that probably correlates with increasing starvation. We also show that autophagy is induced and that the quantity of fat is decreased in starved worms. Our results indicate that the long-term starvation seen in feeding-defective C. elegans mutants activates autophagy, and leads to depletion of fat deposits, small cell size and small body size.

  19. The gravitropism defective 2 mutants of Arabidopsis are deficient in a protein implicated in endocytosis in Caenorhabditis elegans.

    PubMed

    Silady, Rebecca A; Kato, Takehide; Lukowitz, Wolfgang; Sieber, Patrick; Tasaka, Masao; Somerville, Chris R

    2004-10-01

    The gravitropism defective 2 (grv2) mutants of Arabidopsis show reduced shoot phototropism and gravitropism. Amyloplasts in the shoot endodermal cells of grv2 do not sediment to the same degree as in wild type. The GRV2 gene encodes a 277-kD polypeptide that is 42% similar to the Caenorhabditis elegans RME-8 protein, which is required for endocytosis. We hypothesize that a defect in endocytosis may affect both the initial gravity sensing via amyloplasts sedimentation and the subsequent more general tropic growth response.

  20. Hematopoietic, angiogenic and eye defects in Meis1 mutant animals

    PubMed Central

    Hisa, Tomoyuki; Spence, Sally E; Rachel, Rivka A; Fujita, Masami; Nakamura, Takuro; Ward, Jerrold M; Devor-Henneman, Deborah E; Saiki, Yuriko; Kutsuna, Haruo; Tessarollo, Lino; Jenkins, Nancy A; Copeland, Neal G

    2004-01-01

    Meis1 and Hoxa9 expression is upregulated by retroviral integration in murine myeloid leukemias and in human leukemias carrying MLL translocations. Both genes also cooperate to induce leukemia in a mouse leukemia acceleration assay, which can be explained, in part, by their physical interaction with each other as well as the PBX family of homeodomain proteins. Here we show that Meis1-deficient embryos have partially duplicated retinas and smaller lenses than normal. They also fail to produce megakaryocytes, display extensive hemorrhaging, and die by embryonic day 14.5. In addition, Meis1-deficient embryos lack well-formed capillaries, although larger blood vessels are normal. Definitive myeloerythroid lineages are present in the mutant embryos, but the total numbers of colony-forming cells are dramatically reduced. Mutant fetal liver cells also fail to radioprotect lethally irradiated animals and they compete poorly in repopulation assays even though they can repopulate all hematopoietic lineages. These and other studies showing that Meis1 is expressed at high levels in hematopoietic stem cells (HSCs) suggest that Meis1 may also be required for the proliferation/self-renewal of the HSC. PMID:14713950

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

    PubMed

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

    2016-01-01

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

  2. Isolation and characterization of Streptococcus mutans mutants defective in adherence and aggregation.

    PubMed

    Murchison, H; Larrimore, S; Curtiss, R

    1981-12-01

    A method was developed which enriched for mutants of Streptococcus mutans that exhibit defects in adherence to glass, aggregation, or both. Mutants were isolated from derivatives of strains PS14 (serotype c) and 6715 (serotype g) after mutagenesis with either ethyl methane sulfonate or nitrous acid. Cell survival after mutagenesis was kept above 1 to 2% to enhance the probability that mutants resulted from single mutational events. A total of 117 mutants were isolated; they also displayed non-wild-type colony morphology on mitis salivarius agar. These mutants were examined for (i) adherence and aggregation after overnight growth in sucrose-containing medium, (ii) aggregation of nongrowing cells in the presence of 200 microgram of sucrose per ml or 20 microgram of dextran per ml, and (iii) dextranase production on blue dextran agar plates. Although we isolated mutants which exhibited a variation from the parent strain in only one of the traits tested, the majority of mutants exhibited defects in two or more characteristics. Thirty-eight stable mutants of independent origin were categorized into 13 separate phenotypic groups.

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

    PubMed Central

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

    2015-01-01

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

  4. Sim2 Mutants Have Developmental Defects Not Overlapping with Those of Sim1 Mutants

    PubMed Central

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

    2002-01-01

    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

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

    PubMed Central

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

    1996-01-01

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

  6. Disparate peroxisome-related defects in Arabidopsis pex6 and pex26 mutants link peroxisomal retrotranslocation and oil body utilization.

    PubMed

    Gonzalez, Kim L; Fleming, Wendell A; Kao, Yun-Ting; Wright, Zachary J; Venkova, Savina V; Ventura, Meredith J; Bartel, Bonnie

    2017-10-01

    Catabolism of fatty acids stored in oil bodies is essential for seed germination and seedling development in Arabidopsis. This fatty acid breakdown occurs in peroxisomes, organelles that sequester oxidative reactions. Import of peroxisomal enzymes is facilitated by peroxins including PEX5, a receptor that delivers cargo proteins from the cytosol to the peroxisomal matrix. After cargo delivery, a complex of the PEX1 and PEX6 ATPases and the PEX26 tail-anchored membrane protein removes ubiquitinated PEX5 from the peroxisomal membrane. We identified Arabidopsis pex6 and pex26 mutants by screening for inefficient seedling β-oxidation phenotypes. The mutants displayed distinct defects in growth, response to a peroxisomally metabolized auxin precursor, and peroxisomal protein import. The low PEX5 levels in these mutants were increased by treatment with a proteasome inhibitor or by combining pex26 with peroxisome-associated ubiquitination machinery mutants, suggesting that ubiquitinated PEX5 is degraded by the proteasome when the function of PEX6 or PEX26 is reduced. Combining pex26 with mutations that increase PEX5 levels either worsened or improved pex26 physiological and molecular defects, depending on the introduced lesion. Moreover, elevating PEX5 levels via a 35S:PEX5 transgene exacerbated pex26 defects and ameliorated the defects of only a subset of pex6 alleles, implying that decreased PEX5 is not the sole molecular deficiency in these mutants. We found peroxisomes clustered around persisting oil bodies in pex6 and pex26 seedlings, suggesting a role for peroxisomal retrotranslocation machinery in oil body utilization. The disparate phenotypes of these pex alleles may reflect unanticipated functions of the peroxisomal ATPase complex. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  7. Developmental Defects in Mutants of the PsbP Domain Protein 5 in Arabidopsis thaliana

    PubMed Central

    Roose, Johnna L.; Frankel, Laurie K.; Bricker, Terry M.

    2011-01-01

    Plants contain an extensive family of PsbP-related proteins termed PsbP-like (PPL) and PsbP domain (PPD) proteins, which are localized to the thylakoid lumen. The founding member of this family, PsbP, is an established component of the Photosystem II (PS II) enzyme, and the PPL proteins have also been functionally linked to other photosynthetic processes. However, the functions of the remaining seven PPD proteins are unknown. To elucidate the function of the PPD5 protein (At5g11450) in Arabidopsis, we have characterized a mutant T-DNA insertion line (SALK_061118) as well as several RNAi lines designed to suppress the expression of this gene. The functions of the photosynthetic electron transfer reactions are largely unaltered in the ppd5 mutants, except for a modest though significant decrease in NADPH dehydrogenase (NDH) activity. Interestingly, these mutants show striking plant developmental and morphological defects. Relative to the wild-type Col-0 plants, the ppd5 mutants exhibit both increased lateral root branching and defects associated with axillary bud formation. These defects include the formation of additional rosettes originating from axils at the base of the plant as well as aerial rosettes formed at the axils of the first few nodes of the shoot. The root-branching phenotype is chemically complemented by treatment with the synthetic strigolactone, GR24. We propose that the developmental defects observed in the ppd5 mutants are related to a deficiency in strigolactone biosynthesis. PMID:22174848

  8. Azide-resistant mutants in Acinetobacter calcoaceticus A2 are defective in protein secretion.

    PubMed

    Elkeles, A; Rosenberg, E; Ron, E Z

    1994-02-15

    Azide, an inhibitor of ATPase, and a specific inhibitor of protein export was used in order to select for protein secretion mutants in Acinetobacter calcoaceticus A2. Two such mutants were isolated that were azide-resistant and defective in the general protein transport system. The mutation also conferred additional phenotypic changes, including an inability to grow on minimal media or at 40 degrees C. The existence of protein secretion mutants with a selectable phenotype may be useful for the genetic study of protein export.

  9. Characterization of a spontaneous adhesion-defective mutant of Ruminococcus albus strain 20.

    PubMed

    Mosoni, P; Gaillard-Martinie, B

    2001-07-01

    A spontaneous adhesion-defective mutant (mutant D5) of Ruminococcus albus strain 20 was isolated and compared to the parent to investigate the impact of the mutation on cellulolysis and to identify the adhesion mechanism of R. albus. The comparison of kinetics of cellulose degradation by strain 20 and mutant D5 showed that the mutation delayed and reduced bacterial growth on cellulose and cellulose degradation. These results were partly explained by a twofold lower cellulase activity in the mutant than in the parent. The glycocalyx of strain 20, observed by transmission electron microscopy, was large and homogeneous, and linked cells to cellulose. The mutant glycocalyx was aggregated at its periphery and cells attached loosely to cellulose. A glycoprotein of 25 kDa (GP25), present in the membrane fraction and the extracellular medium of strain 20, was not detected in the same fractions of mutant D5. Though glycoprotein GP25 did not bind to cellulose, it may be involved in adhesion as an intermediate component. Different cell-surface features of mutant D5 (cellulases, glycoprotein GP25, glycocalyx) were thus affected, any or all of which may be involved in its adhesion-defective phenotype. These results suggest that adhesion and cellulolysis are linked and that adhesion is a multifactorial phenomenon that involves at least the extracellular glycocalyx.

  10. Isolation of yeast mutants defective for localization of vacuolar vital dyes

    PubMed Central

    Zheng, Bing; Wu, Jennifer N.; Schober, Wendy; Lewis, Dorothy E.; Vida, Thomas

    1998-01-01

    An application of flow cytometric sorting is used for isolation of Saccharomyces cerevisiae mutants that mislocalize vacuolar vital dyes. This screen is based on the ability of a lipophilic styryl compound, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4–64), to label endocytic intermediates from the plasma membrane to the vacuole membrane at 15°C. Cells stained at 15°C for both FM4–64 and carboxydichlorofluorescein diacetate (a vacuolar luminal vital stain), had a pronounced shift in red/green fluorescence from cells stained at 30° or 38°C. Flow cytometric selection based on this characteristic shift allowed the isolation of 16 mutants. These comprised 12 complementation groups, which we have designated SVL for styryl dye vacuolar localization. These groups were put into three classes. Class I mutants contain very large vacuoles; class II mutants have very fragmented vacuoles; and class III mutants show the strongest svl phenotype with punctate/diffuse FM4–64 staining. Limited genetic overlap was observed with previously isolated mutants, namely svl2/vps41, svl6/vps16, and svl7/fab1. The remaining svl mutants appear to represent novel genes, two of which showed temperature-sensitive vacuole staining morphology. Another mutant, svl8, displayed defects in uptake and sorting of phosphatidylcholine and phosphatidylethanolamine. Our flow cytometric strategy may be useful for isolation of other mutants where mislocalization of fluorescent compounds can be detected. PMID:9751732

  11. Mutants of Saccharomyces cerevisiae defective in the farnesylation of Ras proteins.

    PubMed Central

    Goodman, L E; Judd, S R; Farnsworth, C C; Powers, S; Gelb, M H; Glomset, J A; Tamanoi, F

    1990-01-01

    Ras proteins are post-translationally modified by farnesylation. In the present investigation, we identified an activity in crude soluble extracts of yeast cells that catalyzes the transfer of a farnesyl moiety from farnesyl pyrophosphate to yeast RAS2 protein. RAS2 proteins having a C-terminal Cys-Ali-Ali-Xaa sequence (where Ali is an aliphatic amino acid and Xaa is the unspecified C-terminal amino acid) served as substrates for this reaction, whereas RAS2 proteins with an altered or deleted Cys-Ali-Ali-Xaa sequence did not. A yeast mutant, dpr1/ram1, originally isolated as a Ras-processing mutant was shown to be defective in farnesyltransferase activity. In addition, another mutant, ram2, also was defective in the transferase activity. These results demonstrate that at least two genes, DPR1/RAM1 and RAM2, are required for the farnesyltransferase activity in yeast. Images PMID:2124698

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

    PubMed

    Tanaka, Nobuhiro; Uraguchi, Shimpei; Fujiwara, Toru

    2014-01-01

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

  13. Prp8 retinitis pigmentosa mutants cause defects in the transition between the catalytic steps of splicing

    PubMed Central

    Guthrie, Christine

    2016-01-01

    Pre-mRNA splicing must occur with high fidelity and efficiency for proper gene expression. The spliceosome uses DExD/H box helicases to promote on-pathway interactions while simultaneously minimizing errors. Prp8 and Snu114, an EF2-like GTPase, regulate the activity of the Brr2 helicase, promoting RNA unwinding by Brr2 at appropriate points in the splicing cycle and repressing it at others. Mutations linked to retinitis pigmentosa (RP), a disease that causes blindness in humans, map to the Brr2 regulatory region of Prp8. Previous in vitro studies of homologous mutations in Saccharomyces cerevisiae show that Prp8-RP mutants cause defects in spliceosome activation. Here we show that a subset of RP mutations in Prp8 also causes defects in the transition between the first and second catalytic steps of splicing. Though Prp8-RP mutants do not cause defects in splicing fidelity, they result in an overall decrease in splicing efficiency. Furthermore, genetic analyses link Snu114 GTP/GDP occupancy to Prp8-dependent regulation of Brr2. Our results implicate the transition between the first and second catalytic steps as a critical place in the splicing cycle where Prp8-RP mutants influence splicing efficiency. The location of the Prp8-RP mutants, at the “hinge” that links the Prp8 Jab1–MPN regulatory “tail” to the globular portion of the domain, suggests that these Prp8-RP mutants inhibit regulated movement of the Prp8 Jab1/MPN domain into the Brr2 RNA binding channel to transiently inhibit Brr2. Therefore, in Prp8-linked RP, disease likely results not only from defects in spliceosome assembly and activation, but also because of defects in splicing catalysis. PMID:26968627

  14. Prp8 retinitis pigmentosa mutants cause defects in the transition between the catalytic steps of splicing.

    PubMed

    Mayerle, Megan; Guthrie, Christine

    2016-05-01

    Pre-mRNA splicing must occur with high fidelity and efficiency for proper gene expression. The spliceosome uses DExD/H box helicases to promote on-pathway interactions while simultaneously minimizing errors. Prp8 and Snu114, an EF2-like GTPase, regulate the activity of the Brr2 helicase, promoting RNA unwinding by Brr2 at appropriate points in the splicing cycle and repressing it at others. Mutations linked to retinitis pigmentosa (RP), a disease that causes blindness in humans, map to the Brr2 regulatory region of Prp8. Previous in vitro studies of homologous mutations in Saccharomyces cerevisiaes how that Prp8-RP mutants cause defects in spliceosome activation. Here we show that a subset of RP mutations in Prp8 also causes defects in the transition between the first and second catalytic steps of splicing. Though Prp8-RP mutants do not cause defects in splicing fidelity, they result in an overall decrease in splicing efficiency. Furthermore, genetic analyses link Snu114 GTP/GDP occupancy to Prp8-dependent regulation of Brr2. Our results implicate the transition between the first and second catalytic steps as a critical place in the splicing cycle where Prp8-RP mutants influence splicing efficiency. The location of the Prp8-RP mutants, at the "hinge" that links the Prp8 Jab1-MPN regulatory "tail" to the globular portion of the domain, suggests that these Prp8-RP mutants inhibit regulated movement of the Prp8 Jab1/MPN domain into the Brr2 RNA binding channel to transiently inhibit Brr2. Therefore, in Prp8-linked RP, disease likely results not only from defects in spliceosome assembly and activation, but also because of defects in splicing catalysis.

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

    PubMed Central

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

    1992-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  17. Reducing PEX13 expression ameliorates physiological defects of late-acting peroxin mutants

    PubMed Central

    Ratzel, Sarah E.; Lingard, Matthew J.; Woodward, Andrew W.; Bartel, Bonnie

    2010-01-01

    Proteins are targeted to the peroxisome matrix via processes that are mechanistically distinct from those used by other organelles. Protein entry into peroxisomes requires peroxin (PEX) proteins, including early-acting receptor (e.g., PEX5) and docking peroxins (e.g., PEX13 and PEX14) and late-acting PEX5-recycling peroxins (e.g., PEX4 and PEX6). We examined genetic interactions among Arabidopsis peroxin mutants and found that the weak pex13-1 allele had deleterious effects when combined with pex5-1 and pex14-2, defective in early-acting peroxins, as demonstrated by reduced matrix protein import and enhanced physiological defects. In contrast, combining pex13-1 with pex4-1 or pex6-1, which are defective in late-acting peroxins, unexpectedly ameliorated mutant growth defects. Matrix protein import remained impaired in pex4-1 pex13-1 and pex6-1 pex13-1, suggesting that the partial suppression of pex4-1 and pex6-1 physiological defects by a weak pex13 allele may result from restoring the balance between import and export of PEX5 or other proteins that are retrotranslocated from the peroxisome with the assistance of PEX4 and PEX6. Our results suggest that symptoms caused by pex mutants defective in late acting peroxins may result not only from defects in matrix protein import, but also from inefficient removal of PEX5 from the peroxisomal membrane following cargo delivery. PMID:20969679

  18. Reducing PEX13 expression ameliorates physiological defects of late-acting peroxin mutants.

    PubMed

    Ratzel, Sarah E; Lingard, Matthew J; Woodward, Andrew W; Bartel, Bonnie

    2011-01-01

    Proteins are targeted to the peroxisome matrix via processes that are mechanistically distinct from those used by other organelles. Protein entry into peroxisomes requires peroxin (PEX) proteins, including early-acting receptor (e.g. PEX5) and docking peroxins (e.g. PEX13 and PEX14) and late-acting PEX5-recycling peroxins (e.g. PEX4 and PEX6). We examined genetic interactions among Arabidopsis peroxin mutants and found that the weak pex13-1 allele had deleterious effects when combined with pex5-1 and pex14-2, which are defective in early-acting peroxins, as shown by reduced matrix protein import and enhanced physiological defects. In contrast, combining pex13-1 with pex4-1 or pex6-1, which are defective in late-acting peroxins, unexpectedly ameliorated mutant growth defects. Matrix protein import remained impaired in pex4-1 pex13-1 and pex6-1 pex13-1, suggesting that the partial suppression of pex4-1 and pex6-1 physiological defects by a weak pex13 allele may result from restoring the balance between import and export of PEX5 or other proteins that are retrotranslocated from the peroxisome with the assistance of PEX4 and PEX6. Our results suggest that symptoms caused by pex mutants defective in late-acting peroxins may result not only from defects in matrix protein import but also from inefficient removal of PEX5 from the peroxisomal membrane following cargo delivery. © 2010 John Wiley & Sons A/S.

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

    PubMed

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

    2013-10-11

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

  20. Isolation of Magnetospirillum magneticum AMB-1 mutants defective in bacterial magnetic particle synthesis by transposon mutagenesis.

    PubMed

    Wahyudi, A T; Takeyama, H; Matsunaga, T

    2001-01-01

    Nonmagnetic mutants of Magnetospirillum magneticum AMB-1 were recovered following mini-Tn5 transposon mutagenesis. Transconjugants with kanamycin resistance were obtained at a frequency of 2.7 x 10(-7) per recipient. Of 3327 transconjugants, 62 were defective for bacterial magnetic particle (BMP) synthesis. The frequency of independent transposition events for nonmagnetic mutants was about 1.4% in transconjugants. Further analysis of DNA sequences flanking transposon by inverted polymerase chain reaction allowed isolation of at least 10 genes or DNA sequences involved in BMP synthesis in M. magneticum AMB-1.

  1. Isolation and characterization of mutants defective in the cyanide-insensitive respiratory pathway of Pseudomonas aeruginosa.

    PubMed

    Cunningham, L; Williams, H D

    1995-01-01

    The branched respiratory chain of Pseudomonas aeruginosa contains at least two terminal oxidases which are active under normal physiological conditions. One of these, cytochrome co, is a cytochrome c oxidase which is completely inhibited by concentrations of the respiratory inhibitor potassium cyanide as low as 100 microM. The second oxidase, the cyanide-insensitive oxidase, is resistant to cyanide concentrations in excess of 1 mM as well as to sodium azide. In this work, we describe the isolation and characterization of a mutant of P. aeruginosa defective in cyanide-insensitive respiration. This insertion mutant was isolated with mini-D171 (a replication-defective derivative of the P. aeruginosa phage D3112) as a mutagen and by screening the resulting tetracycline-resistant transductants for the loss of ability to grow in the presence of 1 mM sodium azide. Polarographic studies on the NADH-mediated respiration rate of the mutant indicated an approximate 50% loss of activity, and titration of this activity against increasing cyanide concentrations gave a monophasic curve clearly showing the complete loss of cyanide-insensitive respiration. The mutated gene for a mutant affected in the cyanide-insensitive, oxidase-terminated respiratory pathway has been designated cio. We have complemented the azide-sensitive phenotype of this mutant with a wild-type copy of the gene by in vivo cloning with another mini-D element, mini-D386, carried on plasmid pADD386. The complemented cio mutant regained the ability to grow on medium containing 1 mM azide, titration of its NADH oxidase activity with cyanide gave a biphasic curve similar to that of the wild-type organism, and the respiration rate returned to normal levels. Spectral analysis of the cytochrome contents of the membranes of the wild type, the cio mutant, and the complemented mutant suggests that the cio mutant is not defective in any membrane-bound cytochromes and that the complementing gene does not encode a heme

  2. Chloroplast Dysfunction Causes Multiple Defects in Cell Cycle Progression in the Arabidopsis crumpled leaf Mutant1[C][W

    PubMed Central

    Hudik, Elodie; Yoshioka, Yasushi; Domenichini, Séverine; Bourge, Mickaël; Soubigout-Taconnat, Ludivine; Mazubert, Christelle; Yi, Dalong; Bujaldon, Sandrine; Hayashi, Hiroyuki; De Veylder, Lieven; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

    2014-01-01

    The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants. PMID:25037213

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

    PubMed Central

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

    1986-01-01

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

  4. Isolation of Mutants Defective in α-Amylase from Bacillus subtilis: Genetic Analyses

    PubMed Central

    Yamaguchi, Kazuo; Nagata, Yoshiho; Maruo, Bunji

    1974-01-01

    The rate of α-amylase (EC 3.2.1.1) synthesis in Bacillus subtilis is regulated by a gene, amyR, located near a structural gene, amyE, for the enzyme. To construct a fine map of the amyR-amyE region, we isolated 28 mutants defective in α-amylase activity. Eleven mutants out of 28 showed no α-amylase activity, whereas the other 17 showed less α-amylase activity than the parent. Out of 17 partially positive α-amylase mutants, 10 produced temperature-sensitive enzymes, and 4 produced immunologically altered enzymes, two of which are concurrently temperature-sensitive, and 5 produced smaller amounts of α-amylases which are indistinguishable from normal enzyme in their temperature sensitivity and immunological properties. Two out of 11 α-amylase-negative mutants produced material that cross-reacted with anti-amylase serum, and 3 mutants carried suppressible mutations by the suppressor described by Okubo. Mapping data indicate that all 28 mutation sites are located in the amyE region, and none of the groups of the mutants mentioned above contains lesions that are clustered in a single region of amyE. The amyR gene seems most likely to adjoin the terminal region of amyE. PMID:4212116

  5. Genetic complexity of regulatory mutants defective for HLA class II gene expression

    SciTech Connect

    Seidl, C.; Saraiya, C.; Osterweil, Z.; Fu, Y. Ping; Lee, J.S. )

    1992-03-01

    MHC (called HLA in man) class II genes play an essential role in cell-mediated immunity. Absence of HLA class II Ag on B lymphocytes is the basis of some congenital immunodeficiencies (CID). The authors have studied CID by generating transient heterokaryons from cell lines of such patients, and report that the mutations fall into four complementation groups. In addition, fusions with the HLA class II deletion mutant 721.180 indicate that the genetic defects for each group in HLA class II expression map outside the HLA class II region. A small HLA-DRA promoter fragment is sufficient to drive expression of a reporter gene in normal B cell lines, but expression from the same construct is clearly reduced in mutant cell lines representative of all four complementation groups. This confirms earlier results that indicate defective transcription of HLA class II genes in the class II[sup [minus

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  8. A requirement for recombinational repair in Saccharomyces cerevisiae is caused by DNA replication defects of mec1 mutants.

    PubMed Central

    Merrill, B J; Holm, C

    1999-01-01

    To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants. PMID:10511542

  9. Bacillus subtilis ccpA Gene Mutants Specifically Defective in Activation of Acetoin Biosynthesis

    PubMed Central

    Turinsky, Andrew J.; Moir-Blais, Tessa R.; Grundy, Frank J.; Henkin, Tina M.

    2000-01-01

    A large number of carbon source utilization pathways are repressed in Bacillus subtilis by the global regulator CcpA, which also acts as an activator of carbon excretion pathways during growth in media containing glucose. In this study, CcpA mutants defective in transcriptional activation of the alsSD operon, which is involved in acetoin biosynthesis, were identified. These mutants retained normal glucose repression of amyE, encoding α-amylase, and acsA, encoding acetyl-coenzyme A synthetase, and normal activation of ackA, which is involved in acetate excretion; in these ccpA mutants the CcpA functions of activation of the acetate and acetoin excretion pathways appear to be separated. PMID:10986270

  10. Sequence analysis of thymidine kinase-defective mutants of equine herpesvirus-1 (EHV-1).

    PubMed

    Corrochano, L M; Madueño, F; Field, H J; de la Fuente, R

    1993-04-30

    We have amplified, cloned and sequenced the gene encoding the thymidine kinase (TK) of a wild-type strain (Ab4) of equine herpesvirus-1 (EHV-1) and two mutants with defective TK activity isolated for resistance to penciclovir (PCV). One of the mutants, PR1, has suffered a 879-bp deletion which reduces the size of TK to 180 bp. The other mutant, PR3, has an adenine to cytosine mutation resulting in a Lys38-->Thr change. This mutation modifies the amino acid sequence of a domain involved in binding ATP, leading to non-detectable enzymatic activity. Lys38 thus appears to be essential for the activity of the TK of EHV-1.

  11. Recombinant EDA or Sonic Hedgehog rescue the branching defect in Ectodysplasin A pathway mutant salivary glands in vitro.

    PubMed

    Wells, K L; Mou, C; Headon, D J; Tucker, A S

    2010-10-01

    Hypohidrotic ectodermal dysplasia (HED) is characterized by defective ectodermal organ development. This includes the salivary glands (SGs), which have an important role in lubricating the oral cavity. In humans and mice, HED is caused by mutations in Ectodysplasin A (Eda) pathway genes. Various phenotypes of the mutant mouse Eda(Ta/Ta), which lacks the ligand Eda, can be rescued by maternal injection or in vitro culture supplementation with recombinant EDA. However, the response of the SGs to this treatment has not been investigated. Here, we show that the submandibular glands (SMGs) of Eda(Ta/Ta) mice exhibit impaired branching morphogenesis, and that supplementation of Eda(Ta/Ta) SMG explants with recombinant EDA rescues the defect. Supplementation of Edar(dlJ/dlJ) SMGs with recombinant Sonic hedgehog (Shh) also rescues the defect, whereas treatment with recombinant Fgf8 does not. This work is the first to test the ability of putative Eda target molecules to rescue Eda pathway mutant SMGs.

  12. Prevention of neural tube defects in Lrp2 mutant mouse embryos by folic acid supplementation.

    PubMed

    Sabatino, Julia A; Stokes, Bethany A; Zohn, Irene E

    2017-01-20

    Neural tube defects (NTDs) are among the most common structural birth defects in humans and are caused by the complex interaction of genetic and environmental factors. Periconceptional supplementation with folic acid can prevent NTDs in both mouse models and human populations. A better understanding of how genes and environmental factors interact is critical toward development of rational strategies to prevent NTDs. Low density lipoprotein-related protein 2 (Lrp2) is involved in endocytosis of the folic acid receptor among numerous other nutrients and ligands. We determined the effect of iron and/or folic acid supplementation on the penetrance of NTDs in the Lrp2(null) mouse model. The effects of supplementation on folate and iron status were measured in embryos and dams. Periconceptional dietary supplementation with folic acid did not prevent NTDs in Lrp2 mutant embryos, whereas high levels of folic acid supplementation by intraperitoneal injection reduced incidence of NTDs. Importantly, Lrp2(null/+) dams had reduced blood folate levels that improved with daily intraperitoneal injections of folate but not dietary supplementation. On the contrary, iron supplementation had no effect on the penetrance of NTDs in Lrp2 mutant embryos and negated the preventative effect of folic acid supplementation in Lrp2(null/null) mutants. Lrp2 is required for folate homeostasis in heterozygous dams and high levels of supplementation prevents NTDs. Furthermore, high levels of dietary iron supplementation interfered with folic acid supplementation negating the positive effects of supplementation in this model. Birth Defects Research 109:16-26, 2017. © 2016 The Authors Birth Defects Published by Wiley Periodicals, Inc. © 2016 The Authors Birth Defects Research Published by Wiley Periodicals, Inc.

  13. Defective Excision Repair of Pyrimidine Dimers in the Ultraviolet-Sensitive Escherichia coli ras− Mutant

    PubMed Central

    Walker, James R.

    1970-01-01

    The ras− mutant of Escherichia coli K-12 is sensitive to ultraviolet (UV) light but only slightly sensitive to X-irradiation (1.5-fold increase). Other phenotypic properties include normal recombination ability and normal host cell reactivation ability but an abnormally high frequency of UV-induced mutation. The response of the ras− mutant to UV has been studied biochemically. After low doses of UV, the ras− mutant degraded excessive amounts of deoxyribonucleic acid, and long delays in resumption of deoxyribonucleic acid synthesis occurred. Pyrimidine dimers were excised at the normal rate. Although the mutant had the capability of initiating repair replication, the process was not completed after the high UV dose required to allow detection of repair replication. The ras− mutant, after low UV doses, left three to four times as many single-strand breaks not rejoined as did the wild-type strain. PMID:4919983

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

    PubMed Central

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

    2012-01-01

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

  15. Agravitropic mutants of the moss Ceratodon purpureus do not complement mutants having a reversed gravitropic response.

    PubMed

    Cove, David J; Quatrano, Ralph S

    2006-07-01

    New mutants of the moss Ceratodon purpureus have been isolated, which showed abnormal gravitropic responses. The apical cells of protonemal filaments of wild-type strains respond to gravity by growing upwards and are well aligned to the gravity vector. This response only occurs in darkness. Mutants show a range of phenotypes. Some are insensitive to gravity, showing symmetrical growth, while others align to the gravity vector but orient growth downwards. A further class grows in darkness as though it were in light, showing insensitivity to gravity and continued chlorophyll synthesis. Somatic hybrids between mutants and wild-type strains and between pairs of mutants have been selected using transgenic antibiotic resistance as selective markers. Hybrids between wild-type strains and all of the mutants have a wild-type phenotype, and so all mutants therefore have recessive phenotypes. Mutants comprise three complementation groups. One group has a single member, while another has three members. The third has at least 16 members and shows a complex pattern of complementation consistent with a single gene product functioning in both orientation and alignment to gravity, as well as contributing more than one subunit to the mature product.

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

    PubMed

    Samuels, M; Ezzell, R M; Cardozo, T J; Critchley, D R; Coll, J L; Adamson, E D

    1993-05-01

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

  17. Exploiting the triple response of Arabidopsis to identify ethylene-related mutants.

    PubMed Central

    Guzmán, P; Ecker, J R

    1990-01-01

    Alterations in the response of dark-grown seedlings to ethylene (the "triple response") were used to isolate a collection of ethylene-related mutants in Arabidopsis thaliana. Mutants displaying a constitutive response (eto1) were found to produce at least 40 times more ethylene than the wild type. The morphological defects in etiolated eto1-1 seedlings reverted to wild type under conditions in which ethylene biosynthesis or ethylene action were inhibited. Mutants that failed to display the apical hook in the absence of ethylene (his1) exhibited reduced ethylene production. In the presence of exogenous ethylene, hypocotyl and root of etiolated his1-1 seedlings were inhibited in elongation but no apical hook was observed. Mutants that were insensitive to ethylene (ein1 and ein2) produced increased amounts of ethylene, displayed hormone insensitivity in both hypocotyl and root responses, and showed an apical hook. Each of the "triple response" mutants has an effect on the shape of the seedling and on the production of the hormone. These mutants should prove to be useful tools for dissecting the mode of ethylene action in plants. PMID:2152173

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

    PubMed

    Seiler, C; Nicolson, T

    1999-11-15

    Vertebrate mechanosensory hair cells contain a narrow "pericuticular" zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1-43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function. Copyright 1999 John Wiley & Sons, Inc.

  19. A Clonal Genetic Screen for Mutants Causing Defects in Larval Tracheal Morphogenesis in Drosophila

    PubMed Central

    Baer, Magdalena M.; Bilstein, Andreas; Leptin, Maria

    2007-01-01

    The initial establishment of the tracheal network in the Drosophila embryo is beginning to be understood in great detail, both in its genetic control cascades and in its cell biological events. By contrast, the vast expansion of the system during larval growth, with its extensive ramification of preexisting tracheal branches, has been analyzed less well. The mutant phenotypes of many genes involved in this process are probably not easy to reveal, as these genes may be required for other functions at earlier developmental stages. We therefore conducted a screen for defects in individual clonal homozygous mutant cells in the tracheal network of heterozygous larvae using the mosaic analysis with a repressible cell marker (MARCM) system to generate marked, recombinant mitotic clones. We describe the identification of a set of mutants with distinct phenotypic effects. In particular we found a range of defects in terminal cells, including failure in lumen formation and reduced or extensive branching. Other mutations affect cell growth, cell shape, and cell migration. PMID:17603107

  20. Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants.

    PubMed

    Avaro, Sandrine; Belgareh-Touzé, Naïma; Sibella-Argüelles, Carla; Volland, Christiane; Haguenauer-Tsapis, Rosine

    2002-03-15

    We have screened the EUROFAN (European Functional Analysis Network) deletion strain collection for yeast mutants defective in secretory/vacuolar pathways and/or associated biochemical modifications. We used systematic Western immunoblotting to analyse the electrophoretic pattern of several markers of the secretory/vacuolar pathways, the soluble alpha-factor, the periplasmic glycoprotein invertase, the plasma membrane GPI-anchored protein Gas1p, and two vacuolar proteins, the soluble carboxypeptidase Y and the membrane-bound alkaline phosphatase, which are targeted to the vacuole by different pathways. We also used colony immunoblotting to monitor the secretion of carboxypeptidase Y into the medium, to identify disruptants impaired in vacuolar targeting. We identified 25 mutants among the 631 deletion strains. Nine of these mutants were disrupted in genes identified in recent years on the basis of their involvement in trafficking (VPS53, VAC7, VAM6, APM3, SYS1), or glycosylation (ALG12, ALG9, OST4, ROT2). Three of these genes were identified on the basis of trafficking defects by ourselves and others within the EUROFAN project (TLG2, RCY1, MON2). The deletion of ERV29, which encodes a COPII vesicle protein, impaired carboxypeptidase Y trafficking from the endoplasmic reticulum to the Golgi apparatus. We also identified eight unknown ORFs, the deletion of which reduced Golgi glycosylation or impaired the Golgi to vacuole trafficking of carboxypeptidase Y. YJR044c, which we identified as a new VPS gene, encodes a protein with numerous homologues of unknown function in sequence databases.

  1. Equine herpesvirus type 1 mutant defective in glycoprotein E gene as candidate vaccine strain.

    PubMed

    TSUJIMURA, Koji; SHIOSE, Tomoki; YAMANAKA, Takashi; NEMOTO, Manabu; KONDO, Takashi; MATSUMURA, Tomio

    2009-11-01

    An equine herpesvirus type 1 (EHV-1) mutant, DeltagE, defective in glycoprotein E (gE) was evaluated as a modified live virus (MLV) vaccine. Colostrum-deprived Thoroughbred foals inoculated intranasally (i.n.) or intramuscularly (i.m.) with DeltagE did not exhibit any clinical signs of respiratory disease except for a mild nasal discharge in 1 i.n. inoculated foal on Days 1 and 3 post-infection. In contrast, the intranasal inoculation of foals with the revertant of DeltagE resulted in biphasic pyrexia, mucopurulent nasal discharge and swelling of submandibular lymph nodes. These results indicated that gE plays an important role as regards EHV-1 virulence in horses. The ability of DeltagE to protect against wild type EHV-1 challenge infection was assessed using i.m. vaccinated foals. Foals inoculated twice i.m. with 10(5) or 10(6) plaque-forming units (pfu) of DeltagE at an interval of 3 weeks exhibited no clinical evidence of local inflammation, respiratory disease or deleterious systemic responses. Remarkable increases in SN antibody titer to EHV-1 were observed in all vaccinated foals after the 2nd inoculation with DeltagE. Following a wild type EHV-1 challenge infection, vaccinated foals showed milder clinical symptoms than foals vaccinated with a placebo. Specifically, 1 of 3 foals vaccinated with 10(6) pfu of DeltagE exhibited no clinical symptoms other than a mild nasal discharge for 1 day. Additionally, the virus load of nasal shedding and viremia were reduced by vaccination. These results suggest that DeltagE would be a good candidate as an MLV vaccine.

  2. A Serendipitous Mutation Reveals the Severe Virulence Defect of a Klebsiella pneumoniae fepB Mutant

    PubMed Central

    Palacios, Michelle; Broberg, Christopher A.; Walker, Kimberly A.

    2017-01-01

    ABSTRACT Klebsiella pneumoniae is considered a significant public health threat because of the emergence of multidrug-resistant strains and the challenge associated with treating life-threatening infections. Capsule, siderophores, and adhesins have been implicated as virulence determinants of K. pneumoniae, yet we lack a clear understanding of how this pathogen causes disease. In a previous screen for virulence genes, we identified a potential new virulence locus and constructed a mutant (smr) with this locus deleted. In this study, we characterize the smr mutant and show that this mutation renders K. pneumoniae avirulent in a pneumonia model of infection. The smr mutant was expected to have a deletion of three genes, but subsequent genome sequencing indicated that a much larger deletion had occurred. Further analysis of the deleted region indicated that the virulence defect of the smr mutant could be attributed to the loss of FepB, a periplasmic protein required for import of the siderophore enterobactin. Interestingly, a ΔfepB mutant was more attenuated than a mutant unable to synthesize enterobactin, suggesting that additional processes are affected. As FepB is highly conserved among the members of the family Enterobacteriaceae, therapeutic targeting of FepB may be useful for the treatment of Klebsiella and other bacterial infections. IMPORTANCE In addition to having a reputation as the causative agent of several types of hospital-acquired infections, Klebsiella pneumoniae has gained widespread attention as a pathogen with a propensity for acquiring antibiotic resistance. It is capable of causing a range of infections, including urinary tract infections, pneumonia, and sepsis. Because of the rapid emergence of carbapenem resistance among Klebsiella strains, there is a dire need for a better understanding of virulence mechanisms and identification of new drug targets. Here, we identify the periplasmic transporter FepB as one such potential target. PMID

  3. Analysis of a gene that suppresses the morphological defect of bald mutants of Streptomyces griseus.

    PubMed Central

    McCue, L A; Kwak, J; Wang, J; Kendrick, K E

    1996-01-01

    When present in multiple copies, orf1590 restored sporulation to class IIIA bald mutants of Streptomyces griseus, which form sporulation septa and thick spore walls prematurely. The orf1590 alleles from class IIIA bald mutants restored sporulation upon introduction at a high copy number into those same mutants, and the nucleotide sequence of one of these alleles was identical to that of the wild-type strain. We conclude that overexpression of orf1590 suppresses the defect in class IIIA bald mutants. Previous nucleotide sequence and transcript analyses suggested that orf1590 could encode two related proteins, P56 and P49.5, from nested coding sequences. A mutation that prevented the synthesis of P56 without altering the coding sequence for P49.5 eliminated the function of orf1590, as did amino acid substitutions in the putative helix-turn-helix domain located at the N terminus of P56 and absent from P49.5. To determine the coding capacity of orf1590, we analyzed translational fusions between orf1590 and the neo gene from Tn5. Measurement of the expression of fusions to the wild-type and mutant alleles of orf1590 indicated that P56 was the sole product of orf1590 during vegetative growth. Attempts to generate a nonfunctional frameshift mutation in orf1590 were unsuccessful in the absence of a second-site bald mutation, suggesting that orf1590 may be required during vegetative growth by preventing early sporulation. Our results are consistent with the hypothesis that P56 at a high level delays the premature synthesis of sporulation septa and spore walls in class IIIA mutants. PMID:8631675

  4. The maize auxotrophic mutant orange pericarp is defective in duplicate genes for tryptophan synthase beta.

    PubMed Central

    Wright, A D; Moehlenkamp, C A; Perrot, G H; Neuffer, M G; Cone, K C

    1992-01-01

    orange pericarp (orp) is a seedling lethal mutant of maize caused by mutations in the duplicate unlinked recessive loci orp1 and orp2. Mutant seedlings accumulate two tryptophan precursors, anthranilate and indole, suggesting a block in tryptophan biosynthesis. Results from feeding studies and enzyme assays indicate that the orp mutant is defective in tryptophan synthase beta activity. Thus, orp is one of only a few amino acid auxotrophic mutants to be characterized in plants. Two genes encoding tryptophan synthase beta were isolated from maize and sequenced. Both genes encode polypeptides with high homology to tryptophan synthase beta enzymes from other organisms. The cloned genes were mapped by restriction fragment length polymorphism analysis to approximately the same chromosomal locations as the genetically mapped factors orp1 and orp2. RNA analysis indicates that both genes are expressed in all tissues examined from normal plants. Together, the biochemical, genetic, and molecular data verify the identity of orp1 and orp2 as duplicate structural genes for the beta subunit of tryptophan synthase. PMID:1356534

  5. The maize auxotrophic mutant orange pericarp is defective in duplicate genes for tryptophan synthase beta.

    PubMed

    Wright, A D; Moehlenkamp, C A; Perrot, G H; Neuffer, M G; Cone, K C

    1992-06-01

    orange pericarp (orp) is a seedling lethal mutant of maize caused by mutations in the duplicate unlinked recessive loci orp1 and orp2. Mutant seedlings accumulate two tryptophan precursors, anthranilate and indole, suggesting a block in tryptophan biosynthesis. Results from feeding studies and enzyme assays indicate that the orp mutant is defective in tryptophan synthase beta activity. Thus, orp is one of only a few amino acid auxotrophic mutants to be characterized in plants. Two genes encoding tryptophan synthase beta were isolated from maize and sequenced. Both genes encode polypeptides with high homology to tryptophan synthase beta enzymes from other organisms. The cloned genes were mapped by restriction fragment length polymorphism analysis to approximately the same chromosomal locations as the genetically mapped factors orp1 and orp2. RNA analysis indicates that both genes are expressed in all tissues examined from normal plants. Together, the biochemical, genetic, and molecular data verify the identity of orp1 and orp2 as duplicate structural genes for the beta subunit of tryptophan synthase.

  6. Motility defects in Campylobacter jejuni defined gene deletion mutants caused by second-site mutations

    PubMed Central

    de Vries, Stefan P. W.; Gupta, Srishti; Baig, Abiyad; L'Heureux, Joanna; Pont, Elsa; Wolanska, Dominika P.; Maskell, Duncan J.

    2015-01-01

    Genetic variation due to mutation and phase variation has a considerable impact on the commensal and pathogenic behaviours of Campylobacter jejuni. In this study, we provide an example of how second-site mutations can interfere with gene function analysis in C. jejuni. Deletion of the flagellin B gene (flaB) in C. jejuni M1 resulted in mutant clones with inconsistent motility phenotypes. From the flaB mutant clones picked for further analysis, two were motile, one showed intermediate motility and two displayed severely attenuated motility. To determine the molecular basis of this differential motility, a genome resequencing approach was used. Second-site mutations were identified in the severely attenuated and intermediate motility flaB mutant clones: a TA-dinucleotide deletion in fliW and an A deletion in flgD, respectively. Restoration of WT fliW, using a newly developed genetic complementation system, confirmed that the second-site fliW mutation caused the motility defect as opposed to the primary deletion of flaB. This study highlights the importance of (i) screening multiple defined gene deletion mutant clones, (ii) genetic complementation of the gene deletion and ideally (iii) screening for second-site mutations that might interfere with the pathways/mechanisms under study. PMID:26385289

  7. Characterization of T cell mutants with defects in capacitative calcium entry: genetic evidence for the physiological roles of CRAC channels

    PubMed Central

    1995-01-01

    Prolonged Ca2+ influx is an essential signal for the activation of T lymphocytes by antigen. This influx is thought to occur through highly selective Ca2+ release-activated Ca2+ (CRAC) channels that are activated by the depletion of intracellular Ca2+ stores. We have isolated mutants of the Jurkat human T cell line NZdipA to explore the molecular mechanisms that underlie capacitative Ca2+ entry and to allow a genetic test of the functions of CRAC channels in T cells. Five mutant cell lines (CJ-1 through CJ-5) were selected based on their failure to express a lethal diphtheria toxin A chain gene and a lacZ reporter gene driven by NF-AT, a Ca(2+)- and protein kinase C-dependent transcription factor. The rate of Ca2+ influx evoked by thapsigargin was reduced to varying degrees in the mutant cells whereas the dependence of NF-AT/lacZ gene transcription on [Ca2+]i was unaltered, suggesting that the transcriptional defect in these cells is caused by a reduced level of capacitative Ca2+ entry. We examined several factors that determine the rate of Ca2+ entry, including CRAC channel activity, K(+)-channel activity, and Ca2+ clearance mechanisms. The only parameter found to be dramatically altered in most of the mutant lines was the amplitude of the Ca2+ current (ICRAC), which ranged from 1 to 41% of that seen in parental control cells. In each case, the severity of the ICRAC defect was closely correlated with deficits in Ca2+ influx rate and Ca(2-)-dependent gene transcription. Behavior of the mutant cells provides genetic evidence for several roles of ICRAC in T cells. First, mitogenic doses of ionomycin appear to elevate [Ca2+]i primarily by activating CRAC channels. Second, ICRAC promotes the refilling of empty Ca2+ stores. Finally, CRAC channels are solely responsible for the Ca2+ influx that underlies antigen-mediated T cell activation. These mutant cell lines may provide a useful system for isolating, expressing, and exploring the functions of genes involved in

  8. Indirect intergenic suppression of a radiosensitive mutant of Sordaria macrospora defective in sister-chromatid cohesiveness.

    PubMed

    Huynh, A D; Leblon, G; Zickler, D

    1986-01-01

    Six ultra violet (UV) mutageneses were performed on the spo76 UV-sensitive mutant of Sordaria macrospora. Spo76 shows an early centromere cleavage associated with an arrest at the first meiotic division and therefore does not form ascospores. Moreover, it exhibits altered pairing structure (synaptonemal complex), revealing a defect in the sister-chromatid cohesiveness. From 37 revertants which partially restored sporulation, 34 extragenic suppressors of spo76 were isolated. All suppressors are altered in chromosomal pairing but, unlike spo76, show a wild type centromere cleavage. The 34 suppressors were assigned to six different genes and mapped. Only one of the suppressor genes is involved in repair functions.

  9. Cyclic AMP-dependent memory mutants are defective in the food choice behavior of Drosophila.

    PubMed

    Motosaka, Katsunori; Koganezawa, Masayuki; Narikawa, Satoko; Furuyama, Akira; Shinozaki, Kenji; Isono, Kunio; Shimada, Ichiro

    2007-02-01

    Acute choice behavior in ingesting two different concentrations of sucrose in Drosophila is presumed to include learning and memory. Effects on this behavior were examined for four mutations that block associative learning (dunce, rutabaga, amnesiac, and radish). Three of these mutations cause cyclic AMP signaling defects and significantly reduced taste discrimination. The exception was radish, which affects neither. Electrophysiological recordings confirmed that the sensitivity of taste receptors is almost indistinguishable in all flies, whether wild type or mutant. These results suggest that food choice behavior in Drosophila involves central nervous learning and memory operating via cyclic AMP signaling pathways.

  10. Functional expression of human mutant phosphofructokinase in yeast: genetic defects in French Canadian and Swiss patients with phosphofructokinase deficiency.

    PubMed Central

    Raben, N; Exelbert, R; Spiegel, R; Sherman, J B; Nakajima, H; Plotz, P; Heinisch, J

    1995-01-01

    Human phosphofructokinase (PFK) is a tetrameric enzyme, encoded by muscle, liver, and platelet genes. Deficiency of muscle PFK (PFK-M), glycogenosis type VII (Tarui disease), is an autosomal recessive disorder characterized by an exertional myopathy and hemolytic syndrome. Several disease-causing mutations have been identified in the PFK-M gene in Japanese, Ashkenazi Jewish, and Italian patients. We describe the genetic defects in French Canadian and Swiss patients with the disease, and we use a genetically well-defined yeast system devoid of endogenous PFK for structure-function studies of the mutant PFKs. A G-to-A transition at codon 209-in exon 8 of the PFK-M gene, changing an encoded Gly to Asp, is responsible for the disease in a homozygous French Canadian patient. Gly-209-mutated protein is completely inactive in the yeast system. The Swiss patient is a genetic compound, carrying a G-to-A transition at codon 100 in exon 6 (Arg to Gln) and a G-to-A transition at codon 696 in exon 22 (Arg to His). The mutants expressed in yeast generate functional enzyme with modest changes in thermal stability. The advantages and limitations of the yeast system for expression of human mutant PFKs are discussed. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7825568

  11. Genetic studies of an Escherichia coli K-12 temperature-sensitive mutant defective in membrane protein synthesis.

    PubMed Central

    Sato, T; Ohki, M; Yura, T; Ito, K

    1979-01-01

    The mutant divE42(Ts) of Escherichia coli K-12, defective in the synthesis of membrane proteins and in the transcription of the lac operon at high temperature, has been further characterized. It was found that a mutation (divE42) located at about min 22 on the E. coli chromosome map is responsible for the Lac- phenotype and temperature-sensitive growth. The mutation could be contransduced with serC, pyrD, or pyrC by phage P1 at a frequency of 4, 16, or 0.5%, respectively, the gene order being serC-pyrD-ompA-sulA-divE-pyrC. Examination of temperature-independent revertants and Pyr+ transductants revealed that all the mutant phenotypes examined (deficiencies in the increase of activities of some membrane enzymes, expression of the lac operon, and synthesis of several other proteins) are due to a single mutation (divE42) which is recessive to the wild-type (divE+) allele. Protein synthesis in the mutant was also analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis. Synthesis of a number of proteins, including membrane proteins, was found to decrease significantly, whereas that of an elongation factor, EF-Tu, increased upon transfer of a log-phase culture to high temperature (42 degrees C). These effects of temperature shift-up on protein synthesis were evident within 5 min under the conditions used. Images PMID:374381

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed Central

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

    1980-01-01

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

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

    PubMed Central

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

    2008-01-01

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

  15. Reduction of germ cells in the Odysseus null mutant causes male fertility defect in Drosophila melanogaster.

    PubMed

    Cheng, Ya-Jen; Fang, Shu; Tsaur, Shun-Chern; Chen, Yi-Ling; Fu, Hua-Wen; Patel, Nipam H; Ting, Chau-Ti

    2012-01-01

    Odysseus (OdsH) has been identified as a hybrid male sterility gene between Drosophila mauritiana and D. simulans with accelerated evolutionary rate in both expression and DNA sequence. Loss of a testis-specific expression of OdsH causes male fertility defect in D. melanogaster. Yet, the underlying mechanisms at the cellular level are unknown. In an attempt to identify the possible mechanisms and functional roles of OdsH in spermatogenesis, the cell numbers at different developmental stages during spermatogenesis between the OdsH null mutant and wild-type flies were compared. The results showed that the early developing germ cells, including spermatogonia and spermatocytes, were reduced in the OdsH mutant males. In addition, the number of germline stem cells in aged males was also reduced, presumably due to the disruption of germline stem cell maintenance, which resulted in more severe fertility defect. These results suggest that the function of the enhancement of sperm production by OdsH acted across males of all ages.

  16. Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice.

    PubMed

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

    2007-08-23

    Obsessive-compulsive disorder (OCD) is an anxiety-spectrum disorder characterized by persistent intrusive thoughts (obsessions) and repetitive actions (compulsions). Dysfunction of cortico-striato-thalamo-cortical circuitry is implicated in OCD, although the underlying pathogenic mechanisms are unknown. SAP90/PSD95-associated protein 3 (SAPAP3; also known as DLGAP3) is a postsynaptic scaffolding protein at excitatory synapses that is highly expressed in the striatum. Here we show that mice with genetic deletion of Sapap3 exhibit increased anxiety and compulsive grooming behaviour leading to facial hair loss and skin lesions; both behaviours are alleviated by a selective serotonin reuptake inhibitor. Electrophysiological, structural and biochemical studies of Sapap3-mutant mice reveal defects in cortico-striatal synapses. Furthermore, lentiviral-mediated selective expression of Sapap3 in the striatum rescues the synaptic and behavioural defects of Sapap3-mutant mice. These findings demonstrate a critical role for SAPAP3 at cortico-striatal synapses and emphasize the importance of cortico-striatal circuitry in OCD-like behaviours.

  17. A pqr2 mutant encodes a defective polyamine transporter and is negatively affected by ABA for paraquat resistance in Arabidopsis thaliana.

    PubMed

    Dong, Shuchao; Hu, Huizhen; Wang, Youmei; Xu, Zhengdan; Zha, Yi; Cai, Xiwen; Peng, Liangcai; Feng, Shengqiu

    2016-09-01

    Despite the paraquat-resistant mutants that have been reported in plants, this study identified a novel A. thaliana mutant (pqr2) from an XVE inducible activation library based on its resistance to 2 μM paraquat. The pqr2 mutant exhibited a termination mutation in the exon of AT1G31830/PAR1/PQR2, encoded a polyamine uptake transporter AtPUT2/PAR1/PQR2. The PQR2 mutation could largely reduce superoxide accumulation and cell death in the pqr2 plants under paraquat treatment. Moreover, compared with wild type, the pqr2 mutant exhibited much reduced tolerance to putrescine, a classic polyamine compound, which confirmed that PQR2 encoded a defective polyamine transporter. Notably, co-treated with ABA and paraquat, both pqr2 mutant and wild type exhibited a lethal phenotype from seed germination, but the wild type like pqr2 mutant, could remain paraquat-resistance while co-treated with high dosage of Na2WO4, an ABA synthesis inhibitor. Gene expression analysis suggested that ABA signaling should widely regulate paraquat-responsive genes distinctively in wild type and pqr2 mutant. Hence, this study has for the first time reported about ABA negative effect on paraquat-resistance in A. thaliana, providing insight into the ABA signaling involved in the oxidative stress responses induced by paraquat in plants.

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

    PubMed

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

    2010-10-01

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

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

    PubMed Central

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

    2015-01-01

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

  20. An Escherichia coli mutant resistant to phleomycin, bleomycin, and heat inactivation is defective in ubiquinone synthesis.

    PubMed Central

    Collis, C M; Grigg, G W

    1989-01-01

    A mutant of Escherichia coli, selected for resistance to the antibiotic and antitumor agent phleomycin, has been characterized, and the phleomycin resistance determinant has been identified. The mutant is equally resistant to bleomycins. The resistance to phleomycin is strongly dependent on the nature of the C-terminal amine of the drug, with the greatest resistance being shown to phleomycins and bleomycins with the most basic terminal amines. The mutation also confers resistance to the lethal effects of heating at 52 degrees C. Other characteristics of the phleomycin-resistant strain include a slow growth rate, an inability to grow on succinate as the sole carbon source (Suc- phenotype), cross resistance to aminoglycoside antibiotics, and a slight sensitivity to hydrogen peroxide, methyl methanesulfonate, and gamma-irradiation. Some of these characteristics, together with mapping data, suggested that the phleomycin resistance and Suc- determinant probably lies within the ubiF gene coding for an enzyme effecting a step in the biosynthesis of ubiquinone. The phenotypes of known mutants defective in this and other steps of the ubiquinone pathway were found to be closely similar to those of the original phleomycin-resistant strain. PMID:2475481

  1. Genetic mapping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins.

    PubMed Central

    Wretlind, B; Pavlovskis, O R

    1984-01-01

    We isolated 15 mutants of Pseudomonas aeruginosa PAO which were defective in the formation of certain extracellular proteins, such as elastase, staphylolytic enzyme, and lipase ( Xcp mutants). The mutations were mapped on the chromosome by conjugation and transduction. The locations were xcp -1 near 0', with the gene order cys-59- xcp -1- proB , and loci xcp -2, xcp -3, and xcp -31 at 35', with the gene order trpC , D- xcp -3/ xcp -31- xcp -2- argC . Loci xcp -4 and xcp -41 through xcp -44 were cotransducible with proA at 40'; loci xcp -5, xcp -51, xcp -52, and xcp53 were located at 55', with the gene order leu-10- trpF -met-9010- xcp -53- xcp -5/ xcp -51/ xcp+ ++-52, and xcp -6 was located at 65' to 70', between catA and mtu-9002. Nine mutations ( xcp -2, xcp -3, xcp -31, xcp -4, and xcp -41 through xcp -45) caused decreased production of extracellular enzymes. Six strains with mutations xcp -1, xcp -5, xcp -51, xcp -52, xcp -53, and xcp -6 produced cell-bound exoproteins and had defective release mechanisms. The regulation of production of alkaline phosphatase and phospholipase C is different from other exoproteins , such as elastase, but they all seem to share a common release mechanism. Alkaline protease had separate mechanisms for regulation and release, since this protease was found in culture supernatants of all but one of the mutants, and none of the strains had cell-bound enzyme. PMID:6427194

  2. Selective myelin defects in the anterior medullary velum of the taiep mutant rat.

    PubMed

    Song, J; Goetz, B D; Kirvell, S L; Butt, A M; Duncan, I D

    2001-01-01

    The taiep rat is a myelin mutant in which initial hypomyelination is followed by progressive demyelination of the CNS. An in vitro study suggests that accumulation of microtubules within oligodendrocytes is the cause of the taiep myelin defects (Song et al., 1999). In this article, we analyze microtubule accumulation in relation to taiep myelin defects in vivo in the anterior medullary velum (AMV), a CNS tissue that enables entire oligodendrocyte units to be resolved. Immunohistochemical analysis demonstrated notably high levels of beta-tubulin and the microtubule associated protein tau in the somata and processes of taiep oligodendrocytes. This was correlated with markedly reduced expression of the myelin proteins, proteolipid protein (PLP), myelin basic protein (MBP), 2',3 -cyclic nucleotide 3'-phosphodiesterase, and both large (L) and small (S) isoforms of myelin-associated glycoprotein (MAG). Moreover, PLP and L-MAG, which are dependent on the microtubule system for intracellular transport, accumulated in the perinuclear cytoplasm of the taiep oligodendrocyte. The myelin deficit was most marked in the area of the AMV populated by the small somata oligodendrocytes that have fine long processes that support numerous myelin sheaths of small diameter axons. Type III/IV oligodendrocytes, which have large somata and short processes that support a small number of myelin sheaths of large diameter axons, were also affected to a certain degree in compact myelin sheath formation. These results support the hypothesis that myelin loss and oligodendrocyte disruption in the taiep mutant result from a defect in the microtubule system that transports myelin components from the somata to the myelin sheath.

  3. Homologous Recombination Defective Arabidopsis Mutants Exhibit Enhanced Sensitivity to Abscisic Acid

    PubMed Central

    Roy, Sujit; Das, Kali Pada

    2017-01-01

    Abscisic acid (ABA) acts as an important plant hormone in regulating various aspects of plant growth and developmental processes particularly under abiotic stress conditions. An increased ABA level in plant cells inhibits DNA replication and cell division, causing plant growth retardation. In this study, we have investigated the effects of ABA on the growth responses of some major loss-of-function mutants of DNA double-stand break (DSB) repair genes in Arabidopsis during seed germination and early stages of seedling growth for understanding the role of ABA in the induction of genome instability in plants. A comparative analysis of ABA sensitivity of wild-type Arabidopsis and the knockout mutant lines related to DSB sensors, including atatm, atatr, the non-homologous end joining (NHEJ) pathway genes, and mutants related to homologous recombination (HR) pathway genes showed relatively enhanced sensitivity of atatr and HR-related mutants to ABA treatment. The expression levels of HR-related genes were increased in wild-type Arabidopsis (Col-0) during seed germination and early stages of seedling growth. Immunoblotting experiments detected phosphorylation of histone H2AX in wild-type (Col-0) and DSB repair gene mutants after ABA treatment, indicating the activation of DNA damage response due to ABA treatment. Analyses of DSB repair kinetics using comet assay under neutral condition have revealed comparatively slower DSB repair activity in HR mutants. Overall, our results have provided comprehensive information on the possible effect of ABA on DNA repair machinery in plants and also indicated potential functional involvement of HR pathway in repairing ABA induced DNA damage in Arabidopsis. PMID:28046013

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

    PubMed Central

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

    2001-01-01

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

  5. Role of cilia in structural birth defects: insights from ciliopathy mutant mouse models.

    PubMed

    Rao Damerla, Rama; Gabriel, George C; Li, You; Klena, Nikolai T; Liu, Xiaoqin; Chen, Yu; Cui, Cheng; Pazour, Gregory J; Lo, Cecilia W

    2014-06-01

    Structural birth defect (SBD) is a major cause of morbidity and mortality in the newborn period. Although the etiology of SBD is diverse, a wide spectrum of SBD associated with ciliopathies points to the cilium as having a central role in the pathogenesis of SBDs. Ciliopathies are human diseases arising from disruption of cilia structure and/or function. They are associated with developmental anomalies in one or more organ systems and can involve defects in motile cilia, such as those in the airway epithelia or from defects in nonmotile (primary cilia) that have sensory and cell signaling function. Availability of low cost next generation sequencing has allowed for explosion of new knowledge in genetic etiology of ciliopathies. This has led to the appreciation that many genes are shared in common between otherwise clinically distinct ciliopathies. Further insights into the relevance of the cilium in SBD has come from recovery of pathogenic mutations in cilia-related genes from many large-scale mouse forward genetic screens with differing developmental phenotyping focus. Our mouse mutagenesis screen for congenital heart disease (CHD) using noninvasive fetal echocardiography has yielded a marked enrichment for pathogenic mutations in genes required for motile or primary cilia function. These novel mutant mouse models will be invaluable for modeling human ciliopathies and further interrogating the role of the cilium in the pathogenesis of SBD and CHD. Overall, these findings suggest a central role for the cilium in the pathogenesis of a wide spectrum of developmental anomalies associated with CHD and SBDs.

  6. Defective FANCI binding by a fanconi anemia-related FANCD2 mutant.

    PubMed

    Sato, Koichi; Ishiai, Masamichi; Takata, Minoru; Kurumizaka, Hitoshi

    2014-01-01

    FANCD2 is a product of one of the genes associated with Fanconi anemia (FA), a rare recessive disease characterized by bone marrow failure, skeletal malformations, developmental defects, and cancer predisposition. FANCD2 forms a complex with FANCI (ID complex) and is monoubiquitinated, which facilitates the downstream interstrand crosslink (ICL) repair steps, such as ICL unhooking and nucleolytic end resection. In the present study, we focused on the chicken FANCD2 (cFANCD2) mutant harboring the Leu234 to Arg (L234R) substitution. cFANCD2 L234R corresponds to the human FANCD2 L231R mutation identified in an FA patient. We found that cFANCD2 L234R did not complement the defective ICL repair in FANCD2-/- DT40 cells. Purified cFANCD2 L234R did not bind to chicken FANCI, and its monoubiquitination was significantly deficient, probably due to the abnormal ID complex formation. In addition, the histone chaperone activity of cFANCD2 L234R was also defective. These findings may explain some aspects of Fanconi anemia pathogenesis by a FANCD2 missense mutation.

  7. Defective FANCI Binding by a Fanconi Anemia-Related FANCD2 Mutant

    PubMed Central

    Sato, Koichi; Ishiai, Masamichi; Takata, Minoru; Kurumizaka, Hitoshi

    2014-01-01

    FANCD2 is a product of one of the genes associated with Fanconi anemia (FA), a rare recessive disease characterized by bone marrow failure, skeletal malformations, developmental defects, and cancer predisposition. FANCD2 forms a complex with FANCI (ID complex) and is monoubiquitinated, which facilitates the downstream interstrand crosslink (ICL) repair steps, such as ICL unhooking and nucleolytic end resection. In the present study, we focused on the chicken FANCD2 (cFANCD2) mutant harboring the Leu234 to Arg (L234R) substitution. cFANCD2 L234R corresponds to the human FANCD2 L231R mutation identified in an FA patient. We found that cFANCD2 L234R did not complement the defective ICL repair in FANCD2−/− DT40 cells. Purified cFANCD2 L234R did not bind to chicken FANCI, and its monoubiquitination was significantly deficient, probably due to the abnormal ID complex formation. In addition, the histone chaperone activity of cFANCD2 L234R was also defective. These findings may explain some aspects of Fanconi anemia pathogenesis by a FANCD2 missense mutation. PMID:25489943

  8. Heterotaxy and complex structural heart defects in a mutant mouse model of primary ciliary dyskinesia

    PubMed Central

    Tan, Serena Y.; Rosenthal, Julie; Zhao, Xiao-Qing; Francis, Richard J.; Chatterjee, Bishwanath; Sabol, Steven L.; Linask, Kaari L.; Bracero, Luciann; Connelly, Patricia S.; Daniels, Mathew P.; Yu, Qing; Omran, Heymut; Leatherbury, Linda; Lo, Cecilia W.

    2007-01-01

    Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder associated with ciliary defects and situs inversus totalis, the complete mirror image reversal of internal organ situs (positioning). A variable incidence of heterotaxy, or irregular organ situs, also has been reported in PCD patients, but it is not known whether this is elicited by the PCD-causing genetic lesion. We studied a mouse model of PCD with a recessive mutation in Dnahc5, a dynein gene commonly mutated in PCD. Analysis of homozygous mutant embryos from 18 litters yielded 25% with normal organ situs, 35% with situs inversus totalis, and 40% with heterotaxy. Embryos with heterotaxy had complex structural heart defects that included discordant atrioventricular and ventricular outflow situs and atrial/pulmonary isomerisms. Variable combinations of a distinct set of cardiovascular anomalies were observed, including superior-inferior ventricles, great artery alignment defects, and interrupted inferior vena cava with azygos continuation. The surprisingly high incidence of heterotaxy led us to evaluate the diagnosis of PCD. PCD was confirmed by EM, which revealed missing outer dynein arms in the respiratory cilia. Ciliary dyskinesia was observed by videomicroscopy. These findings show that Dnahc5 is required for the specification of left-right asymmetry and suggest that the PCD-causing Dnahc5 mutation may also be associated with heterotaxy. PMID:18037990

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

    PubMed

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

    2015-12-02

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

    Nagahara, Shiori; Takeuchi, Hidenori; Higashiyama, Tetsuya

    2015-03-01

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

  13. Isolation and characterization of a mutant of the marine bacterium Alcanivorax borkumensis SK2 defective in lipid biosynthesis.

    PubMed

    Manilla-Pérez, Efraín; Lange, Alvin Brian; Hetzler, Stephan; Wältermann, Marc; Kalscheuer, Rainer; Steinbüchel, Alexander

    2010-05-01

    In many microorganisms, the key enzyme responsible for catalyzing the last step in triacylglycerol (TAG) and wax ester (WE) biosynthesis is an unspecific acyltransferase which is also referred to as wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT; AtfA). The importance and function of two AtfA homologues (AtfA1 and AtfA2) in the biosynthesis of TAGs and WEs in the hydrocarbon-degrading marine bacterium Alcanivorax borkumensis SK2 have been described recently. However, after the disruption of both the AtfA1 and AtfA2 genes, reduced but substantial accumulation of TAGs was still observed, indicating the existence of an alternative TAG biosynthesis pathway. In this study, transposon-induced mutagenesis was applied to an atfA1 atfA2 double mutant to screen for A. borkumensis mutants totally defective in biosynthesis of neutral lipids in order to identify additional enzymes involved in the biosynthesis of these lipids. At the same time, we have searched for a totally TAG-negative mutant in order to study the function of TAGs in A. borkumensis. Thirteen fluorescence-negative mutants were identified on Nile red ONR7a agar plates and analyzed for their abilities to synthesize lipids. Among these, mutant 2 M(131) was no longer able to synthesize and accumulate TAGs if pyruvate was used as the sole carbon source. The transposon insertion was localized in a gene encoding a putative cytochrome c family protein (ABO_1185). Growth and TAG accumulation experiments showed that the disruption of this gene resulted in the absence of TAGs in 2 M(131) but that growth was not affected. In cells of A. borkumensis SK2 grown on pyruvate as the sole carbon source, TAGs represented about 11% of the dry weight of the cells, while in the mutant 2 M(131), TAGs were not detected by thin-layer and gas chromatography analyses. Starvation and lipid mobilization experiments revealed that the lipids play an important role in the survival of the cells. The

  14. Defects in the ratio of the dynein isoform, DHC11 in the long-flagella mutants of Chlamydomonas reinhardtii.

    PubMed

    Sequeira, Marilyn P; Sinha, Sapna; Motiwalla, Mustafa J; Rao, Venkatramanan G; D'Souza, Jacinta S

    2017-01-22

    The long-flagella mutants (lf1, lf2, lf3 and lf4) of Chlamydomonas reinhardtii are defective in proteins that are required for the assembly of normal flagella, their phenotype being long flagella. In a previous study, we biophysically characterized these mutants for their waveform patterns, swimming speeds, beat frequencies and correlated these parameters with their flagellar lengths. We found an anomaly in this correlation and set out to explore the underlying molecular significance, if any. The diverse inner dynein isoforms are the flagellar motors that convert the chemical energy of ATP into the mechanical energy of motility; we probed the presence of one of these isoforms (DHC11, which might help in bend initiation) in the lf mutants and compared it with the wild-type. Our studies show that the ratio of DHC11 is defective in the long-flagella mutants of Chlamydomonas reinhardtii.

  15. The selection of S. cerevisiae mutants defective in the start event of cell division.

    PubMed

    Reed, S I

    1980-07-01

    Thirty-three temperature-sensitive mutations defective in the start event of the cell division cycle of Saccharomyces cerevisiae were isolated and subjected to preliminary characterization. Complementation studies assigned thes mutations to four complementation groups, one of which, cdc28, has been described previously. Genetic analysis revealed that these complementation groups define single nuclear genes, unlinked to one another. One of the three newly identified genes, cdc37, has been located in the yeast linkage map on chromosome IV, two meiotic map units distal to hom2.--Each mutation produces stage-specific arrest of cell division at start, the same point where mating pheromone interrupts division. After synchronization at start by incubation at the restrictive temperature, the mutants retain the capacity to enlarge and to conjugate.

  16. Accumulation of p53 in a mutant cell line defective in the ubiquitin pathway.

    PubMed Central

    Chowdary, D R; Dermody, J J; Jha, K K; Ozer, H L

    1994-01-01

    The wild-type p53 gene product plays an important role in the control of cell proliferation, differentiation, and survival. Altered function is frequently associated with changes in p53 stability. We have studied the role of the ubiquitination pathway in the degradation of p53, utilizing a temperature-sensitive mutant, ts20, derived from the mouse cell line BALB/c 3T3. We found that wild-type p53 accumulates markedly because of decreased breakdown when cells are shifted to the restrictive temperature. Introduction of sequences encoding the human ubiquitin-activating enzyme E1 corrects the temperature sensitivity defect in ts20 and prevents accumulation of p53. The data therefore strongly indicate that wild-type p53 is degraded intracellularly by the ubiquitin-mediated proteolytic pathway. Images PMID:8114731

  17. Viral DNA Synthesis Defects in Assembly-Competent Rous Sarcoma Virus CA Mutants

    PubMed Central

    Cairns, Tina M.; Craven, Rebecca C.

    2001-01-01

    The major structural protein of the retroviral core (CA) contains a conserved sequence motif shared with the CA-like proteins of distantly related transposable elements. The function of this major region of homology (MHR) has not been defined, in part due to the baffling array of phenotypes in mutants of several viruses and the yeast TY3. This report describes new mutations in the CA protein of Rous sarcoma virus (RSV) that were designed to test whether these different phenotypes might indicate distinct functional subdomains in the MHR. A comparison of 25 substitutions at 10 positions in the RSV conserved motif argues against this possibility. Most of the replacements destroyed virus infectivity, although either of two lethal phenotypes was obtained depending on the residue introduced. At most of the positions, one or more replacements (generally the more conservative substitutions) caused a severe replication defect without having any obvious effects on virus assembly, budding, Gag-Pol and genome incorporation, or protein processing. The mutant particles exhibited a defect in endogenous viral DNA synthesis and showed increased sensitivity of the core proteins to detergent, indicating that the mutations interfere with the formation and/or activity of the virion core. The distribution of these mutations across the MHR, with no evidence of clustering, suggests that the entire region is important for a critical postbudding function. In contrast, a second class of lethal substitutions (those that destroyed virus assembly and release) consists of alterations that are expected to cause severe effects on protein structure by disruption either of the hydrophobic core of the CA carboxyl-terminal domain or of the hydrogen bond network that stabilizes the domain. We suggest that this duality of phenotypes is consistent with a role for the MHR in the maturation process that links the two parts of the life cycle. PMID:11119594

  18. The Saccharomyces cerevisiae start mutant carrying the cdc25 mutation is defective in activation of plasma membrane ATPase by glucose.

    PubMed Central

    Portillo, F; Mazón, M J

    1986-01-01

    Activation of plasma membrane ATPase by the addition of glucose was examined in several cell division cycle mutants of Saccharomyces cerevisiae. The start mutant carrying the cdc25 mutation was shown to be defective in ATPase activation at the restrictive temperature. Genetic analysis showed that lack of growth and defective activation of ATPase at the restrictive temperature were caused by the same mutation. It was also found that CDC25 does not map at the same locus as the structural gene of plasma membrane ATPase (PMA1). We conclude that the product of CDC25 controls the activation of ATPase. PMID:2877973

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

    PubMed Central

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

    2014-01-01

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

  20. Overproduction of stomatal lineage cells in Arabidopsis mutants defective in active DNA demethylation

    PubMed Central

    Yamamuro, Chizuko; Miki, Daisuke; Zheng, Zhimin; Ma, Jun; Wang, Jing; Yang, Zhenbiao; Dong, Juan; Zhu, Jian-Kang

    2014-01-01

    DNA methylation is a reversible epigenetic mark regulating genome stability and function in many eukaryotes. In Arabidopsis, active DNA demethylation depends on the function of the ROS1 subfamily of genes that encode 5-methylcytosine DNA glycosylases/lyases. ROS1-mediated DNA demethylation plays a critical role in the regulation of transgenes, transposable elements and some endogenous genes, but there have been no reports of clear developmental phenotypes in ros1 mutant plants. Here we report that, in the ros1 mutant, the promoter region of the peptide ligand gene EPF2 is hypermethylated, which greatly reduces EPF2 expression and thereby leads to a phenotype of overproduction of stomatal lineage cells. EPF2 gene expression in ros1 is restored and the defective epidermal cell patterning is suppressed by mutations in genes in the RNA-directed DNA methylation pathway. Our results show that active DNA demethylation combats the activity of RNA-directed DNA methylation to influence the initiation of stomatal lineage cells. PMID:24898766

  1. Mutant DnaK chaperones cause ribosome assembly defects in Escherichia coli.

    PubMed Central

    Alix, J H; Guérin, M F

    1993-01-01

    To determine whether the biogenesis of ribosomes in Escherichia coli is the result of the self-assembly of their different constituents or involves the participation of additional factors, we have studied the influence of a chaperone, the product of the gene dnaK, on ribosome assembly in vivo. Using three thermosensitive (ts) mutants carrying the mutations dnaK756-ts, dnaK25-ts, and dnaK103-ts, we have observed the accumulation at nonpermissive temperature (45 degrees C) of ribosomal particles with different sedimentation constants--namely, 45S, 35S, and 25S along with the normal 30S and 50S ribosomal subunits. This is the result of a defect not in thermostability but in ribosome assembly at the nonpermissive temperature. These abnormal ribosomal particles are rescued if the mutant cells are returned to 30 degrees C. Thus, the product of the dnaK gene is implicated in ribosome biogenesis at high temperature. PMID:8105482

  2. Maize reas1 Mutant Stimulates Ribosome Use Efficiency and Triggers Distinct Transcriptional and Translational Responses.

    PubMed

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

    2016-02-01

    Ribosome biogenesis is a fundamental cellular process in all cells. Impaired ribosome biogenesis causes developmental defects; however, its molecular and cellular bases are not fully understood. We cloned a gene responsible for a maize (Zea mays) small seed mutant, dek* (for defective kernel), and found that it encodes Ribosome export associated1 (ZmReas1). Reas1 is an AAA-ATPase that controls 60S ribosome export from the nucleus to the cytoplasm after ribosome maturation. dek* is a weak mutant allele with decreased Reas1 function. In dek* cells, mature 60S ribosome subunits are reduced in the nucleus and cytoplasm, but the proportion of actively translating polyribosomes in cytosol is significantly increased. Reduced phosphorylation of eukaryotic initiation factor 2α and the increased elongation factor 1α level indicate an enhancement of general translational efficiency in dek* cells. The mutation also triggers dramatic changes in differentially transcribed genes and differentially translated RNAs. Discrepancy was observed between differentially transcribed genes and differentially translated RNAs, indicating distinct cellular responses at transcription and translation levels to the stress of defective ribosome processing. DNA replication and nucleosome assembly-related gene expression are selectively suppressed at the translational level, resulting in inhibited cell growth and proliferation in dek* cells. This study provides insight into cellular responses due to impaired ribosome biogenesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

  3. ATP utilization by yeast replication factor C. IV. RFC ATP-binding mutants show defects in DNA replication, DNA repair, and checkpoint regulation.

    PubMed

    Schmidt, S L; Pautz, A L; Burgers, P M

    2001-09-14

    Replication factor C is required to load proliferating cell nuclear antigen onto primer-template junctions, using the energy of ATP hydrolysis. Four of the five RFC genes have consensus ATP-binding motifs. To determine the relative importance of these sites for proper DNA metabolism in the cell, the conserved lysine in the Walker A motif of RFC1, RFC2, RFC3, or RFC4 was mutated to either arginine or glutamic acid. Arginine mutations in all RFC genes tested permitted cell growth, although poor growth was observed for rfc2-K71R. A glutamic acid substitution resulted in lethality in RFC2 and RFC3 but not in RFC1 or RFC4. Most double mutants combining mutations in two RFC genes were inviable. Except for the rfc1-K359R and rfc4-K55E mutants, which were phenotypically similar to wild type in every assay, the mutants were sensitive to DNA-damaging agents. The rfc2-K71R and rfc4-K55R mutants show checkpoint defects, most likely in the intra-S phase checkpoint. Regulation of the damage-inducible RNR3 promoter was impaired in these mutants, and phosphorylation of Rad53p in response to DNA damage was specifically defective when cells were in S phase. No dramatic defects in telomere length regulation were detected in the mutants. These data demonstrate that the ATP binding function of RFC2 is important for both DNA replication and checkpoint function and, for the first time, that RFC4 also plays a role in checkpoint regulation.

  4. Neurophysiological Defects and Neuronal Gene Deregulation in Drosophila mir-124 Mutants

    PubMed Central

    Sun, Kailiang; Westholm, Jakub Orzechowski; Tsurudome, Kazuya; Hagen, Joshua W.; Lu, Yubing; Kohwi, Minoree; Betel, Doron; Gao, Fen-Biao; Haghighi, A. Pejmun; Doe, Chris Q.; Lai, Eric C.

    2012-01-01

    miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets. Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes. To provide genetic insight from Drosophila, we deleted its single mir-124 locus and found that it is dispensable for gross aspects of neural specification and differentiation. On the other hand, we detected a variety of mutant phenotypes that were rescuable by a mir-124 genomic transgene, including short lifespan, increased dendrite variation, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals, purified on the basis of mir-124 promoter activity, revealed broad upregulation of direct miR-124 targets. However, in contrast to the proposed mutual exclusion model for miR-124 function, its functional targets were relatively highly expressed in miR-124–expressing cells and were not enriched in genes annotated with epidermal expression. A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons increases synaptic release at the NMJ, similar to mir-124 mutants. Derepression of the direct miR-124 target network also had many secondary effects, including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature. Altogether, these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology. PMID:22347817

  5. General screening procedure for RNA modificationless mutants: isolation of Escherichia coli strains with specific defects in RNA methylation.

    PubMed Central

    Björk, G R; Kjellin-Stråby, K

    1978-01-01

    A general method for the isolation of mutants of Escherichia coli that are defective in RNA modification is described. The method is based on the fact that RNA with specific undermodifications accumulates under nonpermissive growth conditions and that such a defect can be detected by remodification either in vivo at permissive conditions or in vitro. The method provides a means by which to study mutations affecting essential modification reactions. The usefulness of the method was demonstrated by the isolation of two rRNA and two tRNA methylation defective mutants. Both rRNA mutants accept methyl groups into their 23S rRNA in vitro. Analyses of in vitro methylated 23S rRNA from one of the mutants revealed the presence of several methylated nucleosides, of which 6-methyladenosine was the most abundant (40% of recovered radioactivity). In 23S rRNA from the other mutant, the only product formed in vitro was 5-methylcytidine. The tRNA mutants are characterized in the accompanying paper. Images PMID:342494

  6. Novel hydrotropism mutants of Arabidopsis thaliana and their altered waving response and phototropism.

    PubMed

    Takahashi, Akiko; Kobayashi, Akie; Kakimoto, Yoko; Fujii, Nobuharu; Takahashi, Hideyuki

    2003-10-01

    Roots display positive hydrotropism in response to a moisture gradient, which is important for plants to escape from water stress and regulate the directional growth by interacting with other growth movements such as gravitropism, phototropism and waving response. On Earth, hydrotropism is interfered by gravitropism in particular, so that microgravity conditions or agravitropic mutants have been used for the study of hydrotropism. However, we have recently established an experimental system for the study of hydrotropism in Arabidopsis roots that easily develop hydrotropism in response to moisture gradient by overcoming gravitropism. Using the Arabidopsis system, we isolated hydrotropism mutants named root hydrotropism (rhy). In the present study, we examined the hydrotropism, gravitropism, phototropism, waving response and elongation growth of rhy4 and rhy5 roots that were defective in positive hydrotropism. Interestingly, rhy4 roots curved away from the water source and showed a reduced waving response. Both rhy4 and rhy5 showed normal gravitropism and a slight reduction in phototropism. These results suggest that there is a mutual molecular mechanism underlying hydrotropism, waving response and/or phototropism. Thus, we have obtained novel hydrotropic mutants that will be used for revealing molecular mechanism of root hydrotropism and its interaction with waving response and/or phototropism.

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

    PubMed

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

    2009-12-01

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

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

    PubMed

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

    2015-09-01

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

  9. Developmental Genetics of Chromosome I Spermatogenesis-Defective Mutants in the Nematode Caenorhabditis Elegans

    PubMed Central

    L'Hernault, S. W.; Shakes, D. C.; Ward, S.

    1988-01-01

    Mutations affecting Caenorhabditis elegans spermatogenesis can be used to dissect the processes of meiosis and spermatozoan morphological maturation. We have obtained 23 new chromosome I mutations that affect spermatogenesis (spe mutations). These mutations, together with six previously described mutations, identify 11 complementation groups, of which six are defined by multiple alleles. These spe mutations are all recessive and cause normally self-fertile hermaphrodites to produce unfertilized oocytes that can be fertilized by wild-type male sperm. Five chromosome I mutation/deficiency heterozygotes have similar phenotypes to the homozygote showing that the probable null phenotype of these genes is defective sperm. Spermatogenesis is disrupted at different steps by mutations in these genes. The maturation of 1° spermatocytes is disrupted by mutations in spe-4 and spe-5. Spermatids from spe-8 and spe-12 mutants develop into normal spermatozoa in males, but not in hermaphrodites. fer-6 spermatids are abnormal, and fer-1 spermatids look normal but subsequently become abnormal spermatozoa. Mutations in five genes (fer-7, spe-9, spe-11, spe-13 and spe-15) allow formation of normal looking motile spermatozoa that appear to be defective in either sperm-spermathecal or sperm-oocyte interactions. PMID:3197956

  10. Respiratory chain supercomplexes set the threshold for respiration defects in human mtDNA mutant cybrids.

    PubMed

    D'Aurelio, Marilena; Gajewski, Carl D; Lenaz, Giorgio; Manfredi, Giovanni

    2006-07-01

    Mitochondrial DNA (mtDNA) mutations cause heterogeneous disorders in humans. MtDNA exists in multiple copies per cell, and mutations need to accumulate beyond a critical threshold to cause disease, because coexisting wild-type mtDNA can complement the genetic defect. A better understanding of the molecular determinants of functional complementation among mtDNA molecules could help us shedding some light on the mechanisms modulating the phenotypic expression of mtDNA mutations in mitochondrial diseases. We studied mtDNA complementation in human cells by fusing two cell lines, one containing a homoplasmic mutation in a subunit of respiratory chain complex IV, COX I, and the other a distinct homoplasmic mutation in a subunit of complex III, cytochrome b. Upon cell fusion, respiration is recovered in hybrids cells, indicating that mitochondria fuse and exchange genetic and protein materials. Mitochondrial functional complementation occurs frequently, but with variable efficiency. We have investigated by native gel electrophoresis the molecular organization of the mitochondrial respiratory chain in complementing hybrid cells. We show that the recovery of mitochondrial respiration correlates with the presence of supramolecular structures (supercomplexes) containing complexes I, III and IV. We suggest that critical amounts of complexes III or IV are required in order for supercomplexes to form and provide mitochondrial functional complementation. From these findings, supercomplex assembly emerges as a necessary step for respiration, and its defect sets the threshold for respiratory impairment in mtDNA mutant cells.

  11. Identification of genes involved in fungal responses to strigolactones using mutants from fungal pathogens.

    PubMed

    Belmondo, S; Marschall, R; Tudzynski, P; López Ráez, J A; Artuso, E; Prandi, C; Lanfranco, L

    2016-06-28

    Strigolactones (SLs) as components of root exudates induce hyphal branching of arbuscular mycorrhizal (AM) fungi which is thought to favor the establishment of the beneficial symbiosis. Little is known on how AM fungi respond to SLs. Since AM fungi are poor model systems due to their obligate biotrophism and the lack of genetic transformation protocols, we took advantage of the sensitivity of several phytopathogenic fungi to GR24, a synthetic SLs analog. With the aim to identify the molecular determinants involved in SLs response in AM fungi and assuming conserved mechanisms in the fungal kingdom, we exploited the fungal pathogens Botrytis cinerea and Cryphonectria parasitica, for which mutant collections are available. Exposure of B. cinerea and C. parasitica to GR24 embedded in solid medium led to reduction of fungal radial growth. We set up the screening of a set of well-characterized gene deletion mutants to isolate genotypes with altered responses to SLs. Two B. cinerea mutants (defective of BcTrr1, a thioredoxin reductase and BcLTF1, a GATA transcription factor) turned out to be less responsive to GR24. One feature shared by the two mutants is the overproduction of reactive oxygen species (ROS). Indeed, an oxidizing effect was observed in a B. cinerea strain expressing a redox-sensitive GFP2 in the mitochondrial intermembrane space upon exposure to GR24. ROS and mitochondria are, therefore, emerging as mediators of SLs actions.

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

    PubMed Central

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

    1990-01-01

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

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

    PubMed

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

    2011-01-01

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

  14. Analysis of voltage-gated and synaptic conductances contributing to network excitability defects in the mutant mouse tottering.

    PubMed

    Helekar, S A; Noebels, J L

    1994-01-01

    1. Intracellular current- and voltage-clamp recordings were carried out in CA3 pyramidal neurons from hippocampal slices of adult tg/tg mice and their coisogenic C57BL/6J (+/+) controls with the use of the single-electrode switch-clamp technique. The principal aim of this study was to investigate the mechanisms responsible for the tg gene-linked prolongation (mean 60%) of a giant synaptic response, the potassium-induced paroxysmal depolarizing shift (PDS) at depolarized membrane potentials (Vm -47 to -54 mV) during synchronous network bursting induced by 10 mM potassium ([K+]o). 2. To examine the role of intrinsic voltage-dependent conductances underlying the mutant PDS prolongation, neurons were voltage clamped by the use of microelectrodes filled with 100 mM QX-314 or QX-222 chloride (voltage-gated sodium channel blockers) and 2 M cesium sulphate (potassium channel blocker). The whole-cell currents active during the PDS showed a significantly prolonged duration (mean 34%) at depolarized Vms in tg/tg compared with +/+ cells, indicating that a defect in voltage-dependent conductances is unlikely to completely account for the mutant phenotype. 3. Bath application of 40 microM (DL)-2-aminophosphonovalerate (DL-APV) produced a 30% reduction in PDS duration in both genotypes but failed to significantly alter the tg gene-linked prolongation compared with the wild type. These data indicate that the mutant PDS abnormality does not result from a selective increase of the N-methyl-D-aspartate (NMDA) receptor-mediated excitatory synaptic component. 4. Blockade of gamma-aminobutyric acid-A (GABAA) transmission with picrotoxin (50 microM) or bicuculline (1-5 microM) completely eliminated the difference in PDS duration between the genotypes. Furthermore, although both GABAA receptor antagonists increased the mean PDS duration in +/+ neurons, they did not significantly alter it in tg/tg neurons. These findings are consistent with a reduction in GABAA receptor-mediated synaptic

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

    PubMed

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

    1999-07-15

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

  16. A novel stereocilia defect in sensory hair cells of the deaf mouse mutant Tasmanian devil.

    PubMed

    Erven, Alexandra; Skynner, Michael J; Okumura, Katsuzumi; Takebayashi, Shin-ichiro; Brown, Steve D M; Steel, Karen P; Allen, Nicholas D

    2002-10-01

    Stereocilia are specialized actin-filled, finger-like processes arrayed in rows of graded heights to form a crescent or W-shape on the apical surface of sensory hair cells. The stereocilia are deflected by the vibration of sound, which opens transduction channels and allows an influx of ions to depolarize the hair cell, in turn triggering synaptic activity. The specialized morphology and organization of the stereocilia bundle is crucial in the process of sensory transduction in the inner ear. However, we know little about the development of stereocilia in the mouse and few molecules that are involved in stereocilia maturation are known. We describe here a new mouse mutant with abnormal stereocilia development. The Tasmanian devil (tde) mouse mutation arose by insertional mutagenesis and has been mapped to the middle of chromosome 5. Homozygotes show head-tossing and circling and have raised thresholds for cochlear nerve responses to sound. The gross morphology of the inner ear was normal, but the stereocilia of cochlear and vestibular hair cells are abnormally thin, and they become progressively disorganized with increasing age. Ultimately, the hair cells die. This is the first report of a mutant showing thin stereocilia. The association of thin stereocilia with cochlear dysfunction emphasizes the critical role of stereocilia in auditory transduction, and the discovery of the Tasmanian devil mutant provides a resource for the identification of an essential molecule in hair cell function.

  17. Transcriptomic analysis of swarm motility phenotype of Salmonella enterica serovar Typhimurium mutant defective in periplasmic glucan synthesis

    USDA-ARS?s Scientific Manuscript database

    Movement of food-borne pathogens on moist surfaces enables them to migrate towards more favorable niches and facilitate their survival for extended periods of time. Salmonella enterica serovar Typhimurium mutants defective in OPG synthesis are unable to exhibit motility on moist surfaces (swarming) ...

  18. Cancer-Associated Mutants of RNA Helicase DDX3X Are Defective in RNA-Stimulated ATP Hydrolysis

    DOE PAGES

    Epling, Leslie B.; Grace, Christy R.; Lowe, Brandon R.; ...

    2015-02-25

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

  19. Increased phagocytosis of Mycobacterium marinum mutants defective in lipooligosaccharide production: a structure-activity relationship study.

    PubMed

    Alibaud, Laeticia; Pawelczyk, Jakub; Gannoun-Zaki, Laila; Singh, Vipul K; Rombouts, Yoann; Drancourt, Michel; Dziadek, Jaroslaw; Guérardel, Yann; Kremer, Laurent

    2014-01-03

    Mycobacterium marinum is a waterborne pathogen responsible for tuberculosis-like infections in ectotherms and is an occasional opportunistic human pathogen. In the environment, M. marinum also interacts with amoebae, which may serve as a natural reservoir for this microorganism. However, the description of mycobacterial determinants in the early interaction with macrophages or amoebae remains elusive. Lipooligosaccharides (LOSs) are cell surface-exposed glycolipids capable of modulating the host immune system, suggesting that they may be involved in the early interactions of M. marinum with macrophages. Herein, we addressed whether LOS composition affects the uptake of M. marinum by professional phagocytes. Mutants with various truncated LOS variants were generated, leading to the identification of several previously uncharacterized biosynthetic genes (wbbL2, MMAR_2321, and MMAR_2331). Biochemical and structural approaches allowed resolving the structures of LOS precursors accumulating in this set of mutants. These strains with structurally defined LOS profiles were then used to infect both macrophages and Acanthamoebae. An inverse correlation between LOS completeness and uptake of mycobacteria by phagocytes was found, allowing the proposal of three mutant classes: class I (papA4), devoid of LOS and highly efficiently phagocytosed; class II, accumulating only early LOS intermediates (wbbL2 and MMAR_2331) and efficiently phagocytosed but less than class I mutants; class III, lacking LOS-IV (losA, MMAR_2319, and MMAR_2321) and phagocytosed similarly to the control strain. These results indicate that phagocytosis is conditioned by the LOS pattern and that the LOS pathway used by M. marinum in macrophages is conserved during infection of amoebae.

  20. The Arabidopsis pxa1 Mutant Is Defective in an ATP-Binding Cassette Transporter-Like Protein Required for Peroxisomal Fatty Acid β-Oxidation1

    PubMed Central

    Zolman, Bethany K.; Silva, Illeana D.; Bartel, Bonnie

    2001-01-01

    Peroxisomes are important organelles in plant metabolism, containing all the enzymes required for fatty acid β-oxidation. More than 20 proteins are required for peroxisomal biogenesis and maintenance. The Arabidopsis pxa1 mutant, originally isolated because it is resistant to the auxin indole-3-butyric acid (IBA), developmentally arrests when germinated without supplemental sucrose, suggesting defects in fatty acid β-oxidation. Because IBA is converted to the more abundant auxin, indole-3-acetic acid (IAA), in a mechanism that parallels β-oxidation, the mutant is likely to be IBA resistant because it cannot convert IBA to IAA. Adult pxa1 plants grow slowly compared with wild type, with smaller rosettes, fewer leaves, and shorter inflorescence stems, indicating that PXA1 is important throughout development. We identified the molecular defect in pxa1 using a map-based positional approach. PXA1 encodes a predicted peroxisomal ATP-binding cassette transporter that is 42% identical to the human adrenoleukodystrophy (ALD) protein, which is defective in patients with the demyelinating disorder X-linked ALD. Homology to ALD protein and other human and yeast peroxisomal transporters suggests that PXA1 imports coenzyme A esters of fatty acids and IBA into the peroxisome for β-oxidation. The pxa1 mutant makes fewer lateral roots than wild type, both in response to IBA and without exogenous hormones, suggesting that the IAA derived from IBA during seedling development promotes lateral root formation. PMID:11706205

  1. Glucose-induced regulatory defects in the Saccharomyces cerevisiae byp1 growth initiation mutant and identification of MIG1 as a partial suppressor.

    PubMed Central

    Hohmann, S; Huse, K; Valentin, E; Mbonyi, K; Thevelein, J M; Zimmermann, F K

    1992-01-01

    Saccharomyces cerevisiae byp1-3 mutants displayed a long lag phase when shifted from a nonfermentable carbon source to a medium containing glucose. The byp1-3 mutation also caused several defects in regulatory phenomena which occur during the transition from the derepressed state to the repressed state. As opposed to wild-type cells, the addition of glucose to cells of the byp1-3 mutant grown on nonfermentable carbon sources did not induce a cyclic AMP signal. Fructose-2,6-bisphosphate formation and inactivation of fructose-1,6-bisphosphatase were severely delayed, but trehalase activation was not affected. In addition, the induction of pyruvate decarboxylase both at the level of activity and that of transcription was very slow compared with that in wild-type cells. These pleotropic defects in glucose-induced regulatory phenomena might be responsible for the very long lag phase of byp1-3 cells and the inability of ascospores to initiate growth after germination on glucose media. Screening of a yeast gene library for clones complementing the byp1-3 phenotype resulted in the isolation of a truncated form of the previously described zinc finger transcription repressor MIG1. The entire MIG1 gene and the truncated form suppressed even on a single-copy vector the growth initiation defect but not the regulatory abnormalities of the byp1-3 mutant. MIG1 is not allelic to byp1-3. Images PMID:1597433

  2. A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase.

    PubMed

    Kojima, Kouji; Keta, Sumie; Uesaka, Kazuma; Kato, Akihiro; Takatani, Nobuyuki; Ihara, Kunio; Omata, Tatsuo; Aichi, Makiko

    2016-12-01

    Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.

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

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

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

  5. Defective co-activator recruitment in osteoclasts from microphthalmia-oak ridge mutant mice.

    PubMed

    Sharma, Sudarshana M; Sif, Said; Ostrowski, Michael C; Sankar, Uma

    2009-07-01

    The three basic DNA-binding domain mutations of the microphthalmia-associated transcription factor (Mitf), Mitf(mi/mi), Mitf(or/or), and Mitf(wh/wh) affect osteoclast differentiation with variable penetrance while completely impairing melanocyte development. Mitf(or/or) mice exhibit osteopetrosis that improves with age and their osteoclasts form functional multinuclear osteoclasts, raising the question as to why the Mitf(or/or) mutation results in osteopetrosis. Here we show that Mitf(or/or) osteoclasts express normal levels of acid phosphatase 5 (Acp5) mRNA and significantly lower levels of Cathepsin K (Ctsk) mRNA during receptor activator of nuclear factor kappa B (NFkappaB) ligand (RANKL)-mediated differentiation. Studies using chromatin immunoprecipitation (ChIP) analysis indicate that low levels of Mitf(or/or) protein are recruited to the Ctsk promoter. However, enrichment of Mitf-transcriptional co-activators PU.1 and Brahma-related gene 1 (Brg1) are severely impaired at the Ctsk promoter of Mitf(or/or) osteoclast precursors, indicating that defective recruitment of co-activators by the mutant Mitf(or/or) results in impaired Ctsk expression in osteoclasts. Cathepsin K may thus represent a unique class of Mitf-regulated osteoclast-specific genes that are important for osteoclast function.

  6. Association between congenital defects in papillary outgrowth and functional obstruction in Crim1 mutant mice.

    PubMed

    Wilkinson, Lorine; Kurniawan, Nyoman D; Phua, Yu Leng; Nguyen, Michael J; Li, Joan; Galloway, Graham J; Hashitani, Hikaru; Lang, Richard J; Little, Melissa H

    2012-08-01

    Crim1 hypomorphic (Crim1(KST264/KST264)) mice display progressive renal disease characterized by glomerular defects, leaky peritubular vasculature, and progressive interstitial fibrosis. Here we show that 27% of these mice also present with hydronephrosis, suggesting obstructive nephropathy. Dynamic magnetic resonance imaging using Magnevist showed fast development of hypo-intense signal in the kidneys of Crim1(KST264/KST264) mice, suggesting pooling of filtrate within the renal parenchyma. Rhodamine dextran (10 kDa) clearance was also delayed in Crim1(KST264/KST264) mice. Pyeloureteric peristalsis, while present, was less co-ordinated in Crim1(KST264/KST264) mice. However, isolated renal pelvis preparations suggest normal pelvic smooth muscle contractile responses. An analysis of maturation during the immediate postnatal period [postnatal day (P) 0-15] revealed defects in papillary extension in Crim1({KST264/KST264) mice. While Crim1 expression is weak in pelvic smooth muscle, strong expression is seen in the interstitium and loops of Henle of the extending papilla, commencing at the tip of the P1 papilla and disseminating throughout the papilla by P15. These results, as well as implicating Crim1 in papillary extension and pelvic smooth muscle contractility, highlight the previously unrecognized association between defects in papillary development and progression to chronic kidney disease later in life.

  7. A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

    PubMed Central

    Durack, Juliana; Burke, Thomas P.

    2014-01-01

    The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis. PMID:25535272

  8. Hypersensitivity to mutation and sister-chromatid-exchange induction in CHO cell mutants defective in incising DNA containing UV lesions

    SciTech Connect

    Thompson, L.H.; Brookman, K.W.; Dillehay, L.E.; Mooney, C.L.; Carrano, A.V.

    1982-01-01

    Five UV-sensitive mutant strains of CHO cells representing different genetic complementation groups were analyzed for their ability to perform the incision step of nucleotide excision repair after UV exposure. The assay utilized inhibitors of DNA synthesis to accumulate the short-lived strand breaks resulting from repair incisions. After 6 J/m/sup 2/, each of the mutants showed < 10% of the incision rate of the parental AA8 cells. After 50 J/m/sup 2/, the rate in AA8 was similar to that at 6 J/m/sup 2/, but the rates in the mutants were significantly higher (approx. 20% of the rate of AA8). Thus by this incision assay the mutants were phenotypically indistinguishable. Each of the mutants were hypersensitive to mutation induction at both the hprt and aprt loci by a factor of 10, and in the one strain tested ouabain resistance was induced sevenfold more efficiently than in AA8 cells. Sister chromatid exchange was also induced with sevenfold increased efficiency in the two mutant strains examined. Thus, here CHO mutants resemble xeroderma pigmentosum cells in terms of their incision defects and their hypersensitivity to DNA damage by UV.

  9. 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.

  10. Mutants for Drosophila Isocitrate Dehydrogenase 3b Are Defective in Mitochondrial Function and Larval Cell Death.

    PubMed

    Duncan, Dianne M; Kiefel, Paula; Duncan, Ian

    2017-03-10

    The death of larval salivary gland cells during metamorphosis in Drosophila melanogaster has been a key system for studying steroid controlled programmed cell death. This death is induced by a pulse of the steroid hormone ecdysone that takes place at the end of the prepupal period. For many years, it has been thought that the ecdysone direct response gene Eip93F (E93) plays a critical role in initiating salivary gland cell death. This conclusion was based largely on the finding that the three "type" alleles of E93 cause a near-complete block in salivary gland cell death. Here, we show that these three mutations are in fact allelic to Idh3b, a nearby gene that encodes the β subunit of isocitrate dehydrogenase 3, a mitochondrial enzyme of the tricarboxylic acid (TCA) cycle. The strongest of the Idh3b alleles appears to cause a near-complete block in oxidative phosphorylation, as mitochondria are depolarized in mutant larvae, and development arrests early during cleavage in embryos from homozygous-mutant germline mothers. Idh3b-mutant larval salivary gland cells fail to undergo mitochondrial fragmentation, which normally precedes the death of these cells, and do not initiate autophagy, an early step in the cell death program. These observations suggest a close relationship between the TCA cycle and the initiation of larval cell death. In normal development, tagged Idh3b is released from salivary gland mitochondria during their fragmentation, suggesting that Idh3b may be an apoptogenic factor that functions much like released cytochrome c in mammalian cells.

  11. Mutants for Drosophila Isocitrate Dehydrogenase 3b Are Defective in Mitochondrial Function and Larval Cell Death

    PubMed Central

    Duncan, Dianne M.; Kiefel, Paula; Duncan, Ian

    2017-01-01

    The death of larval salivary gland cells during metamorphosis in Drosophila melanogaster has been a key system for studying steroid controlled programmed cell death. This death is induced by a pulse of the steroid hormone ecdysone that takes place at the end of the prepupal period. For many years, it has been thought that the ecdysone direct response gene Eip93F (E93) plays a critical role in initiating salivary gland cell death. This conclusion was based largely on the finding that the three “type” alleles of E93 cause a near-complete block in salivary gland cell death. Here, we show that these three mutations are in fact allelic to Idh3b, a nearby gene that encodes the β subunit of isocitrate dehydrogenase 3, a mitochondrial enzyme of the tricarboxylic acid (TCA) cycle. The strongest of the Idh3b alleles appears to cause a near-complete block in oxidative phosphorylation, as mitochondria are depolarized in mutant larvae, and development arrests early during cleavage in embryos from homozygous-mutant germline mothers. Idh3b-mutant larval salivary gland cells fail to undergo mitochondrial fragmentation, which normally precedes the death of these cells, and do not initiate autophagy, an early step in the cell death program. These observations suggest a close relationship between the TCA cycle and the initiation of larval cell death. In normal development, tagged Idh3b is released from salivary gland mitochondria during their fragmentation, suggesting that Idh3b may be an apoptogenic factor that functions much like released cytochrome c in mammalian cells. PMID:28104670

  12. Defective Peripheral Nerve Development Is Linked to Abnormal Architecture and Metabolic Activity of Adipose Tissue in Nscl-2 Mutant Mice

    PubMed Central

    Ruschke, Karen; Ebelt, Henning; Klöting, Nora; Boettger, Thomas; Raum, Kay; Blüher, Matthias; Braun, Thomas

    2009-01-01

    Background In mammals the interplay between the peripheral nervous system (PNS) and adipose tissue is widely unexplored. We have employed mice, which develop an adult onset of obesity due to the lack the neuronal specific transcription factor Nscl-2 to investigate the interplay between the nervous system and white adipose tissue (WAT). Methodology Changes in the architecture and innervation of WAT were compared between wildtype, Nscl2−/−, ob/ob and Nscl2−/−//ob/ob mice using morphological methods, immunohistochemistry and flow cytometry. Metabolic alterations in mutant mice and in isolated cells were investigated under basal and stimulated conditions. Principal Findings We found that Nscl-2 mutant mice show a massive reduction of innervation of white epididymal and paired subcutaneous inguinal fat tissue including sensory and autonomic nerves as demonstrated by peripherin and neurofilament staining. Reduction of innervation went along with defects in the formation of the microvasculature, accumulation of cells of the macrophage/preadipocyte lineage, a bimodal distribution of the size of fat cells, and metabolic defects of isolated adipocytes. Despite a relative insulin resistance of white adipose tissue and isolated Nscl-2 mutant adipocytes the serum level of insulin in Nscl-2 mutant mice was only slightly increased. Conclusions We conclude that the reduction of the innervation and vascularization of WAT in Nscl-2 mutant mice leads to the increase of preadipocyte/macrophage-like cells, a bimodal distribution of the size of adipocytes in WAT and an altered metabolic activity of adipocytes. PMID:19436734

  13. Fusion-defective mutants of mouse hepatitis virus A59 contain a mutation in the spike protein cleavage signal.

    PubMed Central

    Gombold, J L; Hingley, S T; Weiss, S R

    1993-01-01

    Infection of primary mouse glial cell cultures with mouse hepatitis virus strain A59 results in a productive, persistent infection, but without any obvious cytopathic effect. Mutant viruses isolated from infected glial cultures 16 to 18 weeks postinfection replicate with kinetics similar to those of wild-type virus but produce small plaques on fibroblasts and cause only minimal levels of cell-to-cell fusion under conditions in which wild type causes nearly complete cell fusion. However, since extensive fusion is present in mutant-infected cells at late times postinfection, the defect is actually a delay in kinetics rather than an absolute block in activity. Addition of trypsin to mutant-infected fibroblast cultures enhanced cell fusion a small (two- to fivefold) but significant degree, indicating that the defect could be due to a lack of cleavage of the viral spike (fusion) protein. Sequencing of portions of the spike genes of six fusion-defective mutants revealed that all contained the same single nucleotide mutation resulting in a substitution of aspartic acid for histidine in the spike cleavage signal. Mutant virions contained only the 180-kDa form of spike protein, suggesting that this mutation prevented the normal proteolytic cleavage of the 180-kDa protein into the 90-kDa subunits. Examination of revertants of the mutants supports this hypothesis. Acquisition of fusion competence correlates with the replacement of the negatively charged aspartic acid with either the wild-type histidine or a nonpolar amino acid and the restoration of spike protein cleavage. These data confirm and extend previous reports concluding cleavage of S is required for efficient cell-cell fusion by mouse hepatitis virus but not for virus-cell fusion (infectivity). Images PMID:8392595

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

    PubMed

    Wright, David J; Tate, Christopher G

    2015-10-01

    The tetracycline antiporter TetA(B) is a member of the Major Facilitator Superfamily which confers tetracycline resistance to cells by coupling the efflux of tetracycline to the influx of protons down their chemical potential gradient. Although it is a medically important transporter, its structure has yet to be determined. One possibility for why this has proven difficult is that the transporter may be conformationally heterogeneous in the purified state. To overcome this, we developed two strategies to rapidly identify TetA(B) mutants that were transport-defective and that could still bind tetracycline. Up to 9 amino acid residues could be deleted from the loop between transmembrane α-helices 6 and 7 with only a slight decrease in affinity of tetracycline binding as measured by isothermal titration calorimetry, although the mutant was transport-defective. Scanning mutagenesis where all the residues between 2 and 389 were mutated to either valine, alanine or glycine (VAG scan) identified 15 mutants that were significantly impaired in tetracycline transport. Of these mutants, 12 showed no evidence of tetracycline binding by isothermal titration calorimetry performed on the purified transporters. In contrast, the mutants G44V and G346V bound tetracycline 4-5 fold more weakly than TetA(B), with Kds of 28 μM and 36 μM, respectively, whereas the mutant R70G bound tetracycline 3-fold more strongly (Kd 2.1 μM). Systematic mutagenesis is thus an effective strategy for isolating transporter mutants that may be conformationally constrained and which represent attractive targets for crystallisation and structure determination. Copyright © 2015. Published by Elsevier B.V.

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

    PubMed Central

    Wright, David J.; Tate, Christopher G.

    2015-01-01

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

  16. Mutant huntingtin, abnormal mitochondrial dynamics, defective axonal transport of mitochondria, and selective synaptic degeneration in Huntington's disease.

    PubMed

    Reddy, P Hemachandra; Shirendeb, Ulziibat P

    2012-02-01

    Huntington's disease (HD) is a progressive, fatal neurodegenerative disease caused by expanded polyglutamine repeats in the HD gene. HD is characterized by chorea, seizures, involuntary movements, dystonia, cognitive decline, intellectual impairment and emotional disturbances. Research into mutant huntingtin (Htt) and mitochondria has found that mutant Htt interacts with the mitochondrial protein dynamin-related protein 1 (Drp1), enhances GTPase Drp1 enzymatic activity, and causes excessive mitochondrial fragmentation and abnormal distribution, leading to defective axonal transport of mitochondria and selective synaptic degeneration. This article summarizes latest developments in HD research and focuses on the role of abnormal mitochondrial dynamics and defective axonal transport in HD neurons. This article also discusses the therapeutic strategies that decrease mitochondrial fragmentation and neuronal damage in HD.

  17. Valosin-containing protein (VCP/p97) inhibitors relieve Mitofusin-dependent mitochondrial defects due to VCP disease mutants.

    PubMed

    Zhang, Ting; Mishra, Prashant; Hay, Bruce A; Chan, David; Guo, Ming

    2017-03-21

    Missense mutations of valosin-containing protein (VCP) cause an autosomal dominant disease known as inclusion body myopathy, Paget disease with frontotemporal dementia (IBMPFD) and other neurodegenerative disorders. The pathological mechanism of IBMPFD is not clear and there is no treatment. We show that endogenous VCP negatively regulates Mitofusin, which is required for outer mitochondrial membrane fusion. Because 90% of IBMPFD patients have myopathy, we generated an in vivo IBMPFD model in adult Drosophila muscle, which recapitulates disease pathologies. We show that common VCP disease mutants act as hyperactive alleles with respect to regulation of Mitofusin. Importantly, VCP inhibitors suppress mitochondrial defects, muscle tissue damage and cell death associated with IBMPFD models in Drosophila. These inhibitors also suppress mitochondrial fusion and respiratory defects in IBMPFD patient fibroblasts. These results suggest that VCP disease mutants cause IBMPFD through a gain-of-function mechanism, and that VCP inhibitors have therapeutic value.

  18. Properties of bacteriophage T4 mutants defective in gene 30 (deoxyribonucleic acid ligase) and the rII gene.

    PubMed

    Karam, J D; Barker, B

    1971-02-01

    In Escherichia coli K-12 strains infected with phage T4 which is defective in gene 30 [deoxyribonucleic acid (DNA) ligase] and in the rII gene (product unknown), near normal levels of DNA and viable phage were produced. Growth of such T4 ligase-rII double mutants was less efficient in E. coli B strains which show the "rapidlysis" phenotype of rII mutations. In pulse-chase experiments coupled with temperature shifts and with inhibition of DNA synthesis, it was observed that DNA synthesized by gene 30-defective phage is more susceptible to breakdown in vivo when the phage is carrying a wild-type rII gene. Breakdown was delayed or inhibited by continued DNA synthesis. Mutations of the rII gene decreased but did not completely abolish the breakdown. T4 ligase-rII double mutants had normal sensitivity to ultraviolet irradiation.

  19. Wing Defects in Drosophila xenicid Mutant Clones Are Caused by C-Terminal Deletion of Additional Sex Combs (Asx)

    PubMed Central

    Bischoff, Kara; Ballew, Anna C.; Simon, Michael A.; O'Reilly, Alana M.

    2009-01-01

    Background The coordinated action of genes that control patterning, cell fate determination, cell size, and cell adhesion is required for proper wing formation in Drosophila. Defects in any of these basic processes can lead to wing aberrations, including blisters. The xenicid mutation was originally identified in a screen designed to uncover regulators of adhesion between wing surfaces [1]. Principal Findings Here, we demonstrate that expression of the βPS integrin or the patterning protein Engrailed are not affected in developing wing imaginal discs in xenicid mutants. Instead, expression of the homeotic protein Ultrabithorax (Ubx) is strongly increased in xenicid mutant cells. Conclusion Our results suggest that upregulation of Ubx transforms cells from a wing blade fate to a haltere fate, and that the presence of haltere cells within the wing blade is the primary defect leading to the adult wing phenotypes observed. PMID:19956620

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  3. Isolation and partial characterization of Rhodopseudomonas sphaeroides mutants defective in the regulation of ribulose bisphosphate carboxylase/oxygenase.

    PubMed

    Weaver, K E; Tabita, F R

    1983-11-01

    Several mutants of Rhodopseudomonas sphaeroides defective in the derepression of the enzyme ribulose 1,5-bisphosphate carboxylase have been isolated by using the unstable Tn5 vectors pJB4JI and pRK340. Transpositional insertion mutants obtained with pJB4JI were demonstrated to be incapable of increasing ribulose 1,5-bisphosphate carboxylase/oxygenase levels when grown on butyrate-bicarbonate medium or under conditions of carbon starvation, whereas the wild-type strain increased activity four- to eightfold. When the wild-type strain was starved for carbon in the presence of chloramphenicol, no derepression was observed. Crude extracts from mutant and wild-type strains had distinct and consistent differences in protein content as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chromatographic evidence indicated that mutants were defective in the regulation of only one of the two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase synthesized by R. sphaeroides.

  4. Sodium Orthovanadate-Resistant Mutants of Saccharomyces Cerevisiae Show Defects in Golgi-Mediated Protein Glycosylation, Sporulation and Detergent Resistance

    PubMed Central

    Kanik-Ennulat, C.; Montalvo, E.; Neff, N.

    1995-01-01

    Orthovanadate is a small toxic molecule that competes with the biologically important oxyanion orthophosphate. Orthovanadate resistance arises spontaneously in Saccharomyces cerevisiae haploid cells by mutation in a number of genes. Mutations selected at 3 mM sodium orthovanadate have different degrees of vanadate resistance, hygromycin sensitivity, detergent sensitivity and sporulation defects. Recessive vanadate-resistant mutants belong to at least six genetic loci. Most mutants are defective in outer chain glycosylation of secreted invertase (van1, van2, van4, van5, van6, VAN7-116 and others), a phenotype found in some MNN or VRG mutants. The phenotypes of these vanadate-resistant mutants are consistent with an alteration in the permeability or specificity of the Golgi apparatus. The previously published VAN1 gene product has a 200 amino acid domain with 40% identity with the MNN9 gene product and 70% identity with the ANP1 gene product. Cells containing the van1-18, mnn9 (vrg6) or anp1 mutations have some phenotypic similarities. The VAN2 gene was isolated and its coding region is identified and reported. It is an essential gene on chromosome XV and its translated amino acid sequence predicts a unique 337 amino acid protein with multiple transmembrane domains. PMID:7672592

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

    PubMed Central

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

    2008-01-01

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

  6. Mutants of PC12 cells with altered cyclic AMP responses

    SciTech Connect

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

    1984-10-01

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

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

    SciTech Connect

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

    1984-02-01

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

  8. A Medicago truncatula Tobacco Retrotransposon Insertion Mutant Collection with Defects in Nodule Development and Symbiotic Nitrogen Fixation1[W][OA

    PubMed Central

    Pislariu, Catalina I.; D. Murray, Jeremy; Wen, JiangQi; Cosson, Viviane; Muni, RajaSekhara Reddy Duvvuru; Wang, Mingyi; A. Benedito, Vagner; Andriankaja, Andry; Cheng, Xiaofei; Jerez, Ivone Torres; Mondy, Samuel; Zhang, Shulan; Taylor, Mark E.; Tadege, Million; Ratet, Pascal; Mysore, Kirankumar S.; Chen, Rujin; Udvardi, Michael K.

    2012-01-01

    A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation. Primary screening of 9,300 mutant lines yielded 317 lines with putative defects in nodule development and/or nitrogen fixation. Of these, 230 lines were rescreened, and 156 lines were confirmed with defective symbiotic nitrogen fixation. Mutants were sorted into six distinct phenotypic categories: 72 nonnodulating mutants (Nod−), 51 mutants with totally ineffective nodules (Nod+ Fix−), 17 mutants with partially ineffective nodules (Nod+ Fix+/−), 27 mutants defective in nodule emergence, elongation, and nitrogen fixation (Nod+/− Fix−), one mutant with delayed and reduced nodulation but effective in nitrogen fixation (dNod+/− Fix+), and 11 supernodulating mutants (Nod++Fix+/−). A total of 2,801 flanking sequence tags were generated from the 156 symbiotic mutant lines. Analysis of flanking sequence tags revealed 14 insertion alleles of the following known symbiotic genes: NODULE INCEPTION (NIN), DOESN’T MAKE INFECTIONS3 (DMI3/CCaMK), ERF REQUIRED FOR NODULATION, and SUPERNUMERARY NODULES (SUNN). In parallel, a polymerase chain reaction-based strategy was used to identify Tnt1 insertions in known symbiotic genes, which revealed 25 additional insertion alleles in the following genes: DMI1, DMI2, DMI3, NIN, NODULATION SIGNALING PATHWAY1 (NSP1), NSP2, SUNN, and SICKLE. Thirty-nine Nod− lines were also screened for arbuscular mycorrhizal symbiosis phenotypes, and 30 mutants exhibited defects in arbuscular mycorrhizal symbiosis. Morphological and developmental features of several new symbiotic mutants are reported. The collection of mutants described here is a source of novel alleles of known symbiotic genes and a resource for cloning novel symbiotic genes via Tnt1 tagging. PMID:22679222

  9. Promoting cooperation through fast response to defection in spatial games

    NASA Astrophysics Data System (ADS)

    Wang, Xu-Wen; Jiang, Luo-Luo; Nie, Sen; Chen, Shi-Ming; Wang, Bing-Hong

    2016-10-01

    Recent experimental research has revealed that the cooperation in dynamic social networks, has significant scope for enhancement because individuals in a social system break the links with defective neighbours. To investigate how the length of defection tolerance affects the cooperation of prisoner’s dilemma game in dynamic ring networks, we study evolution of breaking and rewiring operations for social interaction as a response to the defection strategy. Defection tolerance is measured in terms of the time length that an individual tolerates a defector who continuously adopts the defective strategy. The results show that the dynamic nature of human social networks plays an essential role in promoting cooperation. Interestingly, there exists a critical value of the temptation to defect, below which the system is entirely dominated by cooperators, and a lower value of defection tolerance induces a larger threshold of temptation.

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

    SciTech Connect

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

    2016-04-18

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-02-19

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

  13. Mirror Movement-Like Defects in Startle Behavior of Zebrafish dcc Mutants Are Caused by Aberrant Midline Guidance of Identified Descending Hindbrain Neurons

    PubMed Central

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

    2014-01-01

    Mirror movements are involuntary movements on one side of the body that occur simultaneously with intentional movements on the contralateral side. Humans with heterozygous mutations in the axon guidance receptor DCC display such mirror movements, where unilateral stimulation results in inappropriate bilateral motor output. Currently, it is unclear whether mirror movements are caused by incomplete midline crossing and reduced commissural connectivity of DCC-dependent descending pathways or by aberrant ectopic ipsilateral axonal projections of normally commissural neurons. Here, we show that in response to unilateral tactile stimuli, zebrafish dcc mutant larvae perform involuntary turns on the inappropriate body side. We show that these mirror movement-like deficits are associated with axonal guidance defects of two identified groups of commissural reticulospinal hindbrain neurons. Moreover, we demonstrate that in dcc mutants, axons of these identified neurons frequently fail to cross the midline and instead project ipsilaterally. Whereas laser ablation of these neurons in wild-type animals does not affect turning movements, their ablation in dcc mutants restores turning movements. Thus, our results demonstrate that in dcc mutants, turns on the inappropriate side of the body are caused by aberrant ipsilateral axonal projections, and suggest that aberrant ipsilateral connectivity of a very small number of descending axons is sufficient to induce incorrect movement patterns. PMID:24553931

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

    DOE PAGES

    Bible, Amber; Fletcher, Sarah J; Pelletier, Dale A; ...

    2016-04-18

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

  15. Chemostat cultivation and transcriptional analyses of Clostridium acetobutylicum mutants with defects in the acid and acetone biosynthetic pathways.

    PubMed

    Hönicke, Daniel; Lütke-Eversloh, Tina; Liu, Ziyong; Lehmann, Dörte; Liebl, Wolfgang; Ehrenreich, Armin

    2014-12-01

    Clostridium acetobutylicum is a model organism for the biotechnologically important acetone-butanol-ethanol (ABE) fermentation. With the objective to rationally develop strains with improved butanol production, detailed insights into the physiological and genetic mechanisms of solvent production are required. Therefore, pH-controlled phosphate-limited chemostat cultivation and DNA microarray technology were employed for an in-depth analysis of knockout mutants with defects in the central fermentative metabolism. The set of studied mutants included strains with inactivated phosphotransacetylase (pta), phosphotransbutyrylase (ptb), and acetoacetate decarboxylase (adc) encoding genes, as well as an adc/pta double knockout mutant. A comprehensive physiological characterization of the mutants was performed by continuous cultivation, allowing for a well-defined separation of acidogenic and solventogenic growth, combined with the advantage of the high reproducibility of steady-state conditions. The ptb-negative strain C. acetobutylicum ptb::int(87) exhibited the most striking metabolite profile: Sizable amounts of butanol (29 ± 1.3 mM) were already produced during acidogenic growth. The product patterns of the mutants as well as accompanying transcriptomic data are presented and discussed.

  16. Transcriptomic Analysis of the Swarm Motility Phenotype of Salmonella enterica Serovar Typhimurium Mutant Defective in Periplasmic Glucan Synthesis.

    PubMed

    Bhagwat, Arvind A; Young, Lynn; Smith, Allen D; Bhagwat, Medha

    2017-09-01

    Movement of food-borne pathogens on moist surfaces enables them to migrate towards more favorable niches and facilitate their survival for extended periods of time. Salmonella enterica serovar Typhimurium mutants defective in Osmoregulated periplasmic glucans (OPG) synthesis are unable to exhibit motility on moist surfaces (swarming); however, their mobility in liquid (swim motility) remains unaffected. In order to understand the role of OPG in swarm motility, transcriptomic analysis was performed using cells growing on a moist agar surface. In opgGH deletion mutant, lack of OPG significantly altered transcription of 1039 genes out of total 4712 genes (22%). Introduction of a plasmid-borne copy of opgGH into opgGH deletion mutant restored normal expression of all but 30 genes, indicating a wide-range influence of OPG on gene expression under swarm motility condition. Major pathways that were differentially expressed in opgGH mutants were motility, virulence and invasion, and genes related to the secondary messenger molecule, cyclic di-GMP. These observations provide insights and help explain the pleiotropic nature of OPG mutants such as sub-optimal virulence and competitive organ colonization in mice, biofilm formation, and sensitivity towards detergent stress.

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

    ERIC Educational Resources Information Center

    Doe, Frank J.; Leslie, John F.

    1993-01-01

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

  18. A MUTANT OF YEAST APPARENTLY DEFECTIVE IN THE INITIATION OF PROTEIN SYNTHESIS*

    PubMed Central

    Hartwell, Leland H.; McLaughlin, Calvin S.

    1969-01-01

    A temperature-sensitive mutant of yeast, ts-187, which is apparently unable to initiate the synthesis of new polypeptide chains after a short incubation at the restrictive temperature, is described. The existence of this mutant demonstrates that in eucaryotic cells, as in procaryotic cells, there are processes unique to the initiation of polypeptide chains. PMID:5256225

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

    ERIC Educational Resources Information Center

    Doe, Frank J.; Leslie, John F.

    1993-01-01

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

  20. Isolation of cyanobacterial mutants exhibiting growth defects under microoxic conditions by transposon tagging mutagenesis of Synechocystis sp. PCC 6803.

    PubMed

    Terauchi, Kazuki; Sobue, Riho; Furutani, Yuho; Aoki, Rina; Fujita, Yuichi

    2017-05-12

    Cyanobacteria are photosynthetic prokaryotes that perform oxygenic photosynthesis by extracting electrons from water, with the generation of oxygen as a byproduct. Cyanobacteria use oxygen not only for respiration to produce energy in the dark but also for biosynthesis of various metabolites, such as heme and chlorophyll. Oxygen levels dynamically fluctuate in the field environments, from hyperoxic at daytime to almost anaerobic at night. Thus, adaptation to anaerobiosis should be important for cyanobacteria to survive in low-oxygen and anaerobic environments. However, little is known about the molecular mechanisms of cyanobacterial anaerobiosis because cyanobacteria have been regarded as aerobic organisms. As a first step to elucidate cyanobacterial adaptation mechanisms to low-oxygen environments, we isolated five mutants, T-1-T-5, exhibiting growth defects under microoxic conditions. The mutants were obtained from a transposon-tagged mutant library of the cyanobacterium Synechocystis sp. PCC 6803, which was produced by in vitro transposon tagging of cyanobacterial genomic DNA. Southern blot analysis indicated that a kanamycin resistance gene was inserted in the genome as a single copy. We identified the chromosomal transposon-tagged locus in T-5. Two open reading frames (sll0577 and sll0578) were partially deleted by the insertion of the kanamycin resistance gene in T-5. A reverse transcription polymerase chain reaction suggested that these co-transcribed genes are constitutively expressed under both aerobic and microoxic conditions. Then, we isolated two mutants in which one of the two genes was individually disrupted. Only the mutants partially lacking an intact sll0578 gene showed growth defects under microoxic conditions, whereas it grew normally under aerobic conditions. sll0578 is annotated as purK encoding N(5)-carboxy-aminoimidazole ribonucleotide synthetase involved in purine metabolism. This result implies the unexpected physiological importance of Pur

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

    PubMed Central

    Ventoso, I; Carrasco, L

    1995-01-01

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

  2. Characterization of adenylate cyclase toxin from a mutant of Bordetella pertussis defective in the activator gene, cyaC.

    PubMed

    Hewlett, E L; Gray, M C; Ehrmann, I E; Maloney, N J; Otero, A S; Gray, L; Allietta, M; Szabo, G; Weiss, A A; Barry, E M

    1993-04-15

    Bordetella pertussis adenylate cyclase (AC) toxin has the abilities to 1) enter target cells where it catalyzes cyclic AMP production and 2) lyse sheep erythrocytes, and these abilities require post-translational modification by the product of an accessory gene cyaC (Barry, E. M., Weiss, A. A., Ehrmann, E. E., Gray, M. C., Hewlett, E. L., and Goodwin, M. St. M. (1991) J. Bacteriol. 173, 720-726). In the present study, AC toxin has been purified from an organism with a mutation in cyaC, BPDE386, and evaluated for its physical and functional properties in order to determine the basis for its lack of toxin and hemolytic activities. AC toxin from BPDE386 is indistinguishable from wild-type toxin in enzymatic activity, migration on SDS-polyacrylamide gel electrophoresis, ability to bind calcium, and calcium-dependent conformational change. Although unable to elicit cAMP accumulation, AC toxin from BPDE386 exhibits binding to the surface of Jurkat cells which is comparable to that of wild-type toxin. This target cell interaction is qualitatively different, however, in that 99% of the mutant toxin remains sensitive to trypsin, whereas approximately 20% of cell-associated wild-type toxin enters a trypsin-resistant compartment. To evaluate the ability of this mutant AC toxin to function at its intracellular site of action, the cAMP-stimulated L-type calcium current in frog atrial myocytes was used. Extracellular addition of wild-type toxin results in cAMP-dependent events that include activation of calcium channels and enhancement of calcium current. In contrast, there is no response to externally applied toxin from BPDE386. When injected into the cell interior, however, the AC toxin from BPDE386 is able to produce increases in the calcium current comparable to those observed with wild-type toxin. Although AC toxin from BPDE386 is unaffected in its enzymatic activity, calcium binding, and calcium-dependent conformational change, the mutation in cyaC does result in a toxin

  3. Defective DNA cross-link removal in Chinese hamster cell mutants hypersensitive to bifunctional alkylating agents

    SciTech Connect

    Hoy, C.A.; Thompson, L.H.; Mooney, C.L.; Salazar, E.P.

    1985-04-01

    DNA repair-deficient mutants from five genetic complementation groups isolated previously from Chinese hamster cells were assayed for survival after exposure to the bifunctional alkylating agents mitomycin C or diepoxybutane. Groups 1, 3, and 5 exhibited 1.6- to 3-fold hypersensitivity compared to the wild-type cells, whereas Groups 2 and 4 exhibited extraordinary hypersensitivity. Mutants from Groups 1 and 2 were exposed to 22 other bifunctional alkylating agents in a rapid assay that compared cytotoxicity of the mutants to the wild-type parental strain, AA8. With all but two of the compounds, the Group 2 mutant (UV4) was 15- to 60-fold more sensitive than AA8 or the Group 1 mutant (UV5). UV4 showed only 6-fold hypersensitivity to quinacrine mustard. Alkaline elution measurements showed that this compound produced few DNA interstrand cross-links but numerous strand breaks. Therefore, the extreme hypersensitivity of mutants from Groups 2 and 4 appeared specific for compounds the main cytotoxic lesions of which were DNA cross-links. Mutant UV5 was only 1- to 4-fold hypersensitive to all the compounds. Although the initial number of cross-links was similar for the three cell lines, the efficiency of removal of cross-links was lowest in UV4 and intermediate in UV5. These results suggest that the different levels of sensitivity are specifically related to different efficiencies of DNA cross-link removal. The phenotype of hypersensitivity to both UV radiation and cross-link damage exhibited by the mutants in Groups 2 and 4 appears to differ from those of the known human DNA repair syndromes.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    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.

  6. Organ fusion and defective cuticle function in a lacs1 lacs2 double mutant of Arabidopsis.

    PubMed

    Weng, Hua; Molina, Isabel; Shockey, Jay; Browse, John

    2010-04-01

    As the outermost layer on aerial tissues of the primary plant body, the cuticle plays important roles in plant development and physiology. The major components of the cuticle are cutin and cuticular wax, both of which are composed primarily of fatty acid derivatives synthesized in the epidermal cells. Long-chain acyl-CoA synthetases (LACS) catalyze the formation of long-chain acyl-CoAs and the Arabidopsis genome contains a family of nine genes shown to encode LACS enzymes. LACS2 is required for cutin biosynthesis, as revealed by previous investigations on lacs2 mutants. Here, we characterize lacs1 mutants of Arabidopsis that reveals a role for LACS1 in biosynthesis of cuticular wax components. lacs1 lacs2 double-mutant plants displayed pleiotropic phenotypes including organ fusion, abnormal flower development and reduced seed set; phenotypes not found in either of the parental mutants. The leaf cuticular permeability of lacs1 lacs2 was higher than that of either lacs1 or lacs2 single mutants, as determined by measurements of chlorophyll leaching from leaves immersed in 80% ethanol, staining with toluidine blue dye and direct measurements of water loss. Furthermore, lacs1 lacs2 mutant plants are highly susceptible to drought stress. Our results indicate that a deficiency in cuticular wax synthesis and a deficiency in cutin synthesis together have compounding effects on the functional integrity of the cuticular barrier, compromising the ability of the cuticle to restrict water movement, protect against drought stress and prevent organ fusion.

  7. The basis for colorless hemolymph and cocoons in the Y-gene recessive Bombyx mori mutants: a defect in the cellular uptake of carotenoids.

    PubMed

    Tsuchida, Kozo; Katagiri, Chihiro; Tanaka, Yoshiro; Tabunoki, Hiroko; Sato, Ryoichi; Maekawa, Hideaki; Takada, Naoko; Banno, Yutaka; Fujii, Hiroshi; Wells, Michael A; Jouni, Zeina E

    2004-10-01

    Bombyx mori is an excellent model for the study of carotenoid-binding proteins (CBP). In previous papers, we identified and molecularly characterized a CBP from the Y-gene dominant mutants. In the present study, we attempted to correlate and establish lipid metabolism and distribution in these mutants. When [3H]-triolein was fed to the mutants, typical patterns of uptake of labeled fatty acids from midgut to hemolymph and subsequent delivery to fat body and silk glands were obtained in all mutants. Further analysis of lipid and carotenoid profiles revealed that the yellow coloration in the hemolymph associated with lipophorin is not attributed to a difference in lipophorin concentrations among the mutants, nor to its lipid composition, but rather to its carotenoid content. Lipophorin of the Y+I mutant exhibited the highest concentration of total carotenoids of 55.8 microg/mg lipophorin compared to 3.1 microg/mg in the +Y+I mutant, 1.2 microg/mg in the YI mutant and 0.5 microg/mg in the +YI mutant. Characteristic retention time in HPLC of the different classes of carotenoids of lipophorin identified the presence of lutein as the major chromophore (62-77%), followed by beta-carotenes (22-38%). Although lutein and beta-carotene content of mutants' lipophorin differed significantly, the ratio of lutein to beta-carotene of 3:1 was not different among mutants. Similarly, lipid compositions of mutant silk glands were not significantly different, but carotenoid contents were. The significantly high concentration of lutein in the Y+I mutant silk gland represented more than 160-fold increase compared to +Y+I mutant (p<0.001). In this report, we conclude that lipid metabolism in the mutants is not defected and that the molecular basis for colorless hemolymph and cocoons is a defect in the cellular uptake of lutein associated with the Y-gene recessive mutants.

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

    PubMed Central

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

    2010-01-01

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

  9. [Defects in immune system response by our organisms].

    PubMed

    Español, Teresa

    2005-09-01

    When some of the mechanisms in our immune response system fail, this can be due to external problems such as infections or transplants or due to congenital errors, known as Primary Immunologic Deficiencies. Dr. Español briefly reviews the most important characteristics of our immune response system, and then continues with an analysis of the defects of this system, especially those defects which are classified as Primary Immunologic Deficiencies.

  10. Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides

    PubMed Central

    Karas, Bogumil J.; Wise, Kim S.; Sun, Lijie; Venter, J. Craig; Glass, John I.; Hutchison, Clyde A.; Smith, Hamilton O.; Suzuki, Yo

    2014-01-01

    With only hundreds of genes contained within their genomes, mycoplasmas have become model organisms for precise understanding of cellular processes, as well as platform organisms for predictable engineering of microbial functions for mission-critical applications. Despite the availability of “whole genome writing” in Mycoplasma mycoides, some traditional methods for genetic engineering are underdeveloped in mycoplasmas. Here we demonstrate two facile transposon-mediated approaches for introducing genes into the synthetic cell based on M. mycoides. The marker-less approach involves preparing a fragment containing only a small genomic region of interest with flanking transposase-binding sites, followed by in vitro transposase loading and introduction into the cells. The marker-driven approach involves cloning an open reading frame (ORF) of interest into a vector containing a marker for mycoplasma transformation, as well as sites for transposase loading and random genomic integration. An innovative feature of this construct is to use a single promoter to express the transformation marker and the introduced ORF. The marker-driven approach can be conveniently applied to any exogenous or synthetic gene without any information on the effect of the gene on the strain, whereas the marker-less approach requires that the fragment has a recognizable effect. Using the marker-less method, we found that a region containing the nusG gene rescues a slow growth phenotype of a strain containing a larger deletion encompassing this gene. Using the marker-driven approach, we better defined this finding, thereby establishing that nusG is required for a normal growth rate in synthetic M. mycoides. These methods are suitable for complementation tests to identify genes responsible for assorted functions lacking in deletion mutants. These approaches are also expected to facilitate rapid testing of various natural and engineered genes or gene clusters from numerous sources in M. mycoides

  11. Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides.

    PubMed

    Karas, Bogumil J; Wise, Kim S; Sun, Lijie; Venter, J Craig; Glass, John I; Hutchison, Clyde A; Smith, Hamilton O; Suzuki, Yo

    2014-01-01

    With only hundreds of genes contained within their genomes, mycoplasmas have become model organisms for precise understanding of cellular processes, as well as platform organisms for predictable engineering of microbial functions for mission-critical applications. Despite the availability of "whole genome writing" in Mycoplasma mycoides, some traditional methods for genetic engineering are underdeveloped in mycoplasmas. Here we demonstrate two facile transposon-mediated approaches for introducing genes into the synthetic cell based on M. mycoides. The marker-less approach involves preparing a fragment containing only a small genomic region of interest with flanking transposase-binding sites, followed by in vitro transposase loading and introduction into the cells. The marker-driven approach involves cloning an open reading frame (ORF) of interest into a vector containing a marker for mycoplasma transformation, as well as sites for transposase loading and random genomic integration. An innovative feature of this construct is to use a single promoter to express the transformation marker and the introduced ORF. The marker-driven approach can be conveniently applied to any exogenous or synthetic gene without any information on the effect of the gene on the strain, whereas the marker-less approach requires that the fragment has a recognizable effect. Using the marker-less method, we found that a region containing the nusG gene rescues a slow growth phenotype of a strain containing a larger deletion encompassing this gene. Using the marker-driven approach, we better defined this finding, thereby establishing that nusG is required for a normal growth rate in synthetic M. mycoides. These methods are suitable for complementation tests to identify genes responsible for assorted functions lacking in deletion mutants. These approaches are also expected to facilitate rapid testing of various natural and engineered genes or gene clusters from numerous sources in M. mycoides.

  12. Glucose Starvation Alters Heat Shock Response, Leading to Death of Wild Type Cells and Survival of MAP Kinase Signaling Mutant

    PubMed Central

    Higgins, LeeAnn; Markowski, Todd; Brambl, Robert

    2016-01-01

    A moderate heat shock induces Neurospora crassa to synthesize large quantities of heat shock proteins that are protective against higher, otherwise lethal temperatures. However, wild type cells do not survive when carbohydrate deprivation is added to heat shock. In contrast, a mutant strain defective in a stress-activated protein kinase does survive the combined stresses. In order to understand the basis for this difference in survival, we have determined the relative levels of detected proteins in the mutant and wild type strain during dual stress, and we have identified gene transcripts in both strains whose quantities change in response to heat shock or dual stress. These data and supportive experimental evidence point to reasons for survival of the mutant strain. By using alternative respiratory mechanisms, these cells experience less of the oxidative stress that proves damaging to wild type cells. Of central importance, mutant cells recycle limited resources during dual stress by undergoing autophagy, a process that we find utilized by both wild type and mutant cells during heat shock. Evidence points to inappropriate activation of TORC1, the central metabolic regulator, in wild type cells during dual stress, based upon behavior of an additional signaling mutant and inhibitor studies. PMID:27870869

  13. Glucose Starvation Alters Heat Shock Response, Leading to Death of Wild Type Cells and Survival of MAP Kinase Signaling Mutant.

    PubMed

    Plesofsky, Nora; Higgins, LeeAnn; Markowski, Todd; Brambl, Robert

    2016-01-01

    A moderate heat shock induces Neurospora crassa to synthesize large quantities of heat shock proteins that are protective against higher, otherwise lethal temperatures. However, wild type cells do not survive when carbohydrate deprivation is added to heat shock. In contrast, a mutant strain defective in a stress-activated protein kinase does survive the combined stresses. In order to understand the basis for this difference in survival, we have determined the relative levels of detected proteins in the mutant and wild type strain during dual stress, and we have identified gene transcripts in both strains whose quantities change in response to heat shock or dual stress. These data and supportive experimental evidence point to reasons for survival of the mutant strain. By using alternative respiratory mechanisms, these cells experience less of the oxidative stress that proves damaging to wild type cells. Of central importance, mutant cells recycle limited resources during dual stress by undergoing autophagy, a process that we find utilized by both wild type and mutant cells during heat shock. Evidence points to inappropriate activation of TORC1, the central metabolic regulator, in wild type cells during dual stress, based upon behavior of an additional signaling mutant and inhibitor studies.

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

    PubMed

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

    2015-06-01

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

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

    PubMed

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

    2014-03-01

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

  16. Production of polyhydroxyalkanoates by Escherichia coli mutants with defected mixed acid fermentation pathways.

    PubMed

    Jian, Jia; Zhang, Shao-Qin; Shi, Zhen-Yu; Wang, Wei; Chen, Guo-Qiang; Wu, Qiong

    2010-08-01

    A series of Escherichia coli BW25113 mutants with reduced mixed acid fermentation were constructed. Genes ackA-pta, poxB, ldhA, adhE, and pflB encoding acetate kinase, phosphate acetyltransferase, pyruvate oxidase, D: -lactate dehydrogenase, acetaldehyde dehydrogenase, and pyruvate formate-lyase, respectively, were deleted successively. When grown under microaerobic condition, the mutants reduced approximately 90% acetate excretion after the deletion of genes ackA-pta and poxB. Production of lactate, ethanol, and formate was also significantly reduced after the deletion of genes ldhA, adhE, and pflB, respectively. The accumulation of biomass and poly(3-hydroxybutyrate) (PHB) were significantly enhanced after deleting the mixed acid fermentation. E. coli mutant BWapld with deletions of ackA-pta, poxB, ldhA, and adhE produced twice the cell dry weight (CDW) and 3.5 times of PHB compared with its wild-type under microaerobic conditions. E. coli mutant BWapl with deletions of ackA-pta, poxB, and ldhA also achieved nearly twice CDW and three times of PHB content in comparison to the wild-type during 48 h static cultivation. Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was observed in the mutants under static cultivation. E. coli mutant BWapld could produce approximately 50 wt.% P(3HB-co-3HV) consisting of 5 mol% of 3-hydroxyvalerate (3HV) under aerobic conditions, when the seed culture was inoculated at an appropriate time. When ackA-pta, poxB, ldhA, adhE, and pflB were deleted, E. coli mutant BWapldf accumulated over 70 wt.% P(3HB-co-3HV) consisting of 8 mol% 3HV under aerobic conditions.

  17. Genetic defects of GDF6 in the zebrafish out of sight mutant and in human eye developmental anomalies

    PubMed Central

    2010-01-01

    Background The size of the vertebrate eye and the retina is likely to be controlled at several stages of embryogenesis by mechanisms that affect cell cycle length as well as cell survival. A mutation in the zebrafish out of sight (out) locus results in a particularly severe reduction of eye size. The goal of this study is to characterize the outm233 mutant, and to determine whether mutations in the out gene cause microphthalmia in humans. Results In this study, we show that the severe reduction of eye size in the outm233 mutant is caused by a mutation in the zebrafish gdf6a gene. Despite the small eye size, the overall retinal architecture appears largely intact, and immunohistochemical studies confirm that all major cell types are present in outm233 retinae. Subtle cell fate and patterning changes are present predominantly in amacrine interneurons. Acridine orange and TUNEL staining reveal that the levels of apoptosis are abnormally high in outm233 mutant eyes during early neurogenesis. Mutation analysis of the GDF6 gene in 200 patients with microphthalmia revealed amino acid substitutions in four of them. In two patients additional skeletal defects were observed. Conclusions This study confirms the essential role of GDF6 in the regulation of vertebrate eye size. The reduced eye size in the zebrafish outm233 mutant is likely to be caused by a transient wave of apoptosis at the onset of neurogenesis. Amino acid substitutions in GDF6 were detected in 4 (2%) of 200 patients with microphthalmia. In two patients different skeletal defects were also observed, suggesting pleitrophic effects of GDF6 variants. Parents carrying these variants are asymptomatic, suggesting that GDF6 sequence alterations are likely to contribute to the phenotype, but are not the sole cause of the disease. Variable expressivity and penetrance suggest a complex non-Mendelian inheritance pattern where other genetic factors may influence the outcome of the phenotype. PMID:21070663

  18. Triple point-mutants of hypoxia-inducible factor-1α accelerate in vivo angiogenesis in bone defect regions.

    PubMed

    Li, Chen; Liu, Danping; Zhang, Zheng; Wang, Guoxian; Xu, Na

    2013-11-01

    To investigate the functions of triple point-mutants of hypoxia-inducible factor 1α (HIF1α) in angiogenesis in bone defect regions under normoxic conditions. 1. Triple point-mutations (in amino acids 402, 564, and 803) in the HIF1α coding sequence (CDS) were induced by polymerase chain reaction. The triple mutant HIF1α (402/564/803) was inserted into the adenovirus pAdEasy-1 system for complete viral packaging and titer measurements. 2. For the in vitro experiment, rabbit bone marrow mesenchymal stem cells (MSCs) were divided into four experimental groups. The efficiency of infection was observed by the expression of human renilla reniformis green fluorescent protein (hrGFP). The HIF1α mRNA, protein and VEGF protein expression levels in infected cells in each experimental group were measured. 3. As in the in vivo experiment, the MSCs were divided into four groups and infected with the viral solutions from each complementary in vitro group and cultured under normoxic conditions. The MSCs were used as seed cells and transplanted into an apatite-wollastonite magnetic bioactive glass-ceramic (AW MGC) vector to construct artificial tissue-engineering scaffolds that were then implanted into the in vivo rabbit radial bone defect model. The animals from each group were killed 8 weeks after the surgery, and the tissues from the implantation region were harvested for the evaluation of the angiogenesis. 1. The 402,564, and 803 amino acids in CDS area were point mutated into alanine; three types of recombinant adenovirus were successfully constructed, packaged, and characterized. 2. The expression levels of HIF1α mRNA in A and B groups were significantly higher than those in the C and D groups (P < 0.05). The HIF1α and VEGF protein expression levels in A group were significantly higher than those in the other three groups (P < 0.05). 3. There was prominent angiogenesis in bone defect regions in group A animals. 1. Triple point-mutants of HIF1α efficiently

  19. 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

  20. Genetic screens for Caenorhabditis elegans mutants defective in left/right asymmetric neuronal fate specification.

    PubMed

    Sarin, Sumeet; O'Meara, M Maggie; Flowers, Eileen B; Antonio, Celia; Poole, Richard J; Didiano, Dominic; Johnston, Robert J; Chang, Sarah; Narula, Surinder; Hobert, Oliver

    2007-08-01

    We describe here the results of genetic screens for Caenorhabditis elegans mutants in which a single neuronal fate decision is inappropriately executed. In wild-type animals, the two morphologically bilaterally symmetric gustatory neurons ASE left (ASEL) and ASE right (ASER) undergo a left/right asymmetric diversification in cell fate, manifested by the differential expression of a class of putative chemoreceptors and neuropeptides. Using single cell-specific gfp reporters and screening through a total of almost 120,000 haploid genomes, we isolated 161 mutants that define at least six different classes of mutant phenotypes in which ASEL/R fate is disrupted. Each mutant phenotypic class encompasses one to nine different complementation groups. Besides many alleles of 10 previously described genes, we have identified at least 16 novel "lsy" genes ("laterally symmetric"). Among mutations in known genes, we retrieved four alleles of the miRNA lsy-6 and a gain-of-function mutation in the 3'-UTR of a target of lsy-6, the cog-1 homeobox gene. Using newly found temperature-sensitive alleles of cog-1, we determined that a bistable feedback loop controlling ASEL vs. ASER fate, of which cog-1 is a component, is only transiently required to initiate but not to maintain ASEL and ASER fate. Taken together, our mutant screens identified a broad catalog of genes whose molecular characterization is expected to provide more insight into the complex genetic architecture of a left/right asymmetric neuronal cell fate decision.

  1. An update to the list of mouse mutants with neural tube closure defects and advances toward a complete genetic perspective of neural tube closure.

    PubMed

    Harris, Muriel J; Juriloff, Diana M

    2010-08-01

    The number of mouse mutants and strains with neural tube defects (NTDs) now exceeds 240, including 205 representing specific genes, 30 for unidentified genes, and 9 multifactorial strains. These mutants identify genes needed for embryonic neural tube closure. Reports of 50 new NTD mutants since our 2007 review (Harris and Juriloff, 2007) were considered in relation to the previously reviewed mutants to obtain new insights into mechanisms of NTD etiology. In addition to null mutations, some are hypomorphs or conditional mutants. Some mutations do not cause NTDs on their own, but do so in digenic, trigenic, and oligogenic combinations, an etiology that likely parallels the nature of genetic etiology of human NTDs. Mutants that have only exencephaly are fourfold more frequent than those that have spina bifida aperta with or without exencephaly. Many diverse cellular functions and biochemical pathways are involved; the NTD mutants draw new attention to chromatin modification (epigenetics), the protease-activated receptor cascade, and the ciliopathies. Few mutants directly involve folate metabolism. Prevention of NTDs by maternal folate supplementation has been tested in 13 mutants and reduces NTD frequency in six diverse mutants. Inositol reduces spina bifida aperta frequency in the curly tail mutant, and three new mutants involve inositol metabolism. The many NTD mutants are the foundation for a future complete genetic understanding of the processes of neural fold elevation and fusion along mechanistically distinct cranial-caudal segments of the neural tube, and they point to several candidate processes for study in human NTD etiology.

  2. Flagellar mutants of Chlamydomonas: Studies of radial spoke-defective strains by dikaryon and revertant analysis

    PubMed Central

    Luck, David; Piperno, Gianni; Ramanis, Zenta; Huang, B.

    1977-01-01

    The motility mutant of Chlamydomonas reinhardtii pf14 lacks radial spoke structures in its flagellar axonemes, and 12 proteins present in wild type are missing from a two-dimensional map (isoelectrofocusing/sodium dodecyl sulfate electrophoresis) of its 35S-labeled flagellar proteins. Six of these same proteins are missing in pf1, which lacks spoke-heads. To determine whether any of the missing proteins represent the mutant gene product two experimental approaches have been applied. The first makes use of the fact that gametes of either mutant strain when fused with wild-type gametes to form quadriflagellate dikaryons undergo recovery of flagellar function. Recovery at the molecular level was monitored by prelabeling the mutant proteins with 35S and allowing recovery to occur in the absence of protein synthesis. It is to be expected that the mutant gene product would not be restored as a radioactive protein and that recovery would depend on the assembly of the wild-type counterpart that is not labeled. The second technique makes use of revertants induced by UV irradiation. Dikaryon rescue in the case of pf14 leads to restoration of 11 radioactive components; only protein 3 fails to appear as a radioactive spot. For pf1 only two radioactive proteins are restored; proteins 4, 6, 9, and 10 were not radioactive. Analysis of revertants of pf1 gave evidence (altered map positions) that protein 4 is the mutant gene product. In the case of pf14, analysis of 22 revertants has not provided similar positive evidence that protein 3 is the gene product. Images PMID:269405

  3. Photosynthetic properties of an Arabidopsis thaliana mutant possessing a defective PsbS gene.

    PubMed

    Peterson, R B; Havir, E A

    2001-11-01

    We describe the properties of npq4-9, a new mutant of Arabidopsis thaliana (L.) Heynh. with reduced nonphotochemical quenching (NPQ) capacity that possesses a single amino acid substitution in the PsbS gene encoding PSII-S, a ubiquitous pigment-binding protein associated with photosystem II (PSII) of higher plants. Growth, photosynthetic pigment contents, and levels of the major PSII antenna proteins were not affected by npq4-9. Although the extent of de-epoxidatin of violaxanthin to antheraxanthin plus zeaxanthin for leaves displaying the mutant phenotype equaled or exceeded that observed for the wild type, the relative effectiveness of de-epoxidized xanthophylls in promoting NPQ was consistently lower for the mutant. Energy partitioning in PSII was analyzed in terms of the competition for singlet chlorophyll a among the processes of fluorescence, thermal dissipation, and photochemistry. The key processes of NPQ and photochemistry in open PSII centers are represented by the relative in vivo rate constants kN and kP0, respectively. The magnitude of kP0 in normal leaves declined only slightly with increasing kN, consistent with localization of NPQ primarily in the antenna complex. Conversely, a highly significant linear decline in kP0 with increasing kN was observed for the mutant, consistent with a role for the PSII reaction center in the NPQ mechanism. Although the PSII absorption cross-section for white light was not significantly different relative to that of the wild type, PSII quantum yield was significantly lower in the mutant. The resulting lower capacity for linear electron transport in the mutant primarily affected reduction of terminal acceptors other than CO2. Parallel measurements of fluorescence and in vivo absorbance at 820 nm indicated a consistently higher steady-state level of reduction of PSII acceptors and accumulation of P700+ for the mutant. This suggests that inter-photosystem electron transport in the mutant is restricted either by a higher

  4. Enterohemorrhagic Escherichia coli O157:H7 gal Mutants Are Sensitive to Bacteriophage P1 and Defective in Intestinal Colonization▿

    PubMed Central

    Ho, Theresa Deland; Waldor, Matthew K.

    2007-01-01

    Enterohemorrhagic Escherichia coli (EHEC), especially E. coli O157:H7, is an emerging cause of food-borne illness. Unfortunately, E. coli O157 cannot be genetically manipulated using the generalized transducing phage P1, presumably because its extensive O antigen obscures the P1 receptor, the lipopolysaccharide (LPS) core subunit. The GalE, GalT, GalK, and GalU proteins are necessary for modifying galactose before it can be assembled into the repeating subunit of the O antigen. Here, we constructed E. coli O157:H7 gal mutants which presumably have little or no O antigen. These strains were able to adsorb P1. P1 lysates grown on the gal mutant strains could be used to move chromosomal markers between EHEC strains, thereby facilitating genetic manipulation of E. coli O157:H7. The gal mutants could easily be reverted to a wild-type Gal+ strain using P1 transduction. We found that the O157:H7 galETKM::aad-7 deletion strain was 500-fold less able to colonize the infant rabbit intestine than the isogenic Gal+ parent, although it displayed no growth defect in vitro. Furthermore, in vivo a Gal+ revertant of this mutant outcompeted the galETKM deletion strain to an extent similar to that of the wild type. This suggests that the O157 O antigen is an important intestinal colonization factor. Compared to the wild type, EHEC gal mutants were 100-fold more sensitive to a peptide derived from bactericidal permeability-increasing protein, a bactericidal protein found on the surface of intestinal epithelial cells. Thus, one way in which the O157 O antigen may contribute to EHEC intestinal colonization is to promote resistance to host-derived antimicrobial polypeptides. PMID:17158899

  5. Microstructure defect detection using thermal response

    NASA Astrophysics Data System (ADS)

    Olson, Brandon; Chen, Kuan H.

    2002-04-01

    Detecting thermal and mechanical defects within multilayered microstructures is an important research area within the microdevice community. The detection of material flaws, mechanical damage, and packaging irregularities is often critical to the overall performanc eof the end product. The technique presented hereafter uses a series of surface temperature measurements, generated by a step function heat flux, to determine the thermal properties of a one- dimensional structure. These properties can either be used directly in a design effort, or they can be used as an indicator of problems that may exist within the structure. This technique is essentially non-invasive and it places no requirements on structure size, thus it is predisposed to semiconductor and MEMS applications. The technique exploits a thermal-electrical analog to match a measured thermal resistance pattern with the pattern of a corresponding thermal structure. Typically, the dimensions of the structure and the disturbance amplitude are required for property value determination.

  6. Identification of a malate chemoreceptor in Pseudomonas aeruginosa by screening for chemotaxis defects in an energy taxis-deficient mutant.

    PubMed

    Alvarez-Ortega, Carolina; Harwood, Caroline S

    2007-12-01

    We found that a robust energy taxis response mediated by the Aer receptor can sometimes mask chemotaxis mediated by other methyl-accepting chemotaxis proteins (MCPs) in Pseudomonas aeruginosa. We identified PA2652 as a chemoreceptor for malate by screening aer mcp double mutants by using swarm plate assays.

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

    USDA-ARS?s Scientific Manuscript database

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

  8. Dysregulation of serine biosynthesis contributes to the growth defect of a Mycobacterium tuberculosis crp mutant

    PubMed Central

    Bai, Guangchun; Schaak, Damen D.; Smith, Eric A.; McDonough, Kathleen A.

    2013-01-01

    Summary Mycobacterium tuberculosis CRPMt, encoded by Rv3676 (crp), is a CRP-like transcription factor that binds with the serC – Rv0885 intergenic region. In the present study, we evaluated CRPMt’s regulation of serC and Rv0885 in M. tuberculosis and M. bovis BCG, using site-specific mutagenesis, promoter fusions and RT-PCR. The CRPMt binding site was required for full expression of serC and Rv0885, and expression of both genes was reduced in M. tuberculosis and M. bovis BCG crp mutants. These data show that CRPMt binding directly activates both serC and Rv0885 expression. M. tuberculosis serC restored the ability of an Escherichia coli serC mutant to grow in serine-dropout medium, demonstrating that M. tuberculosis serC encodes a phosphoserine aminotransferase. Serine supplementation, or overexpression of serC, accelerated the growth of M. tuberculosis and M. bovis BCG crp mutants in mycomedium, but not within macrophages. These results establish a role for CRPMt in the regulation of amino acid biosynthesis, and show that reduced serine production contributes to the slow-growth phenotype of M. tuberculosis and M. bovis BCG crp mutants in vitro. Restoration of serine biosynthesis by serC expression will facilitate identification of additional CRPMt-regulated factors required by M. tuberculosis during macrophage and host infection. PMID:21902733

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  11. Monomeric yeast PCNA mutants are defective in interacting with and stimulating the ATPase activity of RFC.

    PubMed

    Ionescu, Costin N; Shea, Kathleen A; Mehra, Rajendra; Prundeanu, Lucia; McAlear, Michael A

    2002-10-29

    Yeast PCNA is a homo-trimeric, ring-shaped DNA polymerase accessory protein that can encircle duplex DNA. The integrity of this multimeric sliding DNA clamp is maintained through the protein-protein interactions at the interfaces of adjacent subunits. To investigate the importance of trimer stability for PCNA function, we introduced single amino acid substitutions at residues (A112T, S135F) that map to opposite ends of the monomeric protein. Recombinant wild-type and mutant PCNAs were purified from E. coli, and they were tested for their properties in vitro. Unlike the stable wild-type PCNA trimers, the mutant PCNA proteins behaved as monomers when diluted to low nanomolar concentrations. In contrast to what has been reported for a monomeric form of the beta clamp in E. coli, the monomeric PCNAs were compromised in their ability to interact with their associated clamp loader, replication factor C (RFC). Similarly, monomeric PCNAs were not effective in stimulating the ATPase activity of RFC. The mutant PCNAs were able to form mixed trimers with wild-type subunits, although these mixed trimers were unstable when loaded onto DNA. They were able to function as weak DNA polymerase delta processivity factors in vitro, and when the monomeric PCNA-41 (A112T, S135F double mutant) allele was introduced as the sole source of PCNA in vivo, the cells were viable and healthy. These pol30-41 mutants were, however, sensitive to UV irradiation and to the DNA damaging agent methylmethane sulfonate, implying that DNA repair pathways have a distinct requirement for stable DNA clamps.

  12. Valosin-containing protein (VCP/p97) inhibitors relieve Mitofusin-dependent mitochondrial defects due to VCP disease mutants

    PubMed Central

    Zhang, Ting; Mishra, Prashant; Hay, Bruce A; Chan, David; Guo, Ming

    2017-01-01

    Missense mutations of valosin-containing protein (VCP) cause an autosomal dominant disease known as inclusion body myopathy, Paget disease with frontotemporal dementia (IBMPFD) and other neurodegenerative disorders. The pathological mechanism of IBMPFD is not clear and there is no treatment. We show that endogenous VCP negatively regulates Mitofusin, which is required for outer mitochondrial membrane fusion. Because 90% of IBMPFD patients have myopathy, we generated an in vivo IBMPFD model in adult Drosophila muscle, which recapitulates disease pathologies. We show that common VCP disease mutants act as hyperactive alleles with respect to regulation of Mitofusin. Importantly, VCP inhibitors suppress mitochondrial defects, muscle tissue damage and cell death associated with IBMPFD models in Drosophila. These inhibitors also suppress mitochondrial fusion and respiratory defects in IBMPFD patient fibroblasts. These results suggest that VCP disease mutants cause IBMPFD through a gain-of-function mechanism, and that VCP inhibitors have therapeutic value. DOI: http://dx.doi.org/10.7554/eLife.17834.001 PMID:28322724

  13. Circadian period lengths of lipid synthesis mutants (cel, chol-1) of Neurospora show defective temperature, but intact pH-compensation.

    PubMed

    Ruoff, Peter; Slewa, Ieda

    2002-05-01

    The influence of extracellular pH on the circadian sporulation rhythm of Neurospora crassa has been investigated for the mutants chol-1 and cel. Both mutants have a defect in the lipid synthesis pathway and require either choline or palmitate, respectively, as supplements for normal growth. The chol-1 and cel mutants also show an impaired temperature-compensation when growing on minimal medium. We investigated the possible correlation between loss of temperature- and pH-compensation in cel and chol-1 similar to the correlation found earlier for the frq7 mutant. Our results show that the cel and the chol-1 mutants, although defective in temperature-compensation have an intact pH-compensation of their circadian rhythms. At present, the products of the frq-locus are the only components of the clock that affect the sporulation rhythm of Neurospora both through pH- and temperature-compensation.

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

    PubMed

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

    2015-06-01

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

  15. Imaging modalities to assess structural birth defects in mutant mouse models.

    PubMed

    Tobita, Kimimasa; Liu, Xiaoqin; Lo, Cecilia W

    2010-09-01

    Assessment of structural birth defects (SBDs) in animal models usually entails conducting detailed necropsy for anatomical defects followed by histological analysis for tissue defects. Recent advances in new imaging technologies have provided the means for rapid phenotyping of SBDs, such as using ultra-high frequency ultrasound biomicroscopy, optical coherence tomography, micro-CT, and micro-MRI. These imaging modalities allow the detailed assessment of organ/tissue structure, and with ultrasound biomicroscopy, structure and function of the cardiovascular system also can be assessed noninvasively, allowing the longitudinal tracking of the fetus in utero. In this review, we briefly discuss the application of these state-of-the-art imaging technologies for phenotyping of SBDs in rodent embryos and fetuses, showing how these imaging modalities may be used for the detection of a wide variety of SBDs.

  16. Endogenic oxidative stress response contributes to glutathione over-accumulation in mutant Saccharomyces cerevisiae Y518.

    PubMed

    Zhu, Yibo; Sun, Jiang; Zhu, Yingyue; Wang, Limei; Qi, Bin

    2015-09-01

    Mechanisms of glutathione (GSH) over-accumulation in mutant Saccharomyces cerevisiae Y518 screened by ultraviolet and nitrosoguanidine-induced random mutagenesis were studied. Y518 accumulated higher levels of GSH and L-cysteine than its wild-type strain. RNA-Seq and pathway enrichment analysis indicated a difference in the expression of key genes involved in cysteine production, the GSH biosynthesis pathway, and antioxidation processes. GSH1, MET17, CYS4, GPX2, CTT1, TRX2, and SOD1 and the transcriptional activators SKN7 and YAP1 were up-regulated in the mutant. Moreover, Y518 showed a dysfunctional respiratory chain resulting from dramatically weakened activity of complex III and significant elevation of intracellular reactive oxygen species (ROS) levels. The supplementation of antimycin A in the culture of the parent strain showed equivalent changes of ROS and GSH level. This study indicates that defective complex III prompts abundant endogenic ROS generation, which triggers an oxidative stress response and upregulation of gene expression associated with GSH biosynthesis. This finding may be helpful for developing new strategies for GSH fermentation process optimization or metabolic engineering.

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

    PubMed Central

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

    2015-01-01

    Brc1, which was first identified as a high-copy, allele-specific suppressor of a mutation impairing the Smc5-Smc6 holocomplex in Schizosaccharomyces pombe, protects genome integrity during normal DNA replication and when cells are exposed to toxic compounds that stall or collapse replication forks. The C-terminal tandem BRCT (BRCA1 C-terminus) domain of fission yeast Brc1 docks with phosphorylated histone H2A (γH2A)-marked chromatin formed by ATR/Rad3 checkpoint kinase at arrested and damaged replication forks; however, how Brc1 functions in relation to other genome protection modules remains unclear. Here, an epistatic mini-array profile reveals critical requirements for Brc1 in mutants that are defective in multiple DNA damage response pathways, including checkpoint signaling by Rad3-Rad26/ATR-ATRIP kinase, DNA repair by Smc5-Smc6 holocomplex, replication fork stabilization by Mrc1/claspin and Swi1-Swi3/Timeless-Tipin, and control of ubiquitin-regulated proteolysis by the COP9 signalosome (CSN). Exogenous genotoxins enhance these negative genetic interactions. Rad52 and RPA foci are increased in CSN-defective cells, and loss of γH2A increases genotoxin sensitivity, indicating a critical role for the γH2A-Brc1 module in stabilizing replication forks in CSN-defective cells. A negative genetic interaction with the Nse6 subunit of Smc5-Smc6 holocomplex indicates that the DNA repair functions of Brc1 and Smc5-Smc6 holocomplex are at least partially independent. Rtt107, the Brc1 homolog in Saccharomyces cerevisiae, has a very different pattern of genetic interactions, indicating evolutionary divergence of functions and DNA damage responses. PMID:25795664

  18. Analysis of the src gene of sarcoma viruses generated by recombination between transformation-defective mutants and quail cellular sequences.

    PubMed Central

    Wang, L H; Moscovici, C; Karess, R E; Hanafusa, H

    1979-01-01

    Tumors were produced in quails about 2 months after injection with a transformation-defective mutant of the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup A (SR-A), that retains a small portion of the src gene. Sarcoma viruses were isolated from each of five such tumors. A transformation-defective mutant which has a nearly complete deletion of the src gene was unable to induce tumors. The avian sarcoma viruses recovered from quail tumors (rASV-Q) had biological properties similar to those of the avian sarcoma viruses previously acquired from chicken tumors (rASV-C); these chicken tumors had been induced by the same transformation-defective mutants. Both rASV-Q and rASV-C transformed cells in culture with similar focus morphology and produced tumors within 7 to 14 days after injection into chickens or quails. The size of rASV-Q genomic RNA was indistinguishable from that of SR-A by polyacrylamide gel electrophoresis. The sequences of rASV-Q RNA genomes were analyzed and compared with those of the parental transformation-defective virus, SR-A and of rASV-C by RNase T1 fingerprinting and oligonucleotide mapping. We found that the src sequences of all five isolates of rASV-Q were identical to each other but different from those of SR-A and rASV-C. Of 13 oligonucleotides of rASV-Q identified as src specific, two were not found in either SR-A or rASV-C RNA. Furthermore, some oligonucleotides present in SR-A or rASV-C or both were absent in rASV-Q. No differences were found for the sequences outside the src region in any of the viruses examined. In addition, rASV-Q-infected cells possessed a 60,000-dalton protein specifically precipitable by rabbit serum raised against SR-D-induced tumors. The facts that the src sequences are essentially the same for rASV's recovered from one animal species and different for rASV's obtained from different species provide conclusive evidence that cellular sequences of normal birds were inserted into the viral genome and supplied to

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

    PubMed

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

    2016-07-01

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

  20. Folding and aggregation of export-defective mutants of the maltose-binding protein.

    PubMed

    Betton, Jean-Michel; Phichith, Denis; Hunke, Sabine

    2002-09-01

    We previously characterized a defective-folding variant of the periplasmic maltose-binding protein, MalE31. To examine the alternative folding pathways open to the MalE31 precursor, we have analyzed the cellular fates of this aggregation-prone protein carrying altered signal sequences. Our results are most easily interpreted by a kinetic competition between exportation, folding, and degradation.

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

    PubMed Central

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

    2015-01-01

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

  2. Purification and characterization of a mutant DnaB protein specifically defective in ATP hydrolysis.

    PubMed Central

    Shrimankar, P; Stordal, L; Maurer, R

    1992-01-01

    The dnaB gene of Escherichia coli encodes an essential DNA replication enzyme. Fueled by the energy derived from the hydrolysis of ATP to ADP+P(i), this enzyme unwinds double-stranded DNA in advance of the DNA polymerase. While doing so, it intermittently stimulates primase to synthesize an RNA primer for an Okazaki fragment. To better understand the structural basis of these and other aspects of DnaB function, we have initiated a study of mutant DnaB proteins. Here, we report the purification and characterization of a mutant DnaB protein (RC231) containing cysteine in place of arginine at residue 231. The mutant protein attains a stable, properly folded structure that allows association of six promoters to form a hexamer, as is also true for wild-type DnaB. Further, the mutant protein interacts with ATP, the nonhydrolyzable ATP analog adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), ADP, and poly(dT), and it stimulates primase action. It is, however, profoundly deficient in ATP hydrolysis, helicase activity, and replication activity at the chromosomal origin of replication. In addition, while general priming reactions with wild-type DnaB and ATP elicited the synthesis of short primers, reactions with DnaB and ATP gamma S or with RC231 and either ATP or ATP gamma S stimulated the synthesis of significantly longer primers. On the basis of these observations, we suggest that primase interacts directly with DnaB throughout primer synthesis during general priming, until dissociation of DnaB from DNA or ATP hydrolysis by DnaB disrupts the interaction and leads to primer termination. Images PMID:1332941

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

    PubMed Central

    Mitra, Mautusi

    2013-01-01

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

  4. Organ fusion and defective shoot development in oni3 mutants of rice

    PubMed Central

    Akiba, Takafumi; Hibara, Ken-Ichiro; Kimura, Fumiko; Tsuda, Katsutoshi; Shibata, Kiko; Ishibashi, Mayu; Moriya, Chihiro; Nakagawa, Kiyotaka; Kurata, Nori; Itoh, Jun-Ichi; Ito, Yukihiro

    2014-01-01

    Maintenance of organ separation is one of the essential phenomena for normal plant development. We have identified and analyzed ONION3 (ONI3), which is required for avoiding organ fusions in rice. Loss-of-function mutations of ONI3, which were identified as mutants with ectopic expression of KNOX genes in leaves and morphologically resembling KNOX overexpressors, showed abnormal organ fusions in developing shoots. The mutant seedlings showed fusions between neighboring organs and also within an organ; they stopped growing soon after germination and subsequently died. ONI3 was shown to encode an enzyme that is most similar to Arabidopsis HOTHEAD and is involved in biosynthesis of long-chain fatty acids. Expression analyses showed that ONI3 was specifically expressed in the outermost cell layer in the shoot apex throughout life cycle, and the oni3 mutants had an aberrant outermost cell layer. Our results together with previous studies suggest that long-chain fatty acids are required for avoiding organ fusions and promoting normal shoot development in rice. PMID:24192297

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

    Perrot-Rechenmann, Catherine; Friml, Jiří

    2016-01-01

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

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

    SciTech Connect

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

    2014-03-07

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

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

    PubMed

    Watts-Williams, Stephanie J; Cavagnaro, Timothy R

    2015-11-01

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

  9. Novel Jbts17 mutant mouse model of Joubert syndrome with cilia transition zone defects and cerebellar and other ciliopathy related anomalies.

    PubMed

    Damerla, Rama Rao; Cui, Cheng; Gabriel, George C; Liu, Xiaoqin; Craige, Branch; Gibbs, Brian C; Francis, Richard; Li, You; Chatterjee, Bishwanath; San Agustin, Jovenal T; Eguether, Thibaut; Subramanian, Ramiah; Witman, George B; Michaud, Jacques L; Pazour, Gregory J; Lo, Cecilia W

    2015-07-15

    Recent studies identified a previously uncharacterized gene C5ORF42 (JBTS17) as a major cause of Joubert syndrome (JBTS), a ciliopathy associated with cerebellar abnormalities and other birth defects. Here we report the first Jbts17 mutant mouse model, Heart Under Glass (Hug), recovered from a forward genetic screen. Exome sequencing identified Hug as a S235P missense mutation in the mouse homolog of JBTS17 (2410089e03rik). Hug mutants exhibit multiple birth defects typical of ciliopathies, including skeletal dysplasia, polydactyly, craniofacial anomalies, kidney cysts and eye defects. Some Hug mutants exhibit congenital heart defects ranging from mild pulmonary stenosis to severe pulmonary atresia. Immunostaining showed JBTS17 is localized in the cilia transition zone. Fibroblasts from Hug mutant mice and a JBTS patient with a JBTS17 mutation showed ciliogenesis defects. Significantly, Hug mutant fibroblasts showed loss of not only JBTS17, but also NPHP1 and CEP290 from the cilia transition zone. Hug mutants exhibited reduced ciliation in the cerebellum. This was associated with reduction in cerebellar foliation. Using a fibroblast wound-healing assay, we showed Hug mutant cells cannot establish cell polarity required for directional cell migration. However, stereocilia patterning was grossly normal in the cochlea, indicating planar cell polarity is not markedly affected. Overall, we showed the JBTS pathophysiology is replicated in the Hug mutant mice harboring a Jbts17 mutation. Our findings demonstrate JBTS17 is a cilia transition zone component that acts upstream of other Joubert syndrome associated transition zone proteins NPHP1 and CEP290, indicating its importance in the pathogenesis of Joubert syndrome.

  10. Novel Jbts17 mutant mouse model of Joubert syndrome with cilia transition zone defects and cerebellar and other ciliopathy related anomalies

    PubMed Central

    Damerla, Rama Rao; Cui, Cheng; Gabriel, George C.; Liu, Xiaoqin; Craige, Branch; Gibbs, Brian C.; Francis, Richard; Li, You; Chatterjee, Bishwanath; San Agustin, Jovenal T.; Eguether, Thibaut; Subramanian, Ramiah; Witman, George B.; Michaud, Jacques L.; Pazour, Gregory J.; Lo, Cecilia W.

    2015-01-01

    Recent studies identified a previously uncharacterized gene C5ORF42 (JBTS17) as a major cause of Joubert syndrome (JBTS), a ciliopathy associated with cerebellar abnormalities and other birth defects. Here we report the first Jbts17 mutant mouse model, Heart Under Glass (Hug), recovered from a forward genetic screen. Exome sequencing identified Hug as a S235P missense mutation in the mouse homolog of JBTS17 (2410089e03rik). Hug mutants exhibit multiple birth defects typical of ciliopathies, including skeletal dysplasia, polydactyly, craniofacial anomalies, kidney cysts and eye defects. Some Hug mutants exhibit congenital heart defects ranging from mild pulmonary stenosis to severe pulmonary atresia. Immunostaining showed JBTS17 is localized in the cilia transition zone. Fibroblasts from Hug mutant mice and a JBTS patient with a JBTS17 mutation showed ciliogenesis defects. Significantly, Hug mutant fibroblasts showed loss of not only JBTS17, but also NPHP1 and CEP290 from the cilia transition zone. Hug mutants exhibited reduced ciliation in the cerebellum. This was associated with reduction in cerebellar foliation. Using a fibroblast wound-healing assay, we showed Hug mutant cells cannot establish cell polarity required for directional cell migration. However, stereocilia patterning was grossly normal in the cochlea, indicating planar cell polarity is not markedly affected. Overall, we showed the JBTS pathophysiology is replicated in the Hug mutant mice harboring a Jbts17 mutation. Our findings demonstrate JBTS17 is a cilia transition zone component that acts upstream of other Joubert syndrome associated transition zone proteins NPHP1 and CEP290, indicating its importance in the pathogenesis of Joubert syndrome. PMID:25877302

  11. Analysis of a Chinese hamster ovary cell mutant with defective mobilization of cholesterol from the plasma membrane to the endoplasmic reticulum.

    PubMed

    Jacobs, N L; Andemariam, B; Underwood, K W; Panchalingam, K; Sternberg, D; Kielian, M; Liscum, L

    1997-10-01

    The factors involved in shuttling cholesterol among cellular membranes have not been defined. Using amphotericin B selection, we previously isolated Chinese hamster ovary cell mutants expressing defects in intracellular cholesterol transport. Complementation analysis among seven mutants identified one cell line, mutant 3-6, with a unique defect. The present analysis revealed three key features of mutant 3-6. First, the movement of cholesterol both from the endoplasmic reticulum and through lysosomes to the plasma membrane was normal. However, when intact 3-6 cells were treated with sphingomyelinase, movement of plasma membrane cholesterol to acyl CoA:cholesterol acyltransferase in the endoplasmic reticulum was defective. Cellular cholesterol was mobilized to this enzyme upon activation by 25-hydroxycholesterol. Second, mutant 3-6 did not utilize endogenously synthesized sterol or low density lipoprotein-derived cholesterol for growth as effectively as parental Chinese hamster ovary cells. Finally, despite normal movement of cholesterol to the plasma membrane, mutant 3-6 was amphotericin B resistant. The plasma membrane cholesterol content was normal as assessed by cholesterol oxidase treatment and Semliki Forest virus fusion, which suggests that the 3-6 mutation alters the organization of cholesterol in the plasma membrane. Our characterization of this mutant cell line should facilitate the identification of gene(s) required for this transport pathway.

  12. Development of natto with germination-defective mutants of Bacillus subtilis (natto).

    PubMed

    Mitsui, Nobuo; Murasawa, Hisashi; Sekiguchi, Junichi

    2009-03-01

    The effects of cortex-lysis related genes with the pdaA, sleB, and cwlD mutations of Bacillus subtilis (natto) NAFM5 on sporulation and germination were investigated. Single or double mutations did not prevent normal sporulation, but did affect germination. Germination was severely inhibited by the double mutation of sleB and cwlD. The quality of natto made with the sleB cwlD double mutant was tested, and the amounts of glutamic acid and ammonia were very similar to those in the wild type. The possibility of industrial development of natto containing a reduced number of viable spores is presented.

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

    PubMed Central

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

    2009-01-01

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

  14. A cysG mutant strain of Rhizobium etli pleiotropically defective in sulfate and nitrate assimilation.

    PubMed Central

    Tate, R; Riccio, A; Iaccarino, M; Patriarca, E J

    1997-01-01

    By its inability to grow on sulfate as the sole sulfur source, a mutant strain (CTNUX8) of Rhizobium etli carrying Tn5 was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a cysG (siroheme synthetase)-homologous gene. By RNase protection assays, it was established that the cysG-like gene had a basal level of expression in thiosulfate- or cysteine-grown cells, which was induced when sulfate or methionine was used. Unlike its wild-type parent (strain CE3), the mutant strain, CTNUX8, was also unable to grow on nitrate as the sole nitrogen source and was unable to induce a high level of nitrite reductase. Despite its pleiotropic phenotype, strain CTNUX8 was able to induce pink, effective (N2-fixing) nodules on the roots of Phaseolus vulgaris plants. However, mixed inoculation experiments showed that strain CTNUX8 is significantly different from the wild type in its ability to nodulate. Our data support the notion that sulfate (or sulfite) is the sulfur source of R. etli in the rhizosphere, while cysteine, methionine, or glutathione is supplied by the root cells to bacteria growing inside the plant. PMID:9393698

  15. Peripheral nervous system defects in erbB2 mutants following genetic rescue of heart development

    PubMed Central

    Woldeyesus, Masresha T.; Britsch, Stefan; Riethmacher, Dieter; Xu, Lan; Sonnenberg-Riethmacher, Eva; Abou-Rebyeh, Faikah; Harvey, Richard; Caroni, Pico; Birchmeier, Carmen

    1999-01-01

    The ErbB2 tyrosine kinase functions as coreceptor for the neuregulin receptors ErbB3 and ErbB4 and can participate in signaling of EGF receptor (ErbB1), interleukin receptor gp130, and G-protein coupled receptors. ErbB2−/− mice die at midgestation because of heart malformation. Here, we report a genetic rescue of their heart development by myocardial expression of erbB2 cDNA that allows survival of the mutants to birth. In rescued erbB2 mutants, Schwann cells are lacking. Motoneurons form and can project to muscle, but nerves are poorly fasciculated and disorganized. Neuromuscular junctions form, as reflected in clustering of AChR and postsynaptic expression of the genes encoding the α-AChR, AChE, ε-AChR, and the RI subunit of the cAMP protein kinase. However, a severe loss of motoneurons on cervical and lumbar, but not on thoracic levels occurs. Our results define the roles of Schwann cells during motoneuron and synapse development, and reveal different survival requirements for distinct motoneuron populations. PMID:10521398

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

    PubMed

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

    2015-06-01

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

  17. Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility

    PubMed Central

    1993-01-01

    Coronin is an actin-binding protein in Dictyostelium discoideum that is enriched at the leading edge of the cells and in projections of the cell surface called crowns. The polypeptide sequence of coronin is distinguished by its similarities to the beta-subunits of trimeric G proteins (E. L. de Hostos, B. Bradtke, F. Lottspeich, R. Guggenheim, and G. Gerisch, 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:4097-4104). To elucidate the in vivo function of coronin, null mutants have been generated by gene replacement. The mutant cells lacking coronin grow and migrate more slowly than wild-type cells. When these cor- cells grow in liquid medium they become multinucleate, indicating a role of coronin in cytokinesis. To explore this role, coronin has been localized in mitotic wild-type cells by immunofluorescence labeling. During separation of the daughter cells, coronin is strongly accumulated at their distal portions including the leading edges. This contrasts with the localization of myosin II in the cleavage furrow and suggests that coronin functions independently of the conventional myosin in facilitating cytokinesis. PMID:8380174

  18. Construction of a lipopolysaccharide reporter cell line and its use in identifying mutants defective in endotoxin, but not TNF-alpha, signal transduction.

    PubMed

    Delude, R L; Yoshimura, A; Ingalls, R R; Golenbock, D T

    1998-09-15

    Gram-negative bacterial LPS is a potent activator of inflammatory responses. The binding of LPS to CD14 initiates signal transduction; however, the molecular processes immediately following this event remain unclear. We engineered an LPS-inducible fibroblast reporter cell line to facilitate the use of molecular genetic techniques to study the LPS signaling pathway. A plasmid containing the human Tac Ag cDNA under transcriptional control of the human E selectin promoter was cotransfected into Chinese hamster ovary (CHO)-K1 cells together with a CD14 expression plasmid. A cell line was obtained, 3E10, which upregulated expression of Tac following stimulation with LPS. Pools of mutagenized cells were exposed to LPS and then labeled with anti-Tac mAb. Cells that failed to up-regulate Tac expression were enriched by flow cytometry. Thirty clonal mutant cell lines were identified that continued to express CD14 and bind LPS, but failed to express Tac or translocate nuclear factor-kappaB (NF-kappaB) following LPS exposure. TNF-alpha-treated mutant cells continued to express Tac and translocate NF-kappaB. An analysis of LPS-induced NF-kappaB activity in heterokaryons derived from polyethylene glycol-fused cell lines indicated that recessive mutations in genes encoding components of the LPS signaling pathway accounted for the signaling defects. To date, two complementation groups have been identified from 11 cell lines analyzed. These data demonstrate that the TNF-alpha signaling pathway diverges from the LPS pathway early in the signal-transduction cascade despite similarities in LPS- and TNF-alpha-induced responses. Identification of the genes affected in these mutant reporter cells should identify heretofore-elusive components of the LPS signaling cascade.

  19. Cell-dependent gag mutants of HIV-1 are crucially defective at the stage of uncoating/reverse transcription in non-permissive cells.

    PubMed

    Koh, K; Miyaura, M; Yoshida, A; Sakurai, A; Fujita, M; Adachi, A

    2000-10-01

    We have previously shown that some of the human immunodeficiency virus type 1 (HIV-1) gag matrix (MA), capsid (CA), and nucleocapsid (NC) mutants display host-cell-dependent replication potential, and that they are defective at the early phase of the virus replication cycle in non-permissive cells. To determine the defective replication stage of the cell-dependent mutants precisely, the processes of virus entry into cells and virus DNA synthesis were monitored by the highly sensitive enzyme-linked immunosorbent assay and polymerase chain reaction amplification analysis. The results obtained indicated that all the cell-dependent MA, CA and NC mutants are defective at the stage of uncoating/reverse transcription, and that a cellular factor(s) is involved in this process.

  20. A Upf3b-mutant mouse model with behavioral and neurogenesis defects.

    PubMed

    Huang, L; Shum, E Y; Jones, S H; Lou, C-H; Dumdie, J; Kim, H; Roberts, A J; Jolly, L A; Espinoza, J L; Skarbrevik, D M; Phan, M H; Cook-Andersen, H; Swerdlow, N R; Gecz, J; Wilkinson, M F

    2017-09-26

    Nonsense-mediated RNA decay (NMD) is a highly conserved and selective RNA degradation pathway that acts on RNAs terminating their reading frames in specific contexts. NMD is regulated in a tissue-specific and developmentally controlled manner, raising the possibility that it influences developmental events. Indeed, loss or depletion of NMD factors have been shown to disrupt developmental events in organisms spanning the phylogenetic scale. In humans, mutations in the NMD factor gene, UPF3B, cause intellectual disability (ID) and are strongly associated with autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD) and schizophrenia (SCZ). Here, we report the generation and characterization of mice harboring a null Upf3b allele. These Upf3b-null mice exhibit deficits in fear-conditioned learning, but not spatial learning. Upf3b-null mice also have a profound defect in prepulse inhibition (PPI), a measure of sensorimotor gating commonly deficient in individuals with SCZ and other brain disorders. Consistent with both their PPI and learning defects, cortical pyramidal neurons from Upf3b-null mice display deficient dendritic spine maturation in vivo. In addition, neural stem cells from Upf3b-null mice have impaired ability to undergo differentiation and require prolonged culture to give rise to functional neurons with electrical activity. RNA sequencing (RNAseq) analysis of the frontal cortex identified UPF3B-regulated RNAs, including direct NMD target transcripts encoding proteins with known functions in neural differentiation, maturation and disease. We suggest Upf3b-null mice serve as a novel model system to decipher cellular and molecular defects underlying ID and neurodevelopmental disorders.Molecular Psychiatry advance online publication, 26 September 2017; doi:10.1038/mp.2017.173.

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

    PubMed

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

    2014-09-01

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

  2. Mechanisms of pharmacological rescue of trafficking defective hERG mutant channels in human long QT syndrome

    PubMed Central

    Gong, Qiuming; Jones, Melanie A.; Zhou, Zhengfeng

    2006-01-01

    Long QT syndrome type 2 is caused by mutations in the human ether-a-go-go-related gene (hERG). We previously reported that the N470D mutation is retained in the endoplasmic reticulum (ER) but can be rescued to the plasma membrane by hERG channel blocker E-4031. The mechanisms of ER retention and how E-4031 rescues the N470D mutant are poorly understood. In this study, we investigated the interaction of hERG channels with ER chaperone protein calnexin. Using coimmunoprecipitation, we showed that the immature forms of both wild type hERG and N470D associated with calnexin. The association required N-linked glycosylation of hERG channels. Pulse-chase analysis revealed that N470D had a prolonged association with calnexin compared to wild type hERG, and E-4031 shortened the time course of calnexin association with N470D. To test whether the prolonged association of N470D with calnexin is due to defective folding of mutant channels, we studied hERG channel folding using trypsin digestion method. We found that N470D and the immature form of wild type hERG were more sensitive to trypsin digestion than the mature form of wild type hERG. In the presence of E-4031, N470D became more resistant to trypsin even in the conditions that its ER-to-Golgi transport was blocked by brefeldin A. These results suggest that defective folding of N470D contributes to its prolonged association with calnexin and ER retention, and that E-4031 may restore proper folding of the N470D channel leading to its cell surface expression. PMID:16361248

  3. Improved Functional Expression of Human Cardiac Kv1.5 Channels and Trafficking-Defective Mutants by Low Temperature Treatment

    PubMed Central

    Toyoda, Futoshi; Matsuura, Hiroshi

    2014-01-01

    We herein investigated the effect of low temperature exposure on the expression, degradation, localization and activity of human Kv1.5 (hKv1.5). In hKv1.5-expressing CHO cells, the currents were significantly increased when cultured at a reduced temperature (28°C) compared to those observed at 37°C. Western blot analysis indicated that the protein levels (both immature and mature proteins) of hKv1.5 were significantly elevated under the hypothermic condition. Treatment with a proteasome inhibitor, MG132, significantly increased the immature, but not the mature, hKv1.5 protein at 37°C, however, there were no changes in either the immature or mature hKv1.5 proteins at low temperature following MG132 exposure. These observations suggest that the enhancement of the mature hKv1.5 protein at reduced temperature may not result from the inhibition of proteolysis. Moreover, the hKv1.5 fluorescence signal in the cells increased significantly on the cell surface at 28°C versus those cultured at 37°C. Importantly, the low temperature treatment markedly shifted the subcellular distribution of the mature hKv1.5, which showed considerable overlap with the trans-Golgi component. Experiments using tunicamycin, an inhibitor of N-glycosylation, indicated that the N-glycosylation of hKv1.5 is more effective at 28°C than at 37°C. Finally, the hypothermic treatment also rescued the protein expression and currents of trafficking-defective hKv1.5 mutants. These results indicate that low temperature exposure stabilizes the protein in the cellular organelles or on the plasma membrane, and modulates its maturation and trafficking, thus enhancing the currents of hKv1.5 and its trafficking defect mutants. PMID:24663680

  4. Characterization of aldehyde ferredoxin oxidoreductase gene defective mutant in Magnetospirillum magneticum AMB-1.

    PubMed

    Wahyudi, Aris Tri; Takeyama, Haruko; Okamura, Yoshiko; Fukuda, Yorikane; Matsunaga, Tadashi

    2003-03-28

    A non-magnetic mutant of Magnetospirillum magneticum AMB-1, designated as NMA21, was generated by mini-Tn5 transposon mutagenesis to identify genes involved in bacterial magnetic particle (BMP) synthesis. Alignment of the DNA sequences flanking the transposon allowed the isolation of an open reading frame (ORF2) within an operon consisting of five genes. The amino acid sequence of ORF2 showed homology with tungsten-containing aldehyde ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus (48% identity and 64% similarity), which functions for aldehyde oxidation. AOR was found to be expressed under microaerobic conditions and localized in the cytoplasm of AMB-1. Iron uptake and growth of NMA21 were lower than wild type. Transmission electron microscopy (TEM) of NMA21 revealed that no BMPs were completely synthesized, but polyhydroxybutyrate (PHB)-like granules were persistently produced. These results indicate that AOR may contribute to ferric iron reduction during BMP synthesis in M. magneticum AMB-1 under microaerobic respiration.

  5. Mutational analysis of BTAF1-TBP interaction: BTAF1 can rescue DNA-binding defective TBP mutants.

    PubMed

    Klejman, Marcin P; Zhao, Xuemei; van Schaik, Frederik M A; Herr, Winship; Timmers, H Th Marc

    2005-01-01

    The BTAF1 transcription factor interacts with TATA-binding protein (TBP) to form the B-TFIID complex, which is involved in RNA polymerase II transcription. Here, we present an extensive mapping study of TBP residues involved in BTAF1 interaction. This shows that residues in the concave, DNA-binding surface of TBP are important for BTAF1 binding. In addition, BTAF1 interacts with residues in helix 2 on the convex side of TBP as assayed in protein-protein and in DNA-binding assays. BTAF1 drastically changes the TATA-box binding specificity of TBP, as it is able to recruit DNA-binding defective TBP mutants to both TATA-containing and TATA-less DNA. Interestingly, other helix 2 interacting factors, such as TFIIA and NC2, can also stabilize mutant TBP binding to DNA. In contrast, TFIIB which interacts with a distinct surface of TBP does not display this activity. Since many proteins contact helix 2 of TBP, this provides a molecular basis for mutually exclusive TBP interactions and stresses the importance of this structural element for eukaryotic transcription.

  6. Defective pairing and synaptonemal complex formation in a Sordaria mutant (spo44) with a translocated segment of the nucleolar organizer.

    PubMed

    Zickler, D; de Lares, L; Moreau, P J; Leblon, G

    1985-01-01

    The recessive meiotic mutant spo44 of Sordaria macrospora, with 90% ascospore abortion, exhibits striking effects on recombination (67% decrease), irregular segregation of the almost unpaired homologues, and a decrease in chiasma frequency in the few cases where bivalents are formed. Three-dimensional reconstructions of ten prophase nuclei indicate that pairing, as judged by the absence of fully formed synaptonemal complexes (SC), is not achieved although lateral elements (LE) assemble. The pairing failure is attributable to defects in the alignment of homologous chromosomes. The leptotene alignment seen in the wild type before SC formation was not observed in the spo44 nuclei. Dense material, considered to be precursor of SC central elements, was found scattered among the LE in two nuclei. The behaviour of spo44 substantiates the hypothesis that chromosome matching and SC formation are separable events. - The total length of the LE in the mutant is the same as in the wild type, but due to variable numbers and length of the individual LE, homologues cannot be lined up. Light microscopic observations indicate that the irregular length and number of LE is due to extensive chromosome breakage. The wild-type function corresponding to spo44 is required for both LE integrity and chromosome matching. Reconstructions of heterozygous nuclei reveal the presence of a supernumerary nucleolar organizer in one arm of chromosome 7. It is suggested that rDNA has been inserted into a gene whose function is involved in pairing or into a controlling sequence that interacts with the pairing process.

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

    PubMed Central

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

    2015-01-01

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

  8. Targeted ANP32E mutant mice do not demonstrate obvious movement defects.

    PubMed

    Wong, Peiyan; Leo, Vonny I; Low, Meijun; Mak, Tak W; Zhang, Xiaodong; Reilly, Patrick T

    2013-01-01

    The ANP32 family of proteins have been implicated in neuronal function through biochemical and cellular biology studies in neurons, as well as by recent behavioural studies of a gene-trapped loss-of-function mutation of Anp32e in mice, particularly with respect to fine motor function. A second targeted allele of the Anp32e, however, did not appear to demonstrate neurological phenotypes. Using a stringently controlled cohort of ten-generation backcrossed, co-caged, sex-matched, littermate pairs, we assayed for potential motor defects in the targeted ANP32E-deficient mice. We found no phenotypic difference in any assays. Since it is unlikely that the gene-trap is a more complete loss-of-function, our results suggest that ANP32E has no appreciable effect on motor functions and that genetic background differences most likely account for the gene-trap phenomena.

  9. ‘Cyclic alopecia’ in Msx2 mutants: defects in hair cycling and hair shaft differentiation

    PubMed Central

    Ma, Liang; Liu, Jian; Wu, Tobey; Plikus, Maksim; Jiang, Ting-Xin; Bi, Qun; Liu, Yi-Hsin; Müller-Röver, Sven; Peters, Heiko; Sundberg, John P.; Maxson, Rob; Maas, Richard L.; Chuong, Cheng-Ming

    2015-01-01

    SUMMARY Msx2-deficient mice exhibit progressive hair loss, starting at P14 and followed by successive cycles of wavelike regrowth and loss. During the hair cycle, Msx2 deficiency shortens anagen phase, but prolongs catagen and telogen. Msx2-deficient hair shafts are structurally abnormal. Molecular analyses suggest a Bmp4/Bmp2/Msx2/Foxn1 acidic hair keratin pathway is involved. These structurally abnormal hairs are easily dislodged in catagen implying a precocious exogen. Deficiency in Msx2 helps to reveal the distinctive skin domains on the same mouse. Each domain cycles asynchronously – although hairs within each skin domain cycle in synchronized waves. Thus, the combinatorial defects in hair cycling and differentiation, together with concealed skin domains, account for the cyclic alopecia phenotype. PMID:12466204

  10. 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

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

    PubMed Central

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

    2015-01-01

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

  12. New nodulation mutants responsible for infection thread development in Lotus japonicus.

    PubMed

    Yano, Koji; Tansengco, Myra L; Hio, Taihei; Higashi, Kuniko; Murooka, Yoshikatsu; Imaizumi-Anraku, Haruko; Kawaguchi, Masayoshi; Hayashi, Makoto

    2006-07-01

    Legume plants develop specialized root organs, the nodules, through a symbiotic interaction with rhizobia. The developmental process of nodulation is triggered by the bacterial microsymbiont but regulated systemically by the host legume plants. Using ethylmethane sulfonate mutagenesis as a tool to identify plant genes involved in symbiotic nodule development, we have isolated and analyzed five nodulation mutants, Ljsym74-3, Ljsym79-2, Ljsym79-3, Ljsym80, and Ljsym82, from the model legume Lotus japonicus. These mutants are defective in developing functional nodules and exhibit nitrogen starvation symptoms after inoculation with Mesorhizobium loti. Detailed observation revealed that infection thread development was aborted in these mutants and the nodules formed were devoid of infected cells. Mapping and complementation tests showed that Ljsym74-3, and Ljsym79-2 and Ljsym79-3, were allelic with reported mutants of L. japonicus, alb1 and crinkle, respectively. The Ljsym82 mutant is unique among the mutants because the infection thread was aborted early in its development. Ljsym74-3 and Ljsym80 were characterized as mutants with thick infection threads in short root hairs. Map-based cloning and molecular characterization of these genes will help us understand the genetic mechanism of infection thread development in L. japonicus.

  13. A Colony Color Assay for Saccharomyces Cerevisiae Mutants Defective in Kinetochore Structure and Function

    PubMed Central

    Perier, F.; Carbon, J.

    1992-01-01

    We have designed a colony color assay for monitoring centromere DNA-protein interactions in yeast (Saccharomyces cerevisiae). The assay is based on the ability of centromere DNA sequences to block (in cis) transcription initiated from a hybrid CEN-GAL1 promoter. Using a IacZ reporter gene under control of the CEN-GAL1 promoter, we screened colonies derived from mutagenized cells for a blue color phenotype indicative of derepression of the hybrid construct. A limited screen in which a 61-bp CEN11 DNA fragment containing an intact CDEIII subregion plus flanking sequences was used as the ``pseudo-operator'' led to the identification of mutations (blu) in three complementation groups. The blu1 mutants exhibited a decrease in activity of the major CEN DNA-binding proteins in vitro. The BLU1 gene was shown to be identical to the previously isolated SPT3 gene, known to be involved in the transcriptional regulation of a subset of yeast genes. Our results indicate that the BLU1/SPT3 gene product may also be required to maintain optimal levels of functional centromere DNA-binding proteins. PMID:1398062

  14. nip, a symbiotic Medicago truncatula mutant that forms root nodules with aberrant infection threads and plant defense-like response.

    PubMed

    Veereshlingam, Harita; Haynes, Janine G; Penmetsa, R Varma; Cook, Douglas R; Sherrier, D Janine; Dickstein, Rebecca

    2004-11-01

    To investigate the legume-Rhizobium symbiosis, we isolated and studied a novel symbiotic mutant of the model legume Medicago truncatula, designated nip (numerous infections and polyphenolics). When grown on nitrogen-free media in the presence of the compatible bacterium Sinorhizobium meliloti, the nip mutant showed nitrogen deficiency symptoms. The mutant failed to form pink nitrogen-fixing nodules that occur in the wild-type symbiosis, but instead developed small bump-like nodules on its roots that were blocked at an early stage of development. Examination of the nip nodules by light microscopy after staining with X-Gal for S. meliloti expressing a constitutive GUS gene, by confocal microscopy following staining with SYTO-13, and by electron microscopy revealed that nip initiated symbiotic interactions and formed nodule primordia and infection threads. The infection threads in nip proliferated abnormally and very rarely deposited rhizobia into plant host cells; rhizobia failed to differentiate further in these cases. nip nodules contained autofluorescent cells and accumulated a brown pigment. Histochemical staining of nip nodules revealed this pigment to be polyphenolic accumulation. RNA blot analyses demonstrated that nip nodules expressed only a subset of genes associated with nodule organogenesis, as well as elevated expression of a host defense-associated phenylalanine ammonia lyase gene. nip plants were observed to have abnormal lateral roots. nip plant root growth and nodulation responded normally to ethylene inhibitors and precursors. Allelism tests showed that nip complements 14 other M. truncatula nodulation mutants but not latd, a mutant with a more severe nodulation phenotype as well as primary and lateral root defects. Thus, the nip mutant defines a new locus, NIP, required for appropriate infection thread development during invasion of the nascent nodule by rhizobia, normal lateral root elongation, and normal regulation of host defense-like responses

  15. Defective erythroid differentiation in miR-451 mutant mice mediated by 14-3-3ζ

    PubMed Central

    Patrick, David M.; Zhang, Cheng C.; Tao, Ye; Yao, Huiyu; Qi, Xiaoxia; Schwartz, Robert J.; Jun-Shen Huang, Lily; Olson, Eric N.

    2010-01-01

    Erythrocyte formation occurs throughout life in response to cytokine signaling. We show that microRNA-451 (miR-451) regulates erythropoiesis in vivo. Mice lacking miR-451 display a reduction in hematrocrit, an erythroid differentiation defect, and ineffective erythropoiesis in response to oxidative stress. 14-3-3ζ, an intracellular regulator of cytokine signaling that is repressed by miR-451, is up-regulated in miR-451−/− erythroblasts, and inhibition of 14-3-3ζ rescues their differentiation defect. These findings reveal an essential role of 14-3-3ζ as a mediator of the proerythroid differentiation actions of miR-451, and highlight the therapeutic potential of miR-451 inhibitors. PMID:20679397

  16. Mutant huntingtin's interaction with mitochondrial protein Drp1 impairs mitochondrial biogenesis and causes defective axonal transport and synaptic degeneration in Huntington's disease.

    PubMed

    Shirendeb, Ulziibat P; Calkins, Marcus J; Manczak, Maria; Anekonda, Vishwanath; Dufour, Brett; McBride, Jodi L; Mao, Peizhong; Reddy, P Hemachandra

    2012-01-15

    The purpose of this study was to investigate the link between mutant huntingtin (Htt) and neuronal damage in relation to mitochondria in Huntington's disease (HD). In an earlier study, we determined the relationship between mutant Htt and mitochondrial dynamics/synaptic viability in HD patients. We found mitochondrial loss, abnormal mitochondrial dynamics and mutant Htt association with mitochondria in HD patients. In the current study, we sought to expand on our previous findings and further elucidate the relationship between mutant Htt and mitochondrial and synaptic deficiencies. We hypothesized that mutant Htt, in association with mitochondria, alters mitochondrial dynamics, leading to mitochondrial fragmentation and defective axonal transport of mitochondria in HD neurons. In this study, using postmortem HD brains and primary neurons from transgenic BACHD mice, we identified mutant Htt interaction with the mitochondrial protein Drp1 and factors that cause abnormal mitochondrial dynamics, including GTPase Drp1 enzymatic activity. Further, using primary neurons from BACHD mice, for the first time, we studied axonal transport of mitochondria and synaptic degeneration. We also investigated the effect of mutant Htt aggregates and oligomers in synaptic and mitochondrial deficiencies in postmortem HD brains and primary neurons from BACHD mice. We found that mutant Htt interacts with Drp1, elevates GTPase Drp1 enzymatic activity, increases abnormal mitochondrial dynamics and results in defective anterograde mitochondrial movement and synaptic deficiencies. These observations support our hypothesis and provide data that can be utilized to develop therapeutic targets that are capable of inhibiting mutant Htt interaction with Drp1, decreasing mitochondrial fragmentation, enhancing axonal transport of mitochondria and protecting synapses from toxic insults caused by mutant Htt.

  17. Suppression of Adipogenesis by Pathogenic Seipin Mutant Is Associated with Inflammatory Response

    PubMed Central

    Takeda, Kosuke; Lim, Xuemei; Sugii, Shigeki; Radda, George K.; Han, Weiping

    2013-01-01

    Background While pathogenic mutations in BSCL2/Seipin cause congenital generalized lipodystrophy, the underlying mechanism is largely unknown. In this study, we investigated whether and how the pathogenic missense A212P mutation of Seipin (Seipin-A212P) inhibits adipogenesis. Methodology/Results We analyzed gene expression and lipid accumulation in stable 3T3-L1 cell lines expressing wild type (3T3-WT), non-lipodystrophic mutants N88S (3T3-N88S) and S90L (3T3-S90L), or lipodystrophic mutant A212P Seipin (3T3-A212P). When treated with adipogenic cocktail, 3T3-WT, 3T3-N88S and 3T3-S90L cells exhibited proper differentiation into mature adipocytes, indistinguishable from control 3T3-L1 cells. In contrast, adipogenesis was significantly impaired in 3T3-A212P cells. The defective adipogenesis in 3T3-A212P cells could be partially rescued by either PPARγ agonist or PPARγ overexpression. Gene expression profiling by microarray revealed that inhibition of adipogenesis was associated with activation of inflammatory genes including IL-6 and iNOS. We further demonstrated that Seipin-A212P expression at pre-differentiation stages significantly activated inflammatory responses by using an inducible expression system. The inflammation-associated inhibition of adipogenesis could be rescued by treatment with anti-inflammatory agents. Conclusions These results suggest that pathogenic Seipin-A212P inhibits adipogenesis and the inhibition is associated with activation of inflammatory pathways at pre-differentiation stages. Use of anti-inflammatory drugs may be a potential strategy for the treatment of lipodystrophy. PMID:23520483

  18. Biochemical defects of mutant nudel alleles causing early developmental arrest or dorsalization of the Drosophila embryo.

    PubMed Central

    LeMosy, E K; Leclerc, C L; Hashimoto, C

    2000-01-01

    The nudel gene of Drosophila is maternally required both for structural integrity of the egg and for dorsoventral patterning of the embryo. It encodes a structurally modular protein that is secreted by ovarian follicle cells. Genetic and molecular studies have suggested that the Nudel protein is also functionally modular, with a serine protease domain that is specifically required for ventral development. Here we describe biochemical and immunolocalization studies that provide insight into the molecular basis for the distinct phenotypes produced by nudel mutations and for the interactions between these alleles. Mutations causing loss of embryonic dorsoventral polarity result in a failure to activate the protease domain of Nudel. Our analyses support previous findings that catalytic activity of the protease domain is required for dorsoventral patterning and that the Nudel protease is auto-activated and reveal an important role for a region adjacent to the protease domain in Nudel protease function. Mutations causing egg fragility and early embryonic arrest result in a significant decrease in extracellular Nudel protein, due to defects in post-translational processing, stability, or secretion. On the basis of these and other studies of serine proteases, we suggest potential mechanisms for the complementary and antagonistic interactions between the nudel alleles. PMID:10628985

  19. DNA-binding-defective mutants of the Epstein-Barr virus lytic switch activator Zta transactivate with altered specificities.

    PubMed Central

    Flemington, E K; Lytle, J P; Cayrol, C; Borras, A M; Speck, S H

    1994-01-01

    The Epstein-Barr virus BRLF1 and BZLF1 genes are the first viral genes transcribed upon induction of the viral lytic cycle. The protein products of both genes (referred to here as Rta and Zta, respectively) activate expression of other viral genes, thereby initiating the lytic cascade. Among the viral antigens expressed upon induction of the lytic cycle, however, Zta is unique in its ability to disrupt viral latency; expression of the BZLF1 gene is both necessary and sufficient for triggering the viral lytic cascade. We have previously shown that Zta can activate its own promoter (Zp), through binding to two Zta recognition sequences (ZIIIA and ZIIIB). Here we describe mutant Zta proteins that do not bind DNA (referred to as Zta DNA-binding mutants [Zdbm]) but retain the ability to transactivate Zp. Consistent with the inability of these mutants to bind DNA, transactivation of Zp by Zdbm is not dependent on the Zta recognition sequences. Instead, transactivation by Zdbm is dependent upon promoter elements that bind cellular factors. An examination of other viral and cellular promoters identified promoters that are weakly responsive or unresponsive to Zdbm. An analysis of a panel of artificial promoters containing one copy of various promoter elements demonstrated a specificity for Zdbm activation that is distinct from that of Zta. These results suggest that non-DNA-binding forms of some transactivators retain the ability to transactivate specific target promoters without direct binding to DNA. Images PMID:8164660

  20. Cell shape and interaction defects in alpha-spectrin mutants of Drosophila melanogaster

    PubMed Central

    1993-01-01

    We show that the alpha-spectrin gene is essential for larval survival and development by characterizing several alpha-spectrin mutations in Drosophila. P-element minigene rescue and sequence analysis were used to identify the alpha-spectrin gene as the l(3)dre3 complementation group of the Dras-Roughened-ecdysoneless region of chromosome 3 (Sliter et al., 1988). Germ line transformants carrying an alpha-spectrin cDNA, whose expression is driven by the ubiquitin promoter, fully rescued the first to second instar lethality characteristic of the l(3)dre3 alleles. The molecular defects in two gamma-ray-induced alleles were identified. One of these mutations, which resulted in second instar lethality, contained a 73-bp deletion in alpha-spectrin segment 22 (starting at amino acid residue 2312), producing a premature stop codon between the two EF hands found in this segment. The second mutation, which resulted in first instar lethality, contained a 20 base pair deletion in the middle of segment 1 (at amino acid residue 92), resulting in a premature stop codon. Examination of the spectrin- deficient larvae revealed a loss of contact between epithelial cells of the gut and disruption of cell-substratum interactions. The most pronounced morphological change was seen in tissues of complex cellular architecture such as the middle midgut where a loss of cell contact between cup-shaped cuprophilic cells and neighboring interstitial cells was accompanied by disorganization of the cuprophilic cell brush borders. Our examination of spectrin deficient larvae suggests that an important role of non-erythroid spectrin is to stabilize cell to cell interactions that are critical for the maintenance of cell shape and subcellular organization within tissues. PMID:8276898

  1. Distinct Innate Responses are Induced by Attenuated Salmonella enterica serovar Typhimurium Mutants

    PubMed Central

    Powell, Daniel A.; Roberts, Lydia M.; Ledvina, Hannah E.; Sempowski, Gregory D.; Curtiss, Roy; Frelinger, Jeffrey A.

    2015-01-01

    Upon bacterial infection the host cells generate a wide variety of cytokines. Genetic attenuation of bacterial physiological pathogens can be accomplished not only by disruption of normal bacterial processes, but also by the loss of the ability to redirect the host immune system. We examined nine attenuated Salmonella Typhimurium mutants for their ability to replicate as well as the cytokines produced after infection of Bone Marrow Derived Macrophages (BMDM). Infection of BMDM with attenuated Salmonella mutants led to host cytokine patterns distinct from those that followed WT infection. Surprisingly, each bacterial mutant had a unique cytokine signature. Because some of the mutants induced an IL-10 response not seen in WT, we examined the role of IL-10 on Salmonella replication. Surprisingly, addition of IL-10 before or concurrent with infection restricted growth of WT Salmonella in BMDM. Bacterial attenuation is not a single process and results in attenuated host responses, which result in unique patterns for each attenuated mutants. PMID:26546408

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

    SciTech Connect

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

    2012-01-01

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

  3. Immune response to Candida albicans is preserved despite defect in O-mannosylation of secretory proteins.

    PubMed

    Corbucci, Cristina; Cenci, Elio; Skrzypek, Franck; Gabrielli, Elena; Mosci, Paolo; Ernst, Joachim F; Bistoni, Francesco; Vecchiarelli, Anna

    2007-12-01

    The PMT gene family in Candida albicans encodes five isoforms of the protein mannosyltransferases that initiate O-mannosylation of secretory proteins. Mutations at the Pmt level have been associated with differences in pathogenicity, e.g. in contrast to pmt5/pmt5, pmt2/PMT2 mutants showed poor virulence. Our objective was to determine whether these differences were related to the capacity of pmt2/PMT2 and pmt5/pmt5 to (i) express differences in selected virulence factors, and (ii) stimulate the natural immune system. The results show that pmt mutants (i) form hyphae in serum, (ii) show defective production of proteases but not of phospholipases with respect to the parental strain, (iii) undergo mycelial transition in the kidneys of hematogenously infected animals, (iv) are phagocytosed and killed by macrophages similar to the parental strain, although neutrophils are unable to destroy pmt5/pmt5, (v) engage TLR4 and stimulate MyD88 leading to NF-kappaB activation, and (vi) stimulate cytokine production by macrophages. Collectively our findings suggest that the defect in protein O-mannosylation in C. albicans cause attenuation of the virulence although the antigenic factors that retain the capacity to stimulate an efficient immune response are preserved.

  4. Light-induced pigment granule migration in the retinular cells of Drosophila melanogaster. Comparison of wild type with ERG-defective mutants

    PubMed Central

    1981-01-01

    The dependence of pigment granule migration (PGM) upon the receptor potential was examined using several strains of electroretinogram (ERG)- defective mutants of Drosophila melanogaster. The mutants that have a defective lamina component but a normal receptor component of the ERG (no on-transient A [nonA] and tan) exhibited normal pigment granule migration. The mutants that have very small or no receptor potentials (certain no receptor potential A [norpA] alleles), on the other hand, exhibited no PGM. In the case of the temperature-sensitive norpA mutant, norpAH52, normal PGM was present at 17 degrees but not at 32 degrees C or above, corresponding to its electrophysiological phenotype. In the transient receptor potential (trp) mutant, whose receptor potential decays to the baseline within a few seconds during a sustained light stimulus, the pigment granules initially moved close to the rhabdomere when light was turned on but moved away after about 5 s during a sustained light stimulus. All these results lend strong support to the notion that PGM is initiated by a light-evoked depolarization of the receptor membrane, i.e., the receptor potential. However, under certain experimental conditions, the receptor potentials failed to induce PGM in the trp mutant. The depolarization of the receptor, thus, appears to be closely associated with PGM but is not a sufficient condition for PGM. PMID:6790662

  5. Export of the outer membrane lipoprotein is defective in secD, secE, and secF mutants of Escherichia coli.

    PubMed Central

    Sugai, M; Wu, H C

    1992-01-01

    The export of major outer membrane lipoprotein has been found to be affected in secD, secE, and secF mutants of Escherichia coli, which are defective in protein export in general. After a shift to the nonpermissive temperature, the kinetics of accumulation of prolipoprotein and pre-OmpA protein was indistinguishable from that of pre-OmpA protein accumulation in the secD and secF mutants but different in the secE mutant. The prolipoprotein accumulated in the secD, secE, and secF mutants at the nonpermissive temperature was not modified with glyceride. We conclude from these results and those of previous studies that the export of lipoprotein requires all common sec gene products except the SecB protein, i.e., the SecA, SecD, SecE, SecF, and SecY proteins. Images PMID:1556071

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

    PubMed

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

    1994-06-11

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

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

    SciTech Connect

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

    2009-12-20

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

  9. Acetylation-defective mutants of Pparγ are associated with decreased lipid synthesis in breast cancer cells

    PubMed Central

    Tian, Lifeng; Wang, Chenguang; Hagen, Fred K.; Gormley, Michael; Addya, Sankar; Soccio, Raymond; Casimiro, Mathew C.; Zhou, Jie; Powell, Michael J.; Xu, Ping; Deng, Haiteng; Sauve, Anthony A.; Pestell, Richard G.

    2014-01-01

    In our prior publications we characterized a conserved acetylation motif (K(R)xxKK) of evolutionarily related nuclear receptors. Recent reports showed that peroxisome proliferator activated receptor gamma (PPARγ) deacetylation by SIRT1 is involved in delaying cellular senescence and maintaining the brown remodeling of white adipose tissue. However, it still remains unknown whether lysyl residues 154 and 155 (K154/155) of the conserved acetylation motif (RIHKK) in Pparγ1 are acetylated. Herein, we demonstrate that Pparγ1 is acetylated and regulated by both endogenous TSA-sensitive and NAD-dependent deacetylases. Acetylation of lysine 154 was identified by mass spectrometry (MS) while deacetylation of lysine 155 by SIRT1 was confirmed by in vitro deacetylation assay. An in vivo labeling assay revealed K154/K155 as bona fide acetylation sites. The conserved acetylation sites of Pparγ1 and the catalytic domain of SIRT1 are both required for the interaction between Pparγ1 and SIRT1. Sirt1 and Pparγ1 converge to govern lipid metabolism in vivo. Acetylation-defective mutants of Pparγ1 were associated with reduced lipid synthesis in ErbB2 overexpressing breast cancer cells. Together, these results suggest that the conserved lysyl residues K154/K155 of Pparγ1 are acetylated and play an important role in lipid synthesis in ErbB2-positive breast cancer cells. PMID:25229978

  10. Tropomyosin-dependent filament formation by a polymerization-defective mutant yeast actin (V266G,L267G).

    PubMed

    Wen, K K; Kuang, B; Rubenstein, P A

    2000-12-22

    A major function of tropomyosin (TPM) in nonmuscle cells may be stabilization of F-actin by binding longitudinally along the actin filament axis. However, no clear evidence exists in vitro that TPM can significantly affect the critical concentration of actin. We previously made a polymerization-defective mutant actin, GG (V266G, L267G). This actin will not polymerize alone at 25 degrees C but will in the presence of phalloidin or beryllium fluoride. With beryllium fluoride, but not phalloidin, this polymerization rescue is cold-sensitive. We show here that GG-actin polymerizability was restored by cardiac tropomyosin and yeast TPM1 and TPM2 at 25 degrees C with rescue efficiency inversely proportional to TPM length (TPM2 > TPM1 > cardiac tropomyosin), indicating the importance of the ends in polymerization rescue. In the presence of TPM, the apparent critical concentration of actin is 5.5 microm, 10-15-fold higher than that of wild type actin but well below that of the GG-actin alone (>20 microm). Non N-acetylated TPMs did not rescue GG-actin polymerization. The TPMs did not prevent cold-induced depolymerization of GG F-actin. TPM-dependent GG-actin polymerization did not occur at temperatures below 20 degrees C. Polymerization rescue may depend initially on the capture of unstable GG-F-actin oligomers by the TPM, resulting in the strengthening of actin monomer-monomer contacts along the filament axis.

  11. Acetylation-defective mutant of Pparγ is associated with decreased lipid synthesis in breast cancer cells.

    PubMed

    Tian, Lifeng; Wang, Chenguang; Hagen, Fred K; Gormley, Michael; Addya, Sankar; Soccio, Raymond; Casimiro, Mathew C; Zhou, Jie; Powell, Michael J; Xu, Ping; Deng, Haiteng; Sauve, Anthony A; Pestell, Richard G

    2014-09-15

    In our prior publications we characterized a conserved acetylation motif (K(R)xxKK) of evolutionarily related nuclear receptors. Recent reports showed that peroxisome proliferator activated receptor gamma (PPARγ) deacetylation by SIRT1 is involved in delaying cellular senescence and maintaining the brown remodeling of white adipose tissue. However, it still remains unknown whether lysyl residues 154 and 155 (K154/155) of the conserved acetylation motif (RIHKK) in Pparγ1 are acetylated. Herein, we demonstrate that Pparγ1 is acetylated and regulated by both endogenous TSA-sensitive and NAD-dependent deacetylases. Acetylation of lysine 154 was identified by mass spectrometry (MS) while deacetylation of lysine 155 by SIRT1 was confirmed by in vitro deacetylation assay. An in vivo labeling assay revealed K154/K155 as bona fide acetylation sites. The conserved acetylation sites of Pparγ1 and the catalytic domain of SIRT1 are both required for the interaction between Pparγ1 and SIRT1. Sirt1 and Pparγ1 converge to govern lipid metabolism in vivo. Acetylation-defective mutants of Pparγ1 were associated with reduced lipid synthesis in ErbB2 overexpressing breast cancer cells. Together, these results suggest that the conserved lysyl residues K154/K155 of Pparγ1 are acetylated and play an important role in lipid synthesis in ErbB2-positive breast cancer cells.

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

    PubMed Central

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

    2002-01-01

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

  13. Diseases Associated with Defective Responses to DNA Damage

    PubMed Central

    O’Driscoll, Mark

    2012-01-01

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

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

    PubMed Central

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

    2002-01-01

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

  15. Selection of chemotaxis mutants of Dictyostelium discoideum

    PubMed Central

    1987-01-01

    A method has been developed for the efficient selection of chemotaxis mutants of Dictyostelium discoideum. Mutants defective in the chemotactic response to folate could be enriched up to 30-fold in one round of selection using a chamber in which a compartment that contained the chemoattractant was separated by a sandwich of four nitrocellulose filters from a compartment that contained buffer. Mutagenized cells were placed in the center of the filter layer and exposed to the attractant gradient built up between the compartments for a period of 3-4 h. While wild-type cells moved through the filters in a wave towards the compartment that contained attractant, mutant cells remained in the filter to which they were applied. After several repetitions of the selection procedure, mutants defective in chemotaxis made up 10% of the total cell population retained in that filter. Mutants exhibiting three types of alterations were collected: motility mutants with either reduced speed of movement, or altered rates of turning; a single mutant defective in production of the attractant- degrading enzyme, folate deaminase; and mutants with normal motility but reduced chemotactic responsiveness. One mutant showed drastically reduced sensitivity in folate-induced cGMP production. Morphogenetic alterations of mutants defective in folate chemotaxis are described. PMID:3793759

  16. Altered gravitropic response, amyloplast sedimentation and circumnutation in the Arabidopsis shoot gravitropism 5 mutant are associated with reduced starch levels.

    PubMed

    Tanimoto, Mimi; Tremblay, Reynald; Colasanti, Joseph

    2008-05-01

    Plants have developed sophisticated gravity sensing mechanisms to interpret environmental signals that are vital for optimum plant growth. Loss of SHOOT GRAVITROPISM 5 (SGR5) gene function has been shown to affect the gravitropic response of Arabidopsis inflorescence stems. SGR5 is a member of the INDETERMINATE DOMAIN (IDD) zinc finger protein family of putative transcription factors. As part of an ongoing functional analysis of Arabidopsis IDD genes (AtIDD) we have extended the characterisation of SGR5, and show that gravity sensing amyloplasts in the shoot endodermis of sgr5 mutants sediment more slowly than wild type, suggesting a defect in gravity perception. This is correlated with lower amyloplast starch levels, which may account for the reduced gravitropic sensitivity in sgr5. Further, we find that sgr5 mutants have a severely attenuated stem circumnutation movement typified by a reduced amplitude and an decreased periodicity. adg1-1 and sex1-1 mutants, which contain no starch or increased starch, respectively, also show alterations in the amplitude and period of circumnutation. Together these results suggest that plant growth movement may depend on starch levels and/or gravity sensing. Overall, we propose that loss of SGR5 regulatory activity affects starch accumulation in Arabidopsis shoot tissues and causes decreased sensitivity to gravity and diminished circumnutational movements.

  17. Bacillus subtilis Mutants with Knockouts of the Genes Encoding Ribonucleases RNase Y and RNase J1 Are Viable, with Major Defects in Cell Morphology, Sporulation, and Competence

    PubMed Central

    Figaro, Sabine; Durand, Sylvain; Gilet, Laetitia; Cayet, Nadège; Sachse, Martin

    2013-01-01

    The genes encoding the ribonucleases RNase J1 and RNase Y have long been considered essential for Bacillus subtilis cell viability, even before there was concrete knowledge of their function as two of the most important enzymes for RNA turnover in this organism. Here we show that this characterization is incorrect and that ΔrnjA and Δrny mutants are both viable. As expected, both strains grow relatively slowly, with doubling times in the hour range in rich medium. Knockout mutants have major defects in their sporulation and competence development programs. Both mutants are hypersensitive to a wide range of antibiotics and have dramatic alterations to their cell morphologies, suggestive of cell envelope defects. Indeed, RNase Y mutants are significantly smaller in diameter than wild-type strains and have a very disordered peptidoglycan layer. Strains lacking RNase J1 form long filaments in tight spirals, reminiscent of mutants of the actin-like proteins (Mre) involved in cell shape determination. Finally, we combined the rnjA and rny mutations with mutations in other components of the degradation machinery and show that many of these strains are also viable. The implications for the two known RNA degradation pathways of B. subtilis are discussed. PMID:23504012

  18. Improved cell survival by the reduction of immediate-early gene expression in replication-defective mutants of herpes simplex virus type 1 but not by mutation of the virion host shutoff function.

    PubMed Central

    Johnson, P A; Wang, M J; Friedmann, T

    1994-01-01

    Derivatives of herpes simplex virus type 1 (HSV-1) have elicited considerable interest as gene transfer vectors because of their ability to infect a wide range of cell types efficiently, including fully differentiated neurons. However, it has been found that infection of many types of cell with vectors derived from replication-defective mutants of HSV-1 is associated with cytopathic effects (CPE). We have previously shown that viral gene expression played an important role in the induction of CPE caused by an HSV-1 mutant deleted for the essential immediate-early gene 3 (IE 3) (P.A. Johnson, A. Miyanohara, F. Levine, T. Cahill, and T. Friedmann, J. Virol. 66:2952-2965, 1992). We have investigated which viral genes might be responsible for CPE by comparing the ability of each of the individual genes expressed by an IE 3 deletion mutant during a nonproductive infection to inhibit biochemical transformation after cotransfection of BHK or CV-1 cells with a selectable marker gene. Transfection of IE genes 1,2, and 4 individually all caused a marked inhibition of colony formation, while transfection of IE 5 and the large subunit of ribonucleotide reductase had little effect. These results suggested that it would be necessary to mutate or reduce the expression of nearly all HSV-1 IE genes to reduce virus-induced CPE. Therefore, we have used VP16 mutants, which are unable to transinduce IE gene expression (C. I. Ace, T. A. McKee, J. M. Ryan, J. M. Cameron, and C. M. Preston, J. Virol. 63:2260-2269, 1989), to derive two replication-defective strains: 14H delta 3, which is deleted for both copies of IE 3, and in 1850 delta 42, which has a deletion in the essential early gene UL42. The IE 3-VP16 double mutant, 14H delta 3, is significantly less toxic than a single IE 3 deletion mutant over a range of multiplicities of infection, as measured in a cell-killing assay, and has an enhanced ability to persist in infected cells in a biologically retrievable form. In contrast, the UL

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

    PubMed

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

    2015-10-01

    Heterozygous mutations in GBA1 gene, encoding for lysosomal enzyme glucocerebrosidase (GCase), are a major risk factor for sporadic Parkinson's disease (PD). Defective GCase has been reported in fibroblasts of GBA1-mutant PD patients and pharmacological chaperone ambroxol has been shown to correct such defect. To further explore this issue, we investigated GCase and elements supporting GCase function and trafficking in fibroblasts from sporadic PD patients--with or without heterozygous GBA1 mutations--and healthy subjects, in basal conditions and following in vitro exposure to ambroxol. We assessed protein levels of GCase, lysosomal integral membrane protein-2 (LIMP-2), which mediates GCase trafficking to lysosomes, GCase endogenous activator saposin (Sap) C and parkin, which is involved in degradation of defective GCase. We also measured activities of GCase and cathepsin D, which cleaves Sap C from precursor prosaposin. GCase activity was reduced in fibroblasts from GBA1-mutant patients and ambroxol corrected this defect. Ambroxol increased cathepsin D activity, GCase and Sap C protein levels in all groups, while LIMP-2 levels were increased only in GBA1-mutant PD fibroblasts. Parkin levels were slightly increased only in the PD group without GBA1 mutations and were not significantly modified by ambroxol. Our study confirms that GCase activity is deficient in fibroblasts of GBA1-mutant PD patients and that ambroxol corrects this defect. The drug increased Sap C and LIMP-2 protein levels, without interfering with parkin. These results confirm that chemical chaperone ambroxol modulates lysosomal markers, further highlighting targets that may be exploited for innovative PD therapeutic strategies. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Novel functions of Stomatal Cytokinesis-Defective 1 (SCD1) in innate immune responses against bacteria.

    PubMed

    Korasick, David A; McMichael, Colleen; Walker, Katie A; Anderson, Jeffrey C; Bednarek, Sebastian Y; Heese, Antje

    2010-07-23

    Eukaryotes employ complex immune mechanisms for protection against microbial pathogens. Here, we identified SCD1 (Stomatal Cytokinesis-Defective 1), previously implicated in growth and development through its role in cytokinesis and polarized cell expansion (Falbel, T. G., Koch, L. M., Nadeau, J. A., Segui-Simarro, J. M., Sack, F. D., and Bednarek, S. Y. (2003) Development 130, 4011-4024) as a novel component of innate immunity. In Arabidopsis, SCD1 is a unique gene encoding for the only protein containing a complete DENN (Differentially Expressed in Normal and Neoplastic cells) domain. The DENN domain is a largely uncharacterized tripartite protein motif conserved among eukaryotic proteins. We show that conditional scd1-1 plants containing a point mutation in a conserved DENN residue affected a subset of signaling responses to some bacterial pathogen-associated molecular patterns (PAMPs). Consistent with increased transcript accumulation of Pathogen-related (PR) genes, scd1-1 plants were more resistant to Pseudomonas syringae pathovar tomato (Pst) DC3000 infection implicating SCD1 as a negative regulator of basal resistance against bacteria. scd1-1 plants were different from known mutants exhibiting constitutive expressor of PR (cpr)-like phenotypes, in that growth impairment of scd1-1 plants was genetically independent of constitutive immune response activation. For scd1-1, shift to elevated temperature or introduction of a mutant allele in Salicylic acid Induction-Deficient 2 (SID2) suppressed constitutive defense response activation. sid2-2 also repressed the resistance phenotype of scd1-1. Temperature shift and sid2-2, however, did not rescue conditional growth and sterility defects of scd1-1. These results implicate SCD1 in multiple cellular pathways, possibly by affecting different proteins. Overall, our studies identified a novel role for eukaryotic DENN proteins in immunity against bacteria.

  1. Novel Functions of Stomatal Cytokinesis-Defective 1 (SCD1) in Innate Immune Responses against Bacteria*

    PubMed Central

    Korasick, David A.; McMichael, Colleen; Walker, Katie A.; Anderson, Jeffrey C.; Bednarek, Sebastian Y.; Heese, Antje

    2010-01-01

    Eukaryotes employ complex immune mechanisms for protection against microbial pathogens. Here, we identified SCD1 (Stomatal Cytokinesis-Defective 1), previously implicated in growth and development through its role in cytokinesis and polarized cell expansion (Falbel, T. G., Koch, L. M., Nadeau, J. A., Segui-Simarro, J. M., Sack, F. D., and Bednarek, S. Y. (2003) Development 130, 4011–4024) as a novel component of innate immunity. In Arabidopsis, SCD1 is a unique gene encoding for the only protein containing a complete DENN (Differentially Expressed in Normal and Neoplastic cells) domain. The DENN domain is a largely uncharacterized tripartite protein motif conserved among eukaryotic proteins. We show that conditional scd1-1 plants containing a point mutation in a conserved DENN residue affected a subset of signaling responses to some bacterial pathogen-associated molecular patterns (PAMPs). Consistent with increased transcript accumulation of Pathogen-related (PR) genes, scd1-1 plants were more resistant to Pseudomonas syringae pathovar tomato (Pst) DC3000 infection implicating SCD1 as a negative regulator of basal resistance against bacteria. scd1-1 plants were different from known mutants exhibiting constitutive expressor of PR (cpr)-like phenotypes, in that growth impairment of scd1-1 plants was genetically independent of constitutive immune response activation. For scd1-1, shift to elevated temperature or introduction of a mutant allele in Salicylic acid Induction-Deficient 2 (SID2) suppressed constitutive defense response activation. sid2-2 also repressed the resistance phenotype of scd1-1. Temperature shift and sid2-2, however, did not rescue conditional growth and sterility defects of scd1-1. These results implicate SCD1 in multiple cellular pathways, possibly by affecting different proteins. Overall, our studies identified a novel role for eukaryotic DENN proteins in immunity against bacteria. PMID:20472560

  2. Regulation of the Bacterial Cell Wall: Analysis of a Mutant of Bacillus subtilis Defective in Biosynthesis of Teichoic Acid

    PubMed Central

    Boylan, R. J.; Mendelson, N. H.; Brooks, D.; Young, F. E.

    1972-01-01

    Bacillus subtilis 168ts-200B is a temperature-sensitive mutant of B. subtilis 168 which grows as rods at 30 C but as irregular spheres at 45 C. Growth at the nonpermissive temperature resulted in a deficiency of teichoic acid in the cell wall. A decrease in teichoic acid synthesis coupled with the rapid turnover of this polymer led to a progressive loss until less than 20% of the level found in wild-type rods remained in spheres. Extracts of cells grown at 45 C contained amounts of the enzymes involved in the biosynthesis and glucosylation of teichoic acids that were equal to or greater than those found in normal rods. Cell walls of the spheres were deficient also in the endogenous autolytic enzyme (N-acyl muramyl-l-alanine amidase). Genetic analysis of the mutant by PBS1-mediated transduction and deoxyribonucleic acid-mediated transformation demonstrated that the lesion responsible for these effects (tag-1) is tightly linked to the genes which regulate the glucosylation of teichoic acid in the mid-portion of the chromosome of B. subtilis. PMID:4622900

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

    PubMed Central

    Allen, M. S.

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  5. Antibody-mediated activation of a defective beta-D-galactosidase: dimeric form of the activatable mutant enzyme.

    PubMed

    Conway de Macario, E; Ellis, J; Guzman, R; Rotman, B

    1978-02-01

    Sedimentation analyses of AMEF, an activatable mutant beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), and the products of its reaction with Fab fragments of activating antibody show that this enzyme exists mainly as 10S dimers. Activation of AMEF by purified antibody resulted in formation of 16S tetramers. A unifying hypothesis postulating a dimer--tetramer equilibrium accounts for this observation as the counterpart of inactivation, which was shown to involve the breakdown of tetramers into inactive subunits [Roth, R. A. & Rotman, B. (1975) Biochem. Biophys. Res. Commun. 67, 1382--1390]. Conditions are described under which AMEF loses the specific antigenic determinant(s) responsible for binding activating antibody, allowing its subsequent use as an absorption to obtain immunologically purified activating antibody,

  6. Antibody-mediated activation of a defective beta-D-galactosidase: dimeric form of the activatable mutant enzyme.

    PubMed Central

    de Macario, E C; Ellis, J; Guzman, R; Rotman, B

    1978-01-01

    Sedimentation analyses of AMEF, an activatable mutant beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), and the products of its reaction with Fab fragments of activating antibody show that this enzyme exists mainly as 10S dimers. Activation of AMEF by purified antibody resulted in formation of 16S tetramers. A unifying hypothesis postulating a dimer--tetramer equilibrium accounts for this observation as the counterpart of inactivation, which was shown to involve the breakdown of tetramers into inactive subunits [Roth, R. A. & Rotman, B. (1975) Biochem. Biophys. Res. Commun. 67, 1382--1390]. Conditions are described under which AMEF loses the specific antigenic determinant(s) responsible for binding activating antibody, allowing its subsequent use as an absorption to obtain immunologically purified activating antibody, PMID:416439

  7. Analysis of combinatorial loss-of-function mutants in the Arabidopsis ethylene receptors reveals that the ers1 etr1 double mutant has severe developmental defects that are EIN2 dependent.

    PubMed

    Hall, Anne E; Bleecker, Anthony B

    2003-09-01

    Ethylene responses in Arabidopsis are controlled by the ETR receptor family. The receptors function as negative regulators of downstream signal transduction components and fall into two distinct subfamilies based on sequence similarity. To clarify the levels of functional redundancy between receptor isoforms, combinatorial mutant lines were generated that included the newly isolated ers1-2 allele. Based on the etiolated seedling growth response, all mutant combinations tested exhibited some constitutive ethylene responsiveness but also remained responsive to exogenous ethylene, indicating that all five receptor isoforms can contribute to signaling and no one receptor subtype is essential. On the other hand, light-grown seedlings and adult ers1 etr1 double mutants exhibited severe phenotypes such as miniature rosette size, delayed flowering, and sterility, revealing a distinct role for subfamily I receptors in light-grown plants. Introduction of an ein2 loss-of-function mutation into the ers1 etr1 double mutant line resulted in plants that phenocopy ein2 single mutants, indicating that all phenotypes observed in the ers1 etr1 double mutant are EIN2 dependent.

  8. Analysis of Combinatorial Loss-of-Function Mutants in the Arabidopsis Ethylene Receptors Reveals That the ers1 etr1 Double Mutant Has Severe Developmental Defects That Are EIN2 Dependent

    PubMed Central

    Hall, Anne E.; Bleecker, Anthony B.

    2003-01-01

    Ethylene responses in Arabidopsis are controlled by the ETR receptor family. The receptors function as negative regulators of downstream signal transduction components and fall into two distinct subfamilies based on sequence similarity. To clarify the levels of functional redundancy between receptor isoforms, combinatorial mutant lines were generated that included the newly isolated ers1-2 allele. Based on the etiolated seedling growth response, all mutant combinations tested exhibited some constitutive ethylene responsiveness but also remained responsive to exogenous ethylene, indicating that all five receptor isoforms can contribute to signaling and no one receptor subtype is essential. On the other hand, light-grown seedlings and adult ers1 etr1 double mutants exhibited severe phenotypes such as miniature rosette size, delayed flowering, and sterility, revealing a distinct role for subfamily I receptors in light-grown plants. Introduction of an ein2 loss-of-function mutation into the ers1 etr1 double mutant line resulted in plants that phenocopy ein2 single mutants, indicating that all phenotypes observed in the ers1 etr1 double mutant are EIN2 dependent. PMID:12953109

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

    PubMed

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

    2012-12-12

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

  10. Enzyme-linked immunosorbent assay antibody responses to a temperature-sensitive mutant of Pseudomonas aeruginosa.

    PubMed Central

    Sordelli, D O; Rojas, R A; Cerquetti, M C; Hooke, A M; Degnan, P J; Bellanti, J A

    1985-01-01

    The serum immunoglobulin G and M responses induced by immunization of mice with temperature-sensitive mutant A/10/25 of Pseudomonas aeruginosa were evaluated by enzyme-linked immunosorbent assay. These antibody responses were immunotype specific, and the immunoglobulin G antibody level, although low, was still significant by day 52 after vaccination. PMID:3930404

  11. Live attenuated herpes simplex virus 2 glycoprotein E deletion mutant as a vaccine candidate defective in neuronal spread.

    PubMed

    Awasthi, Sita; Zumbrun, Elizabeth E; Si, Huaxin; Wang, Fushan; Shaw, Carolyn E; Cai, Michael; Lubinski, John M; Barrett, Shana M; Balliet, John W; Flynn, Jessica A; Casimiro, Danilo R; Bryan, Janine T; Friedman, Harvey M

    2012-04-01

    A herpes simplex virus 2 (HSV-2) glycoprotein E deletion mutant (gE2-del virus) was evaluated as a replication-competent, attenuated live virus vaccine candidate. The gE2-del virus is defective in epithelial cell-to-axon spread and in anterograde transport from the neuron cell body to the axon terminus. In BALB/c and SCID mice, the gE2-del virus caused no death or disease after vaginal, intravascular, or intramuscular inoculation and was 5 orders of magnitude less virulent than wild-type virus when inoculated directly into the brain. No infectious gE2-del virus was recovered from dorsal root ganglia (DRG) after multiple routes of inoculation; however, gE2-del DNA was detected by PCR in lumbosacral DRG at a low copy number in some mice. Importantly, no recurrent vaginal shedding of gE2-del DNA was detected in immunized guinea pigs. Intramuscular immunization outperformed subcutaneous immunization in all parameters evaluated, although individual differences were not significant, and two intramuscular immunizations were more protective than one. Immunized animals had reduced vaginal disease, vaginal titers, DRG infection, recurrent genital lesions, and recurrent vaginal shedding of HSV-2 DNA; however, protection was incomplete. A combined modality immunization using live virus and HSV-2 glycoprotein C and D subunit antigens in guinea pigs did not totally eliminate recurrent lesions or recurrent vaginal shedding of HSV-2 DNA. The gE2-del virus used as an immunotherapeutic vaccine in previously HSV-2-infected guinea pigs greatly reduced the frequency of recurrent genital lesions. Therefore, the gE2-del virus is safe, other than when injected at high titer into the brain, and is efficacious as a prophylactic and immunotherapeutic vaccine.

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

    SciTech Connect

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

    1990-11-01

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

  13. Reversion of lethality and growth defects in Fatiga oxygen-sensor mutant flies by loss of hypoxia-inducible factor-alpha/Sima.

    PubMed

    Centanin, Lázaro; Ratcliffe, Peter J; Wappner, Pablo

    2005-11-01

    Hypoxia-Inducible Factor (HIF) prolyl hydroxylase domains (PHDs) have been proposed to act as sensors that have an important role in oxygen homeostasis. In the presence of oxygen, they hydroxylate two specific prolyl residues in HIF-alpha polypeptides, thereby promoting their proteasomal degradation. So far, however, the developmental consequences of the inactivation of PHDs in higher metazoans have not been reported. Here, we describe novel loss-of-function mutants of fatiga, the gene encoding the Drosophila PHD oxygen sensor, which manifest growth defects and lethality. We also report a null mutation in dHIF-alpha/sima, which is unable to adapt to hypoxia but is fully viable in normoxic conditions. Strikingly, loss-of-function mutations of sima rescued the developmental defects observed in fatiga mutants and enabled survival to adulthood. These results indicate that the main functions of Fatiga in development, including control of cell size, involve the regulation of dHIF/Sima.

  14. Reversion of lethality and growth defects in Fatiga oxygen-sensor mutant flies by loss of Hypoxia-Inducible Factor-α/Sima

    PubMed Central

    Centanin, Lázaro; Ratcliffe, Peter J; Wappner, Pablo

    2005-01-01

    Hypoxia-Inducible Factor (HIF) prolyl hydroxylase domains (PHDs) have been proposed to act as sensors that have an important role in oxygen homeostasis. In the presence of oxygen, they hydroxylate two specific prolyl residues in HIF-α polypeptides, thereby promoting their proteasomal degradation. So far, however, the developmental consequences of the inactivation of PHDs in higher metazoans have not been reported. Here, we describe novel loss-of-function mutants of fatiga, the gene encoding the Drosophila PHD oxygen sensor, which manifest growth defects and lethality. We also report a null mutation in dHIF-α/sima, which is unable to adapt to hypoxia but is fully viable in normoxic conditions. Strikingly, loss-of-function mutations of sima rescued the developmental defects observed in fatiga mutants and enabled survival to adulthood. These results indicate that the main functions of Fatiga in development, including control of cell size, involve the regulation of dHIF/Sima. PMID:16179946

  15. Spermatogenesis-defective (spe) Mutants of the Nematode Caenorhabditis elegans Provide Clues to Solve the Puzzle of Male Germline Functions during Reproduction

    PubMed Central

    Nishimura, Hitoshi; L’Hernault, Steven W.

    2012-01-01

    In most species, each sex produces gametes, usually either sperm or oocytes, from its germline during gametogenesis. The sperm and oocyte subsequently fuse together during fertilization to create the next generation. This review focuses on spermatogenesis and the roles of sperm during fertilization in the nematode Caenorhabditis elegans, where suitable mutants are readily obtained. So far 186 mutants defective in the C. elegans male germline functions have been isolated, and many of these mutations are alleles for one of the ~60 spermatogenesis-defective (spe) genes. Many cloned spe genes are expressed specifically in the male germline, where they play roles during spermatogenesis (spermatid production), spermiogenesis (spermatid activation into spermatozoa), and/or fertilization. Moreover, several spe genes are orthologs of mammalian genes, suggesting that the reproductive processes of the C. elegans and the mammalian male germlines might share common pathways at the molecular level. PMID:20419782

  16. Altered expression of several genes in IIIManL-defective mutants of Streptococcus salivarius demonstrated by two-dimensional gel electrophoresis of cytoplasmic proteins.

    PubMed

    Lapointe, R; Frenette, M; Vadeboncoeur, C

    1993-05-01

    Mannose, glucose and fructose are transported in Streptococcus salivarius by a phosphoenolpyruvate:mannose phosphotransferase system (PTS) which consists of a membrane-bound Enzyme II (EII) and two forms of IIIMan having molecular weights of 38,900 (IIIManH) and 35,200 (IIIManL), respectively. We have previously reported the isolation of spontaneous mutants lacking IIIManL and showed that they exhibit higher beta-galactosidase activity than the parental strain after growth on glucose, and that some of them constitutively express a fructose PTS which is induced by fructose in the parental strain. In an attempt to determine whether the expression of other genes is affected by the mutation and what the physiological link is between them, we examined three S. salivarius IIIManL-defective mutants (strains A37, B31 and G29) and the parental strain using two-dimensional gel electrophoresis after growth of the cells on a variety of sugars. After growth on glucose, five new proteins were detected in the cytoplasm of the three mutants. Two of these proteins were induced in the parental strain by galactose or oligosaccharides containing galactose, and one was specifically induced by melibiose. The other two proteins were not detected in the parental strain under any of the growth conditions tested. Two other proteins were only detected in glucose-grown cells of mutant A37, and a protein associated with the metabolism of fructose was constitutively expressed in mutants B31 and G29. Moreover, we have found that under identical growth conditions the amounts of several other proteins which were detected in the parental strain were either increased or decreased in the mutants. Globally, our results have indicated that (1) the expression of several genes was affected in the spontaneous IIIManL-defective mutants; (2) some of the proteins abnormally produced in the mutants were specifically induced in the parental strain by sugars; (3) the phenotypic modifications observed in the

  17. Transformation-defective mutant of avian myeloblastosis virus that is temperature sensitive for production of transforming protein p45v-myb.

    PubMed Central

    Moscovici, M G; Klempnauer, K H; Symonds, G; Bishop, J M; Moscovici, C

    1985-01-01

    We have characterized a mutant of avian myeloblastosis virus (strain GA907/7) that shows a reduced capacity to transform myelomonocytic cells at the nonpermissive temperature. Myeloblasts transformed by this mutant suffer a substantial decrease in the amount of the transforming protein p45v-myb when shifted from the permissive to the nonpermissive temperature. We presume that the 5- to 10-fold decrease in the amount of p45v-myb causes the loss of the transformed phenotype. The decrease is due to a reduction in the level of v-myb mRNA. Mutant GA907/7 thus provides genetic evidence that p45v-myb is the transforming protein of avian myeloblastosis virus and apparently represents an unusual defect in the production or stability of mRNA. Images PMID:3018515

  18. Molecular basis of the defective heat stress response in Mycobacterium leprae.

    PubMed

    Williams, Diana L; Pittman, Tana L; Deshotel, Mike; Oby-Robinson, Sandra; Smith, Issar; Husson, Robert

    2007-12-01

    Mycobacterium leprae, a major human pathogen, grows poorly at 37 degrees C. The basis for its inability to survive at elevated temperatures was investigated. We determined that M. leprae lacks a protective heat shock response as a result of the lack of transcriptional induction of the alternative sigma factor genes sigE and sigB and the major heat shock operons, HSP70 and HSP60, even though heat shock promoters and regulatory circuits for these genes appear to be intact. M. leprae sigE was found to be capable of complementing the defective heat shock response of mycobacterial sigE knockout mutants only in the presence of a functional mycobacterial sigH, which orchestrates the mycobacterial heat shock response. Since the sigH of M. leprae is a pseudogene, these data support the conclusion that a key aspect of the defective heat shock response in M. leprae is the absence of a functional sigH. In addition, 68% of the genes induced during heat shock in M. tuberculosis were shown to be either absent from the M. leprae genome or were present as pseudogenes. Among these is the hsp/acr2 gene, whose product is essential for M. tuberculosis survival during heat shock. Taken together, these results suggest that the reduced ability of M. leprae to survive at elevated temperatures results from the lack of a functional transcriptional response to heat shock and the absence of a full repertoire of heat stress response genes, including sigH.

  19. Molecular Basis of the Defective Heat Stress Response in Mycobacterium leprae▿ †

    PubMed Central

    Williams, Diana L.; Pittman, Tana L.; Deshotel, Mike; Oby-Robinson, Sandra; Smith, Issar; Husson, Robert

    2007-01-01

    Mycobacterium leprae, a major human pathogen, grows poorly at 37°C. The basis for its inability to survive at elevated temperatures was investigated. We determined that M. leprae lacks a protective heat shock response as a result of the lack of transcriptional induction of the alternative sigma factor genes sigE and sigB and the major heat shock operons, HSP70 and HSP60, even though heat shock promoters and regulatory circuits for these genes appear to be intact. M. leprae sigE was found to be capable of complementing the defective heat shock response of mycobacterial sigE knockout mutants only in the presence of a functional mycobacterial sigH, which orchestrates the mycobacterial heat shock response. Since the sigH of M. leprae is a pseudogene, these data support the conclusion that a key aspect of the defective heat shock response in M. leprae is the absence of a functional sigH. In addition, 68% of the genes induced during heat shock in M. tuberculosis were shown to be either absent from the M. leprae genome or were present as pseudogenes. Among these is the hsp/acr2 gene, whose product is essential for M. tuberculosis survival during heat shock. Taken together, these results suggest that the reduced ability of M. leprae to survive at elevated temperatures results from the lack of a functional transcriptional response to heat shock and the absence of a full repertoire of heat stress response genes, including sigH. PMID:17933896

  20. Pleiotropic aspartate taxis and serine taxis mutants of Escherichia coli.

    PubMed

    Reader, R W; Tso, W W; Springer, M S; Goy, M F; Adler, J

    1979-04-01

    Mutants that at one time were thought to be specifically defective in taxis toward aspartate and related amino acids (tar mutants) or specifically defective in taxis toward serine and related amino acids (tar mutants) are now shown to be pleiotropic in their defects. The tar mutants also lack taxis toward maltose and away from Co2+ and Ni2+. The tsr mutants are altered in their response to a variety of repellents. Double mutants (tar tsr) fail in nearly all chemotactic responses. The tar and tsr mutants provide evidence for two complementary, converging pathways of information flow: certain chemoreceptors feed information into the tar pathway and others into the tsr pathway. The tar and tsr products have been shown to be two different sets of methylated proteins.

  1. hxc2, an Arabidopsis mutant with an altered hypersensitive response to Xanthomonas campestris pv. campestris.

    PubMed

    Godard, F; Lummerzheim, M; Saindrenan, P; Balagué, C; Roby, D

    2000-12-01

    A chemical mutagenized population of Arabidopsis Col-0-gl plants was screened for an altered hypersensitive response (HR) after spray inoculation with an HR-inducing isolate of Xanthomonas campestris pv. campestris (strain 147). Three classes of mutant were identified: those exhibiting an HR- phenotype or partial loss of HR; hyper-responsive mutants showing necrotic lesions rapidly leading to the collapse of leaves; and susceptible mutants. One mutant belonging to the susceptible class, hxc-2, was extensively characterized. The compatible phenotype observed several days after initiation of the interaction was confirmed by measurement of in planta bacterial growth and use of bacterial strains constitutively expressing the GUS reporter gene. In the same way, accumulation of autofluorescent compounds, salicylic acid production and defence gene expression in the mutant were found to be similar to that displayed by the susceptible ecotype. Inoculation of hxc-2 with different avirulent bacteria suggests that the mutation is specific for the interaction with the Xcc 147 strain, although the mutation has been shown to affect a single dominant locus, different from the resistance locus defined by genetic analysis of resistance to Xcc 147. Genetic mapping of the mutation indicated that it is located on chromosome III, defining a previously unknown resistance function in response to X. c. campestris.

  2. Suppression of mutants aberrant in light intensity responses of complementary chromatic adaptation.

    PubMed Central

    Casey, E S; Kehoe, D M; Grossman, A R

    1997-01-01

    Complementary chromatic adaptation is a process in which cyanobacteria alter the pigment protein (phycocyanin and phycoerythrin) composition of their light-harvesting complexes, the phycobilisomes, to help optimize the absorbance of prevalent wavelengths of light in the environment. Several classes of mutants that display aberrant complementary chromatic adaptation have been isolated. One of the mutant classes, designated "blue" or FdB, accumulates high levels of the blue chromoprotein phycocyanin in low-intensity green light, a condition that normally suppresses phycocyanin synthesis. We demonstrate here that the synthesis of the phycocyanin protein and mRNA in the FdB mutants can be suppressed by increasing the intensity of green light. Hence, these mutants have a decreased sensitivity to green light with respect to suppression of phycocyanin synthesis. Although we were unable to complement the blue mutants, we did isolate genes that could suppress the mutant phenotype. These genes, which have been identified previously, encode a histidine kinase sensor and response regulator protein that play key roles in controlling complementary chromatic adaptation. These findings are discussed with respect to the mechanism by which light quality and quantity control the biosynthesis of the phycobilisome. PMID:9226271

  3. Altered Stationary-Phase Response in a Borrelia burgdorferi rpoS Mutant

    PubMed Central

    Elias, Abdallah F.; Bono, James L.; Carroll, James A.; Stewart, Philip; Tilly, Kit; Rosa, Patricia

    2000-01-01

    The homolog of the chromosomally encoded stationary-phase sigma factor RpoS in Borrelia burgdorferi was inactivated using gyrBr as a selectable marker. Two-dimensional nonequilibrium pH gradient electrophoresis of stationary-phase cell lysates identified at least 11 differences between the protein profiles of the rpoS mutant and wild-type organisms. Wild-type B. burgdorferi had a growth phase-dependent resistance to 1 N NaCl, similar to the stationary-phase response reported for other bacteria. The B. burgdorferi rpoS mutant strain was less resistant to osmotic stress in stationary phase than the isogenic rpoS wild-type organism. The results indicate that the B. burgdorferi rpoS homolog influences protein composition and participates in stationary-phase-dependent osmotic resistance. This rpoS mutant will be useful for studying regulation of gene expression in response to changing environmental conditions. PMID:10781562

  4. Expression of a myosin regulatory light chain phosphorylation site mutant complements the cytokinesis and developmental defects of Dictyostelium RMLC null cells.

    PubMed

    Ostrow, B D; Chen, P; Chisholm, R L

    1994-12-01

    In a number of systems phosphorylation of the regulatory light chain (RMLC) of myosin regulates the activity of myosin. In smooth muscle and vertebrate nonmuscle systems RMLC phosphorylation is required for contractile activity. In Dictyostelium discoideum phosphorylation of the RMLC regulates both ATPase activity and motor function. We have determined the site of phosphorylation on the Dictyostelium RMLC and used site-directed mutagenesis to replace the phosphorylated serine with an alanine. The mutant light chain was then expressed in RMLC null Dictyostelium cells (mLCR-) from an actin promoter on an integrating vector. The mutant RMLC was expressed at high levels and associated with the myosin heavy chain. RMLC bearing a ser13ala substitution was not phosphorylated in vitro by purified myosin light chain kinase, nor could phosphate be detected on the mutant RMLC in vivo. The mutant myosin had reduced actin-activated ATPase activity, comparable to fully dephosphorylated myosin. Unexpectedly, expression of the mutant RMLC rescued the primary phenotypic defects of the mlcR- cells to the same extent as did expression of wild-type RMLC. These results suggest that while phosphorylation of the Dictyostelium RMLC appears to be tightly regulated in vivo, it is not essential for myosin-dependent cellular functions.

  5. Defective anti-listerial responses in deciduoma of pseudopregnant mice.

    PubMed Central

    Redline, R. W.; Shea, C. M.; Papaioannou, V. E.; Lu, C. Y.

    1988-01-01

    Two different hormonal regimens to induce pseudopregnancy resulted in a pronounced increase in the susceptibility of the murine uterus to intraluminal injections of Listeria monocytogenes. Preimmunization, which profoundly augments systemic listeria resistance, had no effect on this increased uterine susceptibility. Anti-listerial responses in other organs were unaffected by pseudopregnancy. Animals manifesting increased susceptibility formed distinct uterine swellings in response to the combination of hormones and uterine listeria. These swellings correspond to previously described deciduoma and closely mimic the decidualized endometrium of pregnancy. The nature of the defective response to listeria was investigated by immunocytochemistry. Increased bacterial titers were correlated with an inability of macrophages and T lymphocytes to reach tissue listeria in discrete regions of deciduoma-bearing uteri. Control uteri showed a normal granulomatous pattern of inflammation. These findings closely parallel previous findings in the murine decidua basalis and suggest that properties of decidualized endometrial stromal cells regulate local immune responsiveness. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:3144175

  6. Two types of albino mutants in desert and migratory locusts are caused by gene defects in the same signaling pathway.

    PubMed

    Sugahara, Ryohei; Tanaka, Seiji; Jouraku, Akiya; Shiotsuki, Takahiro

    2017-04-15

    Albinism is caused by mutations in the genes involved in melanin production. Albino nymphs of Locusta migratoria and Schistocerca gregaria reared under crowded conditions are uniformly creamy-white in color. However, nothing is known about the molecular mechanisms underlying this phenomenon in locusts. The albino strain of L. migratoria is known to lack the dark-color-inducing neuropeptide corazonin (Crz). In this study, we report that this albino strain has a 10-base-pair deletion in the gene LmCRZ, which encodes Crz. This mutation was found to cause a frame-shift, resulting in a null mutation in Crz. On the other hand, the albino strain of S. gregaria is known to have an intact Crz. This strain was found to possess a single-nucleotide substitution in the middle of the Crz receptor-encoding gene, SgCRZR, which caused a nonsense mutation, resulting in a truncated receptor. Silencing of SgCRZR in wild-type S. gregaria nymphs greatly reduced the area and intensity of their black patterning, suggesting that the functional defect of SgCRZR likely causes the albinism. The expression level of SgCRZR in the albino S. gregaria was comparable to that in the wild type. Unlike the wild type, the albino strain of this locust did not show a phase-dependent shift in a morphometric trait controlled by Crz. From these results, we conclude that the mutations in LmCRZ and SgCRZR are responsible for the albinism in L. migratoria and S. gregaria, respectively, indicating that the two types of albinism are caused by different genetic defects in the same Crz signaling pathway.

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed

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

    2015-03-01

    Muscle, skeletal, receptor tyrosine kinase (MuSK) is a key organizer at the postsynaptic membrane and critical for proper development and maintenance of the neuromuscular junction. Mutations in MUSK result in congenital myasthenic syndrome (CMS). We hypothesized that the CMS-causing missense mutation (P344R), found within the cysteine-rich domain of the protein, will affect its conformational tertiary structure. Consequently, the protein will misfold, get retained in the endoplasmic reticulum (ER) and lose its biological function through degradation by the highly conserved ER associated degradation (ERAD) machinery. We report that P344R-MuSK mutant is trafficking-deficient when expressed at 37°C in HeLa, COS-7 and HEK293 cell lines. It colocalized with the ER marker calnexin in contrast to wild-type MuSK which localized to the plasma membrane. The N-glycosylation status of P344R-MuSK is that of an immature and not properly post-translationally modified protein. Inhibition of protein synthesis showed that the P344R mutant's half-life is shorter than wild-type MuSK protein. Proteasomal inhibition resulted in the stabilization of the mutant protein. The mutant protein is highly ubiquitinated compared to wild-type confirming targeting for proteasomal degradation. The mutant showed around 50% of its in vivo autophosphorylation activity. P344R-MuSK mutant's trafficking defect is correctable by culturing the expressing cells at 27°C. Moreover, chemical compounds namely 2.5% glycerol, 1% dimethyl sulfoxide, 10 μM thapsigargin and 1 μM curcumin improved the maturation and exit of the mutant protein from the ER. These findings open perspectives for potential therapeutic intervention for patients with CMS harboring the P344R-MuSK mutation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Distinct innate responses are induced by attenuated Salmonella enterica serovar Typhimurium mutants.

    PubMed

    Powell, Daniel A; Roberts, Lydia M; Ledvina, Hannah E; Sempowski, Gregory D; Curtiss, Roy; Frelinger, Jeffrey A

    2016-01-01

    Upon bacterial infection the host cells generate a wide variety of cytokines. Genetic attenuation of bacterial physiological pathogens can be accomplished not only by disruption of normal bacterial processes, but also by the loss of the ability to redirect the host immune system. We examined nine attenuated Salmonella Typhimurium mutants for their ability to replicate as well as the cytokines produced after infection of Bone Marrow Derived Macrophages (BMDM). Infection of BMDM with attenuated Salmonella mutants led to host cytokine patterns distinct from those that followed WT infection. Surprisingly, each bacterial mutant had a unique cytokine signature. Because some of the mutants induced an IL-10 response not seen in WT, we examined the role of IL-10 on Salmonella replication. Surprisingly, addition of IL-10 before or concurrent with infection restricted growth of WT Salmonella in BMDM. Bacterial attenuation is not a single process and results in attenuated host responses, which result in unique patterns for each attenuated mutants. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Arabidopsis thaliana mutant lpsi reveals impairment in the root responses to local phosphate availability.

    PubMed

    Karthikeyan, Athikkattuvalasu S; Jain, Ajay; Nagarajan, Vinay K; Sinilal, Bhaskaran; Sahi, Shivendra V; Raghothama, Kashchandra G

    2014-04-01

    Phosphate (Pi) deficiency triggers local Pi sensing-mediated inhibition of primary root growth and development of root hairs in Arabidopsis (Arabidopsis thaliana). Generation of activation-tagged T-DNA insertion pools of Arabidopsis expressing the luciferase gene (LUC) under high-affinity Pi transporter (Pht1;4) promoter, is an efficient approach for inducing genetic variations that are amenable for visual screening of aberrations in Pi deficiency responses. Putative mutants showing altered LUC expression during Pi deficiency were identified and screened for impairment in local Pi deficiency-mediated inhibition of primary root growth. An isolated mutant was analyzed for growth response, effects of Pi deprivation on Pi content, primary root growth, root hair development, and relative expression levels of Pi starvation-responsive (PSR) genes, and those implicated in starch metabolism and Fe and Zn homeostasis. Pi deprived local phosphate sensing impaired (lpsi) mutant showed impaired primary root growth and attenuated root hair development. Although relative expression levels of PSR genes were comparable, there were significant increases in relative expression levels of IRT1, BAM3 and BAM5 in Pi deprived roots of lpsi compared to those of the wild-type. Better understanding of molecular responses of plants to Pi deficiency or excess will help to develop suitable remediation strategies for soils with excess Pi, which has become an environmental concern. Hence, lpsi mutant will serve as a valuable tool in identifying molecular mechanisms governing adaptation of plants to Pi deficiency.

  11. RhoGDI-binding-defective mutant of Cdc42Hs targets to membranes and activates filopodia formation but does not cycle with the cytosol of mammalian cells.

    PubMed Central

    Gibson, R M; Wilson-Delfosse, A L

    2001-01-01

    We have identified a mutant of the human G-protein Cdc42Hs, R66E, that fails to form a detectable complex with the GDP-dissociation inhibitor RhoGDI in cell-free systems or in intact cells. This point mutant is prenylated, binds guanine nucleotide and interacts with GTPase-activating protein in a manner indistinguishable from wild-type Cdc42Hs. Immunofluorescence localization studies revealed that this RhoGDI-binding-defective mutant is found predominantly in the Golgi apparatus, with a staining pattern similar to that of the wild-type protein. However, unlike wild-type Cdc42Hs, which is distributed in both the microsomal membrane and cytosolic fractions, studies using differential centrifugation show that prenylated R66E Cdc42Hs is found exclusively in association with lipid bilayers. Additionally, whereas the overexpression of RhoGDI results in an apparent translocation of wild-type Cdc42Hs from the Golgi apparatus into the cytosol, identical RhoGDI-overexpression conditions do not alter the Golgi localization of the R66E mutant. Furthermore, overexpression of this RhoGDI-binding-defective mutant of Cdc42Hs seems to activate redistribution of the actin cytoskeleton and filopodia formation in fibroblasts in a manner indistinguishable from the wild-type protein. Taken together, these results suggest that the interaction of Cdc42Hs with RhoGDI is not essential for proper membrane targeting of nascent prenylated Cdc42Hs in mammalian cells; neither is this interaction an essential part of the mechanism by which Cdc42Hs activates filopodia formation. However, it does seem that redistribution of Cdc42Hs to the cytosolic compartment is absolutely dependent on RhoGDI interaction. PMID:11583574

  12. [Study of the olfactory response in the Drosophila homeotic mutant leg-aristae-wing complex gene].

    PubMed

    Simonova, O B; Sukhoverkhova, T I; Zhuravel', D A; Romanova, L G; Korochkin, L I

    2000-11-01

    Antennae are known to be olfactory organs in Drosophila. The leg-aristae-wing complex (lawc) mutation causes a homeotic transformation of the arista (the fifth element of antenna) into tarsal elements. To test how the homeotic transformation of the arista into the tarsus can affect behavior, we studied the olfactory response in the lawc mutants. The data were carefully processed by statistical methods. In spite of a low penetrance of the trait of arista transformation, the mutant flies were found to be approximately half as perceptible to attractant odors than the wild-type flies.

  13. Enhanced use of backup pathways of NHEJ in G2 in Chinese hamster mutant cells with defects in the classical pathway of NHEJ.

    PubMed

    Wu, Wenqi; Wang, Minli; Mussfeldt, Tamara; Iliakis, George

    2008-10-01

    In higher eukaryotes DNA double-strand breaks (DSBs) are repaired by homologous recombination repair (HRR) or nonhomologous end joining (NHEJ). In addition to the DNA-PK dependent pathway of NHEJ (D-NHEJ), cells employ a backup pathway (B-NHEJ) using DNA ligase III and PARP1. We have reported previously that mouse embryo fibroblasts (MEFs) defective in D-NHEJ show enhanced repair of DSBs in G2 not reflecting a contribution of HRR. Here we extend these studies to Chinese hamster mutant cells with defects in the DNA-PKcs, Ku80 or XRCC4 components of D-NHEJ or in the XRCC2 and XRCC3 components of HRR. Using cell sorting to separate cells at defined times after irradiation, we measure repair of DSBs with pulsed-field gel electrophoresis in unperturbed G1- and G2-phase cells. Wild-type cells and mutants of XRCC2 and XRCC3 repair DSBs with similar efficiency in G1 and G2. Mutants of DNA-PKcs, Ku80 and XRCC4 show more pronounced repair in G2 than in G1. These and previously published results provide support for the notion that the increased efficacy of DSB repair in G2 reflects the enhanced function of B-NHEJ, which may be a general feature of rodent cells that also holds for human cells.

  14. Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase.

    PubMed

    Liberi, Giordano; Maffioletti, Giulio; Lucca, Chiara; Chiolo, Irene; Baryshnikova, Anastasia; Cotta-Ramusino, Cecilia; Lopes, Massimo; Pellicioli, Achille; Haber, James E; Foiani, Marco

    2005-02-01

    S-phase cells overcome chromosome lesions through replication-coupled recombination processes that seem to be assisted by recombination-dependent DNA structures and/or replication-related sister chromatid junctions. RecQ helicases, including yeast Sgs1 and human BLM, have been implicated in both replication and recombination and protect genome integrity by preventing unscheduled mitotic recombination events. We have studied the RecQ helicase-mediated mechanisms controlling genome stability by analyzing replication forks encountering a damaged template in sgs1 cells. We show that, in sgs1 mutants, recombination-dependent cruciform structures accumulate at damaged forks. Their accumulation requires Rad51 protein, is counteracted by Srs2 DNA helicase, and does not prevent fork movement. Sgs1, but not Srs2, promotes resolution of these recombination intermediates. A functional Rad53 checkpoint kinase that is known to protect the integrity of the sister chromatid junctions is required for the accumulation of recombination intermediates in sgs1 mutants. Finally, top3 and top3 sgs1 mutants accumulate the same structures as sgs1 cells. We suggest that, in sgs1 cells, the unscheduled accumulation of Rad51-dependent cruciform structures at damaged forks result from defective maturation of recombination-dependent intermediates that originate from the replication-related sister chromatid junctions. Our findings might contribute to explaining some of the recombination defects of BLM cells.

  15. DNA damage response defect in Williams-Beuren syndrome

    PubMed Central

    Guenat, David; Merla, Giuseppe; Deconinck, Eric; Borg, Christophe; Rohrlich, Pierre-Simon

    2017-01-01

    Williams-Beuren syndrome (WBS, no. OMIM 194050) is a rare multisystem genetic disorder caused by a microdeletion on chromosome 7q11.23 and characterized by cardiovascular malformations, mental retardation, and a specific facial dysmorphism. Recently, we reported that a series of non-Hodgkin's lymphoma occurs in children with WBS and thus hypothesized that a predisposition to cancer may be associated with this genetic disorder. The aim of the present study was to ascertain the role played by three genes hemizygously deleted in WBS (RFC2, GTF2I and BAZ1B) in DNA damage response pathways. Cell proliferation, cell cycle analysis, γ-H2A.X induction, and expression of DNA damage response proteins were investigated upon exposure to genotoxic treatments in WBS patient-derived primary fibroblasts and in the 293T cell line treated with specific siRNAs targeting RFC2, GTF2I and BAZ1B. An impaired hydroxyurea-induced phosphorylation of CHK1 was observed in the WBS cells. However, this defective DNA damage response was not associated with an increased sensitivity to genotoxic agents. In addition, depletion of RFC2, GTF2I and BAZ1B using specific siRNAs did not have a significant impact on the DNA damage response in 293T cells. Our results highlight that the ATR-dependent DNA damage response is impaired in WBS patient cells but is also dispensable for viability when these cells undergo a genotoxic stress. The mechanism by which the ATR pathway is impaired in WBS warrants elucidation through further investigation. PMID:28098859

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

    NASA Astrophysics Data System (ADS)

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

    2010-01-01

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

  17. The Proteomic Response to Mutants of the Escherichia coli RNA Degradosome

    DTIC Science & Technology

    2013-01-01

    pathway of RNA degradation. REPORT DOCUMENTATION PAGE (SF298) (Continuation Sheet) Continuation for Block 13 ARO Report Number The proteomic response...of the bacterial proteome and provide the first large-scale proteomic description of the response to perturbation of this major pathway of RNA...truncation mutant11 (rightmost column). Significant function enrichments (adjusted P-value o 0.01, compared to entire E. coli genome) are indicated

  18. An mRNA decapping mutant deficient in P body assembly limits mRNA stabilization in response to osmotic stress

    PubMed Central

    Huch, Susanne; Nissan, Tracy

    2017-01-01

    Yeast is exposed to changing environmental conditions and must adapt its genetic program to provide a homeostatic intracellular environment. An important stress for yeast in the wild is high osmolarity. A key response to this stress is increased mRNA stability primarily by the inhibition of deadenylation. We previously demonstrated that mutations in decapping activators (edc3∆ lsm4∆C), which result in defects in P body assembly, can destabilize mRNA under unstressed conditions. We wished to examine whether mRNA would be destabilized in the edc3∆ lsm4∆C mutant as compared to the wild-type in response to osmotic stress, when P bodies are intense and numerous. Our results show that the edc3∆ lsm4∆C mutant limits the mRNA stability in response to osmotic stress, while the magnitude of stabilization was similar as compared to the wild-type. The reduced mRNA stability in the edc3∆ lsm4∆C mutant was correlated with a shorter PGK1 poly(A) tail. Similarly, the MFA2 mRNA was more rapidly deadenylated as well as significantly stabilized in the ccr4∆ deadenylation mutant in the edc3∆ lsm4∆C background. These results suggest a role for these decapping factors in stabilizing mRNA and may implicate P bodies as sites of reduced mRNA degradation. PMID:28290514

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

    PubMed

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

    2016-03-01

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

  20. Stomatal responses of Argenteum - a mutant of Pisum sativum L. with readily detachable leaf epidermis.

    PubMed

    Jewer, P C; Incoll, L D; Shaw, J

    1982-07-01

    Epidermis is easily detached from both adaxial and abaxial surfaces of leaf four of the Argenteum mutant of Pisum sativum L. The isolated epidermis has stomata with large, easily-measured pores. Hairs and glands are absent. The density of stomata is high and contamination by mesophyll cells is low. In the light and in CO2-free air, stomata in isolated adaxial epidermis of Argenteum mutant opened maximally after 4 h incubation at 25°C. The response of stomata to light was dependent on the concentration of KCl in the incubation medium and was maximal at 50 mol m(-3) KCl. Stomata did not respond to exogenous kinetin, but apertures were reduced by incubation of epidermis on solutions containing between 10(-5) and 10(-1) mol m(-3) abscisic acid (ABA). The responses of stomata of Argenteum mutant to light, exogenous KCl, ABA and kinetin were comparable with those described previously for stomata in isolated epidermis of Commelina communis. A method for preparing viable protoplasts of guard cells from isolated epidermis of Argenteum mutant is described. The response of guard cell protoplasts to light, exogenous KCl, ABA and kinetin were similar to those of stomata in isolated epidermis except that the increase in volume of the protoplasts in response to light was maximal at a lower concentration of KCl (10 mol m(-3)) and that protoplasts responded more rapidly to light than stomata in isolated epidermis. The protoplasts did not respond to exogenous kinetin, but when incubated for 1 h in the light and in CO2-free air on a solution containing 10(-3) mol m(-3) ABA, they decreased in volume by 30%. The advantages of using epidermis from Argenteum mutant for experiments on stomatal movements are discussed.

  1. Histone deacetylase inhibitors correct the cholesterol storage defect in most Niemann-Pick C1 mutant cells.

    PubMed

    Pipalia, Nina H; Subramanian, Kanagaraj; Mao, Shu; Ralph, Harold; Hutt, Darren M; Scott, Samantha M; Balch, William E; Maxfield, Frederick R

    2017-04-01

    Niemann-Pick C (NPC) disease is an autosomal recessive disorder that leads to excessive storage of cholesterol and other lipids in late endosomes and lysosomes. The large majority of NPC disease is caused by mutations in NPC1, a large polytopic membrane protein that functions in late endosomes. There are many disease-associated mutations in NPC1, and most patients are compound heterozygotes. The most common mutation, NPC1(I1061T), has been shown to cause endoplasmic reticulum-associated degradation of the NPC1 protein. Treatment of patient-derived NPC1(I1061T) fibroblasts with histone deacetylase inhibitors (HDACis) vorinostat or panobinostat increases expression of the mutant NPC1 protein and leads to correction of the cholesterol storage. Here, we show that several other human NPC1 mutant fibroblast cell lines can also be corrected by vorinostat or panobinostat and that treatment with vorinostat extends the lifetime of the NPC1(I1061T) protein. To test effects of HDACi on a large number of NPC1 mutants, we engineered a U2OS cell line to suppress NPC1 expression by shRNA and then transiently transfected these cells with 60 different NPC1 mutant constructs. The mutant NPC1 did not significantly reduce cholesterol accumulation, but approximately 85% of the mutants showed reduced cholesterol accumulation when treated with vorinostat or panobinostat. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  2. Thiamine-Auxotrophic Mutants of Pseudomonas fluorescens CHA0 Are Defective in Cell-Cell Signaling and Biocontrol Factor Expression

    PubMed Central

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

    2006-01-01

    In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 × 10−8 M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens. PMID:16597964

  3. Defective excision and postreplication repair of UV-damaged DNA in a recL mutant strain of E. coli K-12.

    PubMed

    Rothman, R H; Clark, A J

    1977-10-24

    The mutation recL152 leads to a reduction of excision repair as measured by an increase in the time required to close uvrA uvrB dependent incision breaks, and by a reduction of host cell reactivation ability. Postreplication repair is also delayed when measured in a uvrB5 recL152 double mutant. Such a determination could not be made using the recL152 single mutant because the excision defect led to an accumulation of breaks in the unlabeled high molecular weight DNA to which the labeled DNA synthesized after irradiation must attach in order to achieve normal high molecular weight. Further, the recL gene product seems to be required to rejoin breaks in parental strand DNA which are generated during postreplication repair, since such gaps accumulate in a recL152 uvrB5 double mutant but not in a recL+ uvrB5 single mutant. We have noticed a striking phenotypic similarity between recL152 and polA1 and suggest that recL152 is required for full in vivo activity of DNA polymerase I.

  4. Mutants defective in the energy-conserving NADH dehydrogenase of Salmonella typhimurium identified by a decrease in energy-dependent proteolysis after carbon starvation.

    PubMed Central

    Archer, C D; Wang, X; Elliott, T

    1993-01-01

    NADH dehydrogenase is the first component of the respiratory chain. It transfers electrons from NADH to ubiquinone and concomitantly establishes a proton motive force across the membrane. Salmonella typhimurium mutants defective in this enzyme were isolated in a screen for strains with increased expression of beta-galactosidase from a hemA-lacZ protein fusion. This unexpected phenotype results from stabilization of the hybrid protein during carbon starvation and is apparently due to an energy requirement for proteolytic attack. Sequence analysis of DNA fragments cloned from an insertion mutant indicates that S. typhimurium has a large cluster of genes encoding the energy-conserving NADH dehydrogenase, similar to one recently described in Paracoccus denitrificans. These findings establish the potential for genetic analysis of a complex enzyme whose function, especially in proton efflux, is poorly understood. Images Fig. 2 PMID:8234329

  5. [Courtship behavior, communicative sound production and resistance to stress in Drosophila mutants with defective agnostic gene, coding for LIMK1].

    PubMed

    Popov, A V; Kaminskaia, A N; Savvateeva-Popova, E V

    2009-01-01

    -motor circuits of the singing center) decreases, and the rate and stability of pacemakers of the pulse and the sine songs increases. So, the sharply elevated LIMK1 and p-cofilin concentrations in the cells of agn(ts3) mutants, in comparison to wild type males, leading to derangement of learning and memory (Medvedeva et al., 2008) are accompanied by decline of motivation of males but do not seriously influence the neuro-motor coordination during singing. The higher resistance of characterictics of their behavior and communicative sound signals to heat shock is in agreement with the fact that the extremely high LIMK1 and P-cofilin concentrations in their cells go down to normal values after HS. At the same time the amyloid aggregations disappear and normally defective learning and memory are restore.

  6. Functional genomics screening utilizing mutant mouse embryonic stem cells identifies novel radiation-response genes.

    PubMed

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

    2015-01-01

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

  7. Characterizing and Targeting Replication Stress Response Defects in Breast Cancer

    DTIC Science & Technology

    2013-08-01

    collaboration with our colleague Dr. Chun Li, an outstanding leader in nanotechnology , we aimed to develop nanoparticles that can carry in vivo imaging...Pɘ.001. Task 4b. To develop nanoparticles to kill RSR-defective breast cancer cells through their binding to the RSR-defect-specific membrane...proteins on cancer cells. We have created the nanoparticles that can specifically bind to RSR-defect breast cells. We will continue to optimize

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

    PubMed Central

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

    2013-01-01

    Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin. Here, we observe that several different oligomeric states of a fusion protein, comprising ALS-associated human G85R SOD1 joined with yellow fluorescent protein (G85R SOD1YFP), which produces ALS in transgenic mice, inhibited anterograde transport when added to squid axoplasm. Inhibition was blocked both by an apoptosis signal-regulating kinase 1 (ASK1; MAPKKK) inhibitor and by a p38 inhibitor, indicating the transport defect is mediated through the MAPK cascade. In further incubations, we observed that addition of the mammalian molecular chaperone Hsc70, abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice, exerted partial correction of the transport defect, associated with diminished phosphorylation of p38. Most striking, the addition of the molecular chaperone Hsp110, in a concentration substoichiometric to the mutant SOD1 protein, completely rescued both the transport defect and the phosphorylation of p38. Hsp110 has been demonstrated to act as a nucleotide exchange factor for Hsc70 and, more recently, to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein, abrogating toxicity. PMID:23509252

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

    PubMed

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

    2013-04-02

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

  10. Differential Impact of LPG-and PG-Deficient Leishmania major Mutants on the Immune Response of Human Dendritic Cells

    PubMed Central

    Jayakumar, Asha; Hickerson, Suzanne; Mostrom, Janet; Turco, Salvatore J.; Beverley, Stephen M.; McDowell, Mary Ann

    2015-01-01

    Background Leishmania major infection induces robust interleukin-12 (IL12) production in human dendritic cells (hDC), ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of Leishmania parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG) and other phosphoglycan-containing molecules (PGs), making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS) responsible for IL12 induction. Methodology/Principal Findings Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating L. major Friedlin V1 mutants defective in LPG alone, (FV1 lpg1-), or generally deficient for all PGs, (FV1 lpg2-). Infection with metacyclic, infective stage, L. major or purified LPG induced high levels of IL12B subunit gene transcripts in hDCs, which was abrogated with FV1 lpg1- infections. In contrast, hDC infections with FV1 lpg2- displayed increased IL12B expression, suggesting other PG-related/LPG2 dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 lpg1-, FV1 lpg2- infections revealed that FV1 lpg1- mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB) and Interferon Regulatory Factor (IRF) mediated transcription. Conclusions/Significance These data suggest that L. major LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring Leishmania surface glycoconjugates that result in modulation of host cellular IL12. PMID:26630499

  11. Isolation and characterization of Pseudomonas putida PpF1 mutants defective in the toluene dioxygenase enzyme system.

    PubMed Central

    Finette, B A; Subramanian, V; Gibson, D T

    1984-01-01

    Pseudomonas putida PpF1 degraded toluene via a dihydrodiol pathway to tricarboxylic acid cycle intermediates. The initial reaction was catalyzed by a multicomponent enzyme, toluene dioxygenase, which oxidized toluene to (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol). The enzyme consisted of three protein components: NADH-ferredoxintol oxidoreductase (reductasetol), ferredoxintol, and a terminal oxygenase which is an iron-sulfur protein (ISPtol). Mutants blocked in each of these components were isolated after mutagenesis with nitrosoguanidine. Mutants occurred as colony morphology variants when grown in the presence of toluene on indicator plates containing agar, mineral salts, a growth-supporting nutrient (arginine), 2,3,5-triphenyltetrazolium chloride (TTC), and Nitro Blue Tetrazolium (NBT). Under these conditions, wild-type colonies appeared large and red as a result of TTC reduction. Colonies of reductasetol mutants were white or white with a light blue center, ferredoxintol strains were light blue with a dark blue center, and strains that lacked ISPtol gave dark blue colonies. Blue color differences in the mutant colonies were due to variations in the extent of NBT reduction. Strains lacking all three components appeared white. Toluene dioxygenase mutants were characterized by assaying toluene dioxygenase activity in crude cell extracts which were complemented with purified preparations of each protein component. Between 40 and 60% of the putative mutants selected from the NBT-TTC indicator plates were unable to grow with toluene as the sole source of carbon and energy. This method should prove extremely useful in isolating mutants in other multicomponent oxygenase enzyme systems. Images PMID:6501223

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

    PubMed Central

    Yeung, M K; Mattingly, S J

    1983-01-01

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

  13. Absence of Cajal-Retzius cells and subplate neurons associated with defects of tangential cell migration from ganglionic eminence in Emx1/2 double mutant cerebral cortex.

    PubMed

    Shinozaki, Koji; Miyagi, Toshihiko; Yoshida, Michio; Miyata, Takaki; Ogawa, Masaharu; Aizawa, Shinichi; Suda, Yoko

    2002-07-01

    Emx1 and Emx2, mouse orthologs of the Drosophila head gap gene, ems, are expressed during corticogenesis. Emx2 null mutants exhibit mild defects in cortical lamination. Segregation of differentiating neurons from proliferative cells is normal for the most part, however, reelin-positive Cajal-Retzius cells are lost by the late embryonic period. Additionally, late-born cortical plate neurons display abnormal position. These types of lamination defects are subtle in the Emx1 mutant cortex. In the present study we show that Emx1 and Emx2 double mutant neocortex is much more severely affected. Thickness of the cerebral wall was diminished with the decrease in cell number. Bromodeoxyuridine uptake in the germinal zone was nearly normal; moreover, no apparent increase in cell death or tetraploid cell number was observed. However, tangential migration of cells from the ganglionic eminence into the neocortex was greatly inhibited. The wild-type ganglionic eminence cells transplanted into Emx1/2-double mutant telencephalon did not move to the cortex. MAP2-positive neuronal bodies and RC2-positive radial glial cells emerged normally, but the laminar structure subsequently formed was completely abnormal. Furthermore, both corticofugal and corticopetal fibers were predominantly absent in the cortex. Most importantly, neither Cajal-Retzius cells nor subplate neurons were found throughout E11.5-E18.5. Thus, this investigation suggests that laminar organization in the cortex or the production of Cajal-Retzius cells and subplate neurons is interrelated to the tangential movement of cells from the ganglionic eminence under the control of Emx1 and Emx2.

  14. Vapb/Amyotrophic lateral sclerosis 8 knock-in mice display slowly progressive motor behavior defects accompanying ER stress and autophagic response.

    PubMed

    Larroquette, Frédérique; Seto, Lesley; Gaub, Perrine L; Kamal, Brishna; Wallis, Deeann; Larivière, Roxanne; Vallée, Joanne; Robitaille, Richard; Tsuda, Hiroshi

    2015-11-15

    Missense mutations (P56S) in Vapb are associated with autosomal dominant motor neuron diseases: amyotrophic lateral sclerosis and lower motor neuron disease. Although transgenic mice overexpressing the mutant vesicle-associated membrane protein-associated protein B (VAPB) protein with neuron-specific promoters have provided some insight into the toxic properties of the mutant proteins, their role in pathogenesis remains unclear. To identify pathological defects in animals expressing the P56S mutant VAPB protein at physiological levels in the appropriate tissues, we have generated Vapb knock-in mice replacing wild-type Vapb gene with P56S mutant Vapb gene and analyzed the resulting pathological phenotypes. Heterozygous P56S Vapb knock-in mice show mild age-dependent defects in motor behaviors as characteristic features of the disease. The homozygous P56S Vapb knock-in mice show more severe defects compared with heterozygous mice reflecting the dominant and dose-dependent effects of P56S mutation. Significantly, the knock-in mice demonstrate accumulation of P56S VAPB protein and ubiquitinated proteins in cytoplasmic inclusions, selectively in motor neurons. The mutant mice demonstrate induction of ER stress and autophagic response in motor neurons before obvious onset of behavioral defects, suggesting that these cellular biological defects might contribute to the initiation of the disease. The P56S Vapb knock-in mice could be a valuable tool to gain a better understanding of the mechanisms by which the disease arises. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. 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.

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

    SciTech Connect

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

    1994-11-01

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

  17. Mutations in the Caenorhabditis elegans orthologs of human genes required for mitochondrial tRNA modification cause similar electron transport chain defects but different nuclear responses.

    PubMed

    Navarro-González, Carmen; Moukadiri, Ismaïl; Villarroya, Magda; López-Pascual, Ernesto; Tuck, Simon; Armengod, M-Eugenia

    2017-07-01

    Several oxidative phosphorylation (OXPHOS) diseases are caused by defects in the post-transcriptional modification of mitochondrial tRNAs (mt-tRNAs). Mutations in MTO1 or GTPBP3 impair the modification of the wobble uridine at position 5 of the pyrimidine ring and cause heart failure. Mutations in TRMU affect modification at position 2 and cause liver disease. Presently, the molecular basis of the diseases and why mutations in the different genes lead to such different clinical symptoms is poorly understood. Here we use Caenorhabditis elegans as a model organism to investigate how defects in the TRMU, GTPBP3 and MTO1 orthologues (designated as mttu-1, mtcu-1, and mtcu-2, respectively) exert their effects. We found that whereas the inactivation of each C. elegans gene is associated with a mild OXPHOS dysfunction, mutations in mtcu-1 or mtcu-2 cause changes in the expression of metabolic and mitochondrial stress response genes that are quite different from those caused by mttu-1 mutations. Our data suggest that retrograde signaling promotes defect-specific metabolic reprogramming, which is able to rescue the OXPHOS dysfunction in the single mutants by stimulating the oxidative tricarboxylic acid cycle flux through complex II. This adaptive response, however, appears to be associated with a biological cost since the single mutant worms exhibit thermosensitivity and decreased fertility and, in the case of mttu-1, longer reproductive cycle. Notably, mttu-1 worms also exhibit increased lifespan. We further show that mtcu-1; mttu-1 and mtcu-2; mttu-1 double mutants display severe growth defects and sterility. The animal models presented here support the idea that the pathological states in humans may initially develop not as a direct consequence of a bioenergetic defect, but from the cell's maladaptive response to the hypomodification status of mt-tRNAs. Our work highlights the important association of the defect-specific metabolic rewiring with the pathological phenotype

  18. Isolation and characterization of Schizosaccharomyces pombe mutants defective in cell wall (1-3)beta-D-glucan.

    PubMed Central

    Ribas, J C; Diaz, M; Duran, A; Perez, P

    1991-01-01

    Schizosaccharomyces pombe thermosensitive mutants requiring the presence of an osmotic stabilizer to survive and grow at a nonpermissive temperature were isolated. The mutants were genetically and biochemically characterized. In all of them, the phenotype segregated in Mendelian fashion as a single gene which coded for a recessive character. Fourteen loci were defined by complementation analysis. Studies of cell wall composition showed a reduction in the amount of cell wall beta-glucan in three strains (JCR1, JCR5, and JCR10) when growing at 37 degrees C. Galactomannan was diminished in two others. Strains JCR1 and JCR5, with mutant alleles cwg1-1 and cwg2-1, respectively, were further studied. The cwg1 locus was mapped on the right arm of chromosome III, 18.06 centimorgans (cM) to the left of the ade5 marker; cwg2 was located on the left arm of chromosome I, 34.6 cM away from the aro5 marker. (1-3)beta-D-Glucan synthase activities from cwg1-1 and cwg2-1 mutant strains grown at 37 degrees C were diminished, as measured in vitro, compared with the wild-type strain; however, Km values and activation by GTP were similar to the wild-type values. Mutant synthases behaved like the wild-type enzyme in terms of thermostability. Analyses of round shape, lytic behavior, and low (1-3)beta-D-glucan synthase activity in cultures derived from ascospores of the same tetrad showed cosegregation of all these characters. Detergent dissociation of (1-3)beta-D-glucan synthase into soluble and particulate fractions and subsequent reconstitution demonstrated that the cwg1-1 mutant was affected in the particulate fraction of the enzymatic activity while cwg2-1 was affected in the soluble component. The antifungal agents Papulacandin B and Aculeacin A had similar effects on the enzymatic activities of the wild type and the cwg2-1 mutant strain, whereas the cwg1-1 mutant, when growing at 37 degrees C, had a more inhibitor-resistant (1,3)beta-D-glucan synthase. It is concluded that the cwg1

  19. High temperature specifically affects the photoprotective responses of chlorophyll b-deficient wheat mutant lines.

    PubMed

    Brestic, Marian; Zivcak, Marek; Kunderlikova, Kristyna; Allakhverdiev, Suleyman I

    2016-12-01

    The effects of high temperature on CO2 assimilation rate, processes associated with photosynthetic electron and proton transport, as well as photoprotective responses, were studied in chlorophyll b-deficient mutant lines (ANK-32A and ANK-32B) and wild type (WT) of wheat (Triticum aestivum L.). Despite the low chlorophyll content and chlorophyll a-to-b ratio, the non-stressed mutant plants had the similar level of CO2 assimilation and photosynthetic responses as WT. However, in ANK mutant plants exposed to prolonged high temperature episode (42 °C for ~10 h), we observed lower CO2 assimilation compared to WT, especially when a high CO2 supply was provided. In all heat-exposed plants, we found approximately the same level of PSII photoinhibition, but the decrease in content of photooxidizable PSI was higher in ANK mutant plants compared to WT. The PSI damage can be well explained by the level of overreduction of PSI acceptor side observed in plants exposed to high temperature, which was, in turn, the result of the insufficient transthylakoid proton gradient associated with low non-photochemical quenching and lack of ability to downregulate the linear electron transport to keep the reduction state of PSI acceptor side low enough. Compared to WT, the ANK mutant lines had lower capacity to drive the cyclic electron transport around PSI in moderate and high light; it confirms the protective role of cyclic electron transport for the protection of PSI against photoinhibition. Our results, however, also suggest that the inactivation of PSI in heat stress conditions can be the protective mechanism against photooxidative damage of chloroplast and cell structures.

  20. Cell-substrate interactions and locomotion of Dictyostelium wild-type and mutants defective in three cytoskeletal proteins: a study using quantitative reflection interference contrast microscopy.

    PubMed Central

    Schindl, M; Wallraff, E; Deubzer, B; Witke, W; Gerisch, G; Sackmann, E

    1995-01-01

    Reflection interference contrast microscopy combined with digital image processing was applied to study the motion of Dictyostelium discoideum cells in their pre-aggregative state on substrata of different adhesiveness (glass, albumin-covered glass, and freshly cleaved mica). The temporal variations of the size and shape of the cell/substratum contact area and the time course of advancement of pseudopods protruding in contact with the substratum were analyzed. The major goal was to study differences between the locomotion of wild-type cells and strains of triple mutants deficient in two F-actin cross-linking proteins (alpha-actinin and the 120-kDa gelation factor) and one F-actin fragmenting protein (severin). The size of contact area, AC, of both wild-type and mutant cells fluctuates between minimum and maximum values on the order of minutes, pointing toward an intrinsic switching mechanism associated with the mechanochemical control system. The fluctuation amplitudes are much larger on freshly cleaved mica than on glass. Wild-type and mutant cells exhibit remarkable differences on mica but not on glass. These differences comprise the population median of AC and alterations in pseudopod protrusion. AC is smaller by a factor of two or more for all mutants. Pseudopods protrude slower and shorter in the mutants. It is concluded that cell shape and pseudopods are destabilized by defects in the actin-skeleton, which can be overcompensated by strongly adhesive substrata. Several features of amoeboid cell locomotion on substrata can be understood on the basis of the minimum bending energy concept of soft adhering shells and by assuming that adhesion induces local alterations of the composite membrane consisting of the protein/lipid bilayer on the cell surface and the underlying actin-cortex. Images FIGURE 3 FIGURE 4 FIGURE 6 FIGURE 8 FIGURE 9 FIGURE 10 FIGURE 11 FIGURE 12 PMID:7756537

  1. Perinuclear Mlp proteins downregulate gene expression in response to a defect in mRNA export.

    PubMed

    Vinciguerra, Patrizia; Iglesias, Nahid; Camblong, Jurgi; Zenklusen, Daniel; Stutz, Françoise

    2005-02-23

    The mRNA export adaptor Yra1p/REF contributes to nascent mRNP assembly and recruitment of the export receptor Mex67p. yra1 mutants exhibit mRNA export defects and a decrease in LacZ reporter and certain endogenous transcripts. The loss of Mlp1p/Mlp2p, two TPR-like proteins attached to nuclear pores, rescues LacZ mRNA levels and increases their appearance in the cytoplasm, without restoring bulk poly(A)+ RNA export. Chromatin immunoprecipitation, FISH and pulse-chase experiments indicate that Mlps downregulate LacZ mRNA synthesis in a yra1 mutant strain. Microarray analyses reveal that Mlp2p also reduces a subset of cellular transcripts in the yra1 mutant. Finally, we show that Yra1p genetically interacts with the shuttling mRNA-binding protein Nab2p and that loss of Mlps rescues the growth defect of yra1 and nab2 but not other mRNA export mutants. We propose that Nab2p and Yra1p are required for proper mRNP docking to the Mlp platform. Defects in Yra1p prevent mRNPs from crossing the Mlp gate and this block negatively feeds back on the transcription of a subset of genes, suggesting that Mlps link mRNA transcription and export.

  2. Characterization of four rice mutants with alterations in the defence response pathway.

    PubMed

    Campbell, M A; Ronald, P C

    2005-01-01

    SUMMARY A fast-neutron mutagenized population of rice seedlings was screened with Magnaporthe grisea, the causal agent of rice blast disease, to identify mutants with alterations in the defence response. Three mutant lines, ebr1, ebr2 and ebr3 (enhanced blast resistance) were identified that display enhanced resistance to M. grisea. ebr1 and ebr3 also confer enhanced resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). ebr3 develops a lesion mimic (LM) phenotype upon inoculation with M. grisea, and the phenotype is also induced by a shift in environmental conditions. The fourth mutant line, ncr1 (necrosis in rice), has an LM phenotype under all conditions tested and lacks enhanced resistance to either M. grisea or Xoo. Complementation testing using the mutant lines ebr3 and ncr1 indicates that the ebr3 and ncr1 loci are nonallelic and recessive. ebr1 and ebr2 display no alterations in expression of the rice pathogenesis-related (PR) genes PBZ1 and PR1, compared to wild-type CO39. ebr3 has an elevated expression of PBZ1 and PR1 only in tissue displaying the LM phenotype. ncr1 strongly expresses PBZ1 in tissue displaying the LM phenotype, whereas PR1 expression in this tissue is similar to wild-type CO39.

  3. Recruitment of DNA replication and damage response proteins to viral replication centers during infection with NS2 mutants of Minute Virus of Mice (MVM).

    PubMed

    Ruiz, Zandra; Mihaylov, Ivailo S; Cotmore, Susan F; Tattersall, Peter

    2011-02-20

    MVM NS2 is essential for viral DNA amplification, but its mechanism of action is unknown. A classification scheme for autonomous parvovirus-associated replication (APAR) center development, based on NS1 distribution, was used to characterize abnormal APAR body maturation in NS2null mutant infections, and their organization examined for defects in host protein recruitment. Since acquisition of known replication factors appeared normal, we looked for differences in invoked DNA damage responses. We observed widespread association of H2AX/MDC1 damage response foci with viral replication centers, and sequestration and complex hyperphosphorylation of RPA(32), which occurred in wildtype and mutant infections. Quantifying these responses by western transfer indicated that both wildtype and NS2 mutant MVM elicited ATM activation, while phosphorylation of ATR, already basally activated in asynchronous A9 cells, was downregulated. We conclude that MVM infection invokes multiple damage responses that influence the APAR environment, but that NS2 does not modify the recruitment of cellular proteins.

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

    PubMed Central

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

    2013-01-01

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

  5. Analysis of Candida albicans Mutants Defective in the Cdk8 Module of Mediator Reveal Links between Metabolism and Biofilm Formation

    PubMed Central

    Lindsay, Allia K.; Morales, Diana K.; Liu, Zhongle; Grahl, Nora; Zhang, Anda; Willger, Sven D.; Myers, Lawrence C.; Hogan, Deborah A.

    2014-01-01

    Candida albicans biofilm formation is a key virulence trait that involves hyphal growth and adhesin expression. Pyocyanin (PYO), a phenazine secreted by Pseudomonas aeruginosa, inhibits both C. albicans biofilm formation and development of wrinkled colonies. Using a genetic screen, we identified two mutants, ssn3Δ/Δ and ssn8Δ/Δ, which continued to wrinkle in the presence of PYO. Ssn8 is a cyclin-like protein and Ssn3 is similar to cyclin-dependent kinases; both proteins are part of the heterotetrameric Cdk8 module that forms a complex with the transcriptional co-regulator, Mediator. Ssn3 kinase activity was also required for PYO sensitivity as a kinase dead mutant maintained a wrinkled colony morphology in the presence of PYO. Furthermore, similar phenotypes were observed in mutants lacking the other two components of the Cdk8 module—Srb8 and Srb9. Through metabolomics analyses and biochemical assays, we showed that a compromised Cdk8 module led to increases in glucose consumption, glycolysis-related transcripts, oxidative metabolism and ATP levels even in the presence of PYO. In the mutant, inhibition of respiration to levels comparable to the PYO-treated wild type inhibited wrinkled colony development. Several lines of evidence suggest that PYO does not act through Cdk8. Lastly, the ssn3 mutant was a hyperbiofilm former, and maintained higher biofilm formation in the presence of PYO than the wild type. Together these data provide novel insights into the role of the Cdk8 module of Mediator in regulation of C. albicans physiology and the links between respiratory activity and both wrinkled colony and biofilm development. PMID:25275466

  6. 49 CFR 210.7 - Responsibility for noise defective railroad equipment.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 4 2011-10-01 2011-10-01 false Responsibility for noise defective railroad...) FEDERAL RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION RAILROAD NOISE EMISSION COMPLIANCE REGULATIONS General Provisions § 210.7 Responsibility for noise defective railroad equipment. Any railroad...

  7. 49 CFR 210.7 - Responsibility for noise defective railroad equipment.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

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

  8. Cleft Palate Defect of Dlx1/2−/− Mutant Mice is Caused by Lack of Vertical Outgrowth in the Posterior Palate

    PubMed Central

    Jeong, Juhee; Cesario, Jeffry; Zhao, Yangu; Burns, Lorel; Westphal, Heiner; Rubenstein, John L. R.

    2014-01-01

    Background Mice lacking the activities of Dlx1 and Dlx2 (Dlx1/2−/−) exhibit cleft palate, one of the most common human congenital defects, but the etiology behind this phenotype has been unknown. Therefore, we analyzed the morphological, cellular, and molecular changes caused by inactivation of Dlx1 and Dlx2 as related to palate development. Results Dlx1/2−/− mutants exhibited lack of vertical growth in the posterior palate during the earliest stage of palatogenesis. We attributed this growth deficiency to reduced cell proliferation. Expression of a cell cycle regulator Ccnd1 was specifically down-regulated in the same region. Previous studies established that the epithelial-mesenchymal signaling loop involving Shh, Bmp4 and Fgf10 is important for cell proliferation and tissue growth during palate development. This signaling loop was disrupted in Dlx1/2−/− palate. Interestingly, however, the decreases in Ccnd1 expression and mitosis in Dlx1/2−/− mutants were independent of this signaling loop. Finally, Dlx1/2 activity was required for normal expression of several transcription factor genes whose mutation results in palate defects. Conclusions The functions of Dlx1 and Dlx2 are crucial for the initial formation of the posterior palatal shelves, and that the Dlx genes lie upstream of multiple signaling molecules and transcription factors important for later stages of palatogenesis. PMID:22972697

  9. Differential protein stability of EGFR mutants determines responsiveness to tyrosine kinase inhibitors

    PubMed Central

    Ray, Paramita; Tan, Yee Sun; Somnay, Vishal; Mehta, Ranjit; Sitto, Merna; Ahsan, Aarif; Nyati, Shyam; Naughton, John P.; Bridges, Alexander; Zhao, Lili; Rehemtulla, Alnawaz; Lawrence, Theodore S.; Ray, Dipankar; Nyati, Mukesh K.

    2016-01-01

    Non-small cell lung cancer (NSCLC) patients carrying specific EGFR kinase activating mutations (L858R, delE746-A750) respond well to tyrosine kinase inhibitors (TKIs). However, drug resistance develops within a year. In about 50% of such patients, acquired drug resistance is attributed to the enrichment of a constitutively active point mutation within the EGFR kinase domain (T790M). To date, differential drug-binding and altered ATP affinities by EGFR mutants have been shown to be responsible for differential TKI response. As it has been reported that EGFR stability plays a role in the survival of EGFR driven cancers, we hypothesized that differential TKI-induced receptor degradation between the sensitive L858R and delE746-A750 and the resistant T790M may also play a role in drug responsiveness. To explore this, we have utilized an EGFR-null CHO overexpression system as well as NSCLC cell lines expressing various EGFR mutants and determined the effects of erlotinib treatment. We found that erlotinib inhibits EGFR phosphorylation in both TKI sensitive and resistant cells, but the protein half-lives of L858R and delE746-A750 were significantly shorter than L858R/T790M. Third generation EGFR kinase inhibitor (AZD9291) inhibits the growth of L858R/T790M-EGFR driven cells and also induces EGFR degradation. Erlotinib treatment induced polyubiquitination and proteasomal degradation, primarily in a c-CBL-independent manner, in TKI sensitive L858R and delE746-A750 mutants when compared to the L858R/T790M mutant, which correlated with drug sensitivity. These data suggest an additional mechanism of TKI resistance, and we postulate that agents that degrade L858R/T790M-EGFR protein may overcome TKI resistance. PMID:27612423

  10. Thiamin-responsive maple-syrup-urine disease: decreased affinity of the mutant branched-chain alpha-keto acid dehydrogenase for alpha-ketoisovalerate and thiamin pyrophosphate.

    PubMed Central

    Chuang, D T; Ku, L S; Cox, R P

    1982-01-01

    The biochemical basis for the therapeutic effects of thiamin in thiamin-responsive maple-syrup-urine disease (MSUD) was investigated in intact and disrupted fibroblast cultures from normals and patients with various forms of MSUD. Decarboxylation of alpha-keto[1-14C]isovalerate (KIV) by intact cells from a thiamin-responsive MSUD patient was at 30-40% of the normal rate with or without thiamin in the incubation medium. Under similar conditions, intact classical MSUD fibroblasts failed to decarboxylate KIV. Branched-chain alpha-keto acid (BCKA) dehydrogenase activity measured in disrupted cells from the thiamin-responsive subject showed sigmoidal kinetics in the absence of thiamin pyrophosphate (TPP), with an increased concentration of substrate needed for half-maximal velocity (K0.5 for KIV = 7 mM vs. 0.05 mM in normal cells). When assayed with 0.2 mM TPP present, the mutant enzyme showed (i) a shift in kinetics to near Michaelis-Menten type as observed with the normal BCKA dehydrogenase and (ii) a lower K0.5 value of 4 mM for KIV, suggesting a TPP-mediated increase in the mutant enzyme's affinity for substrate. By contrast, TPP increased only the Vmax and was without effect on the apparent Km for KIV of the BCKA dehydrogenase from cells of normals and patients with classical MSUD and variant thiamin-responsive MSUD (grade 3). Measurement of the apparent Km for TPP of the BCKA dehydrogenase from thiamin-responsive mutant MSUd cells showed a 16-fold increase in the constant to 25 microM compared to enzymes from normal or classical MSUD cells. These findings demonstrate that the primary defect in the thiamin-responsive MSUD patient is a reduced affinity of the mutant BCKA dehydrogenase for TPP that results in impaired oxidative decarboxylation of BCKA. PMID:6954481

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2006-11-24

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

  13. Periodontal response after tooth movement into intrabony defects.

    PubMed

    Polson, A; Caton, J; Polson, A P; Nyman, S; Novak, J; Reed, B

    1984-04-01

    The present study was undertaken since conflicting evidence exists regarding the effect of such tooth movement on levels of connective tissue attachment. Localized intrabony pockets were produced around isolated incisors in four rhesus monkeys. The root surfaces were planned to the level of the bone at the base of the angular bony defects. An oral hygiene regime was begun and continued for the remainder of the study. The experimental teeth were moved orthodontically into, and through, the original area of the intrabony defect. Two months after cessation of active tooth movement, block specimens were removed for histologic analysis. Control specimens comprised those teeth with induced periodontal defects, but without tooth movement. In specimens not subjected to tooth movement, angular bony defects were present and epithelium lined the root surface to the apical extent of instrumentation. The alveolar bone adjacent to the orthodontically moved teeth no longer had angular defect morphology. On the pressure side, epithelium lined the root surface, was interposed between root surface and bone and terminated at the apical limit of root instrumentation. On the tension side, the crest of the bone was located apical to the level of root planing, and epithelium lined the instrumented portion of the root surface. It was concluded that orthodontic tooth movement into intrabony periodontal defects was without effect upon the levels of connective tissue attachment.

  14. Mutant p63 causes defective expansion of ectodermal progenitor cells and impaired FGF signalling in AEC syndrome.

    PubMed

    Ferone, Giustina; Thomason, Helen A; Antonini, Dario; De Rosa, Laura; Hu, Bing; Gemei, Marica; Zhou, Huiqing; Ambrosio, Raffaele; Rice, David P; Acampora, Dario; van Bokhoven, Hans; Del Vecchio, Luigi; Koster, Maranke I; Tadini, Gianluca; Spencer-Dene, Bradley; Dixon, Michael; Dixon, Jill; Missero, Caterina

    2012-03-01

    Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome, which is characterized by cleft palate and severe defects of the skin, is an autosomal dominant disorder caused by mutations in the gene encoding transcription factor p63. Here, we report the generation of a knock-in mouse model for AEC syndrome (p63(+/L514F) ) that recapitulates the human disorder. The AEC mutation exerts a selective dominant-negative function on wild-type p63 by affecting progenitor cell expansion during ectodermal development leading to a defective epidermal stem cell compartment. These phenotypes are associated with impairment of fibroblast growth factor (FGF) signalling resulting from reduced expression of Fgfr2 and Fgfr3, direct p63 target genes. In parallel, a defective stem cell compartment is observed in humans affected by AEC syndrome and in Fgfr2b(-/-) mice. Restoring Fgfr2b expression in p63(+/L514F) epithelial cells by treatment with FGF7 reactivates downstream mitogen-activated protein kinase signalling and cell proliferation. These findings establish a functional link between FGF signalling and p63 in the expansion of epithelial progenitor cells and provide mechanistic insights into the pathogenesis of AEC syndrome. Copyright © 2012 EMBO Molecular Medicine.

  15. In vitro characterization of 6S RNA release-defective mutants uncovers features of pRNA-dependent release from RNA polymerase in E. coli

    PubMed Central

    Oviedo Ovando, Mariana; Shephard, Lindsay; Unrau, Peter J.

    2014-01-01

    6S RNA is a noncoding RNA that inhibits bacterial transcription by sequestering RNA polymerase holoenzyme (Eσ70) in low-nutrient conditions. This transcriptional block can be relieved by the synthesis of a short product RNA (pRNA) using the 6S RNA as a template. Here, we selected a range of 6S RNA release-defective mutants from a high diversity in vitro pool. Studying the release-defective variant R9-33 uncovered complex interactions between three regions of the 6S RNA. As expected, mutating the transcriptional start site (TSS) slowed and partially inhibited release. Surprisingly, additional mutations near the TSS were found that rescued this effect. Likewise, three mutations in the top strand of the large open bubble (LOB) could considerably slow release but were rescued by the addition of upstream mutations found between a highly conserved “-35” motif and the LOB. Combining the three top strand LOB mutations with mutations near the TSS, however, was particularly effective at preventing release, and this effect could be further enhanced by inclusion of the upstream mutations. Overexpressing R9-33 and a series of milder release-defective mutants in Escherichia coli resulted in a delayed entry into exponential phase together with a decrease in cell survival that correlated well with the severity of the in vitro phenotypes. The complex crosstalk observed between distinct regions of the 6S RNA supports a scrunching type model of 6S RNA release, where at least three regions of the 6S RNA must interact with Eσ70 in a cooperative manner so as to ensure effective pRNA-dependent release. PMID:24681966

  16. Non-invasive, whole-plant imaging of chloroplast movement and chlorophyll fluorescence reveals photosynthetic phenotypes independent of chloroplast photorelocation defects in chloroplast division mutants.

    PubMed

    Dutta, Siddhartha; Cruz, Jeffrey A; Jiao, Yuhua; Chen, Jin; Kramer, David M; Osteryoung, Katherine W

    2015-10-01

    Leaf chloroplast movement is thought to optimize light capture and to minimize photodamage. To better understand the impact of chloroplast movement on photosynthesis, we developed a technique based on the imaging of reflectance from leaf surfaces that enables continuous, high-sensitivity, non-invasive measurements of chloroplast movement in multiple intact plants under white actinic light. We validated the method by measuring photorelocation responses in Arabidopsis chloroplast division mutants with drastically enlarged chloroplasts, and in phototropin mutants with impaired photorelocation but normal chloroplast morphology, under different light regimes. Additionally, we expanded our platform to permit simultaneous image-based measurements of chlorophyll fluorescence and chloroplast movement. We show that chloroplast division mutants with enlarged, less-mobile chloroplasts exhibit greater photosystem II photodamage than is observed in the wild type, particularly under fluctuating high levels of light. Comparison between division mutants and the severe photorelocation mutant phot1-5 phot2-1 showed that these effects are not entirely attributable to diminished photorelocation responses, as previously hypothesized, implying that altered chloroplast morphology affects other photosynthetic processes. Our dual-imaging platform also allowed us to develop a straightforward approach to correct non-photochemical quenching (NPQ) calculations for interference from chloroplast movement. This correction method should be generally useful when fluorescence and reflectance are measured in the same experiments. The corrected data indicate that the energy-dependent (qE) and photoinhibitory (qI) components of NPQ contribute differentially to the NPQ phenotypes of the chloroplast division and photorelocation mutants. This imaging technology thus provides a platform for analyzing the contributions of chloroplast movement, chloroplast morphology and other phenotypic attributes to the

  17. Analysis of Redox Responses During TNT Transformation by Clostridium acetobutylicum ATCC 824 and Mutants Exhibiting Altered Metabolism

    DTIC Science & Technology

    2012-01-01

    relevant for bioremediation studies, and various Clostridium species have been reported to degrade TNT through alternative routes (Ahmad and Hughes 2000...REPORT Analysis of redox responses during TNT transformation by Clostridium acetobutylicum ATCC 824 and mutants exhibiting altered metabolism 14...ABSTRACT 16. SECURITY CLASSIFICATION OF: The transformation of trinitrotoluene (TNT) by several mutant strains of Clostridium acetobutylicum has been

  18. Acidithiobacillus caldus Sulfur Oxidation Model Based on Transcriptome Analysis between the Wild Type and Sulfur Oxygenase Reductase Defective Mutant

    PubMed Central

    Chen, Linxu; Ren, Yilin; Lin, Jianqun; Liu, Xiangmei; Pang, Xin; Lin, Jianqiang

    2012-01-01

    Background Acidithiobacillus caldus (A. caldus) is widely used in bio-leaching. It gains energy and electrons from oxidation of elemental sulfur and reduced inorganic sulfur compounds (RISCs) for carbon dioxide fixation and growth. Genomic analyses suggest that its sulfur oxidation system involves a truncated sulfur oxidation (Sox) system (omitting SoxCD), non-Sox sulfur oxidation system similar to the sulfur oxidation in A. ferrooxidans, and sulfur oxygenase reductase (SOR). The complexity of the sulfur oxidation system of A. caldus generates a big obstacle on the research of its sulfur oxidation mechanism. However, the development of genetic manipulation method for A. caldus in recent years provides powerful tools for constructing genetic mutants to study the sulfur oxidation system. Results An A. caldus mutant lacking the sulfur oxygenase reductase gene (sor) was created and its growth abilities were measured in media using elemental sulfur (S0) and tetrathionate (K2S4O6) as the substrates, respectively. Then, comparative transcriptome analysis (microarrays and real-time quantitative PCR) of the wild type and the Δsor mutant in S0 and K2S4O6 media were employed to detect the differentially expressed genes involved in sulfur oxidation. SOR was concluded to oxidize the cytoplasmic elemental sulfur, but could not couple the sulfur oxidation with the electron transfer chain or substrate-level phosphorylation. Other elemental sulfur oxidation pathways including sulfur diooxygenase (SDO) and heterodisulfide reductase (HDR), the truncated Sox pathway, and the S4I pathway for hydrolysis of tetrathionate and oxidation of thiosulfate in A. caldus are proposed according to expression patterns of sulfur oxidation genes and growth abilities of the wild type and the mutant in different substrates media. Conclusion An integrated sulfur oxidation model with various sulfur oxidation pathways of A. caldus is proposed and the features of this model are summarized. PMID:22984393

  19. Characterization of the defects in bacteriophage T7 DNA synthesis during growth in the Escherichia coli mutant tsnB.

    PubMed Central

    DeWyngaert, M A; Hinkle, D C

    1980-01-01

    The Escherichia coli mutant tsnB (M. Chamberlin, J. Virol. 14:509-516, 1974) is unable to support the growth of bacteriophage T7, although all classes of phage proteins are produced and the host is killed by the infection. During growth in this mutant host, the rate of phage DNA synthesis is reduced and the DNA is not packaged into stable, phagelike particles. The replicating DNA forms concatemers but the very large replicative intermediates (approximately 440S) identified by Paetkau et al. (J. Virol. 22:130-141, 1977) are not detected in T7+-infected tsnB cells. These large structures are formed in tsnB cells infected with a T7 gene 3 (endonuclease) mutant, where normal processing of the large intermediates into shorter concatemers is blocked. At later times during infection of tsnB cells, the replicating DNA accumulates in molecules about 30% shorter than unit length. Analysis of this DNA with a restriction endonuclease indicates that it is missing sequences from the ends (particularly the left end) of the genome. The loss of these specific sequences does not occur during infections with T7 gene 10 (head protein) or gene 19 (maturation protein) mutants. This suggests that the processing of concatemers into unit-length DNA molecules may occur normally in T7 -infected tsnB cells and that the shortened DNA arises from exonucleolytic degradation of the mature DNA molecules. These results are discussed in relation to our recent observation (M. A. DeWyngaert and D. C. Hinkle, J. Biol. Chem. 254:11247-11253, 1979) that E. coli tsnB produces an altered RNA polymerase which is resistance to inhibition by the T7 gene 2 protein. Images PMID:6997508

  20. Characterization of the defects in bacteriophage T7 DNA synthesis during growth in the Escherichia coli mutant tsnB.

    PubMed

    DeWyngaert, M A; Hinkle, D C

    1980-02-01

    The Escherichia coli mutant tsnB (M. Chamberlin, J. Virol. 14:509-516, 1974) is unable to support the growth of bacteriophage T7, although all classes of phage proteins are produced and the host is killed by the infection. During growth in this mutant host, the rate of phage DNA synthesis is reduced and the DNA is not packaged into stable, phagelike particles. The replicating DNA forms concatemers but the very large replicative intermediates (approximately 440S) identified by Paetkau et al. (J. Virol. 22:130-141, 1977) are not detected in T7+-infected tsnB cells. These large structures are formed in tsnB cells infected with a T7 gene 3 (endonuclease) mutant, where normal processing of the large intermediates into shorter concatemers is blocked. At later times during infection of tsnB cells, the replicating DNA accumulates in molecules about 30% shorter than unit length. Analysis of this DNA with a restriction endonuclease indicates that it is missing sequences from the ends (particularly the left end) of the genome. The loss of these specific sequences does not occur during infections with T7 gene 10 (head protein) or gene 19 (maturation protein) mutants. This suggests that the processing of concatemers into unit-length DNA molecules may occur normally in T7 -infected tsnB cells and that the shortened DNA arises from exonucleolytic degradation of the mature DNA molecules. These results are discussed in relation to our recent observation (M. A. DeWyngaert and D. C. Hinkle, J. Biol. Chem. 254:11247-11253, 1979) that E. coli tsnB produces an altered RNA polymerase which is resistance to inhibition by the T7 gene 2 protein.

  1. A Saccharomyces cerevisiae phleomycin-sensitive mutant, ph140, is defective in the RAD6 DNA repair gene.

    PubMed

    He, C H; Masson, J Y; Ramotar, D

    1996-12-01

    The antibiotic bleomycin is used as an anticancer agent for treating a variety of tumours. The antitumour effect of bleomycin is related to its ability to produce lesions such as apurinic/apyrimidinic sites and single- and double-strand breaks in the cellular DNA. Phleomycin is a structurally related form of bleomycin, but it is not used as an anticancer agent. While phleomycin can also damage DNA, neither the exact nature of these DNA lesions nor the cellular process that repairs phleomycin-induced DNA lesions is known. As a first step to understand how eukaryotic cells provide resistance to phleomycin, we used the yeast Saccharomyces cerevisiae as a model system. Several phleomycin-sensitive mutants were generated following gamma-radiation treatment and among these mutants, ph140 was found to be the most sensitive to phleomycin. Molecular analysis revealed that the mutant ph140 harbored a mutation in the DNA repair gene RAD6. Moreover, a functional copy of the RAD6 gene restored full phleomycin resistance to strain ph140. Our findings indicate that the RAD6 protein is essential for yeast cellular resistance to phleomycin.

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

    PubMed Central

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

    1997-01-01

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

  3. Identification of genes associated with copper tolerance in an adhesion-defective mutant of Aeromonas veronii biovar sobria.

    PubMed

    Francki, K T; Chang, B J; Mee, B J; Collignon, P J; Susai, V; Keese, P K

    2000-10-01

    TnphoA mutagenesis was used to identify adhesins of Aeromonas veronii biovar sobria 3767, a strain isolated from a diarrhoeal stool specimen. Six mutants, from a library of 154, exhibited significantly reduced levels of adhesion to HEp-2 cells. Primers to the terminal regions of TnphoA were used for inverse PCR and the product from one mutant was cloned into pBluescript and partial sequence data obtained. Scanning GenBank and EMBL data bases revealed DNA sequence similarity to the copA gene of Pseudomonas syringae pv. tomato which confers resistance to copper and other heavy metals. The transposon was located within the copA gene and the mutant exhibited a reduced tolerance to copper. Primer walking, using the inverse PCR product as a template, revealed three open reading frames (ORFs) copA, B and C in A. veronii biovar sobria 3767. The predicted amino acid sequences of ORFs A and B had significant homology (55 and 34% respectively) to the copA and B proteins of P. syringae. No amino acid or DNA sequence homology existed between ORF C of strain 3767 and any other gene in the data bases scanned. Further analysis of the nucleotide sequence failed to reveal the presence of typical copper regulatory genes within the vicinity of the Aeromonas sequence. The association between copper tolerance and adhesion in A. veronii biovar sobria requires further study.

  4. Drosophila lines with mutant and wild type human TDP-43 replacing the endogenous gene reveals phosphorylation and ubiquitination in mutant lines in the absence of viability or lifespan defects

    PubMed Central

    Chang, Jer-Cherng

    2017-01-01

    Mutations in TDP-43 are associated with proteinaceous inclusions in neurons and are believed to be causative in neurodegenerative diseases such as frontotemporal dementia or amyotrophic lateral sclerosis. Here we describe a Drosophila system where we have engineered the genome to replace the endogenous TDP-43 orthologue with wild type or mutant human TDP-43(hTDP-43). In contrast to other models, these flies express both mutant and wild type hTDP-43 at similar levels to those of the endogenous gene and importantly, no age-related TDP-43 accumulation observed among all the transgenic fly lines. Immunoprecipitation of TDP-43 showed that flies with hTDP-43 mutations had increased levels of ubiquitination and phosphorylation of the hTDP-43 protein. Furthermore, histologically, flies expressing hTDP-43 M337V showed global, robust neuronal staining for phospho-TDP. All three lines: wild type hTDP-43, -G294A and -M337V were homozygous viable, with no defects in development, life span or behaviors observed. The primary behavioral defect was that flies expressing either hTDP-43 G294A or M337V showed a faster decline with age in negative geotaxis. Together, these observations implied that neurons could handle these TDP-43 mutations by phosphorylation- and ubiquitin-dependent proteasome systems, even in a background without the wild type TDP-43. Our findings suggest that these two specific TDP-43 mutations are not inherently toxic, but may require additional environmental or genetic factors to affect longevity or survival. PMID:28686708

  5. Drosophila lines with mutant and wild type human TDP-43 replacing the endogenous gene reveals phosphorylation and ubiquitination in mutant lines in the absence of viability or lifespan defects.

    PubMed

    Chang, Jer-Cherng; Morton, David B

    2017-01-01

    Mutations in TDP-43 are associated with proteinaceous inclusions in neurons and are believed to be causative in neurodegenerative diseases such as frontotemporal dementia or amyotrophic lateral sclerosis. Here we describe a Drosophila system where we have engineered the genome to replace the endogenous TDP-43 orthologue with wild type or mutant human TDP-43(hTDP-43). In contrast to other models, these flies express both mutant and wild type hTDP-43 at similar levels to those of the endogenous gene and importantly, no age-related TDP-43 accumulation observed among all the transgenic fly lines. Immunoprecipitation of TDP-43 showed that flies with hTDP-43 mutations had increased levels of ubiquitination and phosphorylation of the hTDP-43 protein. Furthermore, histologically, flies expressing hTDP-43 M337V showed global, robust neuronal staining for phospho-TDP. All three lines: wild type hTDP-43, -G294A and -M337V were homozygous viable, with no defects in development, life span or behaviors observed. The primary behavioral defect was that flies expressing either hTDP-43 G294A or M337V showed a faster decline with age in negative geotaxis. Together, these observations implied that neurons could handle these TDP-43 mutations by phosphorylation- and ubiquitin-dependent proteasome systems, even in a background without the wild type TDP-43. Our findings suggest that these two specific TDP-43 mutations are not inherently toxic, but may require additional environmental or genetic factors to affect longevity or survival.

  6. Loss-of-Function Mutations in HspR Rescue the Growth Defect of a Mycobacterium tuberculosis Proteasome Accessory Factor E (pafE) Mutant

    PubMed Central

    Jastrab, Jordan B.; Samanovic, Marie I.; Copin, Richard; Shopsin, Bo

    2017-01-01

    ABSTRACT Mycobacterium tuberculosis uses a proteasome to degrade proteins by both ATP-dependent and -independent pathways. While much has been learned about ATP-dependent degradation, relatively little is understood about the ATP-independent pathway, which is controlled by Mycobacterium tuberculosis proteasome accessory factor E (PafE). Recently, we found that a Mycobacterium tuberculosis pafE mutant has slowed growth in vitro and is sensitive to killing by heat stress. However, we did not know if these phenotypes were caused by an inability to degrade the PafE-proteasome substrate HspR (heat shock protein repressor), an inability to degrade any damaged or misfolded proteins, or a defect in another protein quality control pathway. To address this question, we characterized pafE suppressor mutants that grew similarly to pafE+ bacteria under normal culture conditions. All but one suppressor mutant analyzed contained mutations that inactivated HspR function, demonstrating that the slowed growth and heat shock sensitivity of a pafE mutant were caused primarily by the inability of the proteasome to degrade HspR. IMPORTANCE Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required for virulence. We recently discovered a proteasome cofactor, PafE, which is required for the normal growth, heat shock resistance, and full virulence of M. tuberculosis. In this study, we demonstrate that PafE influences this phenotype primarily by promoting the expression of protein chaperone genes that are necessary for surviving proteotoxic stress. PMID:28096448

  7. Secretion defects that activate the phage shock response of Escherichia coli.

    PubMed

    Jones, Susan E; Lloyd, Louise J; Tan, Kum K; Buck, Martin

    2003-11-01

    The phage shock protein (psp) operon of Escherichia coli is induced by membrane-damaging cues. Earlier studies linked defects in secretion across the inner membrane to induction of the psp response. Here we show that defects in yidC and sec secretion induce psp but that defects in tat and srp have no effect. We have also determined the cellular location of PspB and PspD proteins.

  8. Secretion Defects That Activate the Phage Shock Response of Escherichia coli

    PubMed Central

    Jones, Susan E.; Lloyd, Louise J.; Tan, Kum K.; Buck, Martin

    2003-01-01

    The phage shock protein (psp) operon of Escherichia coli is induced by membrane-damaging cues. Earlier studies linked defects in secretion across the inner membrane to induction of the psp response. Here we show that defects in yidC and sec secretion induce psp but that defects in tat and srp have no effect. We have also determined the cellular location of PspB and PspD proteins. PMID:14594846

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

    PubMed

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

    2012-06-01

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

  10. Pseudo-constitutivity of nitrate-responsive genes in nitrate reductase mutants.

    PubMed

    Schinko, Thorsten; Gallmetzer, Andreas; Amillis, Sotiris; Strauss, Joseph

    2013-05-01

    transporter-mediated NO₃⁻ accumulation in NR deficient mutants, originating from traces of nitrate in the media, is responsible for the constitutive expression of NirA-regulated genes, and the associated phenotype is thus termed "pseudo-constitutive". Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Pseudo-constitutivity of nitrate-responsive genes in nitrate reductase mutants

    PubMed Central

    Schinko, Thorsten; Gallmetzer, Andreas; Amillis, Sotiris; Strauss, Joseph

    2013-01-01

    that transporter-mediated NO3- accumulation in NR deficient mutants, originating from traces of nitrate in the media, is responsible for the constitutive expression of NirA-regulated genes, and the associated phenotype is thus termed “pseudo-constitutive”. PMID:23454548

  12. Cystic fibrosis-adapted Pseudomonas aeruginosa quorum sensing lasR mutants cause hyperinflammatory responses.

    PubMed

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

    2015-07-01

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

  13. Genetic analysis of rice mutants responsible for narrow leaf phenotype and reduced vein number.

    PubMed

    Kubo, Fumika Clara; Yasui, Yukiko; Kumamaru, Toshihiro; Sato, Yutaka; Hirano, Hiro-Yuki

    2017-03-17

    Leaves are a major site for photosynthesis and a key determinant of plant architecture. Rice produces thin and slender leaves, which consist of the leaf blade and leaf sheath separated by the lamina joint. Two types of vasculature, the large and small vascular bundles, run in parallel, together with a strong structure, the midrib. In this paper, we examined the function of four genes that regulate the width of the leaf blade and the vein number: NARROW LEAF1 (NAL1), NAL2, NAL3 and NAL7. We backcrossed original mutants of these genes with the standard wild-type rice, Taichung 65. We then compared the effect of each mutation on similar genetic backgrounds and examined genetic interactions of these genes. The nal1 single mutation and the nal2 nal3 double mutation showed a severe effect on leaf width, resulting in very narrow leaves. Although vein number was also reduced in the nal1 and nal2 nal3 mutants, the small vein number was more strongly reduced than the large vein number. In contrast, the nal7 mutation showed a milder effect on leaf width and vein number, and both the large and small veins were similarly affected. Thus, the genes responsible for narrow leaf phenotype seem to play distinct roles. The nal7 mutation showed additive effects on both leaf width and vein number, when combined with the nal1 single or the nal2 nal3 double mutation. In addition, observations of inner tissues revealed that cell differentiation was partially compromised in the nal2 nal3 nal7 mutant, consistent with the severe reduction in leaf width in this triple mutant.

  14. Response to photo-oxidative stress of Pseudomonas aeruginosa PAO1 mutants impaired in different functions.

    PubMed

    Orlandi, Viviana Teresa; Bolognese, Fabrizio; Martegani, Eleonora; Cantaluppi, Vincenzo; Medana, Claudio; Barbieri, Paola

    2017-10-12

    Clinicians often have to deal with infections that are difficult to control because they are caused by superbugs resistant to many antibiotics. Alternatives to antibiotic treatment include antimicrobial photodynamic therapy (aPDT). The photodynamic process causes bacterial death, inducing oxidative stress through the photoactivation of photosensitizer molecules in the presence of oxygen. No PDT-resistant bacteria have been selected to date, thus the response to photo-oxidative stress in non-phototrophic bacteria needs further investigation. The opportunistic pathogen Pseudomonas aeruginosa, in particular, has been shown to be more tolerant to PDT than other micro-organisms. In order to find any genetic determinants involved in PDT-tolerance, a panel of transposon mutants of P. aeruginosa PAO1 involved in the quorum sensing signalling system and membrane cytoplasmic transport were photoinactivated as part of this study. Two pseudomonas quinolone signalling (PQS) knock-out mutants, pqsH(-) and pqsC(-), were as PDT-sensitive as the PAO1 wild-type strains. Two PQS hyperproducer variants, pqsA(-) and rsaL(-), were shown to be more tolerant to photo-oxidative stress than the wild-type strain. In the pqsA(-) mutant, the hyperpigmentation due to the presence of phenazines could protect cells against PDT stress, while in rsaL(-) no pigmentation was detectable. Furthermore, a mutant impaired in an ATP-binding cassette transport involved in maintaining the asymmetry of the outer membrane was significantly more tolerant to photo-oxidative stress than the wild-type strain. These observations support the involvement of quorum sensing and the importance of the bacterial cell envelope when dealing with photo-oxidative stress induced by photodynamic treatment.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  17. Abscisic acid-deficient sit tomato mutant responses to cadmium-induced stress.

    PubMed

    Pompeu, Georgia B; Vilhena, Milca B; Gratão, Priscila L; Carvalho, Rogério F; Rossi, Mônica L; Martinelli, Adriana P; Azevedo, Ricardo A

    2017-03-01

    There is a very effective cross-talk between signals triggered by reactive oxygen species and hormonal responses in plants, activating proteins/enzymes likely to be involved in stress tolerance. Abscisic acid (ABA) is known as a stress hormone that takes part in the integration of signals. This work aimed to characterize the biochemical response and ultrastructural changes induced by cadmium (Cd) in the Micro-Tom (MT) sitiens ABA-deficient mutant (sit) and its wild-type (MT) counterpart. MT and sit plants were grown over a 96-h period in the presence of Cd (0, 10, and 100 μM CdCl2). The overall results indicated increases in lipid peroxidation, hydrogen peroxide content and in the activities of the key antioxidant enzymes such as catalase, glutathione reductase, and ascorbate peroxidase in both genotypes. On the other hand, no alteration was observed in chlorophyll content, while the activity of another antioxidant enzyme, superoxide dismutase, remained constant or even decreased in the presence of Cd. Roots and shoots of the sit mutant and MT were analyzed by light and transmission electron microscopy in order to characterize the structural changes caused by the exposure to this metal. Cd caused a decrease in intercellular spaces in shoots and a decrease in cell size in roots of both genotypes. In leaves, Cd affected organelle shape and internal organization of the thylakoid membranes, whereas noticeable increase in the number of mitochondria and vacuoles in MT and sit roots were observed. These results add new information that should help unravel the relative importance of ABA in regulating the cell responses to stressful conditions induced by Cd apart from providing the first characterization of this mutant to oxidative stress.

  18. Characterization of a temperature-sensitive mutant of mouse FM3A cells defective in DNA replication.

    PubMed Central

    Murakami, Y; Yasuda, H; Miyazawa, H; Hanaoka, F; Yamada, M

    1985-01-01

    The characterization of a temperature-sensitive mutant (tsFT20 strain, dnats) of mouse FM3A cells is reported. After incubation of tsFT20 cells at the nonpermissive temperature (39 degrees C), DNA synthesis ceased with little change in either RNA or protein synthesis. Flow-microfluorometric analysis revealed that the cell cycle of tsFT20 cells grown at 39 degrees C for 16 hr was similar to that of wild-type cells that were synchronized at the G1/S boundary and at S phase by treatment with aphidicolin, a specific inhibitor of DNA polymerase alpha. The DNA polymerase alpha activity of tsFT20 cells measured in crude cell extracts or in purified preparations was inactivated more rapidly at 39 degrees C than the activity of wild-type cells. In the growth revertants of the tsFT20 cell strain, the heat lability of DNA polymerase alpha decreased. These data suggest that tsFT20 is a temperature-sensitive mutant of DNA polymerase alpha or of a factor associated with DNA polymerase alpha that is essential for its activity. PMID:3856858

  19. bioA mutant of Mycobacterium tuberculosis shows severe growth defect and imparts protection against tuberculosis in guinea pigs

    PubMed Central

    Kar, Ritika; Nangpal, Prachi; Mathur, Shubhita; Singh, Swati

    2017-01-01

    Owing to the devastation caused by tuberculosis along with the unsatisfactory performance of the Bacillus Calmette–Guérin (BCG) vaccine, a more efficient vaccine than BCG is required for the global control of tuberculosis. A number of studies have demonstrated an essential role of biotin biosynthesis in the growth and survival of several microorganisms, including mycobacteria, through deletion of the genes involved in de novo biotin biosynthesis. In this study, we demonstrate that a bioA mutant of Mycobacterium tuberculosis (MtbΔbioA) is highly attenuated in the guinea pig model of tuberculosis when administered aerogenically as well as intradermally. Immunization with MtbΔbioA conferred significant protection in guinea pigs against an aerosol challenge with virulent M. tuberculosis, when compared with the unvaccinated animals. Booster immunization with MtbΔbioA offered no advantage over a single immunization. These experiments demonstrate the vaccinogenic potential of the attenuated M. tuberculosis bioA mutant against tuberculosis. PMID:28658275

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

    SciTech Connect

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

    1996-02-20

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

  1. bioA mutant of Mycobacterium tuberculosis shows severe growth defect and imparts protection against tuberculosis in guinea pigs.

    PubMed

    Kar, Ritika; Nangpal, Prachi; Mathur, Shubhita; Singh, Swati; Tyagi, Anil K

    2017-01-01

    Owing to the devastation caused by tuberculosis along with the unsatisfactory performance of the Bacillus Calmette-Guérin (BCG) vaccine, a more efficient vaccine than BCG is required for the global control of tuberculosis. A number of studies have demonstrated an essential role of biotin biosynthesis in the growth and survival of several microorganisms, including mycobacteria, through deletion of the genes involved in de novo biotin biosynthesis. In this study, we demonstrate that a bioA mutant of Mycobacterium tuberculosis (MtbΔbioA) is highly attenuated in the guinea pig model of tuberculosis when administered aerogenically as well as intradermally. Immunization with MtbΔbioA conferred significant protection in guinea pigs against an aerosol challenge with virulent M. tuberculosis, when compared with the unvaccinated animals. Booster immunization with MtbΔbioA offered no advantage over a single immunization. These experiments demonstrate the vaccinogenic potential of the attenuated M. tuberculosis bioA mutant against tuberculosis.

  2. Mechanisms Responsible for a ΦX174 Mutant's Ability To Infect Escherichia coli by Phosphorylation▿

    PubMed Central

    Cox, Jennifer; Putonti, Catherine

    2010-01-01

    The ability for a virus to expand its host range is dependent upon a successful mode of viral entry. As such, the host range of the well-studied ΦX174 bacteriophage is dictated by the presence of a particular lipopolysaccharide (LPS) on the bacterial surface. The mutant ΦX174 strain JACS-K, unlike its ancestor, is capable of infecting both its native host Escherichia coli C and E. coli K-12, which does not have the necessary LPS. The conversion of an alanine to a very reactive threonine on its virion surface was found to be responsible for the strain's expanded host range. PMID:20147402

  3. Mechanisms responsible for a PhiX174 mutant's ability to infect Escherichia coli by phosphorylation.

    PubMed

    Cox, Jennifer; Putonti, Catherine

    2010-05-01

    The ability for a virus to expand its host range is dependent upon a successful mode of viral entry. As such, the host range of the well-studied PhiX174 bacteriophage is dictated by the presence of a particular lipopolysaccharide (LPS) on the bacterial surface. The mutant PhiX174 strain JACS-K, unlike its ancestor, is capable of infecting both its native host Escherichia coli C and E. coli K-12, which does not have the necessary LPS. The conversion of an alanine to a very reactive threonine on its virion surface was found to be responsible for the strain's expanded host range.

  4. Photoelectric response of the N intermediate of bacteriorhodopsin and its mutant T46V.

    PubMed

    Tóth-Boconádi, R; Szabó-Nagy, A; Taneva, S G; Keszthelyi, L

    1999-10-01

    Double flash experiments were performed in order to gain information about the characteristics of the N intermediates of the photocycle of bacteriorhodopsin. The N intermediates of wild-type bacteriorhodopsin and mutant T46V were excited at different delay times after the first laser flash which induced the photocycle and the electric responses were registered. These electric signals revealed that charge motions occurred in both cases, though charge translocation, i.e. H(+) pumping, could not be observed. The delay time dependence of the electric signals is characterized by two distinct processes corresponding to two substates of the N intermediates.

  5. Association of Constitutive Hyperphosphorylation of Hsf1p with a Defective Ethanol Stress Response in Saccharomyces cerevisiae Sake Yeast Strains

    PubMed Central

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

    2012-01-01

    Modern sake yeast strains, which produce high concentrations of ethanol, are unexpectedly sensitive to environmental stress during sake brewing. To reveal the underlying mechanism, we investigated a well-characterized yeast stress response mediated by a heat shock element (HSE) and heat shock transcription factor Hsf1p in Saccharomyces cerevisiae sake yeast. The HSE-lacZ activity of sake yeast during sake fermentation and under acute ethanol stress was severely impaired compared to that of laboratory yeast. Moreover, the Hsf1p of modern sake yeast was highly and constitutively hyperphosphorylated, irrespective of the extracellular stress. Since HSF1 allele replacement did not significantly affect the HSE-mediated ethanol stress response or Hsf1p phosphorylation patterns in either sake or laboratory yeast, the regulatory machinery of Hsf1p is presumed to function differently between these types of yeast. To identify phosphatases whose loss affected the control of Hsf1p, we screened a series of phosphatase gene deletion mutants in a laboratory strain background. Among the 29 mutants, a Δppt1 mutant exhibited constitutive hyperphosphorylation of Hsf1p, similarly to the modern sake yeast strains, which lack the entire PPT1 gene locus. We confirmed that the expression of laboratory yeast-derived functional PPT1 recovered the HSE-mediated stress response of sake yeast. In addition, deletion of PPT1 in laboratory yeast resulted in enhanced fermentation ability. Taken together, these data demonstrate that hyperphosphorylation of Hsf1p caused by loss of the PPT1 gene at least partly accounts for the defective stress response and high ethanol productivity of modern sake yeast strains. PMID:22057870

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

    PubMed

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

    2012-01-01

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

  7. The defective seed5 (des5) mutant: effects on barley seed development and HvDek1, HvCr4, and HvSal1 gene regulation.

    PubMed

    Olsen, Lene T; Divon, Hege H; Al, Ronald; Fosnes, Kjetil; Lid, Stein Erik; Opsahl-Sorteberg, Hilde-Gunn

    2008-01-01

    Barley, one of the major small grain crops, is especially important in climatically demanding agricultural areas of the world, with multiple uses within food, feed, and beverage. The barley endosperm is further of special scientific interest due to its three aleurone cell layers, with the potential of bringing forward the molecular understanding of seed development and cell specification from Arabidopsis and maize. Work done in Arabidopsis and maize indicate the presence of conserved seed developmental pathways where Crinkly4 (Cr4), Defective kernel1 (Dek1), and Supernumerary aleurone layer1 (Sal1) are key players. With the use of microscopy, a comprehensive phenotypic characterization of the barley defective seed5 (des5) mutant is presented here. The analysis further extends to molecular quantification of gene expression changes in the des5 mutant by qRT-PCR. Moreover, full-length genomic sequences of the barley orthologues were generated and these were annotated as HvDek1, HvCr4, and HvSal1. The most striking results in this study are the patchy reduction in number of aleurone cells, rudimentary anticlinal aleurone cell walls, and the specific change of HvCr4 expression compared to HvDek1 and HvSal1. The data presented support the involvement of Hvdes5 in establishing aleurone cells. Finally, how these results might affect the current model of aleurone and epidermal cell identity and development is discussed with a speculation regarding a possible role of Des5 in regulating cell division/ secondary cell wall building.

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

    PubMed

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

    2001-05-01

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

  9. Siderophore production of a periplasmic transport binding protein kinase gene defective mutant of Magnetospirillum magneticum AMB-1.

    PubMed

    Calugay, Ronie J; Okamura, Yoshiko; Wahyudi, Aris Tri; Takeyama, Haruko; Matsunaga, Tadashi

    2004-10-22

    A non-magnetic mutant, NMA61, of the magnetic bacterium Magnetospirillum magneticum AMB-1 was generated by transposon mutagenesis to identify genes involved in magnetosome synthesis. The genomic region of NMA61 interrupted by a Mini-Tn5 transposon was analyzed. The transposon was inserted in an open reading frame (ORF) coding for a periplasmic transport binding protein kinase gene homologue. Three adjacent ORFs and a promoter were identified upstream, indicating that the sequences comprised an operon. Phenotype characterizations showed that the growth inhibition imposed by the exogenous non-assimilable iron chelator nitrilotriacetate was relieved in wild type but not in NMA61, by the addition of the isolated wild type siderophore. Higher concentration of siderophores accumulated in the culture medium of NMA61 than in wild type. These data suggest that the interrupted periplasmic transport binding protein kinase gene homologue is required for siderophore transport into M. magneticum AMB-1.

  10. In vivo and in vitro analysis of ptl1, a yeast ts mutant with a membrane-associated defect in protein translocation.

    PubMed Central

    Toyn, J; Hibbs, A R; Sanz, P; Crowe, J; Meyer, D I

    1988-01-01

    Mutants defective in the ability to translocate proteins across the membrane of the endoplasmic reticulum were selected in Trp- Saccharomyces cerevisiae on the basis of their ability to retain a fusion protein in the cytosol. The fusion comprised the prepro region of prepro-alpha-factor (MF alpha 1) N-terminal to phosphoribosyl anthranilate isomerase (TRP1). The first of the protein translocation mutations, called ptl1, results in temperature-sensitivity of growth and protein translocation. At the non-permissive temperature, precursors to several secretory proteins accumulate in the cytosol. Using this mutant, we demonstrate that the prepro-carboxypeptidase Y that had been accumulated in the cytosol at the non-permissive temperature could be post-translationally translocated into the endoplasmic reticulum when cells were returned to the permissive temperature. This result indicates that post-translational translocation of preproteins across endoplasmic reticulum membranes can occur in vivo. We have also determined that the temperature-sensitive component is membrane-associated in ptl1, and that the membranes derived from this strain show a reversible temperature-sensitive translocation phenotype in vitro. Images PMID:3072198

  11. Iron Inefficiency in Maize Mutant ys1 (Zea mays L. cv Yellow-Stripe) Is Caused by a Defect in Uptake of Iron Phytosiderophores.

    PubMed Central

    Von Wiren, N.; Mori, S.; Marschner, H.; Romheld, V.

    1994-01-01

    To determine the Fe inefficiency factors in the maize mutant ys1 (Zea mays L. cv Yellow Stripe), root exudates of Fe-inefficient ys1 and of two Fe-efficient maize cultivars (Alice, WF9) were collected in axenic nutrient solution cultures. Analysis by thin-layer chromatography and high-performance liquid chromatography revealed that under Fe deficiency ys1 released the phytosiderophore 2[prime]-deoxymugineic acid (DMA) in quantities similar to those of Alice and WF9. Under nonaxenic conditions, DMA released by plants of all three cultivars was rapidly decomposed by microorganisms in the nutrient solution. Uptake experiments with 59Fe-labeled DMA, purified from root exudates of either Fe-deficient Alice or ys1 plants, showed up to 20 times lower uptake and translocation of 59Fe in ys1 than in Alice or WF9 plants. The presence of microorganisms during preculture and short-term uptake experiments had no significant effect on uptake and translocation rates of 59Fe in Alice and ys1 plants. We conclude that Fe inefficiency in the maize mutant ys1 is the result of a defect in the uptake system for Fe-phytosiderophores. PMID:12232304

  12. Sugar Accumulation in Leaves of Arabidopsis sweet11/sweet12 Double Mutants Enhances Priming of the Salicylic Acid-Mediated Defense Response

    PubMed Central

    Gebauer, Pierre; Korn, Martin; Engelsdorf, Timo; Sonnewald, Uwe; Koch, Christian; Voll, Lars M.

    2017-01-01

    sweet11/sweet12 leaves were highly significantly enriched for several GO terms associated with SA signaling and response compared to mock treated wild-type leaves, indicating sugar-mediated priming of the SA pathway in the double mutant. Infection assays with salicylic acid deficient sweet11/sweet12/sid2 triple mutants demonstrated that reduced susceptibility observed in sweet11/sweet12 was entirely dependent on the SA pathway. We suggest a model how defects in phloem loading of sucrose can influence SA priming and hence, compatibility. PMID:28848581

  13. Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair*

    PubMed Central

    Bélanger, François; Angers, Jean-Philippe; Fortier, Émile; Hammond-Martel, Ian; Costantino, Santiago; Drobetsky, Elliot; Wurtele, Hugo

    2016-01-01

    Nucleotide excision repair (NER) is a highly conserved pathway that removes helix-distorting DNA lesions induced by a plethora of mutagens, including UV light. Our laboratory previously demonstrated that human cells deficient in either ATM and Rad3-related (ATR) kinase or translesion DNA polymerase η (i.e. key proteins that promote the completion of DNA replication in response to UV-induced replicative stress) are characterized by profound inhibition of NER exclusively during S phase. Toward elucidating the mechanistic basis of this phenomenon, we developed a novel assay to quantify NER kinetics as a function of cell cycle in the model organism Saccharomyces cerevisiae. Using this assay, we demonstrate that in yeast, deficiency of the ATR homologue Mec1 or of any among several other proteins involved in the cellular response to replicative stress significantly abrogates NER uniquely during S phase. Moreover, initiation of DNA replication is required for manifestation of this defect, and S phase NER proficiency is correlated with the capacity of individual mutants to respond to replicative stress. Importantly, we demonstrate that partial depletion of Rfa1 recapitulates defective S phase-specific NER in wild type yeast; moreover, ectopic RPA1–3 overexpression rescues such deficiency in either ATR- or polymerase η-deficient human cells. Our results strongly suggest that reduction of NER capacity during periods of enhanced replicative stress, ostensibly caused by inordinate sequestration of RPA at stalled DNA replication forks, represents a conserved feature of the multifaceted eukaryotic DNA damage response. PMID:26578521

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

    PubMed

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

    2012-02-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

  17. Analysis of the Ethylene Response in the epinastic Mutant of Tomato1

    PubMed Central

    Barry, Cornelius S.; Fox, Elizabeth A.; Yen, Hsiao-ching; Lee, Sanghyeob; Ying, Tie-jin; Grierson, Donald; Giovannoni, James J.

    2001-01-01

    Ethylene can alter plant morphology due to its effect on cell expansion. The most widely documented example of ethylene-mediated cell expansion is promotion of the “triple response” of seedlings grown in the dark in ethylene. Roots and hypocotyls become shorter and thickened compared with controls due to a reorientation of cell expansion, and curvature of the apical hook is more pronounced. The epinastic (epi) mutant of tomato (Lycopersicon esculentum) has a dark-grown seedling phenotype similar to the triple response even in the absence of ethylene. In addition, in adult plants both the leaves and the petioles display epinastic curvature and there is constitutive expression of an ethylene-inducible chitinase gene. However, petal senescence and abscission and fruit ripening are all normal in epi. A double mutant (epi/epi;Nr/Nr) homozygous for both the recessive epi and dominant ethylene-insensitive Never-ripe loci has the same dark-grown seedling and vegetative phenotypes as epi but possesses the senescence and ripening characteristics of Never-ripe. These data suggest that a subset of ethylene responses controlling vegetative growth and development may be constitutively activated in epi. In addition, the epi locus has been placed on the tomato RFLP map on the long arm of chromosome 4 and does not demonstrate linkage to reported tomato CTR1 homologs. PMID:11553734

  18. Physical and transcriptional map of a 3-Mb region of mouse chromosome 1 containing the gene for the neural tube defect mutant loop-tail (Lp).

    PubMed

    Eddleston, J; Murdoch, J N; Copp, A J; Stanier, P

    1999-03-01

    The Lp mouse mutant provides a model for the severe human neural tube defect (NTD), cranio-rachischisis. To identify the Lp gene, a positional cloning approach has been adopted. Previously, linkage analysis in a large intraspecific backcross was used to map the Lp locus to distal mouse chromosome 1. Here we report a detailed physical map of this region. The interval surrounding Lp has been cloned in a yeast artificial chromosome (YAC) contig consisting of 63 clones spanning approximately 3.2 Mb. Fifty sequence tagged sites (STSs) have been used to construct the contig and establish marker order across the interval. Based on the high level of conserved synteny between distal mouse chromosome 1 and human 1q21-q24, many of these STSs were designed from expressed sequences identified by cross-screening human and mouse databases of expressed sequence tags. Added to other known genes in the region, a total of 29 genes were located and ordered within the contig. Seven novel polymorphisms were identified within the region, allowing refinement of the genetic map and a reduction in the size of the physical interval containing the Lp gene. The Lp interval, between D1Mit113 and Tagln2, can be spanned by two nonchimeric overlapping YACs that define a physical distance of approximately 1 Mb. Within this region, 10 potential candidate genes have been mapped. The materials and genes described here will provide a resource for the identification and further study of the mutated Lp gene that causes this severe neural tube defect and will provide candidates for other defects known to map to the homologous region on human chromosome 1q.

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

    PubMed

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

    2006-09-01

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

  20. The altered gravitropic response of the lazy-2 mutant of tomato is phytochrome regulated.

    PubMed Central

    Gaiser, J C; Lomax, T L

    1993-01-01

    Shoots of the lazy-2 (lz-2) gravitropic mutant of tomato (Lycopersicon esculentum Mill.) have a normal gravitropic response when grown in the dark, but grow downward in response to gravity when grown in the light. Experiments were undertaken to investigate the nature of the light induction of the downward growth of lz-2 shoots. Red light was effective at causing downward growth of hypocotyls of lz-2 seedlings, whereas treatment with blue light did not alter the dark-grown (wild-type) gravity response. Downward growth of lz-2 seedlings is greatest 16 h after a 1-h red light irradiation, after which the seedlings begin to revert to the dark-grown phenotype. lz-2 seedlings irradiated with a far-red light pulse immediately after a red light pulse exhibited no downward growth. However, continuous red or far-red light both resulted in downward growth of lz-2 seedlings. Thus, the light induction of downward growth of lz-2 appears to involve the photoreceptor phytochrome. Fluence-response experiments indicate that the induction of downward growth of lz-2 by red light is a low-fluence phytochrome response, with a possible high-irradiance response component. PMID:11536545

  1. The altered gravitropic response of the lazy-2 mutant of tomato is phytochrome regulated.

    PubMed

    Gaiser, J C; Lomax, T L

    1993-06-01

    Shoots of the lazy-2 (lz-2) gravitropic mutant of tomato (Lycopersicon esculentum Mill.) have a normal gravitropic response when grown in the dark, but grow downward in response to gravity when grown in the light. Experiments were undertaken to investigate the nature of the light induction of the downward growth of lz-2 shoots. Red light was effective at causing downward growth of hypocotyls of lz-2 seedlings, whereas treatment with blue light did not alter the dark-grown (wild-type) gravity response. Downward growth of lz-2 seedlings is greatest 16 h after a 1-h red light irradiation, after which the seedlings begin to revert to the dark-grown phenotype. lz-2 seedlings irradiated with a far-red light pulse immediately after a red light pulse exhibited no downward growth. However, continuous red or far-red light both resulted in downward growth of lz-2 seedlings. Thus, the light induction of downward growth of lz-2 appears to involve the photoreceptor phytochrome. Fluence-response experiments indicate that the induction of downward growth of lz-2 by red light is a low-fluence phytochrome response, with a possible high-irradiance response component.

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

    PubMed Central

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

    2012-01-01

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

  3. Construction of "Toxin Complex" in a Mutant Serotype C Strain of Clostridium botulinum Harboring a Defective Neurotoxin Gene.

    PubMed

    Suzuki, Tomonori; Nagano, Thomas; Niwa, Koichi; Uchino, Masataka; Tomizawa, Motohiro; Sagane, Yoshimasa; Watanabe, Toshihiro

    2017-01-01

    A non-toxigenic mutant of the toxigenic serotype C Clostridium botulinum strain Stockholm (C-St), C-N71, does not produce the botulinum neurotoxin (BoNT). However, the original strain C-St produces botulinum toxin complex, in which BoNT is associated with non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin proteins (HA-70, HA-33, and HA-17). Therefore, in this study, we aimed to elucidate the effects of bont gene knockout on the formation of the "toxin complex." Nucleotide sequence analysis revealed that a premature stop codon was introduced in the bont gene, whereas other genes were not affected by this mutation. Moreover, we successfully purified the "toxin complex" produced by C-N71. The "toxin complex" was identified as a mixture of NTNHA/HA-70/HA-17/HA-33 complexes with intact NTNHA or C-terminally truncated NTNHA, without BoNT. These results indicated that knockout of the bont gene does not affect the formation of the "toxin complex." Since the botulinum toxin complex has been shown to play an important role in oral toxin transport in the human and animal body, a non-neurotoxic "toxin complex" of C-N71 may be valuable for the development of an oral drug delivery system.

  4. Suppressors of spindle checkpoint defect (such) mutants identify new mdf-1/MAD1 interactors in Caenorhabditis elegans.

    PubMed

    Tarailo, Maja; Kitagawa, Risa; Rose, Ann M

    2007-04-01

    The spindle assembly checkpoint (SAC) governs the timing of metaphase-to-anaphase transition and is essential for genome stability. The Caenorhabditis elegans mutant strain gk2 carries a deletion within